Copper-induced Vascular Endothelial Growth Factor Expression and Wound Healing

American Journal of Physiology. Heart and Circulatory Physiology. May, 2002  |  Pubmed ID: 11959648

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. Whereas a direct role of copper to facilitate angiogenesis has been evident two decades ago, the specific targets of copper action remained unclear. This report presents first evidence showing that inducible VEGF expression is sensitive to copper and that the angiogenic potential of copper may be harnessed to accelerate dermal wound contraction and closure. At physiologically relevant concentrations, copper sulfate induced VEGF expression in primary as well as transformed human keratinocytes. Copper shared some of the pathways utilized by hypoxia to regulate VEGF expression. Topical copper sulfate accelerated closure of excisional murine dermal wound allowed to heal by secondary intention. Copper-sensitive pathways regulate key mediators of wound healing such as angiogenesis and extracellular matrix remodeling. Copper-based therapeutics represents a feasible approach to promote dermal wound healing.

Flavonoids Differentially Regulate IFN Gamma-induced ICAM-1 Expression in Human Keratinocytes: Molecular Mechanisms of Action

FEBS Letters. Jun, 2002  |  Pubmed ID: 12044887

The effect of plant flavonoids on intercellular adhesion molecule-1 (ICAM-1) expression in human keratinocyte was investigated. ICAM-1 is known to mediate skin inflammation. Among the flavonoids tested, taxifolin was the most potent in inhibiting interferon gamma (IFN gamma)-induced ICAM-1 protein as well as mRNA expression in human keratinocytes. Much smaller dosages of taxifolin were required in primary keratinocytes compared to HaCaT (immortalized cell) to achieve similar levels of inhibition in the inducible ICAM-1 expression. Regulation of inducible ICAM-1 expression by taxifolin was at transcriptional level by inhibiting the activation of signal transducers and activators of transcription (STAT)1 and protein tyrosine phosphorylation of Janus kinase (JAK)1 suggesting that the JAK-STAT pathway may be the molecular site of action of taxifolin. Finally, taxifolin pre-treatment also potently inhibited IFN gamma-induced ICAM-1 expression in a reconstructed human skin equivalent suggesting therapeutic potential of taxifolin in skin pathological conditions related to increased cell adhesion and inflammation.

Oxidant-induced Vascular Endothelial Growth Factor Expression in Human Keratinocytes and Cutaneous Wound Healing

The Journal of Biological Chemistry. Sep, 2002  |  Pubmed ID: 12068011

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H(2)O(2) potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(2)O(2) response. It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.

Oxygen, Oxidants, and Antioxidants in Wound Healing: an Emerging Paradigm

Annals of the New York Academy of Sciences. May, 2002  |  Pubmed ID: 12074976

Disrupted vasculature and high energy-demand by regenerating tissue results in wound hypoxia. Wound repair may be facilitated by oxygen therapy. Evidence supporting the mode of action of hyperbaric oxygen in promoting wound healing is sketchy, however. Topical oxygen therapy involves local administration of pure oxygen. The advantages of topical oxygen therapy include low cost, the lack of systemic oxygen toxicity, and possibility of home treatment. While this modality of wound care is of outstanding interest, it clearly lacks the support of mechanism-oriented studies. The search for mechanisms by which oxygen supports wound healing has now taken another step. Respiratory burst-derived oxidants support healing. Oxidants serve as cellular messengers to promote healing. Although this information is of outstanding significance to the practice of oxygen therapy, it remains largely unexplored. The search for "natural remedies" has drawn attention to herbals. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. Proanthocyanidins and other tannins facilitate wound healing. A combination of grape seed proanthocyanidin extract and resveratrol facilitates inducible VEGF expression, a key element supporting wound angiogenesis. Strategies to manipulate the redox environment in the wound are likely to be of outstanding significance in wound healing.

Cellular Protection with Proanthocyanidins Derived from Grape Seeds

Annals of the New York Academy of Sciences. May, 2002  |  Pubmed ID: 12074978

Grape seed proanthocyanidins have been reported to possess a broad spectrum of pharmacological and medicinal properties against oxidative stress. We have demonstrated that IH636 proanthocyanidin extract (GSPE) provides excellent protection against free radicals in both in vitro and in vivo models. GSPE had significantly better free radical scavenging ability than vitamins C, E and beta-carotene and demonstrated significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal cells. GSPE protected against tobacco-induced apoptotic cell death in human oral keratinocytes and provided protection against cancer chemotherapeutic drug-induced cytotoxicity in human liver cells by modulating cell cycle/apoptosis regulatory genes such as bcl2, p53 and c-myc. Recently, the bioavailability and mechanistic pathways of cytoprotection by GSPE were examined on acetaminophen-induced hepatotoxicity and nephrotoxicity, amiodarone-induced pulmonary toxicity, doxorubicin-induced cardiotoxicity, DMN-induced immunotoxicity and MOCAP-induced neurotoxicity in mice. Serum chemistry changes, integrity of genomic DNA and histopathology were assessed. GSPE pre-exposure provided near complete protection in terms of serum chemistry changes and DNA damage, as well as abolished apoptotic and necrotic cell death in all tissues. Histopathological examination reconfirmed these findings. GSPE demonstrated concentration-/dose-dependent inhibitory effects on the drug metabolizing enzyme cytochrome P450 2E1, and this may be a major pathway for the anti-toxic potential exerted by GSPE. Furthermore, GSPE treatment significantly decreased TNFalpha-induced adherence of T-cells to HUVEC by inhibiting VCAM-1 expression. These results demonstrate that GSPE is highly bioavailable and may serve as a potential therapeutic tool in protecting multiple target organs from structurally diverse drug- and chemical-induced toxicity.

Functional Genomics: High-density Oligonucleotide Arrays

Methods in Enzymology. 2002  |  Pubmed ID: 12078521

Glutamate-induced C-Src Activation in Neuronal Cells

Methods in Enzymology. 2002  |  Pubmed ID: 12125347

Simultaneous Detection of Tocopherols and Tocotrienols in Biological Samples Using HPLC-coulometric Electrode Array

Methods in Enzymology. 2002  |  Pubmed ID: 12125359

Hemangioma Model for in Vivo Angiogenesis: Inducible Oxidative Stress and MCP-1 Expression in EOMA Cells

Methods in Enzymology. 2002  |  Pubmed ID: 12125369

Dermal Wound Healing Properties of Redox-active Grape Seed Proanthocyanidins

Free Radical Biology & Medicine. Oct, 2002  |  Pubmed ID: 12374620

Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. The wound site is rich in oxidants, such as hydrogen peroxide, mostly contributed by neutrophils and macrophages. We proposed that oxidants in the wound microenvironment support the repair process. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. Previously we have reported that a grape seed proanthycyanidin extract containing 5000 ppm resveratrol (GSPE) potently upregulates oxidant and tumor necrosis factor-alpha inducible VEGF expression in human keratinocytes (Free Radic. Biol. Med. 31:38-42, 2001). Our current objective was to follow up on that finding and test whether GSPE influences dermal wound healing in vivo. First, using a VEGF promoter-driven luciferase reporter construct we observed that the potentiating effect of GSPE on inducible VEGF expression is at the transcriptional level. The reporter assay showed that GSPE alone is able to drive VEGF transcription. Next, two dermal excisional wounds were inflicted on the back of mice and the wounds were left to heal by secondary intention. Topical application of GSPE accelerated wound contraction and closure. GSPE treatment was associated with a more well-defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. GSPE treatment also increased VEGF and tenascin expression in the wound edge tissue. Tissue glutathione oxidation and 4-hydroxynonenal immunostaining results supported that GSPE application enhanced the oxidizing environment at the wound site. Oxidants are known to promote both VEGF as well as tenascin expression. In summary, our current study provides firm evidence to support that topical application of GSPE represents a feasible and productive approach to support dermal wound healing.

Vitamin E Sensitive Genes in the Developing Rat Fetal Brain: a High-density Oligonucleotide Microarray Analysis

FEBS Letters. Oct, 2002  |  Pubmed ID: 12387859

Vitamin E (tocopherols and tocotrienols) is essential for normal neurological function. Recently we have reported that the neuroprotective properties of tocotrienols are much more potent than that of the widely studied tocopherols (Sen, C.K., Khanna, S., Roy, S. and Parker, L. (2000) J. Biol. Chem. 275, 13049-13055). The objective of this study was to evaluate whether (i) oral supplementation of tocotrienols during pregnancy is bioavailable to fetal and mother brains; (ii) short-term change in dietary vitamin E levels of pregnant rats influences gene expression profile of developing fetal brains. We report that dietary tocotrienol is bioavailable to both mother and fetal brains. The enrichment is more in fetal brain tissue. Using a GeneChip microarray expression profiling approach we have identified a specific set of vitamin E sensitive genes in the developing rat fetal brain.

Anti-angiogenic Property of Edible Berries

Free Radical Research. Sep, 2002  |  Pubmed ID: 12448828

Recent studies show that edible berries may have potent chemopreventive properties. Anti-angiogenic approaches to prevent and treat cancer represent a priority area in investigative tumor biology. Vascular endothelial growth factor (VEGF) plays a crucial role for the vascularization of tumors. The vasculature in adult skin remains normally quiescent. However, skin retains the capacity for brisk initiation of angiogenesis during inflammatory skin diseases such as psoriasis and skin cancers. We sought to test the effects of multiple berry extracts on inducible VEGF expression by human HaCaT keratinocytes. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seed, and strawberry) and a grape seed proanthocyanidin extract (GSPE) were studied. The extracts and uptake of their constituents by HaCaT were studied using a multi-channel HPLC-CoulArray approach. Antioxidant activity of the extracts was determined by ORAC. Cranberry, elderberry and raspberry seed samples were observed to possess comparable ORAC values. The antioxidant capacity of these samples was significantly lower than that of the other samples studied. The ORAC values of strawberry powder and GSPE were higher than cranberry, elderberry or raspberry seed but significantly lower than the other samples studied. Wild bilberry and blueberry extracts possessed the highest ORAC values. Each of the berry samples studied significantly inhibited both H2O2 as well as TNF alpha induced VEGF expression by the human keratinocytes. This effect was not shared by other antioxidants such as alpha-tocopherol or GSPE but was commonly shared by pure flavonoids. Matrigel assay using human dermal microvascular endothelial cells showed that edible berries impair angiogenesis.

Presentation of Nitric Oxide Regulates Monocyte Survival Through Effects on Caspase-9 and Caspase-3 Activation

The Journal of Biological Chemistry. Apr, 2003  |  Pubmed ID: 12566444

In the absence of survival factors, blood monocytes undergo spontaneous apoptosis, which involves the activation of caspase-3. Although nitric oxide can block caspase-3 activation and promote cell survival, it can also induce apoptosis. We hypothesized that nitrosothiols that promote protein S-nitrosylation would reduce caspase-3 activation and cell survival, whereas nitric oxide donors (such as 1-propamine 3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA) NONOate and diethylamine (DEA) NONOate) that do not target thiol residues would not. Using human monocytes as a model, we observed that nitrosothiol donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine suppressed caspase-9 and caspase-3 activity and DNA fragmentation. In contrast, PAPA or DEA NONOate did not promote monocyte survival events and appeared to inhibit monocyte survival induced by macrophage colony-stimulating factor. The caspase-3-selective inhibitor DEVD-fluoromethyl ketone reversed DNA fragmentation events, and the caspase-9 inhibitor LEHD-fluoromethyl ketone reversed caspase-3 activity in monocytes treated with PAPA or DEA NONOate in the presence of macrophage colony-stimulating factor. These results were not caused by differences in glutathione levels or the kinetics of nitric oxide release. Moreover, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine directly blocked the activity of recombinant caspase-3, which was reversed by the reducing agent dithiothreitol, whereas PAPA or DEA NONOate did not block the enzymatic activity of caspase-3. These data support the hypothesis that nitrosylation of protein thiol residues by nitric oxide is critical for promoting the survival of human monocytes.

Oxygen Sensing by Primary Cardiac Fibroblasts: a Key Role of P21(Waf1/Cip1/Sdi1)

Circulation Research. Feb, 2003  |  Pubmed ID: 12595337

In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to approximately 1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the PO2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the PO2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-beta1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-beta1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

Molecular Mechanisms of Cardioprotection by a Novel Grape Seed Proanthocyanidin Extract

Mutation Research. Feb-Mar, 2003  |  Pubmed ID: 12628506

Free radicals and oxidative stress play a crucial role in the pathophysiology of a broad spectrum of cardiovascular diseases including congestive heart failure, valvular heart disease, cardiomyopathy, hypertrophy, atherosclerosis and ischemic heart disease. We have demonstrated that IH636 grape seed proanthocyanidin extract (GSPE) provides superior antioxidant efficacy as compared to Vitamins C, E and beta-carotene. A series of studies were conducted using GSPE to demonstrate its cardioprotective ability in animals and humans. GSPE supplementation improved cardiac functional assessment including post-ischemic left ventricular function, reduced myocardial infarct size, reduced ventricular fibrillation (VF) and tachycardia, decreased the amount of reactive oxygen species (ROS) as detected by ESR spectroscopy and reduced malondialdehyde (MDA) formation in the heart perfusate. Cardiomyocyte apoptosis detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining. In concert, the proapoptotic signals mediated by JNK-l and c-fos proteins were also reduced suggesting that the novel cardioprotective properties of GSPE may be at least partially attributed to its ability to block anti-death signaling mediated through the proapoptotic transcription factors and genes such as JNK-1 and c-JUN. In a separate study, GSPE pretreatment significantly inhibited doxorubicin-induced cardiotoxicity as demonstrated by reduced serum creatine kinase (CK) activity, DNA damage and histopathological changes in the cardiac tissue of mice. Concentration-dependent efficacy of GSPE was also assessed in a hamster atherosclerosis model. Approximately 49 and 63% reduction in foam cells, a biomarker of early stage atherosclerosis, were observed following supplementation of 50 and 100 mg GSPE/kg body weight, respectively. A human clinical trial was conducted on hypercholesterolemic subjects. GSPE supplementation significantly reduced oxidized LDL, a biomarker of cardiovascular diseases. Finally, a cDNA microarray study demonstrated significant inhibition of inducible endothelial CD36 expression, a novel cardioregulatory gene, by GSPE. These results demonstrate that GSPE may serve as a potential therapeutic tool in promoting cardiovascular health via a number of novel mechanisms.

Anti-angiogenic Property of Edible Berry in a Model of Hemangioma

FEBS Letters. Jun, 2003  |  Pubmed ID: 12782326

Hemangiomas represent a powerful model to study in vivo angiogenesis. Monocyte chemotactic protein 1 (MCP-1) is known to be responsible for recruiting macrophages to sites of infection or inflammation and facilitate angiogenesis. Recently we have demonstrated that edible berry extracts potently suppress inducible vascular endothelial growth factor expression and in vitro angiogenesis. Comparative analysis of several berry extracts led to the observation that wild blueberry and a berry mix were most effective. Our goal was to follow up on our findings with wild blueberry and the berry mix (OptiBerry). The present work rests on our current finding that these two berry powders significantly inhibit inducible MCP-1 expression in endothelioma cells. Therefore, we sought to examine the effects of wild blueberry and berry mix in an in vivo model of experimental angiogenesis. Reporter studies showed that the berry powders significantly inhibited basal MCP-1 transcription and inducible nuclear factor kappaB transcription. Endothelioma cells pre-treated with berry powders showed diminished ability to form hemangioma. Histological analysis demonstrated markedly decreased infiltration of macrophages in hemangioma of treated mice compared to placebo-treated controls. The current results provide the first in vivo evidence substantiating the anti-angiogenic property of edible berries.

Redox Regulation of Mitochondrial Permeability Transition: Effects of Uncoupler, Lipoic Acid and Its Positively Charged Analog LA-plus and Selenium

BioFactors (Oxford, England). 2003  |  Pubmed ID: 12897451

Molecular Basis of Vitamin E Action: Tocotrienol Modulates 12-lipoxygenase, a Key Mediator of Glutamate-induced Neurodegeneration

The Journal of Biological Chemistry. Oct, 2003  |  Pubmed ID: 12917400

Vitamin E is a generic term for tocopherols and tocotrienols. This work is based on our striking evidence that, in neuronal cells, nanomolar concentrations of alpha-tocotrienol, but not alpha-tocopherol, block glutamate-induced death by suppressing early activation of c-Src kinase (Sen, C. K., Khanna, S., Roy, S., and Packer, L. (2000) J. Biol. Chem. 275, 13049-13055). This study on HT4 and immature primary cortical neurons suggests a central role of 12-lipoxygenase (12-LOX) in executing glutamate-induced neurodegeneration. BL15, an inhibitor of 12-LOX, prevented glutamate-induced neurotoxicity. Moreover, neurons isolated from 12-LOX-deficient mice were observed to be resistant to glutamate-induced death. In the presence of nanomolar alpha-tocotrienol, neurons were resistant to glutamate-, homocysteine-, and l-buthionine sulfoximine-induced toxicity. Long-term time-lapse imaging studies revealed that neurons and their axo-dendritic network are fairly motile under standard culture conditions. Such motility was arrested in response to glutamate challenge. Tocotrienol-treated primary neurons maintained healthy growth and motility even in the presence of excess glutamate. The study of 12-LOX activity and metabolism revealed that this key mediator of glutamate-induced neurodegeneration is subject to control by the nutrient alpha-tocotrienol. In silico docking studies indicated that alpha-tocotrienol may hinder the access of arachidonic acid to the catalytic site of 12-LOX by binding to the opening of a solvent cavity close to the active site. These findings lend further support to alpha-tocotrienol as a potent neuroprotective form of vitamin E.

Revisiting the Essential Role of Oxygen in Wound Healing

American Journal of Surgery. Sep, 2003  |  Pubmed ID: 12946829

Hypoxemia, caused by disrupted vasculature, is a key factor that limits wound healing. Correcting hypoxemia through the administration of supplemental oxygen (O(2)) can have significant beneficial impact on wound healing in the perioperative and outpatient settings. Beyond its role as a nutrient and antibiotic, O(2) may support vital processes such as angiogenesis, cell motility, and extracellular matrix formation. Recent discoveries highlight a novel aspect, addressing the role of O(2) in wound healing via the production of reactive oxygen species (ROS). Almost all wound-related cells possess specialized enzymes that generate ROS (including free radicals and H(2)O(2)) from O(2). Defect in these enzymes is associated with impaired healing. Low wound pO(2) is expected to compromise the function of these enzymes. At low concentrations, ROS serve as cellular messengers to support wound healing. The use of systemic hyperbaric O(2) therapy presents potential advantages, as well as risks. There is evidence to suspect that the use of pressure and systemic pure O(2) may not be essential in wound care. Elimination of these factors by using sub-pure systemic O(2) under normobaric conditions may significantly minimize the risk of O(2) toxicity. Furthermore, opportunities to treat dermal wounds using topical O(2) therapy warrant further investigation. Given that many growth factors require ROS for their function, it is reasonable to assume that approaches to correct wound pO(2) will serve as an effective adjunct in treating chronic wounds.

Significance of Oxygen Therapeutics

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Sep-Oct, 2003  |  Pubmed ID: 12950645

Characterization of Perceived Hyperoxia in Isolated Primary Cardiac Fibroblasts and in the Reoxygenated Heart

The Journal of Biological Chemistry. Nov, 2003  |  Pubmed ID: 12952964

Under normoxic conditions, pO2 ranges from 90 to <3 torr in mammalian organs with the heart at approximately 35 torr (5%) and arterial blood at approximately 100 torr. Thus, "normoxia" for cells is an adjustable variable. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of pO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the pO2 to which cells are adjusted, results in the activation of specific O2-sensitive signal transduction pathways that alter cellular phenotype and function. Thus, reperfusion causes damage to the tissue at the focus of ischemia while triggering remodeling in the peri-infarct region by means of perceived hyperoxia. We reported first evidence demonstrating that perceived hyperoxia triggers the differentiation of cardiac fibroblasts (CF) to myofibroblasts by a p21-dependent mechanism (Roy, S., Khanna, S., Bickerstaff, A. A., Subramanian, S. V., Atalay, M., Bierl, M., Pendyala, S., Levy, D., Sharma, N., Venojarvi, M., Strauch, A., Orosz, C. G., and Sen, C. K. (2003) Circ. Res. 92, 264-271). Here, we sought to characterize the genomic response to perceived hyperoxia in CF using GeneChips trade mark. Candidate genes were identified, confirmed and clustered. Cell cycle- and differentiation-associated genes represented a key target of perceived hyperoxia. Bioinformatics-assisted pathway reconstruction revealed the specific signaling processes that were sensitive to perceived hyperoxia. To test the significance of our in vitro findings, a survival model of rat heart focal ischemia-reperfusion (I-R) was investigated. A significant induction in p21 mRNA expression was observed in I-R tissue. The current results provide a comprehensive molecular definition of perceived hyperoxia in cultured CF. Furthermore, the first evidence demonstrating activation of perceived hyperoxia sensitive genes in the cardiac I-R tissue is presented.

Topical Oxygen As an Adjunct to Wound Healing: a Clinical Case Series

Pathophysiology : the Official Journal of the International Society for Pathophysiology / ISP. Jan, 2003  |  Pubmed ID: 14567939

BACKGROUND: Disrupted vasculature and high energy-demand to support processing and regeneration of wounded tissue are typical characteristics of a wound site. Oxygen delivery is a critical element for the healing of wounds. Clinical experience with adjunctive hyperbaric oxygen therapy in the treatment of chronic wounds have shown that wound hyperoxia increases wound granulation tissue formation and accelerates wound contraction and secondary closure. Nevertheless, the physiologic basis for this modality remains largely unknown. Also, systemic hyperbaric oxygen therapy is associated with risks related to oxygen toxicity. Topical oxygen therapy represents a less explored modality in wound care. The advantages of topical oxygen therapy include low cost, lack of systemic oxygen toxicity, and the ability to receive treatment at home, making the benefits of oxygen therapy available to a much larger population of patients. MATERIALS AND METHODS: Over 9 months, seven surgeons treated 58 wounds in 32 patients with topical oxygen with follow-up ranging from 1 to 8 months. The data presented herein is a retrospective analysis of the results we have achieved using topical oxygen on complex wounds. RESULTS: Thirty-eight wounds in 15 patients healed while on topical oxygen. An additional five wounds in five patients had preoperative oxygen therapy; all wounds initially healed postoperatively. In two patients, wounds recurred post-healing. In ten wounds, topical oxygen had no effect; and two of those patients went on to require limb amputation. There were no complications attributable to topical oxygen. Three patients died during therapy and one died in the first postoperative month from underlying medical problems. Two patients were lost to follow-up. CONCLUSIONS: In this case series, topical oxygen had no detrimental effects on wounds and showed beneficial indications in promoting wound healing.

The General Case for Redox Control of Wound Repair

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Nov-Dec, 2003  |  Pubmed ID: 14617282

The orthodox view has been that reactive oxygen species are primarily damaging to cells. There is general agreement that while high (3%) doses of H(2)O(2) may serve as a clinical disinfectant, its overall effect on healing is not positive. Current work shows that at very low concentrations, reactive oxygen species may regulate cellular signaling pathways by redox-dependent mechanisms. Recent discoveries show that almost all cells of the wound microenvironment contain specialized enzymes that utilize O(2) to generate reactive oxygen species. Numerous aspects of wound healing are subject to redox control. An understanding of how endogenous reactive oxygen species are generated in wound-related cells may influence the healing process and could result in new redox-based therapeutic strategies. Current results with growth factor therapy of wounds have not met clinical expectations. Many of these growth factors, such as platelet-derived growth factor, rely on reactive oxygen species for functioning. Redox-based strategies may serve as effective adjuncts to jump-start healing of chronic wounds. The understanding of wound-site redox biology is also likely to provide novel insights into the fundamental mechanisms that would help to optimize conditions for oxygen therapy. While a window of therapeutic opportunity seems to exist under conditions of low concentrations of reactive oxygen species, high levels may complicate regeneration and remodeling of nascent tissue.

Oxygen: at the Foundation of Wound Healing--introduction

World Journal of Surgery. Mar, 2004  |  Pubmed ID: 14961183

"Wound Healing: Oxygen & Emerging Therapeutics" Columbus, Ohio, September 12-15, 2002. Sponsored by the National Institutes of Health (R13 AR 049171), International Union of Biochemistry & Molecular Biology and UNESCO-Global Network of Molecular & Cell Biology. Conference co-chairs: Chandan K. Sen, the Ohio State University Medical Center and Thomas K. Hunt, University of California-San Francisco. This congress was conceived for two reasons: to consolidate what is known about oxygen in the repair process and to stimulate discussion about new developments of control of healing by redox regulated signaling processes. A historical and evolutionary perspective on the role of oxygen in wound healing--from the classical physiology of oxygen in the wound to the refined concept of redox signaling--is presented.

Reactive Oxygen Species and EGR-1 Gene Expression in Surgical Postoperative Peritoneal Adhesions

World Journal of Surgery. Mar, 2004  |  Pubmed ID: 14961185

Postoperative peritoneal adhesions are common and serious complications of general abdominal and gynecological surgery that can lead to chronic abdominal pain, small-bowel obstruction and infertility. The specific pathophysiology of peritoneal adhesions remains elusive and current treatment is relegated to prevention through meticulous surgical technique and protective physical barriers, gels and solutions. We have reported that reactive oxygen species (ROS), generated by phagocytic cells at the site of tissue injury, serve as major signaling molecules regulating the expression of vascular endothelial growth factor (VEGF) and subsequent wound repair. We hypothesized that peritoneal adhesions are a product of over-healing surgical wounds and that, like in wound healing, ROS are implicated in their pathogenesis. We examined the presence of footprints of ROS and the ROS-inducible angiogenic factor VEGF in human adhesion tissue. An experimental model of peritoneal adhesion was established in rodents to study of the dynamics of ROS-induced gene expression during de novo adhesion tissue formation. Immunohistochemical analysis demonstrated presence of ROS/oxidant and macrophages in human peritoneal tissue. The presence of ROS and ROS-sensitive transcription factor EGR-1 was also evident in an experimental rodent peritoneal adhesion model. Along with ROS, VEGF, and a large number of mature and immature CD31/vWF positive blood vessels were present in the adhesion tissue. These observations are not consistent with the contention that adhesions are non-functional scar tissue. The newly developed rodent model of adhesion may present a useful approach to reproducibly and objectively study molecular mechanisms underlying the dynamic process of de novo adhesion tissue formation.

Perceived Hyperoxia: Oxygen-regulated Signal Transduction Pathways in the Heart

Methods in Enzymology. 2004  |  Pubmed ID: 15063670

Survival Surgery for Coronary Occlusion and Reoxygenation in a Rodent Model

Methods in Enzymology. 2004  |  Pubmed ID: 15063673

Proteomic Analysis of the Mammalian Cell Nucleus

Methods in Enzymology. 2004  |  Pubmed ID: 15063675

Proximal Middle Cerebral Artery Occlusion Surgery for the Study of Ischemia-reoxygenation Injury in the Brain

Methods in Enzymology. 2004  |  Pubmed ID: 15063690

Protocols for Topical and Systemic Oxygen Treatments in Wound Healing

Methods in Enzymology. 2004  |  Pubmed ID: 15063699

DNA Microarray Technology in Nutraceutical and Food Safety

Toxicology Letters. Apr, 2004  |  Pubmed ID: 15068823

The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

Exercise Training Modulates Heat Shock Protein Response in Diabetic Rats

Journal of Applied Physiology (Bethesda, Md. : 1985). Aug, 2004  |  Pubmed ID: 15075301

Strenuous exercise induces oxidative stress and modification of intracellular proteins. Exercise training, however, upregulates endogenous antioxidant defenses and heat shock protein (HSP) expression. In diabetes, perturbations in the endogenous antioxidant and HSP protection have been reported. The aim of this study was to examine the effect of 8 wk of endurance training on HSP expression and oxidative stress markers in the skeletal muscle, heart, and liver of streptozotocin-induced diabetic (SID) and nondiabetic control rats. Induction of diabetes decreased HSP72 expression in heart, liver, and vastus lateralis muscles. SID increased heme oxygenase-1, an oxidative stress-inducible HSP, in liver, red gastrocnemius muscle, and vastus lateralis muscle and glucose-regulated protein 75 in liver. SID increased HSP90 levels in the heart, but levels decreased in the liver. Diabetes induced oxidative stress marker protein carbonyl levels and tissue inflammation. Although endurance training increased the expression of HSP72 in all of the tissues examined, this induction was less pronounced in diabetic rats than in nondiabetic controls. Furthermore, endurance training induced the activation and expression of transcriptional regulator heat shock factor-1 only in nondiabetic control animals. In summary, diabetes may increase susceptibility to oxidative damage and impair HSP protection, but endurance training may offset some of the adverse effects of diabetes by upregulating tissue HSP expression. Our results suggest that diabetes impairs HSP protection, possibly via transcriptionally mediated mechanisms.

A Key Angiogenic Role of Monocyte Chemoattractant Protein-1 in Hemangioendothelioma Proliferation

American Journal of Physiology. Cell Physiology. Oct, 2004  |  Pubmed ID: 15163622

Angiomatous lesions are common in infants and children. Hemangioendotheliomas (HE) represent one type of these lesions. Endothelial cell proliferation and the development of vascular/blood cell-filled spaces are inherent in the growth of HE. Therefore, understanding mechanisms that regulate the proliferation of these lesions should provide key insight into mechanisms regulating angiogenesis. A murine model was used to test the significance of monocyte chemoattractant protein (MCP)-1 in HE proliferation. EOMA cells, a cell line derived from a spontaneously arising murine HE, generate these lesions with 100% efficiency when injected subcutaneously into syngeneic mice. MCP-1 produced by EOMA cells recruit macrophages, which were shown to induce angiogenic behavior in EOMA cells by stimulating transwell migration and inducing sprout formation on type I collagen gels. When EOMA cells were injected into MCP-1(-/-) mice, only 50% of the mice developed tumors, presumably because the low levels of MCP-1 expressed by the injected EOMA cells were enough to overcome any host deficits of this chemokine. When EOMA cells were coinjected with a neutralizing antibody to MCP-1, tumors failed to develop in any of the treated mice, including syngeneic 129P3, C57Bl/6 (wild type), and MCP-1(-/-). These results present the first evidence that MCP-1 is required for HE proliferation and may promote the growth of these lesions by stimulating angiogenic behavior of endothelial cells. This study has produced the first in vivo evidence of a complete response for any neoplasm, specifically a vascular proliferative lesion, to anti-MCP-1 therapy in animals with intact immune systems.

Body Weight and Abdominal Fat Gene Expression Profile in Response to a Novel Hydroxycitric Acid-based Dietary Supplement

Gene Expression. 2004  |  Pubmed ID: 15200237

Obesity is a global public health problem, with about 315 million people worldwide estimated to fall into the WHO-defined obesity categories. Traditional herbal medicines may have some potential in managing obesity. Botanical dietary supplements often contain complex mixtures of phytochemicals that have additive or synergistic interactions. The dried fruit rind of Garcinia cambogia, also known as Malabar tamarind, is a unique source of (-)-hydroxycitric acid (HCA), which exhibits a distinct sour taste and has been safely used for centuries in Southeastern Asia to make meals more filling. Recently it has been demonstrated that HCA-SX or Super Citrimax, a novel derivative of HCA, is safe when taken orally and that HCA-SX is bioavailable in the human plasma as studied by GC-MS. Although HCA-SX has been observed to be conditionally effective in weight management in experimental animals as well as in humans, its mechanism of action remains to be understood. We sought to determine the effects of low-dose oral HCA-SX on the body weight and abdominal fat gene expression profile of Sprague-Dawley rats. We observed that at doses relevant for human consumption dietary HCA-SX significantly contained body weight growth. This response was associated with lowered abdominal fat leptin expression while plasma leptin levels remained unaffected. Repeated high-density microarray analysis of 9960 genes and ESTs present in the fat tissue identified a small set (approximately 1% of all genes screened) of specific genes sensitive to dietary HCA-SX. Other genes, including vital genes transcribing for mitochondrial/nuclear proteins and which are necessary for fundamental support of the tissue, were not affected by HCA-SX. Under the current experimental conditions, HCA-SX proved to be effective in restricting body weight gain in adult rats. Functional characterization of HCA-SX-sensitive genes revealed that upregulation of genes encoding serotonin receptors represent a distinct effect of dietary HCA-SX supplementation.

Tocotrienol: the Natural Vitamin E to Defend the Nervous System?

Annals of the New York Academy of Sciences. Dec, 2004  |  Pubmed ID: 15753140

Vitamin E is essential for normal neurological function. It is the major lipid-soluble, chain-breaking antioxidant in the body, protecting the integrity of membranes by inhibiting lipid peroxidation. Mostly on the basis of symptoms of primary vitamin E deficiency, it has been demonstrated that vitamin E has a central role in maintaining neurological structure and function. Orally supplemented vitamin E reaches the cerebrospinal fluid and brain. Vitamin E is a generic term for all tocopherols and their derivatives having the biological activity of RRR-alpha-tocopherol, the naturally occurring stereoisomer compounds with vitamin E activity. In nature, eight substances have been found to have vitamin E activity: alpha-, beta-, gamma- and delta-tocopherol; and alpha-, beta-, gamma- and delta-tocotrienol. Often, the term vitamin E is synonymously used with alpha-tocopherol. Tocotrienols, formerly known as zeta, , or eta-tocopherols, are similar to tocopherols except that they have an isoprenoid tail with three unsaturation points instead of a saturated phytyl tail. Although tocopherols are predominantly found in corn, soybean, and olive oils, tocotrienols are particularly rich in palm, rice bran, and barley oils. Tocotrienols possess powerful antioxidant, anticancer, and cholesterol-lowering properties. Recently, we have observed that alpha-tocotrienol is multi-fold more potent than alpha-tocopherol in protecting HT4 and primary neuronal cells against toxicity induced by glutamate as well as by a number of other toxins. At nanomolar concentration, tocotrienol, but not tocopherol, completely protected neurons by an antioxidant-independent mechanism. Our current work identifies two major targets of tocotrienol in the neuron: c-Src kinase and 12-lipoxygenase. Dietary supplementation studies have established that tocotrienol, fed orally, does reach the brain. The current findings point towards tocotrienol as a potent neuroprotective form of natural vitamin E.

Hyperbaric Oxygen Therapy Ameliorates Stress-impaired Dermal Wound Healing

Brain, Behavior, and Immunity. May, 2005  |  Pubmed ID: 15797310

Psychological stress has been shown to dysregulate healing in both humans and animals. Studies indicate the possibility for decreased oxygen supply, and increased oxygen demand, in the wounds of the stressed animals. Oxygen is an important mediator of wound healing, and its availability can limit healing rate. Hence, in a mouse model of stress-impaired healing, the hypothesis that hyperbaric oxygen therapy would ameliorate the effect of stress on dermal wound healing was tested. Hyperbaric oxygen therapy (HBO) twice a day during early wound healing significantly ameliorated the effects of stress, bringing healing to near-control levels. There was no significant effect of HBO on the wounds of control animals. Wound inducible nitric oxide synthase (iNOS), modulated by psychological stress and oxygen balance, was studied for gene expression by real-time PCR. Expression of iNOS increased in stressed mice on days 1 (205%; p<.0001), 3 (96%; p<.03), and 5 (249%; p<.03), post-wounding. HBO treatment of the stressed animals decreased iNOS expression by 62.6% (p< .02) day 1 post-wounding. There was no significant effect of HBO on wound healing and iNOS expression in the control animals. Methods aimed at increasing tissue oxygenation, like HBO, have a high therapeutic potential. Their molecular mechanisms, implicated in wound healing, elude clarification due to the lack of appropriate animal models. Our current findings represent the first experimental evidence, demonstrating that HBO corrects stress-impaired dermal wound healing.

Human Genome Screen to Identify the Genetic Basis of the Anti-inflammatory Effects of Boswellia in Microvascular Endothelial Cells

DNA and Cell Biology. Apr, 2005  |  Pubmed ID: 15812241

Inflammatory disorders represent a substantial health problem. Medicinal plants belonging to the Burseraceae family, including Boswellia, are especially known for their anti-inflammatory properties. The gum resin of Boswellia serrata contains boswellic acids, which inhibit leukotriene biosynthesis. A series of chronic inflammatory diseases are perpetuated by leukotrienes. Although Boswellia extract has proven to be anti-inflammatory in clinical trials, the underlying mechanisms remain to be characterized. TNF alpha represents one of the most widely recognized mediators of inflammation. One mechanism by which TNFalpha causes inflammation is by potently inducing the expression of adhesion molecules such as VCAM-1. We sought to test the genetic basis of the antiinflammatory effects of BE (standardized Boswellia extract, 5-Loxin) in a system of TNF alpha-induced gene expression in human microvascular endothelial cells. We conducted the first whole genome screen for TNF alpha- inducible genes in human microvascular cells (HMEC). Acutely, TNF alpha induced 522 genes and downregulated 141 genes in nine out of nine pairwise comparisons. Of the 522 genes induced by TNF alpha in HMEC, 113 genes were clearly sensitive to BE treatment. Such genes directly related to inflammation, cell adhesion, and proteolysis. The robust BE-sensitive candidate genes were then subjected to further processing for the identification of BE-sensitive signaling pathways. The use of resources such as GenMAPP, KEGG, and gene ontology led to the recognition of the primary BE-sensitive TNF alpha-inducible pathways. BE prevented the TNF alpha-induced expression of matrix metalloproteinases. BE also prevented the inducible expression of mediators of apoptosis. Most strikingly, however, TNF alpha-inducible expression of VCAM-1 and ICAM-1 were observed to be sensitive to BE. Realtime PCR studies showed that while TNF alpha potently induced VCAM-1 gene expression, BE completely prevented it. This result confirmed our microarray findings and built a compelling case for the anti-inflammatory property of BE. In an in vivo model of carrageenan-induced rat paw inflammation, we observed a significant antiinflammatory property of BE consistent with our in vitro findings. These findings warrant further research aimed at identifying the signaling mechanisms by which BE exerts its anti-inflammatory effects.

Restraint Stress Alters Lung Gene Expression in an Experimental Influenza A Viral Infection

Journal of Neuroimmunology. May, 2005  |  Pubmed ID: 15833365

In the present study the global effect of restraint stress on gene expression in the murine lung during an experimental influenza A/PR8 viral infection was examined. Gene expression profiling using high density oligonucleotide microarrays revealed that the expression of 95 genes was altered on day 3 post infection (p.i.), while 48 genes were altered on day 7 p.i. Restraint stress reduced and delayed the expression of specific cytokines, cell adhesion molecules and cell surface receptors indicating alterations in cell migration to the site of infection. Furthermore, mapping of the candidate genes to known pathways revealed that genes associated with host defense and immune responses, including chemotaxis and chemokine function, antigen presentation and processing, MHC class II receptor function and inflammation were the major pathways affected by restraint stress.

Relief from a Heavy Heart: Redox-sensitive NF-kappaB As a Therapeutic Target in Managing Cardiac Hypertrophy

American Journal of Physiology. Heart and Circulatory Physiology. Jul, 2005  |  Pubmed ID: 15961373

Access to Exercise and Its Relation to Cardiovascular Health and Gene Expression in Laboratory Animals

Life Sciences. Sep, 2005  |  Pubmed ID: 16002099

The interaction between genes and environment can influence cardiovascular disease (CVD). This 16 month study investigated if genes associated with cardiovascular (CV) regulation were expressed differently in animals having: 1) no access to physical activity or exercise (SED), 2) access to hour-long, twice weekly activity (PA), and 3) access every-other-day to a running wheel (EX). Out of 31,000 genes, a CV subset comprising 44 genes was investigated. Ten genes from this subset were expressed differently in EX compared with SED, and 34 genes were expressed differently in PA compared with SED (p<0.05). Total cholesterol (70+/-8 vs. 101+/-9 mg dl(-1)), triglycerides (104+/-8 vs. 127+/-4 mg dl(-1)), resting systolic blood pressure (130+/-3 vs. 141+/-3 mmHg), mean arterial pressure (110+/-2 vs. 120+/-2 mmHg) and heart rate (380+/-6 vs. 405+/-9 beats min(-1)) were lower in EX compared with SED (p<0.05), but intracellular adhesion molecule levels did not differ among groups. Mean gene expressions for Gja1, Fdft1, Edn1, Cd36, and Hmgb2 differed in animals according to access to physical activity. These genes play roles in heart rate, cholesterol biosynthesis, blood pressure, cell adhesion, and transcription and neurogenesis regulation, respectively. In conclusion, a total of 44 CV genes were expressed differently in SED compared to PA and EX; and SED showed more physiological evidence of CVD.

Dermal Excisional Wound Healing in Pigs Following Treatment with Topically Applied Pure Oxygen

Mutation Research. Nov, 2005  |  Pubmed ID: 16105672

Hypoxia, caused by disrupted vasculature and peripheral vasculopathies, is a key factor that limits dermal wound healing. Factors that can increase oxygen delivery to the regional tissue, such as supplemental oxygen, warmth, and sympathetic blockade, can accelerate healing. Clinical experience with adjunctive hyperbaric oxygen therapy (HBOT) in the treatment of chronic wounds have shown that wound hyperoxia may increase granulation tissue formation and accelerate wound contraction and secondary closure. However, HBOT is not applicable to all wound patients and may pose the risk of oxygen toxicity. Thus, the efficacy of topical oxygen treatment in an experimental setting using the pre-clinical model involving excisional dermal wound in pigs was assessed. Exposure of open dermal wounds to topical oxygen treatment increased tissue pO2 of superficial wound tissue. Repeated treatment accelerated wound closure. Histological studies revealed that the wounds benefited from the treatment. The oxygen treated wounds showed signs of improved angiogenesis and tissue oxygenation. Topically applied pure oxygen has the potential of benefiting some wound types. Further studies testing the potential of topical oxygen in pre-clinical and clinical settings are warranted.

Oxygen: from the Benefits of Inducing VEGF Expression to Managing the Risk of Hyperbaric Stress

Antioxidants & Redox Signaling. Sep-Oct, 2005  |  Pubmed ID: 16115043

Hypoxia limits wound healing. Both normobaric (1 atm) and hyperbaric oxygen (HBO) approaches have been used clinically to oxygenate wound tissue. Recently, we reported that HBO ameliorates stress-induced impairment of dermal healing. We examined the effect of pressure on oxygen-induced vascular endothelial growth factor (VEGF) expression by human HaCaT keratinocytes. Next, we investigated the effect of HBO on whole-body redox and on the ratio of oxidized to reduced glutathione (GSSG/GSH) in the liver, heart, lung, and brain of rats. Superoxygenation (90% O2) of keratinocytes partially arrested cell growth. G2-M growth arrest was substantially augmented by HBO. HBO also caused apoptosis in a small subpopulation. Normobaric oxygen, but not HBO (2 atm), potently induced the expression of VEGF165 and 189. In vivo electron paramagnetic resonance spectroscopy imaging revealed a clear shift of the whole-body redox status toward oxidation in response to HBO. The standard diet of laboratory rats contains excessive (17x human recommended dietary allowance) alpha-tocopherol (E++), which confers exceptional resistance to oxidant insults. People with chronic wounds commonly suffer from under- or malnutrition. We generated vitamin E-deficient (E-) rats by long-term dietary vitamin E restriction. HBO did not raise GSSG/GSH in E++ rats, but post-HBO GSSG/GSH was significantly higher in E- compared with E++. Thus, rats on antioxidant-enriched diet were well protected against HBO. The risk of oxidative stress may negatively impact the net benefits of HBO. This is of special concern for people with inadequate intake of dietary antioxidants. Nutritional antioxidant supplementation may offset HBO-induced oxidative stress.

Wound Site Neutrophil Transcriptome in Response to Psychological Stress in Young Men

Gene Expression. 2005  |  Pubmed ID: 16358416

Communication between the central nervous and the immune system occurs through chemical messengers secreted by nerve cells, endocrine organs, or immune cells. Psychological stressors can disrupt these networks. We have previously observed that disruption of the neuroendocrine immune system adversely influences a broad range of physiological processes including wound healing. Migration of neutrophils to the wound site is an early event that induces a transcriptional activation program, which regulates cellular fate and function, and promotes wound healing. In this study, we have sought to identify stress-sensitive transcripts in wound site neutrophils. A skin blister model was used to collect wound fluid and wound site neutrophils from four young men, experiencing or not examination stress. Self-reported stress was recorded using the Beck Depression Inventory. Stress decreased growth hormone levels at the wound site and was related to impaired wound healing in all subjects. High density microarray analyses were performed using RNA from wound site neutrophils. Results show that psychological stress had an overall suppressive effect on the neutrophil transcriptome. Of the 22,283 transcripts screened, 0.5% were downregulated whereas only under 0.3% were induced by stress in all four out of four subjects. Functionally, stress tilted the genomic balance towards genes encoding proteins responsible for cell cycle arrest, death, and inflammation. Further effort to gain a more comprehensive understanding of the functional significance of such behavior-genome interaction is warranted.

Neuroprotective Properties of the Natural Vitamin E Alpha-tocotrienol

Stroke; a Journal of Cerebral Circulation. Oct, 2005  |  Pubmed ID: 16166580

The current work is based on our previous finding that in neuronal cells, nmol/L concentrations of alpha-tocotrienol (TCT), but not alpha-tocopherol (TCP), blocked glutamate-induced death by suppressing early activation of c-Src kinase and 12-lipoxygenase.

Delivery of Orally Supplemented Alpha-tocotrienol to Vital Organs of Rats and Tocopherol-transport Protein Deficient Mice

Free Radical Biology & Medicine. Nov, 2005  |  Pubmed ID: 16257640

The natural vitamin E tocotrienol (TCT) possesses biological properties not shared by tocopherols (TCP). Nanomolar alpha-TCT, not alpha-TCP, is potently neuroprotective (JBC 275:13049; 278:43508). Tocopherol-transport protein (TTP) represents the primary mechanism for maintaining normal alpha-TCP concentrations in plasma and extrahepatic tissues. TTP primarily transports alpha-TCP and has low affinity for alpha-TCT. There are no studies that have investigated tissue delivery of alpha-TCT when orally gavaged on a long-term basis. A long-term study was conducted to examine the effects of alpha-TCT or alpha-TCP supplementation, either alone or in combination, on tissue levels. Rats were maintained on a vitamin E-deficient diet and gavaged with alpha-TCT or alpha-TCP alone or in combination. Five generations of rats were studied over 60 weeks. TTP-deficient mice were supplemented with TCT and bred to examine tissue delivery of oral alpha-TCT. Orally supplemented alpha-TCT was effectively delivered to most tissues over time. When co-supplemented, alpha-TCP outcompeted alpha-TCT for transport systems delivering vitamin E to tissues. To evaluate the significance of TTP in alpha-TCT delivery to tissues, tissue levels of alpha-TCT in supplemented TTP-deficient mice were studied. alpha-TCT was transported to several vital organs in TTP-deficient mice. alpha-TCT restored fertility in TTP-deficient mice. In sum, orally supplemented alpha-TCT was successfully delivered to several vital organs. The transport efficiency of alpha-TCT to tissues may be maximized by eliminating the co-presence of alpha-TCP in the oral supplement. Examination of whether alpha-TCT may benefit humans suffering from neurological disorders because of congenital TTP deficiency is warranted.

Exercise-induced Oxidative Stress and Muscle Stress Protein Responses in Trotters

European Journal of Applied Physiology. Jan, 2005  |  Pubmed ID: 15221402

Acute exercise induces oxidative stress and heat shock protein (HSP) expression. Information on the protection of stress proteins against oxidant insult and muscle damage during moderate exercise is scanty. We aimed to show how a single bout of moderate exercise affects the markers of oxidative stress and heat shock factor-1 (HSF1; the transcriptional regulator of HSP synthesis), and HSP70, HSP90 and glucose-regulated protein (GRP75) expression in horses. Eight clinically normal and regularly trained standardbred trotters were treadmill-exercised for 45 min at moderate intensity. Blood samples were collected prior to and immediately after exercise and at 4 and 24 h of recovery. Muscle biopsy samples from the middle gluteal muscle were taken before exercise and after 4 h of recovery. Acute exercise did not activate HSF1 or induce expression of HSP70, HSP90 or GRP75 in skeletal muscle. One bout of acute exercise increased protein oxidation, which was measured by protein carbonyls in plasma and muscle, but it did not effect 4-hydroxynonenal protein adducts, which are markers of lipid peroxidation. Furthermore, mild muscle damage was observed 4 h after exercise. Our results showed that horses are susceptible to oxidative stress. One bout of exercise at moderate intensity and duration did not induce HSP responses despite the increased protein oxidation and tissue inflammation in equine muscle.

Transcriptome of the Subcutaneous Adipose Tissue in Response to Oral Supplementation of Type 2 Leprdb Obese Diabetic Mice with Niacin-bound Chromium

Physiological Genomics. Nov, 2006  |  Pubmed ID: 16940432

The effects of oral niacin-bound chromium (NBC) supplementation on the subcutaneous fat tissue of type 2 Lepr(db) obese diabetic mice were examined using high-density comprehensive mouse genome (45,101 probe sets) expression arrays. The influence of such supplementation on the plasma cardiovascular risk factors of these mice was also investigated. Supplementation of NBC had no significant effect on age-dependent weight gain in the Lepr(db) obese diabetic mice. However, NBC lowered total cholesterol (TC), TC-to-HDL ratio, LDL cholesterol, and triglyceride levels while increasing HDL cholesterol in the blood plasma. No effect of NBC supplementation was observed on fasting blood glucose levels. Oral glucose tolerance test revealed a significantly improved clearance of blood glucose between 1 and 2 h of glucose challenge in NBC-supplemented mice. Unbiased genome-wide interrogation demonstrated that NBC resulted in the upregulation of muscle-specific gene expression in the fat tissue. Genes encoding proteins involved in glycolysis, muscle contraction, muscle metabolism, and muscle development were specifically upregulated in response to NBC supplementation. Genes in the adipose tissue that were downregulated in response to NBC supplementation included cell death-inducing DNA fragmentation factor (CIDEA) and uncoupling protein-1, which represent key components involved in the thermogenic role of brown adipose tissue and tocopherol transfer protein, the primary carrier of alpha-tocopherol to adipose tissue. The observation that CIDEA-null mice are resistant to obesity and diabetes suggests that the inhibitory role of NBC on CIDEA expression was favorable. Further studies testing the molecular basis of NBC function and long-term outcomes are warranted.

Natural Vitamin E Alpha-tocotrienol: Retention in Vital Organs in Response to Long-term Oral Supplementation and Withdrawal

Free Radical Research. Jul, 2006  |  Pubmed ID: 16984003

The natural vitamin E tocotrienol (TCT) possesses biological properties not shared by tocopherols (TCP). Nanomolar alpha-TCT, not alpha-TCP, is potently neuroprotective (JBC 275:13049; 278:43508; Stroke 36:2258). The report that the affinity of TTP to bind (alpha-TCT is an order of magnitude lower than that for alpha-TCP questions the bioavailability of orally taken TCT to tissues. Oral supplementation of TCT for 3 years in nine generations of female and male rat was studied. Ten vital organs were examined. To gain insight into the turnover of alpha-TCT in tissues, a subset of supplemented rats was moved to vitamin E deficient diet for 7 weeks. Orally supplemented alpha-TCT was delivered to all vital organs including the brain and spinal cord in significant amounts. In organs such as the skin, adipose and gonads the maximum level of alpha-TCT achieved in response to supplementation was folds higher than baseline values of alpha-TCP in rats maintained on laboratory chow. Females had higher levels of alpha-TCT compared to matched tissues of corresponding males. To gain insight into how quickly alpha-TCT is metabolized in the tissues, washout of alpha-TCT from vital organs was examined. alpha-TCT accumulated in vital organs over more than 2 years was almost completely lost in less than 2 months when the supplementation was stopped. This is in sharp contrast with findings related to alpha-TCP retention. The ability of long-term oral supplementation to maintain and elevate alpha-TCT levels in vital organs together with the rapid elimination of the intact vitamin from all organs studied underscores the need for continuous oral supplementation of TCT.

Safety and Whole-body Antioxidant Potential of a Novel Anthocyanin-rich Formulation of Edible Berries

Molecular and Cellular Biochemistry. Jan, 2006  |  Pubmed ID: 16328973

Edible berry extracts rich in anthocyanins possess a broad spectrum of therapeutic, pharmacologic and anti-carcinogenic properties. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds and strawberry), singly and in combination, were studied in our laboratories for antioxidant efficacy, cytotoxic potential, cellular uptake and anti-angiogenic properties. Combinations of edible berry extracts were evaluated to develop a synergistic formula, OptiBerry, which exhibited high oxygen radical absorbance capacity (ORAC) value, low cytotoxicity and superior anti-angiogenic properties compared to the other combinations tested. The current study sought to determine the broad spectrum safety and antioxidant potential of OptiBerry in vivo. Acute oral LD(50) of OptiBerry was greater than 5 g/kg in rats. Acute dermal LD(50) of OptiBerry was greater than 2 g/kg. No changes in the body weight or adverse effects were observed following necropsy. Primary skin and eye irritation studies were conducted in New Zealand albino rabbits. OptiBerry was classified as slightly irritating to the skin (primary skin irritation index 0.3) and minimally irritating to the eye (maximum mean total score 6.0). The antioxidant potential of OptiBerry was investigated in rats and mice by assessing GSH redox status in tissues as well as by a unique state-of-the-art electron paramagnetic resonance (EPR) imaging of whole-body redox status. A clinically relevant hyperbaric oxygen (HBO) exposure system (2 atm, 2 h) was employed to study the antioxidant properties of OptiBerry. OptiBerry feeding (8 weeks) significantly prevented HBO-induced GSH oxidation in the lung and liver of vitamin E-deficient Sprague Dawley rats. Furthermore, OptiBerry-fed mice, when exposed to HBO, demonstrated significant protection in whole-body HBO-induced oxidation compared to the unfed controls by EPR imaging. Taken together, these results indicate that OptiBerry is reasonably safe and possess antioxidant properties.

Laser Microdissection and Pressure-catapulting Technique to Study Gene Expression in the Reoxygenated Myocardium

American Journal of Physiology. Heart and Circulatory Physiology. Jun, 2006  |  Pubmed ID: 16443670

For focal events such as myocardial infarction, it is important to dissect infarction-induced biological responses as a function of space with respect to the infarct core. Laser microdissection pressure catapulting (LMPC) represents a recent variant of laser capture microdissection that enables robot-assisted rapid capture of catapulted tissue without direct user contact. This work represents the maiden effort to apply laser capture microdissection to study spatially resolved biological responses in myocardial infarction. Infarcted areas of the surviving ischemic-reperfused murine heart were identified using a standardized hematoxylin QS staining procedure. Standard staining techniques fail to preserve tissue RNA. Exposure of the tissue to an aqueous medium (typically used during standard immunohistochemical staining), with or without RNase inhibitors, resulted in a rapid degradation of genes, with approximately 80% loss in the 1st h. Tissue elements (1 x 10(4)-4 x 10(6) microm(2)) captured from infarcted and noninfarcted sites with micrometer-level surgical precision were collected in a chaotropic RNA lysis solution. Isolated RNA was analyzed for quality by microfluidics technology and reverse transcribed to generate high-quality cDNA. Real-time PCR analysis of the cDNA showed marked (200- and 400-fold, respectively) induction of collagen Ia and IIIa at the infarcted site compared with the noninfarcted site. This work reports a sophisticated yet rapid approach to measurement of relative gene expressions from tissue elements captured from spatially resolved microscopic regions in the heart with micrometer-level precision.

Tocotrienols: Vitamin E Beyond Tocopherols

Life Sciences. Mar, 2006  |  Pubmed ID: 16458936

In nature, eight substances have been found to have vitamin E activity: alpha-, beta-, gamma- and delta-tocopherol; and alpha-, beta-, gamma- and delta-tocotrienol. Yet, of all papers on vitamin E listed in PubMed less than 1% relate to tocotrienols. The abundance of alpha-tocopherol in the human body and the comparable efficiency of all vitamin E molecules as antioxidants, led biologists to neglect the non-tocopherol vitamin E molecules as topics for basic and clinical research. Recent developments warrant a serious reconsideration of this conventional wisdom. Tocotrienols possess powerful neuroprotective, anti-cancer and cholesterol lowering properties that are often not exhibited by tocopherols. Current developments in vitamin E research clearly indicate that members of the vitamin E family are not redundant with respect to their biological functions. alpha-Tocotrienol, gamma-tocopherol, and delta-tocotrienol have emerged as vitamin E molecules with functions in health and disease that are clearly distinct from that of alpha-tocopherol. At nanomolar concentration, alpha-tocotrienol, not alpha-tocopherol, prevents neurodegeneration. On a concentration basis, this finding represents the most potent of all biological functions exhibited by any natural vitamin E molecule. An expanding body of evidence support that members of the vitamin E family are functionally unique. In recognition of this fact, title claims in manuscripts should be limited to the specific form of vitamin E studied. For example, evidence for toxicity of a specific form of tocopherol in excess may not be used to conclude that high-dosage "vitamin E" supplementation may increase all-cause mortality. Such conclusion incorrectly implies that tocotrienols are toxic as well under conditions where tocotrienols were not even considered. The current state of knowledge warrants strategic investment into the lesser known forms of vitamin E. This will enable prudent selection of the appropriate vitamin E molecule for studies addressing a specific need.

CDNA Microarray Screening in Food Safety

Toxicology. Apr, 2006  |  Pubmed ID: 16466843

The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests.

Perceived Hyperoxia: Oxygen-induced Remodeling of the Reoxygenated Heart

Cardiovascular Research. Jul, 2006  |  Pubmed ID: 16483558

Focal coronary artery blockage followed by further reperfusion injury is commonly involved in myocardial infarction. The injured heart has some inherent reparative responses. Although such natural healing mechanisms seem to be inefficient, a clear understanding of the underlying principles of myocardial healing holds the key to successful therapy. Under normoxic conditions, pO(2) ranges from 90 to <3 Torr in mammalian organs with the heart at approximately 35 Torr (5%) and arterial blood at approximately 100 Torr. Thus, "normoxia" for cells is an adjustable variable. In response to chronic moderate hypoxia, cells lower their normoxia set-point such that reoxygenation-dependent relative elevation of pO(2) (+DeltapO(2)) results in perceived hyperoxia. Perceived hyperoxia induces differentiation of cardiac fibroblasts to myofibroblasts in the peri-infarct region and represents a significant factor supporting myocardial healing. The oxygen-sensitive signaling pathways involved have been characterized and point towards a central role of p21, TGFbeta and p38MAPK. That low oxygen ambience serves as a cue to trigger angiogenesis is a well-accepted notion. Studies related to perceived hyperoxia establish that the sensing of oxygen environment is not limited to hypoxia. It demonstrates that in addition to being a trigger for injury as is widely recognized, reoxygenation insult has a built-in component of tissue remodeling in the peri-infarct region induced by perceived hyperoxia. Understanding of the underlying mechanisms of this and other myocardial healing responses should prove to be instrumental in developing productive therapeutic approaches to mend the infarcted heart.

Microarray Analysis of Gene Expression: Considerations in Data Mining and Statistical Treatment

Physiological Genomics. May, 2006  |  Pubmed ID: 16554544

DNA microarray represents a powerful tool in biomedical discoveries. Harnessing the potential of this technology depends on the development and appropriate use of data mining and statistical tools. Significant current advances have made microarray data mining more versatile. Researchers are no longer limited to default choices that generate suboptimal results. Conflicting results in repeated experiments can be resolved through attention to the statistical details. In the current dynamic environment, there are many choices and potential pitfalls for researchers who intend to incorporate microarrays as a research tool. This review is intended to provide a simple framework to understand the choices and identify the pitfalls. Specifically, this review article discusses the choice of microarray platform, preprocessing raw data, differential expression and validation, clustering, annotation and functional characterization of genes, and pathway construction in light of emergent concepts and tools.

Transcriptome Analysis of the Ischemia-reperfused Remodeling Myocardium: Temporal Changes in Inflammation and Extracellular Matrix

Physiological Genomics. May, 2006  |  Pubmed ID: 16554547

cDNA microarray analysis was performed to screen 15,000 genes and expressed sequence tags (ESTs) to identify changes in the ischemia-reperfused (I-R) rat myocardial transcriptome in the early (day 2) and late (day 7) inflammatory phases of acute myocardial infarction. Lists of candidate genes that were affected by I-R transiently (2 or 7 days only) or on a more sustained basis (2 and 7 days) were derived. The candidate genes represented three major functional categories: extracellular matrix, apoptosis, and inflammation. To expand on the findings from microarray studies that dealt with the two above-mentioned time points, tissues collected from days 0, 0.25, 2, 3, 5, and 7 after reperfusion were examined. Acute myocardial infarction resulted in upregulation of IL-6 and IL-18. Genes encoding extracellular matrix proteins such as types I and III collagen were upregulated in day 2, and that response progressively grew stronger until day 7 after I-R. Comparable response kinetics was exhibited by the candidate genes of the apoptosis category. Caspases-2, -3, and -8 were induced in response to acute infarction. Compared with the myocardial tissue from the sham-operated rats, tissue collected from the infarct region stained heavily positive for the presence of active caspase-3. Laser microdissection and pressure catapulting technology was applied to harvest infarct and adjacent noninfarct control tissue from a microscopically defined region in the rat myocardium. Taken together, this work presents the first evidence gained from the use of DNA microarrays to understand the molecular mechanisms implicated in the early and late inflammatory phases of the I-R heart.

Regulation of Vascular Responses to Inflammation: Inducible Matrix Metalloproteinase-3 Expression in Human Microvascular Endothelial Cells is Sensitive to Antiinflammatory Boswellia

Antioxidants & Redox Signaling. Mar-Apr, 2006  |  Pubmed ID: 16677108

Endothelial cells are critical elements in the pathophysiology of inflammation. Tumor necrosis factor (TNF) alpha potently induces inflammatory responses in endothelial cells. Recently we have examined the genetic basis of the antiinflammatory effects of Boswellia extract (BE) in a system of TNFalpha-induced gene expression in human microvascular endothelial cells (HMECs). Of the 522 genes induced by TNFalpha in HMECs, 113 genes were sensitive to BE. BE prevented the TNFalpha-induced expression of matrix metalloproteinases (MMPs). In the current work, we sought to test the effects of BE on TNFalpha-inducible MMP expression in HMECs. Acetyl-11-ketobeta- boswellic acid (AKBA) is known to be an active principle in BE. To evaluate the significance of AKBA in the antiinflammatory properties of BE, effects of BE containing either 3% (BE3%) or 30% (BE30%, 5- Loxin) were compared. Pretreatment of HMECs for 2 days with BE potently prevented TNFalpha-induced expression and activity of MMP-3, MMP-10, and MMP-12. In vivo, BE protected against experimental arthritis. In all experiments, both in vitro and in vivo, BE30% was more effective than BE3%. In sum, this work lends support to our previous report that BE has potent antiinflammatory properties both in vitro as well as in vivo.

Postprandial Levels of the Natural Vitamin E Tocotrienol in Human Circulation

Antioxidants & Redox Signaling. May-Jun, 2006  |  Pubmed ID: 16771695

Compared to tocopherols, tocotrienols are poorly understood. The postabsorptive fate of tocotrienol isomers and their association with lipoprotein subfractions was examined. Normocholesterolemic women were subjected to an oral fat challenge supplemented with vitamin E (capsule containing 77 mg alpha-tocotrienol, 96 mg alpha-tocotrienol, 3 mg gamma-tocotrienol, 62 mg alpha-tocopherol, and 96 mg gamma-tocopherol). Plasma samples were collected at every 2 h intervals for up to 8 h following a one-time supplementation. Lipoproteins were measured by NMR spectroscopy, and subfractions of lipoproteins were isolated by density gradient ultracentrifugation. The maximal alpha-tocotrienol concentrations in supplemented individuals averaged approximately 3 microM in blood plasma, 1.7 microM in LDL, 0.9 microM in triglyceride-rich lipoprotein, and 0.5 microM in HDL. The peak plasma level corresponded to 12- to 30-fold more than the concentration of alpha-tocotrienol required to completely prevent stroke-related neurodegeneration. Tocotrienols were detected in the blood plasma and all lipoprotein subfractions studied postprandially.

Oxygen-sensitive Reset of Hypoxia-inducible Factor Transactivation Response: Prolyl Hydroxylases Tune the Biological Normoxic Set Point

Free Radical Biology & Medicine. Jun, 2006  |  Pubmed ID: 16785028

Cellular O(2) sensing enables physiological adjustments to variations in tissue pO(2). Under basal conditions, cells are adjusted to an O(2) environment biologically read as normoxia. Any sharp departure from that state of normoxia triggers O(2)-sensitive biological responses. The stabilization of hypoxia-inducible factor (HIF) signifies a robust biological readout of hypoxia. In the presence of sufficient O(2), HIF is hydroxylated and degraded. HIF prolyl hydroxylation is catalyzed by prolyl hydroxylase isoenzymes PHD1, 2, and 3. Using HT22 neurons stably transfected with a HIF reporter construct, we tested a novel hypothesis postulating that biological cells are capable of resetting their normoxic set point by O(2)-sensitive changes in PHD expression. Results of this study show that the pO(2) of the mouse brain cortex was 35 mm Hg or 5% O(2). Exposure of HT22, adjusted to growing in 20% O(2), to 5% O(2) resulted in HIF-driven transcription. However, cells adjusted to growing in 5% O(2) did not report hypoxia. Cells adjusted to growing in 30% O(2) reported hypoxia when acutely exposed to room air culture conditions. When grown under high O(2) conditions, cells reset their normoxic set point upward by down-regulating the expression of PHD1-3. When grown under low O(2) conditions, cells reset their normoxic set point downward by inducing the expression of PHD1-3. Exposure of mice in vivo to a hypoxic 10% O(2) environment lowered blood as well as brain pO(2). Such hypoxic exposure induced PHD1-3. Exposure of mice to a hyperoxic 50% O(2) ambience repressed the expression of PHD1-3, indicating that O(2)-sensitive regulation of PHD expression is effective in the brain in vivo. siRNA dependent knockdown of PHD expression revealed that O(2)-sensitive regulation of PHD may contribute to tuning the normoxic set point in biological cells.

Heat Shock Protein 60 Response to Exercise in Diabetes: Effects of Alpha-lipoic Acid Supplementation

Journal of Diabetes and Its Complications. Jul-Aug, 2006  |  Pubmed ID: 16798478

The pathophysiology of diabetes includes oxidative stress and impaired heat shock protein (HSP) expression. We studied the effects of alpha-lipoic acid (LA) supplementation for 8 weeks and acute exercise on HSP60 expression and the oxidative stress marker 4-hydroxynonenal adducts (4-HNE) in streptozotocin-induced diabetic (SID) and nondiabetic control rats. Diabetes was associated with decreased HSP60 in the heart and increased levels of HSP60 and 4-HNE in the liver. LA increased HSP60 in the liver of control and diabetic rats and decreased 4-HNE in the liver and heart. Acute exercise increased liver 4-HNE, which was offset by LA. In conclusion, diabetes induced oxidative stress and impaired myocardial HSP60 expression, while LA partially offsets these alterations in a tissue-specific manner.

Dietary Antioxidants for the Athlete

Current Sports Medicine Reports. Jun, 2006  |  Pubmed ID: 16822339

Physical exercise induces oxidative stress and tissue damage. Although a basal level of reactive oxygen species (ROS) is required to drive redox signaling and numerous physiologic processes, excess ROS during exercise may have adverse implications on health and performance. Antioxidant nutrients may be helpful in that regard. Caution should be exercised against excess antioxidant supplements, however. This article presents a digest for sports practitioners. The following three recommendations are made: 1) it is important to determine the individual antioxidant need of each athlete performing a specific sport; 2) multinutrient preparations, as opposed to megadoses of any single form of nutrient, seem to be a more prudent path to choose; and 3) for outcomes of antioxidant supplementation, performance should not be the only criteria. Overall well being of the athlete, faster recovery, and minimization of injury time could all be affected by antioxidant therapy.

Joint International Conference on EPR Spectroscopy and Wound Healing

Antioxidants & Redox Signaling. Jul-Aug, 2006  |  Pubmed ID: 16910786

The International Conference on Electron Paramagnetic Resonance Spectroscopy and Imaging of Biological Systems (EPR 2005) was held September 4 through 9, 2005, at Columbus, Ohio, U.S.A. Nearly 200 participants from 16 countries presented recent advances on the use of EPR technology to study biologic processes, with an emphasis on human health. For the first time, the EPR conference allied with the Wound Healing Conference, and the alliance opened an avenue for successful amalgamation of the basic biomedical and clinical aspects of wound healing with EPR technology and vice versa. This should lead to emerging applications of EPR technology in biomedical research and clinical practice.

Characterization of the Potent Neuroprotective Properties of the Natural Vitamin E Alpha-tocotrienol

Journal of Neurochemistry. Sep, 2006  |  Pubmed ID: 16923160

The natural vitamin E tocotrienols possess properties not shared by tocopherols. Nanomolar alpha-tocotrienol, not alpha-tocopherol, is potently neuroprotective. On a concentration basis, this finding represents the most potent of all biological functions exhibited by any natural vitamin E molecule. We sought to dissect the antioxidant-independent and -dependent neuroprotective properties of alpha-tocotrienol by using two different triggers of neurotoxicity, homocysteic acid (HCA) and linoleic acid. Both HCA and linoleic acid caused neurotoxicity with comparable features, such as increased ratio of oxidized to reduced glutathione GSSG/GSH, raised intracellular calcium concentration and compromised mitochondrial membrane potential. Mechanisms underlying HCA-induced neurodegeneration were comparable to those in the path implicated in glutamate-induced neurotoxicity. Inducible activation of c-Src and 12-lipoxygenase (12-Lox) represented early events in that pathway. Overexpression of active c-Src or 12-Lox sensitized cells to HCA-induced death. Nanomolar alpha-tocotrienol was protective. Knock-down of c-Src or 12-Lox attenuated HCA-induced neurotoxicity. Oxidative stress represented a late event in HCA-induced death. The observation that micromolar, but not nanomolar, alpha-tocotrienol functions as an antioxidant was verified in a model involving linoleic acid-induced oxidative stress and cell death. Oral supplementation of alpha-tocotrienol to humans results in a peak plasma concentration of 3 microm. Thus, oral alpha-tocotrienol may be neuroprotective by antioxidant-independent as well as antioxidant-dependent mechanisms.

Dermal Wound Healing is Subject to Redox Control

Molecular Therapy : the Journal of the American Society of Gene Therapy. Jan, 2006  |  Pubmed ID: 16126008

Previously we have reported in vitro evidence suggesting that that H2O2 may support wound healing by inducing VEGF expression in human keratinocytes (C. K. Sen et al., 2002, J. Biol. Chem.277, 33284-33290). Here, we test the significance of H2O2 in regulating wound healing in vivo. Using the Hunt-Schilling cylinder approach we present the first evidence that the wound site contains micromolar concentrations of H2O2. At the wound site, low concentrations of H2O2 supported the healing process, especially in p47(phox)- and MCP-1-deficient mice in which endogenous H2O2 generation is impaired. Higher doses of H2O2 adversely influenced healing. At low concentrations, H2O2 facilitated wound angiogenesis in vivo. H2O2 induced FAK phosphorylation both in wound-edge tissue in vivo and in human dermal microvascular endothelial cells. H2O2 induced site-specific (Tyr-925 and Tyr-861) phosphorylation of FAK. Other sites, including the Tyr-397 autophosphorylation site, were insensitive to H2O2. Adenoviral gene delivery of catalase impaired wound angiogenesis and closure. Catalase overexpression slowed tissue remodeling as evidenced by a more incomplete narrowing of the hyperproliferative epithelium region and incomplete eschar formation. Taken together, this work presents the first in vivo evidence indicating that strategies to influence the redox environment of the wound site may have a bearing on healing outcomes.

DNA Microarray Technology in the Evaluation of Weight Management Potential of a Novel Calcium-potassium Salt of (-)-hydroxycitric Acid

Toxicology Mechanisms and Methods. 2006  |  Pubmed ID: 20021004

Quality and quantity of diet and nutrients are key factors of human health and disease prevention. Molecular diagnostics and cellular signaling play a fundamental role in the usefulness of novel nutraceuticals and functional foods. Increasing knowledge of the genes and molecules involved in the development of obesity is creating new methods of obesity regulation. Traditional herbal medicines may have some potential in weight management. Botanical dietary supplements often contain complex mixtures of phytochemicals that have additive or synergistic interactions. Evidence from numerous human and animal dietary studies has demonstrated the potential therapeutic effects of traditional herbal medicines in controlling obesity. We analyzed the effects of low-dose oral administration of calcium-potassium salt of (-)-hydroxycitric acid (HCA-SX) on the body weight and abdominal fat transcriptome in rats. HCA-SX restricted body weight gain in rats and lowered abdominal fat leptin expression. High-density microarray analysis of 9960 genes and ESTs present in the fat tissue identified a small set of specific genes sensitive to dietary HCA-SX. Mitochondrial/nuclear proteins necessary for fundamental support of the tissue were not affected by HCA-SX, further demonstrating its safety. Functional characterization of HCA-SX sensitive genes revealed that up-regulation of genes encoding serotonin receptors represents a distinct effect of HCA-SX on appetite suppression.

12(S)-hydroperoxyeicosatetraenoic Acid (12-HETE) Increases Mitochondrial Nitric Oxide by Increasing Intramitochondrial Calcium

Archives of Biochemistry and Biophysics. Dec, 2007  |  Pubmed ID: 17963719

12(S)-hydroxyeicosatetraenoic acid (12-HETE) is one of the metabolites of arachidonic acid involved in pathological conditions associated with mitochondria and oxidative stress. The present study tested effects of 12-HETE on mitochondrial functions. In isolated rat heart mitochondria, 12-HETE increases intramitochondrial ionized calcium concentration that stimulates mitochondrial nitric oxide (NO) synthase (mtNOS) activity. mtNOS-derived NO causes mitochondrial dysfunctions by decreasing mitochondrial respiration and transmembrane potential. mtNOS-derived NO also produces peroxynitrite that induces release of cytochrome c and stimulates aggregation of mitochondria. Similarly, in HL-1 cardiac myocytes, 12-HETE increases intramitochondrial calcium and mitochondrial NO, and induces apoptosis. The present study suggests a novel mechanism for 12-HETE toxicity.

Harnessing Hypoxic Adaptation to Prevent, Treat, and Repair Stroke

Journal of Molecular Medicine (Berlin, Germany). Dec, 2007  |  Pubmed ID: 18043901

The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functions as diverse as bladder control and creative thinking. Chemical and electrical transmission in the nervous system is rapidly disrupted in stroke as a result of hypoxia and hypoglycemia. Despite being highly evolved in its architecture, the human brain appears to utilize phylogenetically conserved homeostatic strategies to combat hypoxia and ischemia. Specifically, several converging lines of inquiry have demonstrated that the transcription factor hypoxia-inducible factor-1 (HIF1-1) mediates the activation of a large cassette of genes involved in adaptation to hypoxia in surviving neurons after stroke. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of one of its sensors (e.g., inhibition of HIF prolyl 4 hydroxylases) leads to profound sparing of brain tissue and enhanced recovery of function. In this review, we discuss the potential mechanisms that could subserve protective and restorative effects of augmenting hypoxic adaptation in the brain. The strategy appears to involve HIF-dependent and HIF-independent pathways and more than 70 genes and proteins activated transcriptionally and post-transcriptionally that can act at cellular, local, and system levels to compensate for oxygen insufficiency. The breadth and depth of this homeostatic program offers a hopeful alternative to the current pessimism towards stroke therapeutics.

Leveraging an Information Warehouse to Create Translational Research Environment for Wound Care Center

AMIA ... Annual Symposium Proceedings / AMIA Symposium. AMIA Symposium. 2007  |  Pubmed ID: 18694039

In order to discover new biomarkers and therapeutic agents for personalized wound care, a vast amount of clinical information is collected and stored at The Ohio State University Medical Center (OSUMC) Comprehensive Wound Center (CWC). The Information Warehouse (IW) group at OSUMC has developed and implemented a comprehensive data collection network and analysis pipeline to support clinical, translational and outcomes research, and cost analyses that can be converted into clinical best practices for wound care.

The Role of the NADPH Oxidase Complex, P38 MAPK, and Akt in Regulating Human Monocyte/macrophage Survival

American Journal of Respiratory Cell and Molecular Biology. Jan, 2007  |  Pubmed ID: 16931806

M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)-induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF-stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/macrophage survival. Macrophages from p47(phox-/-) mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogen-activated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF-stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF-induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.

Mitochondrial Nitric Oxide Synthase

Frontiers in Bioscience : a Journal and Virtual Library. 2007  |  Pubmed ID: 17127362

Nitric oxide (NO) regulates several cellular functions via reversible regulation of mitochondrial respiration. Nitric oxide also reacts with mitochondrial superoxide anion to produce the potent oxidative species peroxynitrite that irreversibly hinders mitochondrial activities. Recent findings demonstrating that mitochondria produce NO via mitochondrial NO synthase (mtNOS) has intrigued several laboratories revealing crucial roles for mtNOS-derived NO and peroxynitrite in regulating the functions of mitochondria, cells and organs. The present article reviews the current understanding of the interactions between mitochondria, and NO and peroxynitrite.

Laser Microdissection and Capture of Pure Cardiomyocytes and Fibroblasts from Infarcted Heart Regions: Perceived Hyperoxia Induces P21 in Peri-infarct Myocytes

American Journal of Physiology. Heart and Circulatory Physiology. Mar, 2007  |  Pubmed ID: 17158647

Myocardial infarction caused by ischemia-reperfusion in the coronary vasculature is a focal event characterized by an infarct-core, bordering peri-infarct zone and remote noninfarct zone. Recently, we have reported the first technique, based on laser microdissection pressure catapulting (LMPC), enabling the dissection of infarction-induced biological responses in multicellular regions of the heart. Molecular mechanisms in play at the peri-infarct zone are central to myocardial healing. At the infarct site, myocytes are more sensitive to insult than robust fibroblasts. Understanding of cell-specific responses in the said zones is therefore critical. In this work, we describe the first technique to collect the myocardial tissue with a single-cell resolution. The infarcted myocardium was identified by using a truncated hematoxylin-eosin stain. Cell elements from the infarct, peri-infarct, and noninfarct zones were collected in a chaotropic RNA lysis solution with micron-level surgical precision. Isolated RNA was analyzed for quality by employing microfluidics technology and reverse transcribed to generate cDNA. Purity of the collected specimen was established by real-time PCR analyses of cell-specific genes. Previously, we have reported that the oxygen-sensitive induction of p21/Cip1/Waf1/Sdi1 in cardiac fibroblasts in the peri-infarct zone plays a vital role in myocardial remodeling. Using the novel LMPC technique developed herein, we confirmed that finding and report for the first time that the induction of p21 in the peri-infarct zone is not limited to fibroblasts but is also evident in myocytes. This work presents the first account of an analytical technique that applies the LMPC technology to study myocardial remodeling with a cell-type specific resolution.

Alpha-lipoic Acid Modulates Heat Shock Factor-1 Expression in Streptozotocin-induced Diabetic Rat Kidney

Antioxidants & Redox Signaling. Apr, 2007  |  Pubmed ID: 17280490

Increased oxidative stress and impaired heat shock protein (HSP) synthesis may contribute to diabetic nephropathy. The question of whether 8-week thiol antioxidant alpha-lipoic acid (LA) supplementation modulates HSP response and oxidative stress was studied in the kidney of streptozotocin-induced diabetic (SID) and nondiabetic rats. SID caused a histological mesangial expansion, tubular dilatation, and increased levels of transforming growth factor-beta (TGF-beta), a mediator of glomerulosclerosis. SID increased 4-hydroxynonenal (4-HNE) protein adduct formation, a marker of lipid peroxidation, and heme oxygenase-1 (HO-1), also a marker of oxidative stress. Moreover, SID increased the DNA-binding activity of heat shock factor-1 (HSF-1) and expression of heat shock protein 60 (HSP60). In contrast, LA supplementation partially reversed histological findings of glomerulosclerosis and decreased TGF-beta. LA also increased HSF-1 and decreased HO-1 protein expression, without affecting 4-HNE protein adduct levels. At the mRNA level, LA increased expression of HSF-1, HSP90, and glucose-regulated protein (GRP75) in both control and diabetic animals and HSP72 in SID rats. However, LA supplementation did not affect these HSPs at the protein level. These findings suggest that in addition to its antiglomerulosclerotic effects, LA can induce cytoprotective response in SID.

MiRNA: Licensed to Kill the Messenger

DNA and Cell Biology. Apr, 2007  |  Pubmed ID: 17465885

Current developments have brought non-coding genes under limelight together with their better-known siblings, the coding genes or mRNA. The 2006 Nobel Prize in Physiology or Medicine was awarded to Andrew Fire and Craig Mello for their 1998 discovery that double-stranded RNA triggers suppression of gene activity in a homology-dependent manner, a process named RNA interference (RNAi). Post-transcriptional regulation of genes was generally regarded as an odd regulatory mechanism for several years until it was learnt that regulatory trans-acting antisense RNAs exist in several species. Identification of a large number of small RNA molecules called microRNAs (miRNAs) elevated the overall field of biomedical RNAi to the striking level of current recognition. miRNAs represent a class of endogenous small ( approximately 22 nucleotides) RNA molecules that can repress protein synthesis. It is estimated that there are over 600 miRNAs in mammalian cells, and that about 30% of all genes are regulated by miRNA. Current understanding of the molecular mechanism of any disease would be incomplete without factoring in the functional significance of miRNA. In the category of the futuristic RNAi drugs, miRNA-based therapies are promising. The field has progressed rapidly as it relates to cancer research (highlighted in DNA and Cell Biology Volume 26, Number 4), while development in most other areas (highlighted in DNA and Cell Biology Volume 26, Number 3) of biomedical research remains in its infancy, offering significant opportunity for researchers. Approaches to interfere with miRNA function in vivo offer novel therapeutic opportunities. Lessons in gene therapy have taught us that tinkering with the genetic machinery comes with its own set of risks, especially in a clinical setting. miRNA-based therapies are also subject to such risks, which need to be prudently managed. Having acknowledged the potential risk, we have to recognize that new knowledge about the functional roles of miRNA is revolutionizing cell biology and will have a major impact on biomedical research imminently.

MicroRNA in Cutaneous Wound Healing: a New Paradigm

DNA and Cell Biology. Apr, 2007  |  Pubmed ID: 17465889

Repair of a defect in the human skin is a highly orchestrated physiological process involving numerous factors that act in a temporally resolved synergistic manner to re-establish barrier function by regenerating new skin. The inducible expression and repression of genes represents a key component of this regenerative process. MicroRNAs (miRNAs) are approximately 22-nucleotide-long endogenously expressed non-coding RNAs that regulate the expression of gene products by inhibition of translation and/or transcription in animals. miRNAs play a key role in skin morphogenesis and in regulating angiogenesis. The vascular endothelial growth factor signaling path seems to be under repressor control by miRNAs. Mature miRNA-dependent mechanisms impair angiogenesis in vivo. It is critically important to recognize that the understanding of cutaneous wound healing is incomplete without appreciating the functional significance of wound-induced miRNA. Ongoing work in our laboratory has led to the observation that the cutaneous wound healing process involves changes in the expression of specific miRNA at specific phases of wound healing. We hypothesize that dysregulation of specific miRNA is critical in derailing the healing sequence in chronic problem wounds. If tested positive, this hypothesis is likely to lead to completely novel diagnostic and therapeutic strategies for the treatment of problem wounds.

Algorithms for Mapping of MRNA Targets for MicroRNA

DNA and Cell Biology. Apr, 2007  |  Pubmed ID: 17465893

MicroRNAs (miRNAs) are involved in human health and disease as endogenous suppressors of the translation of coding genes. At this early point of time in miRNA biology, it is important to identify specific cognate mRNA targets for miRNA. Investigation of the significance of miRNAs in disease processes relies on algorithms that hypothetically link specific miRNAs to their putative target genes. The development of such algorithms represents a hot area of research in biomedical informatics. Lack of biological data linking specific miRNAs to their respective mRNA targets represents the most serious limitation at this time. This article presents a concise review addressing the most popular concepts underlying state-of-the-art algorithms and principles aimed at target mapping for specific miRNAs. Strategies for improvement of the current bioinformatics tools and effective approaches for biological validation are discussed.

Tocotrienols in Health and Disease: the Other Half of the Natural Vitamin E Family

Molecular Aspects of Medicine. Oct-Dec, 2007  |  Pubmed ID: 17507086

Tocochromanols encompass a group of compounds with vitamin E activity essential for human nutrition. Structurally, natural vitamin E includes eight chemically distinct molecules: alpha-, beta-, gamma- and delta-tocopherol; and alpha-, beta-, gamma- and delta-tocotrienol. Symptoms caused by alpha-tocopherol deficiency can be alleviated by tocotrienols. Thus, tocotrienols may be viewed as being members of the natural vitamin E family not only structurally but also functionally. Palm oil and rice bran oil represent two major nutritional sources of natural tocotrienol. Taken orally, tocotrienols are bioavailable to all vital organs. The tocotrienol forms of natural vitamin E possesses powerful hypocholesterolemic, anti-cancer and neuroprotective properties that are often not exhibited by tocopherols. Oral tocotrienol protects against stroke-associated brain damage in vivo. Disappointments with outcomes-based clinical studies testing the efficacy of alpha-tocopherol need to be handled with caution and prudence recognizing the untapped opportunities offered by the other forms of natural vitamin E. Although tocotrienols represent half of the natural vitamin E family, work on tocotrienols account for roughly 1% of the total literature on vitamin E. The current state of knowledge warrants strategic investment into investigating the lesser known forms of vitamin E.

Aerobically Derived Lactate Stimulates Revascularization and Tissue Repair Via Redox Mechanisms

Antioxidants & Redox Signaling. Aug, 2007  |  Pubmed ID: 17567242

Hypoxia serves as a physiologic cue to drive an angiogenic response via HIF-dependent mechanisms. Interestingly, minor elevation of lactate levels in the tissue produces the same effect under aerobic conditions. Aerobic glycolysis contributes to lactate accumulation in the presence of oxygen, especially under inflammatory conditions. We previously postulated that aerobic lactate accumulation, already known to stimulate collagen deposition, will also stimulate angiogenesis. If substantiated, this concept would advance understanding of wound healing and aerobic angiogenesis because lactate accumulation has many aerobic sources. In this study, Matrigel plugs containing a powdered, hydrolyzable lactate polymer were implanted into the subcutaneous space of mice. Lactate monomer concentrations in the implant were consistent with wound levels for more than 11 days. They induced little inflammation but considerable VEGF production and were highly angiogenic, as opposed to controls. Arterial hypoxia abrogated angiogenesis. Furthermore, inhibition of lactate dehydrogenase by using oxamate also prevented the angiogenic effects of lactate. Lactate monomer, at concentrations found in cutaneous wounds, stabilized HIF-1alpha and increased VEGF levels in aerobically cultured human endothelial cells. Accumulated lactate, therefore, appears to convey the impression of "metabolic need" for vascularization, even in well-oxygenated and pH-neutral conditions. Lactate and oxygen together stimulate angiogenesis and matrix deposition.

Regulation of C-Src Activity in Glutamate-induced Neurodegeneration

The Journal of Biological Chemistry. Aug, 2007  |  Pubmed ID: 17569670

c-Src is heavily expressed in the brain and in human neural tissues. Our pursuit for characterization of the neuroprotective mechanisms of tocotrienols led to the first evidence demonstrating that rapid c-Src activation plays a central role in executing glutamate-induced neurodegeneration. It is now known that Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. Here, we sought to examine the mechanisms that regulate inducible c-Src activity in glutamate-challenged HT4 neural cells and primary cortical neurons. Knockdown of c-Src protected cells against glutamate-induced loss of viability. Consistently, microinjection of siRNA against c-Src protected cells against glutamate. Using overexpression and knockdown approaches, we noted that SHP-1 may be implicated in glutamate-induced c-Src activation. Following such activation, Cbp and caveolin-1 were phosphorylated and associated with Csk. Csk was translocated to the membrane where it down-regulated glutamate-induced c-Src activity by catalyzing the inhibitory phosphorylation of a tyrosine residue in c-Src. Findings of this study present a new paradigm that addresses the regulation of c-Src under neurodegenerative conditions.

Tocotrienols: the Emerging Face of Natural Vitamin E

Vitamins and Hormones. 2007  |  Pubmed ID: 17628176

Natural vitamin E includes eight chemically distinct molecules: alpha-, beta-, gamma-, and delta-tocopherols and alpha-, beta-, gamma-, and delta-tocotrienols. More than 95% of all studies on vitamin E are directed toward the specific study of alpha-tocopherol. The other forms of natural vitamin E remain poorly understood. The abundance of alpha-tocopherol in the human body and the comparable efficiency of all vitamin E molecules as antioxidants led biologists to neglect the non-tocopherol vitamin E molecules as topics for basic and clinical research. Recent developments warrant a serious reconsideration of this conventional wisdom. The tocotrienol subfamily of natural vitamin E possesses powerful neuroprotective, anticancer, and cholesterol-lowering properties that are often not exhibited by tocopherols. Current developments in vitamin E research clearly indicate that members of the vitamin E family are not redundant with respect to their biological functions. alpha-Tocotrienol, gamma-tocopherol, and delta-tocotrienol have emerged as vitamin E molecules with functions in health and disease that are clearly distinct from that of alpha-tocopherol. At nanomolar concentration, alpha-tocotrienol, not alpha-tocopherol, prevents neurodegeneration. On a concentration basis, this finding represents the most potent of all biological functions exhibited by any natural vitamin E molecule. Recently, it has been suggested that the safe dose of various tocotrienols for human consumption is 200-1000/day. A rapidly expanding body of evidence supports that members of the vitamin E family are functionally unique. In recognition of this fact, title claims in publications should be limited to the specific form of vitamin E studied. For example, evidence for toxicity of a specific form of tocopherol in excess may not be used to conclude that high-dosage "vitamin E" supplementation may increase all-cause mortality. Such conclusion incorrectly implies that tocotrienols are toxic as well under conditions where tocotrienols were not even considered. The current state of knowledge warrants strategic investment into the lesser known forms of vitamin E. This will enable prudent selection of the appropriate vitamin E molecule for studies addressing a specific health need.

Transcriptome of Primary Adipocytes from Obese Women in Response to a Novel Hydroxycitric Acid-based Dietary Supplement

DNA and Cell Biology. Sep, 2007  |  Pubmed ID: 17708719

Obesity is a global public health problem. Traditional herbal medicines may have some potential in managing obesity. The dried fruit rind of Garcinia cambogia, also known as Malabar tamarind, is a unique source of (-)-hydroxycitric acid (HCA), which exhibits a distinct sour taste and has been safely used for centuries in Southeastern Asia to make meals more filling. Recently it has been demonstrated that when taken orally, a novel, highly soluble calcium/potassium salt of HCA (HCA-SX) is safe and bioavailable in the human plasma. Although HCA-SX seems to be conditionally effective in weight management in experimental animals and in humans, its mechanism of action remains unclear.

Transcriptome-wide Analysis of Blood Vessels Laser Captured from Human Skin and Chronic Wound-edge Tissue

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2007  |  Pubmed ID: 17728400

Chronic wounds represent a substantial public health problem. The development of tools that would enable sophisticated scrutiny of clinical wound tissue material is highly desirable. This work presents evidence enabling rapid specific identification and laser capture of blood vessels from human tissue in a manner which lends itself to successful high-density (U133A) microarray analysis. Such screening of transcriptome followed by real-time PCR and immunohistochemical verification of candidate genes and their corresponding products were performed by using 3 mm biopsies. Of the 18,400 transcripts and variants screened, a focused set of 53 up-regulated and 24 down-regulated genes were noted in wound-derived blood vessels compared with blood vessels from intact human skin. The mean abundance of periostin in wound-site blood vessels was 96-fold higher. Periostin is known to be induced in response to vascular injury and its expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Forty-fold higher expression of heparan sulfate 6-O-endosulfatase1 (Sulf1) was noted in wound-site vessels. Sulf1 has been recently recognized to be anti-angiogenic. During embryonic vasculogenesis, CD24 expression is down-regulated in human embryonic stem cells. Wound-site vessels had lower CD24 expression. The findings of this work provide a unique opportunity to appreciate the striking contrast in the transcriptome composition in blood vessels collected from the intact skin and from the wound-edge tissue. Sets of genes with known vascular functions but never connected to wound healing were identified to be differentially expressed in wound-derived blood vessels paving the way for innovative clinically relevant hypotheses.

P21waf1/cip1/sdi1 As a Central Regulator of Inducible Smooth Muscle Actin Expression and Differentiation of Cardiac Fibroblasts to Myofibroblasts

Molecular Biology of the Cell. Dec, 2007  |  Pubmed ID: 17881730

The phenotypic switch of cardiac fibroblasts (CFs) to myofibroblasts is essential for normal and pathological wound healing. Relative hyperoxic challenge during reoxygenation causes myocardial remodeling. Here, we sought to characterize the novel O(2)-sensitive molecular mechanisms responsible for triggering the differentiation of CFs to myofibroblasts. Exposure of CFs to hyperoxic challenge-induced transcription of smooth muscle actin (SMA) and enhanced the stability of both Acta2 transcript as well as of SMA protein. Both p21 deficiency as well as knockdown blunted hyperoxia-induced Acta2 and SMA response. Strikingly, overexpression of p21 alone markedly induced differentiation of CFs under normoxia. Overexpression of p21 alone induced SMA transcription by down-regulating YB1 and independent of TGFbeta1. In vivo, hyperoxic challenge induced p21-dependent differentiation of CFs to myofibroblasts in the infarct boundary region of ischemia-reperfused heart. Tissue elements were laser-captured from infarct boundary and from a noninfarct region 0.5 mm away. Reperfusion caused marked p21 induction in the infarct region. Acta2 as well as SMA expression were markedly up-regulated in CF-rich infarct boundary region. Of note, ischemia-reperfusion-induced up-regulation of Acta2 in the infarct region was completely abrogated in p21-deficient mice. This observation establishes p21 as a central regulator of reperfusion-induced phenotypic switch of CFs to myofibroblasts.

Minimally Invasive Neuroradiologic Model of Preclinical Transient Middle Cerebral Artery Occlusion in Canines

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2008  |  Pubmed ID: 18779582

Stroke is currently the third leading cause of death in the United States, with approximately 780,000 Americans affected by a new or recurring stroke each year. Although a variety of therapeutic approaches have shown promise in small-animal models of stroke, the vast majority of clinical trials to test the efficacy of such modalities have failed. To bridge the translational gap between laboratory and clinical research, we developed a preclinical model of acute ischemic stroke in dogs. Using a minimally invasive endovascular approach, a platinum coil was intravascularly guided through the vertebrobasilar system under C-arm fluoroscopy to occlude the M1 segment of the middle cerebral artery (MCA) for 1 h. The approach included femoral artery catheterization to access the MCA and therefore eliminated the occurrence of head trauma associated with other preclinical stroke models relying on transorbital or craniectomy approaches. After 1 h of focal MCA ischemia, the coil was retrieved to cause reperfusion, which was verified by arteriograms. At 24 h, T2-weighted coronal magnetic resonance (MR) images were acquired and processed for three-dimensional reconstruction of the brain and its vasculature. Infarction, limited to the area at risk, was noted. Two independent observers calculated the mean percentage hemispherical lesion volumes as follows: observer 1, 30.9 +/- 2.1%; observer 2, 31.2 +/- 4.3%. Infarct-affected changes in histology were determined by hematoxylin and eosin as well as by Fluoro-Jade staining. This work reports the successful development of a powerful preclinical model of stroke that lends itself to the study of biologic mechanisms as well as to testing experimental therapeutics.

Assessment of Wound-site Redox Environment and the Significance of Rac2 in Cutaneous Healing

Free Radical Biology & Medicine. Feb, 2008  |  Pubmed ID: 18068132

We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.

Redox Regulation of the VEGF Signaling Path and Tissue Vascularization: Hydrogen Peroxide, the Common Link Between Physical Exercise and Cutaneous Wound Healing

Free Radical Biology & Medicine. Jan, 2008  |  Pubmed ID: 18191754

Vascularization, under physiological or pathophysiological conditions, typically takes place by one or more of the following processes: angiogenesis, vasculogenesis, arteriogenesis, and lymphangiogenesis. Although all of these mechanisms of vascularization have sufficient contrasting features to warrant consideration under separate cover, one common feature shared by all is their sensitivity to the VEGF signaling pathway. Conditions such as wound healing and physical exercise result in increased production of reactive oxygen species such as H(2)O(2), and both are associated with increased tissue vascularization. Understanding these two scenarios of adult tissue vascularization in tandem offers the potential to unlock the significance of redox regulation of the VEGF signaling pathway. Does H(2)O(2) support tissue vascularization? H(2)O(2) induces the expression of the most angiogenic form of VEGF, VEGF-A, by a HIF-independent and Sp1-dependent mechanism. Ligation of VEGF-A to VEGFR2 results in signal transduction leading to tissue vascularization. Such ligation generates H(2)O(2) via an NADPH oxidase-dependent mechanism. Disruption of VEGF-VEGFR2 ligation-dependent H(2)O(2) production or decomposition of such H(2)O(2) stalls VEGFR2 signaling. Numerous antioxidants exhibit antiangiogenic properties. Current evidence lends firm credence to the hypothesis that low-level endogenous H(2)O(2) supports vascular growth.

Redox Signals in Wound Healing

Biochimica Et Biophysica Acta. Nov, 2008  |  Pubmed ID: 18249195

Physical trauma represents one of the most primitive challenges that threatened survival. Healing a problem wound requires a multi-faceted comprehensive approach. First and foremost, the wound environment will have to be made receptive to therapies. Second, the appropriate therapeutic regimen needs to be identified and provided while managing systemic limitations that could secondarily limit the healing response. Unfortunately, most current solutions seem to aim at designing therapeutic regimen with little or no consideration of the specific details of the wound environment and systemic limitations. One factor that is centrally important in making the wound environment receptive is correction of wound hypoxia. Recent work have identified that oxygen is not only required to disinfect wounds and fuel healing but that oxygen-dependent redox-sensitive signaling processes represent an integral component of the healing cascade. Over a decade ago, it was proposed that in biological systems oxidants are not necessarily always the triggers for oxidative damage and that oxidants such as H2O2 could actually serve as signaling messengers and drive several aspects of cellular signaling. Today, that concept is much more developed and mature. Evidence supporting the role of oxidants such as H2O2 as signaling messenger is compelling. A complete understanding of the continuum between the classical and emergent roles of oxygen requires a thorough consideration of current concepts in redox biology. The objective of this review is to describe our current understanding of how redox-sensitive processes may drive dermal tissue repair.

Evidence for the Involvement of MiRNA in Redox Regulated Angiogenic Response of Human Microvascular Endothelial Cells

Arteriosclerosis, Thrombosis, and Vascular Biology. Mar, 2008  |  Pubmed ID: 18258815

A Dicer knockdown approach was used to test the significance of miRNA in regulating the redox state and angiogenic response of human microvascular endothelial cells (HMECs).

HSP27 Regulates P53 Transcriptional Activity in Doxorubicin-treated Fibroblasts and Cardiac H9c2 Cells: P21 Upregulation and G2/M Phase Cell Cycle Arrest

American Journal of Physiology. Heart and Circulatory Physiology. Apr, 2008  |  Pubmed ID: 18263706

Treatment of cancer patients with anthracyclin-based chemotherapeutic drugs induces congestive heart failure by a mechanism involving p53. However, it is not known how p53 aggravates doxorubicin (Dox)-induced toxicity in the heart. On the basis of in vitro acute toxicity assay using heat shock factor-1 (HSF-1) wild-type (HSF-1(+/+)) and HSF-1-knockout (HSF-1(-/-)) mouse embryonic fibroblasts and neonatal rat cardiomyocyte-derived H9c2 cells, we demonstrate a novel mechanism whereby heat shock protein 27 (HSP27) regulates transcriptional activity of p53 in Dox-treated cells. Inhibition of p53 by pifithrin-alpha (PFT-alpha) provided different levels of protection from Dox that correlate with HSP27 levels in these cells. In HSF-1(+/+) cells, PFT-alpha attenuated Dox-induced toxicity. However, in HSF-1(-/-) cells (which express a very low level of HSP27 compared with HSF-1(+/+) cells), there was no such attenuation, indicating an important role of HSP27 in p53-dependent cell death. On the other hand, immunoprecipitation of p53 was found to coimmunoprecipitate HSP27 and vice versa (confirmed by Western blotting and matrix-assisted laser desorption/ionization time of flight), demonstrating HSP27 binding to p53 in Dox-treated cells. Moreover, upregulation of p21 was observed in HSF-1(+/+) and H9c2 cells, indicating that HSP27 binding transactivates p53 and enhances transcription of p21 in response to Dox treatment. Further analysis with flow cytometry showed that increased expression of p21 results in G(2)/M phase cell cycle arrest in Dox-treated cells. Overall, HSP27 binding to p53 attenuated the cellular toxicity by upregulating p21 and prevented cell death.

Wound Angiogenesis As a Function of Tissue Oxygen Tension: a Mathematical Model

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2008  |  Pubmed ID: 18272493

Wound healing represents a well orchestrated reparative response that is induced by injuries. Angiogenesis plays a central role in wound healing. In this work, we sought to develop the first mathematical model directed at addressing the role of tissue oxygen tension on cutaneous wound healing. Key components of the developed model include capillary tips, capillary sprouts, fibroblasts, inflammatory cells, chemoattractants, oxygen, and the extracellular matrix. The model consists of a system of nonlinear partial differential equations describing the interactions in space and time of these variables. The simulated results agree with the reported literature on the biology of wound healing. The proposed model represents a useful tool to analyze strategies for improved healing and generate a hypothesis for experimental testing.

Characterization of the Structural and Functional Changes in the Myocardium Following Focal Ischemia-reperfusion Injury

American Journal of Physiology. Heart and Circulatory Physiology. Jun, 2008  |  Pubmed ID: 18375718

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation (r2 = 0.85) between measured MRI and triphenyltetrazolium chloride (TTC) infarct volumes was noted. Our observation that until day 3 post-I/R the infarct size as measured by TTC staining and MRI was much larger than that of the myocyte-silent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MRI and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time.

Topical Oxygen Therapy Induces Vascular Endothelial Growth Factor Expression and Improves Closure of Clinically Presented Chronic Wounds

Clinical and Experimental Pharmacology & Physiology. Aug, 2008  |  Pubmed ID: 18430064

1. Chronic wounds, especially in diabetics, represent a serious threat to human health. 2. Correcting a compromised state of tissue oxygenation by the administration of supplemental O(2) is known to benefit wound healing. Beyond its role as a nutrient and antibiotic, O(2) supports wound healing by driving redox signaling. 3. Hyperbaric oxygen (HBO) therapy is widely used and approved by Center for Medicare and Medicaid Services to treat specific ulcerations. The current literature supports the notion that approaches to topically oxygenate wounds may be productive. 4. Here, we present the results of two simultaneous studies testing the effects of HBO and portable topical oxygen (TO) therapies. These two therapeutic approaches have several contrasting features. 5. In total, 1854 patients were screened in outpatient wound clinics for non-randomized enrolments into the HBO (n = 32; 31% diabetic) and TO (n = 25; 52% diabetic) studies. 6. Under the conditions of the present study, HBO treatment seemed to benefit some wounds while not benefiting others. Overall, HBO did not result in statistically significant improvements in wound size in the given population over the time monitored in the present study. 7. However, TO significantly improved wound size. Among the three O(2)-sensitive genes (VEGF, TGFbeta1 and COL1A1) studied in wound edge tissue biopsies, TO treatment was associated with higher VEGF165 expression in healing wounds. Expression of the other genes mentioned was not affected by TO. There was no significant change in the expression levels of any of genes studied in patients in the HBO study. This establishes a link between VEGF gene expression and healing outcome for TO therapy. 8. Taken together, the present study provides evidence demonstrating that TO treatment benefits wound healing in patients suffering from chronic wounds. Treatment with TO is associated with an induction of VEGF expression in wound edge tissue and an improvement in wound size.

Characterization of the Acute Temporal Changes in Excisional Murine Cutaneous Wound Inflammation by Screening of the Wound-edge Transcriptome

Physiological Genomics. Jul, 2008  |  Pubmed ID: 18460641

This work represents a maiden effort to systematically screen the transcriptome of the healing wound-edge tissue temporally using high-density GeneChips. Changes during the acute inflammatory phase of murine excisional wounds were characterized histologically. Sets of genes that significantly changed in expression during healing could be segregated into the following five sets: up-early (6-24 h; cytokine-cytokine receptor interaction pathway), up-intermediary (12-96 h; leukocyte-endothelial interaction pathway), up-late (48-96 h; cell-cycle pathway), down-early (6-12 h; purine metabolism) and down-intermediary (12-96 h; oxidative phosphorylation pathway). Results from microarray and real-time PCR analyses were consistent. Results listing all genes that were significantly changed at any specific time point were further mined for cell-type (neutrophils, macrophages, endothelial, fibroblasts, and pluripotent stem cells) specificity. Candidate genes were also clustered on the basis of their functional annotation, linking them to inflammation, angiogenesis, reactive oxygen species (ROS), or extracellular matrix (ECM) categories. Rapid induction of genes encoding NADPH oxidase subunits and downregulation of catalase in response to wounding is consistent with the fact that low levels of endogenous H2O2 is required for wound healing. Angiogenic genes, previously not connected to cutaneous wound healing, that were induced in the healing wound-edge included adiponectin, epiregulin, angiomotin, Nogo, and VEGF-B. This study provides a digested database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of cutaneous wound healing comprehensively.

Redox-active Antioxidant Modulation of Lipid Signaling in Vascular Endothelial Cells: Vitamin C Induces Activation of Phospholipase D Through Phospholipase A2, Lipoxygenase, and Cyclooxygenase

Molecular and Cellular Biochemistry. Aug, 2008  |  Pubmed ID: 18496733

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.

Differentiation of Bone Marrow Mesenchymal Stem Cells into the Smooth Muscle Lineage by Blocking ERK/MAPK Signaling Pathway

Stem Cells and Development. Oct, 2008  |  Pubmed ID: 18564029

Smooth muscle cells (SMCs) are major components of blood vessels and other hollow visceral organs required for tissue engineering of these organs. This study aims to evaluate whether adult bone marrow-derived mesenchymal stem cells (BMMSCs), multipotent cells, can be converted into SMCs. We examined the ERK/MAPK pathway as it exerts anti-myogenic signals in SMCs. Undifferentiated BMMSCs express most SMC marker genes, albeit mainly at low levels, except smooth muscle myosin heavy chain (SMMHC), the most definitive marker of differentiated SMC. The treatment of BMMSC with MEK inhibitor up-regulated the expression of alpha-smooth muscle actin (ASMA), h-caldesmon, and SMMHC in BMMSC in low serum condition. MEK inhibitor-treated BMMSC also contracted a collagen gel in response to endothelin. Interestingly, inhibition of MEK induced myocardin expression in BMMSC. In conclusion, BMMSCs treated MEK inhibitor gain a SMC-like phenotype with ligand-induced cell contractility to endothelin in vitro. This approach has obvious implications for cell therapeutics and tissue engineering of hollow visceral organs such as blood vessels.

Nutrigenomic Basis of Beneficial Effects of Chromium(III) on Obesity and Diabetes

Molecular and Cellular Biochemistry. Oct, 2008  |  Pubmed ID: 18636317

Insulin resistance has been shown to be the major contributing factor to the metabolic syndrome, which comprises a cluster of risk factors for metabolic aberrations such as obesity, dyslipidemia, hypertension, and hyperglycemia. Additionally, insulin resistance has been associated with the occurrence of cardiovascular disease and type 2 diabetes. Epidemiological studies indicate that obesity and diabetes have become alarmingly prevalent in recent years. Substantial evidence suggests that dietary interventions and regular exercise greatly improve body mass index and lipid profile as well as alleviate insulin resistance. Therefore, dietary supplements such as insulin-sensitizing agents may be beneficial in the prevention and treatment of obesity and type 2 diabetes. Numerous in vitro and in vivo studies suggest that chromium supplements, particularly niacin-bound chromium or chromium-nicotinate, may be effective in attenuating insulin resistance and lowering plasma cholesterol levels. Utilizing the powerful technology of nutrigenomics to identify the genes regulated by chromium supplementation may shed some light on the underlying mechanisms of chromium-gene interactions, and thus provide strategies to mitigate and prevent insulin-resistance-related disorders.

Temperature Measurement on Tissue Surface During Laser Irradiation

Medical & Biological Engineering & Computing. Feb, 2008  |  Pubmed ID: 17891430

Tissue surface temperature distribution on the treatment site can serve as an indicator for the effectiveness of a photothermal therapy. In this study, both infrared thermography and theoretical simulation were used to determine the surface temperature distribution during laser irradiation of both gel phantom and animal tumors. Selective photothermal interaction was attempted by using intratumoral indocyanine green enhancement and irradiation via a near-infrared laser. An immunoadjuvant was also used to enhance immunological responses during tumor treatment. Monte Carlo method for tissue absorption of light and finite difference method for heat diffusion in tissue were used to simulate the temperature distribution during the selective laser photothermal interaction. An infrared camera was used to capture the thermal images during the laser treatment and the surface temperature was determined. Our findings show that the theoretical and experimental results are in good agreement and that the surface temperature of irradiated tissue can be controlled with appropriate dye and adjuvant enhancement. These results can be used to control the laser tumor treatment parameters and to optimize the treatment outcome. More importantly, when used with immunotherapy as a precursor of immunological responses, the selective photothermal treatment can be guided by the tissue temperature profiles both in the tumor and on the surface.

Redox Modification of Ryanodine Receptors Contributes to Sarcoplasmic Reticulum Ca2+ Leak in Chronic Heart Failure

Circulation Research. Dec, 2008  |  Pubmed ID: 19008475

Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

Oral Administration of Blueberry Inhibits Angiogenic Tumor Growth and Enhances Survival of Mice with Endothelial Cell Neoplasm

Antioxidants & Redox Signaling. Jan, 2009  |  Pubmed ID: 18817478

Endothelial cell neoplasms are the most common soft tissue tumor in infants. Subcutaneous injection of spontaneously transformed murine endothelial (EOMA) cells results in development of hemangioendothelioma (HE). We have previously shown that blueberry extract (BBE) treatment of EOMA cells in vitro prior to injection in vivo can significantly inhibit the incidence and size of developing HE. In this study, we sought to determine whether oral BBE could be effective in managing HE and to investigate the mechanisms through which BBE exerts its effects on endothelial cells. A dose-dependent decrease in HE tumor size was observed in mice receiving daily oral gavage feeds of BBE. Kaplan-Meier survival curve showed significantly enhanced survival for mice with HE tumors given BBE, compared to control. BBE treatment of EOMA cells inhibited both c-Jun N-terminal kinase (JNK) and NF-kappaB signaling pathways that culminate in monocyte chemoattractant protein-1 (MCP-1) expression required for HE development. Antiangiogenic effects of BBE on EOMA cells included decreased proliferation by BrdU assay, decreased sprouting on Matrigel, and decreased transwell migration. Thus, this work provides first evidence demonstrating that BBE can limit tumor formation through antiangiogenic effects and inhibition of JNK and NF-kappaB signaling pathways. Oral administration of BBE represents a potential therapeutic antiangiogenic strategy for treating endothelial cell neoplasms in children.

Neuroprotective and Antiinflammatory Properties of a Novel Demethylated Curcuminoid

Antioxidants & Redox Signaling. Mar, 2009  |  Pubmed ID: 18724833

A demethylated derivative of curcumin (DC; 67.8% bisdemethylcurcumin, 20.7% demethylmonodemethoxycurcumin, 5.86% bisdemethoxycurcumin, 2.58% demethylcurcumin) was prepared by using a 95% extract of curcumin (C(95); 72.2% curcumin, 18.8% monodemethoxycurcumin, 4.5% bisdemethoxycurcumin). DC increased glutathione and reduced reactive oxygen species (ROS) in HT4 neuronal cells. In a model of glutamate-induced death of HT4, DC was more effective than C(95) in neuroprotection. The protective effects of DC were retained even when DC was withdrawn from culture media after pretreatment. DC treatment, unlike an equal dose of C(95), completely spared glutamate-induced loss of cellular GSH. Both DC and C(95) prevented glutamate-induced elevation of cellular ROS but failed to attenuate glutamate-induced elevation of intracellular calcium. In human microvascular endothelial cells (HMECs) challenged with TNF-alpha, GeneChip analysis revealed that only a subcluster of 23 TNF-alpha-inducible genes were uniquely sensitive to C(95). In sharp contrast, 1,065 TNF-alpha-inducible genes were sensitive to DC but not to C(95), suggesting that DC was more effective in antagonizing the effects of TNF-alpha on HMECs. Functional analysis identified that the genes uniquely sensitive to DC belonged in four functional categories: cytokine-receptor interaction, focal adhesion, cell adhesion, and apoptosis. Real-time PCR as well as ELISA studies demonstrated that TNF-alpha-inducible CXCL10 and CXCL11 expression was sensitive to DC but not to C(95). Flow-cytometry studies recognized ICAM-1 and VCAM-1 as TNF-alpha-inducible adhesion molecules that were uniquely sensitive to DC. Taken together, DC exhibited promising neuroprotective and antiinflammatory properties that must be characterized in vivo.

Gadolinium-containing Phosphatidylserine Liposomes for Molecular Imaging of Atherosclerosis

Journal of Lipid Research. Nov, 2009  |  Pubmed ID: 19017616

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE(-/-) mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.

Diabetes Impairs Exercise Training-associated Thioredoxin Response and Glutathione Status in Rat Brain

Journal of Applied Physiology (Bethesda, Md. : 1985). Feb, 2009  |  Pubmed ID: 19074570

Regular exercise plays an important preventive and therapeutic role in oxidative stress-associated diseases such as diabetes and its complications. Thiol antioxidants including thioredoxin (TRX) and glutathione (GSH) have a crucial role in controlling cellular redox status. In this study, the effects of 8 wk of exercise training on brain TRX and GSH systems, and antioxidant enzymes were tested in rats with or without streptozotocin-induced diabetes. We found that in untrained animals, the levels of TRX-1 (TRX1) protein and activity, and thioredoxin-interacting protein (TXNip) were similar in diabetic and nondiabetic animals. Exercise training, however, increased TRX1 protein in nondiabetic animals without affecting TXNip levels, whereas diabetes inhibited the effect of training on TRX1 protein and also increased TXNip mRNA. In addition, the proportion of oxidized glutathione (GSSG) to total GSH was increased in animals with diabetes, indicating altered redox status and possibly increased oxidative stress. Glutathione peroxidase-1 (GPX1) levels were not affected by diabetes or exercise training, although diabetes increased total GPX activity. Both diabetes and exercise training decreased glutathione reductase (GRD) activity and cytosolic superoxide dismutase (Cu,Zn-SOD) levels. Nevertheless, diabetes or training had no effect on Cu,Zn-SOD mRNA, Mn-SOD protein, total SOD activity, or catalase mRNA, protein, or activity. Our findings suggest that exercise training increases TRX1 levels in brain without a concomitant rise in TXNip, and that experimental diabetes is associated with an incomplete TRX response to training. Increased oxidative stress may be both a cause and a consequence of perturbed antioxidant defenses in the diabetic brain.

MicroRNA Expression in Response to Murine Myocardial Infarction: MiR-21 Regulates Fibroblast Metalloprotease-2 Via Phosphatase and Tensin Homologue

Cardiovascular Research. Apr, 2009  |  Pubmed ID: 19147652

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA. Encoded in the genome of most eukaryotes, miRNAs have been proposed to regulate specifically up to 90% of human genes through a process known as miRNA-guided RNA silencing. For the first time, we sought to test how myocardial ischaemia-reperfusion (IR) changes miR expression.

Wound Healing Essentials: Let There Be Oxygen

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Jan-Feb, 2009  |  Pubmed ID: 19152646

The state of wound oxygenation is a key determinant of healing outcomes. From a diagnostic standpoint, measurements of wound oxygenation are commonly used to guide treatment planning such as amputation decision. In preventive applications, optimizing wound perfusion and providing supplemental O(2) in the perioperative period reduces the incidence of postoperative infections. Correction of wound pO(2) may, by itself, trigger some healing responses. Importantly, approaches to correct wound pO(2) favorably influence outcomes of other therapies such as responsiveness to growth factors and acceptance of grafts. Chronic ischemic wounds are essentially hypoxic. Primarily based on the tumor literature, hypoxia is generally viewed as being angiogenic. This is true with the condition that hypoxia be acute and mild to modest in magnitude. Extreme near-anoxic hypoxia, as commonly noted in problem wounds, is not compatible with tissue repair. Adequate wound tissue oxygenation is required but may not be sufficient to favorably influence healing outcomes. Success in wound care may be improved by a personalized health care approach. The key lies in our ability to specifically identify the key limitations of a given wound and in developing a multifaceted strategy to specifically address those limitations. In considering approaches to oxygenate the wound tissue it is important to recognize that both too little as well as too much may impede the healing process. Oxygen dosing based on the specific need of a wound therefore seems prudent. Therapeutic approaches targeting the oxygen sensing and redox signaling pathways are promising.

Heat Shock Proteins in Diabetes and Wound Healing

Current Protein & Peptide Science. Feb, 2009  |  Pubmed ID: 19275675

The heat shock proteins (HSPs), originally identified as heat-inducible gene products, are a highly conserved family of proteins that respond to a wide variety of stress. Although HSPs are among the most abundant intracellular proteins, they are expressed at low levels under normal physiological conditions, and show marked induction in response to various stressors. HSPs function primarily as molecular chaperones, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific pathways for degradation. By modulating inflammation, wound debris clearance, cell proliferation, migration and collagen synthesis, HSPs are essential for normal wound healing of the skin. In this review, our goal is to discuss the role and clinical implications of HSP with respect to skin wound healing and diabetes. The numerous defects in the function of HSPs associated with diabetes could contribute to the commonly observed complications and delayed wound healing in diabetics. Several physical, pharmacological and genetic approaches may be considered to address HSP-directed therapies both in the laboratory and in the clinics.

Inhibition of Vascular Smooth-muscle Cell Proliferation and Arterial Restenosis by HO-3867, a Novel Synthetic Curcuminoid, Through Up-regulation of PTEN Expression

The Journal of Pharmacology and Experimental Therapeutics. Jun, 2009  |  Pubmed ID: 19276401

Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, has been shown to play a vital role in vascular smooth muscle cell (SMC) proliferation and hence is a potential therapeutic target to inhibit vascular remodeling. The goal of this study was to evaluate the efficacy and mechanism of HO-3867 [((3E,5E)-3,5-bis[(4-fluorophenyl)methylidene]-1-[(1-hydroxy-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl]piperidin-4-one)], a new synthetic curcuminoid, in the inhibition of vascular SMC proliferation and restenosis. Experiments were performed using human aortic SMCs and a rat carotid artery balloon injury model. HO-3867 (10 microM) significantly inhibited the proliferation of serum-stimulated SMCs by inducing cell cycle arrest at the G(1) phase (72% at 24 h) and apoptosis (at 48 h). HO-3867 significantly increased the phosphorylated and total levels of PTEN in SMCs. Suppression of PTEN expression by PTEN-small interfering RNA transfection reduced p53 and p21 levels and increased extracellular signal-regulated kinase 1/2 phosphorylation, resulting in decreased apoptosis. Conversely, overexpression of PTEN by cDNA transfection activated caspase-3 and increased apoptosis. Furthermore, HO-3867 significantly down-regulated matrix metalloproteinase (MMP)-2, MMP-9, and nuclear factor (NF)-kappaB expressions in SMCs. Finally, HO-3867 inhibited arterial neointimal hyperplasia through overexpression of PTEN and down-regulation of MMPs and NF-kappaB proteins. HO-3867 is a potent drug, capable of overexpressing PTEN, which is a key target in the prevention of vascular remodeling, including restenosis.

Characterization of a Preclinical Model of Chronic Ischemic Wound

Physiological Genomics. May, 2009  |  Pubmed ID: 19293328

Chronic ischemic wounds presenting at wound clinics are heterogeneous with respect to etiology, age of the wound, and other factors complicating wound healing. In addition, there are ethical challenges associated with collecting repeated biopsies from a patient to develop an understanding of the temporal dynamics of the mechanisms underlying chronic wounds. The need for a preclinical model of ischemic wound is therefore compelling. The porcine model is widely accepted as an excellent preclinical model for human wounds. A full-thickness bipedicle flap approach was adopted to cause skin ischemia. Closure of excisional wounds placed on ischemic tissue was severely impaired resulting in chronic wounds. Histologically, ischemic wounds suffered from impaired re-epithelialization, delayed macrophage recruitment and poorer endothelial cell abundance and organization. Compared with the pair-matched nonischemic wound, unique aspects of the ischemic wound biology were examined on days 3, 7, 14, and 28 by systematic screening of the wound tissue transcriptome using high-density porcine GeneChips. Ischemia markedly potentiated the expression of arginase-1, a cytosolic enzyme that metabolizes the precursor of nitric oxide l-arginine. Ischemia also induced the SOD2 in the wound tissue perhaps as survival response of the challenged tissue. Human chronic wounds also demonstrated elevated expression of SOD2 and arginase-1. This study provides a thorough database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of ischemic chronic wounds in a preclinical setting.

Evidence-based Recommendations for the Use of Topical Oxygen Therapy in the Treatment of Lower Extremity Wounds

The International Journal of Lower Extremity Wounds. Jun, 2009  |  Pubmed ID: 19443899

Topical oxygen therapy provides another tool in the armamentarium of clinicians treating refractory lower extremity wounds. Devices suitable for providing topical oxygen therapy in a clinical setting have recently become available. This article reviews the evidence to justify the use of this treatment modality, including in vitro, preclinical data, and clinical data. It also provides a protocol for how to administer topical oxygen therapy as well as guidance on patient selection and management to optimize outcomes. Randomized controlled trials are not yet reported and clearly necessary. The current body of evidence suggests that topical oxygen therapy may be considered as a second line of therapy for refractory wounds.

Remodeling of the Ischemia-reperfused Murine Heart: 11.7-T Cardiac Magnetic Resonance Imaging of Contrast-enhanced Infarct Patches and Transmurality

Antioxidants & Redox Signaling. Aug, 2009  |  Pubmed ID: 19450139

Our laboratory has published the first evidence obtained from fast low-angle-shot cine magnetic resonance imaging (11.7 T) studies demonstrating secondary myocyte death after ischemia/reperfusion (IR) of the murine heart. This work provides the first evidence from 11.7-T magnet-assisted pixel-level analysis of the post-IR murine myocardial infarct patches. Changes in function of the remodeling heart were examined in tandem. IR compromised cardiac function and induced LV hypertrophy. During recovery, the IR-induced increase in LV mass was partly offset. IR-induced wall thinning was noted in the anterior aspect of LV and at the diametrically opposite end. Infarct size was observed to be largest on post-IR days 3 and 7. With time (day 28), however, the infarct size was significantly reduced. IR-induced absolute signal-intensity enhancement was highest on post-IR days 3 and 7. As a function of post-IR time, signal-intensity enhancement was attenuated. The threshold of hyperenhanced tissue resulted in delineation of contours that identified necrotic (bona fide infarct) and reversibly injured infarct patches. The study of infarct transmurality indicated that whereas the permanently injured tissue volume remained unchanged, part of the reversibly injured infarct patch recovered in 4 weeks after IR. The approach validated in the current study is powerful in noninvasively monitoring remodeling of the post-IR beating murine myocardium.

Alpha-Lipoic Acid Modulates Thiol Antioxidant Defenses and Attenuates Exercise-induced Oxidative Stress in Standardbred Trotters

Free Radical Research. Aug, 2009  |  Pubmed ID: 19548154

Several micronutrient supplementation strategies are used to cope with oxidative stress, although their benefits have recently been questioned. The aim of the present study was to examine the effects of DL-alpha-lipoic acid (LA) in response to acute exercise and during recovery in horses. Six standardbred trotters were tested on the treadmill before and after 5-week LA supplementation (25 mg/kg body weight/day). According to electron paramagnetic resonance measurements, strenuous aerobic exercise increased significantly free radical formation in the gluteus medius muscle, which was prevented by LA supplementation. The activities of thioredoxin reductase and glutathione reductase in muscle were significantly increased in LA-treated horses, but neither LA nor exercise affected muscle thioredoxin activity. LA increased the concentration of total glutathione in muscle at rest and during recovery. Treatment with LA blunted the exercise-induced increase in plasma oxygen radical absorbance capacity and decreased the post-exercise levels of lipid hydroperoxides in plasma and malondialdehyde in plasma and in muscle. These findings suggest that LA enhances thiol antioxidant defences and decreases exercise-induced oxidative stress in skeletal muscle.

Micromanaging Vascular Biology: Tiny MicroRNAs Play Big Band

Journal of Vascular Research. 2009  |  Pubmed ID: 19571573

Micro-RNAs (miRNAs) are estimated to regulate 30% of the human genome primarily through translational repression. In 2005-2008, the first series of observations establishing the key significance of miRNAs in the regulation of vascular biology came from experimental studies involved in arresting miRNA biogenesis to deplete the miRNA pools of vascular tissues and cells. Dicer-dependent biogenesis of miRNA is required for blood vessel development during embryogenesis and wound healing. miRNAs regulate redox signaling in endothelial cells, a key regulator of vascular cell biology. miRNAs that regulate angiogenesis include miRNA 17-5p, cluster 17-92, 21, 27a&b, 126, 130a, 210, 221, 222, 378 and the let7 family. miRNAs also represent a new therapeutic target for the treatment of proliferative vascular diseases as well as hypertension. Evidence supporting the regulation of inducible adhesion molecules by miRNA supports a role of miRNAs in regulating vascular inflammation. Productive strategies to safely up-regulate as well as down-regulate miRNAs in vivo are in place and being tested for their value in disease intervention. Prudent targeting of non-coding genes such as miRNAs, which in turn regulates large sets of coding genes, holds promise in gene therapy. Recent developments in miRNA biology offer lucrative opportunities to manage vascular health.

Fabrication and Characterization of Prosurvival Growth Factor Releasing, Anisotropic Scaffolds for Enhanced Mesenchymal Stem Cell Survival/growth and Orientation

Biomacromolecules. Sep, 2009  |  Pubmed ID: 19689108

Scaffolds that not only mimic the mechanical and structural properties of the target tissue but also support cell survival/growth are likely necessary for the development of mechanically functional cardiovascular tissues. To reach these goals, we have generated scaffolds that are elastic to approximate soft tissue mechanical properties, are nanofibrous to mimic fibrous nature of extracellular matrix (ECM), have aligned structure to guide cellular alignment, and are capable of releasing insulin-like growth factor (IGF-1) to administrate cellular growth and survival. We have developed a technique that can quickly fabricate (<3 h) such scaffolds by simultaneously electrospinning elastase-sensitive polyurethaneurea nanofibers, encapsulating IGF-1 into poly(lactide-co-glycolide) (PLGA) microspheres and assembling them into scaffolds. Scaffold morphology, mechanical properties, degradation with or without elastase, and bioactivity of the released IGF-1 were assessed. The scaffolds had degree of alignment approximately 70%. They were flexible and relatively strong, with tensile strengths of 3.4-11.1 MPa, elongations at break of 71-88%, and moduli of 2.3-7.9 MPa at the alignment direction. IGF-1 release profile and bioactivity were dependent on PLGA content and molecular weight and IGF-1 loading. The released IGF-1 remained bioactive for 4 weeks. The fabricated nanofibers were elastase-sensitive with weight remaining <59% after a 4-week degradation in the presence of elastase. Mesenchymal stem cells (MSCs) were seeded on the scaffolds and cultured either under normal culture conditions (21% O(2), 5% CO(2), and 20% fetal bovine serum (FBS)) or hypoxia/nutrient starvation conditions (5% O(2), 5% CO(2), and 1% FBS) to evaluate the effect of IGF-1 loading on cell growth and survival. Under normal culture conditions, MSCs were found to align on the scaffolds with a degree of alignment matching that of the scaffold. The IGF-1 loaded scaffolds enhanced MSC growth during a 7-day culture period, with higher IGF-1 content showing better stimulus effect. Under hypoxia/nutrient starvation conditions, the IGF-1 loaded scaffolds were found to significantly improve MSC survival.

Translational Informatics: Enabling High-throughput Research Paradigms

Physiological Genomics. Nov, 2009  |  Pubmed ID: 19737991

A common thread throughout the clinical and translational research domains is the need to collect, manage, integrate, analyze, and disseminate large-scale, heterogeneous biomedical data sets. However, well-established and broadly adopted theoretical and practical frameworks and models intended to address such needs are conspicuously absent in the published literature or other reputable knowledge sources. Instead, the development and execution of multidisciplinary, clinical, or translational studies are significantly limited by the propagation of "silos" of both data and expertise. Motivated by this fundamental challenge, we report upon the current state and evolution of biomedical informatics as it pertains to the conduct of high-throughput clinical and translational research and will present both a conceptual and practical framework for the design and execution of informatics-enabled studies. The objective of presenting such findings and constructs is to provide the clinical and translational research community with a common frame of reference for discussing and expanding upon such models and methodologies.

A Mathematical Model of Ischemic Cutaneous Wounds

Proceedings of the National Academy of Sciences of the United States of America. Sep, 2009  |  Pubmed ID: 19805373

Chronic wounds represent a major public health problem affecting 6.5 million people in the United States. Ischemia, primarily caused by peripheral artery diseases, represents a major complicating factor in cutaneous wound healing. In this work, we sought to develop a mathematical model of ischemic dermal wounds. The model consists of a coupled system of partial differential equations in the partially healed region, with the wound boundary as a free boundary. The extracellular matrix (ECM) is assumed to be viscoelastic, and the free boundary moves with the velocity of the ECM at the boundary. The model equations involve the concentration of oxygen, PDGF and VEGF, the densities of macrophages, fibroblasts, capillary tips and sprouts, and the density and velocity of the ECM. Simulations of the model demonstrate how ischemic conditions may limit macrophage recruitment to the wound-site and impair wound closure. The results are in general agreement with experimental findings.

Human Skin Wounds: a Major and Snowballing Threat to Public Health and the Economy

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Nov-Dec, 2009  |  Pubmed ID: 19903300

ABSTRACT In the United States, chronic wounds affect 6.5 million patients. An estimated excess of US$25 billion is spent annually on treatment of chronic wounds and the burden is rapidly growing due to increasing health care costs, an aging population and a sharp rise in the incidence of diabetes and obesity worldwide. The annual wound care products market is projected to reach $15.3 billion by 2010. Chronic wounds are rarely seen in individuals who are otherwise healthy. In fact, chronic wound patients frequently suffer from "highly branded" diseases such as diabetes and obesity. This seems to have overshadowed the significance of wounds per se as a major health problem. For example, NIH's Research Portfolio Online Reporting Tool (RePORT; http://report.nih.gov/), directed at providing access to estimates of funding for various disease conditions does list several rare diseases but does not list wounds. Forty million inpatient surgical procedures were performed in the United States in 2000, followed closely by 31.5 million outpatient surgeries. The need for post-surgical wound care is sharply on the rise. Emergency wound care in an acute setting has major significance not only in a war setting but also in homeland preparedness against natural disasters as well as against terrorism attacks. An additional burden of wound healing is the problem of skin scarring, a $12 billion annual market. The immense economic and social impact of wounds in our society calls for allocation of a higher level of attention and resources to understand biological mechanisms underlying cutaneous wound complications.

Injectable, Highly Flexible, and Thermosensitive Hydrogels Capable of Delivering Superoxide Dismutase

Biomacromolecules. Dec, 2009  |  Pubmed ID: 19919046

Injectable hydrogels are attractive for cell and drug delivery. In this work, we synthesized a family of injectable, biodegradable, fast gelling and thermosensitive hydrogels based on N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), dimethyl-gamma-butyrolactone acrylate (DBA), and 2-hydroxyethyl methacrylate-poly(trimethylene carbontate) (HEMAPTMC) macromer. Type I collagen was composited with the hydrogels to improve their biocompatibility. The hydrogel copolymer solutions were readily injectable at 4 degrees C. The solutions exhibited thermal transition temperatures ranging from 23.6 to 24.5 degrees C and were capable of gelation within 7 s at 37 degrees C to form highly flexible and soft hydrogels with moduli from 39 to 119 KPa and breaking strains >1000%, depending on the copolymer composition and collagen addition. After 2 weeks incubation in PBS, the hydrogels demonstrated weight losses ranging from 10-20%. The completely degraded hydrogels had thermal transition temperatures >40 degrees C and were soluble at body temperature. Superoxide dismutase (SOD) was encapsulated in the hydrogels for the purpose of capturing superoxide within the inflammatory tissue after being delivered in vivo. The hydrogels demonstrated a sustained release profile during a 21-day release period. The release kinetics was dependent on the SOD loading, collagen addition, hydrogel degradation and water content. The released SOD remained bioactive during the entire release period. To test in vitro if the loaded SOD could protect cells encapsulated within the hydrogel from attack by superoxide, human mesenchymal stem cells (MSC) were encapsulated in SOD-loaded hydrogels and cultured in medium containing superoxide generated by activated macrophages. It was found that SOD loading largely suppressed superoxide penetration into the hydrogel and cell membrane. Under normal culture conditions, SOD loading stimulated MSC growth. The SOD-loaded hydrogel exhibited significantly higher cell numbers than the non-SOD loaded hydrogel during a 7-day culture period. These results demonstrated that the developed hydrogels could be used as delivery vehicles for stem cell therapy and drug delivery.

Injectable, Rapid Gelling and Highly Flexible Hydrogel Composites As Growth Factor and Cell Carriers

Acta Biomaterialia. Jun, 2010  |  Pubmed ID: 20004745

A family of injectable, rapid gelling and highly flexible hydrogel composites capable of releasing insulin-like growth factor (IGF-1) and delivering mesenchymal stromal cell (MSC) were developed. Hydrogel composites were fabricated from Type I collagen, chondroitin sulfate (CS) and a thermosensitive and degradable hydrogel copolymer based on N-isopropylacrylamide, acrylic acid, N-acryloxysuccinimide and a macromer poly(trimethylene carbonate)-hydroxyethyl methacrylate. The hydrogel copolymer was gellable at body temperature before degradation and soluble at body temperature after degradation. Hydrogel composites exhibited LCSTs around room temperature. They could easily be injected through a 26-gauge needle at 4 degrees C, and were capable of gelling within 6s at 37 degrees C to form highly flexible gels with moduli matching those of the rat and human myocardium. The hydrogel composites showed good oxygen permeability; the oxygen pressure within the hydrogel composites was similar to that in the air. The effects of collagen and CS contents on LCST, gelation time, injectability, mechanical properties and degradation properties were investigated. IGF-1 was loaded into the hydrogel composites for enhanced cell survival/growth. The released IGF-1 remained bioactive during a 2-week release period. Small fraction of CS in the hydrogel composites significantly decreased IGF-1 release rate. The release kinetics appeared to be controlled mainly by hydrogel composite water content, degradation and interaction with IGF-1. Human MSC adhesion on the hydrogel composites was comparable to that on the tissue culture plate. MSCs were encapsulated in the hydrogel composites and were found to grow inside during a 7-day culture period. IGF-1 loading significantly accelerated MSC growth. RT-PCR analysis demonstrated that MSCs maintained their multipotent differentiation potential in hydrogel composites with and without IGF-1. These injectable and rapid gelling hydrogel composites demonstrated attractive properties for serving as growth factor and cell carriers for cardiovascular tissue engineering applications.

Nanomolar Vitamin E Alpha-tocotrienol Inhibits Glutamate-induced Activation of Phospholipase A2 and Causes Neuroprotection

Journal of Neurochemistry. Mar, 2010  |  Pubmed ID: 20028458

Our previous works have elucidated that the 12-lipoxygenase pathway is directly implicated in glutamate-induced neural cell death, and that such that toxicity is prevented by nM concentrations of the natural vitamin E alpha-tocotrienol (TCT). In the current study we tested the hypothesis that phospholipase A(2) (PLA(2)) activity is sensitive to glutamate and mobilizes arachidonic acid (AA), a substrate for 12-lipoxygenase. Furthermore, we examined whether TCT regulates glutamate-inducible PLA(2) activity in neural cells. Glutamate challenge induced the release of [(3)H]AA from HT4 neural cells. Such response was attenuated by calcium chelators (EGTA and BAPTA), cytosolic PLA(2) (cPLA(2))-specific inhibitor (AACOCF(3)) as well as TCT at 250 nM. Glutamate also caused the elevation of free polyunsaturated fatty acid (AA and docosahexaenoic acid) levels and disappearance of phospholipid-esterified AA in neural cells. Furthermore, glutamate induced a time-dependent translocation and enhanced serine phosphorylation of cPLA(2) in the cells. These effects of glutamate on fatty acid levels and on cPLA(2) were significantly attenuated by nM TCT. The observations that AACOCF(3), transient knock-down of cPLA(2) as well as TCT significantly protected against the glutamate-induced death of neural cells implicate cPLA(2) as a TCT-sensitive mediator of glutamate induced neural cell death. This work presents first evidence recognizing glutamate-induced changes in cPLA(2) as a novel mechanism responsible for neuroprotection observed in response to nanomolar concentrations of TCT.

Oxygen-sensitive Outcomes and Gene Expression in Acute Ischemic Stroke

Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. Jul, 2010  |  Pubmed ID: 20145654

Acute ischemic stroke (AIS) results in focal deprivation of blood-borne factors, one of them being oxygen. The purpose of this study was two-fold: (1) to identify therapeutic conditions for supplemental oxygen in AIS and (2) to use transcriptome-wide screening toward uncovering oxygen-sensitive mechanisms. Transient MCAO in rodents was used to delineate the therapeutic potential of normobaric (NBO, 100% O(2), 1ATA) and hyperbaric oxygen (HBO, 100% O(2), 2ATA) during ischemia (iNBO, iHBO) and after reperfusion (rNBO, rHBO). Stroke lesion was quantified using 4.7 T MRI at 48 h. Supplemental oxygen during AIS significantly attenuated percent stroke hemisphere lesion volume as compared with that in room air (RA) controls, whereas identical treatment immediately after reperfusion exacerbated lesion volume (RA=22.4+/-1.8, iNBO=9.9+/-3.6, iHBO=6.6+/-4.8, rNBO=29.8+/-3.6, rHBO=35.4+/-7.6). iNBO and iHBO corrected penumbra tissue pO(2) during AIS as measured by EPR oxymetry. Unbiased query of oxygen-sensitive transcriptome in stroke-affected tissue identified 5,769 differentially expressed genes. Candidate genes were verified by real-time PCR using neurons laser-captured from the stroke-affected somatosensory cortex. Directed microarray analysis showed that supplemental oxygen limited leukocyte accumulation to the infarct site by attenuation of stroke-inducible proinflammatory chemokine response. The findings provide key information relevant to understanding oxygen-dependent molecular mechanisms in the AIS-affected brain.

Macrophage Dysfunction Impairs Resolution of Inflammation in the Wounds of Diabetic Mice

PloS One. 2010  |  Pubmed ID: 20209061

Chronic inflammation is a characteristic feature of diabetic cutaneous wounds. We sought to delineate novel mechanisms involved in the impairment of resolution of inflammation in diabetic cutaneous wounds. At the wound-site, efficient dead cell clearance (efferocytosis) is a pre-requisite for the timely resolution of inflammation and successful healing.

Hypoxia Inducible MicroRNA 210 Attenuates Keratinocyte Proliferation and Impairs Closure in a Murine Model of Ischemic Wounds

Proceedings of the National Academy of Sciences of the United States of America. Apr, 2010  |  Pubmed ID: 20308562

Ischemia complicates wound closure. Here, we are unique in presenting a murine ischemic wound model that is based on bipedicle flap approach. Using this model of ischemic wounds we have sought to elucidate how microRNAs may be implicated in limiting wound re-epithelialization under hypoxia, a major component of ischemia. Ischemia, evaluated by laser Doppler as well as hyperspectral imaging, limited blood flow and lowered tissue oxygen saturation. EPR oximetry demonstrated that the ischemic wound tissue had pO(2) <10 mm Hg. Ischemic wounds suffered from compromised macrophage recruitment and delayed wound epithelialization. Specifically, epithelial proliferation, as determined by Ki67 staining, was compromised. In vivo imaging showed massive hypoxia inducible factor-1alpha (HIF-1alpha) stabilization in ischemic wounds, where HIF-1alpha induced miR-210 expression that, in turn, silenced its target E2F3, which was markedly down-regulated in the wound-edge tissue of ischemic wounds. E2F3 was recognized as a key facilitator of cell proliferation. In keratinocytes, knock-down of E2F3 limited cell proliferation. Forced stabilization of HIF-1alpha using Ad-VP16- HIF-1alpha under normoxic conditions up-regulated miR-210 expression, down-regulated E2F3, and limited cell proliferation. Studies using cellular delivery of miR-210 antagomir and mimic demonstrated a key role of miR-210 in limiting keratinocyte proliferation. In summary, these results are unique in presenting evidence demonstrating that the hypoxia component of ischemia may limit wound re-epithelialization by stabilizing HIF-1alpha, which induces miR-210 expression, resulting in the down-regulation of the cell-cycle regulatory protein E2F3.

Fra-2 Mediates Oxygen-sensitive Induction of Transforming Growth Factor Beta in Cardiac Fibroblasts

Cardiovascular Research. Sep, 2010  |  Pubmed ID: 20427335

In the ischaemia-reperfused heart, transforming growth factor beta (TGFbeta) proteins trigger the differentiation of cardiac fibroblasts (CFs) contributing to fibrosis. Reoxygenation of the heart, in addition to being a trigger for reperfusion injury, induces tissue remodelling by hyperoxia-sensitive signalling processes involving TGFbeta. Here, we sought to characterize the molecular mechanisms responsible for the O(2)-sensitive transcriptional induction of TGFbeta in murine CF and to test the significance of such findings in the infarcted myocardium in vivo using laser capture microdissection.

Acute Exercise and Thioredoxin-1 in Rat Brain, and Alpha-lipoic Acid and Thioredoxin-interacting Protein Response, in Diabetes

International Journal of Sport Nutrition and Exercise Metabolism. Jun, 2010  |  Pubmed ID: 20601738

Thioredoxin (TRX) is a protein disulfide reductase that plays an important role in many thiol-dependent cellular reductive processes, antioxidant protection, and signal transduction. Moreover, TRX reduces and maintains the function of many proteins during oxidative stress, which is increased in diabetes. The authors recently reported that diabetes impairs brain redox status and TRX response to exercise training. As a continuation of their studies, they hypothesized that alpha-lipoic acid, a natural thiol antioxidant, has a favorable effect on the brain TRX and glutathione (GSH) system in diabetes. Streptozotocin-induced diabetes was used as a chronic model and exhaustive exercise as an acute model for disrupted redox balance. Half the diabetic and nondiabetic animals were subjected to a bout of exhaustive exercise after 8 wk with or without lipoic acid and analyzed for key thiol antioxidants. Lipoic acid neither altered diabetes-induced oxidative stress as assessed by the increased ratio of oxidized to total GSH nor had any impact on the antioxidant protein response to exercise. However, lipoic acid increased mRNA of TRX-interacting protein, an inhibitor of TRX-1, and glutaredoxin-1 in diabetes. Exercise increased TRX-1 mRNA in both diabetic and nondiabetic animals but had no effect on TRX-1 protein. Cytosolic superoxide dismutase mRNA was only increased in diabetes, whereas exercise increased the protein levels in nondiabetic animals. The findings suggest that exhaustive exercise induces mRNA of TRX-1 in the brain and that lipoic acid cannot prevent diabetes-induced disturbances in GSH homeostasis. Because lipoic acid increased TRX-interacting protein transcription in diabetes, high doses may impair TRX-1 homeostasis.

Endothelial Dysfunction in the Microcirculation of Patients with Obstructive Sleep Apnea

American Journal of Respiratory and Critical Care Medicine. Dec, 2010  |  Pubmed ID: 20656942

Obstructive sleep apnea (OSA) is a risk factor for cardiovascular disease. We hypothesized that patients with OSA and no cardiovascular disease have oxidant-related microcirculatory endothelial dysfunction.

Palm Oil-derived Natural Vitamin E Alpha-tocotrienol in Brain Health and Disease

Journal of the American College of Nutrition. Jun, 2010  |  Pubmed ID: 20823491

A growing body of research supports that members of the vitamin E family are not redundant with respect to their biological function. Palm oil derived from Elaeis guineensis represents the richest source of the lesser characterized vitamin E, alpha-tocotrienol. One of 8 naturally occurring and chemically distinct vitamin E analogs, alpha-tocotrienol possesses unique biological activity that is independent of its potent antioxidant capacity. Current developments in alpha-tocotrienol research demonstrate neuroprotective properties for the lipid-soluble vitamin in brain tissue rich in polyunsaturated fatty acids (PUFAs). Arachidonic acid (AA), one of the most abundant PUFAs of the central nervous system, is highly susceptible to oxidative metabolism under pathologic conditions. Cleaved from the membrane phospholipid bilayer by cytosolic phospholipase A(2), AA is metabolized by both enzymatic and nonenzymatic pathways. A number of neurodegenerative conditions in the human brain are associated with disturbed PUFA metabolism of AA, including acute ischemic stroke. Palm oil-derived alpha-tocotrienol at nanomolar concentrations has been shown to attenuate both enzymatic and nonenzymatic mediators of AA metabolism and neurodegeneration. On a concentration basis, this represents the most potent of all biological functions exhibited by any natural vitamin E molecule. Despite such therapeutic potential, the scientific literature on tocotrienols accounts for roughly 1% of the total literature on vitamin E, thus warranting further investment and investigation.

Reactive Oxygen Species Mediate Oxidized Low-density Lipoprotein-induced Inhibition of Oct-4 Expression and Endothelial Differentiation of Bone Marrow Stem Cells

Antioxidants & Redox Signaling. Dec, 2010  |  Pubmed ID: 20836655

This study was to test the hypothesis that oxidized low-density lipoprotein (ox-LDL) modified the behavior of bone marrow stem cells, including proliferation, Oct-4 expression, and their endothelial differentiation through reactive oxygen species (ROS) formation in vitro. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL with or without the antioxidant N-acetylcysteine (NAC). Ox-LDL generated a significant amount of ROS in the culture system as measured with electron paramagnetic resonance spectroscopy, and substantially inhibited the proliferation, Oct-4 expression, and endothelial differentiation of MAPCs. ROS production from ox-LDL in the culture system was completely prevented by NAC (1 mM). NAC treatment completely restored endothelial differentiation potential of MAPCs that was diminished by low-dose ox-LDL. NAC also significantly, but not completely, reversed the inhibitory effect of ox-LDL on proliferation and Oct-4 expression in MAPCs. NAC treatment only slightly restored Akt phosphorylation impaired by ox-LDL in the cells. ROS formation was important in the action of ox-LDL on MAPCs, including Oct-4 expression, proliferation, and endothelial differentiation. However, other mechanism(s) like Akt signaling and apoptosis might also play a critical role in mediating the effect of ox-LDL on these cells.

Oxygenation State As a Driver of Myofibroblast Differentiation and Wound Contraction: Hypoxia Impairs Wound Closure

The Journal of Investigative Dermatology. Dec, 2010  |  Pubmed ID: 21068734

Myofibroblasts are ubiquitous in the human body and may form from the differentiation of fibroblasts, epithelial cells, endothelial cells, and mononuclear cells, among others. Their clinical significance could be substantial, depending on biomedical context. Myofibroblasts help contract open skin wounds, but they could also be key drivers of fibrosis across numerous tissue systems and support tumor invasiveness. Understanding the molecular events underlying myofibroblast formation is significant for many human diseases. In this issue, Modarressi et al. address the significance of wound tissue hypoxia in impairing wound contraction by compromising myofibroblast formation. They present compelling evidence indicating tissue hypoxia conflicts with wound closure. We are reminded that correcting wound tissue hypoxia is critical for the tissue's response to other therapeutic interventions.

Differential Roles of Hypoxia Inducible Factor Subunits in Multipotential Stromal Cells Under Hypoxic Condition

Journal of Cellular Biochemistry. Nov, 2010  |  Pubmed ID: 21104912

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition. © 2010 Wiley-Liss, Inc.

Alpha-lipoic Acid Does Not Alter Stress Protein Response to Acute Exercise in Diabetic Brain

Cell Biochemistry and Function. Dec, 2010  |  Pubmed ID: 21104931

Heat shock proteins (HSPs) are molecular chaperones which may act protective in cerebrovascular insults and peripheral diabetic neuropathy. We hypothesized that alpha-lipoic acid (LA), a natural thiol antioxidant, may enhance brain HSP response in diabetes. Rats with or without streptozotocin-induced diabetes were treated with LA or saline for 8 weeks. Half of the rats were subjected to exhaustive exercise to investigate HSP induction, and the brain tissue was analyzed. Diabetes increased constitutive HSC70 mRNA, and decreased HSP90 and glucose-regulated protein 75 (GRP75) mRNA without affecting protein levels. Exercise increased HSP90 protein and mRNA, and also GRP75 and heme oxygenase-1 (HO-1) mRNA only in non-diabetic animals. LA had no significant effect on brain HSPs, although LA increased HSC70 and HO-1 mRNA in diabetic animals and decreased HSC70 mRNA in non-diabetic animals. Eukaryotic translation elongation factor-2, essential for protein synthesis, was decreased by diabetes and suggesting a mechanism for the impaired HSP response related to translocation of the nascent chain during protein synthesis. LA supplementation does not offset the adverse effects of diabetes on brain HSP mRNA expression. Diabetes may impair HSP translation through elongation factors related to nascent chain translocation and subsequent responses to acute stress.

Detection of Macrophages Via Paramagnetic Vesicles Incorporating Oxidatively Tailored Cholesterol Ester: an Approach for Atherosclerosis Imaging

Nanomedicine (London, England). Nov, 2010  |  Pubmed ID: 21128718

Macrophages play a key role in the initiation, progression and complications of atherosclerosis. In this article we describe the synthesis of biocompatible, paramagnetic, fluorescent phosphatidylserine vesicles containing cholesterol ester with a free carboxylic acid function and its use for targeted imaging of macrophages.

Prevalence of Obstructive Sleep Apnea in Patients with Chronic Wounds

Journal of Clinical Sleep Medicine : JCSM : Official Publication of the American Academy of Sleep Medicine. Dec, 2010  |  Pubmed ID: 21206743

Chronic non-healing wounds are a major human and economic burden. Obstructive sleep apnea (OSA) is prevalent in patients with obesity, diabetes, aging, and cardiovascular disease, all of which are risk factors for chronic wounds. We hypothesized that OSA would have more prevalence in patients of a wound center than the general middle-aged population.

Differential Roles of Hypoxia Inducible Factor Subunits in Multipotential Stromal Cells Under Hypoxic Condition

Journal of Cellular Biochemistry. Mar, 2011  |  Pubmed ID: 21328454

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF, and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition.

MicroRNAs As New Maestro Conducting the Expanding Symphony Orchestra of Regenerative and Reparative Medicine

Physiological Genomics. May, 2011  |  Pubmed ID: 21467158

The human genome encodes 1,048 microRNAs (miRNAs). These miRNAs regulate virtually all biological processes. Leaving ignominy on the significance miRNAs behind we are approaching a new era in tissue repair where an ever expanding orchestra of events that enable tissue repair and regeneration seems to be conducted by miRNAs as the maestro. microRNAs are emerging as molecular rheostats that fine-tune and switch regulatory circuits governing tissue repair. Key elements of tissue repair such as stem cell biology, inflammation, hypoxia-response, and angiogenesis are all under the sophisticated control of a network of specific mRNAs. Embryonic stem cells lacking miRNAs lose their "stemness." Manipulation of specific cellular miRNAs helps enhance reprogramming of somatic cells to an embryonic stem cell-like phenotype helping generate inducible pluripotent stem cells. Expression of miRNAs is subject to control by epigenetic factors. Such control influences the balance between proliferation and differentiation of stem cells. Angiomirs regulate various aspects of angiogenesis, such as proliferation, migration, and morphogenesis of endothelial cells. MiRNAs play a key role in resolution of inflammation. Hypoxia-inducible mRNAs or hypoxamirs suppress mitochondrial respiration, cause cell cycle arrest, and interfere with growth factor signaling. miRNA-210 is a good example in this category that impairs wound closure. As fine tools enabling specific and temporally controlled manipulation of cell-specific miRNAs emerge, miRNA-based therapies hold promise in facilitating tissue repair. Treatment of skin wounds has lower barriers because it lends itself to local delivery of miRNA mimics and antagonizing agents minimizing risks associated with systemic off-target toxicity.

Particulate β-glucan Induces TNF-α Production in Wound Macrophages Via a Redox-sensitive NF-κβ-dependent Pathway

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. May-Jun, 2011  |  Pubmed ID: 21518092

Glucans are known to promote wound repair. Noncellulosic β-glucans are recognized as potent immunological activators. β-Glucans are generally safe and are known to attenuate the rate of postoperative infection. Glyc101 is a particulate β-glucan isolated from Saccharomyces cerevisiae. In this study, the hypothesis that Glyc101 regulates wound macrophage function was tested. Glyc101 induced tumor necrosis factor (TNF) α transcription in macrophages isolated from murine wound site. Multiplex assay identified interleukin (IL)-10 and TNFα as two cytokines that are induced by Glyc101 in human blood monocyte-derived macrophages. Glyc101-induced TNFα production was observed to be mediated via the TLR-2 and dectin-1 receptors, receptor tyrosine kinases and NFκB activation. In murine wound macrophages, Glyc101 potentiated phorbol 12-myristate 13-acetate-induced respiratory burst. In vivo, implantation of Glyc101-enriched polyvinyl alcohol-sponges at the wound-site induced TNFα expression in macrophages. Consistently, Glyc101 induced TNFα expression in wound-site macrophages isolated from two patients with chronic wounds. These observations establish the translational significance of the net findings of this study. Activation of wound macrophages by Glyc101 represents one of the potential mechanisms by which this β-glucan may benefit chronic wounds where inefficient inflammatory response is one of the underlying causes of impaired healing.

Tocotrienol Vitamin E Protects Against Preclinical Canine Ischemic Stroke by Inducing Arteriogenesis

Journal of Cerebral Blood Flow and Metabolism : Official Journal of the International Society of Cerebral Blood Flow and Metabolism. Nov, 2011  |  Pubmed ID: 21673716

Vitamin E consists of tocopherols and tocotrienols, in which α-tocotrienol is the most potent neuroprotective form that is also effective in protecting against stroke in rodents. As neuroprotective agents alone are insufficient to protect against stroke, we sought to test the effects of tocotrienol on the cerebrovascular circulation during ischemic stroke using a preclinical model that enables fluoroscopy-guided angiography. Mongrel canines (mean weight=26.3±3.2 kg) were supplemented with tocotrienol-enriched (TE) supplement (200 mg b.i.d, n=11) or vehicle placebo (n=9) for 10 weeks before inducing transient middle cerebral artery (MCA) occlusion. Magnetic resonance imaging was performed 1 hour and 24 hours post reperfusion to assess stroke-induced lesion volume. Tocotrienol-enriched supplementation significantly attenuated ischemic stroke-induced lesion volume (P<0.005). Furthermore, TE prevented loss of white matter fiber tract connectivity after stroke as evident by probabilistic tractography. Post hoc analysis of cerebral angiograms during MCA occlusion revealed that TE-supplemented canines had improved cerebrovascular collateral circulation to the ischemic MCA territory (P<0.05). Tocotrienol-enriched supplementation induced arteriogenic tissue inhibitor of metalloprotease 1 and subsequently attenuated the activity of matrix metalloproteinase-2. Outcomes of the current preclinical trial set the stage for a clinical trial testing the effects of TE in patients who have suffered from transient ischemic attack and are therefore at a high risk for stroke.

Natural Vitamin E α-tocotrienol Protects Against Ischemic Stroke by Induction of Multidrug Resistance-associated Protein 1

Stroke; a Journal of Cerebral Circulation. Aug, 2011  |  Pubmed ID: 21719775

α-Tocotrienol (TCT) represents the most potent neuroprotective form of natural vitamin E that is Generally Recognized As Safe certified by the U.S. Food and Drug Administration. This work addresses a novel molecular mechanism by which α-TCT may be protective against stroke in vivo. Elevation of intracellular oxidized glutathione (GSSG) triggers neural cell death. Multidrug resistance-associated protein 1 (MRP1), a key mediator of intracellular oxidized glutathione efflux from neural cells, may therefore possess neuroprotective functions.

Mitochondrially Targeted α-tocopheryl Succinate is Antiangiogenic: Potential Benefit Against Tumor Angiogenesis but Caution Against Wound Healing

Antioxidants & Redox Signaling. Dec, 2011  |  Pubmed ID: 21902599

AIMS: A plausible strategy to reduce tumor progress is the inhibition of angiogenesis. Therefore, agents that efficiently suppress angiogenesis can be used for tumor suppression. We tested the antiangiogenic potential of a mitochondrially targeted analog of α-tocopheryl succinate (MitoVES), a compound with high propensity to induce apoptosis. RESULTS: MitoVES was found to efficiently kill proliferating endothelial cells (ECs) but not contact-arrested ECs or ECs deficient in mitochondrial DNA, and suppressed angiogenesis in vitro by inducing accumulation of reactive oxygen species and induction of apoptosis in proliferating/angiogenic ECs. Resistance of arrested ECs was ascribed, at least in part, to the lower mitochondrial inner transmembrane potential compared with the proliferating ECs, thus resulting in the lower level of mitochondrial uptake of MitoVES. Shorter-chain homologs of MitoVES were less efficient in angiogenesis inhibition, thus suggesting a molecular mechanism of its activity. Finally, MitoVES was found to suppress HER2-positive breast carcinomas in a transgenic mouse as well as inhibit tumor angiogenesis. The antiangiogenic efficacy of MitoVES was corroborated by its inhibitory activity on wound healing in vivo. INNOVATION AND CONCLUSION: We conclude that MitoVES, a mitochondrially targeted analog of α-tocopheryl succinate, is an efficient antiangiogenic agent of potential clinical relevance, exerting considerably higher activity than its untargeted counterpart. MitoVES may be helpful against cancer but may compromise wound healing.

Tocotrienols: the Lesser Known Form of Natural Vitamin E

Indian Journal of Experimental Biology. Oct, 2011  |  Pubmed ID: 22013739

A recent and growing body of research has shown that members of this vitamin E family posses unique biologic functions. Tocotrienols have garnered much of this recent attention, and in particular alpha-tocotrienol has been shown to be the most potent neuroprotective form of vitamin E. Protection exclusively mediated through tocotrienols has been arbitrated to many mechanisms including inhibition of 12-LOX, c-Src, PLA2 and through up-regulation of MRP1. Further, tocotrienols have recently been shown to induce arteriogenesis through induction of TIMP1 and decreased activation of MMP2. However, the unique therapeutic potential of tocotrienols is not limited to neuroprotection. Tocotrienols have been shown to have molecular targets including: apoptotic regulators, cytokines, adhesion molecules, enzymes, kinases, receptors, transcription factors, and growth factors. In spite of this large and unique therapeutic potential, scientific literature on tocotrienols only accounts for approximately 1% of vitamin E research. Given the potential of tocotrienols and relatively scant literature, further investigation is warranted.

Platelet-rich Fibrin Matrix Improves Wound Angiogenesis Via Inducing Endothelial Cell Proliferation

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Nov, 2011  |  Pubmed ID: 22092846

The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.

MiRNA in Innate Immune Responses: Novel Players in Wound Inflammation

Physiological Genomics. May, 2011  |  Pubmed ID: 21139022

Chronic wounds represent a major and rising socioeconomic threat affecting over 6.5 million people in the United States costing in excess of US $25 billion annually. Wound healing is a physiological response to injury that is conserved across tissue systems. In humans, wounding is followed by instant response aimed at hemostasis, which in turn provides the foundation for inflammatory processes that closely follow. Inflammation is helpful and a prerequisite for healing as long as it is mounted and resolved in a timely manner. Chronic inflammation derails the healing cascade resulting in impaired wound closure. Disruption of Dicer, the RNase III enzyme that generates functional miRNAs, has a major impact on the overall immune system. Emerging studies indicate that miRNAs, especially miR-21, miR-146a/b, and miR-155, play a key role in regulating several hubs that orchestrate the inflammatory process. Direct evidence from studies addressing wound inflammation being limited, the current work represents a digest of the relevant literature that is aimed at unveiling the potential significance of miRNAs in the regulation of wound inflammation. Such treatment would help establish new paradigms highlighting a central role of miRs in the understanding and management of dysregulated inflammation as noted in conjunction with chronic wounds.

MiR-200b Targets Ets-1 and is Down-regulated by Hypoxia to Induce Angiogenic Response of Endothelial Cells

The Journal of Biological Chemistry. Jan, 2011  |  Pubmed ID: 21081489

The miR-200 family plays a crucial role in epithelial to mesenchymal transition via controlling cell migration and polarity. We hypothesized that miR-200b, one miR-200 family member, could regulate angiogenic responses via modulating endothelial cell migration. Delivery of the miR-200b mimic in human microvascular endothelial cells (HMECs) suppressed the angiogenic response, whereas miR-200b-depleted HMECs exhibited elevated angiogenesis in vitro, as evidenced by Matrigel® tube formation and cell migration. Using in silico studies, miR target reporter assay, and Western blot analysis revealed that v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1), a crucial angiogenesis-related transcription factor, serves as a novel direct target of miR-200b. Knocking down endogenous Ets-1 simulated an anti-angiogenic response of the miR-200b mimic-transfected cells. Certain Ets-1-associated genes, namely matrix metalloproteinase 1 and vascular endothelial growth factor receptor 2, were negatively regulated by miR-200b. Overexpression of Ets-1 rescued miR-200b-dependent impairment in angiogenic response and suppression of Ets-1-associated gene expression. Both hypoxia as well as HIF-1α stabilization inhibited miR-200b expression and elevated Ets-1 expression. Experiments to identify how miR-200b modulates angiogenesis under a low oxygen environment illustrated that hypoxia-induced miR-200b down-regulation de-repressed Ets-1 expression to promote angiogenesis. This study provides the first evidence that hypoxia-sensitive miR-200b is involved in induction of angiogenesis via directly targeting Ets-1 in HMECs.

MiR-210: the Master Hypoxamir

Microcirculation (New York, N.Y. : 1994). Dec, 2011  |  Pubmed ID: 22171547

MicroRNAs (miRs) are small non-coding RNAs implicated mainly in post-transcriptional gene silencing by interacting with the unstranslated region of the transcript. miR-210 represents a major hypoxia-inducible miRs, also known as hypoxamirs, which is ubiquitously expressed in a wide range of cells, serving versatile functions. This review article summarizes the current progress on biogenesis of miR-210 and its physiological roles including arrest of cell proliferation, repression of mitochondrial respiration, arrest of DNA repair, vascular biology, and angiogenesis. Given the fact that miR-210 is aberrantly expressed in a number of diseases such as tumor progression, myocardial infarction and cutaneous ischemic wounds, miR-210 could serve as an excellent candidate for prognostic purposes and therapeutic intervention. With the advancement of computational prediction, high-throughput target validation methodology, sequencing, proteomic analysis and microarray, it is anticipated that more down-stream targets of miR-210 and its-associated biological consequences under hypoxia will be unveiled establishing miR-210 as a major hub in the biology of hypoxia-response.

MiRNA in Wound Inflammation and Angiogenesis

Microcirculation (New York, N.Y. : 1994). Dec, 2011  |  Pubmed ID: 22211762

Chronic wounds represent a rising health and economic burden to our society. Emerging studies indicate that miRNAs play a key role in regulating several hubs that orchestrate the wound inflammation and angiogenesis processes. Of interest to wound inflammation are the regulatory loops where inflammatory mediators elicited following injury, are regulated by miRNAs as well as regulate miRNA expression. Adequate angiogenesis is a key determinant of success in ischemic wound repair. Hypoxia and cellular redox state are among the key factors that drive wound angiogenesis. We provided first evidence demonstrating that miRNAs regulate cellular redox environment via a NADPH oxidase dependent mechanism in human microvascular endothelial cells (HMECs). We further demonstrated that hypoxia-sensitive miR-200b is involved in induction of angiogenesis by directly targeting Ets-1 in HMECs. These studies points towards a potential role of miRNA in wound angiogenesis. miRNA-based therapeutics represents one of the major commercial hot spots in today's biotechnology market space. Understanding the significance of miRs in wound inflammation and angiogenesis may help design therapeutic strategies for management of chronic non-healing wounds.

An Endovascular Canine Middle Cerebral Artery Occlusion Model for the Study of Leptomeningeal Collateral Recruitment

Investigative Radiology. Jan, 2011  |  Pubmed ID: 20856126

This work aimed to refine a large animal in minimally invasive reversible middle cerebral artery (MCA) occlusion (MCAO) model to account for leptomeningeal collateral formation.

MicroRNAs in Skin and Wound Healing

Physiological Genomics. May, 2011  |  Pubmed ID: 20959495

MicroRNAs (miRNAs) are small endogenous RNA molecules ∼22 nt in length. miRNAs are capable of posttranscriptional gene regulation by binding to their target messenger RNAs (mRNAs), leading to mRNA degradation or suppression of translation. miRNAs have recently been shown to play pivotal roles in skin development and are linked to various skin pathologies, cancer, and wound healing. This review focuses on the role of miRNAs in cutaneous biology, the various methods of miRNA modulation, and the therapeutic opportunities in treatment of skin diseases and wound healing.

Commitment to Intellectual Honesty and Personal Responsibility

Antioxidants & Redox Signaling. Apr, 2012  |  Pubmed ID: 22239129

Abstract Scientific integrity represents the core of the research enterprise and the sharing of scientific information. It is this commitment to intellectual honesty and to responsible conduct and reporting of research that propels the successful advancement of knowledge. This afternoon I received a notification from officials of the University of Connecticut indicating that their investigation on research misconduct has found Antioxidants and Redox Signaling (ARS) Co-editor Professor Dipak K. Das guilty of fabrication and falsification of data. To demonstrate its commitment to protecting the integrity of science, ARS has terminated Dr. Das's position as Co-editor effective today. The report on findings of the investigation has identified that two articles published in ARS by the Das laboratory suffer from fabricated data. Both articles have been retracted effective today. Formal retraction notices will be issued on the Publisher's website ( www.liebertonline.com/ars ) and published in the Journal imminently. These actions reinforce the high standards necessary to advance the science that underpins the value that ARS brings to its community. Antioxid. Redox Signal. 16, 635.

Oral Tocotrienols Are Transported to Human Tissues and Delay the Progression of the Model for End-Stage Liver Disease Score in Patients

The Journal of Nutrition. Feb, 2012  |  Pubmed ID: 22298568

The natural vitamin E family is composed of 8 members equally divided into 2 classes: tocopherols (TCP) and tocotrienols (TE). A growing body of evidence suggests TE possess potent biological activity not shared by TCP. The primary objective of this work was to determine the concentrations of TE (200 mg mixed TE, b.i.d.) and TCP [200 mg α-TCP, b.i.d.)] in vital tissues and organs of adult humans receiving oral supplementation. Eighty human participants were studied. Skin and blood vitamin E concentrations were determined from healthy participants following 12 wk of oral supplementation of TE or TCP. Vital organ vitamin E levels were determined by HPLC in adipose, brain, cardiac muscle, and liver of surgical patients following oral TE or TCP supplementation (mean duration, 20 wk; range, 1-96 wk). Oral supplementation of TE significantly increased the TE tissue concentrations in blood, skin, adipose, brain, cardiac muscle, and liver over time. α-TE was delivered to human brain at a concentration reported to be neuroprotective in experimental models of stroke. In prospective liver transplantation patients, oral TE lowered the Model for end-stage liver disease (MELD) score in 50% of patients supplemented, whereas only 20% of TCP-supplemented patients demonstrated a reduction in MELD score. This work provides, to our knowledge, the first evidence demonstrating that orally supplemented TE are transported to vital organs of adult humans. The findings of this study, in the context of the current literature, lay the foundation for Phase II clinical trials testing the efficacy of TE against stroke and end-stage liver disease in humans.

Propagation of Cutaneous Thermal Injury: A Mathematical Model

Wound Repair and Regeneration : Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. Jan, 2012  |  Pubmed ID: 22211391

Cutaneous burn wounds represent a significant public health problem with 500,000 patients per year in the USA seeking medical attention. Immediately after skin burn injury, the volume of the wound burn expands due to a cascade of chemical reactions, including lipid peroxidation chain reactions. Such expansion threatens life and is therefore highly clinically significant. Based on these chemical reactions, the present paper develops for the first time a three-dimensional mathematical model to quantify the propagation of tissue damage within 12 hours post initial burn. We use the model to investigate the effect of supplemental antioxidant vitamin E for intercepting propagation. We show, for example, that if tissue levels of vitamin E tocotrienol are increased, postburn, by five times then this would slow down the lipid peroxide propagation by at least 50%. We chose the alpha-tocotrienol form of vitamin E as it is a potent inhibitor of 12-lipoxygenase, which is known to propagate oxidative lipid damage. Our model is formulated in terms of differential equations, and sensitivity analysis is performed on the parameters to ensure the robustness of the results.

Rebound Peer Review: a Viable Recourse for Aggrieved Authors?

Antioxidants & Redox Signaling. Feb, 2012  |  Pubmed ID: 22098370

Scholarly peer review represents the linchpin of academic publishing. Recognized benefits of the peer review system are manifold. Critics raise several valid concerns that deserve attention. Several studies show that the current peer review system lacks robustness and is subject to bias in favor of well-established research groups and "mainstream" theories. Hypotheses that harmonize with that of the leaders in the field are more likely to be accepted for publication in prestigious journals than heretic or radical ones. Then, there is the risk posed by the potentially unscrupulous reviewer. Alternatives to traditional peer review have been tried but the outcomes fall much short of expectations. Postreview rejection can be equally frustrating for the author and editor particularly when they are victims of limitations of the blinded forms of review. To provide recourse for authors who felt that their work has been rejected not because of the quality of science but because of the constraints of the peer review system, ARS introduces a rebound track for peer review ( www.liebertpub.com/ars ). The rebound peer review track is a two-tier process that represents a hybrid of partially blinded and open peer review systems. The goal is to make sure that every author has the opportunity to rescue their rejected work which they feel may have been victimized by the glitches of the current peer review system. I invite affected authors to make full use of this experimental mechanism so we know whether the rebound peer review should prevail as a viable recourse.