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.

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.

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

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.

Would Healing: Oxygen and Emerging Therapeutics. Columbus, Ohio, September 12-15, 2002

Antioxidants & Redox Signaling. Dec, 2002  |  Pubmed ID: 12580193

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.

Peroxisomal Membrane Monocarboxylate Transporters: Evidence for a Redox Shuttle System?

Biochemical and Biophysical Research Communications. Apr, 2003  |  Pubmed ID: 12705896

One of the many functions of liver peroxisomes is the beta-oxidation of long-chain fatty acids. It is essential for the continuation of peroxisomal beta-oxidation that a redox shuttle system exist across the peroxisomal membrane to reoxidize NADH. We propose that this redox shuttle system consists of a substrate cycle between lactate and pyruvate. Here we present evidence that purified peroxisomal membranes contain both monocarboxylate transporter 1 (MCT 1) and MCT 2 and that along with peroxisomal lactate dehydrogenase (pLDH) form a Peroxisomal Lactate Shuttle. Peroxisomal beta-oxidation was greatly stimulated by the addition of pyruvate and this increase was partially inhibited by the addition of the MCT blocker alpha-cyano-4-hydroxycinnamate (CINN). We also found that peroxisomes generated lactate in the presence of pyruvate. Together these data provide compelling that the Peroxisome Lactate Shuttle helps maintain organelle redox and the proper functioning of peroxisomal beta-oxidation.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

Oxygen-inducible Glutamate Oxaloacetate Transaminase As Protective Switch Transforming Neurotoxic Glutamate to Metabolic Fuel During Acute Ischemic Stroke

Antioxidants & Redox Signaling. May, 2011  |  Pubmed ID: 21361730

This work rests on our previous report (J Cereb Blood Flow Metab 30: 1275-1287, 2010) recognizing that glutamate (Glu) oxaloacetate transaminase (GOT) is induced when brain tissue hypoxia is corrected during acute ischemic stroke (AIS). GOT can metabolize Glu into tricarboxylic acid cycle intermediates and may therefore be useful to harness excess neurotoxic extracellular Glu during AIS as a metabolic substrate. We report that in cultured neural cells challenged with hypoglycemia, extracellular Glu can support cell survival as long as there is sufficient oxygenation. This effect is abrogated by GOT knockdown. In a rodent model of AIS, supplemental oxygen (100% O(2) inhaled) during ischemia significantly increased GOT expression and activity in the stroke-affected brain tissue and prevented loss of ATP. Biochemical analyses and in vivo magnetic resonance spectroscopy during stroke demonstrated that such elevated GOT decreased Glu levels at the stroke-affected site. In vivo lentiviral gene delivery of GOT minimized lesion volume, whereas GOT knockdown worsened stroke outcomes. Thus, brain tissue GOT emerges as a novel target in managing stroke outcomes. This work demonstrates that correction of hypoxia during AIS can help clear extracellular neurotoxic Glu by enabling utilization of this amino acid as a metabolic fuel to support survival of the hypoglycemic brain tissue. Strategies to mitigate extracellular Glu-mediated neurodegeneration via blocking receptor-mediated excitotoxicity have failed in clinical trials. We introduce the concept that under hypoglycemic conditions extracellular Glu can be transformed from a neurotoxin to a survival factor by GOT, provided there is sufficient oxygen to sustain cellular respiration.

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.

Significance of Brain Tissue Oxygenation and the Arachidonic Acid Cascade in Stroke

Antioxidants & Redox Signaling. May, 2011  |  Pubmed ID: 20673202

The significance of the hypoxia component of stroke injury is highlighted by hypermetabolic brain tissue enriched with arachidonic acid (AA), a 22:6n-3 polyunsaturated fatty acid. In an ischemic stroke environment in which cerebral blood flow is arrested, oxygen-starved brain tissue initiates the rapid cleavage of AA from the membrane phospholipid bilayer. Once free, AA undergoes both enzyme-independent and enzyme-mediated oxidative metabolism, resulting in the formation of number of biologically active metabolites which themselves contribute to pathological stroke outcomes. This review is intended to examine two divergent roles of molecular dioxygen in brain tissue as (1) a substrate for life-sustaining homeostatic metabolism of glucose and (2) a substrate for pathogenic metabolism of AA under conditions of stroke. Recent developments in research concerning supplemental oxygen therapy as an intervention to correct the hypoxic component of stroke injury are discussed.

MicroRNA in Ischemic Stroke Etiology and Pathology

Physiological Genomics. May, 2011  |  Pubmed ID: 20841499

Small, noncoding, microRNAs (miRNAs) have emerged as key mediators of posttranscriptional gene silencing in both pathogenic and pathological aspects of ischemic stroke biology. In stroke etiology, miRNA have distinct expression patterns that modulate pathogenic processes including atherosclerosis (miR-21, miR-126), hyperlipidemia (miR-33, miR-125a-5p), hypertension (miR-155), and plaque rupture (miR-222, miR-210). Following focal cerebral ischemia, significant changes in the miRNA transcriptome, independent of an effect on expression of miRNA machinery, implicate miRNA in the pathological cascade of events that include blood brain barrier disruption (miR-15a) and caspase mediated cell death signaling (miR-497). Early activation of miR-200 family members improves neural cell survival via prolyl hydroxylase mRNA silencing and subsequent HIF-1α stabilization. Pro- (miR-125b) and anti-inflammatory (miR-26a, -34a, -145, and let-7b) miRNA may also be manipulated to positively influence stroke outcomes. Recent examples of successfully implemented miRNA-therapeutics direct the future of gene therapy and offer new therapeutic strategies by regulating large sets of genes in related pathways of the ischemic stroke cascade.

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.

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.

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.

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.