Systemic sclerosis is characterised by three main pathological features namely auto-immunity, vasculopathy and an overgrowth of connective tissues in the skin and visceral organs including lung, heart, kidney and gastrointestinal tract. Currently, there is no intervention directed towards fibrosis in systemic sclerosis. Blocking immune response with an immunosuppressive drug like cyclophosphamide remains the mainstream approach for systemic sclerosis but this tends to compromise endogenous production of anti-fibrotic cytokine mediators like prostanoids and leukotrienes; such treatment may therefore exacerbate the fibroproliferative phase of systemic sclerosis This article is protected by copyright. All rights reserved.
Prostacyclin (PGI2) that is released from the vascular endothelium plays an important role in vasodilatation and thrombo-resistance, and it has long been suspected to protect cell survival. How it does so has never been clear. Recently, it has been shown that the NADPH oxidase 4 (Nox4) improves endothelial cell functions and promotes angiogenesis in vivo, but it was not known how to boost Nox4 therapeutically to exploit its protective functions in the vasculature. Here, we identified such a stimulus. Results: The selective and stable prostacyclin receptor (IP-R) agonist cicaprost increases the expression of Nox4 in human endothelial cells of several types, including endothelial progenitor cells. The elevation of cellular cyclic-AMP increased Nox4 expression and H2O2 production and prevented endothelial cell apoptosis. We delineate the intracellular signaling that promotes cytoprotection: Cicaprost acts via the IP-R/protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein pathway. Importantly, the up-regulation of Nox4 by cicaprost also enhanced endothelial cell proliferation, migration, and angiogenesis, with all effects being substantially decreased by Nox4 gene silencing. Finally, cicaprost enhanced the growth of blood vessels into subcutaneous sponges implanted in mice, an effect that was also blocked by Nox4 gene silencing.
Heme oxygenases, namely heme oxygenase-1 and heme oxygenase-2, have important biological functions in vascular homeostasis. Heme oxygenase and its catabolic products, including bilirubin and carbon monoxide, have been implicated in blood pressure regulation. Increased expression of heme oxygenase exerts multiple protective actions against hypertension-induced vascular injuries. However, the underlining mechanisms of these effects are not entirely clear. We and others have demonstrated that heme oxygenase-1 can modulate production of reactive oxygen species, both in vivo and in vitro, from NADPH oxidase, a major enzymatic source of reactive oxygen species generation in the vascular wall. NADPH oxidase has been implicated in the development of hypertension and hypertension-related organ injuries. In this mini review, we summarize our current understanding of the interactions between heme oxygenase and NADPH oxidase, and we propose that modulation of NADPH oxidase activity by heme oxygenase could be a potential mechanism of the beneficial effects of heme oxygenase in hypertension.
Angiogenesis, the formation of new blood vessels, is a key physiological event in organ development and tissue responses to hypoxia but is also involved in pathophysiologies such as tumour growth and retinopathies. Understanding the molecular mechanisms involved is important to design strategies for therapeutic intervention. One important regulator of angiogenesis is transforming growth factor-?1 (TGF-?1). In addition, reactive oxygen species (ROS) and the ROS-forming NADPH oxidase type 4 (Nox4) have been implicated as additional regulators such as during hypoxia. Here, we show that both processes are indeed mechanistically linked. TGF-?1-stimulated Nox4 expression and ROS formation in endothelial cells. In cells from Nox4-deficient mice, TGF-?1-induced cell proliferation, migration and tube formation were abolished. In vivo, TGF-?1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice. Thus, endothelial cells are regulated by a TGF-?1 signalling pathway involving Nox4-derived ROS to promote angiogenesis. In order to abrogate pathological angiogenesis triggered by a multitude of factors, such as TGF-?1 and hypoxia, Nox4 may thus be an ideal therapeutic target.
Hypoxia in the tumor microenvironment triggers differential signaling pathways for tumor survival. In this study, we characterize the involvement of hypoxia and reactive oxygen species (ROS) generation in the antineoplastic mechanism of proopiomelanocortin (POMC) gene delivery in a mouse B16-F10 melanoma model in vivo and in vitro. Histological analysis revealed increased TUNEL-positive cells and enhanced hypoxic activities in melanoma treated with adenovirus encoding POMC (Ad-POMC) but not control vector. Because the apoptotic cells were detected mainly in regions distant from blood vessels, it was hypothesized that POMC therapy might render melanoma cells vulnerable to hypoxic insult. Using a hypoxic chamber or cobalt chloride (CoCl2), we showed that POMC gene delivery elicited apoptosis and caspase-3 activation in cultured B16-F10 cells only under hypoxic conditions. The apoptosis induced by POMC gene delivery was associated with elevated ROS generation in vitro and in vivo. Blocking ROS generation using the antioxidant N-acetyl-l-cysteine abolished the apoptosis and caspase-3 activities induced by POMC gene delivery and hypoxia. We further showed that POMC-derived melanocortins, including ?-MSH, ?-MSH, and ACTH, but not ?-MSH, contributed to POMC-induced apoptosis and ROS generation during hypoxia. To elucidate the source of ROS generation, application of the NADPH oxidase inhibitor diphenyleneiodonium attenuated ?-MSH-induced apoptosis and ROS generation, implicating the proapoptotic role of NADPH oxidase in POMC action. Of the NADPH oxidase isoforms, only Nox4 was expressed in B16-F10 cells, and Nox4 was also elevated in Ad-POMC-treated melanoma tissues. Silencing Nox4 gene expression with Nox4 siRNA suppressed the stimulatory effect of ?-MSH-induced ROS generation and cell apoptosis during hypoxia. In summary, we demonstrate that POMC gene delivery suppressed melanoma growth by inducing apoptosis, which was at least partly dependent on Nox4 upregulation.
Uncontrolled fibrosis in organs like heart, kidney, liver and lung is detrimental and may lead to end-stage organ failure. Currently there is no effective treatment for fibrotic disorders. Transforming growth factor (TGF)-? has a fundamental role in orchestrating the process of fibrogenesis; however, interventions directly targeting TGF-? would have undesired systemic side effects due to the multiple physiological functions of TGF-?. Further characterization of the downstream signaling pathway(s) involved in TGF-?-mediated fibrosis may lead to discovery of novel treatment strategies for fibrotic disorders. Accumulating evidence suggests that Nox4 NADPH oxidase may be an important downstream effector in mediating TGF-?-induced fibrosis, while NADPH oxidase-dependent redox signaling may in turn regulate TGF-?/Smad signaling in a feed-forward manner. It is proposed that pharmacological inhibition of the Nox4 function may represent a novel approach in treatment of fibrotic disorders.
The proliferation of new blood vessels in the retina is a leading cause of vision impairment. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is involved in cell signaling for ischemia-induced angiogenesis, but its role in retinal neovascularization is unclear. We have analyzed the dependence of retinal neovascularization on the Nox2 isoform in oxygen-induced retinopathy (OIR) in mice.
The prognosis of malignant melanoma is poor due to high incidence of metastasis, underscoring the demand for development of novel therapeutic strategies. Stress hormone pro-opiomelanocortin (POMC) is the precursor for several anti-inflammatory peptides that hold promise for management of cancer-related diseases. The present study evaluated the antimetastatic potential and mechanism of POMC therapy for metastatic melanoma. Adenovirus-mediated POMC gene delivery potently inhibited the invasiveness of human and mouse melanoma cells. Moreover, after induction of lung metastasis, systemic POMC expression significantly reduced the foci formation and neovascularization in lungs. Mechanistic studies revealed that POMC therapy inhibited the epithelial-mesenchymal transition (EMT) of melanoma cells by upregulation of E-cadherin and downregulation of vimentin and ?-smooth muscle actin (?-SMA). In addition, microarray analysis unveiled POMC gene transfer reduced the mRNA level of multiple prometastatic factors, including hepatoma-derived growth factor (HDGF). Cell culture and immunohistochemical studies further confirmed that POMC gene delivery significantly decreased the expression of HDGF in melanoma cells and tissues. Despite stimulating the invasion and EMT, exogenous HDGF supply only partially attenuated the POMC-mediated invasion inhibition and EMT change in melanoma cells. Finally, we delineated the contribution of melanocortins to POMC-induced inhibition of invasion, HDGF downregulation, and E-cadherin upregulation. Together, these results indicate that HDGF downregulation participates in POMC-induced suppression of metastasis and EMT in melanoma.
Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200) showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn) expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.
The anti-inflammatory peptide annexin-1 binds to formyl peptide receptors (FPR) but little is known about its mechanism of action in the vasculature. Here we investigate the effect of annexin peptide Ac2-26 on NADPH oxidase activity induced by tumour necrosis factor alpha (TNF?) in human endothelial cells. Superoxide release and intracellular reactive oxygen species (ROS) production from NADPH oxidase was measured with lucigenin-enhanced chemiluminescence and 2,7-dichlorodihydrofluorescein diacetate, respectively. Expression of NADPH oxidase subunits and intracellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) were determined by real-time PCR and Western blot analysis. Promoter activity of nuclear factor kappa B (NF?B) was measured by luciferase activity assay. TNF? stimulated NADPH-dependent superoxide release, total ROS formation and expression of ICAM-1and VCAM-1. Pre-treatment with N-terminal peptide of annexin-1 (Ac2-26, 0.5-1.5 µM) reduced all these effects, and the inhibition was blocked by the FPRL-1 antagonist WRW4. Furthermore, TNF?-induced NF?B promoter activity was attenuated by both Ac2-26 and NADPH oxidase inhibitor diphenyliodonium (DPI). Surprisingly, Nox4 gene expression was reduced by TNF? whilst expression of Nox2, p22phox and p67phox remained unchanged. Inhibition of NADPH oxidase activity by either dominant negative Rac1 (N17Rac1) or DPI significantly attenuated TNF?-induced ICAM-1and VCAM-1 expression. Ac2-26 failed to suppress further TNF?-induced expression of ICAM-1 and VCAM-1 in N17Rac1-transfected cells. Thus, Ac2-26 peptide inhibits TNF?-activated, Rac1-dependent NADPH oxidase derived ROS formation, attenuates NF?B pathways and ICAM-1 and VCAM-1 expression in endothelial cells. This suggests that Ac2-26 peptide blocks NADPH oxidase activity and has anti-inflammatory properties in the vasculature which contributes to modulate in reperfusion injury inflammation and vascular disease.
Damage to peripheral nerves following trauma or neurodegenerative diseases often results in various sensory and motor abnormalities and chronic neuropathic pain. The loss of neurotrophic factor support has been proposed to contribute to the development of peripheral neuropathy. The main objective of this study was to investigate the protective effect of glial cell line-derived neurotrophic factor (GDNF) using peripheral gene delivery in a rat model of constriction-induced peripheral nerve injury. In this study, it was shown that mechanical and thermal hypersensitivity increased on the injured limb at day 7 after chronic constrictive injury (CCI) was induced. The neurological changes were correlated with the structural changes and loss of GDNF/Akt signaling, particularly in the distal stump of the injured sciatic nerve. Subsequently, recombinant adenovirus was employed to evaluate the potential of intramuscular GDNF gene delivery to alleviate the CCI-induced nerve degeneration ad neuropathic pain. After CCI for 3 days, intramuscular injection of adenovirus encoding GDNF (Ad-GDNF) restored the protein level and activity of GDNF/Akt signaling pathway in the sciatic nerve. This was associated with an improved myelination profile and behavioral outcomes in animals with CCI. In conclusion, the present study demonstrates the involvement of GDNF loss in the pathogenesis of CCI-induced neuropathic pain and the therapeutic potential of intramuscular GDNF gene delivery for the treatment of peripheral nerve degeneration.
Pro-opiomelanocortin (POMC) is a precursor of various neuropeptides. POMC-derived neuropeptides are potent inflammation inhibitors and immunosuppressants. Evidence that osteoarthritis (OA) is an inflammatory disease is accumulating. We assessed whether intra-articular gene delivery of POMC ameliorates experimentally induced OA in a rat model. OA was induced in Wistar rats by anterior cruciate ligament-transection (ACLT) in the knee of one hind limb. Adenoviral vector encoding human POMC (AdPOMC) was injected intra-articularly into the knee joints after ACLT. The transgene expression and the inflammatory responses were evaluated using immunoblotting, immunohistochemistry and enzyme-linked immunosorbent assay. The treated joints were assessed histologically for manifestations of the disease. Human POMC was expressed in the chondrocytes and synovial membrane after the intra-articular injection. POMC gene transfer reduced nuclear factor-?B activity and the levels of interleukin-1? in HTB-94 chondrosarcoma cells and Raw 264.7 macrophages; it also reduced microvessel density in the synovium. Histological examination showed that symptoms of OA in AdPOMC-treated rats were less severe than in rats treated with either empty adenoviral vector (AdNull) or normal saline. Intra-articular injection of adenoviral vectors expressing POMC significantly suppressed the progression and severity of OA, and reduced inflammatory responses and angiogenesis. POMC gene delivery may offer novel therapeutic approach for treating OA.
Malignant melanoma is one of the leading causes of cancer mortality worldwide, underlining the need for effective novel therapies. In this study, the therapeutic efficacy and mechanism of systemic pro-opiomelanocortin (POMC) therapy were evaluated in mice bearing established melanoma. Injection of adenovirus encoding POMC (Ad-POMC) led to hepatic POMC overexpression and elevated adrenocorticotropin (ACTH) levels in the circulation. Systemic POMC therapy significantly attenuated the growth of established melanoma and prolonged the survival of tumor-bearing mice. Histological analysis revealed that systemic POMC therapy induced melanogenic differentiation while reducing melanoma growth. In addition, POMC therapy also elicited a significant reduction in the neovascular network of melanoma. Last, we demonstrated that POMC-derived peptides, including ACTH, ?-melanocyte-stimulating hormone (?-MSH), and ?-MSH, are involved in POMC-mediated melanogenic differentiation and angiogenesis inhibition. In summary, systemic POMC therapy suppresses melanoma growth via induction of melanogenic differentiation and angiogenesis blockade, thereby demonstrating its potential as a novel treatment modality for melanoma.
Hepatoma-derived growth factor (HDGF) is a neurotrophic factor found in mouse spinal cord and hippocampal neurons. In various malignant tumors, the role of HDGF in tumor progression and its use as a diagnostic biomarker or therapeutic target have been extensively explored. However, the prognostic function and mitogenic role of HDGF in gliomagenesis are yet to be verified. In this study, we found a significant incidence of HDGF prevalence between the different pathological types and stages of glioma in 105 patients. We also found a prognostic significance in 41 glioblastoma multiforme (GBM) patients, with prevalence of nuclear HDGF predicting short survival of patients with GBM after surgery. To delineate the mitogenic role of HDGF in gliomagenesis, an adenoviral-expressed HDGF small interfering RNA (Ad-HDGF siRNA) was used to knock down expression of nuclear HDGF. After knocking down nuclear HDGF expression in human GBM cells, cell growth and cell invasion and induction on apoptosis by caspase-3 activation were significantly inhibited. We conclude that HDGF is a mitogenic growth factor in glioma progression and can be a useful prognostic marker for GBM and therapeutic target for clinical management of glioma in the future.
Deprivation of neurotrophic factors contributes to the pathogenesis of diabetic neuropathy. However, the role of glial cell-derived neurotrophic factor (GDNF) in the pathogenesis of diabetic neuropathy remains unclear. The present study evaluated the pathogenic role of GDNF deficiency and the therapeutic potential of GDNF gene transfer for diabetic neuropathy. After injection of streptozotocin (STZ) for 2 weeks, diabetic rats displayed significant alteration in electrophysiological parameters, which was associated with structural changes and defective myelination in the sciatic nerves. The early diabetic neuropathy was accompanied by attenuation of the GDNF/GFRalpha1/Akt signaling cascade and depletion of sensory neuropeptides in the peripheral nerves. After detection of neuropathy, intramuscular GDNF gene transfer reversed the deficiency of GDNF/Akt signaling in the sciatic nerve and improved the neurological functions of diabetic rats. Moreover, GDNF gene delivery alleviated the axonal demyelination and restored the sensory neuropeptide levels in the sciatic nerve of diabetic rats. In summary, peripheral GDNF gene delivery ameliorates the diabetes-induced downregulation of the GDNF signaling complex in the peripheral nervous system and holds promises for treatment of diabetic neuropathy.
Choroidal neovascularization (CNV) is the leading cause of blindness in patients with age-related macular degeneration (AMD). This study evaluated the inhibitory effect of vasostatin (VS), an endogenous angiogenesis inhibitor, on CNV.
Neurogenic inflammation frequently causes acute plasma leakage in airways and life-threatening pulmonary edema. However, limited strategies are available to alleviate neurogenic inflammation. Proopiomelanocortin (POMC) is the precursor of anti-inflammatory melanocortins, which have been proposed of therapeutic potential for various inflammatory diseases. The present study aimed to evaluate whether peripheral POMC expression ameliorated capsaicin-induced acute neurogenic inflammation in rat trachea. Prophylactic POMC expression was achieved by intravenous injection of adenovirus encoding POMC (Ad-POMC), which led to POMC expression in livers and elevated plasma adrenocorticotropin levels for approximately 60 days. After gene delivery for 7 days, neurogenic inflammation was induced in rats by capsaicin injection. The extent of capsaicin-evoked plasma leakage in trachea was alleviated in Ad-POMC-treated rats compared with animals of control groups (P < 0.01). Moreover, the number of endothelial gaps in tracheal venules was also significantly decreased in Ad-POMC-treated animals (P < 0.01). Prophylactic POMC expression, however, did not alter the basal substance P (SP) expression or the capsaicin-induced SP elevation in trachea and circulation. Instead, cell cultures studies revealed that POMC overexpression or application of POMC-derived melanocortins potently inhibited the SP-induced migration of endothelial cells (P < 0.01), thereby possibly contributing to the attenuation of endothelial gap formation and plasma leakage. The present study indicates that the anti-inflammatory POMC gene vector or melanocortins may constitute a therapeutic alternative for neurogenic inflammation.
The synthesis of extracellular matrix including collagen during wound healing responses involves signaling via reactive oxygen species (ROS). We hypothesized that NADPH oxidase isoform Nox4 facilitates the stimulatory effects of the profibrotic cytokine transforming growth factor (TGF) ?(1) on collagen production in vitro and in vivo. TGF?(1) stimulated collagen synthesis and hydrogen peroxide generation in mouse cardiac fibroblasts, and both responses were attenuated by a scavenger of superoxide and hydrogen peroxide (EUK-134). Furthermore, by expressing a dominant negative form of Nox4 (Adv-Nox4(?NADPH)) in fibroblasts, TGF?(1)-induced hydrogen peroxide production and collagen production were abrogated, suggesting that Nox4-dependent ROS are important for TGF?(1) signaling in collagen production. This was confirmed by the inhibitory effect of an adenovirus carrying siRNA targeting Nox4 (Adv-Nox4i) on TGF?(1)-induced collagen synthesis and expression of activated myofibroblasts marker smooth muscle alpha actin. Finally we used a mouse model of subcutaneous sponge implant to examine the role of Nox4 in the local stimulatory effects of TGF?(1) on collagen accumulation in vivo. TGF?(1)-induced collagen accumulation was significantly reduced when the sponges were instilled with Adv-Nox4(?NADPH). In conclusion, Nox4 acts as an intermediary in the signaling of TGF?(1) to facilitate collagen synthesis.
Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 ?m porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.
Both reactive oxygen species (ROS) and Forkhead box O (FOXO) family transcription factors are involved in the regulation of adipogenic differentiation of preadipocytes and stem cells. While FOXO has a pivotal role in maintaining cellular redox homeostasis, the interactions between ROS and FOXO during adipogenesis are not clear. Here we examined how ROS and FOXO regulate adipogenesis in human adipose-derived stem cells (hASC). The identity of isolated cells was confirmed by their surface marker expression pattern typical for human mesenchymal stem cells (positive for CD29, CD44, CD73, CD90, and CD105, negative for CD45 and CD31). Using a standard adipogenic cocktail consisting of insulin, dexamethasone, indomethacin, and 3-Isobutyl-1-methylanxthine (IDII), adipogenesis was induced in hASC, which was accompanied by ROS generation. Scavenging ROS production with N-acetyl-L-cysteine or EUK-8, a catalytic mimetic of superoxide dismutase (SOD) and catalase, inhibited IDII-induced adipogenesis. We then mimicked IDII-induced oxidative stress through a lentiviral overexpression of Nox4 and an exogenous application of hydrogen peroxide in hASC and both manipulations significantly enhanced adipogenesis without changing the adipogenic differentiation rate. These data suggest that ROS promoted lipid accumulation in hASC undergoing adipogenesis. Antioxidant enzymes, including SOD2, catalase, and glutathione peroxidase were upregulated by IDII during adipogenesis, and these effects were blunted by FOXO1 silencing, which also suppressed significantly IDII-induced adipogenesis. Our findings demonstrated a balance of ROS generation and endogenous antioxidants in cells undergoing adipogenesis. Approaches targeting ROS and/or FOXO1 in adipocytes may bring new strategies to prevent and treat obesity and metabolic syndrome.
Hepatoma-derived growth factor (HDGF) stimulates the migration, invasion and metastasis in several types of cancer cells. However, the mechanism underlying HDGF-stimulated migration remains unclear. In this study, we investigated the influence of HDGF on cytoskeleton remodeling and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in non-transformed NIH/3T3 cells. Exogenous HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression and Akt phosphorylation in dose- and time-dependent manners. Application of LY294002, a PI3K inhibitor, attenuated the HDGF-induced migration, dorsal ruffles and podosome rosettes formation. Consistently, the HDGF-overexpressing NIH/3T3 transfectants exhibited significantly increased motility and elevated PI3K/Akt activities, which were repressed by LY294002 or adenovirus-mediated overexpression of endogenous PI3K antagonist, PTEN. In summary, HDGF elicits the activation of PI3K/Akt signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration.
Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. Previously, our studies have shown a significant mass of vascularized cardiac tissue can be generated using a vascularized tissue engineering chamber approach in nude rats. In this present study, syngeneic rats were investigated as an animal model for cardiac tissue engineering using the arteriovenous loop (AVL) chamber in the presence of a functional immune system. Neonatal cardiomyocytes implanted into the AVL chamber survived and assembled into a contractile flap confirming the basic features we previously showed in growing a cardiac construct. There was no significant loss of the assembled cardiac muscle from immune response. The engineered cardiac muscle flaps (ECMFs) formed were transplanted to the neck vessels of the same animal using a microsurgical technique, and all transplanted tissues remained contractile. The cardiac muscle volume of the control and transplant groups was estimated with histomorphometry using desmin and ?-sarcomeric actin immunostaining, and there were no significant differences between the two groups. Finally, utilizing a novel model of transplantation, the ECMFs were transplanted to the heart of a recipient syngeneic rat as a vascularized tissue. The cardiac muscle within the transplanted ECMF was shown to survive and remain contractile for the 4-week post-transplantation period, and importantly, the cardiomyocytes retained the elongated, striated appearance of a mature phenotype. This study demonstrated the proof of concept for transplanting tissue-engineered cardiac muscle as a vascularized cardiac construct.
How signaling via reactive oxygen species (ROS) influences skin pigmentation is unclear. We have investigated how NADPH oxidase-derived ROS modulates the expression of the key pigment "melanin" synthesizing enzymes in B16 mouse melanoma cells. A melanin inducer ?-melanocyte-stimulating hormone (?-MSH) caused ROS generation that was inhibited by the NADPH oxidase inhibitor Diphenyleneiodonium (DPI) and was insensitive to antagonists of other ROS-producing enzyme systems including mitochondrial enzymes, cycloxygenase, and xanthine oxidase. NADPH oxidase 4 (Nox4) was found to be the most abundant isoform expressed in B16 cells, and its gene levels, as well as ROS generation, were enhanced by ?-MSH. Interestingly, silencing Nox4 gene expression with Nox4 siRNA augmented melanin formation under basal conditions and after ?-MSH stimulation, demonstrating that constitutive or stimulated Nox4-dependent ROS inhibits melanin formation. This process may be mediated by targeting the promoter region of a melanin synthesizing enzyme tyrosinase, because Nox4 siRNA enhanced tyrosinase promoter activity. Moreover, inhibition of tyrosinase mRNA expression in Nox4 siRNA-treated cells by blocking de novo mRNA and protein synthesis with actinomycin D and cycloheximide respectively indicates that Nox4 repression induces melanogenesis by increasing tyrosinase gene expression. We also found that ?-MSH activated its downstream signal transducer microphthalmia-associated transcription factor (MITF) to stimulate Nox4 gene expression. We thus identified a novel mechanism by MITF signaling that in turn stimulates Nox4 to drive ROS generation, thereby repressing melanin synthesis. Such sequence of actions appears to act as an internal feedback mechanism to fine-tune melanin synthesis in response to exogenous challenges such as UV radiation.
Surface treatment to a biomaterial surface has been shown to modify and help cell growth. Our aim was to determine the best surface-modified system for the treatment of limbal stem cell deficiency, which would facilitate expansion of autologous limbal epithelial cells while maintaining cultivated epithelial cells in a less differentiated state. Commercially available contact lenses were variously surface-modified by plasma polymerization with ratios of acrylic acid to octadiene tested at 100% acrylic acid, 50:50% acrylic acid: octadiene and 100% octadiene to produce high-, mid-, and low-, and no-acid. X-ray photoelectron spectroscopy (XPS) was used to analyse the chemical composition of the plasma polymer deposited layer. Limbal explants cultured on high acid modified contact lenses outgrew more cells. Immunofluorescence and RT2-PCR-Array results indicated that higher acrylic acid content can also help maintain progenitor cells during ex vivo expansion of epithelial cells. This study provides the first evidence for the ability of high acid modified contact lenses to preserve the stemness and to be used as substrates for the culture of limbal cells in the treatment of Limbal stem cell deficiency (LSCD).
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