The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic pro     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelia (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic properties. Increased NO-generation in a cell may select mutant p53 cells and contribute to tumour angiogenesis by upregulating VEGF. In addition, NO may modulate tumour DNA repair mechanisms by upregulating p53, poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase (DNA-PK). An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease.     

Nitric oxide-dependent synthesis of vascular endothelial growth factor is impaired by high glucose

Synthesis of vascular endothelial growth factor (VEGF), the major angiogenic molecule, is induced by nitric oxide (NO) in various cell types, including vascular smooth muscle cells (VSMC). Therefore, compounds which inhibit NO generation can also influence VEGF synthesis. Here we investigated the effect of increased glucose concentration (25 mM vs. 5.5 mM) on cytokine-induced VEGF synthesis in rat VSMC. The cells growing in the medium containing 5.5 mM glucose and exposed to IL-1-beta, TNF-alpha and IFN-gamma induced expression of an inducible isoform of nitric oxide synthase (NOS II). This is followed by generation of NO and the concomitant expression of VEGF gene and release of VEGF protein. In contrast, 25 mM glucose impaired induction of NOS II expression and thus NO synthesis was lower than in 5.5 mM glucose. Consequently, the VEGF promoter activation was attenuated, resulting in decreased mRNA synthesis and lower production of VEGF protein. The results indicate that abnormally high concentrations of glucose can impair generation of NO and the NO-dependent VEGF synthesis. This may play a role in the development and progression of vascular dysfunctions in cardiovascular diseases.     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases(NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interest-ingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting theetiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and hasbeen associated with tumour grade, proliferation rate and expression of important signaling componentsassociated with cancer development such as the oestrogen receptor. It appears that high levels of NOSexpression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumorcells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxicallytherefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic properties.Increased NO-generation in a cell may select mutant p53 cells and contribute to tumour angiogenesis byupregulating VEGF. In addition, NO may modulate tumour DNA repair mechanisms by upregulating p53,poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase (DNA-PK). An understand-ing at the molecular level of the role of NO in cancer will have profound therapeutic implications for thediagnosis and treatment of disease.     

Macrophages derived from septic mice modulate nitric oxide synthase and angiogenic mediators in the heart

Macrophages (Mps) can exert the defense against invading pathogens. During sepsis, bacterial lipopolisaccharide (LPS) activates the production of inflammatory mediators by Mps. Nitric oxide synthase (NOS) derived‐nitric oxide (NO) is one of them. Besides, Mps may produce pro‐angiogenic molecules such as vascular endothelial growth factor‐A (VEGF‐A) and metalloproteinases (MMPs). The mechanisms involved in the cardiac neovascular response by Mps during sepsis are not completely known. We investigated the ability of LPS‐treated Mps from septic mice to modulate the behavior of cardiac cells as producers of NO and angiogenic molecules. In vivo LPS treatment (0.1 mg/mouse) increased NO production more than fourfold and induced de novo NOS2 expression in Mps. Immunoblotting assays also showed an induction in VEGF‐A and MMP‐9 expression in lysates obtained from LPS‐treated Mps, and MMP‐9 activity was detected by zymography in cell supernatants. LPS‐activated Mps co‐cultured with normal heart induced the expression of CD31 and VEGF‐A in heart homogenates and increased MMP‐9 activity in the supernatants. By immunohistochemistry, we detected new blood vessel formation in hearts cultured with LPS treated Mps. When LPS‐stimulated Mps were co‐cultured with isolated cardiomyocytes in a transwell assay, the expression of NOS2, VEGF‐A and MMP‐9 was induced in cardiac cells. In addition, MMP‐9 activity was up‐regulated in the supernatant of cardiomyocytes. The latter was due to NOS2 induction in Mps from in vivo LPS‐treated mice. In conclusion LPS‐treated Mps are inducers of inflammatory/angiogenic mediators in cardiac cells, which could be triggering neovascularization, as an attempt to improve cardiac performance in sepsis. J. Cell. Physiol. 228: 1584–1593, 2013. © 2013 Wiley Periodicals, Inc.     

Nitric oxide synthase-cyclooxygenase interactions are involved in tumor cell angiogenesis and migration

Nitric oxide (NO), produced by distinct nitric oxide synthase (NOS) isoforms, and prostaglandins generated by expression of cyclooxygenases are important mediators in tumor progression. Previous studies have shown that NO can influence the formation of prostaglandin E2 (PGE2). We provide evidence that NO, derived from iNOS and eNOS activity in LMM3 murine mammary adenocarcinoma cell line, is involved in tumor angiogenesis and in tumor cell migration. LMM3 cells that also stimulate their neovascularization activity and migration liberate high basal amounts of PGE2. There is large amount of evidence that postulates positive regulatory interactions between NOS and cyclooxygenase (COX) isoforms. We here show that, in the LMM3 cell line, while PGE2 exerts a positive modulation on NOS activity, NO closes the loop with a negative feed back on COX activity. We also provide evidence of a positive regulatory effect of protein tyrosine kinases on NOS as well as on COX enzymatic functions affecting tumor induced angiogenesis and cell migration.     

Nitric oxide is proangiogenic in the retina and choroid

Nitric oxide (NO) has been shown to have proangiogenic or antiangiogenic effects depending upon the setting. In this study, we used mice with targeted deletion of one of the three isoforms of nitric oxide synthase (NOS) to investigate the effects of NO in ocular neovascularization. In transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors, deficiency of any of the three isoforms caused a significant decrease in subretinal neovascularization, but no alteration of VEGF expression. In mice with laser-induced rupture of Bruch's membrane, deficiency of inducible NOS (iNOS) or neuronal NOS (nNOS), but not endothelial NOS (eNOS), caused a significant decrease in choroidal neovascularization. In mice with oxygen-induced ischemic retinopathy, deficiency of eNOS, but not iNOS or nNOS caused a significant decrease in retinal neovascularization and decreased expression of VEGF. These data suggest that NO contributes to both retinal and choroidal neovascularization and that different isoforms of NOS are involved in different settings and different disease processes. A broad spectrum NOS inhibitor may have therapeutic potential for treatment of both retinal and choroidal neovascularization.     

Endogenous production of nitric oxide by vascular endothelial growth factor down-regulates proliferation of choriocarcinoma cells

The trophoblast-like choriocarcinoma cell line BeWo expresses a receptor for vascular endothelial growth factor (VEGF) and proliferates in response to VEGF. Nitric oxide (NO) seems to play a key role in the VEGF-induced proliferation of endothelial cells but the NO mechanistic regulation of VEGF-stimulated trophoblast proliferation is presently unclear. We assessed the effect of exogenous VEGF on BeWo cell proliferation by [3H]thymidine incorporation. The VEGF-induced proliferation of BeWo cells was significantly increased by the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine methyl ester (L-NAME), but was inhibited by the NO donor, sodium nitroprusside. Treatment of the cells with 10 ng/ml of VEGF increased not only eNOS expression but also NO production. The extracellular signal-regulated kinase (Erk) of the mitogen-activated protein kinase (MAPK) family was activated by VEGF as demonstrated by the phosphorylation of Erk in Western blots. The effects of VEGF on NO production and the expression of endothelial NOS (eNOS) were attenuated by treating BeWo cells with the selective inhibitor of MAPK kinase, PD98059. VEGF-stimulated proliferation of BeWo cells was inhibited by the tyrosine kinase inhibitor genistein but increased by PD98059. Other kinase inhibitors, including LY294002 (phosphoinositide 3-kinase inhibitor) and SB203580 (P38 MAPK inhibitor), had no effect on the proliferation of the cells and NO production. These results indicate that endogenous NO production down-regulates the VEGF-stimulated proliferation of BeWo cells and that the activation of Erk plays an important role in this mechanism.     

Transforming growth factor-beta1 stimulates vascular endothelial growth factor 164 via mitogen-activated protein kinase kinase 3-p38alpha and p38delta mitogen-activated protein kinase-dependent pathway in murine mesangial cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inducer of extracellular matrix synthesis leading to progressive glomerular fibrosis. The intracellular signaling mechanisms involved in this process remain incompletely understood. The p38 mitogen-activated protein kinase (MAPK) is a major stress signal transducing pathway that is rapidly activated by TGF-beta1 in mesangial cells. We have previously demonstrated MKK3 as the immediate upstream MAPK kinase required for selective activation of p38 MAPK isoforms, p38alpha and p38delta, and stimulation of pro-alpha1(I) collagen by TGF-beta1 in murine mesangial cells. In this study, we further sought to determine MAPK kinase 3 (MKK3)-dependent TGF-beta1 responses by gene expression profiling analysis utilizing mesangial cells isolated from Mkk3-/- mice compared with Mkk3+/+ controls. Interestingly, vascular endothelial growth factor (VEGF) was identified as a TGF-beta1-induced gene affected by deletion of Mkk3. VEGF is a well known endothelial mitogen, whose actions in nonendothelial cell types are still not well understood. We confirmed that TGF-beta1 increased VEGF mRNA and protein synthesis of VEGF164 and VEGF188 isoforms in wild-type mesangial cells. However, in the Mkk3-/- mesangial cells, both TGF-beta1-induced VEGF mRNA and VEGF164 protein expression were inhibited, whereas TGF-beta1-induced VEGF188 protein expression was unaffected. Furthermore, transfection of dominant negative mutants of p38alpha and p38delta resulted in marked inhibition of TGF-beta1-induced VEGF164 expression but not VEGF188, and treatment with recombinant mouse VEGF164 increased collagen and fibronectin mRNA expression in mesangial cells. Taken together, our findings suggest a critical role for the MKK3-p38alpha and p38delta MAPK pathway in mediating VEGF164 isoform-specific stimulation by TGF-beta1 in mesangial cells. Further, VEGF164 stimulates collagen and fibronectin expression in mesangial cells and thus in turn enhances TGF-beta1-induced extracellular matrix and may play an important role in progressive glomerular fibrosis.     

Arginine metabolic pathways involved in the modulation of tumor-induced angiogenesis by macrophages

Neovascularization, an essential step for tumor progression and metastasis development, can be modulated by the presence of macrophages (Mps) in the tumor microenvironment. The ability of Mps to regulate the angiogenicity of the LMM3 tumor cell line was studied. Peritoneal Mps from LMM3 tumor-bearing mice (TMps) potentiate in vivo LMM3 angiogenicity. These results were confirmed by CD31 immunoblotting assays. The activity of TMps depended on nitric oxide synthase (NOS) and arginase (A) activity. By immunoblotting we evidenced that AI and AII isoforms were up-regulated in TMps while the inducible and neuronal NOS isoforms were highly expressed in normal Mps. TMps might positively modulate tumor growth by stimulating angiogenic cascade mainly through polyamine synthesis.     

Fibulin-5 antagonizes vascular endothelial growth factor (VEGF) signaling and angiogenic sprouting by endothelial cells

Fibulin-5 (FBLN-5) is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. It is also a gene target of TGF-beta in fibroblasts and endothelial cells that regulates cell proliferation and motility in a context-specific manner. Whereas FBLN-5 expression is low in adult vasculature, its expression is high in developing and injured vasculature, implicating FBLN-5 in regulating angiogenesis and endothelial cell function. We show here that TGF-beta stimulates FBLN-5 expression in endothelial cells, and that this response was inhibited by coadministration of the proangiogenic factor, VEGF. FBLN-5 expression was downregulated significantly during endothelial cell tubulogenesis, implying that FBLN-5 expression antagonizes angiogenesis. Accordingly, FBLN-5 overexpression in or recombinant FBLN-5 treatment of endothelial cells abrogated their ability to undergo angiogenic sprouting, doing so by inhibiting endothelial cell proliferation and invasion through Matrigel matrices. Moreover, FBLN-5 antagonized VEGF signaling in endothelial cells, as well as enhanced their expression of the antiangiogenic factor, thrombospondin-1. Finally, the ability of FBLN-5 to antagonize angiogenic processes was determined to be independent of its integrin-binding RGD motif. Collectively, our findings establish FBLN-5 as a novel antagonist of angiogenesis and endothelial cell activities, and offer new insights into why tumorigenesis downregulates FBLN-5 expression.     

Knockdown of the inducible nitric oxide synthase (NOS2) splicing variant S3 promotes autophagic cell death from nitrosative stress in SW480 human colon cancer cells

Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.     

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.     

Angiogenesis modulators expression in culture cell lines positives for HPV-16 oncoproteins

Altered angiogenesis response is observed in patients with cervical cancer. In this study we examined whether Human Papilloma Virus (HPV) positive epithelial cells are able to produce angiogenic modulators. When added to human umbilical vein endothelial cells (HUVEC) the media conditioned by HPV-16 positive cells was able to induce proliferation, whereas a contrary effect was observed for media derived from non-tumorigenic keratinocytes. The analyses of angiogenesis modulator's mRNA levels result in a decrease of the antiangiogenic factors TSP-1 and 2 in HPV-16 positive cells. In contrast the expression of the pro-angiogenic molecules: bFGF, IL-8, TGF-beta, TNFalpha, and VEGF were higher in these cells as compared to control keratinocytes. Furthermore the pattern of VEGF isoforms observed in the cells positive for the viral genome point to a preferential induction of the VEGF(189) isoform. We therefore conclude that cervical cancer cells expressing HPV-16 genome are able to contribute to the pro-angiogenic response that might support tumor growth and invasion of the surrounding tissues.     

Nitric oxide attenuates epithelial-mesenchymal transition in alveolar epithelial cells

Patients with interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF) and bronchopulmonary dysplasia (BPD), suffer from lung fibrosis secondary to myofibroblast-mediated excessive ECM deposition and destruction of lung architecture. Transforming growth factor (TGF)-beta1 induces epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) to myofibroblasts both in vitro and in vivo. Inhaled nitric oxide (NO) attenuates ECM accumulation, enhances lung growth, and decreases alveolar myofibroblast number in experimental models. We therefore hypothesized that NO attenuates TGF-beta1-induced EMT in cultured AEC. Studies of the capacity for endogenous NO production in AEC revealed that endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) are expressed and active in AEC. Total NOS activity was 1.3 pmol x mg protein(-1) x min(-1) with 67% derived from eNOS. TGF-beta1 (50 pM) suppressed eNOS expression by more than 60% and activity by 83% but did not affect iNOS expression or activity. Inhibition of endogenous NOS with l-NAME led to spontaneous EMT, manifested by increased alpha-smooth muscle actin (alpha-SMA) expression and a fibroblast-like morphology. Provision of exogenous NO to TGF-beta1-treated AEC decreased stress fiber-associated alpha-SMA expression and decreased collagen I expression by 80%. NO-treated AEC also retained an epithelial morphology and expressed increased lamellar protein, E-cadherin, and pro-surfactant protein B compared with those treated with TGF-beta alone. These findings indicate that NO serves a critical role in preserving an epithelial phenotype and in attenuating EMT in AEC. NO-mediated regulation of AEC fate may have important implications in the pathophysiology and treatment of diseases such as IPF and BPD.     

Induction of angiogenesis by expression of soluble type II transforming growth factor-beta receptor in mouse hepatoma.

The biological effect of transforming growth factor-beta (TGF-beta) is cell type-specific and complex. The precise role of TGF-beta is not clear in vivo. To elucidate the regulation mechanism of endogenous TGF-beta on hepatoma progression, we modified the MH129F mouse hepatoma cell with a retroviral vector encoding the extracellular region of type II TGF-beta receptor (TRII). Soluble TRII (TRIIs) blocked TGF-beta binding to TRII on the membrane of hepatoma cells. Growth of MH129F cells was inhibited by TGF-beta1 treatment; however, soluble TRII-overexpressing cells (MH129F/TRIIs) did not show any change in proliferation after TGF-beta1 treatment. MH129F/TRIIs cells also increased vascular endothelial growth factor (VEGF) expression, endothelial cell migration, and tube formation. Implantation of MH129F/TRIIs cells into C3H/He mice showed the significantly enhanced tumor formation. According to Western blot and protein kinase C assay, the expression of VEGF, KDR/flk-1 receptor, and endothelial nitric-oxide synthase was enhanced, and the phosphorylation activity of protein kinase C was increased up to 3.7-fold in MH129F/TRIIs tumors. Finally, a PECAM-1-stained intratumoral vessel was shown to be 4.2-fold higher in the MH129F/TRIIs tumor. These results indicate that VEGF expression is up-regulated by a blockade of endogenous TGF-beta signaling in TGF-beta-sensitive hepatoma cells and then stimulates angiogenesis and tumorigenicity. Therefore, we suggest that endogenous TGF-beta is a major regulator of the VEGF/flk-1-mediated angiogenesis pathway in hepatoma progression.     

Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 cells by TGF-beta isoforms

Proliferation of mesenchymal precursors of osteogenic and chondrogenic cells and migration of these precursors to repair sites are important early steps in bone repair. Transforming growth factor-beta (TGF-beta) has been implicated in the promotion of bone repair and may have a role in these processes. Three isoforms of TGF-beta, TGF-beta1, -beta2, and -beta3, are expressed in fracture healing, however, their specific roles in the repair process are unknown. Differential actions of the TGF-beta isoforms on early events of bone repair were explored in the multipotent mesenchymal precursor cell line, C3H10T1/2. Cell migration was determined using a modified Boyden chamber in response to concentrations of each isoform ranging from 10(-12) to 10(-9) g/ml. All three isoforms demonstrated a dose-dependent chemotactic stimulation of untreated C3H10T1/2 cells. Checkerboard assays indicated that all three isoforms also stimulated chemokinesis of the untreated cells. C3H10T1/2 cells treated with all-trans-retinoic acid (ATRA) and expressing relatively higher levels of osteoblastic gene markers such as alkaline phosphatase and collagen type I, lower levels of chondrocytic gene markers collagen type II and aggrecan, and unchanged levels of the adipose marker adipsin did not demonstrate significant chemokinesis or chemotaxis in response to TGF-beta1 or -beta3 at concentrations ranging from 10(-12) to 10(-9) g/ml. In the ATRA-treated cells, TGF-beta2 stimulated a significant increase in chemotaxis only at the highest concentration tested. Cell proliferation was assessed by mitochondrial dehydrogenase activity and cell counts at TGF-beta concentrations from 10(-11) to 10(-8) g/ml. None of the TGF-beta isoforms stimulated cell proliferation in untreated or ATRA-treated C3H10T1/2 cells. Analysis of TGF-beta receptors (TGF-betaR1, -betaR2, and -betaR3) showed a 1.6- to 2.8-fold decrease in mRNA expression of these receptors in ATRA-treated cells. In conclusion: (1) while all three TGF-beta isoforms stimulate chemotaxis/chemokinesis of multipotent C3H10T1/2 cells, TGF-beta1 and -beta3 do not stimulate chemotaxis in C3H10T1/2 cells treated with ATRA while TGF-beta2 stimulated chemotaxis only at the highest concentration tested. (2) TGF-beta isoforms do not appear to stimulate cell proliferation in C3H10T1/2 cells in either a multipotent state or after ATRA treatment when expressing higher levels of alkaline phosphatase and collagen type I gene markers. (3) Decrease in mRNA expression for TGF-betaR1, -betaR2, and -betaR3 upon ATRA treatment could potentially explain the lack of chemotaxis/chemokinesis in these cells expressing higher levels of alkaline phosphatase and collagen type I.     

Green tea modulation of inducible nitric oxide synthase in hypoxic/reoxygenated cardiomyocytes

Hypoxia/reoxygenation (H/R) is one of the causes of the increased expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes. Since an aberrant NOS induction has detrimental consequences, we evaluated the effect of a green tea extract (GTE) on the NOS induction and activity in H/R-cardiomyocytes to define a nutritional strategy. Cultured rat cardiomyocytes were exposed to H/R in the presence of two concentrations of a green tea extract (GTE), which is reported to inhibit NOS expression and activity in different cells. In cultured cardiomyocytes two NOS isoforms were constitutively expressed, but only iNOS was induced by H/R. GTE supplementation at the lowest concentration, comparable to that in human plasma after dietary consumption, was ineffective, while the highest, comparable to that achievable by dietary supplements, counteracted the effect of H/R on iNOS induction and activity. It is necessary to verify in humans the relationship between the modulation of NO production and green tea dietary consumption.