Hypoxia-induced expression of HIF-1alpha and its target genes in umbilical venous endothelial cells of Tibetans and immigrant Han

The better adaptation of native Tibetans to hypoxia is thought to be partly due to improved umbilical circulation, which results in reduced pre- and postnatal fatalities. We hypothesized that the difference in umbilical circulation between native Tibetans and other high-altitude inhabitants was due to differences in the expression of hypoxia-induced factor (HIF-1) and its target genes vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). We tested this hypothesis by examining the effect of hypoxia on the expression of HIF-1alpha, VEGF, and iNOS in cultured umbilical venous endothelial cells (UVECs) from native Tibetans and immigrant Hans. UVECs were collected and cultured under hypoxic (0.5% oxygen) or normoxic conditions for 2, 4, 12 and 24 h. The mRNA levels of HIF-1alpha, VEGF, endothelial nitric oxide synthase (eNOS) and iNOS and the protein level of HIF-1alpha were determined with RT-PCR and Western blot analyses, respectively. In both immigrant Han and Tibetans, HIF-1alpha mRNA was constitutively expressed under normoxic condition, and remained constant after hypoxic exposure. In contrast, HIF-1alpha protein was undetectable under normoxic condition, but underwent dynamic changes in response to hypoxia. It was induced at 4 h, peaked at 12 h, and remained elevated at 24 h. Concurrent with the induction of HIF-1alpha protein, the mRNA levels of VEGF and iNOS were also up-regulated whereas that of eNOS was down-regulated. The lack of a hypoxia-related difference in the expression of HIF-1alpha and its target genes suggests that HIF-1alpha does not play a critical role in high altitude adaptation. Alternative mechanisms may be responsible for the better adaptation of native Tibetans.     

Hypoxia-induced regulation of nitric oxide synthase in cardiac endothelial cells and myocytes and the role of the PI3-K/PKB pathway

The roles of endothelial nitric oxide synthase (eNOS), and its putative association with protein kinase B (PKB), and inducible nitric oxide synthase (iNOS) are not well characterized in hypoxic cardiac cells and there is a lack of studies that measure nitric oxide (NO) directly. Objective To measure NO production in cardiomyocytes and cardiac microvascular endothelial cells (CMECs) under baseline and hypoxic conditions and to evaluate the expression, regulation and activation of eNOS, iNOS and PKB. The effect of PI3-K/PKB inhibition on NO production and eNOS expression/activation was also investigated. Methods Adult rat cardiomyocytes and rat CMECs were made hypoxic by cell pelleting and low PO₂ incubation. Intracellular NO was measured by FACS analysis of DAF-2/DA fluorescence, and eNOS, iNOS and PKB were evaluated by Western blotting or flow cytometry. Upstream PKB inhibition was achieved with wortmannin. Results (1) NO levels increased in both cell types after exposure to hypoxia. (2) In hypoxic CMECs, eNOS was upregulated and activated, no iNOS expression was observed and PKB was activated. (3) In myocytes, hypoxia did not affect eNOS expression, but increased its activation. Activated PKB also increased during hypoxia. FACS analysis showed increased iNOS in hypoxic myocytes. (4) Wortmannin resulted in decreased hypoxia-induced NO production and reduced activated eNOS levels. Conclusions Cardiomyocytes and CMECs show increased NO production during hypoxia. eNOS seems to be the main NOS isoform involved as source of the increased NO generation, although there may be a role for iNOS and other non-eNOS sources of NO in the hypoxic myocytes. Hypoxia-induced PKB and eNOS activation occurred simultaneously in both cell types, and the PI3-K/PKB pathway was associated with hypoxia-induced NO production via eNOS activation.     

iNOS expression inhibits hypoxia-inducible factor-1 activity

Hypoxia-inducible factor-1 (HIF-1) activates genes important in vascular function such as vascular endothelial growth factor (VEGF), erythropoietin (EPO), and inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a free radical gas that mediates a number of cellular processes, including regulation of gene expression, vasodilatation, and neurotransmission. Here we demonstrate that iNOS expression inhibits HIF-1 activity under hypoxia in C6 glioma cells transfected with an iNOS gene and a VEGF promoter-driven luciferase gene. HIF-1 induction of VEGF-luciferase activity in C6 cell is also inhibited by sodium nitroprusside (SNP). Furthermore, pretreatment of C6 cells with N-acetyl-l-cysteine (NAC), an antioxidant, nullified the inhibitory effect of iNOS on HIF-1 binding. These results demonstrate that NO generated by iNOS expression inhibits HIF-1 activity in hypoxic C6 cells and suggest a negative feedback loop in the HIF-1 --> iNOS cascade.     

Expression and role of factor inhibiting hypoxia-inducible factor-1 in pulmonary arteries of rat with hypoxia-induced hypertension

Hypoxia-inducible factor-1alpha subunit (HIF-1alpha) plays a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH) by transactivating it' target genes. As an oxygen-sensitive attenuator, factor inhibiting HIF-1 (FIH) hydroxylates a conserved asparagine residue within the C-terminal transactivation domain of HIF-1alpha under normoxia and moderate hypoxia. FIH protein is downregulated in response to hypoxia, but its dynamic expression and role during the development of HPH remains unclear. In this study, an HPH rat model was established. The mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. The pulmonary artery remodeling index became evident after 7 d of hypoxia, while the right ventricular hypertrophy index became significant after 14 d of hypoxia. The messenger RNA (mRNA) and protein expression of HIF-1alpha and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1alpha, were markedly upregulated after exposure to hypoxia in pulmonary arteries. FIH protein in lung tissues declined after 7 d of hypoxia and continued to decline through the duration of hypoxia. FIH mRNA had few changes after exposure to hypoxia compared with after exposure to normoxia. In hypoxic rats, FIH protein showed significant negative correlation with VEGF mRNA and VEGF protein. FIH protein was negatively correlated with mean pulmonary arterial pressure, pulmonary artery remodeling index and right ventricular hypertrophy index. Taken together, our results suggest that, in the pulmonary arteries of rat exposed to moderate hypoxia, a time-dependent decrease in FIH protein may contribute to the development of rat HPH by enhancing the transactivation of HIF-1alpha target genes such as VEGF.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. HIF-1 alpha overexpression may be beneficial in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic heart disease. To address this issue, human peripheral blood mononuclear cells (PBMNCs) were induced to differentiate into endothelial progenitor cells (EPCs), and then were transfected with either an HIF-1 alpha-expressing or a control vector and cultured under normoxia or hypoxia. Hypoxia-induced HIF-1 alpha mRNA and protein expression was increased after HIF-1 alpha transfection. This was accompanied by VEGF mRNA induction and increased VEGF secretion. Hypoxia-stimulated VEGF mRNA induction was significantly abrogated by HIF-1 alpha-specific siRNA. Functional studies showed that HIF-1 alpha overexpression further promoted hypoxia-induced EPC differentiation, proliferation and migration. The expressions of endothelial cell markers CD31, VEGFR2 (Flk-1) and eNOS as well as VEGF and NO secretions were also increased. Furthermore, in an in vivo model of hindlimb ischemia, HIF-1 alpha-transfected EPCs homed to the site of ischemia. A higher revascularization potential was also demonstrated by increased capillary density at the injury site. Our results revealed that endothelial progenitor cells ex vivo modification by hypoxia inducible factor-1 alpha gene transfection is feasible and may offer significant advantages in terms of EPC expansion and treatment efficacy.     

Increased expression of HIF-1alpha, nNOS, and VEGF in the cerebral cortex of anemic rats

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial Pa(O(2)) values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 +/- 2 g/l. In anemic rats, cerebral cortical HIF-1alpha protein levels were increased, relative to controls (1.7 +/- 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 +/- 0.2- and 1.5 +/- 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1alpha and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 +/- 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1alpha protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.     

Effects of hypoxia exposure on hepatic cytochrome P450 1A (CYP1A) expression in Atlantic croaker: molecular mechanisms of CYP1A down-regulation

Hypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), N(ω)-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.     

PGE1 analog alprostadil induces VEGF and eNOS expression in endothelial cells

Endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor 1-alpha (HIF-1alpha) are important regulators of endothelial function, which plays a role in the pathophysiology of heart failure (HF). PGE1 analog treatment in patients with HF elicits beneficial hemodynamic effects, but the precise mechanisms have not been investigated. We have investigated the effects of the PGE1 analog alprostadil on eNOS, VEGF, and HIF-1alpha expression in human umbilical vein endothelial cells (HUVEC) using RT-PCR and immunoblotting under normoxic and hypoxic conditions. In addition, we studied protein expression by immunohistochemical staining in explanted hearts from patients with end-stage HF, treated or untreated with systemic alprostadil. Alprostadil causes an upregulation of eNOS and VEGF protein and mRNA expression in HUVEC and decreases HIF-1alpha. Hypoxia potently increased eNOS, VEGF, and HIF-1alpha synthesis. The alprostadil-induced upregulation of eNOS and VEGF was prevented by inhibition of MAPKs with PD-98056 or U-0126. Consistently, the expression of eNOS and VEGF was increased, and HIF-1alpha was reduced in failing hearts treated with alprostadil. The potent effects of alprostadil on endothelial VEGF and eNOS synthesis may be useful for patients with HF where endothelial dysfunction is involved in the disease process.     

Stabilization of hypoxia-inducible factor-1alpha is involved in the hypoxic stimuli-induced expression of vascular endothelial growth factor in osteoblastic cells

It has been suggested that blood vessel formation is an important event coupled to bone formation. The expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, has been shown to be greatly stimulated in osteoblasts by hypoxic stimuli such as deprivation of oxygen and treatment with cobalt. In other cell types, hypoxia-inducible factor-1 (HIF-1) that binds hypoxia-response element (HRE) has been shown to mediate gene expression induced by hypoxic stimuli. In this study, we investigated the effects of hypoxic stimuli on HIF-1, HRE, and VEGF in osteoblastic cell lines. Exposure of these cells to hypoxia or cobalt resulted in a great increase in the protein level of HIF-1alpha and the gene expression of VEGF. Transforming growth factor-beta1, prostaglandin E2, dexamethasone, and 1,25-dihydroxyvitamin D3 that have been shown to regulate VEGF gene expression in osteoblasts had no effect on HIF-1alpha induction. Blocking the enzymatic activity of phosphatidylinositol 3-kinase, p38, MEK-1 did not have any effect on the cobalt-stimulated increase of HIF-1alpha in these cells. In contrast, N-acetylcysteine (NAC), a scavenger of reactive oxygen species, abolished the cobalt induction of HIF-1alpha and that of the VEGF and a HRE-driven reporter genes. However, the hypoxia responses were not affected by NAC. These findings suggest that hypoxia and cobalt can induce VEGF gene expression in osteoblasts by increasing the level of HIF-1alpha protein through different mechanisms.     

Activation of Hsp90-eNOS and increased NO generation attenuate respiration of hypoxia-treated endothelial cells

Hypoxia induces various adoptive signaling in cells that can cause several physiological changes. In the present work, we have observed that exposure of bovine aortic endothelial cells (BAECs) to extreme hypoxia (1-5% O(2)) attenuates cellular respiration by a mechanism involving heat shock protein 90 (Hsp90) and endothelial nitric oxide (NO) synthase (eNOS), so that the cells are conditioned to consume less oxygen and survive in prolonged hypoxic conditions. BAECs, exposed to 1% O(2), showed a reduced respiration compared with 21% O(2)-maintained cells. Western blot analysis showed an increase in the association of Hsp90-eNOS and enhanced NO generation on hypoxia exposure, whereas there was no significant accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). The addition of inhibitors of Hsp90, phosphatidylinositol 3-kinase, and NOS significantly alleviated this hypoxia-induced attenuation of respiration. Thus we conclude that hypoxia-induced excess NO and its derivatives such as ONOO(-) cause inhibition of the electron transport chain and attenuate O(2) demand, leading to cell survival at extreme hypoxia. More importantly, such an attenuation is found to be independent of HIF-1alpha, which is otherwise thought to be the key regulator of respiration in hypoxia-exposed cells, through a nonphosphorylative glycolytic pathway. The present mechanistic insight will be helpful to understand the difference in the magnitude of endothelial dysfunction.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.