HIF-1alpha, HIF-2alpha and VHL mRNA expression in different cell lines during hypoxia

Hypoxia inducible factor-1alpha (HIF-1alpha) mRNA expression is significantly decreased under hypoxia in different cell lines exposed directly to hypoxia or treated with dimethyloxalylglycine which mimics hypoxic effects under normoxic conditions. However, the decreased expression of HIF-1alpha mRNA is accompanied by an increase of HIF-1alpha protein (pHIF-1alpha) level as well as by overexpression of known HIF-dependent genes (VEGF, Glut1, PFKFB-3 and PFKFB-4) under hypoxic conditions or with the use of dimethyloxalylglycine. Expression of HIF-1alpha mRNA also depends on iron because desferrioxamine and cobalt chloride produce similar to hypoxia effects on the levels of this mRNA. It was shown that HIF-1alpha mRNA expression did not change significantly in some cell lines (SKBR3, MDA-MB468 and BT549) under hypoxia. However, in these cell lines hypoxia decreases expression of HIF-2alpha mRNA, another member of HIF-alpha gene family, as a result of cell specific regulation of HIF-alpha genes under hypoxia. Moreover, hypoxia slightly induces expression of PFKFB-4 mRNA in SKBR3, MDA-MB468 and BT549 as compared to other cell lines where this effect of hypoxia was much stronger and adaptation to hypoxia is controlled by HIF-1alpha. Hypoxia slightly reduces expression of tumor suppressor VHL which targets HIF-1alpha for ubiquitination. Thus, our results clearly demonstrated down regulation of HIF-1alpha or HIF-2alpha in different cell lines by hypoxia.     

Stimulation of HIF-1alpha, HIF-2alpha, and VEGF by prolyl 4-hydroxylase inhibition in human lung endothelial and epithelial cells

Diminished alveolar and vascular development is characteristic of bronchopulmonary dysplasia (BPD) affecting many preterm newborns. Hypoxia promotes angiogenic responses in developing lung via, for example, vascular endothelial growth factor (VEGF). To determine if prolyl 4-hydroxylase (PHD) inhibition could augment hypoxia-inducible factors (HIFs) and expression of angiogenic proteins essential for lung development, HIF-1alpha and -2alpha proteins were assessed in human developing and adult lung microvascular endothelial cells and alveolar epithelial-like cells treated with either the HIF-PHD-selective inhibitor PHI-1 or the nonselective PHD inhibitors dimethyloxaloylglycine (DMOG) and deferoxamine (DFO). PHI-1 stimulated HIF-1alpha and -2alpha equally or more effectively than did DMOG or DFO, enhanced VEGF release, and elevated glucose consumption, whereas it was considerably less cytotoxic than DMOG or DFO. Moreover, VEGF receptor Flt-1 levels increased, whereas KDR/Flk-1 decreased. PHI-1 treatment also increased PHD-2, but not PHD-1 or -3, protein. These results provide proof of principle that HIF stimulation and modulation of HIF-regulated angiogenic proteins through PHI-1 treatment are feasible, effective, and nontoxic in human lung cells, suggesting the use of PHI-1 to enhance angiogenesis and lung growth in evolving BPD.     

Myocardial hypoxia-inducible HIF-1alpha, VEGF, and GLUT1 gene expression is associated with microvascular and ICAM-1 heterogeneity during endotoxemia

The systemic inflammatory response to infection is the leading cause of mortality in North American intensive-care units. Although much is known about inflammatory mediators, the relationships between microregional inflammation, microvascular heterogeneity, hypoxia, hypoxia-inducible gene expression, and myocardial dysfunction are unknown. Male Sprague-Dawley rats were injected intraperitoneally with LPS to test the hypothesis that sepsis-induced local inflammation and increased microvascular heterogeneity are spatially and temporally associated with hypoxia, hypoxia-inducible gene expression, and decreased left-ventricular contractility. Using a combination of three-dimensional microvascular imaging, tissue Po(2), and pressure-volume conductance measurements, we found that 5 h after LPS, minimum oxygen-diffusion distances increased (P < 0.05), whereas tissue oxygenation and contractility both decreased (P < 0.05) in the left ventricle. Real-time RT-PCR analysis revealed that the hypoxia-inducible genes hypoxia-inducible factor (HIF)-1alpha, VEGF, and glucose transporter (GLUT) 1 were all upregulated (P < 0.05) in the left ventricle. Tissue regions expressing ICAM-1, obtained by using laser-capture microdissection, had increased HIF-1alpha and GLUT1 (P < 0.05) gene expression. VEGF gene expression was more diffuse. In LPS rats, GLUT1 gene expression correlated (P < 0.05) with left-ventricular contractility. In 5-h hypoxic cardiomyocytes, we found strong transient HIF-1alpha, weak VEGF, and greater prolonged GLUT1 gene expression. By comparison, the HIF-1alpha-GLUT1 gene-induction pattern was reversed in the left ventricle of LPS rats. Together, these results show that LPS induces hypoxia in the left ventricle associated with increased microvascular heterogeneity and decreased contractility. HIF-1alpha and GLUT1 gene induction are related to a heterogeneous ICAM-1 expression and may be cardioprotective during the onset of septic injury.     

Oligomycin inhibits HIF-1alpha expression in hypoxic tumor cells

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of cellular responses to reduced oxygen availability. The contribution of mitochondria in regulation of HIF-1 in hypoxic cells has received recent attention. We demonstrate that inhibition of electron transport complexes I, III, and IV diminished hypoxic HIF-1 accumulation in different tumor cell lines. Hypoxia-induced HIF-1 accumulation was not prevented by the antioxidants Trolox and N-acetyl-cysteine. Oligomycin, inhibitor of F₀F₁-ATPase, prevented hypoxia-induced HIF-1 protein accumulation and had no effect on HIF-1 induction by hypoxia-mimicking agents desferrioxamine or dimethyloxalylglycine. The inhibitory effect of mitochondrial respiratory chain inhibitors and oligomycin on hypoxic HIF-1 content was pronounced in cells exposed to hypoxia (1.5% O₂) but decreased markedly when cells were exposed to severe oxygen deprivation (anoxia). Taken together, these results do not support the role for mitochondrial reactive oxygen species in HIF-1 regulation, but rather suggest that inhibition of electron transport chain and impaired oxygen consumption affect HIF-1 accumulation in hypoxic cells indirectly via effects on prolyl hydroxylase function. hypoxia-inducible factor 1; oxygen sensing     

HIF-1alpha cytoplasmic accumulation is associated with cell death in old rat cerebral cortex exposed to intermittent hypoxia

Intermittent hypoxia, followed by reoxygenation, determines the production of reactive oxygen species (ROS), which may lead to accelerated aging and to the appearance of age-related diseases. The rise in ROS levels might constitute a stress-stimulus activating specific redox-sensitive signalling pathways, so inducing either damaging or protective functions. Here, we report that in old rat cerebral cortex exposed to hypoxia, the accumulation in the cytoplasm of hypoxic inducible factor 1alpha (HIF-1alpha)--the master regulator of oxygen homeostasis--concomitant with p66(Shc) activation and reduced IkBalpha phosphorylation is associated with tissue apoptosis or necrosis. In young cerebral cortex, we hypothesize that the hypoxic damage may be reversible, based on our demonstration of elevated HIF-1alpha levels, combined with a low level of IkBalpha phosphorylation, a decrease in IAP-1 and a lack of major change in Bcl2 family proteins. These observations are associated with a low level of cell death induced by hypoxia, suggesting that HIF-1alpha activation in cortical neurons may produce rescue proteins in response to intermittent hypoxia.     

TGF-beta1、HIF-1alpha、VEGF在胃癌组织及癌旁组织中的表达及临床意义

目的：研究TGF-beta1（转化生长因子-beta1）、HIF-1alpha（低氧诱导因子-1alpha）、VEGF（血管内皮生长因子）在胃癌组织及癌旁组织中 的表达及临床意义。方法：选取于我院就诊的160 例胃癌手术患者切除的组织,采用免疫组化技术检测手术切除的胃癌组织中的 TGF-beta 1、HIF-1alpha及VEGF的表达,分析其与患者临床病理参数的关系。结果：免疫组化结果显示：TGF-beta1、HIF-1alpha及VEGF 在胃 癌组织中的表达均高于癌旁组织,差异均有统计学意义（P<0.05）;TGF-beta1、HIF-1alpha及VEGF 的表达均与肿瘤分期、淋巴结转移及 浸润深度有关（P<0.05）;VEGF的表达分别与TGF-beta1、HIF-1alpgha的表达呈相关关系（P<0.05）。结论：TGF-beta1、HIF-1alpha及VEGF在胃 癌组织中的表达与胃癌的病理学特征有关,检测TGF-beta1、HIF-1alpha及VEGF的表达将有助于临床诊治胃癌患者。     

Sex-related dimorphic response of HIF-1 alpha expression in myocardial ischemia

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) plays a role in a number of cell-protective pathways after ischemia. There are clear sex-related differences in the remodeling process, and hearts from males tend to dilate in response to pathological loads and ischemia to a greater degree than do hearts from females. Thus we hypothesized that there would be a sex-related dimorphic response of HIF-1 alpha to an ischemic event. Male and female rats were euthanized 5 and 24 h after coronary ligation (M-MI and F-MI; MI, myocardial ischemia), and HIF-1 alpha expression was determined by immunohistochemistry, Western blot, and quantitative RT-PCR. Sham-operated male and female animals served as controls (M-SH and F-SH). In the ischemic area, histochemical analysis at 5 h showed that HIF was expressed in 33% of cell nuclei in M-MI and in 55% in F-MI. At 24 h, HIF expression increased to 49% in M-MI and to 82% in F-MI (P < 0.05 vs. SH and also M-MI vs. F-MI). This difference was not only statistically significant between the two sexes at 24 h but also within each sex at 5 and 24 h after ligation. Western blots confirmed that, at 24 h after ischemia, HIF protein increased significantly in both male and female hearts relative to sham-operated animals but that the increase in females was 60% greater than that seen in males. mRNA expression of HIF was significantly increased at 24 h in F-MI versus M-MI and sham-operated animals. Expression of downstream HIF target genes (heme oxygenase and brain natriuretic peptide) was increased in proportion to the levels of HIF expression. These data suggest a novel cellular mechanism to explain the sex-related dimorphic response to ischemia and also the possibility that exogenous modulation of HIF might represent a new therapeutic approach to preventing left ventricular remodeling.     

Hypoxia regulates avian cardiac Arnt and HIF-1alpha mRNA expression

The aryl hydrocarbon receptor nuclear translocator (Arnt) and hypoxia-inducible factor (HIF)-1alpha mediate cellular responses to hypoxia. We investigated the ability of hypoxia to regulate Arnt and HIF-1alpha mRNA in the heart in vivo. We cloned avian Arnt, developed an in vivo model of chronic cardiac hypoxia, and measured expression of cardiac Arnt and HIF-1alpha mRNA by quantitative RT-PCR. Chronic hypoxic exposure (24 h to 15% O(2)) of day 9 chick embryos resulted in a 30-fold increase in covalent binding of (3)H-misonidazole, a hypoxic tissue marker, to cardiac tissue, and a 2-fold induction of cardiac inducible nitric oxide synthase mRNA, compared to normoxic controls. In this same model, cardiac Arnt mRNA expression decreased by 35%, while HIF-1alpha mRNA expression increased 400%. These data suggest that regulation of Arnt and HIF-1alpha mRNA expression may contribute to the physiological responses of the heart during prolonged hypoxia.