A simple method to induce hypoxia-induced vascular endothelial growth factor-A (VEGF-A) expression in T24 human bladder cancer cells

Angiogenesis is an essential process for the establishment, development, and dissemination of several malignant tumors including bladder cancer. The hypoxic condition promotes the stabilization of hypoxia-inducible factor 1 alpha (HIF-1α), which translocates to the nucleus to mediate angiogenic factors including the vascular endothelial growth factor A (VEGF-A). AnaeroGen system was developed for microbiology area to create a low oxygen tension required to the growth of anaerobic bacteria. Here, we hypothesized the use of AnaeroGen system to induce hypoxia in T24 human bladder carcinoma cells, in order to promote the overexpression of VEGF-A. T24 cells were cultured in six-well plates containing McCoy medium. Exposures of T24 cells to hypoxia for 1, 8, 24, and 48 h were performed using the Oxoid AnaeroGen system, while T24 cells under normoxia were used as control. The expression of VEGF-A and HIF-1α was analyzed by real-time PCR. ELISA for HIF-1α was carried out. The VEGF-A expression increased significantly by Oxoid AnaeroGen-induced hypoxia in a time-depending manner, reaching the peak in 48 h of hypoxia. Although HIF-1α mRNA was not changed, HIF-1α protein was increased in the presence of hypoxia, reaching a peak at 8 h. These results demonstrated that the Oxoid AnaeroGen system is a simple method to expose T24 cells to hypoxia and efficiently to upregulate VEGF expression in T24 cells.     

Hyperbaric oxygen preconditioning promotes angiogenesis in rat liver after partial hepatectomy

Hyperbaric oxygen preconditioning (HBO-PC) increases the level of HIF-1alpha (hypoxia inducible factor-1alpha) and its target gene VEGF (vascular endothelial growth factor) which is involved in angiogenesis. Liver regeneration is an angiogenesis-dependent process. We hypothesized that HIF-1alpha and VEGF mediated the angiogenesis effect of HBO-PC on regenerating rat liver. Male Sprague Dawley rats received HBO-PC followed by 70% partial hepatectomy. Proliferation of hepatocytes and endothelial cells was evaluated by BrdU (bromodeoxyuridine) staining. Microvascular density was assessed by immunohistochemistry. mRNA expression of HIF-1alpha was assessed by quantitative RT-PCR and protein levels of HIF-1alpha and VEGF were assessed by western blot. HIF-1alpha DNA-binding activity was determined with an ELISA-based kit. HBO-PC increased the proliferation index of endothelial cells and microvascular density at 48 h after partial hepatectomy. The protein level and DNA-binding activity of HIF-1alpha and the protein level of VEGF were increased by HBO-PC before and after partial hepatectomy. Partial hepatectomy alone also increased proliferation index and the expressions of HIF-1alpha and VEGF. Our results indicated that the angiogenesis effect of HBO-PC on liver after partial hepatectomy could be achieved by increased HIF-1alpha activity and VEGF expression. However, the angiogenic effect of HBO-PC is moderate and HBO-PC failed to produce additional effect on the enhancement of HIF-1alpha and VEGF induced by partial hepatectomy alone.     

动物抗低氧胁迫相关基因的表达调控机制

体内氧浓度的稳定是机体维持自身功能的一个必要条件。在低氧条件下,机体内部在低氧信号的刺激下形成一个强大的防御体系以保护自己的组织。在采取防御的过程中,低氧诱导因子-1 (hypoxia inducible factor-1,HIF-1)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、促红细胞生成素(erythropoietin, EPO)、核因子-κB (nuclear factor-κB, NF-κB)等基因表达上调。HIF-1是一个与低氧胁迫相关的转录因子,它的激活与体内氧浓度相关。VEGF是HIF-1下游的一个靶基因,它是至今发现的一个在促血管新生方面起着最关键作用的因子。NF-κB能够抑制由低氧引起的细胞凋亡。以上这些基因在动物抗低氧胁迫方面起着重要作用,综述了低氧条件下HIF-1、VEGF、EPO、NF-κB的功能、表达特性以及调控机制。     

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.