Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion

Hypoxia and hypoxia-inducible factor-1 (HIF-1) play a critical role in glioblastoma multiforme (GBMs). CXCR4 is involved in angiogenesis and is upregulated by HIF-1alpha. CXCR4 is a chemokine receptor for stromal cell-derived factor-1 (SDF-1)alpha, also known as CXCL12. We hypothesized that CXCR4 would be upregulated by hypoxia in GBMs. First, we investigated the expression of HIF-1alpha and CXCR4 in GBMs. CXCR4 was consistently found colocalized with HIF-1alpha expression in pseudopalisading glioma cells around areas of necrosis. In addition, angiogenic tumor vessels were strongly positive for CXCR4. Next, we tested the in vitro effect of hypoxia and vascular endothelial growth factor (VEGF) on the expression of CXCR4 in glioma cell lines and in human brain microvascular endothelial cells (HBMECs). Exposure to hypoxia induced significant expression of CXCR4 and HIF-1alpha in glioma cells, whereas treatment with exogenous VEGF increased CXCR4 expression in HBMECs. We also transfected U87MG glioma cells with an HIF-1alpha construct and observed that CXCR4 was upregulated in these cells even in normoxic conditions. We then used a lentivirus-mediated shRNA expression vector directed against HIF-1alpha. When exposed to hypoxia, infected cells failed to show HIF-1alpha and CXCR4 upregulation. We performed migration assays under normoxic and hypoxic conditions in the presence or absence of AMD3100, a CXCR4 inhibitor. There was a significant increase in the migration of U87MG and LN308 glioma cells in hypoxic conditions, which was inhibited in the presence of AMD3100. These studies demonstrate the critical role played by hypoxia and CXCR4 in glioma cell migration. Based on these studies, we suggest that hypoxia regulates CXCR4 in GBMs at two levels. First, through HIF-1alpha in the pseudopalisading tumor cells themselves and, secondly, by the VEGF-stimulated angiogenic response in HBMECs. We believe this knowledge may lead to a potentially important two-pronged therapy against GBM progression using chemotherapy targeting CXCR4.     

Inducible expression of endothelial PAS domain protein-1 by hypoxia in human lung adenocarcinoma A549 cells. Role of Src family kinases-dependent pathway.

Hypoxia is a potent inducer of tumor angiogenesis, the process of which is mostly mediated by induction of vascular endothelial growth factor (VEGF). In this study, we investigated the effect of hypoxia on the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and endothelial PAS domain protein-1 (EPAS1). These two similar but distinct basic helix-loop-helix-PAS proteins have been postulated to activate VEGF expression in response to hypoxia. We showed that EPAS1, but not HIF-1alpha, is abundantly expressed in human lung adenocarcinoma A549 cells. Exposure of cultured A549 cells to hypoxia increased EPAS1 mRNA and protein levels. A specific inhibitor for Src family kinases, PP1, abolished the hypoxia-induced expression of EPAS1. Transient transfection assays revealed that forced expression of EPAS1 increased the reporter gene activity driven by EPAS1 promoter as well as by VEGF promoter. Finally, overexpression of EPAS1 by infection of adenoviral vector expressing EPAS1 cDNA evidently induced the endogenous EPAS1 gene expression. Together, these data demonstrate Src family kinases mediate the hypoxia-mediated EPAS1 gene expression, which in turn positively autoregulates its own expression. Given an EPAS1 as a potent activator of the VEGF gene, these findings will provide a novel insight into the mechanisms underlying the enhancement of growth property of EPAS1-expressing tumor cells under the hypoxic environment.     

Actin-sequestering protein,thymosin beta-4, is a novel hypoxia responsive regulator

Angiogenesis is induced by soluble factors such as vascular endothelial growth factor (VEGF) released from tumor cells in hypoxia. It enhances solid tumor growth and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and 121 in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor growth and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.     

Intussusceptive microvascular growth in human glioma

Intussusceptive microvascular growth (IMG), which occurs by splitting of the existing vasculature by transluminal pillars or transendothelial bridges, has been demonstrated in several tumors such as colon and mammary carcinomas, melanoma and B-cell non-Hodgkin’s lymphomas. In this study, we have correlated in human glioma the extent of angiogenesis, evaluated as microvascular density, the immunoreactivity of tumor cells to vascular endothelial growth factor (VEGF), vessel diameter and IMG to the tumor stage. Results demonstrate for the first time a relationship in human glioma progression between angiogenesis, VEGF immunoreactivity of tumor cells, vessel diameter and the number of connections of intraluminal tissue folds with the opposite vascular wall, expression of IMG and suggest that IMG could be a mechanism of compensatory vascular growth occurring in human glioma. The advantages are that (1) blood vessels are generated more rapidly; (2) it is energetically and metabolically more economic; (3) the capillaries thereby formed are less leaky.     

缺氧微环境对胶质瘤细胞U251迁移性的影响

目的：探讨二氯化钴模拟化学缺氧的细胞微环境对人胶质瘤U251细胞增殖与迁移性的影响及其机制。方法胶质瘤U251细胞的培养基中加入二氯化钴模拟细胞缺氧,通过MTS检测细胞增殖,通过划痕实验检测细胞迁移,通过实时荧光定量PCR检测U251细胞表达血管内皮生长因子水平,采用免疫印迹检测细胞内缺氧诱导因子HIF-1α、动力蛋白RhoA与Cdc42、粘附分子E-cadherin与β-catenin表达,以及该过程中信号转导因子STAT3的磷酸化水平。结果二氯化钴不会促进U251细胞增殖,能显著促进细胞迁移;缺氧条件下U251细胞内VEGF水平升高, E-cadherin、β-catenin表达降低, HIF-1α表达和STAT3磷酸化水平随时间上调。结论二氯化钴模拟缺氧微环境可通过抑制U251细胞间粘附分子E-cadherin、β-catenin表达和提高VEGF-a水平,促进胶质瘤细胞迁移及周围血管生成,增强胶质瘤迁移与转移能力,细胞内信号转导分子HIF-1α和STAT3的激活在该过程中起重要作用。     

2-methoxyestradiol inhibits hypoxia-inducible factor-1{alpha} and suppresses growth of lesions in a mouse model of endometriosis

Endometriosis, the presence of ectopic endometrial tissue outside the uterine cavity, is a common disease affecting women during their reproductive years. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Nevertheless, angiogenesis plays an essential role in the pathogenesis of the disease, making it a potential novel target for therapy. In the current study, we demonstrate in an established mouse model of endometriosis that transient hypoxia in transplanted endometriosis-like lesions results in the up-regulation of hypoxia-inducible factor-1alpha (HIF-1alpha), leading to the expression of vascular endothelial growth factor (VEGF), a key player in endometriosis-associated angiogenesis. Systemic treatment with the angiogenesis inhibitor 2-methoxyestradiol suppressed HIF-1alpha expression in vivo, resulting in a decreased downstream expression of HIF-1alpha target genes, such as for VEGF, phosphoglycerate kinase, and glucose transporter-1. 2-Methoxyestradiol also suppressed VEGF-induced vascular permeability, as demonstrated in a modified Miles assay. Finally, systemic treatment with 2-methoxyestradiol significantly inhibited the growth of endometriosis-like lesions in a dose-dependent manner. In conclusion, hypoxia appears to play an important role in the pathogenesis of endometriosis and endometriosis-associated angiogenesis, and the angiogenesis inhibitor 2-methoxyestradiol may be a potential candidate for systemic treatment in the future.     

HIF-1α和VEGF在胃癌中的表达及其相关性研究

目的 通过对不同分化和高低转移的胃癌组织中HIF-1α和VEGF表达情况的检测,揭示HIF-1α和VEGF与胃癌恶化程度及转移的关系.方法 用免疫组织化学法(SP法)检测HIF-1α和VEGF在胃癌组织中的表达.结果 HIF-1α和VEGF的表达均高于正常组织.HIF-1α和VEGF在胃癌中的表达与分化、转移相关.HIF-1α和VEGF在胃癌中的表达是正相关.结论 HIF-1α和VEGF与胃癌的分化、转移密切相关.HIF-1α与VEGF在胃癌中的表达密切正相关.两种因子高表达提示预后不良,具有一定的临床诊断意义.     

Basic FGF augments hypoxia induced HIF-1-alpha expression and VEGF release in T47D breast cancer cells

AIM: Both hypoxia inducible factor 1 (HIF-1) and basic fibroblast growth factor (bFGF) play important roles in tumour angiogenesis. This study was designed to clarify the cooperative effect of these two mediators in induction of vascular endothelial cell growth factor (VEGF) release from breast cancer and probe possible mechanisms involved. METHODS: Release of VEGF from a breast cancer cell line (T47D) was quantitated by enzyme linked immunosorbent assay (ELISA). Expression of HIF-1 and ERK was assayed using Western blotting. Transient transfection and dual luciferase reporter assay were used to study HIF-1 transactivity. RESULTS: The data showed that hypoxia induced the expression of HIF-1alpha protein, the transactivity of HIF-1 and the release of VEGF. bFGF further augmented these hypoxic inductions. The PI3K pathway was required for these processes as demonstrated by application of PI3Kinase inhibitor (LY294002) or mutant construct transfections. In contrast, the MEK1 inhibitor PD98059 showed no effect on either activation of HIF-1 or VEGF release, which is in agreement with our finding that ERK1/2 was not activated by hypoxia. Under hypoxic conditions, bFGF activated the MEK1/ERK pathway. PD98059 blocked the activation of ERK1/2 and suppressed bFGF-induced HIF-1 transactivity, yet the protein expression of HIF-1alpha or VEGF release was not affected by PD98059. CONCLUSION: bFGF augments hypoxia induced VEGF release mainly through the PI3K pathway and partly depending on HIF-1 activity. Elucidation of this mechanism may provide a new target for anti-angiogenesis in cancer therapy.