乙型肝炎病毒x蛋白与缺氧诱导因子-1α在肝癌中的表达及可能的调节机制

目的检测乙型肝炎病毒x蛋白(HBx)与缺氧诱导因子-1(HIF-1)α在肝癌中的表达,探讨正常氧和缺氧状态下,HBx对HIF-1α可能的调节机制。方法采用免疫组织化学染色方法检测78份原发性HCC组织标本中HBx和HIF-1α的表达,用SPSS10.0进行相关性分析;免疫荧光和Western blot检测常氧和缺氧条件下,HepG2及稳定转染HBx基因的HepG2细胞(HepG2- X)中HIF-1α的表达;流式细胞术检测常氧和缺氧状态下HepG2及HepG2-X细胞活性氧(ROS)的含量。结果78份肝癌组织标本中,HBx和HIF-1α免疫组织化学染色阳性率分别为74.36%(58/78)和69.23%(54/78),两者表达呈正相关(r=0.636,P<0.05)。免疫荧光检测表明:常氧状态下, HepG2细胞中HIF-1α的表达阴性而HepG2-X中表达阳性,主要位于细胞浆,部分位于细胞核,而缺氧状态下,HepG2和HepG2-X细胞的细胞质和细胞核均有表达。Western blot检测显示:常氧状态下,HepG2细胞中HIF-α几乎无表达,而HepG2-X明显表达。两者在缺氧1 h开始均表达,8 h达到高峰,16 h后逐渐下降,测量两者缺氧8 h时的表达,发现HepG2-X中HIF-α的表达增高。流式细胞术检测细胞ROS含量显示:在常氧状态下,HepG2-X细胞中ROS含量明显高于HepG2细胞。在缺氧状态下,二者ROS含量无显著差异,但均明显高于常氧状态下HepG2细胞的ROS含量。结论HBx及HIF-1α在人肝细胞肝癌组织中广泛表达,并显著正相关;常氧或缺氧状态下,HBx均可上调HIF-1α在HepG2细胞中表达,并且HBx对HIF-1α的这种调节作用可能通过ROS通路实现。     

Retinal endothelial angiogenic activity: effects of hypoxia and glial (Müller) cells

OBJECTIVE: To explore the impact of retinal glial (Müller) cells on survival and neovascularization-related activities of cultured retinal endothelial cells under normoxic and hypoxic conditions. METHODS: Bovine retinal endothelial cells (BRECs) were cultured under normoxia or hypoxia (0.5% O2) either alone, together with the human Müller cell line MIO-M1, or in normoxia- or hypoxia-conditioned media of MIO-M1 cells. Cell number, proliferation, apoptotic cell death, and migration of BRECs were determined. RESULTS: Exposure of BRECs to hypoxia for 24 h decreased the number of adherent cells and the proliferation rate, but increased apoptosis and cell migration. Increased apoptosis and decreased proliferation of the BRECs occurred also in the presence of conditioned media of MIO-M1 cells. Under normoxic conditions, co-culture with MIO-M1 cells resulted in increased proliferation, but decreased apoptosis and migration rates of BRECs. Under hypoxic conditions, the Müller cells released elevated amounts of VEGF but their presence decreased proliferation, apoptosis and the migration rates of BRECs. CONCLUSIONS: Hypoxia inhibits the proliferation of retinal endothelial cells. Müller cells release soluble mediators that enhance this hypoxia-mediated effect but, under certain conditions (i.e., in co-culture), may protect retinal endothelial cells from apoptosis, thus supporting their survival. Altogether the findings indicate that the key signal necessary to trigger retinal endothelial proliferation under hypoxia remains to be determined.     

Testosterone-stimulated growth of the rat prostate may be driven by tissue hypoxia and hypoxia-inducible factor-1alpha

Testosterone-stimulated growth of the ventral prostate (VP) in castrated rats is preceded by angiogenesis, but the mechanisms coordinating vascular and tissue growth are unknown. Adult rats were castrated and some treated with testosterone. Tissue hypoxia was studied morphologically using the hypoxia marker pimonidazole (Hypoxyprobe), hypoxia-inducible factor-1 (HIF-1) alpha, vascular endothelial growth factor (VEGF), and carbonicanhydrase 9 (CA-9) levels by western blotting and quantitative RT-PCR. In the intact untreated prostate, most glands were unstained by the hypoxia marker but already 1 day after castration most epithelial cells in the VP were stained. Seven days after castration prostate glands were apparently normoxic again, and HIF-1alpha, VEGF, and CA-9 were decreased. Treatment of 7-day castrated rats with testosterone resulted in increased epithelial hypoxyprobe staining and increased HIF-1alpha, VEGF, and CA-9 levels. The transient increase in tissue hypoxia after testosterone treatment is probably caused by a temporary mismatch between oxygen consumption and supply. Treatment of prostate epithelial cells in vitro under normoxic conditions also increased HIF-1alpha, and this could be blocked if epidermal growth factor receptor (EGFR) signaling was blocked with gefitinib. In vivo gefitinib could, however, not block the testosterone induced increase in HIF-1alpha. Testosterone may thus induce HIF-1alpha and its downstream angiogenesis promoting genes by at least two mechanisms, hypoxia and EGFR signaling. Transient epithelial cell hypoxia could by rapidly increasing HIF-1alpha and VEGF be an essential coordinator of testosterone-stimulated vascular and glandular growth.     

Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype

We previously reported that adenosine A2B receptor activation stimulates angiogenesis. Because hypoxia is a potent stimulus for the release of both adenosine and angiogenic factors, we tested the hypothesis that hypoxia alters the expression of adenosine receptors toward an "angiogenic" phenotype. We used human umbilical vein endothelial cells (HUVECs) and bronchial smooth muscle cells (BSMCs) because, under normoxic conditions, adenosine does not release vascular endothelial growth factor (VEGF). HUVECs expressed a characteristic A2A phenotype (the selective A2A agonist CGS21680 was as potent as the nonselective agonist 5'-N-ethylcarboxamidoadenosine [NECA] in generating cAMP). Hypoxia (4.6% O2, 3 hours) decreased A2A mRNA from 1.56+/-0.3% to 0.16+/-0.01% of beta-actin expression but increased A2B mRNA from 0.08+/-0.01% to 0.27+/-0.05%. Consistent with changes in receptor expression, CGS21680 failed to increase cAMP in hypoxic HUVECs, whereas NECA remained active (A2B phenotype), and NECA increased VEGF release from 9.5+/-1.0 to 14.2+/-1.2 pg/mL (P<0.05), indicating that increased A2B receptors were functionally coupled to upregulation of VEGF. Hypoxia had similar effects on BSMCs, increasing A2B mRNA by 2.4+/-0.3-fold, from 0.42+/-0.04% to 1.00+/-0.13% of beta-actin. Whereas NECA had no effect on VEGF release in normoxic BSMCs, it increased VEGF release in hypoxic BSMCs, from 74.6+/-9.6 to 188.3+/-16.7 pg/mL (P<0.01), and a selective A2B antagonist, CVT-6694, inhibited this increase. A2B receptors activated a VEGF reporter made unresponsive to hypoxia by mutating its hypoxia-inducible factor-1 (HIF-1) binding element, indicating a mechanism independent of HIF-1. In conclusion, hypoxia modulates the expression of adenosine receptors in human endothelial and smooth muscle cells toward an A2B"angiogenic" phenotype.     

Hypoxia stimulates proliferation of human hepatoma cells through the induction of hexokinase II expression

BACKGROUND/AIMS: In a hypoxic state, a glycolytic system is operating as a salvage pathway of generating ATP, and hexokinase II, the first enzyme in this system, might be over-expressed in hepatocellular carcinomas (HCCs). This study was to evaluate if hexokinase II is participating in HCC cell survival in a hypoxic state, and to analyze the mechanism of cell death caused by hexokinase II-specific inhibition. METHODS: Human hepatoma cell lines were grown either in a normoxic or hypoxic condition. Hexokinase II and hypoxia-inducible factor-1alpha (HIF-1alpha) expression were evaluated using immunoblot techniques. Cell growth was assessed using the MTS assay. Apoptotic signaling cascades were explored by immunoblot analysis. RESULTS: Hypoxia stimulated HCC cellular growth through HIF-1alpha-dependent induction of hexokinase II expression. The hexokinase II-specific inhibitor, 3-bromopyruvate, significantly suppressed cellular growth in a hypoxic state compared to cells in a normoxic condition. This suppression was due to the induction of apoptosis through activating mitochondrial apoptotic signaling cascades. CONCLUSIONS: This study demonstrates that hypoxia stimulates HCC cellular growth through hexokinase II induction, and its inhibition induces apoptotic cell death. Therefore, hexokinase II induction may participate in HCC progression and the blockage of this enzyme may therapeutically be efficacious in human HCCs.