Hypoxia regulates vascular endothelial growth factor receptor KDR/Flk gene expression through adenosine A2 receptors in retinal capillary endothelial cells.
Hypoxia regulates vascular endothelial growth factor receptor KDR/Flk gene expression through adenosine A2 receptors in retinal capillary endothelial cells.
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发表时间：
1996-06
影响因子：
4.4
通讯作者：
H. Takagi;G. King;N. Ferrara;L. P. Aiello
中科院分区：
文献类型：
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作者：
H. Takagi;G. King;N. Ferrara;L. P. Aiello
血管内皮生长因子（VEGF）是一种内皮细胞特异性血管生成因子，在许多缺血性视网膜病变中起重要作用。作者在体外培养的牛视网膜内皮细胞（BREC）中研究了两种已知的VEGF受体（KDR和Flt）的缺氧基因调控及其机制。方法采用计算机控制的红外线水套式CO2培养箱，将融合的BREC单层暴露于不同的氧浓度。采用北方印迹法和125 I-VEGF结合分析法检测缺氧对VEGF受体mRNA和蛋白表达的影响。结果北方印迹法检测到BREC中KDR的表达，而Flt的表达不明显。低氧以剂量和时间依赖性方式降低KDR基因表达，在暴露于0%氧气24小时后观察到最大抑制至常氧对照的0.5 +/- 0.2%（P = 0.019），并且在氧气浓度低于5%时具有显著抑制作用。3小时后，氧呼吸阻断使KDR mRNA表达降低至对照的58% +/- 7.1%（P = 0.001）。CPA是一种稳定的腺苷A1受体（A1 R）激动剂，在A1 R刺激浓度下不影响KDR mRNA表达，但在与A2 R反应的较高浓度下，它使KDR mRNA水平降低至对照的30% +/- 4.9%（P = 0.002）。腺苷A2受体（A2 R）激动剂DPMA以剂量依赖性方式降低KDR mRNA，EC 50为5至10 nM。A1 R拮抗剂8-环戊基-1，3-二丙基黄嘌呤和8-苯基茶碱在A1 R抑制浓度下不抑制KDR mRNA的缺氧反应，但在A2 R有效剂量下抑制反应（P = 0.001）。A2 R拮抗剂CSC在10 μ M时抑制KDR缺氧反应42% +/- 7.8%（P = 0.008）。在暴露于1%氧气24小时后，与BREC的特异性VEGF结合从每毫克蛋白15.1% +/- 0.3%降低至12.7% +/- 0.4%（P < 0.001）。相比之下，长期暴露于1%氧气（72小时）导致VEGF结合从每毫克蛋白13.5% +/- 1.1%增加到18.3% +/- 0.8%（P < 0.001）。Scatchard分析检测到缺氧暴露30小时后受体结合位点减少而结合亲和力无变化，但表明缺氧暴露72小时后特异性结合位点增加（4.2 +/- 0.6 x 10（4）位点/细胞至6.7 +/- 1.0 x 10（4）位点/细胞，P = 0.049），受体亲和力无变化。结论：缺氧诱导KDR mRNA水平和VEGF结合位点的初步下降，这是通过腺苷与A2 R的结合介导的。然而，暴露于长时间的缺氧导致VEGF结合位点的增加，其机制尚未确定。
PURPOSE Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic factor that serves an important role in numerous ischemic retinopathies. The authors studied the hypoxic gene regulation of two known VEGF receptors (KDR and Flt) and its mechanism in cultured bovine retinal endothelial cells (BREC). METHODS Confluent monolayers of BREC were exposed to various oxygen concentrations using a computer-controlled, infrared, water-jacked CO2 incubator with reduced oxygen control. Northern blot analysis and 125I-VEGF binding analysis were used to identify hypoxia-induced alterations of VEGF receptor at mRNA and protein levels. RESULTS KDR was detectable by Northern blot analysis in BREC, whereas Flt was not. Hypoxia decreased KDR gene expression in a dose-and time-dependent manner with maximal inhibition to 0.5 +/- 0.2% (P = 0.019) of normoxic control observed after 24 hours exposure to 0% oxygen and with significant inhibition at oxygen concentrations below 5%. Blockade of oxygen respiration decreased KDR mRNA expression to 58% +/- 7.1% of control (P = 0.001) after 3 hours. CPA, a stable adenosine A1 receptor (A1R) agonist, did not affect KDR mRNA expression at A1R stimulatory concentrations, but it decreased KDR mRNA levels to 30% +/- 4.9% (P = 0.002) of control at higher concentrations that react with A2R. DPMA, an adenosine A2 receptor (A2R) agonist, decreased KDR mRNA in a dose-dependent manner with an EC50 of 5 to 10 nM. A1R antagonists, 8-cyclolentyl-1,3-dipropylxanthine and 8-phenyltheophylline, did not inhibit the hypoxic response of KDR mRNA at A1R inhibitory concentrations but did inhibit the response at A2R effective doses (P = 0.001). The A2R antagonist, CSC, inhibited the KDR hypoxic response by 42% +/- 7.8% (P = 0.008) at 10 microM. Specific VEGF binding to BREC was decreased from 15.1% +/- 0.3% to 12.7% +/- 0.4% per milligram protein (P < 0.001) after exposure to 1% oxygen for 24 hours. In contrast, long-term exposure to 1% oxygen (72 hours) resulted in an increase of VEGF binding from 13.5% +/- 1.1% to 18.3% +/- 0.8% per milligram protein (P < 0.001). Scatchard analysis detected a decrease of receptor binding sites without change in binding affinity after 30 hours of exposure to hypoxia but demonstrated an increase in specific binding sites (4.2 +/- 0.6 x 10(4) sites/cell to 6.7 +/- 1.0 x 10(4) sites/cell, P = 0.049) with unaltered receptor affinity after 72 hours of hypoxic exposure. CONCLUSIONS These data suggest that hypoxia induces an initial decline in KDR mRNA levels and VEGF binding sites as mediated through adenosine binding to the A2R. Exposure to prolonged periods of hypoxia, however, results in an increase in VEGF binding sites by an as yet unidentified mechanism.