Forooghian Farzin, Razavi Rozita, Timms Lee
Department of Ophthalmology and Vision Sciences and Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
Br J Ophthalmol. 2007 Oct;91(10):1406-10. doi: 10.1136/bjo.2007.123125. Epub 2007 Jun 13.
Hypoxia-inducible factor (HIF) is a common transcription factor for many angiogenic proteins. Retinal pigment epithelial (RPE) cells are an important source of angiogenic factors in the retina. The expression of HIF, its regulation by proline hydroxylase (PHD) enzymes, and its downstream regulation of angiogenic factors like vascular endothelial growth factor (VEGF) and erythropoietin (EPO) was studied in RPE cells in order to determine some of the molecular mechanisms underlying ischaemic retinal disease.
ARPE-19 cells were cultured for various times under hypoxic conditions. Cellular HIF and PHD isoforms were analysed and quantified using western blot and densitometry. VEGF and EPO secreted into the media were assayed using enzyme-linked immunosorbent assay (ELISA). Messenger RNA (mRNA) was quantified using real-time quantitative reverse transcriptase polymerase chain reaction (qPCR). RNA interference was achieved using siRNA techniques.
HIF-1 alpha was readily produced by ARPE-19 cells under hypoxia, but HIF-2 alpha and HIF-3 alpha could not be detected even after HIF-1 alpha silencing. HIF-1 alpha protein levels showed an increasing trend for the first 24 h while HIF-1 alpha mRNA levels fluctuated during this time. After 36 h HIF-1 alpha protein levels declined to baseline levels, a change that was coincident with a rise in both PHD2 and PHD3. Silencing HIF-1 alpha significantly decreased VEGF secretion. Significant production of EPO could not be detected at the protein or mRNA level.
HIF-1 alpha appears to be the main isoform of HIF functioning in ARPE-19 cells. Under hypoxia, HIF-1 alpha levels are likely self-regulated by a feedback loop that involves both transcriptional and post-translational mechanisms. VEGF production by human RPE cells is regulated by HIF-1 alpha. EPO was not produced in significant amounts by RPE cells under hypoxic conditions, suggesting that other cells and/or transcription factors in the retina are responsible for its production.
缺氧诱导因子（HIF）是多种血管生成蛋白的常见转录因子。视网膜色素上皮（RPE）细胞是视网膜中血管生成因子的重要来源。对RPE细胞中HIF的表达、脯氨酸羟化酶（PHD）对其的调控以及其对血管内皮生长因子（VEGF）和促红细胞生成素（EPO）等血管生成因子的下游调控进行了研究，以确定缺血性视网膜疾病的一些分子机制。
将ARPE - 19细胞在缺氧条件下培养不同时间。使用蛋白质免疫印迹法和光密度测定法分析并定量细胞中的HIF和PHD亚型。使用酶联免疫吸附测定（ELISA）法检测分泌到培养基中的VEGF和EPO。使用实时定量逆转录聚合酶链反应（qPCR）对信使核糖核酸（mRNA）进行定量。使用小干扰RNA（siRNA）技术实现RNA干扰。
ARPE - 19细胞在缺氧条件下容易产生HIF - 1α，但即使在HIF - 1α沉默后也检测不到HIF - 2α和HIF - 3α。HIF - 1α蛋白水平在最初24小时呈上升趋势，而在此期间HIF - 1α mRNA水平波动。36小时后，HIF - 1α蛋白水平降至基线水平，这一变化与PHD2和PHD3的升高同时发生。沉默HIF - 1α显著降低VEGF分泌。在蛋白质或mRNA水平均未检测到明显的EPO产生。
HIF - 1α似乎是在ARPE - 19细胞中发挥作用的HIF主要亚型。在缺氧条件下，HIF - 1α水平可能通过涉及转录和翻译后机制的反馈环进行自我调节。人RPE细胞的VEGF产生受HIF - 1α调控。在缺氧条件下，RPE细胞未大量产生EPO，这表明视网膜中的其他细胞和/或转录因子负责其产生。
