| Background:Vision impairment, disability, and blindness are major public health problems. Significant suffering, disability, loss of productivity, and lower quality of life can affect millions of people. Retinopathies are ocular diseases in which deterioration of the retina is initiated by abnormal neovascularization, resulting in vision loss. Vascular retinopathies are the leading causes of visual disability and blindness worldwide. Pathological growth of new blood vessels in pre-retinal region is the hallmark of retinopathies. There is increasing evidence that endothelial progenital cells (EPCs)make a major contribution to both vascular repair and dysfunction and that this is dependent on environmental cues. A critical role has been identified for these cells in proper retinal repair, their dependence on hypoxia-regulated factors such as stromal derived factor (SDF-1) and vascular endothelial growth factor(VEGF) for their recruitment to ischemic sites, and their dysfunction in diabetic retinopathy. Cells that express CD34 (CD34+) are capable of differentiating into endothelial cells and forming vascular structures while cells that express CD 14 (CD14+) participate in "vascular mimicry" and fail to form a patent vasculature.Objective:To characterize the effect of oxygen concentration on the expression and cellular localization of VEGFR-1, VEGFR-2 and CXCR4 receptors on endothelial progenitor cells and modulate development of neovascularization.Materials and Methods:Endothelial progenitor cells were divided into 21 groups: Cells of group 0(3 groups) were analyzed immediately after thawing as control, the cells of other 18 groups were seperately incubated at 37℃ in 5% O2 (as normoxia, 3 groups),0.2% O2 (as hypoxia,3 groups) and 20%(as hyperoxia,3 groups) O2 in a humidified airtight glove box hypoxia chamber (Coy labs) and flushed with nitrogen and 5% carbon dioxide. We chose 4h and 24h as observed time point.Experiments were performed for eight times.VEGFR-1, VEGFR-2 and CXCR4 receptor expression was determined by Flow cytometric analysis, RT-PCR analysis and Western Blot analysis.Western Blot analysis to determine protein levels of VEGFR-1, VEGFR-2 and CXCR4 in EPCs:Equal quantity of whole cell lysate fractionationed by SDS-PAGE and blotted on to a nitrocellulose membrane was stained with antibodies to VEGFR-1, VEGFR-2 and CXCR4. Signals were developed using ECL Plus and band intensity quantified using Ultra Quant software.RT- PCR analysis:RNA was reverse transcribed and PCR performed to analyze the Mrna exression of VEGFR-1, VEGFR-2 and CXCR4 in EPCs.Results:1.FACs analysis:VEGFR-1 & VEGFR-2 surface expression in normoxic cells were sustained at 4 and 24h treatments. CXCR4 expression in EPCs treated with normoxic & hypoxic levels of oxygen decreased with increase in exposure time. Increased exposure (24 h) to hypoxia directly correlated with increased VEGFR-1 & VEGFR-2 levels in EPCs compared to 4 h. In cells under hyperoxic condition VEGFR-1 levels decreased with 24 h treatment while both CXCR4 & VEGFR-2 levels increased.2. Western blotting analysis:VEGFR-1 protein levels of the receptors were increased at 4h in all the three treatment groups. Only EPCs from hypoxic environment sustained the same expression for VEGFR-1 protein after 24 h.的 creased VEGFR-2 protein was recorded in EPCs from hyperoxic environment for both 4 & 24h. CXCR4 protein level was not affected by hypoxic or hyperoxic oxygen levels.3.Hyperoxia induced decreased mRNA expression levels for VEGFR-1 and VEGFR-2 at 24h in EPCs.Conclusions:1.Transient changes in oxygen environment have a profound effect for VEGFR-1, VEGFR-2 and CXCR4 on endothelial progenitor cells.2. Oxygen concentration may determine the differentiation status of EPCs through the regulation of hypoxia regulated factors, offers potential therapeutic targets to retinal neovascularization. |
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