PURPOSE: We determined if hypoxia-inducible factor-1α (HIF-1α) and Roundabout4 (Robo4) colocalized in fibrovascular membranes (FVM) from patients with proliferative diabetic retinopathy (PDR), and investigated the regulation of HIF-1α on Robo4 in microvascular endothelial cells under normoxic and hypoxic conditions in vitro. METHODS: Immunofluorescence and confocal laser scanning microscopy were done to analyze the colocalization of HIF-1α and Robo4 in the FVM. Expression of HIF-1α was knocked down by small interfering RNA (siRNA) technology to study its effects on Robo4 expression of human retinal endothelial cells (HREC) and human dermal microvascular endothelial cells (HDMEC) under normoxic and/or hypoxic conditions. Full-length human HIF-1α gene was transfected into HREC and HDMEC using GFP lentivirus vectors to overexpress HIF-1α under normoxic conditions. The HIF-1α and Robo4 mRNA and protein expressions were quantified by real-time PCR and Western blot. A cell proliferation, migration assay, and flow cytometry were used to analyze the effect of HIF-1α regulation on Robo4 in HREC under hypoxic conditions. RESULTS: Colocalization of HIF-1α and Robo4 in vessels of FVM was confirmed by immunofluorescence staining. Knockdown of HIF-1α expression by siRNA in the HREC and HDMEC inhibited Robo4 expression in mRNA and protein level, while overexpressed HIF-1α increased Robo4 mRNA and protein expression. Silencing HIF-1α in endothelial cells under hypoxic conditions inhibited cell invasion and proliferation, which showed that HIF-1α and Robo4 overexpression due to hypoxic conditions correlated with HREC migration and proliferation. CONCLUSIONS: Both HIF-1α and Robo4 may have a vital role during the formation of FVM. The increased or decreased expression of Robo4 by stimulation or knockdown of HIF-1α suggesting that Robo4 is positively regulated by HIF-1α under normoxic and hypoxic conditions in microvascular endothelial cells in vitro. The HIF-1α gene promotes HREC invasion and proliferation by transcriptionally upregulating Robo4 under hypoxic conditions.
PURPOSE: We determined if hypoxia-inducible factor-1α (HIF-1α) and Roundabout4 (Robo4) colocalized in fibrovascular membranes (FVM) from patients with proliferative diabetic retinopathy (PDR), and investigated the regulation of HIF-1α on Robo4 in microvascular endothelial cells under normoxic and hypoxic conditions in vitro. METHODS: Immunofluorescence and confocal laser scanning microscopy were done to analyze the colocalization of HIF-1α and Robo4 in the FVM. Expression of HIF-1α was knocked down by small interfering RNA (siRNA) technology to study its effects on Robo4 expression of human retinal endothelial cells (HREC) and human dermal microvascular endothelial cells (HDMEC) under normoxic and/or hypoxic conditions. Full-length human HIF-1α gene was transfected into HREC and HDMEC using GFP lentivirus vectors to overexpress HIF-1α under normoxic conditions. The HIF-1α and Robo4 mRNA and protein expressions were quantified by real-time PCR and Western blot. A cell proliferation, migration assay, and flow cytometry were used to analyze the effect of HIF-1α regulation on Robo4 in HREC under hypoxic conditions. RESULTS: Colocalization of HIF-1α and Robo4 in vessels of FVM was confirmed by immunofluorescence staining. Knockdown of HIF-1α expression by siRNA in the HREC and HDMEC inhibited Robo4 expression in mRNA and protein level, while overexpressed HIF-1α increased Robo4 mRNA and protein expression. Silencing HIF-1α in endothelial cells under hypoxic conditions inhibited cell invasion and proliferation, which showed that HIF-1α and Robo4 overexpression due to hypoxic conditions correlated with HREC migration and proliferation. CONCLUSIONS: Both HIF-1α and Robo4 may have a vital role during the formation of FVM. The increased or decreased expression of Robo4 by stimulation or knockdown of HIF-1α suggesting that Robo4 is positively regulated by HIF-1α under normoxic and hypoxic conditions in microvascular endothelial cells in vitro. The HIF-1α gene promotes HREC invasion and proliferation by transcriptionally upregulating Robo4 under hypoxic conditions.