Expression of Robo4 in the fibrovascular membranes from patients with proliferative diabetic retinopathy and its role in RF/6A and RPE cells

PURPOSE Robo4, a member of the roundabout (Robo) family, acts as a neuronal guidance receptor and plays some role in vasculogenesis and angiogenesis. This study investigated the effect of Robo4 on the formation of fibrovascular membranes (FVMs) from patients with proliferative diabetic retinopathy and its roles in choroid-retina endothelial (RF/6A) and human retinal pigment epithelial (RPE) cells. METHODS RT-PCR and immunohistochemistry were used to determine the levels of mRNA and the presence and distribution of Robo4 in FVMs. Small interfering RNA (siRNA) technology was used to knock down Robo4 expression and to study its effects on RF/6A and RPE cells in vitro. Cell proliferation, migration, spreading, cycling, and apoptosis were assessed with MTT assay, Boyden chamber assay, immunocytochemistry, and flow cytometry. Tube formation by RF/6A on Matrigel was also analyzed. RESULTS The level of Robo4 mRNA was high in FVMs. Robo4 was expressed in the vessels and fibrous-like tissue co-immunostained for CD31 and GFAP, respectively. Robo4 siRNA knockdown inhibited cell proliferation and migration. Tube formation by RF/6A cells was also disturbed. Under hypoxic conditions, more apoptotic cells were evident among the knockdown cells than among the control cells (p<0.01). CONCLUSIONS Robo4 may play a role in the formation of FVMs. Silencing the expression of Robo4 in RF/6A and RPE cells inhibited their proliferation and reduced their tolerance of hypoxic conditions, suggesting physiologic functions of Robo4 in the cells of the retina.