Investigation Of The Effect Of Robo4on Endothelial Permeability

Blood-retinal barrier (BRB) serves as a selective partition between the retina and thecirculation and maintains the highly specialized environment. BRB includes innerblood–retinal barrier (iBRB) and outer blood–retinal barrier (oBRB). Endothelial cells thatline retinal capillaries form an inner blood–retinal barrier that prevents leakage of proteinand fluid into the inner retina. Disruption of iBRB characterized by capillary leakage is amajor cause of visual loss in a number of human ocular disorders including diabeticretinopathy (DR), retinopathy of prematurity (ROP) and Coats disease. Therefore, a betterunderstanding of the mechanism underlying the regulation of retinal endothelial barrierintegrity is of potential physiological and pharmacological significance.Increased permeability of iBRB may occur through two pathways, the paracellularpathway and the transcellular pathway. The paracellular route is governed by tight junction(TJ) and is usually the main route of increased endothelial barrier permeability. Occludin andzonula occludens (ZO)-1are key tight junction proteins. Appropriate protein content and theproper localization of these proteins within TJ are essential in establishing a tight barrierbetween endothelial cells. It has been suggested that rearrangement and polymerization ofactin filaments may influence the function of TJ thereby affecting endothelial permeability.Robo family (Roundabout family) are conservative transmembrane proteins. Researchesshow that Robo family proteins play an important role in the directing axon guidance andneuron cell migration. Recently, Robo4(magic roundabout, MRB), a new member of Robo family, has been found, which is endothelial cell-specific and expressed at sites of activeangiogenesis. Further, some signaling pathways have been reported to be involved in Robo4signaling in cell migration. Recent studies have suggested that Robo4inhibits pathologicalangiogenesis by inhibiting vascular endothelial growth factor (VEGF) downstreanmsignaling. Although Robo4+/+mice exhibited decreased basal vascular permeability in mousemodels of oxygen-induced retinopathy (OIR), there have been no report on the completemechanism of how Robo4exerts its effect on the basal permeability in retinal endothelialcells.To further elucidate the function of Robo4, expression of Robo4was reduced by applyingRNA interference in cultured human retinal vascular endothelial cells (HRVECs). The role ofRobo4depletion on the modulation of tight junction proteins was also investigated toattempt to understand the mechanism by which Robo4affects endothelial barrier properties.In the first part, Robo4expression was measured by western blot in HRVECs treated withadvanced glycation end product (AGE) which induced retinal endothelial hyperpermeability.The results show that AGE stimulation suppressed the amount of Robo4expression inHRVECs in a time-dependent manner and a dose-dependent manner. To investigate the roleof Robo4in endothelial permeability, Robo4function was inhibited with specific siRNA.HRVECs were grown to confluence on transwell filters and endothelial barrier permeabilitywas tested by observing diffusion of dextran in HRVEC monolayers. In this experiment,Robo4knockdown resulted in hyperpermeability when compared with the control groups(P<0.05). These results support the hypothesis that the depletion of Robo4by siRNA has apotential to induce endothelial hyperpermeability and disturb the retinal endothelial barrier. It has been suggested that TJ proteins and actin filaments, which binds occludin throughZO-1at TJ play an essential role in regulating the endothelial barrier integrity. In the secondpart, western blot analysis of both occludin and ZO-1showed decreased expression inRobo4-depleted cells (P<0.001). In addition, rearrangement of cell cortical cytoskeletal wasexamined by labeling polymeric F-actin with rhodamine phalloidin. Robo4-depleted cellsshowed excessive stress fiber formation and F-actin on cellular boundaries was attenuated.These results indicate that the depletion of Robo4disrupts TJ and increases permeability ofendothelial barrier in part by affecting the expression levels of ZO-1, occludin, andrearrangement of F-actin.Actin reorganization is regulated by a diverse group of actin-binding proteins, whichincludes the actin-depolymerization factor (ADF)/cofilin family of proteins. LIM kinase(LIMK)1plays a critical role in stimulus-induced actin reorganization by phosphorylatingcofilin. In the last part, the phosphorylation status of cofilin was investigated by western blot.The total amount of cofilin did not differ in the three groups (P>0.05), however, an increasedlevel of phosphorylated cofilin was observed in Robo4siRNA-treated group (P<0.01).Western blot analysis demonstrated significant increase in the level of LIMK1inRobo4-depleted cells (P<0.01), suggesting that LIMK1may be involved in Robo4depletion-induced cofilin phosphorylation. To further investigate whether the changes incofilin phosphorylation in Robo4-depleted cells are due to promotion of LIMK1activity,HRVECs were co-transfected with Robo4siRNA and LIMK1siRNA. LIMK1siRNAmarkedly suppressed cofilin phosphorylation induced by the depletion of Robo4(P<0.001).Furthermore, depletion of LIMK1suppressed the formation of excessive stress fibers in Robo4-depleted cells. Interestingly, western blot analysis showed that LIMK1knockdowncould increase expression levels of occludin (P<0.001) and ZO-1(P<0.05) in Robo4depleted cells. These results suggest that LIMK1knockdown resulted in TJ reformation.Finally the effect of LIMK1siRNA on HRVEC monolayers’ permeability in Robo4-depletedcells was tested and it was found that LIMK1siRNA reverted hyperpermeability induced byRobo4depletion (P<0.05), suggesting that Robo4can regulate actin dynamics via aLIMK1-cofilin pathway. The above findings are strongly indicative of a signaling pathwaythat involves LIMK1/cofilin, which leads to endothelial hyperpermeability.Taken together, this study demonstrated an important role of Robo4in retinal endothelialbarrier integrity. Knockdown of Robo4expression in HRVECs induced endothelialhyperpermeability associated with the down-regulation of ZO-1, occludin, and therearrangement of F-actin and that LIMK1/cofilin signal transduction system may beinvolved in the modulating process.