Effect of Robo4 on Retinal Endothelial Permeability

Abstract

Purpose: To examine the role of Roundabout 4 (Robo4) in retinal endothelial permeability and analyze the structural events that lead to barrier disruption.

Materials and methods: Small interfering RNA (siRNA) technology was used to knockdown Robo4 expression to study its effects on the permeability of human retinal vascular endothelial cells (HRVECs) in vitro. The endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the HRVEC monolayers. The impact of Robo4 siRNA on the expression of tight junction (TJ) proteins and the activation of LIM-kinase (LIMK)/cofilin pathway were measured by western blotting. The change of actin cytoskeleton was detected using indirect immunofluorescence.

Results: Robo4 siRNA increased the permeability of HRVEC monolayers. The expression levels of TJ-associated proteins occludin and zonula occludens (ZO)-1 were suppressed in Robo4-depleted cells. In addition, there was rearrangement of F-actin in HRVECs. These processes were induced through increased activity in the LIMK/cofilin pathway which coincided with a disruption in the barrier properties of retinal endothelial monolayers.

Conclusions: Knockdown of Robo4 expression in HRVECs induced endothelial hyperpermeability associated with the downregulation of ZO-1, occludin, and the rearrangement of F-actin and that LIM-kinase 1 (LIMK1)/cofilin signal transduction system may be involved in the modulating process.

KEYWORDS::

• Roundabout 4 (Robo4)
• Small interfering RNA (siRNA)
• Endothelial permeability
• Tight junction
• LIM-kinase (LIMK)/cofilin

ACKNOWLEDGMENTS

This work was supported by all the members of Key Laboratory for Molecular Enzymology & Engineering, Ministry of Education in Jilin University, and Central Laboratory of the Second Hospital in Jilin University.

Declaration of interest: This work was supported by the National Natural Science Foundation of China (Grant No. 30973265); Key Projects of Science and Technology Development Plan of Jilin province (Grant No. 20090453); Research Fund for Key Specialty-Ophthalmology Construction Program of Jilin Department of Health. The authors reports no conflict of interest. The authors alone are responsible for the content and writing of the paper.