Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro.

Gang Zhang,Xinyue Qin,Yu Wang,Rong Wang,Ke Cheng,Qi Li,Rongrong Zhang

doi:10.3389/fneur.2017.00504

Abstract

Repulsive guidance molecule a (RGMa) is a major neuron guidance factor in central nervous systems. We previously found that inhibition of RGMa could greatly enhance neural function rehabilitation in rats after MCAO/reperfusion. Neuron guidance factors are often regulators of angiogenesis. However, the effect of RGMa on angiogenesis and its mechanisms remain to be determined. Here, we investigated the effect of RGMa on endothelial cell (EC) proliferation, migration, tube formation, and cytoskeleton reassembly. The addition of recombinant RGMa significantly decreased the proliferation, migration, and tube formation of ECs. It also decreased the level of phosphorylated focal adhesion kinase (p-FAK Tyr397). Furthermore, the F-actin of the cytoskeleton assembly was obviously suppressed, with decreased filopodia and lamellipodia after the addition of RGMa. Knockout of neogenin or Unc5b significantly diminished RGMa’s inhibition of EC migration, tube formation, and cytoskeleton reassembly. RGMa-induced p-FAK (Tyr397) decrease was also abolished by knockout of neogenin or Unc5b. These results indicate that RGMa may be a negative regulator of angiogenesis through downregulating VEGF and p-FAK (Tyr397) via neogenin and Unc5b in vitro.

Highlights

Acute ischemic stroke is a leading cause of severe cognitive impairment, physical disability, and mortality worldwide
Repulsive guidance molecule a (RGMa) Expression Increased in primary human umbilical artery endothelial cell (HUAEC), Human Umbilical Vein Endothelial Cells (HUVECs), and Rat Brain Microvascular Endothelial Cells (RBMECs) Stimulated with vascular endothelial growth factor (VEGF)
RGMa mRNA (Figures 1G–I) and protein levels (Figures 1J–L) were significantly increased in endothelial cell (EC) after the addition of VEGF for 40 min. These findings indicate that RGMa may be a negative regulator in angiogenesis

Summary

Introduction

Acute ischemic stroke is a leading cause of severe cognitive impairment, physical disability, and mortality worldwide. RGMa Inhibits Angiogenesis reason, novel effective approaches for stroke therapy need to be explored. Repulsive guidance molecule a (RGMa) is a key regulator of many cell processes, including neural guidance [5], cell differentiation, migration, and adhesion [6]. We previously found that RGMa was significantly increased and induced growth-cone collapse in an MCAO/reperfusion rat model [10]. Our previous study demonstrated that RGMa induced growth-cone collapse via activation of the ROCK/CRMP-2 and GSK-3β/CRMP-2 pathways [10]. Yamashita et al reported that RGMa suppressed angiogenesis of human umbilical artery endothelial cells (HUAECs) in vitro and in a Matrigel plug model in vivo [19]. The present study aimed to explore the role and mechanisms of RGMa in angiogenesis

Methods

Results

Conclusion