Perlecan Domain V Induces VEGf Secretion in Brain Endothelial Cells through Integrin α5β1 and ERK-Dependent Signaling Pathways

Douglas N Clarke,Lisa Auckland,Christi Parham,Andrezj Fertala,Jill Roberts,Michael Kahle,Courtney S Shaw,Boyeon Lee,Abraham Al Ahmad,Gregory J Bix

doi:10.1371/journal.pone.0045257

Abstract

Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α5β1 integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV’s angio-modulatory activity outside of the brain, binds poorly to α5β1 and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV’s DGR sequence as an important element for the interaction of DV with α5β1. Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV’s induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV’s mechanism of action on BECs, and further support its potential as a novel stroke therapy.

Highlights

Stroke is the leading cause of long term disability and a major cause of death within the United States, with an average fatality rate slightly over 134,000 deaths/year and an overall cost of over $7 billion/year [1]
Domain V (DV) Binding to a5b1 Integrin is Partially Mediated by its DGR Sequence
Because we observed a significant difference in our Kd values of WT DV and D3904A DV, we asked whether this mutation had any functional significance in our proliferation assays

Summary

Introduction

Stroke is the leading cause of long term disability and a major cause of death within the United States, with an average fatality rate slightly over 134,000 deaths/year and an overall cost of over $7 billion/year [1]. There is rapid proteolysis of the extracellular matrix (ECM) as well as dramatic changes in the expression of ECM receptors, cell-bound integrins, in the infarct core and ischemic penumbra regions [3,4,5]. Within this context, we hypothesized that the brain ECM may play a role in poststroke brain repair. Domain V (DV), the C-terminal fragment of perlecan, has anti-angiogenic activity outside of the brain following cleavage from perlecan, and is referred to as endorepellin [9,10]. LG3, the 24 kDa C-terminal portion of DV, has been reported to be responsible for DV’s anti-angiogenic activity [11]

Methods

Results

Conclusion