Shock Wave Enhances Angiogenesis through VEGFR2 Activation and Recycling.

Tien-Hung Huang,Tsung-Cheng Yin,Cheuk-Kwan Sun,Mel S Lee,Yi-Ling Chen,Hon-Kan Yip,Ching-Jen Wang

doi:10.2119/molmed.2016.00108

Abstract

Although low-energy shock wave (SW) is adopted to treat ischemic diseases because of its pro-angiogenic properties, the underlying mechanism remains unclear. This study aimed at testing whether SW-induced angiogenesis may be through endothelial vascular endothelial growth factor receptor 2 (VEGFR2) signaling and trafficking. Phosphorylation of VEGFR2-Akt-eNOS axis and production of nitric oxide (NO) were determined in human umbilical vein endothelial cells (HUVECs) treated with SW. Carotid artery in ob/ob mice was treated with SW before evaluation with sprouting assay. Critical limb ischemia was induced in ob/ob mice to evaluate blood flow recovery after SW treatment. Tube formation and migration assays were also performed with/without SW treatment in the presence/absence of SU5416 (VEGFR2 kinase inhibitor) and siRNA-driven silencing of VEGFR2. Chloroquine was used for disrupting endosome, and Rab11a controlling slow endocytic recycling was silenced with siRNA in vitro. Following SW treatment, augmented ligand-independent phosphorylation in VEGFR2-Akt-eNOS axis and endogenous NO production, increased cellular migration and tube formation, elevated sprouting of carotid artery and blood flow in ischemic limb in ob/ob mice were noted. Moreover, SU5416 and VEGFR2 silencing both inhibited SW-induced angiogenesis. SW-induced angiogenesis, which was accompanied by increased VEGFR2 protein expression without transcriptional change, was suppressed by chloroquine and Rab11a silencing. We concluded that SW enhanced angiogenesis via ligand-independent activation of VEGFR2 and further prolonged through endosome-to-plasma membrane recycling in endothelial cells.

Highlights

Shock wave (SW) therapy is a noninvasive mechanical treatment strategy that has been applied clinically for decades
In the processes of stimulation generated by blood flow-induced shear stress, vascular endothelial growth factor receptor 2 (VEGFR2) acts as a mechanotransducer to transmit activated signal through ligand-independent phosphorylation, thereby leading to vasodilation via endothelial nitric oxide synthase activation [12] and plays a predominate role in VEGFR2-induced angiogenesis [13]
The number of nitric oxide-converted fluorescent cells in the shock wave (SW) group was significantly higher than that in the control group (P = 0.0025) (Figure 1E). These results indicated that ligand-independent activation of VEGFR2-Akt-endothelial nitric oxide synthase (eNOS) axis, which plays a predominant role in angiogenesis, was revealed in human umbilical vein endothelial cells (HUVECs) post SW treatment

Summary

Introduction

Shock wave (SW) therapy is a noninvasive mechanical treatment strategy that has been applied clinically for decades. In the processes of stimulation generated by blood flow-induced shear stress, vascular endothelial growth factor receptor 2 (VEGFR2) acts as a mechanotransducer to transmit activated signal through ligand-independent phosphorylation, thereby leading to vasodilation via endothelial nitric oxide synthase (eNOS) activation [12] and plays a predominate role in VEGFR2-induced angiogenesis [13]. Similar to laminar shear stress in endothelial cells, SW has been reported to lead to RESEARCH ARTICLE mechanosensory complex formation and Akt/eNOS phosphorylation [14]. VEGFR2 undergoes constitutive endosome-to-plasma membrane recycling in endothelial cells [15]. Receptor traffic has been reported to be affected by external environmental factors including infection, aging and degenerative diseases [17] as well as stimulation by fluid shear stress in renal proximal tubular cells [18]. Whether SWinduced angiogenesis is associated with VEGFR2 recycling is still unknown

Objectives

Methods

Results

Conclusion