Acute Microvascular Action of Vascular Endothelial Growth Factor in Skeletal Muscle Ischemia/Reperfusion Injury

Abstract

The purpose of this study was to investigate the acute action of vascular endothelial growth factor (VEGF) in the microcirculation of skeletal muscle subject to ischemia/reperfusion in vivo and to determine the role of nitric oxide synthase in VEGF-induced microvascular protection. A vascular pedicle isolated rat cremaster muscle model coupled with local intraarterial infusion technique was used. Each muscle underwent 4 hours of zero-flow warm ischemia followed by 2 hours of reperfusion. Femoral artery cannulation was performed before reperfusion. The infusate was administered by continuous infusion into the arterial tree of the muscle beginning at 1 minute before reperfusion and at the rate of 0.1 ml/hour throughout the entire reperfusion period. Three groups were designed: (1) the ischemia/reperfusion group, with infusion normal saline; (2) the VEGF plus ische-mia/reperfusion group, with infusion of recombinant human VEGF165 protein; and (3) the L-NA plus VEGF plus ischemia/reperfusion group, with infusion of N-nitro-L-arginine (L-NA; a nonselective nitric oxide synthase antagonist) mixed with VEGF165 protein. After 2 hours of reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion, and endothelium-dependent and endothelium-independent vasodilatation were performed. The muscle was harvested and processed for reverse-transcriptase polymerase chain reaction for measuring eNOS and endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) gene expression. Reperfusion caused significant microvascular alterations including vasoconstriction, poor capillary perfusion, and endothelial dysfunction in the skeletal muscle. These alterations were significantly attenuated by intraarterial infusion of VEGF during reperfusion, but the beneficial effect of VEGF was reduced significantly by coadministration of L-NA. Reverse-transcriptase polymerase chain reaction study revealed that ischemia/reperfusion depressed eNOS mRNA expression but enhanced iNOS mRNA expression. Intraarterial infusion of VEGF during reperfusion amplified mRNA expression of eNOS but not of iNOS. Local intraarterial infusion of VEGF produced significant microvascular protection from skeletal muscle ischemia/reperfusion injury. The VEGF-induced enhancement of eNOS may play an important mechanistic role.