Gene transfer of CGRP enhanced angiogenesis in rat ischemic hindlimb

【Author】

ZHENG Shuai,LI Wen-Jing,BAI Xue,XU Ming-Jiang,WANG Xian Department of Physiology and Pathophysiology,School of Basic Medical Sciences,Peking University Health Science Center,Beijing 100083,China

【Abstract】

<正>Calcitonin gene-related peptide(CGRP),a 37-amino acid peptide,exists in the end of peripheral sensory nerves.Previous studies from our and others have shown that CGRP prevents cells from ischemia injury of heart and kidney by mediating ischemia preconditioning,but its effect on angiogenesis remains elusive.In this study we sought to explore CGRP effects on angiogenesis in rat ischemic hindlimb.For in vivo study,hindlimb ischemia model of male SD rats was prepared,and adenovirus vectors encoding CGRP(Adv-CGRP)or GFP were injected into the ischemic hindlimb muscle.The laser doppler perfusion scanner(LDPI)was used to monitor the limbs blood perfusion at day 1,day 3,day 7,day 14,day 21 and day 28 after CGRP gene transfer.The human umbilical vein endothelial cells(HUVECs)were isolated and cultured,and P3-P5 generations were used for scratching assay and matrigel assay under CGRP treatment in vitro.LDPI determination showed that the Adv-CGRP injected rats significantly increased blood perfusion recovery at day 14,day 21 and day 28(0.5358±0.0528 vs 0.6801±0.0336, 0.7089±0.0553 vs 0.8834±0.0449 and 0.7221±0.0392 vs 0.8861±0.0310,respectively,P<0.05,n=10).Scratching assay showed that CGRP(100 nmol/L,6 h)enhanced HUVECs migration(n=4,P<0.05).CGRP at 1-100 nmol/L for 2.5 h also significantly increased the numbers of tube-like structure for about 1.3,1.4 and 1.5 folds vs control on matrigel(n=3,P<0.05).The Western blot results revealed that CGRP 100 nmol/L promoted AMPK activation for about 1.5 folds in HUVECs.CGRP-enhanced tube-like structure formation could be inhibited by AMPK inhibitor, compound C.AICAR,AMPK agonist,had similar effect as CGRP.These results indicate that CGRP facilitates angiogenesis in rat ischemic hindlimb probably through promoting endothelial cells migration and tube-like structure by,at least in part,AMPK activation-dependent mechanism.

【Keywords】

CGRP;;angiogenesis;;hindlimb ischemia;;AMPK

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