Effect Of CD151–AAA ^194-196 In A Rat Ischemia Hind-limb And The Mechanism Involved

ObjectiveAngiogenesis, the outgrowth of new microvascular structures from the preexisting vasculature, contribute to the pathogenesis of many disorders, including ischemic diseases and cancer . Essential mechanisms for angiogenesis remains controversial, that integrins play important role in angiogenesis may be acceptability. Integrins are heterodimeric adhesion receptors for extracellular matrix proteins, consisting of anα-subunit and aβ-subunit. The integrinsα3β1 andα6β1 specifically bind to laminins, it form complexes with tetraspanins.CD151, a tetraspanin protein family member, contains two extracellular loops and two short cytoplasmic tails, it is linked closely withα3 /α6 integrins through the site-specific QRD^194-196 in large extracellular loop.The formation of CD151-α3 /α6 Integrins complex modulate cell migration, cell morphology, cell adhesion strengthening and may contribute to cell signaling. CD151 has been shown to stimulate the development of collateral vessels in animal models of hind lamb and myocardial ischemia . Previous study show antibodies to CD151 inhibit the motility of endothelial cells, similar effect was also observed with antibodies toα3 integrin. Based on the above findings, we hypothesize that cutting off the links between CD151 and laminin-binding integrins (α3β1,α6β1) could inhibit angiogenesis. In this study we construct CD151–QRD194–196→AAA mutant, then use rAAV for delivery of the human CD151 gene and it's mutant into rat muscle tissues. By observing the role of them in a rat hind-limb ischemia model and investigating signaling pathways, we can better explore the molecular mechanisms of CD151-induced angiogenesis. FAK-MAPKs, FAK-PI3K and Rac/Cdc42 pathways will be evaluated.Methods1. pAAV-GFP,pAAV-CD151 and pAAV-CD151–QRD194–196→AAA were constructed. The Recombinant adeno-associated virus rAAV–GFP,rAAV-CD151 and rAAV-CD151-AAA^194-196 were prepared using a triple-plasmid co-transfection method in 293 cell lines. The titer of rAAV was determined by the quantitative DNA dot-blot hybridization.2. Male Wistar adult rats weighing 200-250 g were randomly divided into 5 groups of 6 rats each, saline, rAAV-GFP, rAAV-CD151, and rAAV-CD151–AAA^194-196 (1.5×1011 pfu in 1 mL of phosphate buffered saline) were injected (100μL/site/rat) into 4 major muscle sites: adductor (3 sites), quadriceps (3 sites), semimembranous (2 sites), and gastrocnemius (2 sites). After 1 week of transduction, the rat hind-limb ischemia model was established as previously described. Briefly, the rats were anesthetized as previously detailed, and an incision was made extending inferiorly from the left inguinal ligament to a point just proximal to the knee. The femoral artery was then completely excised from its proximal origin to the point where it bifurcates into the saphenous and popliteal arteries. Sham-operated animals received an equivalent volume of sterile saline and underwent the same surgical procedure without Femoral artery resection. 5 weeks after viral administration, The skin temperature of the hind-limb was measured and the capillary density of the ischemic muscle was observed by anti-CD34 antibody immunochemical staining. The expression of CDl 51 in the ischemic tissue was detected by Western blot and immunohistochemistry. In addition, Western blot analysis for FAK,PI3K,Akt,eNOS,ERK and Rac1/Cdc42 was performed.Results1. Successfully construct pAAV-CD151-AAA^194-196 mutant. The titer of virus was determined by Northern blot.2. The expression of CD151 and CD151 mutant protein were increased significantly in rAAV-CD151 and rAAV-CD151–AAA194–196 groups compared with other groups. There were no significant difference of the expression between the rAAV-CD151 and rAAV-CD151–AAA194–196 groups. But the average skin temperature and the capillary density in rAAV-CD151 group were higher than those in rAAV-CD151–AAA194–196 group. There were no significant differences in the saline, rAAV-GFP and rAAV-CD151–AAA194–196 groups, the densities of capillaries and average skin temperature were similar in the 3 groups. In addition, CD151 could activate the FAK-MAPKs, FAK-PI3K and Rac1/Cdc42 pathways. However, expression of CD151–AAA194–196 could not have the above results.Conclusion1. Successfully construct rAAV-CD151-AAA^194-196.2. This study suggested that CD151 is linked withα3/α6 integrins to form a functional complex and activates FAK-MAPKs, FAK-PI3K and Rac1/Cdc42 signaling pathways, then promotes angiogenesis. The formation of CD151-α3/α6 integrins complex is the Starting mechanisms of CD151-induced angiogenesis.3. Our study suggested that CD151 associated withα3/α6 integrins through the site-specific QRD^194-196 in large extracellular loop.