| Background & Objective:Coronary heart disease seriously endanger human health, novel therapies have beendeveloped recently to stimulate collateral vessel formation and to improve muscles function.Microvascular angiogensis and coronary collateral vessels developed to ameliorate thefunction of the damaged heart.Integrins are important cell surface adhesion molecules,it mediates cells andcell-mediated extracellular matrix interactions and plays an important role in a series ofbiological activities such as cell proliferation, differentiation and cell migration. Fourtransmembrane superfamily (TM4SF) is the integrin-associated protein of. Extracellularsignal to intracellular signal transduction is mostly through the model ofintegrin-Tetraspanin-PKC complex.Our group launched a number of researches in CD151 in angiogenesis:①Inapplication of Matrigel model,we found that CD151 was observed in the maintenance ofcell morphology and in the role of vitro angiogenesis and when given the anti-CD151antibody and anti-α6β1, it was inhibited significantly;②CD151 recombinantadeno-associated virus transfection of human umbilical vein endothelial cells, cellproliferation, migration and tube-like structure formation was significantly enhanced;③CD151 transfected rat ischemic hind limb function, microvascular proliferation andformation of collateral circulation were significantly stronger than non-transfected rat;④in the rat model of myocardial infarction CD151 transfected with recombinant adeno-associated virus, myocardial blood capillary density increased significantly, theindicators of hemodynamics of left ventricular peak systolic pressure (LVPSP) andmaximum left ventricular pressure rate of rise and fall (±dp/dtmax) were markedlyimproved.Research tells us that CD 151 can improve myocardial blood supply and heart functionin small animal model, then in the big animal model whether CD151 plays to improvemyocardial blood supply and heart function effectivly? Studies found that cardiac functionin preservation needs the absolute number of cardiomyocyte, cardiomyocyte apoptosisoccurred in non-infarct area is the main reason for the deterioration of cardiac function,transmembrane 4 superfamily members CD53 and CD9 have been confirmed to play animportant role in apoptosis, then whether CD151 inhibited apoptosis of myocardial cells,thereby improving cardiac function? This experiment, we build CD151 recombinantadeno-associated virus, then it is transfected into myocardial infarction model in miniatureswine, CD151 expression was observed. We also observe the impact of CD151 onmyocardial perfusion, cardiac function and cardiac apoptosis.Methods and results:pAAV-CD151 and pAAV-antiCD151 were constructed, rAAV were produced bytransfections of 293 cells, the titer of virus was determined by Nothern blot.The acute myocardial infarction (AMI) model was established by ligation of the leftanterior coronary artery in 1-month aged minipigs. The surviving minpigs were groupedrandomly as Control (n=4), GFP group (n=6), CD151 group (n=6) and antiCD151 group(n=6). rAAV-GFP, rAAV-CD151, and rAAV-antiCD 151 virus were delivered by directinjection in myocardium.without operated group was taken as non-infarction control. Eightweeks after AMI,the following endpoints were measured: 1. CD151 mRNA was detectedusing RT-PCR; 2. Western blot analysis for CD151 was performed; 3. The formation ofcollateral circulation was evaluated by coronary arteriography; 4. 13N-NH3 PET/CT toevaluate blood perfusion and compute the area of infarct; 5. Minipigs heart function andleftventricular remodeling were assessed by echocardiographic evalutian; 6. Cardiomyocytesapoptosis was examined with TUNEL assay; 7. The expressions of bax, and bcl-2 geneswere examined by reverse transcription polymerase chain reaction and the expressions ofbax, and bcl-2 proteins were examined by western blot. 1. The acute myocardial infarction(AMI) model by ligation of the left anteriorcoronary artery in minipigs were established successfully. 2. Our RT-PCR showed thattransfection with rAAV-CD151 could apparently lead to CD151 overexpression in minipigmyocardium; immunblot analysis showed that in comparion with the Control (OD value1.11±0.10) and antiCD151 (OD value 0.73±0.21), the expression of CD151 group(ODvalue 2.38±0.12) was significantly incresed (P＜0.05); 3. Coronary angiography showedthat 8 weeks after myocardial infarction, there forms a small number of collateral vessels,in GFP group the level of collateral vessels varies from 0 to 3 in CD151 group, but there isnearly no collateral vessels newly formed in antiCD 151 group; 4.13N-NH3 PET myocardialperfusion showed that myocardium blood flow in CD151 transfection group after 8 weeksafter has been significantly improved, while defect size is decreased significantly (P＜0.05)comparing to the GFP group; the defect area in the antiCD151 Group is significantly, larger(P＜0.05) comparing to the CD151 group. The percentage of defect 8 weeks after theregional area of the total imaging is GFP group (21.13±3.6)%, CD151 group (15.34±3.3)%, antiCD151 (35.45±5.7)%; 5. Echocardiography showed that, contractile functionfactors: EF, FS,△T,△IVST in CD151 group were significantly higher than GFP groupand lower than Control group(P＜0.05), ALWTd, 1VSTd in CD151 group was less thickercompared with the control group(P＞0.05); 6. TUNEL-positive staining of cardiomyocyteswas significantly reduced in the group of swine receiving rAAV-CD151 compared with thegroup receiving rAAV-GFP or rAAV-antiCD151, whereas there shows no statisticallysignificant difference between the CD151 and control groups; 7. CD151 gene deliverysignificantly reduced the ratio of of pro-apoptotic Bax gene versus anti-apoptotic Bcl-2gene compared with the GFP and antiCD151 groups (CD151, 0.41±0.06 vs GFP, 2.33±0.03;antiCD151, 4.01±0.02). The ratio between the CD151 and Control groups shows nostatistically significant difference (CD151, 0.41±0.06; Control, 0.39±0.04), immunblotanalysis showed that in comparion with the GFP (OD value 3.21±0.12) andantiCD151(OD value 3.53±0.09) groups, the ratio of bax and bcl-2 proteins in groups ofCD151 group (OD value 1.13±0.11) and Control (OD value 0.93±0.04) wassignificantly decresed (P＜0.05), with no significant difference between CD151 andControl groups. Conclusions:1. rAAV-CD151 mediated CD151 gene delivery induced an efficient and stableexpression in minipig myocardium.2. CD151 upregulation stimulates improves blood perfusion and reduces the size ofinfarct area in a minipig myocardial models.3. CD151 blocks the post-infarction deterioration of cardiac functions and preventscardiomyocytes from apoptosis, this may due to angiogenesis and to the decrease ofbax/bcl-2 mRNA ratio and the expression of bax and bcl-2 proteins. |
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