| Background: The tissue repair after myocardial infarction (MI) is the determinativefactorof ventricular remodeling. The recent studies showed that Phenytoin (PHT)through mediated paracrine effect can accelerate wound healing. Our lab research hadalready conformed that Phenytoin (PHT) could accelerate tissue repair after MI, andthe possible mechanism may be that PHT can induce paracrine effect throughactivation of macrophage. Macrophage is the important resource of various cytokinesand growth factors. The implantation of macrophage early after MI can enhanceneovascularization, tissue repair, and improve ventricular remodeling and cardiacfunction. But the mechanism of the role of PHT and macrophage mediated paracrineeffect in tissue repair after MI is unclear.Objective: experimental ischemia-reperfusion (I/R) in Wistar rats was induced bycoronary ligation and reperfusion. After early MI, The animals were respectivelyreceived intramyocardial injection of peritoneal macrophages, and PHT-prestimulatingperitoneal macrophages, or peritoneal injection of PHT 14 days after I/R. In order toinvestigate the role and mechanism of macrophage and PHT mediated paracrine effectin the tissue repair and ventricular remodeling, we observe the changes of thehistopathology, molecular biology and hemodynamics by above influencing factors.Method: In the experiment in vitro, the peritoneal macrophages were adherentlycultured, and parts of cultured macrophages were prestimulated by PHT, finally all ofmacrophages were labeled by fluorescent dye DAPI before implantation. In theexperiment in vivo, the survival animals induced experimental I/R were randomlydistributed into Sham group (n=12), control group (AMI group, n=23), Phenytoingroup (PHT group, n=17), macrophage group (MΦgroup, n=20), phenytoin-prestimulating macrophage group (MΦ-PHT group, n=16). Animals in MΦgroup andMΦ-PHT group were respectively received macrophages or PHT-prestimulating macrophages (1×105, 100μL) by intramyocardial injection within the central andperi-ischemia area on the reperfusion, while AMI and PHT group received incompleteDMEM (100μL). In addition, animals in PHT group were intraperitoneallyadministrated by PHT 75mg/kg per day. At 7 days after the induction of I/R,Histology examination were analyzed including collagen volume fraction (CVF),index of expansion, infarct size and so on. Proportion of collagen fiber was identifiedby picrosirius staining plus polarized microscopy. Arterioles density and capillarydensity in infarct region were respectively evaluated by anti-α-SMA and anti-vWFimmunofluorescent staining. Cardiocyte cross-sectional area (CSA) in non-infarctregion was assessed by anti-WGA immunofluorescent staining. The levels of proteinexpression of vascular endothelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), matrix metalloproteinase-2 (MMP-2), tissue inhibitor ofmetalloproteinase-1 (TIMP-1), tissue inhibitor of metaltoproteinase-2 (TIMP-2)within infarct region 7 days after MI were determined by Western blot. Thehemodynamic measurement were performed before the remaining rats were sacrificed28 days after MI. histopathogic analysis 28 days after MI were similar to 7 days afterMI.Result: 1. in vitro pure macrophages and PHT-stimulating macrophages were bothadherent in the culture stably, and could express CD68 antigen. By DAPI staining, thenuclear of macrophage were labeled successfully.2. in vivo experiment: 1) DAPI-labeled macrophage were located within the infarct region at 1 day after MI. 2)Compared to AMI group, LV+dp/dtmax in MΦ-PHT group was significantly increasedat 28th day after MI. LV+dp/dtmax in MΦ-PHT group was more than MΦgroup at 28thday after MI. 3) The index that ratio of ventricle and body weight (V/BW), ratio ofleft ventricle and body weight (LV/BW) and ratio of right ventricle and body weight(RV/BW) were not significantly different between each group. 4) Compared with AMIgroup, Index of Expansion at 7th day and 28th day were all decreased in PHT group, MΦgroup and MΦ-PHT group; Infarct Size of MΦ-PHT group at 28th day was fewerthan AMI group. Compared with AMI group, Infarct Size at 7th day and 28th day inPHT group had down tendency. Compared with MΦgroup, Infarct Size at.7th day and28th day in MΦ-PHT group had down tendency. Fibrosis area in AMI group at 7th daywas more than in MΦgroup and MΦ-PHT group. Fibrosis area in AMI group at 28thday was also more than in PHT group, MΦgroup and MΦ-PHT group. Comparedwith MΦgroup, Fibrosis area at 28th day in MΦ-PHT group was decreased. Comparedwith AMI group, CSA within non-infarct region at 7th day were reduced in MΦgroupand MΦ-PHT group, while PHT group had down tendency. CSA within non-infarctregion at 28th day in PHT group, MΦgroup and MΦ-PHT group were smaller thanAMI group. 5) Compared with AMI group, CVF in infarct zone at 7th day and 28th daywere larger in PHT group, MΦgroup and MΦ-PHT group. CVF in infarct zone at 7thday in MΦ-PHT group was larger than in MΦgroup. Compared with AMI group,CVF in non-infarct zone at 7th day and 28th day were both smaller in MΦgroup andMΦ-PHT group. 6) Compared with AMI group, Proportion of Collagen Fiber ininfarct zone at 7th day and 28th day had increased in PHT group, MΦgroup andMΦ-PHT group. Proportion of Collagen Fiber in infarct zone at 7th day and 28th day inMΦ-PHT group were higher than MΦgroup. 7) Compared with AMI group, arterioledensity in infarct zone at 7th day and 28th day had increased in PHT group, MΦgroupand MΦ-PHT group. Compared with MΦgroup, MΦ-PHT group at 7th day and 28thday had increasing tendency. 8) Compared with AMI group, capillary density ininfarct zone at 7th day and 28th day had increasing tendency in PHT group. Comparedwith MΦgroup, MΦ-PHT group at 7th day and 28th day also had increasing tendency.9) Compared with AMI group, the levels of bFGF, MMP-2, TIMP-1 and TIMP-2protein expression in infarct zone at 7th day were significantly increased in MΦ-PHTgroup. MMP-2 protein expression in infarct zone at 7th day in PHT group and MΦgroup were fewer than in AMI group. Compared with MΦgroup, the levels of VEGF,bFGF, MMP-2, TIMP-1 and TIMP-2 protein expression in infarct zone at 7th day were significantly increased in MΦ-PHT group. The ratio of MMP-2/TIMP-2 in AMI groupwas more than in Sham group.Conclusion: 1. Phenytoin through mediated paracrine effect can accelerate collagendeposition, collagen maturation, and promote neovascularization, cardiac repair,therefore can improve ventricular remodeling after MI. 2. Macrophage ofintramyocardial implantation through the above mechanism can accelerate tissuerepair and improve ventricular remodeling. 3. Phenytoin-prestimulating macrophagethrough mediated paracrine effect can significantly generate synergistic effect oncardiac repair after MI, and the mechanism may be the up-regulation of growth factorsand key enzymes involved in tissue repair through the paracrine effect. For this reason,phenytoin could accelerate tissue repair and improve ventricular remodeling and leftventricular systolic function more effectively than pure macrophage implantation.
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