| Background: Myocardial infarction(MI) results in large-scale loss of cardiomyocytes and left ventricular(LV) remodeling, which is a major factor in the progression of heart failure. Cell-based cardiac repair offers the promise of rebuilding the injured heart. Both experimental studies and clinical trials on MI have shown the improvement in cardiac function by stem cells therapy. The mechanism underlying this therapeutic effect is considered to be the stem cells mediated paracrine effect. Phenytoin(PHT) is a new category of wound healing agent in addition to its antiepileptic activity. In vitro studies implied that PHT could accelerate the autocrine and paracrine activity of growth factors by up-regulating the related receptors, i. e. the paracrine effect of PHT. The recent study by Zhou et al. tested the effect of PHT on the early phase of post-infarction ventricular remodeling in rat and showed that PHT could accelerate the healing process in the infarcted region by stimulating collagen accumulation. However the underlying mechanisms are not fully understood.Objective: Experimental MI was induced in rat by 60-min coronary ligation and reperfusion. The survival animals received intramyocardial injection of DAPI-labeled MSCs, intraperitoneal injection of PHT or the combining application of both. By observing the changes of neovascularization, LV remodeling as well as evaluating cardiac function, we sought to explore the combining effect of MSCs and PHT on cardiac repair and the relationships between MSCs mediated and PHT mediated paracrine effect.Methods: In in vitro experiments, mesenchymal stem cells(MSCs) were isolated from bone marrow and cultured based on plastic adherence. MSCs frorn passage 3 or 4 were labeled with DAPI before transplantation. In the second series of experiments following coronary ligation and reperfusion in 56 Wistar rat, periinfarct area of left ventricles were randomly injected with DMEM/F12(control and PHT groups) or MSCs (MSCs and MSCs+PHT groups). PHT was injected intraperitoneally 75mg/kg/day for consecutive 14 days after MI. Histological examination was performed by picrosirius staining plus polarized microscopy for collagen analysis.Capillary density and arterioles density were assessed within infarct and periinfarct area by vWF andα-SMA fluorescent staining, respectively. The protein levels of VEGF, bFGF, MMP-2 and TIMP-1 within infarct area were determined by western blot 7 days after MI. Hemodynamic measurements were performed to assess heart function 28 days after MI.Results: 1) DAPI-labeled MSCs were mainly located within periinfarct and infarct area. Some MSCs were positive for vWF andα-SMA, suggesting MSCs developing endothelial cell and smooth muscle cell phenotypes. 2) Compared to control group, PHT group had smaller infarct size and LV dimension as well as increased wall thickness of infarct area 28 days after MI(P＜0.05), much more significant results were observed in MSCs group and MSCs+PHT group. Cardiomyocyte cross sectional area (CSA) of interventricular septum (IVS) was significantly reduced in PHT group, MSCs group and MSCs+PHT group versus control group (430.72+70.59μm~2, 438.74±44.15μm~2, 429.44±58.50μm~2, respectively vs. 518.25±51.55μm~2). 3) PHT group had less collagen content within infarct area versus control group (56.20±8.61％vs. 71.40±6.44％, P＜0.05), but the proportion of thick collagen fibers, i.e. mature collagen fibers was similar to that in control group 28 days after MI. MSCs group and MSCs+PHT group not only had less collagen content(51.064±5.88％and 49.39±2.29％, respectively, P＜0.05) but also less mature collagen fibers within infarct area, compared with control group. Collagen content within IVS was less in PHT group, MSCs group and MSCs+PHT group, compared with control group 28 days after MI (P＜0.05). 4) MSCs group and MSCs+PHT group increased the density of capillary and arterioles within periinfarct area both 7 days and 28 days after MI, meanwhile PHT group had higher vessel density than in control group only 28 days after MI. Capillary density in MSCs+PHT group was even higher than in PHT group both 7days and 28 days after MI(160.00±34.16/mm~2 vs. 110.00±23.66/mm~2 on 7 days, 100.00±19.15/mm~2 vs. 71.67±16.02/mm~2 on 28 days, P＜0.05). 5) PHT group, MSCs group and MSCs+PHT group had similar protein levels of VEGF, bFGF and MMP-2 to control group within infarct area 7 days after MI. However, PHT group reduced protein level of TIMP-1.6) Hemodynamic measurements showed no significant differences among control group, PHT group, MSCs group or MSCs+PHT group.Conclusions: 1) MSCs transplantation can accelerate infarct healing, angiogenesis, vasculogenesis and improve LV remodeling through paracrine effect. However, the maturation process of newly formed collagen within infarct area is markedly inhibited. 2) PHT can also accelerate infarct healing, angiogenesis and attenuate LV remodeling through paracrine mechanism. Yet the effect is inferior to that by cell transplantation. 3) PHT promotes the deposition and maturation of newly formed collagen within infarct area without significant effect on collagen metabolism in non-infarct area, which is also attributable to paracrine activity. 4) The combining effect of MSCs and PHT includes reinforced neovascularization as well as healing acceleration and LV remodeling attenuation, signifying the synergism of combining application. 5) The combining application of MSCs and PHT remarkably inhibits the maturation of newly formed collagen within infarct area, indicating the antagonism between MSCs and PHT.
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