A Role Of Matrix Elasticity In Defining Optimal Time Frame Of Bone Marrow Mononuclear Cells Transplantation For Acute Myocardial Infarction

PREFACEDetermining which time point is optimal for bone marrow-derived cells transplantation for acute myocardial infarction(AMI) has attracted a great deal of attention.Studies have verified the interaction between cell treatment effect and transfer timing and have suggested that the optimal time frame for bone marrow-derived cells therapy might be about one week after AMI.However,the potential mechanism underlying the time-dependent therapeutic response remains unclear.Recently,a growing body of in vitro evidence has suggested that stem cells are able to feel and respond to the stiffness of their microenvironment to commit to a relevant lineage,indicating that soft matrices that mimic brain are neurogenic,stiffer matrices that mimic muscle are myogenic and comparatively rigid matrices that mimic collagenous bone prove osteogenic.Simultaneously,considering the fact that the myocardium post-infarction experiences a time-dependent stiffness change from flexible to rigid as a result of myocardial remodelling following tissue necrosis and massive extracellular matrix deposition,we presume that the myocardial stiffness within a certain time frame (possibly one week post-AMI) after infarction might provide a more favourable physical microenvironment for the phenotypic plasticity and functional specification of engrafted bone marrow-derived cells committed to some cell lineages,such as endothelial cells,vascular smooth muscle cells or cardiomyocytes.The beneficial effect facilitates angiogenesis and myocardiogenesis in the infarcted heart,and subsequently leads to more amelioration of cardiac functions.The present study aimed to verify the scientific hypothesis.If the hypothesis were true,it would be of great help to understand the mechanism underlying the optimal timing for bone marrow-derived cells transplantation and to establish a direction for the time selection of cell therapy.PART ONE Impact of Timing on Efficacy and Safety of Bone Marrow Mononuclear Cells Transplantation in Acute Myocardial Infarction: A Pooled Subgroup Analysis of Randomised Controlled TrialsObjectives To investigate the impact of timing on efficacy and safety of bone marrow mononuclear cells(BMMNCs) transfer in patients with acute myocardial infarction (AMI) by a pooled subgroup analysis of randomised controlled trials.MethodsA systematic literature search of PubMed,MEDLINE and Cochrane EBM databases was made on randomised controlled trials with at least 3-month follow-up data for patients with AMI undergoing emergent percutaneous coronary intervention(PCI) and receiving BMMNCs transfer thereafter.ResultsA total of 7 trials with 660 patients were available for analysis.Compared to baseline level,BMMNCs transfer at day 4 to 7 post AMI significantly improved left ventricular ejection fraction(LVEF)(4.63%increase,95%confidence interval[CI] 1.00%to 8.26%,p=0.01),reduced left ventricular end-systolic volumes(LVESV) (95%CI—0.53 to—0.02,p=0.03),and decreased the incidences of revascularization (odd ratio[OR]=0.60,95%CI 0.37 to 0.97,p=0.04) and the cumulative clinical events of death or recurrent myocardial infarction(OR=0.32,95%CI 0.11 to 0.95,p =0.04),death,recurrent myocardial infarction,culprit artery restenosis or ventricular arrhythmia(OR=0.59,95%CI 0.36 to 0.96,p=0.03) while these improvements did not reach statistical significance in emergent transfer trials(within 24 hours post AMI).Compared with emergent transfer,BMMNCs therapy at day 4 to 7 also significantly reduced the incidence of revascularization(p for interaction=0.02).ConclusionsTiming of cell administration might play an important role in the therapeutic response and safety in AMI patients.BMMNCs transfer at day 4 to 7 post AMI was superior to that within 24 hours in improving LVEF,decreasing LVESV and reducing the incidence of revascularization.PART TWO Time Courses of Myocardial Elasticity,Serum Vascular Endothelial Growth Factor Concentrations and Cardiac Function in Mice with Experimental Myocardial Infarction ObjectivesTo investigate the time course of myocardial elasticity,serum vascular endothelial growth factor(VEGF) concentrations and infracted heart function after acute myocardial infarction(AMI) in mice,which makes it possible to take further analyses for the effects of them on the biologic behaviors of transferred cells.MethodForty BALB/c mice(6 weeks old,weight 20-25g) were made into murine AMI models by the ligation of left coronary artery and eight sham operations were regarded as the control.Echocardiography and pressure-volume conductance catheter technique were used to evaluate cardiac function and left ventricular pressure at 1 hour,24 hours, 7 days,14 days and 28 days post AMI,respectively.Thereafter serum VEGF concentrations were detected by ELISA technique.Elastic modulus of infarcted myocardium were detected by atomic force microscope,and hematoxylin and eosin and Mallary staining of paraffin section were performed to observe the time-dependent pathologic changes of infarcted heart.Comparisons of continuous variables between two groups were performed by one-way ANOVA.The criticalα-level for these analyses was set at p＜0.05.Data analyses were performed by SPSS 11.5 software.ResultsThe elastic modulus(E) of myocardium at 1 hour post infarction showed a decrease tendency compared to the normal myocardium(16.60 kPa vs.17.94 kPa,p＞0.05),and the tendency became statistically significant by hour 24 post AMI(E=4.21 kPa, p＜0.001).Thereafter the stiffness of infarcted myocardium gradually increased and had significant differences compared with the three former groups(day 7 post AMI: E=31.38 kPa;day 14:E=53.23 kPa;day 28:E=90.22 kPa;all p＜0.001).In contrast, serum VEGF expression significantly increased by 1 hour after myocardial infarction (49.44 pg/ml vs.38.58 pg/ml,p=0.024) and reached peak concentration at hour 24 (96.30 pg/ml),with statistically significant differences compared to other groups(all p＜0.01).Following the peaking,serum VEGF concentrations gradually decreased and reached normal level by day 14 post AMI(43.89 pg/ml,vs.the control,p＞0.10). Pathological tissue staining showed that,compared with other groups,the injured hearts at hour 24 post AMI had more significant inflammatory cells infiltration following massive myonecrosis.By day 7,the removal of the necrotic myocytes was paralleled by a reduction of inflammatory cells,with the start of immature fibrosis scar formation and complete scar formation by 28 days post infarction.These time-related changes in the above biological and physical parameters were coincidence with those in left ventricular function and pressure.LVEF,FS,and LVESP showed the lowest level at 24 hours post AMI among all the groups(LVEF: 0.50;FS:0.25;LVESP:99.30mmHg;all p＜0.01),meanwhile LVEDP reached the first peak value(12.35 mmHg vs.3.89 mmHg[control group],p＜0.01).Conclusion:Elastic modulus of mice myocardium,serum VEGF concentrations and cardiac function after AMI presents time-dependent changes and remains high consistency among them.Elastic modulus of infarcted myocardium experienced a time-dependent process of decreasing at first and then increasing.It reached the bottom at hour 24 post AMI,meanwhile,serum VEGF reached peak concentration and cardiac function remains the worst degrees at the moment.PART THREE The impacts of matrix elasticity and VEGF on differentiation of bone marrow mononuclear cells to endothelial progenitor cellsObjectivesTo investigated the impacts of matrix elasticity and VEGF on specification of bone marrow mononuclear cells(BMMNCs) along endothelial progenitor cells(EPC) by cells culture in the medium with different VEGF concentrations and matrix stiffness similar to the elasticity of infarcted myocardium at different stages of infarction.MethodsThe flexibility of polyacrylamide gel with different acrylamide/bisacrylamide ratios was determined by atomic force microscope to bolt the elastic matrix that could mimick the elastic modulus of infarcted myocardium of hour 1,hour 24,day 7,day 14 and day 28 post AMI.BMMNCs were isolated and cultured in the medium with the above elastic modulus and different VEGF concentrations(0ng/ml,2.5ng/ml,10ng/ml and 20ng/ml).Cellular immunophenotypes(CD133,VEGFR2,CD45 and UEA-1) and uptake of acetylated LDL(ac-LDL),which were specified to EPC,were detected by immunofluorescent technique and flow cytometry.The data based on same elasticity or VEGF concentration were analyzed using one-way ANOVA.The interactive effects of matrix elasticity and VEGF on cell differentiation were measured using two-way ANOVA.The criticalα-level for these analyses was set at p＜0.05. Data analyses were performed by SPSS 11.5 software.ResultsElastic modulus of the bolted gels was approximately 4kPa,15kPa,42kPa and 72 kPa that could mimick that of infarcted myocardium at hour 24,hour 1,day 7 to 14 and day 14 to 28 post AMI,respectively.Ratio of double positive cells with FITC-UEA-1/DiI-AcLDL had no significant differences among the above flexibility in the culture condition with 10ng/ml to 20ng/ml VEGF(all p＞0.05).With the decrease of VEGF concentrations to 2.5ng/ml in the culture medium,the difference in ratio of double positive cells started to be significant between 42kPa and 15 kPa (72.44%vs.52.44%,p=0.04),and the beneficial effects of 42kPa on promoting cell differentiation became more significant when VEGF concentration was decreased to 0ng/ml.Furthermore,the interactive effect of matrix elasticity and VEGF concentration on stimulating endothelial lineage differentiation was demonstrated by two-way ANOVA(p＜0.01).Regardless of the impacts of VEGF concentrations, 42kPa showed the more favorable effects,whether on absolute numbers or ratio of positive cells,than the others elasticity.Likewise,after controlling the impacts of matrix elasticity,VEGF concentration of 2.5ng/ml seems to be more beneficial both in numbers and in ratio of double positive cells than other concentrations.Moreover, flow cytometry analyses for cells cultured in the medium with 2.5ng/ml VEGF showed that,expression of specified cell surface antigens of EPC still remains the highest level in 42kPa group(CD45(-)/CD133(+)/VEGFR2(+):1.94%).ConclusionsMatrix elasticity plays an important role on promoting the specification of BMMNCs along EPC.Elastic modulus of 42 kPa,corresponding to of myocardial stiffness at day 7 to 14 post AMI,has a more beneficial effect on pro-differentiation compared with other elasticities.Moreover,an interactive effect on stimulating endothelial lineage differentiation might exist between matrix elasticity and VEGF concentration. PART FOUR Optimal Timing of Bone Marrow Mononuclear Cells Transplantation for Acute Myocardial Infarction in Mice and the Potential MechanismsObjectivesTo elucidate the optimal time frame for bone marrow mononuclear cells(BMMNCs) transplantation for acute myocardial infarction(AMI) in mice and the potential mechanisms by detecting capillary density in cell transplantation area and cardiac function after cell injection at different time points post AMI.MethodsBMMNCs were isolated from BALB/c mice by density gradient centrifugation.AMI animal models were prepared by coronary artery ligation of BALB/c mice.Isolated BMMNCs were directly injected into infarct area at hour 1,hour 24,day 7,day 14 and day 28 post AMI.Injection of M199 medium at corresponding time points was regarded as the control.Two months postinfarct,cardiac function and left ventrieular pressure were measured by echocardiography and pressure-volume conductance catheter technique.Meanwhile,capillary density in the injected area was detected by observing the expression of vWF using immunohistochemical method.Comparisons of continuous variables between cell transfer groups and control groups were performed by independent t test,and comparisons of absolute changes in related parameters between two groups by one-way ANOVA.The criticalα-level for these analyses was set at p＜0.05.Data analyses were performed by SPSS 11.5 software.ResultsCapillary density within cell transplantation area in hour 24,day 7 and day 14 injection group was more than the control(control groups:1.32/HP;hour 24,day 7, day 14 injection groups:2.60/HP,4.60/HP,3.80/HP,respectively;all p value＜0.001).Among all the injection groups,day 7 and day 14 injection groups had the highest capillary density.Likewise,the beneficial effects of BMMNCs transplantation at day 7 and day 14 post AMI on absolute changes from control data(△) in LVEF, FS,LVEDP,LVESP and±dp/dt were also demonstrated in present study.The effects of cell transplantation on the above parameters were more favorable in day 7 injection group than hour 24 transfer group(△LVEF:20.95%vs.11.41%,p=0.025;△FS: 13.47%vs.7.13%,p=0.023;△LVEDP:-15.94 mmHg vs.-8.17 mmHg,p＜0.05; +△dp/dt:9001.02mmHg/s vs.4891.53 mmHg/s;p＜0.05),whereas there was no significant differences between day 7 injection group and day 14 injection group (△LVEF:18.82%;△FS:11.63%;△LVEDP:-15.34 mmHg;△LVESP:9.19 mmHg; -△dp/dt:-4650.71 mmHg/s;+△dp/dt:8265.35 mmHg/s;all p value＜0.05).There existed high linear correlations between these parameters(△LVEF,△FS,△LVEDP and△LVESP) and the capillary densities(all r＞0.75,all p＜0.01).Moreover, improvements of LVIDd and LVIDs also showed the more beneficial tendencies in day 7 injection group and day 14 injection group,with no significant differences when compared to the others cell injection groups.ConclusionsThe optimal time frame for BMMNCs therapy for AMI occurs within the period from day 7 to day 14 after the infarction.Cell transplantation at the moment is able to promote the improvements in injured heart function utmost,which might be associated with the highest density of capillary within transplantation area possibly due to the more favorable physical microenvironment(elasticity of infarcted myocardium).