Cyclosporine A-nanoparti Cles Emulsion Enhances Therapeutic Benefit Of Adipose Tissue-derived Stem Cells Transplantation Afteracute Myocardial Infarction In A Swine Model

Background:Limited transplanted stem cells survival has been observed due toapoptosis and necrosis. Thus, the challenge for cardiac cell therapy is to strengthendonor cells for the harsh microenvironment of the infarcted heart. Cyclosporine A(CsA), an immunosuppressive agent widely used to reduce rejection aftertransplantation.Some tudies suggest that CsA has been shown to inhibit-apoptosis andhave a favorable impact on myocardial ischemia-reperfusion injury(MIRI). However,Severe adverse effects of long-term use of CsA are increasingly recognized,including hepatotoxic and nephrotoxic, limited success in the clinical use. Dependingon the successful targeting drug delivery system(TDDS)of cancer chemotherapy,thepresent work was to make cyclosporine (CsA) incorporated into cyclosporine Ananoparticles emulsion(CsA-NP). CsA-NP is supposed to improve the absorptioncharacteristics, thus leading to a reduction in the doses administered, toxic and sub-therapeutic levels.Objective:Therefore, the present study examined whether the CsA-NP enhancestherapeutic benefit of adipose tissue-derived stem cells (ASCs) transplantation afteracute and possible mechanisms.Methods: The first part: CsA–NP emulsion are prepared by the high pressurehomogenization method.The products were evaluated by measuring drug loading withHPLC, particle sizing by dynamic light scattering (DLS).The second part: To investigated the effect of CsA-NP on the proliferation anddifferentiation of the ASCs in vitro. ASCs were harvested from porcine inguinaladipose tissue undergoing isolated and culture.The molecular phenotypes of ASCswere analysed by flow cytometry. The proliferation and differentiation of ASCs weremeasured by direct cell counting and MTTassay, respectively.The third part: To investigate whether CsA-NP could protect the ASCs againstapoptosis and possible mechanisms in a model of oxidative stress-induced consistingof hydrogen peroxide (H2O2) in vitro. Apoptosis of ASCs was assessed using anAnnexin-VFITC/PI apoptosis kit.Cell activity was determined by CCK-8assay.Caspase-3activity was detected by applying a caspase-3assay kit. Expression ofcytochrome c was investigated using Western blot analysis.The fourth part: Delayed-enhanced magnetic resonance imaging (DE-MRI)evaluate cardiac function and viability of miniswine myocardial infarction model bypercutaneous balloon occlusion method.The fifth part: A total of17swine survived after AMI were divided into fourgroups: group control (n=5); group CsA-NP(n=4); group ASCs(n=4); group CsA-NP+ASCs(n=4).ASCs or PBS were delivered by intracoronary injection after AMI1week later. At0,8weeks after transplantation, DE-MRI studies were performed. Theinfarcted myocardium and implanted cells were studied histologically.Results: The first part: The prepared with the optimized formulation and preparationtechnique was stable with uniform partical size distribution and the dimension of50%emulsion droplets was less than162nm. The second part: The results showed that ASCs were positive for CD29,CD44,CD90and CD105,but negative for CD31,CD34,CD45and HLA-DR. We found that theproliferation of ASCs was stimulated by0.01–1mg/ml CsA-NP and unaffected by5mg/ml CsA-NP (P<0.01),and the best proliferative effect was observed atconcentration of0.5mg/ml.The third part: Apoptotic by H2O2induced were inhibited by pre-treatment withCsA-NP at concentration of0.1-10mg/ml for24h, and the best effect was observedat concentration of5mg/m(lP<0.001). While examining the mechanisms of CsA-NPin protecting ASCs apoptosis, we found that the collapse of mitochondria membranepotential (DCm) induced by H2O2was partially reversed by CsA-NP (0.1-10mg/ml).Pre-treatment with CsA-NP (0.1-10mg/ml)down-regulation of activation ofcaspase-3.Furthermore, Western-blot showed that CsA-NP (5mg/ml) completelyinhibited the H2O2-induced release of cytochrome C.The fourth part: Seventeen swine survived after AMI,and the success rate reached54.8%(17/31). MRI showed all intervention animals had infarcts size10.2±2.9(cm3).Intervention animals had significantly increased end-diastolic and end-systolicvolumes(P<0.05), and decreased stroke volume, ejection fraction and cardiac output(P<0.001). These MRI values were matched to microsphere values obtained fromshort-axis slices at pathology.The fifth part: DE-MRI demonstrated LVEDV and LVESV increased in ASCs andCsA-NP+ASCs groups(P<0.05).Moreover, LVEF increased significantly in theCsA-NP+ASCs group(P<0.01) compared with the CsA-NP and ASCs only,while theinfact size and the thickness of the ventricular wall decreased significantly. Thevascular density of the infarct core and infarct border zone also improved in CsA-NP+ASCs group (P<0.01)but not in other groups. The CsA-NP+ASCs group was furtherconfirmed by DAPI and immunofluorescent staining that there were an increasedexpression of cardiomyocyte markers such asα-actin, cTn-T, and vWF than that ofASCs group(P<0.01).Masson thichrome stain showed that there was less fibrosis withmore surviving myocardium in CsA-NP+ASCs and ASCs group than that in controlgroup(P<0.01and P<0.05,respectively).TUNEL assay indicated that administration significantly decreased cell apoptosis in peri-infarct myocardium in CsA-NP+ASCsand CsA-NP group(P<0.01and P<0.05,respectively).Expression level of caspase-3and cytochrome C in CsA-NP+ASCs and CsA-NP group significantly decreased(P<0.05).Conclusions: CsA-NP enhances transplantation of ASCs in swines can improve thecondition of left ventricular remodeling and recover the cardiac functions after AMI.The improvement of cardiac functions is related to the inhibition of cellapoptosis,promotion ASCs proliferation, differentiation and survival,as well asincrease of blood vessels in cardiac muscle tissues after ASCs transplantationcombination with CsA-NP.