Transplantation Of Mesenchymal Stem Cells Overexpressing MiR126Enhances Ischemic Angiogenesis Via AKT/ERK-related Pathway

BackgroudIschemic heart disease which triggers dysfunction and the death of cardiomyocytes is the most common cause of death throughout the world despite continued advances in the prevention and treatment of coronary artery disease. The symptoms of ischemic heart disease are angina, myocardial infarction, arrhythmia and heart failure, which are the consequence of decreased patency of atherosclerotic vessels. The mortality of heart failure can reach60%within one year for patients in New York Heart Association functional class IV.Over the past decades,improvements in medical therapy, percutaneous transluminal coronary angioplasty(PTCA), coronary artery bypass surgery(CABG) and heart transplantation, have dramatically improved the prognosis of ischemic heart disease. However, all the methods including drugs, thrombolysis and revascularization are not able to influence the dead myocardium. Many researchers devote to studies in the field of myocardium regeneration and repairing insulted myocardium with stem cells.During AMI repairs, coronary collateral formed which could improve myocardial perfusion, circulation(CC)often decrease myocardial necrosis, restore hibernating myocardial viability and reduce left ventricular function disorder. As self neovascularisation is insufficient for compensation myocardial ischemia induced from CHD, stimulating small vessels to develop in ischemic region and promoting to form CC become attractive and reasonable strategy. As study of the angiogenesis and VGF development, theraputic angiogenesis has become a new approach to treat IHD. This strategy is designed to promote the development of supplemental collateral blood vessels that will constitute endogenous bypass conduits around occluded native arteries, a strategy termed "therapeutic angiogenesis."Recentely,a few studys on MSCs to treat MI in animal experiment have proven that MSCs have the ability of differentiating into cardiac myocytes, living long time, promoting angiogenesis, and improving haemodynamics. However, some problems, for example, how to further augment the implanted cells survival and other functions to overcome the limitations for their clinical applications, are still to be explored. Since the knowledge of a variety of biological processes, including developmental timing, signal transduction, tissue differentiation and maintenance, Antiapoptosis, Anti-inflammation and neoangiogenesis under miRNAs regulate, new therapeutic targets for ischemic heart disease may identified in these non-coding RNAs.MiR-126is an endothelial cell-specific miRNA that plays an essential role in neoangiogenesis following MI and in maintenance of vascular integrity [94]. MiR-126functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1and phosphoinositol-3kinase regulatory subunit2(PIK3R2/p85-beta). MiR-126represses the expression of spredl and PIK.3R2, which negatively regulate VEGF signaling via the ERK and AKT pathways, respectively. Thus, in the absence of miR-126, Spred-1/PI3k expression is elevated, resulting in repression of angiogenic signaling. Conversely, miR-126overexpression relieves the repressive influence of Spred-1/PI3k on the signaling pathways activated by VEGF and FGF, favoring angiogenesis. It is reasonable to speculate that miRNA126, a novel but practical regulator of stem cell implantation for ischemic heart disease, introduce a promising strategy for the treatment of myocardial infarction. Part Ⅰ. MSCs isolation, culture and identificationObjectiveThis study was to establish the experimental method for isolating, culturing, proliferating and inducing mice bone marrow mesenchymal stem cells in vitro.MethodsWe isolated MSCs from animal bone marrow and separated MSCs by Percoll Centrifugation.MSCs were cultured and subcultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) with10%selected fetal calf serum. Cell growth patterns were evaluated by MTT test. Cell cycle and surface antigenic features were analyzed by flow cytometry technique.Results:To isolate MSCs from bone marrow aspiration, the samples were fractionated with Percoll by density gradient centrifugation for mononuclear cell isolation. Through replacing culture medium, the non-adherent cells were removed and the remaining tightly adhered cells were MSCs. The morphology of cultured MSCs was fibroblast-like form. Primary cultured BMSCs adhered to and stretched into polygon or spindle within24hours. The first3days was the relative inhibition period, and then the cell proliferation accelerated gradually in a pattern of logarithm and reached the period of plateau by Day7or so, with90% cell coverage. The number of P3BMSCs can reach1062weeks later, and that can satisfy the required number of cell transplantation. The signs of BMSCs with positive CD29, CD44, CD90and negative CD34, CD45were detected by the flow cytometry, and that is consisted with the characteristics of BMSCs.Conclusions:In this study, we believed the adherent cells in cultured medium were mainly MSCs obtained by Percoll fractionation. Numerous molecular markers of MSCs were characterized, but unique marker of MSCs and any marker of blood cells and endothelial cells were not identified. MSCs showed stable growth in vitro, easy survival in subculture and rapid proliferation in culture medium. Part II Transfection of miR-126in mesenchymal stem cellsand its expressionObject:Construct Lentivirus vector to express miR-126, produce pseudoviral particles and determine its titer.Methods:Lentiviral MicroRNAs expression vector was constructed with Gateway system. Mature miR126, TRE promoter and eGFP sequences were inserted into plasmids to produce pUp-TRE, pDown-miR126and pTail-IRES/eGFP; scramble sequence was set as negative control pLV.EX3d.P/puro-TRE> miR126>IRES/eGFP was obtained with incubation of donors and accepter vectors catalyzed by LR clonase. Plasmid was then sequenced and purified for lentivirus envelope.Envelope helper plasmids:pLV/helper-SL3, pLV/helper-SL4, pLV/helper-SL5, with pLV.EX3d.P/puro-TRE-miR126-IRES/eGFP or pLVrtTA/neo which contains the imperative elements for virus packaging, were co-transfected into293T cells with lipofectamine2000, according to the manufacture’s instructions for the generation of Lenti-miR126-eGFP/puro or Lenti-rtTA/neo respectively.To perform lentiviral infections, the mice MSCs cells were plated at40%-50%confluence and incubated overnight. Cells were firstly treated by Lenti-rtTA/neo, Selection was terminated when control cells were completely dead and antibiotic free medium were used for propagation. Neomycin resistant cells were then infected by Lenti-miR126-eGFP/puro and grown with2μg/ml Puromycin. Double resistance cells were ultimately obtained, and2μg/ml doxycycline was added to medium and intrigue expression of miR126.Results:(1) MiR-126genomic sequence was amplified and lentiviral expression vector were constructed.(2) LV-miR-126pseudovirus was packaged and the titer was determined:(3)293cell infected by LV-miR-126can over-express miR-126.Conclusion:LV-miR-126infection can enhance the expression level of miR-126in293cell lines. Part III MiR126promote the expression of ERK and AKT in MSCsObjective:To evaluate the role of miR-126in ERKI and AKT signal transduction pathways.Methods:Total RNA was extracted from cells with All-in-One miRNA qRT-PCR Detection Kit according to the manufacturer’s instructions. Expression of miR126was detected by quantitative real-time RT-PCR using the All-in-OneTM miRNA qPCR Primer. The relative levels of miR126transcripts were normalized to the control U6mRNA. Relative gene expression was quantified using the GraphPad Prism4.0software and expressed as%of the control.The miR126transfected and nontransfected MSCs were collected separately for ERK1and PERK1、AKT and pAKT Western blot assay. Chemiluminent detection was performed with the ECL kit. Bands on Western blots were quantified by densitometry using Quantity One software.Results:Results uncovered that under higher expression of miR126, ERK1and pERK1、 AKT、pAKT and capillarynetwork density were dramatically increased (P＜0.01). Conclusions:MiR-126represses the expression of spredl and PIK3R2, which negatively regulate VEGF signaling via the ERK and AKT pathways, respectively. Part IV Transplantation of mesenchymal stem cells overexpressing MiR126Enhances Ischemic angiogenesis via AKT/ERK-related pathwayObjective:To whether a investigate new strategy that combines MSCs transplantation and ex vivo miR-126transferring with lentiviral vectors was more therapeutically efficient than MSCs cell therapy alone in a C57mice Myocardial infarction modelMethods:Mice models of myocardial infarction were established by ligation of coronary artery. The ligated animals were randomly divided into three groups (15in each) and, after2weeks, were intramyocardial injected at the heart infarct zone with miR126-transfected MSCs (miR126-MSCs Group) MSCs (MSCs group), or medium (PBS group).Six weeks later, histological study and Echocardiographic assessment were performed.Results:Capillary density of the infarcted region was significantly improved in the miR126-MSCs group compared with the MSC group and the PBS group (both P＜0.01). Western blot showed that ERK1,pERK1, AKT and pAKT gene were dramatically enhanced in the miR126-MSC group compared with the MSC group and the PBS group (both P<0.05) and. Echocardi ography showed MiR126led to sustained improvement in cardiac function, as assessed by left ventricle ejection fraction (LVEF) and fraction of shortening(FS).Conclusions:The present study shows that Mesenchymal stem cells overexpressing of MiR126transplantation therapy induced more potent angiogenesis and collateral vessel formation than MSC cell therapy alone. We put forward the hypothesis that transplantation of MSCs transfected with mi R126can improve angiogenesis and cardiac function in infarcted area of mice heart, which may be due to stimulation of AKT/ERK-related pathway.