Hypoxia-preconditioned Bone Marrow Mesenchymal Stem Cells Improves Transplantation Therapy After Heart Ischemia Of Rats

Background:The ischemic damage resulting from myocardial infarction(MI)induces the irreversible loss of cardiomyocytes,scar formation,and may ultimately result in congestive heart failure.Bone marrow mesenchymal stem cells (MSCs)are multipotent adult stem cells residing within the bone marrow microenvironment.These stem cells are able to differentiate into endothelial cells,vascular smooth muscle cells,and perhaps even cardiac-like myocytes when transplanted into the ischemic heart.Both animal and clinical studies have provided evidence that MSC transplantation can improve cardiac function through possible myogenesis and angiogenesis following MI.The injection of viable cells directly into the infarcted myocardium,or cellular cardiomyoplasty,results in beneficial effects such as angiogenesis, cardiac regeneration,and cardiac functional preservation in experimental MI animals and MI patients.A major dilemma in stem cell therapy for ischemic heart diseases is the low survival rate of transplanted cells.For example, most implanted cells may die within 4 days after transplantation into the ischemic heart.Endogenous and environmental factors,such as the inflammatory response,may contribute to cell death,and apoptosis has been identified as an important mechanism resulting in damage to implanted cells. Thus,improving grafted cell survival after transplantation is critical for enhancing the efficacy and efficiency of stem cell therapy.Angiogenesis remains one of the putative mechanisms in cardiac functional recovery after MI and stem cell transplantation.Stimulating angiogenesis showed therapeutic effects in the ischemic heart disease. Transplanted MSC can stimulate angiogenesis after MI by secreting multiple angiogenic cytokines and differentiating into endothelial cells.It can be reasoned that enhancing the MSC's ability to promote angiogenesis will further increase the therapeutic potential of MSC transplantation after MI.Hypoxic preconditioning(HP)by sublethal hypoxic insult stimulates an endogenous mechanism resulting in multiple protective responses including protein expressions that protect against future lethal hypoxia and other insults. HP has been used as a powerful tool for protecting cells from necrotic and apoptotic death and enhancing angiogenesis.Cell transplantation therapy and HP have been intensively studied but largely as separate research topics.Whether HP can enhance MSC survival and angiogenic ability,whether HP-MSC transplantation can promote infracted heart function recovery is still obscure.Objectives:This study explored the novel strategy of hypoxic preconditioning (HP)bone marrow mesenchymal stem cells(MSC)before transplantation into the infarcted heart in order to promote their survival and therapeutic potential of MSC transplantation after myocardial ischemia(MI).Methods:MSC from green fluorescent protein(GFP)transgenic mice were cultured under normoxic(N-MSC)or hypoxic(0.5%O₂ for 24 hrs)(HP-MSC) condition.Expression of growth factors and anti-apoptotic genes were examined by Western blotting.To determine the vulnerability of N-MSCs and HP-MSCs to apoptosis,cells in culture were subjected to 24 hours of normoxia or sublethal hypoxia followed by 24 hours of serum deprivation.Cells were then stained with trypan blue and caspase-3 to assess cell death and apoptosis,respectively.To assess whether HP might protect MSCs in vivo, Hoechst 33342-labeled N-MSC or HP-MSC were intramyocardially injected into 5 sites of peri-infarct region of rats 30 min after permanent MI. Twenty-four hrs and 72 hrs later,the animals were sacrificed and apoptosis of implanted cells was identified by the ratio of caspase-3/Hoechst/GFP co-labeled cells vs.total transplanted cells pre-labeled with Hoechst and GFP, cell death was identified by the ratio of TUNEL/Hoechst positive cells vs.total transplanted cells.6 weeks after transplantation,angiogenesis was determined by CD31 and smooth muscle actin staining;Masson trichrome and hematoxylin-eosin staining were performed to assess infarct size;and cardiac functional recovery of ischemic heart was measured by the left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP),the maximum dp/dt and minimum dp/dt.Results:HP increased expression of pro-survival/pro-angiogenesis factors including hypoxia-inducible factor 1,angiopoietin-1,vascular endothelial growth factor and its receptor FIk-1,erythropoietin and its receptor EPO-R, NF-KB,Bcl-2,Bcl-xL,while suppressed caspase-3 activation.Serum deprivation markedly increased cell death and apoptosis,which was significantly reduced in HP-MSC group compared with that of the N-BMSC group.Twenty-four hrs and 72 hrs after transplantation,the death and apoptosis of implanted cells were much less in HP-MSC transplantation group compared with N-MSC transplantation group.6 weeks after MI,MHC staining showed few transplanted cells developed to cardiac like myocytes.Both the total vessel density/area and arteriole density/area in both MSC transplantation groups were greater than that in the ischemia-only group. Between the two transplantation groups,significantly greater total vessel density/area and arteriole density/area were observed in the HP-MSC group than in the N-MSC group.The infarct size was reduced in both groups that received MSC transplantation;however,rats receiving HP-MSC transplantation showed the smallest infarct size.MSC transplantation helped to prevent the negative LV remodeling.Rats that received N-MSC had significantly increased LVSP and the absolute values of±dp/dt compared with the control group.Even greater cardiac functional benefits were found in HP-MSC transplantation group,showing higher LVSP,improved±dp/dt, and near normal level of LVEDP.Conclusions:Hypoxic preconditioning significantly enhances MSC's capability to repair infarcted myocardium,attributable to reduced apoptosis of implanted cells,increased angiogenesis/vascularization,and paracrine effects.