The Effect Of Bone Marrow Mesenchymal Stem Cell Transplantation On Diabetic Cardiomyopathy

With the incidence increased grandually of the diabetes mellitus (DM), diabetic complications become the hotpoint of the research. Diabetic cardio-myopathy (DCM) is a serious complication of diabetes which was first considered by Prof. Rubier in 1972 . Diabetic cardiomyopathy often accompanied the cardiac dysfunction. Studies show that energy metabolic disorders, abnormal function and increased apoptosis of myocardial cells also associated with diabetes. Some cytokines and inflammation factors which contribute to myocardial fibrosis are abnormal expressed in diabetic myocardium. Diabetic microvascular complications and neuropathy also play very important roles in cardiomyopathy. The results above indicate that the physiological disfunction of myocardium and ventricular remodling play critical roles in the pathogenesis of diabetic cardiomyopathy.Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Because of their advantages, such as ease of obtaining by a simple routine bone marrow aspiration, ability to self-renew and pluri-potential of differentiation, MSCs have been considered as one of the most promising candidates of autologous cells for both cell therapy and gene therapy ofmesenchymal tissues. MSCs can differentiate into cells of various phenotypes in the infarcted myocardium, participating in myogenesis and angiogenesis, and also secret lots of cytokines and vascular growth factors, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor-1(HGF-1) and adrenomedullin(AM). MSC transplantation prevented left ventricular remodeling of ischemic heart, which also inhibited myocardial fibrosis in a rat model of dilated cardiomyopathy and attenuated the increased expression of MMP-2 and MMP-9. But little is known about the therapeutic potential of MSCs for DCM and an experiment would be needed to test the hypothesis that cell transplantation may be of value in the setting of diabetic heart disease. The aims of our study were to investigate (1) whether systemic intravenous delivery of MSCs can colonize in the diabetic myocarium and (2) whether MSCs transplantation induces angiogenesis, attenuates the remodeling and improves the cardiac function of DCM.Methods80 Male Sprague-Dawley rats aged 7-8 weeks (160-180g body weight) were used , obtained from Medical Institute Animal Center of Zhejiang University. DM was induced with a single intraperitoneal injection of streptozotocin (60mg/kg in 0.1mol/L citrate buffer , PH 4. 5, Sigma), while the control group received saline of the same volume. Serum glucose levels were measured every week during the experiment and all animals with levels <300mg/dl were excluded from the DCM group . 8 weeks later, the DCM were established.We humanely killed Male Sprague-Dawley rats aged 8 weeks and harvested bone marrow by crushing and flushing their femurae and tibiae with phosphate-buffered saline (PBS). Nucleated cells were isolated with a density gradient method, and then introduced into 25 cm flask (Falcon, Oxnard, CA, USA) and cultured (at a density of 5×10⁷ cell/ml) at 37°C in humid air with 5 % CO₂ in Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA)containing 10% fetal bovine serum (Gibco, USA), penicillin (100 U/ml) and streptomycin (100 mg/ml). A small number of cells developed visible colonies by 5 to 7 days. The medium was replaced every 4 days and nonadherent hematopoietic cells were removed . The adherent, spindle-shaped MSCs expanded and purified after 3 to 4 passages after the cells were first plated. MSCs were tested by fluorescence-activating cell sorting before transplantation. MSCs were incubated in 50μg/ml 4'6-Diamidino-2-Phenylindole (DAPI) overnight before injection into the femoral vein. The number of transplantedcells was 5×10⁶ cells in 1ml volume . The untreated DCM and control group also received lml PBS, each group about 10 rats.4 weeks after transplantation, we evaluated the cardiac conditions by echocardiography, histopathologic analysis including arteriolar density and and fibrosis area, reverse transcription polymerase chain reaction (RT-PCR) analysis for matrix metalloproteinase(MMP)-2, MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1, western blot analysis for Troponin T, and zymography analysis for of MMP-2 activity. ResultsCharacterization of Cultured MSCsMost cultured MSCs expressed CD44 and CD90, whereas they were negative for CD45. Baseline Characteristics of DCM and Control Group before transplantationBefore MSC transplantation, blood glucose was elevated obviously in DCM group (P<0.01), which gained less weight than control group (P<0.01). Left ventricular end-diastolic dimensions (LVDd) of the 2 groups were similar but were greater in DCM group when corrected for body weight (P<0.01). LVPW, relative wall thickness (RWT) and FS were lower in DCM group (P<0.05, P<0.05 and P<0.01 respectively). Left atrial dimension (LAD) was greater in DCM group, although the change did not reach statistical significance. Baseline characteristics of 3 groups 4 weeks after transplantation4 weeks after MSC transplantation, the levels of blood glucose were stillhigher in DCM group and DCM+MSC group (P<0.01 versus control respectively). LVDd of 3 groups were similar but LVDd and heart weight were greater in DCM group when corrected for body weight (P<0.01 and P<0.05 versus control respectively), which were atennuated by MSC transplatation, although the effect did not reach statistical significance. LVPW and RWT were lower in DCM group (P<0.01 and P<0.05 versus control respectively), which were improved by MSC transplantation, although only the improvement in LVPW reached statistical significance (P<0.05). The differences of interventricular septal diastolic thickness (IVSd), LAD and FS among the 3 groups did not reach statistical significance. Myogenesis and Angiogenesis Induced by MSCsBlue fluorescence - labeled MSCs were found in the myocardium 4 weeks after transplantation. Immunofluorescence demonstrated that transplanted MSCs were positive for the cardiac markers cardiac TnT and myosin heavy chain. These results suggest that a small number of transplanted MSCs can express troponin T and myosin heavy chain.The number of arterioles in the myocardium of DCM group decreased significantly compared with the control group (2.71 ± 0.29 arterioles/0.2mm² versus 5. 46±0. 61 arterioles/0.2mm², p<0.01), which was attenuated by MSC transplantation (4.38±0.25 arterioles/0.2mm² in DCM+MSC group, p<0.05 versus DCM group) . Reduction of Myocardial Fibrosis by MSC TransplantationMasson's trichrome staining demonstrated obvious interstitial and perivascular fibrosis in the myocardium of DCM group compared with Control and MSC group .. Quantification of fibrosis area showed significant increase of fibrosis in DCM group (p<0.01 versus control group), while MSC transplantation attenuated fibrosis progression in diabetic heart , although the change did not reach statistical significance.The activity of MMP-2 was decreased in DCM group compared with control group (72±4% of control, p<0. 01), which was increased by MSC transplantation(97±3% of control in DCM+MSC group, p<0.01 versus DCM group). The mRNA levels of MMP-2 were quite low in 3 groups, and no difference was found.The mRNA levels of MMP-9 were significanly increased in DCM group (p<0. 01 versus control group), which was attenuated by MSC transplantation (p<0. 01 versus DCM group), although the mRNA level of MMP-9 was still higher than control group (p<0.01). The mRNA levels of TIMP-1 were significantly decreased in DCM group (p<0. 01 versus control group), which was increased by MSC transplantation, although the change did not reach statistical significance.Expression of TnT by western blotWestern blot demonstrated that the expression of TnT was dereased in DCM group compared with control group (p<0.01 ), which was slightly increased by MSC transplantation , although the change did not reach statistical significance . ConclusionIn our study, we have demonstrated the following effects of MSC transplantation on diabetic myocardium:(1)systemic intravenous delivery of MSCs can colonize in the diabetic myocarium;(2) MSCs induce angiogenesis and myogenesis;(3) MSCs attenuate the cardiac remodeling and induce changes of MMPs and TIMP. Intravenous MSC transplantation induces angiogenesis and attenuates the remodeling of DCM.