Bone Marrow Mesenchymal Seem Cells With Support Of Ultrasound-Mediated Microbubbles And Bispecific Antibody Prevent Myocardial Fibrosis And PⅢNP Predicts Early Prognosis And Cardiac Remodeling In Coronary Heart Disease Patients With Metabolic Syndrome

Myocardial fibrosis is becoming one of the most important problems in cardiology. Stem cells have unique advantages in the treatment of myocardial damage. Medical, interventional and surgical intervention have made delectable advancement in revascularization, and improve the symptoms of myocardial fibrosis partly. But myocardial cell lack reproductive activity. How to repair the infarct myocardium and fibrotic heart is a hard problem. From 20th Century 90's, with the development of stem cell study[3], stem cell with multi-directional differentiation and self-duplication ability shine new light on the therapy of cardiovascular disease.In cellular cardiomyoplasty, bone marrow mesenchymal stem cells (BMSC), hemopoietic stem cell, endothelial progenitor cell, embryonic stem cell, skeletal myoblast are the most commonly used stem cells. BMSC have many desirable characteristics, including low immunogenicity, liability for isolation and amplification, no- ethics debate[4], and multiple differentiation[5, 10], making these cells one of the most frequently used stem cells for the repairing and reconstruction of injured myocardium[6].In investigations of cellular cardiomyoplasty, it was found that BMSC transplantation via the peripheral vein pathway reduced local trauma and had a high feasibility as compared to alternative treatments. However, the repairing efficacy is limited by the migratory number and colonization amount of targeted stem cells[7, 8]. And its repairing mechanism remains unclear.BMSC transplantation via vein is safety and easy to approach, but low efficacy. So it is important to find a way to improve the efficiency. Meanwhile, the new way would be a better choice if it can increase the homing number of BMSC and promote the homing niche. Microbubble could improve the microenvironment in the target site for homing, which is helpful for the survival of stem cells. Microbubble act as cavitation nuclei or enhancer in myocardium. Once excited by a high peak pressure ultrasound pulse, the mechanical effects, like shock wave and microstream, released from cavitation could produce a series of bioeffects, contributing to sonoporation, microvascular rupture and hematoma[8]. Ultrasound-targeted microbubble destruction can effectively deliver hepatocyte growth factor into the infracted myocardium and facilitate angiogenesis, which provides a novel way in the gene therapy of myocardial infarction[9]. Targeted delivery of bone marrow mononuclearcell by ultrasound-targeted microbubble to the myocardium of the cardiomyopathic hamster increased the capillary densities and regional blood flow and inhibited cardiac remodeling.CD29 is the molecular marker that shows a positive rate of more than 99% on the surface of BMSC, and AMLCA can specifically recognize injured myocardium[9]. Prepared CD29×AMLCA is the connecting bridge between injured myocardium and BMSC. Therefore, CD29×AMLCA can bind to both BMSC and injured myocardium, and it can act as the bridge between them. In order to promote the homing of BMSC and understand its repairing mechanism, we combined bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB) to guide BMSC to improve the homing number of BMSC and evaluate the therapeutic efficacy of this treatment on myocardial fibrosis.Besides the animal study of BMSC preventing myocardial fibrosis, we also explored the role of plasma procollagenⅢN-terminal peptide (PⅢNP) in predicting the prognosis and cardiac remodeling in coronary heart disease (CHD) patients with metabolic syndrome (MS). In the CHD population, many patients also have MS[11] , resulting in an urgent need for objective parameters and predictors of the prognosis and the extent of cardiac remodeling in these patients. Recent studies have revealed that imbalanced metabolism of interstitial collagen provides a morphological foundation for cardiac remodeling, and the plasma collagen marker procollagenⅢN-terminal peptide (PⅢNP) is associated with chronic heart failure [12] and acute myocardial infarction [13]. CHD patients with MS often have a high risk of chronic heart failure, and cardiac remodeling may result in the deterioration of cardiac function, arterial compliance and exercise tolerance. However, it is still not known what role PⅢNP plays in these patients. Likewise, the association between PⅢNP and arterial atherosclerotic diseases and other risk factors remains unclear. The purpose of this cohort study was to explore the role of plasma PⅢNP in predicting the prognosis and cardiac remodeling in CHD patients with MS.The study included the following five parts:PARTⅠ: ESTABLISHMENT OF ISOPROTERENOL-INDUCED MYOCARDIAL FIBROSIS AND MECHANISM OF COLLAGEN DEPOSITIONObjective To establish a manipulation-easy animal model of myocardial fibrosis and investigate the mechanism of collagen deposition. Methods Twenty BALB/c mice were randomly divided into two groups: experiment group and control group. Mice in experiment group were subjected to hypodermic injection with isoproterenol at 50 mg/kg. The injection was carried out twice a day for continuous 10 days. Physiological saline was injected with the same method in control group. Forty-five days after injection, collagen distribution was observed using sirius red staining. Expression levels of matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1) in myocardium were detected by fluorescent qRT-PCR. The expression of laminin(LN) in heart, liver and kidney was detected by immunohistochemistry. Results Experiment group had increased levels of MMP-9, TIMP-1, LN and collagen deposition in myocardium than control group(p<0.05). The expression of LN in liver and kidney showed no difference(p>0.05). Conclusion Isoproterenol-induced collagen deposition in the experiment is a reproducible, handy and credible method to establish animal model of myocardial fibrosis. MMP-TIMP imbalance is involved in the mechanism.PART II:ISOLATION, CULTURE AND IDENTIFICATION OF BONE MARROW MESENCHYMAL STEM CELLS FROM MOUSEObjective To establish a stable method for isolation and culture of bone marrow mesenchymal stem cells(BMSC) from mouse, and morphological and functional indexes and cellular phenotype were used to identify the cultured BMSC. Methods Mouse limbs were sterilely isolated.The BMSC were enriched and expanded by using bone marrow adherent culture.Morphology of the BMSC was examined under light microscopy. The surface antigens CD29, CD44, CD117 phenotype was analyzed by flow cytometry. The ability of osteogenic and adipogenic diferentiation of the BMSC under diferent induction conditions was assessed.Results BMSC could obtain by the adherence separation method. The in vitro cultured BMSC grew in adherence and most of them grew in parallel arrays or a whirlpool-like pattern. The cells had a fusiform or polygonal shape. The expression of CD29,CD44,CD117 was 99.94%, 99.28%, 1.35%, and BMSC could be induced to differentiate into osteoblast and adipocytes cells. Conclusion BMSC isolated by the adherence separation method have a good homogeneity.PARTⅢ: BISPECIFIC ANTIBODY AND ULTRASOUND-MEDIATED MICROBUBBLES PROMOTE BONE MARROW MESENCHYMAL STEM CELLS HOMING EFFICIENCY AND PREVENT MYOCARDIAL FIBROSISObjective To investigate the efficacy of myocardial fibrosis intervention using bone marrow mesenchymal stem cells (BMSC) with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). Methods BiAb (anti-CD29×anti-myosin light chain antibody (AMLCA)) was prepared and combined with isolated BMSC from male mice and transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein, followed by ultrasound-mediated microbubbles (BMSC+BiAb+MB). This study included seven groups: BMSC+BiAb+MB, BMSC, BiAb, MB, BMSC+BiAb, untreated, and control groups. Five weeks after treatment, expression levels of sex-determining region of Y-chromosome (SRY), matrix metalloproteinases-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent qRT-PCR. Collagen distribution was observed using sirius red staining. The protein expression of signal transducer and activators of transcription 1 (STAT1) and STAT3 was detected by Western blot. Results The highest homing number of BMSC was in the BMSC+BiAb+MB group, second highest in the BMSC+BiAb group, and lowest in BMSC alone. Compared to the untreated group, BMSC+BiAb+MB, BMSC+BiAb, and BMSC groups had decreased levels of MMP-9, TIMP-1, STAT1, and collagen deposition, and increased levels of STAT3. Up-regulated STAT3 and down-regulated TIMP-1 were significantly different in BMSC+BiAb+MB as compared to BMSC alone or BMSC+BiAb. Conclusion Homing rate and repairing efficacy of BMSC improved with treatment utilizing a combination of BiAb and MB. BMSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway. PART IV: BONE MARROW MESENCHYMAL STEM CELLS PREVENT MYOCARDIAL DIBROSIS VIA THE POSSIBLE DIGNALING PATHWAYObjective To investigate the potential mechanism of myocardial fibrosis intervention using BMSC. Methods Isolated BMSC from male mice were transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein (treated group). This study included three groups: treated, untreated, and control groups. Five weeks after transplantation, expression levels of nuclear factor-κb(NF-κb), heat shock protein-70(HSP-70), proliferating cell nuclear antigen(PCNA) and caspase-3 in myocardium were detected by immunohistochemistry. Results BMSC can home to fibroid heart. Compared to the untreated group, treated group had decreased levels of NF-κb, HSP-70, caspase-3 and increased levels of PCNA ( p<0.05 ) . Conclusion BMSC transplantation can improve collagen deposition in injured myocardium after homing, the mechanism that may be related to the NF-κb, HSP-70, caspase-3 and PCNA mediated signaling pathway. PART V: PROCOLLAGENⅢN-TERMINAL PEPTIDE PREDICTS SHORT-TERM PROGNOSIS AND CARDIAC REMODELING IN CORONARY HEART DISEASE PATIENTS WITH METABOLIC SYNDROMEObjective To explore the role of plasma procollagenⅢN-terminal peptide (PⅢNP) in predicting the prognosis and cardiac remodeling in CHD patients with MS. Methods One hundred and eight patients were classified into high and low PⅢNP groups according to the median value of plasma PⅢNP. Cardiovascular examinations including echocardiogram, carotid color ultrasound examination, coronary angiography and 6-minute walking test (6MWT) were carried out before and after a one-year follow-up. Readmission for cardiac and cerebrovascular events was assessed during the follow-up period. Results Plasma PⅢNP level was significantly correlated with age(β=0.686,p<0.001), high-sensitivity C-reactive protein (hs-CRP)(β=0.194,p=0.001), and body mass index (BMI)(β=0.108,p=0.023) in a multiple stepwise regression model. There was a positive correlation between LnPⅢNP and a raised left ventricular mass index(LVMI) in partial correlation analysis. The COX proportional hazard model analysis indicated that the level of PIIINP, left ventricular ejection fraction, and hs-CRP were independent predictors of cardio-cerebral readmission during follow-up. A PIIINP value of 4.0μg/L was the best threshold value for determining the need for readmission. Conclusion PIIINP levels rise with increases in age, hs-CRP and BMI in CHD patients with MS, and a high level of PIIINP indicates recent deterioration of cardiac remodeling and exercise tolerance, as well as a poor prognosis.