Yingjie Wei

Low level laser irradiation precondition to create friendly milieu of infarcted myocardium and enhance early survival of transplanted bone marrow cells

We suggested that low‐level laser irradiation (LLLI) precondition prior to cell transplantation might remodel the hostile milieu of infarcted myocardium and subsequently enhance early survival and therapeutic potential of implanted bone marrow mesenchymal stem cells (BMSCs). Therefore, in this study we wanted to address: (1) whether LLLI pre‐treatment change the local cardiac micro‐environment after myocardial infarction (MI) and (2) whether the LLLI preconditions enhance early cell survival and thus improve therapeutic angiogenesis and heart function. MI was induced by left anterior descending artery ligation in female rats. A 635 nm, 5 mW diode laser was performed with energy density of 0.96 J/cm2 for 150 sec. for the purpose of myocardial precondition. Three weeks later, qualified rats were randomly received with LLLI precondition (n= 26) or without LLLI precondition (n= 27) for LLLI precondition study. Rats that received thoracotomy without coronary ligation were served as sham group (n= 24). In the cell survival study, rats were randomly divided into 4 groups: serum‐free culture media injection (n= 8), LLLI precondition and culture media injection (n= 8), 2 million male BMSCs transplantation without LLLI pre‐treatment (n= 26) and 2 million male BMSCs transplantation with LLLI precondition (n= 25) group, respectively. Vascular endothelial growth factor (VEGF), glucose‐regulated protein 78 (GRP78), superoxide dismutase (SOD) and malondialdehyde (MDA) in the infarcted myocardium were evaluated by Western blotting, real‐time PCR and colorimetry, respectively, at 1 hr, 1 day and 1 week after laser irradiation. Cell survival was assayed with quantitative real‐time PCR to identify Y chromosome gene and apoptosis was assayed with transferase‐mediated dUTP end labelling staining. Capillary density, myogenic differentiation and left ventricular function were tested by immunohistochemistry and echocardiography, respectively, at 1 week. After LLLI precondition, increased VEGF and GRP78 expression, as well as the enhanced SOD activity and inhibited MDA production, was observed. Compared with BMSC transplantation and culture media injection group, although there was no difference in the improved heart function and myogenic differentiation, LLLI precondition significantly enhanced early cell survival rate by 2‐fold, decreased the apoptotic percentage of implanted BMSCs in infarcted myocardium and thus increased the number of newly formed capillaries. Taken together, LLLI precondition could be a novel non‐invasive approach for intraoperative cell transplantation to enhance cell early survival and therapeutic potential.

Do mitochondria contribute to left ventricular non-compaction cardiomyopathy? New findings from myocardium of patients with left ventricular non-compaction cardiomyopathy.

BACKGROUND Left ventricular non-compaction cardiomyopathy (LVNC) is a rare congenital cardiomyopathy that is associated with mutations in mitochondrial DNA (mtDNA), however, no study of myocardium mtDNA of LVNC patients has been reported. To identify novel candidate mtDNA variants that may be responsible for the pathogenesis of LVNC, myocardial specimens were examined to investigate pathogenic mtDNA variants. MATERIALS AND METHODS Samples from six patients who were diagnosed with LVNC and underwent heart transplantation were analyzed. The sequence and copy number of mtDNA from these samples were determined by Sanger sequencing and fluorescence-based quantitative polymerase chain reaction, respectively. RESULTS Myocardial mtDNA sequences analysis revealed 227 substitution variants, including 157 coding variants and 70 non-coding variants. An m.9856T>C (Ile217Thr) mutation in MT-CO3 from one LVNC patient was found to be a non-haplogroup associated variant, and was rare in the mtDB Human Mitochondrial Genome Database, suggesting that the variant may be pathogenic. And there was statistically significant difference in mtDNA copy number between LVNC patients and normal control subjects. Electron microscopy (EM) of left ventricular myocardium showed abnormality in mitochondrial morphology and disordered sarcomeric organization. CONCLUSION The identification of mtDNA sequence variants in myocardial specimens may be helpful for further investigation of the underlying pathogenic implications of myocardial mtDNA mutations in LVNC. However, measurement of mtDNA copy number showed that there was lower mtDNA content in myocardium of LVNC patients than in normal controls (P<0.01). Lower mtDNA copy number and morphological abnormalities of mitochondria suggested mitochondrial dysfunction that may be associated with etiology of LVNC.

Intramyocardial injection of tannic acid attenuates postinfarction remodeling: A novel approach to stabilize the breaking extracellular matrix

OBJECTIVE Myocardial infarction is associated with early matrix metalloproteinase activation and extracellular matrix degradation. We tested the hypothesis that stabilizing the original extracellular matrix of the infarcted left ventricle with local injection of tannic acid would preserve cardiac structure and function. METHODS In vitro cytotoxicity of tannic acid was performed first; myocardial infarction model was induced by ligation of the left anterior descending branch in rats. Tannic acid was intramyocardially injected into infarcted site 24 hours after myocardial infarction (n = 30), and saline solution was injected in the same way as in the control (n = 30). The matrix metalloproteinase activity from tannic acid/saline solution-treated tissues was assayed by gelatin zymography 24 hours and 1 week after the treatment. The collagen content in the infarcted area was evaluated by hydroxyproline colorimetry assay 1 and 4 weeks after the treatment. Left ventricular structure and function were also evaluated with echocardiography, hemodynamics, and histologic examination. RESULTS Tannic acid at a concentration of 0.05% had minimal cytotoxic effects on cultured cardiomyocytes and thus was subsequently chosen as the optimal concentration for injection. Compared with the saline solution injection group, tannic acid treatment inhibited the matrix metalloproteinase-2/-9 activity and increased the collagen content at the early post-myocardial infarction stage (48.6 +/- 7.2 vs 37.3 +/- 6 microg/mg dry weight). Tannic acid treatment also significantly reduced infarct expansion (infarct expansion index: 1.04 +/- 0.15 vs 1.42 +/- 0.21) and left ventricular dilatation at 4 weeks after infarction. Although tannic acid treatment improved fractional shortening (26% +/- 2.4% vs 23.3% +/- 3.2%), it failed to alter blood pressure (systolic blood pressure: 93.8 +/- 8.2 vs 90.6 +/- 8.5 mm Hg) and rate of pressure rise. CONCLUSIONS Local delivery of tannic acid prevents collagen matrix degradation via cross-linking fibrous collagen and inhibiting matrix metalloproteinase activity but does not improve the intrinsic contractile function of myocardium. This treatment may be helpful to attenuate the adverse topographic remodeling after acute myocardial infarction.

Cell survival and redistribution after transplantation into damaged myocardium.

•  Introduction •  Poor cell survival limits the efficacy of cell transplantation •  Extracardiac cell redistribution and consequent safety issues •  A hybrid approach to support cell survival and decrease cell escape

Association of angiotensin-converting enzyme I/D polymorphism with heart failure: a meta-analysis

Heart failure (HF) is a complex clinical syndrome and is thought to have a genetic basis. Numerous case–control studies have investigated the association between heart failure and polymorphisms in candidate genes. Most studies focused on the angiotensin-converting enzyme insertion/deletion (ACE I/D) polymorphism, however, the results were inconsistent because of small studies and heterogeneous samples. The objective was to assess the association between the ACE I/D polymorphism and HF. We performed a meta-analysis of all case–control studies that evaluated the association between ACE I/D polymorphism and HF in humans. Studies were identified in the PUBMED and EMBASE databases, reviews, and reference lists of relevant articles. Two reviewers independently assessed the studies. Seventeen case–control studies with a total of 5576 participants were included in the meta-analysis, including 2453 cases with HF and 3123 controls. The heterogeneity between studies was significant. No association was found under all the four genetic models (D vs. I, DD vs. ID and II, DD and ID vs. II, DD vs. ID). Subgroup analyses for ischemic HF (IHF) and HF because of dilated cardiomyopathy (DHF) also showed no significant association between ACE I/D polymorphism and HF. No significant association between the ACE I/D polymorphism and risk of HF was found in this meta-analysis. The future studies should focus on large-scale prospective and case–control studies which designed to investigate gene–gene and gene–environment interactions to shed light on the genetics of HF.

Dermatopontin promotes adhesion, spreading and migration of cardiac fibroblasts in vitro

Dermatopontin (DPT), an extracellular matrix (ECM) protein, has been previously shown to be upregulated in the infarct zone of experimentally induced myocardial infarction (MI) rats. However, the accurate role that DPT exerts in the ventricular remodeling process after MI remains poorly understood. In this study, we evaluated the expression pattern of DPT mRNA and protein as well as its secretion in cultured neonatal rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) under conditions of hypoxia and serum deprivation (hypoxia/SD). Further, we tested the possible roles of DPT in CFs adhesion, spreading, migration and proliferation, which greatly promote the ventricular remodeling process after MI. Results showed that hypoxia/SD stimulated DPT expression and secretion in CMs and CFs and that DPT promoted adhesion, spreading and migration of CFs whereas had no effect on CFs proliferation. In addition, functional blocking antibodies specific for integrin α3 and β1 significantly reduced CFs adhesion and migration that DPT induced, suggesting that integrin α3β1 is at least one receptor for CFs adhesion and migration to DPT. These results implicated that DPT participates in the ventricular remodeling process after MI and may act as a potential therapeutic target for ventricular remodeling.

Profiling of differentially expressed microRNAs in arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a kind of primary cardiomyopathy characterized by the fibro-fatty replacement of right ventricular myocardium. Currently, myocardial microRNAs have been reported to play critical role in the pathophysiology of cardiovascular pathophysiology. So far, the profiling of microRNAs in ARVC has not been described. In this study, we applied S-Poly (T) Plus method to investigate the expression profile of microRNAs in 24 ARVC patients heart samples. The tissue levels of 1078 human microRNAs were assessed and were compared with levels in a group of 24 healthy controls. Analysis of the area under the receiver operating characteristic curve (ROC) supported the 21 validated microRNAs to be miRNA signatures of ARVC, eleven microRNAs were significantly increased in ARVC heart tissues and ten microRNAs were significantly decreased. After functional enrichment analysis, miR-21-5p and miR-135b were correlated with Wnt and Hippo pathway, which might involve in the molecular pathophysiology of ARVC. Overall, our data suggested that myocardial microRNAs were involved in the pathophysiology of ARVC, miR-21-5p and miR-135b were significantly associated with both the myocardium adipose and fibrosis, which was a potential disease pathway for ARVC and might to be useful as therapeutic targets for ARVC.

Tissue Extracts From Infarcted Myocardium of Rats in Promoting the Differentiation of Bone Marrow Stromal Cells Into Cardiomyocyte-like Cells

OBJECTIVE To investigate whether cardiac tissue extracts from rats could mimic the cardiac microenvironment and act as a natural inducer in promoting the differentiation of bone marrow stromal cells (BMSCs) into cardiomyocytes. METHODS Three kinds of tissue extract or cell lysate [infarcted myocardial tissue extract (IMTE), normal myocardial tissue extract (NMTE) and cultured neonatal myocardial lysate (NML)] were employed to induce BMSCs into cardiomyocyte-like cells. The cells were harvested at each time point for reverse transcription-polymerase chain reaction (RT-PCR) detection, immunocytochemical analysis, and transmission electron microscopy. RESULTS After a 7-day induction, BMSCs were enlarged and polygonal in morphology. Myofilaments, striated sarcomeres, Z-lines, and more mitochondia were observed under transmission electron microscope. Elevated expression levels of cardiac-specific genes and proteins were also confirmed by RT-PCR and immunocytochemistry. Moreover, IMTE showed a greater capacity of differentiating BMSCs into cardiomyocyte-like cells. CONCLUSIONS Cardiac tissue extracts, especially IMTE, can effectively differentiate BMSCs into cardiomyocyte-like cells.

CRP promotes MMP-10 expression via c-Raf/MEK/ERK and JAK1/ERK pathways in cardiomyocytes

C-reactive protein (CRP) was reported to be a predictor for left ventricular (LV) remodeling. Matrix metalloproteinase (MMP)-10 participates in the LV remodeling process. However, the intrinsic relationship between CRP and MMP-10 in cardiomyocytes remains unclear. The purpose of this study is to observe whether CRP may promote MMP-10 expression, and if so, to clarify signaling pathways to be involved in CRP-induced MMP-10 expression in cardiomyocytes. We observed in cultured cardiomyocytes that CRP at a dose of 5 μg/ml increased MMP-10 expression and activity in a time-dependent manner, as measured by real-time polymerase chain reaction (PCR), western blots, and casein zymography analysis. We hypothesized that signal pathways of mitogen-activated protein kinases (MAPKs) and Janus kinases (JAKs)/signal transducers and activators of transcription (STATs) might be involved in CRP-induced MMP-10 expression. Our results showed that CRP markedly activated c-Raf/MEK/ERK and JAK1/ERK signaling pathways but not JAK1/STAT3 signaling pathway by using the phosphor-specific antibodies against these pathways, and blockages of c-Raf/MEK/ERK and JAK1/ERK signaling pathways by the specific ERK1/2 inhibitor U0126 and JAK1 inhibitor piceatannol could significantly decrease CRP-induced MMP-10 expression. In addition, we demonstrated that the DNA binding sites of AP-1 and STAT3 in the nucleus of cardiomyocytes mediated CRP-induced MMP-10 expression. In conclusion, we demonstrated that CRP promoted MMP-10 expression and activity in cardiomyocytes, and clarified that c-Raf/MEK/ERK and JAK1/ERK signaling pathways were involved in MMP-10 expression regulation via activation of DNA binding sites for AP-1 and STAT3 in cardiomyocytes. Our findings suggest that CRP acts as a predictor for LV remodeling might be associated with its promotion effect on MMP-10 expression and activity.

Type-specific dysregulation of matrix metalloproteinases and their tissue inhibitors in end-stage heart failure patients: relationship between MMP-10 and LV remodelling.

Although past studies observed the changes of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in end‐stage heart failure (HF) patients, a consistent and clear pattern of type‐specific MMPs and/or TIMPs has yet to be further defined. In this study, proteomic approach of human protein antibody arrays was used to compare MMP and TIMP expression levels of left ventricular (LV) myocardial samples from end‐stage HF patients due to dilated cardiomyopathy (DCM) with those from age‐ and sex‐ matched non‐failing patients. Western blot analysis, immunohistochemistry and ELISA were used for validation of our results. We observed that MMP‐10 and ‐7 abundance increased, accompanied by decreased TIMP‐4 in DCM failing hearts (n= 8) compared with non‐failing hearts (n= 8). The results were further validated in a cohort of 34 end‐stage HF patients derived from three forms of cardiomyopathies. Cardiac and plasma MMP‐10 levels were positively correlated with the LV end‐diastolic dimension in this HF cohort. In addition, we observed that insulin‐like growth factor‐2 promoted MMP‐10 production in neonatal rat cardiomyocytes. In conclusion, this study demonstrated a selective up‐regulation of MMP‐10 and ‐7 along with a discordant change of TIMP‐4, and a positive correlation between MMP‐10 levels and the degree of LV dilation in end‐stage HF patients. Our findings suggest that type‐specific dysregulation of MMPs and TIMPs is associated with LV remodelling in end‐stage HF patients, and MMP‐10 may act as a novel biomarker for LV remodelling.