Jianfeng Hou

In Vitro Effects of Low-Level Laser Irradiation for Bone Marrow Mesenchymal Stem Cells: Proliferation, Growth Factors Secretion and Myogenic Differentiation

Bone marrow derived mesenchymal stem cells (BMSCs) have shown to be an appealing source for cell therapy and tissue engineering. Previous studies have confirmed that the application of low‐level laser irradiation (LLLI) could affect the cellular process. However, little is known about the effects of LLLI on BMSCs. The aim of this study was designed to investigate the influence of LLLI at different energy densities on BMSCs proliferation, secretion and myogenic differentiation.

MicroRNA-193 pro-proliferation effects for bone mesenchymal stem cells after low-level laser irradiation treatment through inhibitor of growth family, member 5.

The enhanced proliferation of mesenchymal stem cells (MSCs) can be helpful for the clinical translation of cell therapy. Low-level laser irradiation (LLLI) has been demonstrated as regulating MSC proliferation. MicroRNAs (miRNAs) are involved in various pathophysiologic processes in stem cells, but the role of miRNAs in the LLLI-based promotion of MSC proliferation remains unclear. We found that the proliferation level and cell cycle-associated genes in MSCs were increased after LLLI treatment in a time-dependent manner. Microarray assays revealed subsets of miRNAs to be differentially regulated, and these dynamic changes were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) after LLLI. miR-193 was the most highly up-regulated miRNA, and the change in it was related with the proliferation level. Gain-loss function experiments demonstrated that miR-193 could regulate the proliferation of MSCs, including humans and rats, but could not affect the apoptosis and differentiation level. Blockade of miR-193 repressed the MSC proliferation induced by LLLI. By qRT-PCR, we found that miR-193, in particular, regulated cyclin-dependent kinase 2 (CDK2) expression. Bioinformatic analyses and luciferase reporter assays revealed that inhibitor of growth family, member 5 (ING5) could be the best target of miR-193 to functionally regulate proliferation and CDK2 activity, and the mRNA and protein level of ING5 was regulated by miR-193. Furthermore, the ING5 inhibited by small interfering RNA (siRNA) could up-regulate the proliferation of MSCs and the expression of CDK2. Taken together, these results strongly suggest that miR-193 plays a critical part in MSC proliferation in response to LLLI stimulation, which is potentially amenable to therapeutic manipulation for clinical application.

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.

Low-level laser irradiation alters cardiac cytokine expression following acute myocardial infarction: a potential mechanism for laser therapy.

OBJECTIVES Low-level laser irradiation (LLLI) has the potential of exerting cardioprotective effect following myocardial infarction (MI). The authors hypothesized that LLLI could influence the expression of cardiac cytokines and contribute to the reversal of ventricular remodeling. BACKGROUND LLLI regulates the expression of cytokines after tissue damage. However, little is known concerning the alteration of the cardiac cytokine expression profile after LLLI. METHODS MI was created by coronary ligation. The surviving rats were divided randomly into laser and control groups. 33 rats were exposed to a diode laser (635  nm, 5  mW, CW, laser, beam spot size 0.8  cm(2), 6  mW/cm(2), 150  sec, 0.8  J, 1J/cm(2)) as laser group. Another 33 rats received only coronary ligation and served as control group. 28 rats received a thoracotomy without coronary ligation (sham group). One day after laser irradiation, 5 rats from each group were sacrificed and the heart tissues were analyzed by cytokine antibody arrays. Enzyme-linked immunosorbent assay (ELISA) was performed to confirm its reliability. Two weeks after MI, cardiac function and structure were evaluated by echocardiography and histological study. RESULTS Cytokine antibody array indicated 4 cytokines were significantly changed after laser therapy. ELISA confirmed that granulocyte-macrophage colony stimulating factor and fractalkine were the cytokines involved in the response to therapeutic laser irradiation. However, there was no difference in cytokine release between various groups at 2 weeks after MI. Although LLLI did not improve the damaged heart function, it did reduce the infarct area expansion. CONCLUSIONS The antibody-based protein array technology was applied for screening the cytokine expression profile following MI, with or without laser irradiation. The expression of multiple cytokines was regulated in the acute phase after LLLI. Our results revealed a potential novel mechanism for applying laser therapy to the treatment of heart disease.

Alteration of Parasympathetic/Sympathetic Ratio in the Infarcted Myocardium After Schwann Cell Transplantation Modified Electrophysiological Function of Heart A Novel Antiarrhythmic Therapy

Background— Neural remodeling after myocardial infarction (MI) may cause fatal ventricular arrhythmia. Schwann cells (SCs), which are important for neurogenesis, are dramatically reduced after MI. We investigated the feasibility of modifying nervous system regeneration after MI and the efficacy by which it may prevent ventricular arrhythmia following SC transplantation. Methods and Results— Immediately after creation of MI, syngenic Lewis rats were randomized into cell transplantation (n=80) and control groups (n=72). SCs were isolated from sciatic nerves, and 5×106 cells were intramyocardially injected into the infarct region. Expression levels of myocardial nerve growth factor, vascular endothelial growth factor, growth-associated protein 43, connexin 43, and laminin in the SC group were significantly higher than control at 7 and 14 days after cell transplantation. Immunohistochemical staining illustrated increases in sympathetic and parasympathetic nerves in both groups. However, SC transplantation significantly increased the parasympathetic/sympathetic ratio at 14 days after cell injection. Dynamic electrocardiography and programmed electric stimulation were also performed. The SCs significantly decreased the low-/high-frequency ratio and arrhythmia score of programmed electric stimulation-induced ventricular arrhythmia at 2 weeks after cell injection. However, SCs did not restore heart function. Conclusion— Transplanted SCs in the infarcted myocardium secrete multiple biological molecules, which alter the ratio of parasympathetic/sympathetic nerve density to normalize irritable myocardium. SC transplantation might be a novel cell-based antiarrhythmic therapy following MI.

Impact of Renal Dysfunction on Long-Term Survival After Isolated Coronary Artery Bypass Surgery

BACKGROUND Preoperative renal dysfunction has been an important predictor for adverse cardiovascular events after coronary artery bypass grafting (CABG). In the past, serum creatinine was widely used to assess renal function. Until recently, estimated glomerular filtration rate (eGFR) was recommended in evaluating renal function. The Cockcroft-Gault formula and the Modification of Diet in Renal Disease (MDRD) equation are two widely used formulas in clinical practice. Which method best predicts long-term outcome after CABG is still unknown. This study compared the predictive effectiveness of the Cockcroft-Gault formula, the MDRD equation, and serum creatinine level for in-hospital and long-term mortality. METHODS We retrospectively reviewed data collected from 5559 patients who underwent isolated CABG at Fuwai Hospital from January 1999 to December 2005. The main outcomes were in-hospital and long-term mortality. Receiver operating characteristic (ROC) curves and Cox analysis were used for the comparison. RESULTS Mean follow-up was 56.5 +/- 24.6 months. ROC curve analysis showed that the Cockcroft-Gault formula had the greatest accuracy for predicting in-hospital mortality (area under the curve, 0.755; p < 0.001). Multivariate analysis confirmed that the eGFR based on the Cockcroft-Gault formula was an independent predictor of in-hospital (odds ratio, 4.51, p < 0.001) and long-term (hazard ratio, 1.54; p = 0.003) mortality. Both formulas were better than the serum creatinine level. CONCLUSIONS Both formulas could provide a better measure of risk assessment than serum creatinine for in-hospital and long-term mortality. The Cockcroft-Gault formula was better than the MDRD equation for predicting in-hospital mortality.

Comparison between drug-eluting stents and coronary artery bypass grafting for unprotected left main coronary artery disease: a meta-analysis of two randomized trials and thirteen observational studies.

Objective: The clinical outcomes for unprotected left main coronary artery (LMCA) between coronary artery bypass grafting (CABG) and drug-eluting stents (DES) are still controversial. The objective was to compare safety and efficacy between DES and CABG for unprotected LMCA. Methods: Electronic databases and article references were systematically searched (2000–2010) to access relevant studies. Results: Fifteen studies with 5,479 patients were finally involved in the present study. The mortality was similar in DES and CABG groups at 1 year [odds ratio (95% confidence interval): 0.71 (0.5–1.03)], 2 years [1.28 (0.93–1.76)], 3 years [0.88 (0.53–1.46)], 4 years [0.46 (0.18–1.17)], and 5 years [1.16 (0.85–1.57)]. No significant difference was found between DES and CABG in the risk of composite endpoint of death, myocardial infarction and cerebrovascular events during 5 years’ follow-up [1 year, 0.95 (0.63–1.43); 2 years, 1.34 (0.67–2.57); 3 years, 1.06 (0.59–1.90); 4 years, 0.53 (0.26–1.08); 5 years, 0.83 (0.33–2.07)]. However, the risk of repeat revascularization was significantly higher in the DES group than the CABG group at 1 year [5.00 (2.85–8.77)], 2 years [4.79 (2.72–8.45)], 3 years [5.72 (3.07–10.65)], 4 years [2.16 (1.17–4.01)], and 5 years [5.65 (3.44–9.27)]. Conclusion: Our meta-analysis indicates that there are no significant differences in the safety between CABG and DES in patients with unprotected LMCA in the 5 years after intervention. But CABG is superior to DES for repeat revascularization.

Preoperative high-sensitivity C-reactive protein predicts depression in patients undergoing coronary artery bypass surgery: A single-center prospective observational study

OBJECTIVE Our objective was to reveal the contribution of preoperative high-sensitivity C-reactive protein (hsCRP) levels to the risk of depression in patients undergoing coronary artery bypass grafting (CABG). METHODS In this study, 232 consecutive patients undergoing elective CABG were evaluated for depression utilizing the 9-item Patient Health Questionnaire scale at least 3 days before surgery and 6 months after CABG. In addition, peripheral blood samples were collected at baseline and the circulation levels of hsCRP were measured. RESULTS The preoperative and postoperative rate of depression was 18.1%. Interestingly, preoperative depression was independently associated with women (odds ratio [OR], 2.49; 95% confidence interval [CI], 1.13-5.50) and log-transformed hsCRP (OR, 1.16; 95% CI, 1.06-1.27) after adjusting for various factors using logistic regression, including age, sex, and university-level education. Postoperative depression was only associated with log-transformed hsCRP (OR, 1.15; 95% CI, 1.05-1.25) in these patients. Log-transformed hsCRP remained associated with future depression after adjusting for postoperative medications or major adverse cardiovascular events during the follow-up period. CONCLUSIONS The present study is the first to report elevated serum hsCRP is an independent predictor for depression in CABG patients not only preoperatively but also up to 6 months after surgery. These clinical findings may reveal a potential target for improving prognosis in CABG patients with depression.

Vascularized Atrial Tissue Patch Cardiomyoplasty With Omentopexy Improves Cardiac Performance After Myocardial Infarction

BACKGROUND The tissue-engineered cardiac patch can alleviate ventricular remodeling and improve functional recovery in experimental myocardial infarction. However, the size of the engineered patch is limited due to insufficient vascularization. This study evaluated the effects of autologous atrial tissue patch cardiomyoplasty and omentopexy in rats with myocardial infarction. METHODS Myocardial infarction was induced by left coronary artery ligation in Sprague-Dawley rats. Three weeks later, either a patch of left atrium (A group) or omentum (O group) or both (OA group) were placed over the infarct zone. The atrial tissue patch was harvested from the autologous left atrial appendage along its long axis. The rats in the Control group received rethoracotomy only. After 4 weeks, the survival of the transplanted atrial tissue patch, ventricular remodeling, and cardiac performance were examined. RESULTS After 4 weeks, surviving myocardium was only detected in the OA group, as indicated by immunolabeling of cardiac troponin-I. Compared with the Control group, only animals in the OA group showed improved heart function assessed by left ventricular ejection fraction (57.9% ± 5.8% vs 47.5% ± 4.5%, p < 0.05) and left ventricular fractional shortening (25.2% ± 3.6% vs 20.7% ± 2.0%, p < 0.05). The histologic analysis demonstrated increased scar thickness in the OA group. This was accompanied by increased angiogenesis of the border zone but decreased expression and activity of matrix metalloproteinase and endothelin-1 levels. CONCLUSIONS The omentopexy supported the survival of the autologous atrial tissue patch, which resulted in attenuated ventricular remodeling and restoration of heart function in rats with myocardial infarction. Our findings might represent a novel therapeutic strategy for heart failure.

Cardiomyocyte cytokinesis score: a potential method for cardiomyocyte proliferation

One of the most important indicators of myocardial regeneration is cardiomyocyte proliferation. However, it is difficult to distinguish cardiomyocytes in the regenerating stage from binucleated or multinucleated myocytes by conventional morphometric techniques. As cell cycle progression (CCP) scores have been successfully applied to the evaluation of the proliferation of cancer cells, we sought to establish a multi‐gene score to evaluate cardiomyocyte proliferation in this study. Given the disturbances of nuclear division without cell division that occurs in cardiomyocytes, ten cytokinesis‐correlated genes (Anln, Aurkb, Cenpa, Kif4, Kif23, Prc1, RhoA, Spin1, TACC2, and CDC42) were chosen to establish the cardiomyocyte cytokinesis score (CC‐Score). The expression levels of these genes in H9C2 rat cardiomyoblast cells, the proliferation of which were stimulated or inhibited, were detected using qRT‐PCR. To confirm the feasibility of the CC‐Score system, four conventional methods for evaluating cardiomyocyte proliferation, including the MTT assay, BrdU assay, immunofluorescence, and flow cytometry analysis, were used in each group. The results of the CC‐Score in the assessment of the proliferation of H9C2 cells were consistent with those of four commonly used proliferative assay methods. We conclude that the CC‐Score can be used to assess the proliferation status of H9C2 cells, and suggest that the CC‐Score may be a potential method for the assessment of cardiomyocyte proliferation in myocardial regeneration. However, validation studies utilizing primary cultured rat cardiomyocytes and heart tissue are warranted.