Chuan-Jue Cui

In Vitro Effects of Pirfenidone on Cardiac Fibroblasts: Proliferation, Myofibroblast Differentiation, Migration and Cytokine Secretion

Cardiac fibroblasts (CFs) are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remodeling such as proliferation, myofibroblast differentiation, migration and cytokine secretion. Fibroblasts were isolated from neonatal rat hearts and bioassays were performed to determine the effects of pirfenidone on fibroblast function. We demonstrated that treatment of CFs with pirfenidone resulted in decreased proliferation, and attenuated fibroblast α-smooth muscle actin expression and collagen contractility. Boyden chamber assay illustrated that pirfenidone inhibited fibroblast migration ability, probably by decreasing the ratio of matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1. Furthermore, pirfenidone attenuated the synthesis and secretion of transforming growth factor-β1 but elevated that of interleukin-10. These direct and pleiotropic effects of pirfenidone on cardiac fibroblasts point to its potential use in the treatment of adverse myocardial remodeling.

Upregulated expression of cardiac ankyrin repeat protein in human failing hearts due to arrhythmogenic right ventricular cardiomyopathy

Expression of cardiac ankyrin repeat protein (CARP) is augmented in heart failure due to dilated or ischaemic cardiomyopathy. It is unclear whether CARP is upregulated in heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC). In the present study, we investigated the expression pattern of CARP and the correlation between CARP and the well‐known heart failure marker pro‐atrial natriuretic peptide (proANP) in ARVC failing hearts.

Positive correlation of plasma PCSK9 levels with HbA1c in patients with type 2 diabetes

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been demonstrated to be involved in not only lipid metabolism but also glucose homeostasis. Glycated haemoglobin (HbA1c) is a ‘gold standard’ for monitoring long‐term glycaemic control. However, the correlation of plasma PCSK9 levels with HbA1c remains undetermined.

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.

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.

Familial Hypercholesterolemia Phenotype in Chinese Patients Undergoing Coronary Angiography.

Objective— Familial hypercholesterolemia (FH) is characterized by an elevated low-density lipoprotein cholesterol and increased risk of premature coronary artery disease. However, the general picture and mutational spectrum of FH in China are far from recognized, representing a missed opportunity for the investigation. Approach and Results— A total of 8050 patients undergoing coronary angiography were enrolled. The diagnosis of clinical FH was made using Dutch Lipid Clinic Network criteria, and the information of relatives was obtained by inquiring for the probands or from their own medical records of certain clinics/hospitals. Molecular analysis of FH was performed using target exome sequencing in LDLR (low-density lipoprotein cholesterol receptor gene), APOB (apolipoprotein B gene), and PCSK9 (proprotein convertase subtilisin/kexin type 9 gene). As a result, 3.5% of the patients with definite/probable FH phenotype (definite 1.0% and probable 2.5%) were identified. Women FH had fewer premature coronary artery disease (women <60, or men <55 years of age) when compared with men FH (70.6% versus 82.7%; P<0.001), whereas angiographic extension of coronary artery disease was significantly increased with FH diagnosis in both men and women (P<0.001). Patterns of medication use in definite/probable FH were as follows: nontreated, 20.6%; low intensity, 6.0%; moderate intensity, 68.3%; and high intensity, 5.0%. However, none of them had achieved the low-density lipoprotein cholesterol <100 mg/dL. Additionally, mutational analysis was performed in 245 definite/probable FH cases, and risk variants were identified in 115 patients, giving a detection rate of 46.9%. Conclusions— We showed firsthand a common identification but poor treatment of patients with FH phenotype in Chinese coronary angiography patients. Genetic data in our FH cases might contribute to update the frequency and spectrum of Chinese FH scenarios.

Up-regulated lipocalin-2 in pulmonary hypertension involving in pulmonary artery SMC resistance to apoptosis.

A key feature of pulmonary hypertension (PH) is the remodeling of small pulmonary arteries due to abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. However, the cellular mechanisms underlying how PASMCs in the pathological condition of pulmonary hypertension become resistant to apoptosis remain unknown. It was recently reported that lipocalin 2 (Lcn2) is up-regulated in a wide array of malignant conditions, which facilitates tumorigenesis partly by inhibiting cell apoptosis. In this study, we observed that the expression levels of Lcn2 were significantly elevated in a rat PH model induced with monocrotaline and in patients with congenital heart disease-associated PH (CHD-PH) when compared with respective control. Therefore, we hypothesize that Lcn2 could regulate human PASMC (HPASMC) apoptosis through a mechanism. By the detection of DNA fragmentation using the TUNEL assay, the detection of Annexin V/PI-positive cells using flow cytometry, and the detection of cleaved caspase-3 and caspase-3 activity, we observed that Lcn2 significantly inhibited HPASMC apoptosis induced by serum withdrawal and H2O2 treatment. We also observed that Lcn2 down-regulated the proapoptotic protein Bax, decreased the levels of cellular ROS, and up-regulated the expression of superoxide dismutases (SOD1 and SOD2). In conclusion, Lcn2 significantly inhibits HPASMC apoptosis induced by oxidative stress via decreased intracellular ROS and elevated SODs. Up-regulation of Lcn2 in a rat PH model and CHD-PH patients may be involved in the pathological process of PH.

HDL subfractions and very early CAD: novel findings from untreated patients in a Chinese cohort.

Coronary artery disease (CAD) in very young individuals is a rare disease associated with poor prognosis. However, the role of specific lipoprotein subfractions in very young CAD patients (≤45 years) is not established yet. A total of 734 consecutive CAD subjects were enrolled and were classified as very early (n = 81, ≤45), early (n = 304, male: 45–55; female: 45–65), and late (n = 349, male: >55; female: >65) groups. Meanwhile, a group of non-CAD subjects were also enrolled as controls (n = 56, ≤45). The lipoprotein separation was performed using Lipoprint System. As a result, the very early CAD patients have lower large high-density lipoprotein (HDL) subfraction and higher small low-density lipoprotein (LDL) subfraction (p < 0.05). Although body mass index was inversely related to large HDL subfraction, overweight did not influence its association with very early CAD. In the logistic regression analysis, large HDL was inversely [OR 95% CI: 0.872 (0.825–0.922)] while small LDL was positively [1.038 (1.008–1.069)] related to very early CAD. However, after adjusting potential confounders, the association was only significant for large HDL [0.899 (0.848–0.954)]. This study firstly demonstrated that large HDL subfraction was negatively related to very early CAD suggestive of its important role in very early CAD incidence.

Large HDL Subfraction But Not HDL-C Is Closely Linked With Risk Factors, Coronary Severity and Outcomes in a Cohort of Nontreated Patients With Stable Coronary Artery Disease: A Prospective Observational Study.

AbstractHigh-density lipoprotein (HDL) is highly heterogeneous in its size and composition. Till now, the link of HDL subfractions to coronary risk is less clear. We aimed to investigate the associations of HDL subfractions with traditional risk factors (RFs), coronary severity, and outcomes in a cohort of nontreated patients with stable coronary artery disease (CAD).We prospectively enrolled 591 eligible patients. Baseline HDL subfractions were separated by Lipoprint system. HDL subfractions (large, medium, and small) and HDL-cholesterol (HDL-C) levels were dichotomized into low and high group according to the 50 percentile. Coronary severity was evaluated by SYNTAX, Gensini, and Jeopardy scoring systems. Patients were followed up annually for major adverse cardiovascular events (MACEs). Cox proportional hazards’ models were used to evaluate the risk of HDL subfractions on MACEs.Patients with high large HDL-C levels had a decreased number of RFs. Significantly, large HDL-C levels were negatively associated with coronary severity assessed by SYNTAX and Gensini score (both P < 0.05). New MACEs occurred in 67 (11.6%) patients during a median 17.0 months follow-up. Moreover, the log-rank test revealed that there was a significant difference between high and low large HDL-C groups in event-free survival analysis (P = 0.013), but no differences were observed in total HDL-C groups and medium or small HDL-C groups (both P > 0.05). In particular, the multivariate Cox-proportional hazards model revealed that high large HDL-C was associated with lower MACEs risk (hazard ratio [95% confidence interval] 0.531 [0.295–0.959]) independent of potential confounders.Higher large HDL-C but not medium, small, or total HDL-C is associated with lower cardiovascular risk, highlighting the potential beneficial of HDL subfractionation.