Ji-Chang Zhang

Cardioprotective effects of peroxisome proliferator activated receptor γ activators on acute myocarditis: anti-inflammatory actions associated with nuclear factor κB blockade

Objective: To test the hypothesis that activation of peroxisome proliferator activated receptor γ (PPAR-γ) reduces experimental autoimmune myocarditis (EAM) associated with inhibitor κB (IκB) α induction, blockade of nuclear factor κB (NF-κB), and inhibition of inflammatory cytokine expression. Methods: EAM was induced in Lewis rats by immunisation with porcine cardiac myosin. PPAR-γ activators 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and pioglitazone (PIO) were administered to rats with EAM. Results: Enhanced PPAR-γ expression was prominently stained in the nuclear and perinuclear regions of infiltrating inflammatory cells. Administration of 15d-PGJ2 and PIO greatly reduced the severity of myocarditis and suppressed myocardial mRNA and protein expression of inflammatory cytokines in rats with EAM. In addition, treatment with PPAR-γ activators enhanced IκB concentrations in the cytoplasmic fractions and nuclear fractions from inflammatory myocardium. Concurrently, NF-κB was greatly activated in myocarditis; this activation was blocked in the 15d-PGJ2 treated and PIO treated groups. Conclusions: PPAR-γ may have a role in the pathophysiology of EAM. Because an increase in IκB expression and inhibition of translocation of the NF-κB subunit p65 to the nucleus in inflammatory cells correlated with the protective effects of PPAR-γ activators, these results suggest that PPAR-γ activators act sequentially through PPAR-γ activation, IκB induction, blockade of NF-κB activation, and inhibition of inflammatory cytokine expression. These results suggest that PPAR-γ activators such as 15d-PGJ2 and PIO may have the potential to modulate human inflammatory heart diseases such as myocarditis.

Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1

Insulin-like growth factor-1 (IGF-1) is an important regulator of cardiomyocyte homeostasis and cardiac structure, and the prosurvival and antiapoptotic effects of IGF-1 have been investigated. However, the effect of microRNA-320 (miR-320) in ischemia and reperfusion (I/R) by targeting IGF-1 is rarely discussed. We investigated the role of miR-320 in I/R injury. A total of 192 healthy female Wistar rats were divided into eight groups (n = 24). Rat heart I/R model was established. Hemodynamics, infarct size weight (ISW), heart function, and rat cardiomyocyte apoptosis were measured. Hypoxia-reoxygenation (H/R) in rat cardiomyocyte was used to simulate the I/R process. The mRNA levels of miR-320 and IGF-1, and proteins levels of IGF-1, IGF-1R, p-IGF-1R, p-ASK1, p-JNK, p-p38, Bcl-2, Bax and Caspase-3 were measured. In vivo inhibition of miR-320 expression significantly increased IGF-1 and IGF-1R mRNA levels, elevated the absolute values of SBP, DBP, MAP, ± dp/dtmax, LVEF and LVFS, decreased ISW, LVESD and LVEDd and the number of TUNEL positive cells, lowered the levels of p-ASK1, p-JNK, p-p38, Bax and Caspase-3 and increased expression of Bcl-2 compared to the I/R + NC group. Compared to H/R + NC group in vitro, miR-320 inhibition increased IGF-1 mRNA levels, inhibited cardiomyocyte apoptosis, down-regulated p-ASK, p-JNK, p-p38, Bax and Caspase-3 levels, and up-regulated Bcl-2 level. MiR-320 inhibition target elevated IGF-1 mRNA and protein levels, suppress early cardiomyocyte apoptosis of I/R, and inhibited ASK1-JNK/p38 pathway, which provides a new target for clinical study of I/R injury.

The Protective Effect of MicroRNA-320 on Left Ventricular Remodeling after Myocardial Ischemia-Reperfusion Injury in the Rat Model

The primary objective of this study investigated the role of microRNA-320 (miR-320) on left ventricular remodeling in the rat model of myocardial ischemia-reperfusion (I/R) injury, and we intended to explore the myocardial mechanism of miR-320-mediated myocardium protection. We collected 120 male Wistar rats (240–280 g) in this study and then randomly divided them into three groups: (1) sham surgery group (sham group: n = 40); (2) ischemia-reperfusion model group (I/R group: n = 40); and (3) I/R model with antagomir-320 group (I/R + antagomir-320 group: n = 40). Value changes of heart function in transesophageal echocardiography were recorded at various time points (day 1, day 3, day 7, day 15 and day 30) after surgery in each group. Myocardial sections were stained with hematoxylin and eosin (H&E) and examined with optical microscope. The degree of myocardial fibrosis was assessed by Sirius Red staining. Terminal dUTP nick end-labeling (TUNEL) and qRT-PCR methods were used to measure the apoptosis rate and to determine the miR-320 expression levels in myocardial tissues. Transesophageal echocardiography showed that the values of left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular systolic pressure (LVSP) and ±dp/dtmax in the I/R group were obviously lower than those in the sham group, while the left ventricular end-diastolic pressure (LVEDP) value was higher than that in the sham group. The values of LVEF, LVFS, LVSP and ±dp/dtmax showed a gradual decrease in the I/R group, while the LVEDP value showed an up tendency along with the extension of reperfusion time. The H&E staining revealed that rat myocardial tissue in the I/R group presented extensive myocardial damage; for the I/R + antagomir-320 group, however, the degree of damage in myocardial cells was obviously better than that of the I/R group. The Sirius Red staining results showed that the degree of myocardial fibrosis in the I/R group was more severe along with the extension of the time of reperfusion. For the I/R + antagomir-320 group, the degree of myocardial fibrosis was less severe than that in the I/R group. Tissues samples in both the sham and I/R + antagomir-320 groups showed a lower apoptosis rate compared to I/R group. The qRT-PCR results indicated that miR-320 expression in the I/R group was significantly higher than that in both the sham and I/R + antagomir-320 groups. The expression level of miR-320 is significantly up-regulated in the rat model of myocardial I/R injury, and it may be implicated in the prevention of myocardial I/R injury-triggered left ventricular remodeling.

MicroRNA-130a alleviates human coronary artery endothelial cell injury and inflammatory responses by targeting PTEN via activating PI3K/Akt/eNOS signaling pathway

Our study aims to investigate the roles of microRNA-130a (miR-130a) in human coronary artery endothelial cells (HCAECs) injury and inflammatory responses by targeting PTEN through the PI3K/Akt/eNOS signaling pathway. HCAECs were treated with 1.0 mmol/L homocysteine (HCY) and assigned into eight groups: the blank group, the negative control (NC) group, the miR-130a mimics group, the miR-130a inhibitors group, the si-PTEN group, the Wortmannin group, the miR-130a inhibitors + si-PTEN group and the miR-130a mimics + Wortmannin group. Luciferase reporter gene assay was used to validate the relationship between miR-130a and PTEN. The expressions of miR-130a, PTEN and PI3K/Akt/eNOS signaling pathway-related proteins were detected by qRT-PCR assay and Western blotting. MTT assay and Hoechst 33258 staining were adopted to testify cell growth and apoptosis. The NO kit assay was used to detect the NO release. ELISA was conducted to measure serum cytokine levels. Luciferase reporter gene assay confirmed the target relationship between miR-130a and PTEN. Compared with the blank and NC groups, the miR-130a mimics and si-PTEN groups showed significant increases in the expressions of PI3K/Akt/eNOS signaling pathway-related proteins, cell viability and the NO release, while serum cytokine levels and cell apoptosis were decreased; by contrast, an opposite trend was observed in miR-130a inhibitors and Wortmannin groups. However, no significant difference was found in the miR-130a inhibitors + si-PTEN and miR-130a mimics + Wortmannin groups when compared with the blank group. These results indicate that miR-130a could alleviate HCAECs injury and inflammatory responses by down-regulating PTEN and activating PI3K/Akt/eNOS signaling pathway.

Anti‐Apoptotic Effect of MicroRNA‐30b in Early Phase of Rat Myocardial Ischemia‐Reperfusion Injury Model

This study aimed to investigate the effect of microRNA‐30b (miR‐30b) in rat myocardial ischemic‐reperfusion (I/R) injury model. We randomly divided Sprague–Dawley (SD) rats (n = 80) into five groups: 1) control group; 2) miR‐30b group; 3) sham‐operated group; 4) I/R group, and 5) I/R+miR‐30b group. Real‐time quantitative polymerase chain reaction, immunohistochemical staining and Western blot analysis were conducted. TUNEL assay was employed for testing cardiomyocyte apoptosis. Our results showed that miR‐30b levels were down‐regulated in I/R group and I/R + miR‐30b group compared with sham‐operated group (both P < 0.05). However, miR‐30b level in I/R + miR‐30b group was higher than I/R group (P < 0.05). Markedly, the apoptotic rate in I/R group showed highest in I/R group (P < 0.05). Additionally, the results illustrated that protein levels of Bcl‐2, Bax, and caspase‐3 were at higher levels in ischemic regions in I/R group, comparing to sham‐operated group (all P < 0.05), while Bcl‐2/Bax was reduced (P < 0.05). Bcl‐2 level and Bcl‐2/Bax were obviously increased in I/R + miR‐30b group by comparison with I/R group, and expression levels of Bax and caspase‐3 were down‐regulated (all P < 0.05). We also found that in I/R + miR‐30b group, KRAS level was apparently lower and p‐AKT level was higher by comparing with I/R group (both P < 0.05). Our study indicated that miR‐30b overexpression had anti‐apoptotic effect on early phase of rat myocardial ischemia injury model through targeting KRAS and activating the Ras/Akt pathway. J. Cell. Biochem. 116: 2610–2619, 2015.

MicroRNA-210 Modulates the Cellular Energy Metabolism Shift During H2O2-Induced Oxidative Stress by Repressing ISCU in H9c2 Cardiomyocytes

Background/Aims: The myocardial energy metabolism shift is one of the most important pathological features of ischemic heart disease (IHD). Although several microRNAs (miRs) are involved in the regulation of myocardial energy metabolism, their exact effects and underlying mechanisms remain unclear. The aim of this study was to investigate whether microRNA(miR-210) regulates the energy metabolism shift during oxidative stress in H9c2 cardiomyocytes. Methods: Cell survival was analyzed via CCK assay. The energy metabolism shift was detected by lactate assay, ATP assay and RT2 profiler glucose metabolism PCR array. Protein and mRNA expression levels were determined by western blot and qPCR. We also used kits to detect the activity of Complex I, Sirt3 and the NAD+/NADH ratio. Results: We determined that miR-210 promoted the energy metabolism shift. The iron-sulfur cluster assembly protein (ISCU) was a target of miR-210. Additionally, we detected the activity of complex I and found that miR-210 inhibits mitochondrial respiration. Interestingly, miR-210 may also indirectly regulate SIRT3 by regulating ISCU. Conclusion: Our results confirm that miR-210 is essential and sufficient for modulating the cellular energy metabolism shift during H2O2-induced oxidative stress in H9c2 cardiomyocytes by targeting ISCU.

Inhibition of long noncoding RNA IGF2AS promotes angiogenesis in type 2 diabetes

BACKGROUND In this study, the angiogenic effect of long noncoding RNA (lncRNA), insulin growth factor 2 antisense (IGF2AS) in type 2 diabetes was evaluated. METHODS Between Wistar rat and Goto-Kakizaki (GK) rat, a genetic model of type 2 diabetes, mRNA expressions of IGF2AS and IGF2 in myocardial microvascular endothelial (mMVE) cells were compared by qRT-PCR. In GK mMVE cells, IGF2AS was inhibited by siRNA. Its effects on cell proliferation and invasion were evaluated by MTT and wound-healing assays. Also, changes of IGF2, VEGF and IGF1 in siRNA-transfected GK mMVE cells were evaluated by qRT-PCR and western blot. In IGF2AS-inhibited GK mMVE cells, IGF2 was further downregulated to evaluate its role in IGF2AS-associated angiogenic regulation, using MTT, wound-healing qRT-PCR and western blot assays, respectively. RESULTS IGF2AS was upregulated, whereas IGF2 was downregulated, in diabetic GK mMVE cells. IGF2AS inhibition augmented proliferation and invasion in GK mMVE cells. It also upregulated IGF2 and VEGF (not IGF1) at both molecular and protein levels. Conversely, IGF2 downregulation upregulated IGF2AS and reversely inhibited angiogenic effect of IGF2AS inhibition in GK mMVE cells. It also downregulated VEGF but had no effect on IGF1. CONCLUSION IGF2AS inhibition has angiogenic effect in diabetic GK mMVE cells. The functions of IGF2AS in type 2 diabetes are very likely through the inverse regulation of IGF2, but independent of IGF1.

Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

BackgroundOxidative stress can induce cell injury in vascular endothelial cells, which is the initial event in the development of atherosclerosis. Although quantitative real-time polymerase chain reaction (qRT-PCR) has been widely used in gene expression studies in oxidative stress injuries, using carefully validated reference genes has not received sufficient attention in related studies. The objective of this study, therefore, was to select a set of stably expressed reference genes for use in qRT-PCR normalization in oxidative stress injuries in human umbilical vein endothelial cells (HUVECs) induced by hydrogen peroxide (H2O2).ResultsUsing geNorm analysis, we found that five stably expressed reference genes were sufficient for normalization in qRT-PCR analysis in HUVECs treated with H2O2. Genes with the most stable expression according to geNorm were U6, TFRC, RPLP0, GAPDH, and ACTB, and according to NormFinder were ALAS1, TFRC, U6, GAPDH, and ACTB.ConclusionTaken together, our study demonstrated that the expression stability of reference genes may differ according to the statistical program used. U6, TFRC, RPLP0, GAPDH, and ACTB was the optimal set of reference genes for studies on gene expression performed by qRT-PCR assays in HUVECs under oxidative stress study.

Protection of Human Umbilical Vein Endothelial Cells against Oxidative Stress by MicroRNA-210

Oxidative stress induces endothelial cell apoptosis and promotes atherosclerosis development. MicroRNA-210 (miR-210) is linked with apoptosis in different cell types. This study aimed to investigate the role of miR-210 in human umbilical vein endothelial cells (HUVECs) under oxidative stress and to determine the underlying mechanism. HUVECs were treated with different concentrations of hydrogen peroxide (H2O2), and cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ATP assay. To evaluate the role of miR-210 in H2O2-mediated apoptosis, gain-and-loss-of-function approaches were used, and the effects on apoptosis and reactive oxygen species (ROS) level were assayed using flow cytometry. Moreover, miR-210 expression was detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and expression of the following apoptosis-related genes was assessed by qRT-PCR and Western blot at the RNA and protein level, respectively: caspase-8-associated protein 2 (CASP8AP2), caspase-8, and caspase-3. The results showed that H2O2 induced apoptosis in HUVECs in a dose-dependent manner and increased miR-210 expression. Overexpression of miR-210 inhibited apoptosis and reduced ROS level in HUVECs treated with H2O2. Furthermore, miR-210 downregulated CASP8AP2 and related downstream caspases at protein level. Thus, under oxidative stress, miR-210 has a prosurvival and antiapoptotic effect on HUVECs by reducing ROS generation and downregulating the CASP8AP2 pathway.

Initial clinical experience of CrossBoss catheter for in-stent chronic total occlusion lesions: A case report

Background:The CrossBoss coronary chronic total occlusion (CTO) crossing catheter has been demonstrated to have greatly improved the success rate of crossing CTO lesions, but there are no published data on its application for in-stent CTO lesions. Methods:In the current study, we retrospectively reviewed the clinical data of 8 patients with in-stent CTO lesions that were managed with the CrossBoss catheter and herein we report the efficacy and safety of the CrossBoss crossing and re-entry system for this clinically challenging condition. Results:The CrossBoss catheter was used for 8 patients with in-stent CTO lesions, which resulted in success in 6 cases and failure in 2 cases, with a 75% success rate. Of the 6 patients with successful treatment, 5 cases had the occlusive lesions crossed with the CrossBoss catheter through a proximal lumen-to-distal lumen approach, whereas the remaining case had his occlusive lesions penetrated by the CrossBoss catheter and the guidewire. Two cases failed in treatment as the CrossBoss catheter could not cross the occlusive lesions. The 6 cases with successful treatment included 3 cases with occlusive lesions in the left anterior descending artery, 1 case with occlusive lesions in the obtuse marginal branches, and 2 cases with occlusive lesions in the right coronary artery, and the 2 cases with failure in treatment had their occlusive lesions in the right coronary artery. In addition, patients with a higher Japan chronic total occlusion score were found to have a lower success rate of crossing the occlusive lesions. None of the patients developed complications. Conclusion:Our study demonstrates that the CrossBoss catheter has a high success rate and is safe for in-stent CTOs and can be recommended for this rather clinically challenging condition.