Zhang M

NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy in a Type 2 Diabetes Rat Model

Background Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved. Methods Type 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3 inflammasome activation. Results Diabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure, fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1β (IL-1β). Evidence for pyroptosis was found in vivo, and the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo did not attenuate systemic metabolic disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflammasome, and mature IL-1β in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3 inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1β. Conclusion NLRP3 inflammasome contributed to the development of DCM. NF-κB and TXNIP mediated the ROS-induced caspase-1 and IL-1β activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a protective effect on DCM.

Adiponectin Inhibits Lipopolysaccharide-Induced Adventitial Fibroblast Migration and Transition to Myofibroblasts via AdipoR1-AMPK-iNOS Pathway

Adiponectin is an important antiatherogenic adipocytokine that inhibits inflammation, insulin resistance, and oxide stress. Inflammation in the vascular adventitia is a crucial factor in the pathogenesis of atherosclerosis. Adventitial fibroblasts (AFs) can proliferate, divide into myofibroblasts, and migrate to the intima to become a new component of atherosclerotic plaque under inflammation and atherosclerosis. We investigated whether adiponectin might prevent AFs from proliferating, migrating, and transforming into myofibroblasts. Cultured AFs were stimulated with lipopolysaccharide (LPS) in the presence or absence of adiponectin. Methyl thiazolyl tetrazolium assay and migration and scratch-wound assays demonstrated that adiponectin reduced the AF proliferation and migration induced by LPS, respectively, whereas treatment with AdipoR1 small interfering (si) RNA (siAdipoR1), AMP-activated protein kinase (AMPK) siRNA (siAMPK), and an AMPK inhibitor reversed the effect. Immunocytochemistry and Western blot revealed that adiponectin reduced the transition of AFs to myofibroblasts, and treatment with siAdipoR1, siAMPK, and the AMPK inhibitor increased the transition. RT-PCR, Western blotting, and nitric oxide (NO) assay showed that adiponectin reduces induced NO synthase (iNOS) and nitrotyrosine expression and NO and ONOO(-) production induced by LPS. Treatment with siAdipoR1, siAMPK, and the AMPK inhibitor significantly attenuated adiponectin-induced phosphorylation of AMPK and its downstream target acetyl-coenzyme A carboxylase and up-regulated iNOS mRNA and protein expression, which resulted in a marked increase of NO and ONOO(-) production. In apolipoprotein E-deficient mice, immunohistochemistry of treated vascular adventitia showed that both iNOS expression and ONOO(-) production could be reversed with an adenovirus-adiponectin vector. Taken together, these results suggest that adiponectin reduces LPS-induced NO production and nitrosative stress and prevents AFs from proliferating, transforming to myoflbroblasts, and migrating to the intima, thus worsening atherosclerosis, by inhibiting the AdipoR1-AMPK-iNOS pathway in AFs.

Inhibition of high-mobility group box 1 improves myocardial fibrosis and dysfunction in diabetic cardiomyopathy

BACKGROUND High-mobility group box 1 (HMGB1) is an important mediator of the inflammatory response. Its expression is increased in diabetic cardiomyopathy (DCM), but its role is unclear. We investigated the potential role and mechanism of HMGB1 in diabetes-induced myocardial fibrosis and dysfunction in mice. METHODS In vivo, type 1 diabetes was induced by streptozotocin (STZ) in mice. HMGB1 expression was knocked down by lentivirus-mediated short-hairpin RNA (shRNA). Cardiac function was assessed by echocardiography. Total collagen deposition was assessed by Massons trichrome and Picrosirius red staining. HMGB1, collagen I and III, and transforming growth factor β1 (TGF-β1) expression was quantified by immunostaining and western bolt analysis. In vitro, isolated neonatal cardiac fibroblasts were treated with high glucose (HG) or recombinant HMGB1 (rHMGB1). Pharmacologic (neutralizing anti-HMGB1 antibody) or genetic (shRNA-HMGB1) inhibition of HMGB1 was used to investigate the role of HMGB1 in HG-induced functional changes of cardiac fibroblasts. RESULTS In vivo, HMGB1 was diffusely expressed in the myocardium of diabetic mice. HMGB1 silencing ameliorated left ventricular dysfunction and remodeling and decreased collagen deposition in diabetic mice. In vitro, HG induced HMGB1 translocation and secretion in both viable cardiomyocytes and fibroblasts. Administration of rHMGB1 dose-dependently increased the expression of collagens I and III and TGF-β1 in cardiac fibroblasts. HMGB1 inhibition reduced HG-induced collagen production, matrix metalloproteinase (MMP) activities, proliferation, and activated mitogen-activated protein kinase signaling in cardiac fibroblasts. CONCLUSIONS HMGB1 inhibition could alleviate cardiac fibrosis and remodeling in diabetic cardiomyopathy. Inhibition of HMGB1 might have therapeutic potential in the treatment of the disease.

Arginase I Attenuates Inflammatory Cytokine Secretion Induced by Lipopolysaccharide in Vascular Smooth Muscle Cells

Objective—Inflammation plays an important role in atherosclerosis. Arginase I (Arg I) promotes the proliferation of vascular smooth muscle cells; however, the effect of Arg I on inflammation remains unknown. The present study investigated the role of Arg I in inflammation in vitro and in vivo. Methods and Results—Quantitative reverse transcription–polymerase chain reaction and Western blot analysis demonstrated that Arg I inhibited tumor necrosis factor-&agr; production induced by lipopolysaccharide in human aortic smooth muscle cells. Inducible nitric oxide synthase substrate competition and nuclear factor-&kgr;B activation were main contributors to lipopolysaccharide-mediated inflammatory cytokine generation. However, Arg I could attenuate the function of inducible nitric oxide synthase and inhibit the subsequent nuclear factor-&kgr;B activation, leading to inhibition of tumor necrosis factor-&agr; generation. Furthermore, upregulation of Arg I significantly decreased macrophage infiltration and inflammation in atherosclerotic plaque of rabbits, whereas downregulation of Arg I aggravated these adverse effects. Conclusion—The results indicate the antiinflammatory effects of Arg I and suggest an unexpected beneficial role of Arg I in inflammatory disease.

MicroRNA-7a/b protects against cardiac myocyte injury in ischemia/reperfusion by targeting poly(ADP-ribose) polymerase.

Objectives MicroRNA-7 (miR-7) is highly connected to cancerous cell proliferation and metastasis. It is also involved in myocardial ischemia-reperfusion (I/R) injury and is upregulated in cardiomyocyte under simulated I/R (SI/R). We aimed to investigate the role of miR-7 during myocardial I/R injury in vitro and in vivo and a possible gene target. Methods and Results Real-time PCR revealed that miR-7a/b expression was upregulated in H9c2 cells after SI/R. Flow cytometry showed SI/R-induced cell apoptosis was decreased with miR-7a/b mimic transfection but increased with miR-7a/b inhibitor in H9c2 cells. In a rat cardiac I/R injury model, infarct size determination and TUNEL assay revealed that miR-7a/b mimic decreased but miR-7a/b inhibitor increased cardiac infarct size and cardiomyocyte apoptosis as compared with controls. We previously identified an important gene connected with cell apoptosis -- poly(ADP-ribose) polymerase (PARP) -- as a candidate target for miR-7a/b and verified the target by luciferase reporter activity assay and western blot analysis. Conclusions miR-7a/b is sensitive to I/R injury and protects myocardial cells against I/R-induced apoptosis by negatively regulating PARP expression in vivo and in vitro. miR-7a/b may provide a new therapeutic approach for treatment of myocardial I/R injury. Poly(ADP-ribose) polymerase.

HMGB1 mediates hyperglycaemia‐induced cardiomyocyte apoptosis via ERK/Ets‐1 signalling pathway

Apoptosis is a key event involved in diabetic cardiomyopathy. The expression of high mobility group box 1 protein (HMGB1) is up‐regulated in diabetic mice. However, the molecular mechanism of high glucose (HG)‐induced cardiomyocyte apoptosis remains obscure. We aimed to determine the role of HMGB1 in HG‐induced apoptosis of cardiomyocytes. Treating neonatal primary cardiomyocytes with HG increased cell apoptosis, which was accompanied by elevated levels of HMGB1. Inhibition of HMGB1 by short‐hairpin RNA significantly decreased HG‐induced cell apoptosis by reducing caspase‐3 activation and ratio of Bcl2‐associated X protein to B‐cell lymphoma/leukemia‐2 (bax/bcl‐2). Furthermore, HG activated E26 transformation‐specific sequence‐1 (Ets‐1), and HMGB1 inhibition attenuated HG‐induced activation of Ets‐1 via extracellular signal‐regulated kinase 1/2 (ERK1/2) signalling. In addition, inhibition of Ets‐1 significantly decreased HG‐induced cardiomyocyte apoptosis. Similar results were observed in streptozotocin‐treated diabetic mice. Inhibition of HMGB1 by short‐hairpin RNA markedly decreased myocardial cell apoptosis and activation of ERK and Ets‐1 in diabetic mice. In conclusion, inhibition of HMGB1 may protect against hyperglycaemia‐induced cardiomyocyte apoptosis by down‐regulating ERK‐dependent activation of Ets‐1.

Traditional Chinese medication for cardiovascular disease

Traditional Chinese medication (TCM) is increasingly used to treat cardiovascular disease (CVD) in China and some other Asian countries. However, therapeutic efficacy and adverse effects of TCM are difficult to evaluate because few large-scale, randomized controlled trials (RCTs) enrolling patients with CVD have been performed. In this Review, we critically examine the current evidence on the cardiovascular effects of TCM. We reviewed 68 RCTs that included a total of 16,171 patients. The methodological quality of the trials was generally low. Only three reports described adverse cardiovascular events specifically, although in most studies TCM was associated with significant improvements in surrogate end points for hypertension, coronary heart disease, cardiac arrhythmias, and heart failure. The risk of adverse effects was not increased compared with no intervention, placebo, or Western medications. However, whether TCM is effective in reducing the all-cause or cardiovascular mortality in patients with CVD remains unknown and must be tested in large-scale RCTs with adverse cardiovascular events as primary end points.

Effects and mechanisms of PPARα activator fenofibrate on myocardial remodelling in hypertension

Although peroxisome proliferator‐activated receptor α (PPARα) is highly expressed in the heart, the effects of PPARα on cardiac remodelling and the underlying mechanisms are unclear. The present study was undertaken to test the hypothesis that PPARα activator fenofibrate plays a key role in left ventricular hypertrophic remodelling via the formation of c‐fos/c‐jun heterodimers in spontaneous hypertensive rats (SHRs). Twenty‐four male 8‐week‐old SHRs were randomly divided into two groups, one group treated with oral saline (n= 10) and another treated with oral fenofibrate (60 mg.kg−1.d−1, n= 14). Ten same‐aged Wistar–Kyoto (WKY) rats were selected as a normal control group. Using echocardiography, immunohistochemistry, co‐immunoprecipitation, Western blot analysis and real‐time RT‐PCR, we showed that the left ventricular wall thickness and significantly reduced and left ventricular diastolic function improved in SHRs treated with fenofibrate compared with SHRs treated with saline. Similarly, the excessive collagen deposition and the up‐regulation of collagen I, collagen III, c‐fos and c‐jun seen in SHRs receiving saline were significantly attenuated in SHRs receiving fenofibrate. In addition, fenofibrate markedly decreased the expression of AP‐1 and c‐fos/c‐jun heterodimers (P < 0.01). These results demonstrated that PPARα activator fenofibrate may exert a protective effect on cardiac remodelling in SHRs by decreasing the expression of c‐fos and c‐jun and suppressing the formation of c‐fos/c‐jun heterodimers, which may further inhibit transcription of the downstream genes involved in the pathogenesis of left ventricular hypertrophy induced by hypertension.

Antagonist of microRNA-21 improves balloon injury-induced rat iliac artery remodeling by regulating proliferation and apoptosis of adventitial fibroblasts and myofibroblasts.

Molecular pathways involved in adventitial fibroblasts (AFs) and myofibroblasts (MFs) proliferation and apoptosis contribute to vascular remodeling. MicroRNA‐21 (miR‐21) plays an important role in regulating cellular proliferation and apoptosis of many cell types; however, the effect of miR‐21 on AFs and MFs is still unknown. In this study, we found that miR‐21 was expressed in AFs and overexpressed in MFs. Inhibition of miR‐21 decreased proliferation and increased apoptosis of AFs and MFs, and overexpression of miR‐21 with pre‐miR‐21 had the reverse effect. Programmed cell death 4 (PDCD4), related to cell proliferation and apoptosis, was validated as a direct target of miR‐21 by dual‐luciferase reporter assay and gain and loss of function of miR‐21 in AFs and MFs. PDCD4 knockdown with siRNA partly rescued the reduced proliferation with miR‐21 inhibition and alleviated the increased apoptosis induced by miR‐21 inhibition in AFs and MFs. Moreover, increasing PDCD4 expression by miR‐21 inhibition significantly decreased JNK/c‐Jun activity. In contrast, decreasing PDCD4 expression by pre‐miR‐21 treatment increased JNK/c‐Jun activity, while the effect of miR‐21 inhibition on JNK/c‐Jun activity could be rescued by PDCD4 siRNA. Moreover, miR‐21 inhibition could regulate proliferation and apoptosis of vascular AFs and MFs in vivo. Furthermore, miR‐21 inhibition reversed vascular remodeling induced by balloon injury. In summary, our findings demonstrate that miR‐21 may have a critical role in regulating proliferation and apoptosis of AFs and MFs, and PDCD4 is a functional target gene involved in the miR‐21‐mediated cellular effects in vascular remodeling by a miR‐21/PDCD4/JNK/c‐Jun pathway. J. Cell. Biochem. 113: 2989–3001, 2012.

Association of Plasma Angiotensin-(1–7) Level and Left Ventricular Function in Patients with Type 2 Diabetes Mellitus

Background We recently found that overexpression of angiotensin (Ang)-converting enzyme 2, which metabolizes Ang-II to Ang-(1–7) and Ang-I to Ang-(1–9), may prevent diabetes-induced left ventricular remodeling and dysfunction in rats. Our objective was to evaluate the association of plasma Ang-(1–7) level and left ventricular function in patients with type 2 diabetes mellitus. Methodology/Principal Findings We measured the left ventricular ejection fraction (EF), ratio of early to late left ventricular filling velocity (E/A) and ratio of early diastolic mitral inflow to annular velocity (E/Ea) by ultrasonography in 110 patients with type 2 diabetes mellitus for more than 5 years. Anthropometric and fasting blood values were obtained from medical records. The plasma Ang-(1-7) level in patients with a poor EF (<50%) was significantly lower than that in patients with EF ≥50%; the level in patients with E/A <1 was significantly lower than that in patients with E/A ≥1; and the level in patients with E/Ea >15 was significantly lower than that in patients with E/Ea ≤15. Ang-(1–7) level was negatively correlated with E/Ea and Log-N-terminal pro-B-type natriuretic peptide and positively with EF and E/A. Stepwise multiple regression analysis revealed that Ang-(1–7), hemoglobin A1c and Ang-II levels as well as duration of diabetes predicted EF; Ang-(1–7) level, fasting blood glucose, low-density lipoprotein cholesterol level and duration of diabetes predicted E/A; and Ang-(1–7) and hemoglobin A1c levels predicted E/Ea. Conclusions/Significance Plasma Ang-(1–7) level is independently associated with left ventricular function in patients with type 2 diabetes mellitus and may be a biomarker for assessing cardiac function in such patients.