Fengshuang An

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.

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.

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.

ptx3, a key component of innate immunity, is induced by saa via fprl1‐mediated signaling in HAECs

Serum amyloid A (SAA) is regarded as an important acute phase protein in coronary artery diseases. However, its involvement in the immune response of atherosclerosis is poorly understood. The present study was designed to investigate the influence of SAA on the secretion of long pentraxin 3 (PTX3), a key component of innate immunity, in human aortic endothelial cells (HAECs). Our study revealed that recombinant SAA up‐regulated PTX3 production in a remarkable dose‐ and time‐dependent manner and the activation of formyl peptide receptor‐like 1 (FPRL1) was crucial for SAA‐induced expression of PTX3 in HAECs. Meanwhile, SAA‐induced PTX3 production could be significantly down‐regulated by using the specific siRNA sequences for Jun N‐terminal kinases (JNK). Furthermore, the activation of activator protein‐1 (AP‐1) was necessary for the up‐regulation of PTX3 expression. We also found that the activation of nuclear factor‐kappa B (NF‐κB) played an important role in this process. Our findings demonstrate that SAA up‐regulates PTX3 production via FPRL1 significantly, and thus, contributes to the inflammatory pathogenesis of atherosclerosis. J. Cell. Biochem. 112: 2097–2105, 2011.

Serum amyloid A stimulates lipoprotein-associated phospholipase A2 expression in vitro and in vivo

OBJECTIVES Although lipoprotein-associated phospholipase A2 (Lp-PLA2) has been regarded as a biomarker and a causative agent for acute coronary events recently, the mechanism of the regulation of Lp-PLA2 has not been fully elucidated yet. This study was aimed to investigate the influence of serum amyloid A (SAA) on the expression of Lp-PLA2 in THP-1 cells and ApoE-deficient (ApoE(-/-)) mice. METHODS THP-1 cells were stimulated by SAA and the mRNA and protein expression of Lp-PLA2 was detected. ApoE(-/-) mice were intravenously injected with murine SAA1 lentivirus. Formyl peptide receptor like-1 (FPRL1) agonist (WKYMVm) and inhibitor (WRW(4)), mitogen-activated protein kinases (MAPKs) inhibitors, and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist and inhibitor were used to investigate the mechanism of regulation of Lp-PLA2. RESULTS Recombinant SAA up-regulated Lp-PLA2 expression in a dose and time-dependent manner in THP-1 cells. Immunohistochemical analysis of aortic root of ApoE(-/-) mice also demonstrated that the expression of Lp-PLA2 was up-regulated significantly with SAA treatment. WRW(4) decreased SAA-induced Lp-PLA2 production; while WKYMVm could induce Lp-PLA2 expression. ERK1/2, JNK1/2, and p38 inhibition reduced SAA-induced Lp-PLA2 production. Furthermore, the results suggested the activation of PPAR-γ played a crucial role in this process. CONCLUSION These results demonstrate that SAA up-regulates Lp-PLA2 production significantly via a FPRL1/MAPKs./PPAR-γ signaling pathway.

Association of nucleotide-binding oligomerization domain-like receptor 3 inflammasome and adverse clinical outcomes in patients with idiopathic dilated cardiomyopathy.

Abstract Background: The nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome is a multiprotein complex consisting of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1. In peripheral blood mononuclear cells (PBMCs), NLRP3 can activate interleukin-1β (IL-1β), important in the chronic inflammatory process of idiopathic dilated cardiomyopathy (IDCM). Therefore, the NLRP3 inflammasome in PBMCs may be involved in the pathogenesis of IDCM. We evaluated the association of circulating levels of NLRP3 inflammasome and cardiac function in patients with IDCM and 6-month rehospitalization. Methods: We enrolled 54 patients with IDCM and 20 healthy volunteers and analyzed left ventricle ejection fraction (LVEF), electrocardiography findings and circulating levels of NLRP3, ASC, caspase-1, IL-1β, N terminal-pro type B natriuretic peptide (NT-pro BNP) and blood values. Patients were followed up for 6 months. Results: On admission and discharge, the circulating levels of NLRP3, ASC, caspase-1 and IL-1β were higher in IDCM patients than healthy controls (all p<0.05). In patients, NLRP3 mRNA level was associated with LVEF, NT-pro BNP level and monocyte count (all p<0.05). LVEF at admission and mRNA levels of NLRP3 and IL-1β at discharge were independent risk factors of 6-month rehospitalization for patients. High NLRP3 mRNA level was associated with cumulative rehospitalization rate (p<0.05). Conclusions: NLRP3 level in PBMCs may be associated with cardiac function and rehospitalization in IDCM patients.

Visfatin Destabilizes Atherosclerotic Plaques in Apolipoprotein E–Deficient Mice

Objectives Although there is evidence that visfatin is associated with atherogenesis, the effect of visfatin on plaque stability has not yet been explored. Methods In vivo, vulnerable plaques were established by carotid collar placement in apolipoprotein E–deficient (ApoE−/−) mice, and lentivirus expressing visfatin (lenti-visfatin) was locally infused in the carotid artery. The lipid, macrophage, smooth muscle cell (SMC) and collagen levels were evaluated, and the vulnerability index was calculated. In vitro, RAW264.7 cells were stimulated with visfatin, and the MMPs expressions were assessed by western blot and immunofluorescence. And the mechanism that involved in visfatin-induced MMP-8 production was investigated. Results Transfection with lenti-visfatin significantly promoted the expression of visfatin which mainly expressed in macrophages in the plaque. Lenti-visfatin transfection significantly promoted the accumulation of lipids and macrophages, modulated the phenotypes of smooth muscle cells and decreased the collagen levels in the plaques, which significantly decreased the plaque stability. Simultaneously, transfection with lenti-visfatin significantly up-regulated the expression of MMP-8 in vivo, as well as MMP-1, MMP-2 and MMP-9. Recombinant visfatin dose- and time-dependently up-regulated the in vitro expression of MMP-8 in macrophages. Visfatin promoted the translocation of NF-κB, and inhibition of NF-κB significantly reduced visfatin-induced MMP-8 production. Conclusions Visfatin increased MMP-8 expression, promoted collagen degradation and increased the plaques vulnerability index.

Effects of extended-release felodipine on endothelial vasoactive substances in patients with essential hypertension.

Abstract Background: Endothelial dysfunction plays a significant role in the pathogenesis of essential hypertension (EH). This trial was undertaken to reveal the effects of extended-release felodipine on endothelial vasoactive substances in EH patients. Methods: A colorimetric chemical method was employed to measure the level of nitric oxide (NO) and nitric oxide synthase (NOS), while radioimmunoassay was employed to measure endothelin (ET), angiotensin-II (Ang-II), thromboxane A2 (TXA2) and prostacyclin I2 (PGI2) in plasma of the subjects. Group 1 consisted of 120 patients who were treated with a 4-week mono-therapy of felodipine. Group 2 consisted of 70 patients who were participating in fitness programs during this period. Another group comprising 80 individuals was selected as controls. Data from both the starting point and the ending point were collected and analyzed. Results: After a 4-week mono-therapy of extended-release felodipine in Group 1, the levels of plasma ET, Ang-II and TXA2 decreased significantly, while levels of NO, NOS and PGI2 did not noticeably change. In Group 2, there were almost no changes in levels of ET, Ang-II, TXA2, NO, NOS and PGI2. Conclusions: From these results, we conclude that felodipine reduced blood pressure by decreasing the secretion of ET, Ang-II and TXA2. Consequently, felodipine can revitalize the endothelial function in EH patients. Clin Chem Lab Med 2008;46:393–5.

Safety of the neprilysin/renin-angiotensin system inhibitor LCZ696

OBJECTIVES The combined neprilysin/rennin-angiotensin system inhibitor sacubitril/valsartan (LCZ696) has shown its superiority over ACEI/ARB therapy. In view of the existing concern of its adverse effects, we aimed to provide evidence of the safety of the new drug. RESULTS A total of 6 randomized trials with 11,821 subjects were included in this analysis. No significant differences were found in any adverse effects between LCZ696 and ACEI/ARB or placebo groups. LCZ696 significantly decreased the risks of serious adverse events and death compared with ACEI/ARB. LCZ696 also significantly decrease the risk of discontinuation of treatment for any adverse event no matter compared with ACEI/ARB or a placebo. LCZ696 significantly increased the risk of angioedema and dizziness, while it decreased the risk of renal dysfunction and bronchitis. There was no difference for hypotension, hyperkalemia, cough, upper respiratory tract inflammation, diarrhoea, back pain, nasopharyngitis, headache and influenza between the LCZ696 group and the ACEI/ARB group. MATERIALS AND METHODS A meta-analysis of eligible studies that used LCZ696 in heart failure and hypertension was performed. Embase, PubMed and the Cochrane Library were searched for randomized controlled trials (RCTs) with data on any adverse effects, serious adverse events, discontinuation of treatment for any adverse event, death, angioedema, hypotension, hyperkalemia, and other adverse effects to perform this meta-analysis. CONCLUSIONS In addition to the beneficial effect of LCZ696 on end point events, the available evidences showed that LCZ696 was associated with less drug-risks than a placebo and ACEI/ARB.Objectives The combined neprilysin/rennin-angiotensin system inhibitor sacubitril/valsartan (LCZ696) has shown its superiority over ACEI/ARB therapy. In view of the existing concern of its adverse effects, we aimed to provide evidence of the safety of the new drug. Results A total of 6 randomized trials with 11,821 subjects were included in this analysis. No significant differences were found in any adverse effects between LCZ696 and ACEI/ARB or placebo groups. LCZ696 significantly decreased the risks of serious adverse events and death compared with ACEI/ARB. LCZ696 also significantly decrease the risk of discontinuation of treatment for any adverse event no matter compared with ACEI/ARB or a placebo. LCZ696 significantly increased the risk of angioedema and dizziness, while it decreased the risk of renal dysfunction and bronchitis. There was no difference for hypotension, hyperkalemia, cough, upper respiratory tract inflammation, diarrhoea, back pain, nasopharyngitis, headache and influenza between the LCZ696 group and the ACEI/ARB group. Materials and Methods A meta-analysis of eligible studies that used LCZ696 in heart failure and hypertension was performed. Embase, PubMed and the Cochrane Library were searched for randomized controlled trials (RCTs) with data on any adverse effects, serious adverse events, discontinuation of treatment for any adverse event, death, angioedema, hypotension, hyperkalemia, and other adverse effects to perform this meta-analysis. Conclusions In addition to the beneficial effect of LCZ696 on end point events, the available evidences showed that LCZ696 was associated with less drug-risks than a placebo and ACEI/ARB.

Prolyl hydroxylase 3 overexpression accelerates the progression of atherosclerosis in ApoE-/- mice.

PHD3 belongs to the family of 2-oxoglutarate and iron-dependent dioxygenases and is a critical regulator of HIF-1α. Its expression is increased in cardiovascular diseases such as cardiomyopathy, myocardial ischemia-reperfusion injury, and congestive heart failure. However, the association between PHD3 and atherosclerosis has not been clearly elucidated. In the present study, we investigated the potential effect and mechanism of PHD3 in apolipoprotein E-deficient (ApoE-/-) mice. Murine PHD3 lentivirus and shRNA -PHD3 lentivirus were constructed and injected intravenously into ApoE-/- mice fed on a high fat diet. The aortic atherosclerotic lesion area was larger with PHD3 over-expression. With increased PHD3 levels, macrophages and smooth muscle cells were enhanced. The apoptosis of atherosclerotic plaques revealed an increase when PHD3 was elevated. Furthermore, the expression of intercellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), monocyte chemotactic protein 1 (MCP-1), interleukin-1beta (IL-1β) and tumor necrosis factor-α(TNF-α) were upregulated with PHD3 over-expression. In vitro, we explored the specific signaling pathway of PHD3 in HUVECs. PHD3 over-expression is associated with activation of ERK1/2 and JNK phosphorylation of MAPK signaling pathway. PHD3 inhibition decreased the apoptosis of HUVECs treated with ox-LDL (50 μg/ml). Our study suggests that PHD3 is not only a regulator of HIF-1α but also an active participant in atherogenesis.