Xiuren Gao

Atrial Fibrillation Induces Myocardial Fibrosis Through Angiotensin II Type 1 Receptor–Specific Arkadia-Mediated Downregulation of Smad7

Rationale: Tachycardia-induced atrial fibrosis is a hallmark of structural remodeling of atrial fibrillation (AF). The molecular mechanisms underlying the AF-induced atrial fibrosis remain unclear. Objective: To determine the role of angiotensin II (Ang II)/Ang II type 1 (AT1) receptor–coupled transforming growth factor (TGF)-&bgr;1/Smad signaling pathway in the AF-induced atrial fibrosis. Methods and Results: Rapid atrial pacing (1000 ppm) was applied to the left atrium of rabbit heart to induce atrial fibrillation and fibrosis. Quantitative PCR and Western blot analysis revealed that rapid atrial pacing caused a marked increase in the expression of Ang II, TGF-&bgr;1, phosphorylated Smad2/3 (P-Smad2/3), Arkadia, and hydroxyproline synthesis. However, the expression of Smad7, a key endogenous antagonist of the TGF-&bgr;1/Smad-mediated fibrosis, was significantly decreased. These changes were dose-dependently reversed by AT1 receptor antagonist losartan, implicating the involvement of AF-induced release of Ang II and activation of AT1 receptor–specific pathway. In the adult rabbit cardiac fibroblasts, Ang II increased the expression of TGF-&bgr;1, P-Smad2/3, Smad4, Arkadia, and collagen I synthesis and significantly reduced Smad7 expression. These effects of Ang II were reversed by losartan but not by the AT2 antagonist (PD123319). In addition, extracellular signal-regulated kinase inhibitor and anti–TGF-&bgr;1 antibody also blocked the Ang II–induced downregulation of Smad7. Silencing of Smad7 gene by small interfering RNA abolished the antagonism of losartan on the fibrogenic effects of Ang II on cardiac fibroblasts, whereas overexpression of Smad7 blocked Ang II–induced increase in collagen I synthesis. Conclusions: Ang II/AT1 receptor–specific activation of Arkadia-mediated poly-ubiquitination and degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-&bgr;1/Smad signaling and serves as a key mechanism for AF-induced atrial fibrosis.

Angiotensin II increases collagen I expression via transforming growth factor-beta1 and extracellular signal-regulated kinase in cardiac fibroblasts

Angiotensin II is a powerful mediator to induce cardiac remodeling and fibrosis. Transforming growth factor-beta1 (TGF-beta1) and extracellular signal-regulated kinase (ERK) have been implicated in the angiotensin II-induced cardiac fibrosis. However, the signaling pathways for this angiotensin II effect and the interaction between ERK and the TGF-beta1 signaling in this effect have not been well-illustrated. Cardiac fibroblasts were prepared from the ventricles of adult male Sprague-Dawley rats. They were treated with 1 microM angiotensin II in the presence or absence of losartan (angiotensin II AT(1) receptor antagonist), PD123319 (angiotensin II AT(2) receptor antagonist), an anti-TGF-beta1 antibody or PD98059 (ERK inhibitor). The cells were collected for Western blotting and reverse transcription-polymerase chain reaction. Angiotensin II caused a significant increase of the expression of TGF-beta(1), ERK1, phosphorylated-Smad2/3, Smad4 and collagen I. This increase was attenuated by losartan but was not affected by PD123319. An anti-TGF-beta(1) antibody and PD98059 diminished angiotensin II-induced Smad2/3 phosphorylation and the expression of Smad7 and collagen I. Our results suggest that angiotensin II induces collagen I expression through angiotensin II AT(1) receptor-TGF-beta(1)-Smads signaling pathway in cardiac fibroblasts. ERK, by regulating Smads signaling, also participated in the angiotensin II-induced collagen I expression.

Efficacy of different doses of aspirin in decreasing blood levels of inflammatory markers in patients with cardiovascular metabolic syndrome

Objectives Inflammation and platelet aggregation and activation are key processes in the initiation of a cardiovascular event. Patients with metabolic syndrome have a high risk of cardiovascular events. This study determined whether small and medium doses of aspirin have anti‐inflammation and antiplatelet aggregation effects in patients with metabolic syndrome.

Fluvastatin decreases cardiac fibrosis possibly through regulation of TGF-β1/Smad 7 expression in the spontaneously hypertensive rats

Statins ameliorate myocardial fibrosis after myocardial infarction. We designed this study to determine whether fluvastatin reduced hypertension-induced myocardial hypertrophy and fibrosis and whether these fluvastatin effects involved transforming growth factor beta1 (TGF-beta1) and Smad 7, factors known to play a role in the myocardial hypertrophy and fibrosis. We randomized 14 week old spontaneously hypertensive rats (SHRs) to receiving vehicle or 5-20 mg/kg/day fluvastatin for 8 weeks. Wistar Kyoto (WKY) rats receiving vehicle or 10 mg/kg/day fluvastatin were also studied. SHRs had an increased blood pressure, left ventricular hypertrophy and fibrosis compared with WKY rats. SHRs also had an elevated TGF-beta1 expression and a decreased Smad 7 expression. These changes in SHRs were dose-dependently attenuated by fluvastatin. For example, the hydroxyproline content was 3.2+/-0.1, 4.0+/-0.1 and 3.5+/-0.1 microg/mg heart and the Smad 7 protein expression was 5.1+/-0.6, 1.0+/-0.1 and 4.1+/-0.7 arbitrary units for WKY rats, SHRs and SHRs receiving 20 mg/kg/day fluvastatin, respectively. The hydroxyproline content in the SHRs treated with or without fluvastatin was positively correlated with the left ventricular mass index, systolic blood pressure and the amount of TGF-beta1 proteins and negatively correlated with the Smad 7 expression level. The left ventricular mass index was positively correlated with the systolic blood pressure. Fluvastatin did not alter the blood pressure, left ventricular mass index and collagen content of WKY rats. These results suggest that fluvastatin reduces hypertension-induced myocardial hypertrophy and fibrosis. These effects may involve an increased expression of Smad 7 and a decreased expression of TFG-beta1. Our results call for clinical studies to evaluate these fluvastatin effects in hypertensive patients.

Angiotensin II upregulates Kv1.5 expression through ROS-dependent transforming growth factor-beta1 and extracellular signal-regulated kinase 1/2 signalings in neonatal rat atrial myocytes

Kv1.5 potassium channel represents a promising target for atrial fibrillation (AF) therapy. During AF, the renin-angiotensin system is markedly activated. Recent evidence indicates that angiotensin II (Ang II) can upregulate Kv1.5 channel, but the mechanism remains unknown. In this study, we report that Ang II-mediated transforming growth factor-beta1 (TGF-β1)/Smad2/3 and extracellular signal-regulated kinase (ERK) 1/2 signalings are involved in atrial Kv1.5 expression. In neonatal rat atrial myocytes, quantitative PCR and Western blotting revealed that Ang II upregulated TGF-β1, synapse-associated protein 97 (SAP97) and Kv1.5 expression in a time- and concentration-dependent manner. The Ang II-induced upregulation of Kv1.5, SAP97 and phosphorylated Smad2/3 (P-Smad2/3) were reversed by the Ang II type 1 (AT1) receptor antagonist losartan, an anti-TGF-β1 antibody and the ERK 1/2 inhibitor PD98059 but not by the AT2 receptor antagonist PD123319. mRNA knockdown of either Smad2 or Smad3 blocked Ang II-induced expression of Kv1.5 and SAP97. These data suggest that AT1 receptor/TGF-β1/P-Smad2/3 and ERK 1/2 signalings are involved in Ang II-induced Kv1.5 and SAP97 expression. Flow cytometry and Western blotting revealed that losartan and the anti-TGF-β1 antibody diminished Ang II-induced reactive oxygen species (ROS) generation and that the antioxidants diphenyleneiodonium and N-acetyl cysteine inhibited Ang II-induced expression of P-Smad2/3, phosphorylated ERK (P-ERK) 1/2, Kv1.5, SAP97, suggesting that ROS participate in Kv1.5 and SAP97 regulation by modulating Ang II-induced P-Smad2/3 and P-ERK 1/2 expression. In conclusion, we demonstrate that ROS-dependent Ang II/AT1 receptor/TGF-β1/P-Smad2/3 and Ang II/ERK 1/2 signalings are involved in atrial Kv1.5 and SAP97 expression. Antioxidants would be beneficial for AF treatment through inhibiting atrial Kv1.5 expression.

The secretion patterns and roles of cardiac and circulating arginine vasopressin during the development of heart failure

OBJECTIVE The aim of this study is to investigate local cardiac and circulating AVP secretion during heart failure and to determine whether AVP mediates ventricular remodeling. METHODS We assessed cardiac function and AVP levels of post-myocardial infarction (MI) heart-failure rats 3 weeks (n = 10), 4 weeks (n = 10), 6 weeks (n = 10), 9 weeks (n = 15) after the proximal left anterior descending coronary artery (LAD) ligation. Ten sham-operated rats were used as the control group. In vitro, cardiac microvascular endothelial cells (CMECs) were initiated from isolated Wistar rat hearts and subjected to Ang II to induce AVP expression and secretion. Besides, the effects of AVP stimulation on CMECs and cardiac fibroblasts (CFs) were studied using methylthiazol tetrazolium assay, Western blotting and real-time PCR. RESULTS With cardiac dysfunction, plasma and local cardiac AVP, aldosterone levels increased over time, peaking at 9 weeks post-MI. AVP levels were negatively correlated with serum Na(+) and LVEF but positively correlated with LVEDD and myocardial hydroxyproline. In CMECs treated with Ang II, AVP mRNA and protein expression increased. In addition, AVP promoted CFs proliferation and up-regulated the expression of endothelin-1 and connective tissue growth factor. CONCLUSION CMECs are the cellular sources of elevated local heart AVP stimulated with Ang II/AT1. An intrinsic cardiac AVP system exists. Local cardiac and circulating AVP secretion were enhanced by deteriorating cardiac function. AVP may promote ventricular remodeling. Thus, AVP could be an important mediator of myocardial fibrosis in late-stage heart failure.

Effects of hydrochlorothiazide on cardiac remodeling in a rat model of myocardial infarction-induced congestive heart failure.

Heart failure is a major cause of morbidity and mortality worldwide. Diuretics are regarded as the first-line treatment for patients with heart failure because they provide symptomatic relief. However, the specific benefits of diuretics and their effects on heart failure survival remain unclear. This study was designed to investigate the potential of hydrochlorothiazide to improve cardiac remodeling compared with spironolactone. Heart failure was produced by ligation of the left anterior descending coronary artery in male Sprague-Dawley rats. Two weeks after coronary artery ligation, 55 rats were randomly divided into four groups: sham-operated group (n=10), control group (n=15), hydrochlorothiazide group (12.5 mg/kg/day, n=15) and spironolactone group (20 mg/kg/day, n=15). Cardiac function was assessed by echocardiography and Millar catheter after treatment with drugs for 8 weeks. Compared with the control group, ejection fraction and left ventricular end-systolic pressure were significantly improved in the hydrochlorothiazide and spironolactone treatment groups (P<0.05). In addition, hydrochlorothiazide and spironolactone reduced collagen volume fraction and proinflammatory cytokine levels. Moreover, gene and protein expression of TGF-β1, Smad2, Smad3 and Smad7 (P<0.05) were also reduced. Nevertheless, no significant differences were observed between the hydrochlorothiazide and spironolactone groups. These results suggest that hydrochlorothiazide improves cardiac remodeling as effectively as spironolactone by reducing proinflammatory cytokine levels and inhibiting the TGF-β signaling pathway in post-myocardial infarction congestive heart failure. Moreover, the effects of the drugs on the TGF-β signaling pathway are likely to result from inhibited TGF-β and R-Smads expression rather than increased Inhibitory-Smad7 expression.

Changes in cell autophagy and apoptosis during age-related left ventricular remodeling in mice and their potential mechanisms.

Cardiac structures and functions change with advanced age, but the underlying mechanisms are not well understood. Autophagy and apoptosis play important roles in the process of cardiac remodeling. This study was designed to explore changes in cell autophagy and apoptosis during age-related left ventricular remodeling and to determine whether the mitogen-activated protein kinase (MAPK) pathway is an underlying mechanism. Eight 5-month-old (adult group) and eight 24-month-old male C57bl/6 mice (aged group) were studied. The heart mass index, left ventricular mass index and hydroxyproline content of both groups were compared. Western Blotting was used to quantitate the protein expression of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, caspase-3, B-cell leukemia-2 (Bcl-2) and MAPKs in the left ventricles of adult and aged mice. Our results showed that the heart mass index, left ventricular mass index and hydroxyproline content in the left ventricles of the aged mice were increased significantly compared with the adult mice, indicating that left ventricular remodeling occurs with aging. The expression of LC3 and Beclin-1 in the left ventricles of aged mice were decreased significantly compared to adult mice. Meanwhile, the level of myocardial caspase-3 in adult mice remained the same in aged mice, and the level of myocardial Bcl-2 increased significantly in aged mice. There were no differences in the expression level of myocardial extracellular signal-regulated kinase 1/2 (ERK1/2), activated/phospho-ERK1/2, c-Jun N-terminal kinase 1/2 (JNK1/2) and p38 between aged and adult mice. However, the expression of myocardial activated/phospho-JNK1/2 increased significantly in aged mice, while activated/phospho-p38 decreased significantly. These findings indicate that autophagy decreases without a concurrent change in apoptosis during age-related left ventricular remodeling in mice. The MAPK pathway may be involved in the regulation of age-related left ventricular remodeling by modulating autophagy.

Downregulation of VEGF and upregulation of TL1A expression induce HUVEC apoptosis in response to high glucose stimuli

High glucose‑induced endothelial cell apoptosis is considered to be the initiator of diabetes‑associated vascular complications. Experiments in vivo and in vitro have demonstrated that high glucose levels contribute to the apoptosis of endothelial cells by mediating cellular dysfunction and metabolic disorder via the production of various cytokines. As the most important endogenous vascular regulators, the balance between pro‑proliferative effector vascular endothelial growth factor (VEGF) and anti‑proliferative effector tumor necrosis factor‑like cytokine 1A (TL1A) is important in the modulation of endothelial cell survival and proliferation, and neovascularization. The present study aimed to explore whether the imbalance between VEGF and TL1A affected the apoptosis of human umbilical vein endothelial cells (HUVECs) exposed to high glucose conditions and then further investigated the potential mechanism. The results showed that the downregulation of VEGF in combination with the upregulation of TL1A in response to high glucose levels led to enhanced HUVEC apoptosis. Further experiments revealed that silencing high glucose‑induced TL1A expression using TL1A small interfering (si)RNA or the overexpression of VEGF by transfection with VEGF DNA resulted in a reduced HUVEC apoptosis rate compared with the controls. The effects occurred by attenuating and activating the phosphoinositide 3‑kinase/Akt/endothelial nitric oxide synthase pathway, respectively. In addition, VEGF and TL1A inhibited each other in hyperglycemia. In conclusion, these findings provide theoretical support for the further investigation of novel therapeutic strategies designed to maintain the balance between VEGF and TL1A and, thus, to prevent the onset and progression of endothelial cell apoptosis in response to high glucose stimuli.

Decreased intralymphocytic magnesium content is associated with diastolic heart dysfunction in patients with essential hypertension.

Diastolic heart failure (DHF) is a clinical syndrome characterized by the symptoms and signs of heart failure, preserved ejection fraction (EF) and abnormal diastolic function [1]. Hypertension is the most common risk factor and the principal precursor for DHF [2]. Hypertension-induced left ventricular (LV) hypertrophy that includes myocardial hypertrophy and abnormal accumulation of fibrillar collagen causes a decreased compliance and LV diastolic dysfunction. It is estimated that 20–60% of patients with heart failure have preserved EF [3,4]. Unfortunately, in contrast to the situation of heart failure with low EF, the mortality and morbidity of DHF have not been significantly decreased in the past decades [5,6]. Magnesium is the second most abundant intracellular cation. As a cofactor formany enzymes,magnesiumplays a role in various biological functions including energy metabolism and muscle contraction [7]. It is known that activation of the rennin–angiotensin–aldosterone system and the use of diuretics in patients with essential hypertension or cardiac failure may deplete serum potassium and affect intracellular magnesium homeostasis [8,9]. We have shown the importance of maintaining magnesium homeostasis in reducing arrhythmia and myocardial hypertrophy in patients with heart failure or hypertension [10,11]. However, the role of magnesium homeostasis in diastolic heart dysfunction is not known yet. Thus, we designed this study to address this issue. Since myocardium of patients is usually not easily accessible, we measured intralymphocytic magnesium content to reflect the magnesium content in myocardial cells. This was a single center prospective study that conformed to the ethical principles outlined in the Declaration of Helsinki. The study protocol was approved by the ethics committee of the First Affiliated Hospital of the SunYat-SenUniversity (Guangzhou, China). Consecutive 206 essential hypertension patients with diastolic heart dysfunction documented by Doppler echocardiography from November 2007 to October 2009 were enrolled. Patients with diastolic heart dysfunction and clinical signs and symptoms of heart failure as described in the European Society of Cardiology Guidelines published in 2008 [12] were attributed to DHF group (group A). Those free of signs and symptoms of