Role of α1D‐adrenoceptors in vascular wall hypertrophy during angiotensin II‐induced hypertension

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Angiotensin‐(1–7) treatment ameliorates angiotensin II‐induced apoptosis of human umbilical vein endothelial cells

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A distinct AMP‐activated protein kinase phosphorylation site characterizes cardiac hypertrophy induced by l‐thyroxine and angiotensin II

1. The purpose of the present study was to evaluate differences in the AMP‐activated protein kinase (AMPK) phosphorylation sites in cardiac hypertrophy induced by l ‐thyroxine and angiotensin (Ang) II. 2. Cardiac hypertrophy was induced in wild‐type and AMPKα2‐knockout mice by treatment with 1 mg/kg, i.p., thyroxine or 1.44 mg/kg per day AngII for 14 days. The phenotype of the hypertrophy was evaluated using echocardiographic measurments and histological analyses. The phosphorylation of AMPK at α‐Ser^485/491 and α‐Thr¹⁷² was determined by western blot analysis. 3. In wild‐type mice, the phosphorylation of AMPKα‐Ser^485/491 was significantly elevated in the AngII‐treated group, but not in the thyroxine‐reated group, compared with the vehicle control group. In contrast, the phosphorylation of AMPKα‐Thr¹⁷² was significantly increased by thyroxine, but not AngII, treatment compared with the vehicle control group. Furthermore, knockout of the AMPKα2 subunit abolished phosphorylation at the α‐Ser^485/491 site and significantly suppressed phosphorylation at the α‐Thr¹⁷² site, resulting in alleviation of thyroxine‐ but not AngII‐induced hypertrophy. 4. In conclusion, l ‐thyroxine and AngII induce the phosphorylation of distinct sites of AMPK in cardiac hypertrophy. Phosphorylation of AMPK α‐Thr¹⁷² may contribute to thyroxine‐induced cardiac hypertrophy.     

Synergism of simvastatin with losartan prevents angiotensin II‐induced cardiomyocyte apoptosis in vitro

Objectives Increasing evidence suggests that cardiomyocyte apoptosis has an important role in the transition from compensatory cardiac remodelling to heart failure. The synergistic effect of statins (3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase inhibitors) and angiotensin II (Ang II) type 1 receptor antagonists reduces the incidence of cardiovascular events. However, the anti‐apoptotic potential of the synergism between losartan and simvastatin in heart failure remains unexplored. Here, we demonstrate that Ang II‐induced apoptosis is prevented by losartan and simvastatin in neonatal cardiomyocytes. Methods The in‐vitro cardiomyocyte apoptosis model was established by co‐culturing neonate rat cardiomyocytes with Ang II. Cell viability was analysed by the MTT assay. Cell apoptosis was evaluated using fluorescence microscopy and flow cytometry. Apoptosis‐related proteins Bax and Bcl‐2 expressions were measured by flow cytometry detection. Key findings Incubation with 10^?7 m Ang II for 48 h increased cardiomyocyte apoptosis and decreased cell viability. Losartan (10^?5 m ) and simvastatin (10^?5 m ), either alone or in combination, significantly decreased Ang II‐induced cardiomyocyte apoptosis and increased cell viability. The q values calculated by the probability sum test were 1.31 for cardiomyocyte apoptosis and 1.21 for cell viability. Ang II induced a significant increase in Bax protein expression, whereas Bcl‐2 protein expression was decreased. Losartan alone or in combination with simvastatin blocked the increased Bax expression and increased Bcl‐2 expression. However, simvastatin had no such effect. Conclusions Our data provide the first evidence that synergism of simvastatin with losartan prevents angiotensin II‐induced cardiomyocyte apoptosis in vitro. Synergism between simvastatin and losartan may provide a new therapeutic approach to the prevention of cardiac remodelling.     

Celecoxib,but not rofecoxib or naproxen,attenuates cardiac hypertrophy and fibrosis induced in vitro by angiotensin and aldosterone

1. Cyclo‐oxygenase (COX)‐2 inhibitors and other non‐steroidal anti‐inflammatory drugs (NSAIDs) have been implicated in increased cardiovascular events. However, the direct effects of these drugs on cardiac function have not been explored extensively. Given the important role of the renin–angiotensin–aldosterone system (RAAS) in cardiac remodelling, we sought to determine the effect of COX‐2 inhibitors and non‐specific (NS‐) NSAIDs on RAAS‐induced cardiac hypertrophy and fibrosis in neonatal rat cardiac myocytes (NCM) and fibroblasts (NCF) isolated from 1–2‐day‐old Sprague‐Dawley rat pups. 2. The NCM were pretreated for 2 h with COX‐2 inhibitors (celecoxib or rofecoxib) or NS‐NSAIDs (naproxen; all at 0.1–10 μmol/L) before being stimulated with 10 μmol/L aldosterone for 72 h or with 0.1 μmol/L angiotensin (Ang) II for 60 h. Hypertrophy of NCM was assessed by [³H]‐leucine incorporation. 3. The NCF were pretreated with COX‐2 inhibitors or naproxen as described for NCM before being stimulated with 0.1 μmol/L AngII for 48 h. Collagen synthesis was subsequently assayed by [³H]‐proline incorporation. 4. Pooled cryopreserved male and female rat hepatocytes were treated with or without COX‐2 inhibitors for 1 h before 1 nmol/L aldosterone (～540 pg/mL) was added to all wells. Cells were incubated for a further 60 min and culture media harvested by centrifugation. Human hepatic HepG2 cells were treated with compounds with or without serum starvation for 48 h. All cells were pretreated with COX‐2 inhibitors for 2 h before the addition of aldosterone. Cell culture media were harvested after a further 3, 18, 24 or 48 h incubation. Aldosterone concentrations in the culture media were determined by enzyme immunoassay. 5. Aldosterone‐ and AngII‐stimulated NCM hypertrophy was inhibited by celecoxib, but not by rofecoxib or naproxen. In NCF, AngII‐stimulated collagen synthesis was inhibited by celecoxib and, to a lesser extent, by rofecoxib, whereas naproxen had no effect. The COX‐2 inhibitors inhibited aldosterone uptake and/or metabolism by rat hepatocytes, but had no effect in human hepatic HepG2 cells. 6. These results demonstrate a potential antiremodelling effect of selective COX‐2 inhibitors in the setting of RAAS stimulation in cardiac cells, whereas naproxen has no effect.     

Phosphorylation of GATA4 at serine 105 is required for left ventricular remodelling process in angiotensin II‐induced hypertension in rats

In this study, we investigated whether local intramyocardial GATA4 overexpression affects the left ventricular (LV) remodelling process and the importance of phosphorylation at serine 105 (S105) for the actions of GATA4 in an angiotensin II (AngII)‐induced hypertension rat model. Adenoviral constructs overexpressing wild‐type GATA4 or GATA4 mutated at S105 were delivered into the anterior LV free wall. AngII (33.3 µg/kg/h) was administered via subcutaneously implanted minipumps. Cardiac function and structure were examined by echocardiography, followed by histological immunostainings of LV sections and gene expression measurements by RT‐qPCR. The effects of GATA4 on cultured neonatal rat ventricular fibroblasts were evaluated. In AngII‐induced hypertension, GATA4 overexpression repressed fibrotic gene expression, reversed the hypertrophic adult‐to‐foetal isoform switch of myofibrillar genes and prevented apoptosis, whereas histological fibrosis was not affected. Overexpression of GATA4 mutated at S105 resulted in LV chamber dilatation, cardiac dysfunction and had minor effects on expression of myocardial remodelling genes. Fibrotic gene expression in cardiac fibroblasts was differently affected by overexpression of wild‐type or mutated GATA4. Our results indicate that GATA4 reduces AngII‐induced responses by interfering with pro‐fibrotic and hypertrophic gene expressions. GATA4 actions on LV remodelling and fibroblasts are dependent on phosphorylation site S105.     

Estrogen and ERα enhanced β‐catenin degradation and suppressed its downstream target genes to block the metastatic function of HA22T hepatocellular carcinoma cells via modulating GSK‐3β and β‐TrCP expression

In our previous experiments, we found β‐catenin was highly expressed in the tumor area with high invasive ability and poor prognosis. In this study, we have examined the mechanism by which ERα regulates β‐catenin expression as well as the metastasis ability of hepatocellular cancer HA22T cells. To identify whether the anticancer effect of estrogen and ERα is mediated through suppression of β‐catenin expression, we co‐transfected pCMV‐β‐catenin and ERα into HA22T cells, and determined the cell motility by wound healing, invasion, and migration assays. Results showed that estrogen and/or ERα inhibited β‐catenin gene expression and repressed HA22T cell motility demonstrated that similar data was observed in cells expressing the ERα stable clone. Moreover, we examined the protein‐protein interaction between ERα and β‐catenin by immunostain, co‐immunoprecipitation, and Western blotting. E2 enhanced the binding of ERα with β‐catenin and then triggered β‐catenin to bind with E3 ligase (βTrCP) to promote β‐catenin degradation. Finally by employing systematic ChIP studies, we showed ERα can interact directly with the β‐catenin promoter region following E2 treatment. All our results reveal that estrogen and ERα blocked metastatic function of HA22T cells by modulating GSK3β and βTrCP expression and further enhanced β‐catenin degradation and suppressed its downstream target genes. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 519–529, 2017.     

Intrarenal alterations of the angiotensin‐converting enzyme type 2/angiotensin 1–7 complex of the renin‐angiotensin system do not alter the course of malignant hypertension in Cyp1a1‐Ren‐2 transgenic rats

The role of the intrarenal renin‐angiotensin system (RAS) in the pathophysiology of malignant hypertension is not fully understood. Accumulating evidence indicates that the recently discovered vasodilator axis of the RAS, angiotensin‐converting enzyme (ACE) type 2 (ACE2)/angiotensin 1–7 (ANG 1–7), constitutes an endogenous system counterbalancing the hypertensiogenic axis, ACE/angiotensin II (ANG II)/AT1 receptor. This study aimed to evaluate the role of the intrarenal vasodilator RAS axis in the pathophysiology of ANG II‐dependent malignant hypertension in Cyp1a1‐Ren‐2 transgenic rats. ANG II‐dependent malignant hypertension was induced by 13 days′ dietary administration of indole‐3‐carbinol (I3C), a natural xenobiotic that activates the mouse renin gene in Cyp1a1‐Ren‐2 transgenic rats. It was hypothesized that pharmacologically‐induced inhibition of the ACE2/ANG 1–7 complex should aggravate, and activation of this axis should attenuate, the course of ANG II‐dependent malignant hypertension. Blood pressure (BP) was monitored by radiotelemetry. ACE2 inhibitor (DX 600, 0.2 μg/day) and ACE2 activator (DIZE, 1 mg/day) were administrated via osmotic minipumps. Even though ACE2 inhibitor significantly decreased and ACE2 activator increased intrarenal ANG 1–7 concentrations, the course of BP, as well as of albuminuria, cardiac hypertrophy and renal glomerular damage, were not altered. It was shown that intrarenal alterations in the ACE2/ANG 1–7 complex did not significantly modify the course of malignant hypertension in I3C‐induced Cyp1a1‐Ren‐2 transgenic rats. Thus, in our experimental setting alterations of this intrarenal vasodilator complex of the RAS do not significantly modify the form of malignant hypertension that clearly depends on the inappropriately increased activity of the ACE/ANG II/AT1 receptor axis.     

Oral administration of alcalase potato protein hydrolysate‐APPH attenuates high fat diet‐induced cardiac complications via TGF‐β/GSN axis in aging rats

Consumption of high fat diet (HFD) is associated with increased cardiovascular risk factors among elderly people. Aging and obesity induced‐cardiac remodeling includes hypertrophy and fibrosis. Gelsolin (GSN) induces cardiac hypertrophy and TGF‐β, a key cytokine, which induces fibrosis. The relationship between TGF‐β and GSN in aging induced cardiac remodeling is still unknown. We evaluated the expressions of TGF‐β and GSN in HFD fed 22 months old aging SD rats, followed by the administration of either probucol or alcalase potato protein hydrolysate (APPH). Western blotting and Masson trichrome staining showed that APPH (45 and 75 mg/kg/day) and probucol (500 mg/kg/day) treatments significantly reduced the aging and HFD‐induced hypertrophy and fibrosis. Echocardiograph showed that the performance of the hearts was improved in APPH, and probucol treated HFD aging rats. Serum from all rats was collected and H9c2 cells were cultured with collected serums separately. The GSN dependent hypertrophy was inhibited with an exogenous TGF‐β in H9c2 cells cultured in HFD+ APPH treated serum. Thus, we propose that along with its role in cardiac fibrosis, TGF‐β also acts as an upstream activator of GSN dependent hypertrophy. Hence, TGF‐β in serum could be a promising therapeutic target for cardiac remodeling in aging and/or obese subjects.     

Angiotensin II type 1 receptor blockade attenuates gefitinib-induced cardiac hypertrophy via adjusting angiotensin II-mediated oxidative stress and JNK/P38 MAPK pathway in a rat model

Gefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR), used for the treatment of advanced or metastatic non-small cell lung cancer. Recently, studies proved that Gefitinib-induced cardiotoxicity through induction of oxidative stress leads to cardiac hypertrophy. The current study was conducted to understand the mechanisms underlying gefitinib-induced cardiac hypertrophy through studying the roles of angiotensin II (AngII), oxidative stress, and mitogen-activated protein kinase (MAPK) pathway. Male Wistar albino rats were treated with valsartan, gefitinib, or both for four weeks. Blood samples were collected for AngII and cardiac markers measurement, and hearts were harvested for histological study and biochemical analysis. Gefitinib caused histological changes in the cardiac tissues and increased levels of cardiac hypertrophy markers, AngII and its receptors. Blocking of AngII type 1 receptor (AT1R) via valsartan protected hearts and normalized cardiac markers, AngII levels, and the expression of its receptors during gefitinib treatment. valsartan attenuated gefitinib-induced NADPH oxidase and oxidative stress leading to down-regulation of JNK/p38-MAPK pathway. Collectively, AT1R blockade adjusted AngII-induced NADPH oxidase and JNK/p38-MAPK leading to attenuation of gefitinib-induced cardiac hypertrophy. This study found a pivotal role of AngII/AT1R signaling in gefitinib-induced cardiac hypertrophy, which may provide novel approaches in the management of EGFRIs-induced cardiotoxicity.     

ATDC promotes the growth and invasion of hepatocellular carcinoma cells by modulating GSK‐3β/Wnt/β‐catenin signalling

Accumulating evidence has suggested that the ataxia telangiectasia group D complementing (ATDC) gene is an emerging cancer‐related gene in multiple human cancer types. However, little is known about the role of ATDC in hepatocellular carcinoma (HCC). In this study, we aimed to investigate the expression level, biological function and underlying mechanism of ATDC in HCC. The expression of ATDC in HCC cells was detected by quantitative real‐time polymerase chain reaction and western blot analysis. Cell growth was determined by cell counting kit‐8 assay and colony formation assay. Cell invasion was assessed by Transwell invasion assay. The activation status of Wnt/β‐catenin signalling was evaluated by the luciferase reporter assay. Functional experiments showed that the silencing of ATDC expression significantly suppressed the growth and invasion of HCC cells, whereas the overexpression of ATDC promoted the growth and invasion of HCC cells in vitro. Moreover, we showed that ATDC overexpression promoted the phosphorylation of glycogen synthase kinase (GSK)‐3β and resulted in the activation of Wnt/β‐catenin signalling. Notably, the inhibition of GSK‐3β activity significantly abrogated the tumour suppressive effect of ATDC silencing, while the silencing of β‐catenin partially reversed the oncogenic effect of ATDC overexpression. Taken together, these findings reveal an oncogenic role of ATDC in HCC and show that the suppression of ATDC impedes the growth and invasion of HCC cells associated with the inactivation of Wnt/β‐catenin signalling. Our study suggests that ATDC may serve as a potential therapeutic target for HCC.     

14‐3‐3 inhibits insulin‐like growth factor‐I‐induced proliferation of cardiac fibroblasts via a phosphatidylinositol 3‐kinase‐dependent pathway

1. Insulin‐like growth factor (IGF)‐I plays an important role in the pathogenesis of heart disease and has been shown to strongly induce the proliferation of cardiac fibroblasts (CFs). It remains unknown whether 14‐3‐3 proteins, which are associated the regulation of signal transduction, affect IGF‐I‐induced CF proliferation. 2. In the present study, we investigated the effects of 14‐3‐3 proteins on CF proliferation in response to IGF‐I. Proliferation of CFs was determined by cell counting and a bromodeoxyuridine incorporation assay. Phosphorylation of signalling molecules was evaluated by western blottling. Activity of nuclear factor of activated T cells (NFAT) was examined using a dual luciferase reporter gene assay and immunofluorescence. 3. It was found that adenovirus‐mediated transfection of YFP‐R18 peptide (AdR18), a known inhibitor of 14‐3‐3, significantly enhanced IGF‐I‐induced CF proliferation. This potentiation arose from an increase in phosphorylation of phosphatidylinositol 3‐kinase (PI3‐K) and AKT (protein kinase B), inactivation of glycogen synthesis kinase (GSK) 3β and increased NFAT activity. 4. Collectively, the results of the present study suggest that 14‐3‐3 proteins inhibit IGF‐I‐induced CF proliferation via a PI3‐K‐dependent NFAT signalling pathway. This finding may contribute to our understanding of the function of 14‐3‐3 proteins in the heart.     

Resveratrol attenuates hyperoxia‐induced oxidative stress,inflammation and fibrosis and suppresses Wnt/β‐catenin signalling in lungs of neonatal rats

Although survival rate of infants born prematurely has been raised by supplemental oxygen treatment, it is followed by high morbidity of hyperoxia‐induced bronchopulmonary dysplasia. In this study, the effect of resveratrol on the lung injury was evaluated in hyperoxia‐exposed rats of preterm birth. The results demonstrated that hyperoxia led to thickened alveolar wall, simplified alveolar architecture and fibrosis. In addition, elevated methane dicarboxylic aldehyde level, decreased glutathione level and superoxide dismutase activity were also found in hyperoxic lungs, as well as the increased tumor necrosis factor‐α, interleukin‐1β and interleukin‐6 in the bronchoalveolar lavage fluid. Fibrotic‐associated proteins transforming growth factor‐β1, α‐smooth muscle actin, collagen I and fibronectin deposition were also found in interstitial substance of lungs. Furthermore, Wnt/β‐catenin signalling was found to be active in hyperoxia‐induced lungs. In addition, expression of SP‐C was increased and T1α was decreased in hyperoxia‐exposed lungs. Resveratrol intraperitoneal administration alleviated hyperoxia‐induced histological injury of lungs, regulated redox balance, decreased pro‐inflammatory cytokine release, and down‐regulated expression of fibrotic‐associated proteins. Furthermore, Wnt/β‐catenin signalling was also suppressed by resveratrol, as represented by diminished expression of lymphoid enhancer factor‐1, Wnt induced signalling protein‐1 and cyclin D1. In addition, the increase of SP‐C and decrease of T1α expression was prevented as well. The present study showed that resveratrol could protect lungs from hyperoxia‐induced injury through its antioxidant, anti‐inflammatory and anti‐fibrotic effects. The transdifferentiation of alveolar epithelial type II cells to alveolar epithelial type I cells promotion and Wnt/β‐catenin signalling suppression are also involved in the protective effect.     

Regulation of cardiac angiotensin‐converting enzyme and angiotensin AT1 receptor gene expression in Npr1 gene‐disrupted mice

1. Understanding of the regulatory mechanisms of gene expression in the control of blood pressure and fluid volume is a key issue in cardiovascular medicine. Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signalling antagonizes the physiological and pathophysiological effects mediated by the renin–angiotensin–aldosterone system (RAAS) in the regulation of cardiovascular homeostasis. 2. The targeted‐disruption of the Npr1 gene (coding for GC‐A/PRA) leads to activation of the cardiac RAAS involved in the hypertrophic remodelling process, which influences cardiac size, expression of pro‐inflammatory cytokine genes and the behaviour of various hypertrophy marker genes. The Npr1 gene‐knockout (Npr1^?/?) mice exhibit 35–40 mmHg higher systolic blood pressure and a significantly greater heart weight to bodyweight ratio than wild‐type (Npr1^+/+) mice. 3. The expression of both angiotensin‐converting enzyme (ACE) and angiotensin II AT_1a receptors are significantly increased in hearts from Npr1^?/? mice compared with hearts from Npr1^+/+ mice. In parallel, the expression of interleukin‐6 and tumour necrosis factor‐α is also markedly increased in hearts from Npr1^?/? mice. 4. These findings indicate that disruption of NPRA/cGMP signalling leads to augmented expression of the cardiac RAAS in conjunction with pro‐inflammatory cytokines in Npr1‐null mutant mice, which promotes the development of cardiac hypertrophy and remodelling.     

BPA‐toxicity via superoxide anion overload and a deficit in β‐catenin signaling in human bone mesenchymal stem cells

Bisphenol A (BPA), used in the manufacture of products based on polycarbonate plastics and epoxy resins, is well known as an endocrine‐disrupting monomer. In the current study, BPA increased cytotoxicity in hBMSCs in a dose‐ and time‐dependent manner, concomitantly with increased lipid peroxidation. Increased cell death in BPA‐treated cells was markedly blocked by pretreatment with the superoxide dismutase mimetic MnTBAP and MnTMPyP, but not by catalase, glutathione, the glutathione peroxidase mimetic ebselen, the NOS inhibitor NAME, or the xanthine oxidase inhibitor allopurinol. Furthermore, the decline in nuclear β‐catenin and cyclin D1 levels in hBMSCs exposed to BPA was reversed by MnTBAP treatment. Finally, treatment of hBMSCs with the GSK3β inhibitor LiCl₂ increased nuclear β‐catenin levels and significantly attenuated cytotoxicity compared with BPA treatment. Our current results in hBMSCs exposed to BPA suggest that BPA causes a disturbance in β‐catenin signaling via a superoxide anion overload. © 2016 The Authors Environmental Toxicology Published by Wiley Periodicals, Inc. Environ Toxicol 32: 344–352, 2017.