AngII-induced TGF-β1 Research Articles

Liensinine improves AngII-induced vascular remodeling via MAPK/TGF-β1/Smad2/3 signaling

Ethnopharmacological relevanceLiensinine(Lien, C37H42N2O6) is an alkaloid compound from plumula nelumbinis that demonstrates an antihypertensive effect. The protective effects of Lien on target organs during hypertension are still unclear.Aim of the study: This study aimed to understand the mechanism of Lien during the treatment of hypertension, with emphasis on vascular protection. Materials and methodsLien was extracted and isolated from plumula nelumbinis for further study. In vivo model of Ang II-induced hypertension, non-invasive sphygmomanometer was used to detect the blood pressure in and out of the context of Lien intervention. Ultrasound was used to detect the abdominal aorta pulse wave and media thickness of hypertensive mice, and RNA sequencing was used to detect the differential genes and pathways of blood vessels. The intersection of Lien and MAPK protein molecules was detected by molecular interconnecting technique. The pathological conditions of abdominal aorta vessels of mice were observed by HE staining. The expression of PCNA, α-SMA, Collagen Type Ⅰ and Collagen Type Ⅲ proteins were detected by IHC. The collagen expression in the abdominal aorta was detected by Sirius red staining. The MAPK/TGF-β1/Smad2/3 signaling and the protein expression of PCNA and α-SMA was detected by Western blot. In vitro, MAPK/TGF-β1/Smad2/3 signaling and the protein expression of PCNA and α-SMA were detected by Western blot, and the expression of α-SMA was detected by immunofluorescence; ELISA was used to detect the effect of ERK/MAPK inhibitor PD98059 on Ang Ⅱ-induced TGF-β1secrete; and the detection TGF-β1and α-SMA protein expression by Western blot; Western blot was used to detect the effect of ERK/MAPK stimulant12-O-tetradecanoyl phorbol-13-acetate (TPA) on the protein expression of TGF-β1 and α-SMA. ResultsLien displayed an antihypertensive effect on Ang Ⅱ-induced hypertension, reducing the pulse wave conduction velocity of the abdominal aorta and the thickness of the abdominal aorta vessel wall, ultimately improving the pathological state of blood vessels. RNA sequencing further indicated that the differential pathways expressed in the abdominal aorta of hypertensive mice were enriched in proliferation-related markers compared with the Control group. The profile of differentially expressed pathways was ultimately reversed by Lien. Particularly, MAPK protein demonstrated good binding with the Lien molecule. In vivo, Lien inhibited Ang Ⅱ-induced abdominal aorta wall thickening, reduced collagen deposition in the ventral aortic vessel, and prevented the occurrence of vascular remodeling by inhibiting MAPK/TGF-β1/Smad2/3 signaling activation. In addition, Lien inhibited the activation of Ang II-induced MAPK and TGF-β1/Smad2/3 signaling, attenuating the expression of PCNA and inhibiting the reduction of α-SMA, collectively playing a role in the inhibition of Ang Ⅱ-induced hypertensive vascular remodeling. PD98059 alone could inhibit Ang Ⅱ-induced elevation of TGF-β1 and the decrease of α-SMA expression. Further, PD98059 combined with Lien had no discrepancy with the inhibitors alone. Simultaneously TPA alone could significantly increase the expression of TGF-β1 and decrease the expression of α-SMA. Further, Lien could inhibit the effect of TPA. ConclusionThis study helped clarify the protective mechanism of Lien during hypertension, elucidating its role as an inhibitor of vascular remodeling and providing an experimental basis for the research and development of novel antihypertensive therapies.

Irisin attenuates angiotensin II-induced atrial fibrillation and atrial fibrosis via LOXL2 and TGFβ1/Smad2/3 signaling pathways.

Irisin was reported as a cardioprotective and anti-oxidative effector, while the effect on atrial fibrosis is unknown. The current research examined irisin's function in atrial fibrillation (AF); atrial fibrosis brought on by Ang II can be suppressed, thus lessening the risk of developing AF. 246 individuals were enrolled in the present case-control study. Chinese AF patients (n=126), 83 of whom were paroxysmal AF (PAF), 43 patients with persistent AF (PeAF), and 120 healthy controls. Saline or Ang II (2.0 mg/kg/day) was subcutaneously injected into healthy male C57BL/6 mice for four weeks. Once daily for four weeks, intraperitoneal injections of exogenous irisin (500 g/kg/day) were administered. In comparison to PAF patients and healthy controls (all P<0.05), PeAF patients had significantly higher rates of heart failure (HF), large left atrial size (LAD), hypertrophic protein B-type natriuretic peptide (BNP), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-terminal telopeptide of type I collagen (CTX-I), and transforming growth factor beta-1 (TGF-β1), while superoxide dismutase (SOD) level was low. Expression of irisin was decreased in AF patients' serum and Ang II-infused mice. Exogenous irisin dramatically reduced apoptosis, atrial fibrosis, atrial inflammation, and the susceptibility to AF caused by Ang II. In the atrial tissue, irisin inhibited Ang II-induced fibroblast transdifferentiation, LOXL2, TGF-β1, collagen production, and phosphorylation of Smad2/3. The study results speculated that irisin could be a potential AF target, and it inhibited atrial fibrosis and significantly impaired increased AF susceptibility through inactivation of LOXL2 and the TGF-β/Smad pathway.

Sauchinone Protects Renal Mesangial Cell Dysfunction against Angiotensin II by Improving Renal Fibrosis and Inflammation.

Abnormal and excessive growth of mesangial cells is important in the pathophysiologic processes of diabetes-associated interstitial fibrosis and glomerulosclerosis, leading to diabetic nephropathy, which eventually turns into end-stage renal disease. Sauchinone, a biologically-active lignan isolated from aerial parts of Saururus chinensis, has anti-inflammatory and anti-viral activities effects on various cell types. However, there are no studies reporting the effects of sauchinone on diabetic nephropathy. The present study aims to investigate the role of sauchinone in mesangial cell proliferation and fibrosis induced by angiotensin II, as well as the underlying mechanisms of these processes. Human renal mesangial cells were induced by angiotensin II (AngII, 10 μM) in the presence or absence of sauchinone (0.1–1 μM) and incubated for 48 h. In this study, we found that AngII induced mesangial cell proliferation, while treatment with sauchinone inhibited the cell proliferation in a dose-dependent manner. Pre-treatment with sauchinone induced down-regulation of cyclins/CDKs and up-regulation of CDK inhibitor, p21, and p27kip1 expression. In addition, AngII-enhanced expression of fibrosis biomarkers such as fibronectin, collagen IV, and connective tissue growth factor (CTGF), which was markedly attenuated by sauchinone. Sauchinone also decreased AngII-induced TGF-β1 and Smad-2, Smad-3, and Smad-4 expression. This study further revealed that sauchinone ameliorated AngII-induced mesangial inflammation through disturbing activation of inflammatory factors, and NLRP3 inflammasome, which is composed of the NLRP3 protein, procaspase-1, and apoptosis-associated speck-like protein containing a CARD (ASC). Moreover, pretreatment of sauchinone inhibited NF-κB translocation and ROS production in AngII-exposed mesangial cells. These data suggest that sauchinone has a protective effect on renal proliferation, fibrosis and inflammation. Therefore, sauchinone might be a potential pharmacological agent in prevention of AngII-induced renal damage leading to diabetic nephropathy.

IMM-H007 improves heart function via reducing cardiac fibrosis

Cardiac dysfunction is a pathological state characterized by damaged ability of the left ventricle (LV) to either eject or fill blood accompanied by cardiac hypertrophy and fibrosis. IMM-H007, an adenosine derivative, is an activator of AMP-Activated Protein Kinase (AMPK). AMPK can decrease the transforming growth factor-β1 (TGF-β1) expression during fibrosis. Therefore, we hypothesized that IMM-H007 contributed to cardiac dysfunction by mediating cardiac fibrosis. To test this hypothesis, we used angiotensin II (AngII)-induced cardiac remodeling model treated with IMM-H007 or vehicle. Echocardiography measurements showed that IMM-H007 significantly improved heart function indicated by increased LV ejection fraction (%LVEF) and LV fractional shortening (%LVFS). Histological staining and qRT-PCR analysis revealed that IMM-H007 markedly reduced AngII-induced cardiac fibroblast activation (α-smooth muscle actin and periostin) and matrix protein production (Collagen I and Collagen III). However, IMM-H007 did not affect AngII-induced cardiac hypertrophy. Immunoblotting analysis revealed that IMM-H007 activated AMPK, decreased the expression of TGF-β1, and inhibited the activation of Smad2 in heart tissues. In mouse primary cultured cardiac fibroblasts, pharmacological activation of AMPK by IMM-H007 significantly reduced AngII-induced TGF-β1 expression as well. Consistently, in human cardiac fibroblasts-adult ventricular (HCF-av), IMM-H007 activated AMPK and markedly suppressed AngII-induced TGF-β1 expression. These results together reveal that IMM-H007 improves heart function, and alleviates AngII-induced cardiac fibrosis by regulating AMPK-TGF-β1 signaling. These findings suggest IMM-H007 as a potential drug for treating cardiac dysfunction.

The cofilin phosphatase slingshot homolog 1 restrains angiotensin II-induced vascular hypertrophy and fibrosis in vivo

The dual specificity phosphatase slingshot homolog 1 (SSH1) contributes to actin remodeling by dephosphorylating and activating the actin-severing protein cofilin. The reorganization of the actin cytoskeleton has been implicated in chronic hypertension and the subsequent mechano-adaptive rearrangement of vessel wall components. Therefore, using a novel Ssh1−/− mouse model, we investigated the potential role of SSH1 in angiotensin II (Ang II)-induced hypertension, and vascular remodeling. We found that loss of SSH1 did not produce overt phenotypic changes and that baseline blood pressures as well as heart rates were comparable between Ssh1+/+ and Ssh1−/− mice. Although 14 days of Ang II treatment equally increased systolic blood pressure in both genotypes, histological assessment of aortic samples indicated that medial thickening was exacerbated by the loss of SSH1. Consequently, reverse-transcription quantitative PCR analysis of the transcripts from Ang II-infused animals confirmed increased aortic expression levels of fibronectin, and osteopontin in Ssh1−/− when compared to wild-type mice. Mechanistically, our data suggest that fibrosis in SSH1-deficient mice occurs by a process that involves aberrant responses to Ang II-induced TGFβ1. Taken together, our work indicates that Ang II-dependent fibrotic gene expression and vascular remodeling, but not the Ang II-induced pressor response, are modulated by SSH1-mediated signaling pathways and SSH1 activity is protective against Ang II-induced remodeling in the vasculature.

Xanthine Oxidase Inhibition by Febuxostat in Macrophages Suppresses Angiotensin II-Induced Aortic Fibrosis.

BACKGROUNDSeveral reports from basic researches and clinical studies have suggested that xanthine oxidase (XO) inhibitors have suppressive effects on cardiovascular diseases. However, the roles of a XO inhibitor, febuxostat (FEB), in the pathogenesis of vascular remodeling and hypertension independent of the serum uric acid level remain unclear.METHODSTo induce vascular remodeling in mice, angiotensin II (Ang II) was infused for 2 weeks with a subcutaneously implanted osmotic minipump. FEB was administered every day during Ang II infusion. Aortic fibrosis was assessed by elastica van Gieson staining. Mouse macrophage RAW264.7 cells (RAW) and mouse embryonic fibroblasts were used for in vitro studies.RESULTSFEB suppressed Ang II-induced blood pressure elevation and aortic fibrosis. Immunostaining showed that Ang II-induced macrophage infiltration in the aorta tended to be suppressed by FEB, and XO was mainly colocalized in macrophages, not in fibroblasts. Transforming growth factor-β1 (TGF-β1) mRNA expression was induced in the aorta in the Ang II alone group, but not in the Ang II + FEB group. Ang II induced α-smooth muscle actin-positive fibroblasts in the aortic wall, but FEB suppressed them. XO expression and activity were induced by Ang II stimulation alone but not by Ang II + FEB in RAW. FEB suppressed Ang II-induced TGF-β1 mRNA expression in RAW.CONCLUSIONSOur results suggested that FEB ameliorates Ang II-induced aortic fibrosis via suppressing macrophage-derived TGF-β1 expression.

AdipoRon, an adiponectin receptor agonist, attenuates cardiac remodeling induced by pressure overload.

AdipoRon, a small-molecule adiponectin receptor (AdipoR) agonist, has been reported to be implicated in cardiovascular diseases. However, its role in pressure-overload-induced cardiac remodeling is still elusive. To elucidate the role of AdipoRon in the pathogenesis of cardiac remodeling in vivo and vitro, in the left ventricle of human end-stage heart failure, the expression of AdipoR2 is upregulated. Meanwhile, increased expression of AdipoR2 was also observed in mice failing hearts. Oral administration of AdipoRon alleviated cardiac hypertrophy and fibrosis induced by pressure overload, as evidenced by the beneficial change of cross-sectional area of cardiomyocytes, heart weight-to-body weight ratio, gene expression of hypertrophic markers, ventricle collagen ratio, and cardiac function. The AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-β1 expression and cardiac fibroblast differentiation, and these inhibitory effects were abrogated by treatment with the AMPK inhibitor Compound C. Consistent with the above results, AdipoRon abolished the ability to retard AngII-induced TGF-β1 expression in AMPKα2-/- cardiac fibroblasts. In AMPKα2-/- mice subjected to aortic banding, AdipoRon abolished the protective effect, as indicated by increased cross-sectional area, cardiac collagen ratio, and cardiac dysfunction. Our results demonstrated that AdipoR2 expression was markedly increased in the failing hearts. AdipoRon inhibited TGF-β1 expression and myofibroblast differentiation in AMPKα-dependent manner in vitro. In line with the vitro results, AMPKα2-/- mice markedly abrogated the inhibitory effects of AdipoRon in cardiac remodeling. These results indicated AdipoRon may hold promise of an effective therapy against pressure-overload-induced cardiac remodeling. KEY MESSAGES: • The increased expression of AdipoR2 is observed in human and mice failing hearts, the changeable expression of AdipoR suggests the possible role of AdipoR in cardiac remodeling. • Oral administration of AdipoRon alleviates cardiac hypertrophy and fibrosis induced by pressure overload, and AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-β1 expression and cardiac fibroblast differentiation. • These findings provide new mechanistic insight and open new therapeutic pathways for heart failure.

Long noncoding RNA MALAT1 mediates cardiac fibrosis in experimental postinfarct myocardium mice model.

Cardiac fibrosis is a pathological remodeling response to myocardial infarction (MI) and impairs cardiac contractility. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is increased in patients with MI. However, the functions of MALAT1 in cardiac fibrosis have not been elucidated. This study elucidates the roles of MALAT1 in MI and the underlying mechanisms. The MI model was established by artificial coronary artery occlusion in mice. Western blot analysis and quantitative reverse transcription-polymerase chain reaction were performed to analyze protein expression and RNA expression, respectively. Cardiac function was measured by echocardiography. Masson's trichrome staining was used to exhibit the fibrotic area in MI hearts. Cardiac fibroblasts were isolated from newborn pups, and cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Upregulation of MALAT1 and downregulation of microRNA-145 (miR-145) were induced in MI heart and angiotensin II (AngII)-treated cardiac fibroblasts, and the inhibition of miR-145 expression was reversed by MALAT1 depletion. Knockdown MALAT1 ameliorated MI-impaired cardiac function and prevented AngII-induced fibroblast proliferation, collagen production, and α-SMA expression in cardiac fibroblasts. MALAT1 stability and transforming growth factor-β1 (TGF-β1) activity were regulated by miR-145. AngII-induced TGF-β1 activity in cardiac fibroblasts was blocked by MALAT1 knockdown. Based on these results, we concluded that lncRNA MALAT1 promotes cardiac fibrosis and deteriorates cardiac function post-MI by regulating TGF-β1 activity via miR-145.

Inhibition of (pro)renin Receptor Contributes to Renoprotective Effects of Angiotensin II Type 1 Receptor Blockade in Diabetic Nephropathy.

Aims: Renal renin-angiotensin system (RAS) plays a pivotal role in the development of diabetic nephropathy (DN). Angiotensin II (Ang II) type 1 receptor (AT1R) blockade elevates (pro)renin, which may bind to (pro)renin receptor (PRR) and exert receptor-mediated, angiotensin-independent profibrotic effects. We therefore investigated whether PRR activation leads to the limited anti-fibrotic effects of AT1R blockade on DN, and whether PRR inhibition might ameliorate progression of DN.Methods: To address the issue, the expression of RAS components was tested in different stages of streptozotocin (STZ)-induced diabetic rats (6, 12, and 24 weeks) and 6-week AT1R blockade (losartan) treated diabetic rats. Using the blocker for PRR, the handle region peptide (HRP) of prorenin, the effects of PRR on high glucose or Ang II-induced proliferative and profibrotic actions were evaluated by measurement of cell proliferation, matrix metalloproteinase-2 (MMP-2) activity, activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and transforming growth factor-β1 (TGF-β1) expression in rat mesangial cells (MCs).Results: PRR was downregulated in the kidneys of different stages of diabetic rats (6, 12, and 24 weeks). Moreover, 6-week losartan treatment further suppressed PRR expression via upregulating AT2R, and ameliorated diabetic renal injury. HRP inhibited high glucose and Ang II-induced proliferative and profibrotic effects in MCs through suppressing TGF-β1 expression and activating MMP-2. Meanwhile, HRP enhanced losartan's anti-fibrotic effects through further inhibiting phosphorylation of ERK1/2 and TGF-β1 expression. Moreover, the inhibitive effect of HRP on Ang II-induced TGF-β1 expression depended on the regulation of PRR expression by AT2R.Conclusions: Our findings suggest that inhibition of PRR contributes to renoprotection against diabetic nephropathy by AT1R blockade.

Metformin attenuates angiotensin II-induced TGFβ1 expression by targeting hepatocyte nuclear factor-4-α.

Metformin, a small molecule, antihyperglycaemic agent, is a well-known activator of AMP-activated protein kinase (AMPK) and protects against cardiac fibrosis. However, the underlying mechanisms remain elusive. TGFβ1 is a key cytokine mediating cardiac fibrosis. Here, we investigated the effects of metformin on TGFβ1 production induced by angiotensin II (AngII) and the underlying mechanisms. Wild-type and AMPKα2-/- C57BL/6 mice were injected s.c. with metformin or saline and infused with AngII (3mg·kg-1 ·day-1 ) for 7days. Adult mouse cardiac fibroblasts (CFs) were isolated for in vitro experiments. In CFs, metformin inhibited AngII-induced TGFβ1 expression via AMPK activation. Analysis using bioinformatics predicted a potential hepatocyte nuclear factor 4α (HNF4α)-binding site in the promoter region of the Tgfb1 gene. Overexpressing HNF4α increased TGFβ1 expression in CFs. HNF4α siRNA attenuated AngII-induced TGFβ1 production and cardiac fibrosis in vitro and in vivo. Metformin inhibited the AngII-induced increases in HNF4α protein expression and binding to the Tgfb1 promoter in CFs. In vivo, metformin blocked the AngII-induced increase in cardiac HNF4α protein levels in wild-type mice but not in AMPKα2-/- mice. Consequently, metformin inhibited AngII-induced TGFβ1 production and cardiac fibrosis in wild-type mice but not in AMPKα2-/- mice. HNF4α mediates AngII-induced TGFβ1 transcription and cardiac fibrosis. Metformin inhibits AngII-induced HNF4α expression via AMPK activation, thus decreasing TGFβ1 transcription and cardiac fibrosis. These findings reveal a novel antifibrotic mechanism of action of metformin and identify HNF4α as a new potential therapeutic target for cardiac fibrosis. This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Adiponectin attenuates Ang Ⅱ-induced TGFβ1 production in human mesangial cells via an AMPK-dependent pathway.

Glomerulosclerosis is a key element in end-stage renal failure. Angiotensin II (Ang II) plays an important role in modulating cell growth and extracellular matrix (ECM) synthesis and degradation. Adiponectin, a protein derived from adipocytes, is primarily involved in regulating glucose levels and fatty acid break down. It has recently been shown to have antiatherosclerotic and anti-inflammatory properties. However, the role of adiponectin as a renoprotective agent has not been fully explored. We herein examine the effect of adiponectin on Ang II-induced TGFβ1 and ECM production in human renal mesangial cells (HRMCs) and explore the signaling pathway involved. In this study, we found that both adiponectin receptor 1 and adiponectin receptor 2 are expressed in HRMCs. Adiponectin (10 μg/mL) attenuated the stimulatory effect of Ang II on TGF-β1 and fibronectin. Furthermore, adiponectin activated the AMP-activated protein kinase (AMPK), and the AMPK-specific inhibitor (compound C) blocked AMPK activation. We also determined that compound C blocked the inhibitory effect of adiponectin on Ang II-stimulated TGFβ1 and fibronectin production. In summary, these results demonstrate that adiponectin suppresses Ang II-induced synthesis of ECM in mesangial cells via activation of the AMPK pathway. Based on our data, we suggest that this mechanism could delay the progression of kidney disease.

NF-κB Mediates the Survival of Corneal Myofibroblast Induced by Angiotensin II

To investigate the role of angiotensin II (Ang II) in the regulation of corneal myofibroblast apoptosis and the possible signaling pathway. Rabbit corneal myofibroblasts were cultured in vitro and the cell phenotype was identified by expression of α-smooth muscle actin (α-SMA) and formation of F-actin. The expression of Ang II type I receptor (AT1R) in keratocytes and corneal myofibroblasts were detected by immunofluorescence staining and Western blot. The effect of Ang II on corneal myofibroblast apoptosis induced by serum starvation and TNFα plus cycloheximide (CHX) was examined by TUNEL, Hoechst 33258 staining, and caspase 3/7 activity assay. The effect of Ang II on nuclear factor-κB (NF-κB)-dependent DNA binding activity and transcriptional activity was studied by electrophoresis mobility shift assay (EMSA) and luciferase reporter assay, respectively. Ang II-induced TGFβ1 secretion by corneal myofibroblasts was determined by ELISA. Ang II type I receptor expression was more abundant in corneal myofibroblasts compared with keratocytes. Ang II reduced corneal myofibroblasts apoptotic response to serum starvation or treatment with TNFα plus CHX. This protective effect was attenuated in the presence of AT1R antagonist losartan or NF-κB-specific inhibitor Bay11-7082. Ang II increased NF-κB-dependent DNA-binding activity and transcriptional activity, and also increased TGFβ1 production by corneal myofibroblasts. Ang II induces corneal myofibroblasts resistance to apoptosis via activating NF-κB signaling pathway, and thus should be further investigated as a possible target for therapy of corneal fibrosis.

Effect of sodium tanshinone ⅡA sulfonate on AngⅡ-induced atrial fibroblast collagen synthesis and TGF-β1 activation

Effect of sodium tanshinone ⅡA sulfonate on AngⅡ-induced atrial fibroblast collagen synthesis and TGF-β1 activation

Overexpression of SIRT1 in vascular smooth muscle cells attenuates angiotensin II-induced vascular remodeling and hypertension in mice

Angiotensin II (AngII) induces the development of vascular hypertrophy and hypertension. We have shown previously that overexpression of class III deacetylase SIRT1 inhibits AngII-induced hypertrophy in vascular smooth muscle cells (VSMCs). However, the direct role of SIRT1 in VSMCs in response to AngII infusion in vivo remains unclear. Here, we found that the expression and activity of SIRT1 in mouse aortas was decreased significantly by AngII infusion. VSMC-specific SIRT1 transgene (SV-Tg) prevented the increase in systolic blood pressure (SBP) caused by AngII infusion without affecting heart function in mice. SIRT1 overexpression alleviated vascular remodeling in mouse thoracic and renal aortas induced by AngII infusion, and significantly inhibited reactive oxygen species (ROS) generation, vascular inflammation, and collagen synthesis in arterial walls. Reduced expression of transforming growth factor-β 1 (TGF-β1) was also observed in the aortas of AngII-infused SV-Tg mice. Moreover, SIRT1 overexpression decreased AngII-increased binding of nuclear factor-κB on its specific binding sites on TGF-β1 promoter. Taken together, these data demonstrate that SIRT1 overexpression in VSMCs reduces SBP and inhibits AngII-induced vascular remodeling in mice. The inhibition of vascular remodeling contributes, at least in part, to the antihypertensive effect of SIRT1. SIRT1 is reduced in aortas of AngII-infused hypertensive mice. SIRT1 VSMC transgene alleviates AngII-increased systolic blood pressure. SIRT1 VSMC transgene attenuates AngII-induced vascular remodeling. VSMC SIRT1 overexpression inhibits remodeling-related pathological changes. VSMC SIRT1 overexpression reduces AngII-induced TGF-β1 expression.

Krüppel-Like Factor 4 Transcriptionally Regulates TGF-β1 and Contributes to Cardiac Myofibroblast Differentiation

Angiotensin II (Ang II) plays a major role in the pathogenesis of cardiac fibrosis in hypertension. It is known that Ang II induces TGF-β1 expression. How transcription mediates Ang II-induced TGF-β1 expression, as well as its contribution to cardiac fibrosis, is unknown. We studied the role of Krüppel-like family transcription factors in Ang II-induced myofibroblast formation. We found that among the Krüppel-like family members, Krüppel-like factor 4 (Klf4) was the highest expressed form in isolated cardiac fibroblasts after Ang II treatment. Klf4 increased expression of α-SMA and collagen, as well as increased myofibroblast formation. ChIP assays showed that Klf4 specifically bound to the TGF-β1 promoter. Deletion and mutagenesis analysis showed that the sites at −184∼−180 bp and −45∼−41 bp in the TGF-β1 promoter were responsible for Klf4 transactivation of the TGF-β1 promoter. Our studies demonstrate that Klf4 plays a pivotal role in Ang II-induced cardiac myofibroblast differentiation and collagen synthesis through transcriptional upregulation of TGF-β1.

Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-β1 pathways in cardiac fibroblasts

Angiotensin II (AngII) is involved in extracellular matrix (ECM) accumulation contributing to heart failure. Periostin, a 90 kDa ECM protein, is a key regulator of cardiac fibrosis, and its expression is significantly higher in failing hearts. We determined the modulatory effect of AngII on periostin level and explored the possible signal transduction mechanism. AngII (400 ng/kg/min) or normal saline was infused subcutaneously for 28 days into rats; AngII antagonism was with losartan (10 mg/kg/day orally). AngII infusion induced cardiac fibrosis and increased periostin expression, which was attenuated by losartan. In cultured adult rat cardiac fibroblasts, AngII promoted the mRNA and protein expression of periostin. AngII provoked activation of cAMP response element-binding protein (CREB), and CREB small interfering RNA (siRNA) suppressed AngII-induced periostin expression. Inhibition of p38 mitogen-activated protein kinase (p38 MAPK) with SB202190 attenuated AngII-induced CREB activation and periostin expression. Transfection with Ras guanyl-releasing protein 1 siRNA or RasN17 dominant-negative plasmid prevented AngII-induced p38 MAPK phosphorylation and periostin expression. Transforming growth factor (TGF)-β1 antibody decreased the stimulatory effect of AngII on periostin expression. The extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 attenuated AngII-induced TGF-β1 expression, Smad2/3 nuclear accumulation, and periostin expression. The activation of the Ras/p38 MAPK/CREB pathway is required for AngII-induced periostin expression. ERK1/2 also participates in AngII-induced periostin expression by regulating TGF-β1/Smad signalling.

Angiotensin II-induced differentiation of adipose tissue-derived mesenchymal stem cells to smooth muscle-like cells

Angiotensin II (Ang II) is involved in the development of cardiovascular disease and vascular remodeling. In this study, we demonstrate that treatment of human adipose tissue-derived mesenchymal stem cells (hADSCs) with Ang II increased the expression of smooth muscle-specific genes, including α-smooth muscle actin (α-SMA), calponin, h-caldesmon, and smooth muscle myosin heavy chain (SM-MHC), and also elicited the secretion of transforming growth factor-β1 (TGF-β1) and delayed phosphorylation of Smad2. The Ang II-induced expression of α-SMA and delayed phosphorylation of Smad2 were blocked by pretreatment of the cells with a TGF-β type I receptor kinase inhibitor, SB-431542, small interference RNA-mediated depletion of endogenous Smad2, and adenoviral expression of Smad7. Furthermore, the Ang II-induced TGF-β1 secretion, α-SMA expression, and delayed phosphorylation of Smad2 in hADSCs were abrogated by the MEK inhibitor U0126, suggesting a pivotal role of MEK/ERK pathway in the Ang II-induced activation of TGF-β1-Smad2 signaling pathway. The smooth muscle-like cells which were differentiated from hADSCs by Ang II treatment exhibited contraction in response to 60 mM KCl. These results suggest that Ang II induces differentiation of hADSCs to contractile smooth muscle-like cells through ERK-dependent activation of the autocrine TGF-β1-Smad2 crosstalk pathway.

Angiotensin II Mediates High Glucose-Induced TGF-β1 and Fibronectin Upregulation in HPMC through Reactive Oxygen Species

To demonstrate the presence of an independent renin-angiotensin system (RAS) in the peritoneum and to determine the role of locally produced angiotensin (Ang) II in high glucose-induced upregulation of transforming growth factor (TGF)-beta1 and fibronectin by human peritoneal mesothelial cells (HPMC). In cultured HPMC, the expression of mRNAs for angiotensinogen, angiotensin-converting enzyme (ACE), Ang II type 1 receptor (AT1), and TGF-beta1 was evaluated by real-time polymerase chain reaction; ACE, AT1, and fibronectin proteins by Western blot analysis; and Ang I, Ang II, and TGF-beta1 proteins by ELISA. Dichlorofluorescein (DCF)-sensitive cellular reactive oxygen species (ROS) were measured by fluorometry. HPMC constitutively expressed all the components of RAS, and 50 mmol/L D-glucose (high glucose) significantly increased angiotensinogen, ACE, and AT1 mRNAs and ACE, AT1, and Ang II proteins. Ang II increased TGF-beta1 and fibronectin protein expression and DCF-sensitive cellular ROS. Losartan prevented Ang II-induced increase in cellular ROS. Both losartan and captopril inhibited high glucose-induced upregulation of TGF-beta1 and fibronectin expression in HPMC in a dose-dependent manner. Antioxidant catalase and NADPH oxidase inhibitor diphenyleneiodinium effectively inhibited Ang II-induced TGF-beta1 and fibronectin protein expression. The present data demonstrate that HPMC constitutively express RAS, that Ang II produced by HPMC mediates high glucose-induced upregulation of TGF-beta1 and fibronectin expression, and that Ang II-induced TGF-beta1 and fibronectin expression in HPMC is mediated by NADPH oxidase-dependent ROS. These data suggest that locally produced Ang II and ROS in the peritoneum may be potential therapeutic targets in peritoneal fibrosis during long-term peritoneal dialysis.