II-induced Cardiac Fibroblast Proliferation Research Articles

Biomimetic nanoparticles loaded lutein functionalized by macrophage membrane for targeted amelioration pressure overload-induced cardiac fibrosis

Lutein is a strong antioxidant with anti-inflammatory, anti-oxidative and cardioprotective effects and could be a promising candidate for the treatment of hypertensive heart disease (HHD), but is not clinically appealing because of its low oral bioavailability and main distribution in the eyes. To address this, a biomimetic drug delivery system-MMLNPs was established by coating macrophage membranes (MMs) onto lutein-loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles (LNPs). This study characterized the physical properties of biomimetic nanoparticles and examined the targeting capability, therapeutic effects and mechanism, and biosecurity of administering them for cardiac fibrosis therapy in the transverse aortic constriction (TAC) model and in vitro. Transmission electron microscope mapping and dynamic light scattering analysis proved that MMLNPs were spherical nanoparticles camouflaged by a layer of cell membrane and had negative zeta potential. Confocal laser scanning microscopy and flow cytometry analysis showed that MMs on the biomimetic nanoparticles hindered the phagocytosis of macrophages and facilitated the targeting of activated endothelial cells. Ex vivo fluorescence imaging experiments demonstrated the targeting of biomimetic nanoparticles to the injured heart. EdU assay indicated that MMLNPs have the same potential to inhibit angiotensin (Ang) II-induced cardiac fibroblast proliferation as free lutein. Furthermore, echocardiography showed that MMLNPs improved cardiac function and structure, and Masson staining and western blotting showed that MMLNPs ameliorated cardiac fibrosis. We found MMLNPs inhibited the interleukin (IL)-11/ERK signaling pathway which was up-regulated in the TAC model compared to the sham-operated mouse. Biochemical testing and hematoxylin and eosin staining proved that the long-term use of MMLNPs lacked biological toxicity. Collectively, MMLNPs might be a promising nanodrug delivery approach to attenuate pressure overload (PO)-induced cardiac fibrosis.

Puerarin attenuates angiotensin II-induced cardiac fibroblast proliferation via the promotion of catalase activity and the inhibition of hydrogen peroxide-dependent Rac-1 activation

The aims of the present study were to evaluate the effects of puerarin on angiotensin II-induced cardiac fibroblast proliferation and to explore the molecular mechanisms of action. Considering the role of H2O2 in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, we hypothesized that modulating catalase activity would be a potential target in regulating the redox-sensitive pathways. Our results showed that the activation of Rac1 was dependent on the levels of intracellular H2O2. Puerarin blocked the phosphorylation of extracellular regulated protein kinases (ERK)1/2, abolished activator protein (AP)-1 binding activity, and eventually attenuated cardiac fibroblast proliferation through the inhibition of H2O2-dependent Rac1 activation. Further studies revealed that angiotensin II treatment resulted in decreased catalase protein expression and enzyme activity, which was disrupted by puerarin via the upregulation of catalase protein expression at the transcriptional level and the prolonged protein degradation. These findings indicated that the anti-proliferation mechanism of puerarin was mainly through blocking angiontensin II-triggered downregulation of catalase expression and H2O2-dependent Rac1 activation.

Anti-proliferative effect of the extract of Guangzao (Fructus Choerospondiatis) on cultured rat cardiac fibroblasts

ObjectiveTo ascertain if total flavonoids of Guangzao (Fructus Choerospondiatis) (TFFC) extracted from Guangzao (Fructus Choerospondiatis) can inhibit angiotensin II-induced proliferation of cardiac fibroblasts (CFs). MethodsCFs were cultured by the differential attachment method. A model of cell proliferation was established by stimulation with Ang II. Cardiac fibroblasts growth was determined using a hemocytometer. Cell proliferation was detected by methyl thiazole tetrazolium. Lactate dehydrogenase activity was measured by chemical colorimetric method. ResultsProliferation of TFFC-treated (25, 50, 100 mg/L) fibroblasts was significantly less than that of cells in the angiotensin II group (P < 0.01), and TFFC inhibited proliferation in a dose-dependent manner. These inhibitory effects were partly blocked by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME) and 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ). ConclusionTFFC inhibited angiotensin II-induced proliferation of cardiac fibroblasts via a mechanism that probably involves activation of the NO-cyclic guanosine monophosphate signaling pathway.

PKC δ and βII regulate angiotensin II-mediated fibrosis through p38: a mechanism of RV fibrosis in pulmonary hypertension.

Pulmonary hypertension (PH) eventually leads to right ventricular (RV) fibrosis and dysfunction that is associated with increased morbidity and mortality. Although angiotensin II plays an important role in RV remodeling associated with hypoxic PH, the molecular mechanisms underlying RV fibrosis in PH largely remain unresolved. We hypothesized that PKC-p38 signaling is involved in RV collagen accumulation in PH and in response to angiotensin II stimulation. Adult male Sprague-Dawley rats were exposed to 3 wk of normoxia or hypoxia (10% FiO2 ) as a model of PH. Hypoxic rats developed RV hypertrophy and fibrosis associated with an increase in PKC βII and δ protein expression and p38 dephosphorylation in freshly isolated RV cardiac fibroblasts. Further mechanistic studies were performed in cultured primary cardiac fibroblasts stimulated with angiotensin II, a key activator of ventricular fibrosis in PH. Angiotensin II induced a reduction in p38 phosphorylation that was attenuated following chemical inhibition of PKC βII and δ. Molecular and chemical inhibition of PKC βII and δ abrogated angiotensin II-induced cardiac fibroblast proliferation and collagen deposition in vitro. The effects of PKC inhibition on proliferation and fibrosis were reversed by chemical inhibition of p38. Conversely, constitutive activation of p38 attenuated angiotensin II-induced increase of cardiac fibroblast proliferation and collagen accumulation. PKC βII- and δ-dependent inactivation of p38 regulates cardiac fibroblast proliferation and collagen deposition in response to angiotensin II, which suggests that the PKC-p38 signaling in cardiac fibroblasts may be involved and important in the pathophysiology of RV fibrosis in PH.

Dietary capsaicin ameliorates pressure overload-induced cardiac hypertrophy and fibrosis through the transient receptor potential vanilloid type 1.

Dietary capsaicin plays a protective role in hypertension, atherosclerosis, obesity, and hyperlipidemia through activating the transient receptor potential vanilloid type 1 (TRPV1), a nonselective cation channel. This study was designed to investigate the role of capsaicin in cardiac hypertrophy and fibrosis in a pressure overload model. TRPV1 knockout (KO) mice and their wild-type (WT) littermates, aged 8 weeks, were randomly divided into sham and aortic banding surgery groups and were fed with chow or chow plus capsaicin for 10 weeks. Dietary capsaicin significantly attenuates pressure overload-induced increase in heart weight index, enlargement of ventricular volume, decrease in cardiac function, and increase in cardiac fibrosis in WT mice. However, these effects of capsaicin were absent in TRPV1 KO mice. Additionally, capsaicin blunted pressure overload-induced upregulation of transforming growth factor β, connective tissue growth factor, and the phosphorylation of Smad2/3 in WT mice but not in TRPV1 KO mice. Moreover, capsaicin attenuated pressure overload-induced overexpression of metalloproteinase (MMP)-2, MMP-9 and MMP-13 in WT mice but not in TRPV1 KO mice. Capsaicin also attenuated angiotensin II-induced proliferation of cardiac fibroblasts from mice with the TRPV1 channel. Our results suggest that dietary capsaicin protects against cardiac hypertrophy and fibrosis in pressure overload mice through TRPV1.

The critical role of Sestrin 1 in regulating the proliferation of cardiac fibroblasts

The proliferation of cardiac fibroblasts is pivotal in the development of cardiac fibrosis. Sestrin 1, which functions as antioxidant, plays diverse roles in the regulation of proliferation and cellular injury that is induced by oxidative stress. However, little is known regarding the impact of Sestrin 1 on the proliferation of cardiac fibroblasts. In the present study, with knockdown of Sestrin 1 by siRNA, we surveyed the effect of Sestrin 1 on cardiac fibroblast proliferation. Downregulation of Sestrin 1 promotes Ang II-induced proliferation of cardiac fibroblasts, leading to increased DNA synthesis and collagen production. Moreover, in the absence of Ang II, a similar phenotype to the basal condition was detected with silencing of Sestrin 1. Further analysis of the pro-proliferating signals revealed that knockdown of Sestrin 1 significantly activated ERK1/2 and mTOR, meanwhile, downregulation of Sestrin 1 also enhanced the expression of collagen type I and CTGF, which play important role in the cardiac fibrosis. Consistent with the antioxidant property of Sestrin 1, we determined that the proliferation induced by silence of Sestrin 1 was accompanied by a remarkably enhanced production of reactive oxygen species (ROS). However, diminishing ROS by NAC, a potent antioxidant, could only partly repress the pro-proliferative effect of Sestrin 1-downregulation. Consequently, our study demonstrated that Sestrin 1 plays an important role in the proliferation of cardiac fibroblasts, and the effect could be partly mediated by decreased oxidative stress.

PPAR-γ activation by rosiglitazone suppresses angiotensin II-mediated proliferation and phenotypictransition in cardiac fibroblasts via inhibition of activation of activator protein 1

Cardiac fibroblasts play an important role in myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands have been reported to have a number of cardioprotective properties. However, the mechanism underlying this protective effect has not yet been elucidated. The purpose of the present study was to investigate the effect of rosiglitazone on angiotensin II-induced cardiac fibroblast proliferation and differentiation. Cardiac fibroblasts were stimulated with angiotensin II (10–7M) in the presence or absence of rosiglitazone (10–5nM). Pretreatment of cardiac fibroblasts with rosiglitazone significantly inhibited angiotensin II-induced cardiac fibroblast proliferation and profibrotic phenotypes differentiation and, thus, reduced the overall production of collagen components. PPAR-γ antagonist GW9662 significantly inhibited these effects of rosiglitazone, suggesting that these effects of rosiglitazone were PPAR-γ-dependent. To investigate the mechanisms involved, we found that PPAR-γ activation by rosiglitazone inhibited the formation of c-fos/c-jun heterodimers and expression of activator protein 1 induced by ANG II and thus inhibited transcription of the downstream genes involved in CFs proliferation and differentiation. Our data suggests PPAR-γ activation could have an anti-fibrotic effect through limiting cardiac fibroblast proliferation and differentiation, which are the critical steps in the pathogenesis of cardiac fibrosis.

Effects of Chinese herbal medicine Xuefu Zhuyu Decoction on angiotensin Ⅱ-induced proliferation and extracellular matrix synthesis of rat cardiac fibroblasts

To investigate the effects of Xuefu Zhuyu Decoction, a compound Chinese herbal medicine, on the proliferation and extracellular matrix synthesis of cardiac fibroblasts (CFs) from neonatal rats induced by angiotensin II, and to explore the mechanism of Xuefu Zhuyu Decoction in improving myocardial fibrosis. CFs derived from neonatal rats were cultured with enzymatic dissociation to establish a cell model of cardiac fibrosis induced by angiotensin II in vitro. CFs were divided into 6 groups, including blank control group, angiotensin II group, valsartan-medicated serum group, and 5%, 10% and 15% Xuefu Zhuyu decoction-medicated serum group. CFs in the blank control group were incubated with normal rat serum; CFs in the other groups were stimulated with angiotensin II at a dose of 10-6 mol/L, and then incubated with normal rat serum and 5%, 10% and 15% Xuefu Zhuyu decoction-medicated serum for 24 h, respectively. Methyl thiazolyl tetrazolium assay was employed to measure CF proliferation, and collagen synthesis was detected by analysis of the hydroxyproline in CFs. Radioimmunoassay was used to measure concentrations of procollagen III and hyaluronic acid. Enzyme-linked immunosorbent assay was used to measure fibronectin expression. Compared with the blank control group, angiotensin II induced cardiac fibroblast proliferation, stimulated DNA and collagen syntheses (P<0.01), and increased fibronectin, hyaluronic acid and procollagen III concentrations (P<0.01). Xuefu Zhuyu decoction-medicated serum inhibited CF proliferation and DNA and collagen syntheses (P<0.05), and decreased the concentrations of fibroectin, hyaluronic acid and procollagen III (P<0.01). Angiotensin II can induce cardiac fibrosis, stimulate cardiac fibroblast proliferation and extracellular matrix synthesis, while Xuefu Zhuyu Decoction can prevent cardiac fibrosis, the mechanism of which may be related to inhibiting CF proliferation and extracellular matrix synthesis.

E0094 Research of triptolide on Ang II-induced neonatal cardiac fibroblasts proliferation and collagen synthesis

ObjectivesTo observe whether TP can inhibit proliferation and collagen synthesis of cardiac fibroblasts and clarify the possible mechanisms.MethodsDifferential attachment technique to obtain and cultivate neonatal SD rat cardiac fibroblasts. Experimental...

17β-estradiol inhibits angiotensin II-induced cardiac myofibroblast differentiation

Cardiac fibroblasts play an important role in myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Estrogen has been reported to have a number of cardioprotective properties. However, it is unclear whether estrogen affects cardiac fibroblast differentiation. The purpose of the present study was to investigate the effect of estrogen on angiotensin II-induced cardiac fibroblast proliferation and differentiation. Cardiac fibroblasts were stimulated with angiotensin II (1 μM) in the presence or absence of 17β-estradiol (100 nM). Pretreatment of cardiac fibroblasts with 17β-estradiol significantly inhibited angiotensin II-induced cardiac fibroblast proliferation and differentiation (indicated by a reduction in alpha-smooth muscle actin (α-SMA) expression) by 25% and 20%. Pretreatment of 17β-estradiol significantly reduced angiotensin II-increased levels of phospho-p38 mitogen-activated protein kinase (MAPK) by 40% and nuclear factor-κB (NF-κB) binding activity in cardiac fibroblasts by 55%. Our data suggests estrogen could have an anti-fibrotic effect through limiting cardiac fibroblast proliferation and differentiation, which are the critical steps in the pathogenesis of cardiac fibrosis.

L-Carnitine attenuates angiotensin II-induced proliferation of cardiac fibroblasts: role of NADPH oxidase inhibition and decreased sphingosine-1-phosphate generation

The heart is unable to synthesize l-carnitine and is strictly dependent on the l-carnitine provided by the blood stream; however, additional studies are needed to better understand the mechanism of l-carnitine supplementation to the heart. The aim of this study was to evaluate the effects of l-carnitine on angiotensin II (Ang II)-induced cardiac fibroblast proliferation and to explore its intracellular mechanism(s). Cultured rat cardiac fibroblasts were pretreated with l-carnitine (1-30 mM) then stimulated with Ang II (100 nM). Ang II increased fibroblast proliferation and endothelin-1 expression, which were partially inhibited by l-carnitine. l-Carnitine also attenuated Ang II-induced NADPH oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, activator protein-1-mediated reporter activity and sphingosine-1-phosphate generation. In addition, l-carnitine increased prostacyclin (PGI 2) generation in cardiac fibroblasts. siRNA transfection of PGI 2 synthase significantly reduced l-carnitine-induced PGI 2 and its anti-proliferation effects on cardiac fibroblasts. Furthermore, blockading potential PGI 2 receptors, including immunoprecipitation (IP) receptors and peroxisome proliferator-activated receptors alpha (PPARα) and delta , revealed that siRNA-mediated blockage of PPARα considerably reduced the anti-proliferation effect of l-carnitine. In summary, these results suggest that l-carnitine attenuates Ang II-induced effects (including NADPH oxidase activation, sphingosine-1-phosphate generation and cell proliferation) in part through PGI 2 and PPARα-signaling pathways.

Involvement of reactive oxygen species in urotensin II-induced proliferation of cardiac fibroblasts

Urotensin II, a cyclic dodecapeptide, has recently been demonstrated to play an important role in cardiac remodeling and fibrosis. Cardiac fibroblast is the cell type known to proliferate during cardiac fibrosis and to produce the excess matrix proteins characteristic of cardiac remodeling. However, the effect of urotensin II on cardiac fibroblast proliferation and the intracellular mechanisms remain to be clarified. Cultured neonatal rat cardiac fibroblasts were stimulated with urotensin II, cell proliferation and the reactive oxygen species generation were examined. We also examined the effects of antioxidant pretreatment on urotensin II-induced cell proliferation, extracellular signal-regulated kinase phosphorylation, and the tyrosine phosphorylation of epidermal growth factor receptor, to elucidate the redox-sensitive pathway in urotensin II-induced cell proliferation. Urotensin II-increased cell proliferation and intracellular reactive oxygen species levels which were inhibited by antioxidants N-acetylcysteine, and the flavin inhibitor diphenyleneiodonium. Urotensin II potently activated the tyrosine phosphorylation of epidermal growth factor receptors and extracellular signal-regulated kinase. Pretreatment of cells with U0126, an inhibitor of the upstream activator of mitogen-activated protein kinase kinase, or with AG1478, a selective epidermal growth factor receptor kinase inhibitor, reduced the urotensin II-increased extracellular signal-regulated kinase phosphorylation. Antioxidants, U0126, and AG1478, all significantly inhibited urotensin II-increased cell proliferation in cardiac fibroblasts. Our data suggest that the redox-sensitive intracellular signaling pathway plays a role in urotensin II-induced proliferation in rat cardiac fibroblasts.