Jin-Yu Wang

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

AIMS 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. METHODS AND RESULTS 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. CONCLUSION 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.

CTRP3 attenuates post-infarct cardiac fibrosis by targeting Smad3 activation and inhibiting myofibroblast differentiation.

C1q/tumor necrosis factor-related protein-3 (CTRP3) is a novel adipokine with modulation effects on metabolism, inflammation, and cardiovascular system. This study aimed to investigate the effect of CTRP3 on cardiac fibrosis and its underlying mechanism. The myocardial expression of CTRP3 was significantly decreased after myocardial infarction (MI). Adenovirus-delivered CTRP3 supplement attenuated myocardial hypertrophy, improved cardiac function, inhibited interstitial fibrosis, and decreased the number of myofibroblasts post-MI. In cultured adult rat cardiac fibroblasts (CFs), CTRP3 attenuated cell proliferation; migration; and the expression of connective tissue growth factor, collagen I, and collagen III induced by transforming growth factor (TGF)-β1. Moreover, CTRP3 inhibited whereas CTRP3 small interfering RNA (siRNA) facilitated the expression of α-SMA and profibrotic molecules induced by TGF-β1. CTRP3 also attenuated TGF-β1-induced Smad3 phosphorylation, nuclear translocation, and interaction with p300. CTRP3 increased the phosphorylation of AMP-activated protein kinase (AMPK) and Akt in both rat hearts and CFs. Adenine 9-β-d-arabinofuranoside (AraA), an AMPK inhibitor, abolished the protective effect of CTRP3 against TGF-β1-induced profibrotic response and Smad3 activation. Taken together, CTRP3 attenuates cardiac fibrosis by inhibiting myofibroblast differentiation and the subsequent extracellular matrix production. AMPK is required for the anti-fibrotic effect of CTRP3 through targeting Smad3 activation and inhibiting myofibroblast differentiation.Key messageCTRP3 alleviates cardiac fibrosis in a rat post-MI model and in cardiac fibroblasts.CTRP3 inhibits fibroblast-to-myofibroblast differentiation.CTRP3 exerts anti-fibrotic effect through targeting Smad3 activation.AMPK mediates the anti-fibrotic effect of CTRP3 by inhibition of Smad3 activation.

Overexpression of C1q/Tumor Necrosis Factor–Related Protein-3 Promotes Phosphate-Induced Vascular Smooth Muscle Cell Calcification Both In Vivo and In Vitro

Objective—Vascular calcification is highly correlated with increased cardiovascular morbidity and mortality. C1q/tumor necrosis factor–related protein-3 (CTRP3) is a newly identified adipokine that plays important roles in cardiovascular system. Here, we investigated the role of CTRP3 in vascular calcification and its underlying mechanism. Approach and Results—Adenine-induced chronic renal failure rat model was used to mimic the process of arterial medial calcification. The level of CTRP3 was elevated in serum and abdominal aorta of chronic renal failure rats. Periadventitial gene delivery of CTRP3 significantly accelerated the calcification of abdominal aorta and arterial ring. In cultured vascular smooth muscle cells (VSMCs), CTRP3 increased &bgr;-glycerophosphate–induced calcium deposition and alkaline phosphatase activity. Although CTRP3 alone was not sufficient to induce calcification in VSMCs, it upregulated the expression of osteogenic marker genes including runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2, and osteopontin. CTRP3 further enhanced &bgr;-glycerophosphate–induced downregulation of smooth muscle &agr;-actin and smooth muscle 22&agr;, while augmenting osteogenic marker expression in VSMCs induced by &bgr;-glycerophosphate. In contrast, knockdown of CTRP3 in VSMCs potently suppressed &bgr;-glycerophosphate–induced calcification. Mechanistically, knockdown of Runx2 inhibited CTRP3-promoted VSMC calcification. CTRP3 increased extracellular signal–regulated kinase 1/2 phosphorylation and reactive oxygen species production. Preincubation with U0126, an extracellular signal–regulated kinase 1/2 upstream kinase inhibitor, had no effect on CTRP3-induced reactive oxygen species production. However, pretreatment with N-acetyl-L-cysteine, a reactive oxygen species scavenger, suppressed CTRP3-induced extracellular signal–regulated kinase 1/2 phosphorylation. Both N-acetyl-L-cysteine and U0126 significantly inhibited CTRP3-induced upregulation of Runx2 and calcified nodule formation. Conclusions—CTRP3 promotes vascular calcification by enhancing phosphate-induced osteogenic transition of VSMC through reactive oxygen species–extracellular signal–regulated kinase 1/2–Runx2 pathway.

Angiotensin II Reduces Cardiac AdipoR1 Expression through AT1 Receptor/ROS/ERK1/2/c-Myc Pathway

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.

Apelin induces vascular smooth muscle cells migration via a PI3K/Akt/FoxO3a/MMP-2 pathway.

Apelin is an adipokine that has a critical role in the development of atherosclerosis, which may offer potential for therapy. Because migration of vascular smooth muscle cells (VSMCs) is a key event in the development of atherosclerosis, understanding its effect on the atherosclerotic vasculature is needed. Here we investigated the effect of apelin on VSMC migration and the possible signaling mechanism. In cultured rat VSMCs, apelin dose- and time-dependently promoted VSMC migration. Apelin increased the phosphorylation of Akt, whereas LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and an Akt1/2 kinase inhibitor blocked the apelin-induced VSMC migration. Apelin dose-dependently induced phosphorylation of Forkhead box O3a (FoxO3a) and promoted its translocation from the nucleus to cytoplasm, which were blocked by LY294002 and Akt1/2 kinase inhibitor. Furthermore, apelin increased matrix metalloproteinase 2 (MMP-2) expression and gelatinolytic activity. Overexpression of a constitutively active, phosphorylation-resistant mutant, TM-FoxO3a, in VSMCs abrogated the effect of apelin on MMP-2 expression and VSMC migration. ARP101, an inhibitor of MMP-2, suppressed apelin-induced VSMC migration. Moreover, the levels of apelin, phosphorylated Akt, FoxO3a, and MMP-2 were higher in human carotid-artery atherosclerotic plaque than in adjacent normal vessels. We demonstrate that PI3K/Akt/FoxO3a signaling may be involved in apelin inducing VSMC migration. Phosphorylation of FoxO3a plays a central role in mediating the apelin-induced MMP-2 activation and VSMC migration.

Globular CTRP3 promotes mitochondrial biogenesis in cardiomyocytes through AMPK/PGC-1α pathway.

BACKGROUND Mitochondrial biogenesis is crucial for the maintenance of mitochondrial function and cellular homeostasis. C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine that owns multiple functions on metabolic and cardiovascular diseases. However, whether CTRP3 affects mitochondrial biogenesis in cardiomyocytes remains unknown. METHODS Neonatal rat ventricular myocytes were cultured and treated with globular CTRP3 (gCTRP3). The expression of mitochondrial biogenesis related genes was measured by real-time PCR and western blot analysis. Mitochondrial morphology was assessed by a transmission electron microscope. ATP content, oxygen consumption rate (OCR), and sirtuin1 activity were measured with commercial kits. RESULTS gCTRP3 increased the expression of peroxisome proliferators activated receptor-γ co-activator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), NRF-2, mitochondrial transcription factor A (TFAM), cytochrome B, and oxidative phosphorylation complexes III and V, and increased mitochondrial cristae components and OCR. Additionally, gCTRP3 enhanced mitochondrial DNA copy number and ATP content, while the induction was inhibited by knockdown of PGC-1α via small interfering RNA. gCTRP3 increased phosphorylation of AMP-activated protein kinase (AMPK), whereas adenine 9-β-d-arabinofuranoside (AraA), an AMPK inhibitor, attenuated gCTRP3-mediated induction of NRF-1, TFAM, and complexes III and V. gCTRP3 increased both the expression and activity of sirtuin1, whereas inhibition of sirtuin1 by EX-527 attenuated gCTRP3-induced responses. Meanwhile, gCTRP3-mediated activation of sirtuin1 was attenuated by AraA. Moreover, gCTRP3 restored the reduction of sirtuin1, PGC-1α, NRF-1, complex III and ATP content induced by hypoxia-reoxygenation injury. CONCLUSION CTRP3 promotes mitochondrial biogenesis in cardiomyocytes via AMPK/PGC-1α pathway. GENERAL SIGNIFICANCE CTRP3 is an endogenous modulator for mitochondrial biogenesis, and may protect cardiomyocytes by ameliorating mitochondrial dysfunction.

Decreased adiponectin level is associated with aggressive phenotype of tongue squamous cell carcinoma

Circulating adiponectin levels are inversely associated with risk of various obesity‐related cancers. However, the effect of adiponectin on carcinogenesis and progression of tongue squamous cell carcinoma (TSCC) remains unknown. We measured serum adiponectin levels in 59 patients with TSCC and 50 healthy controls. Expression of adiponectin and its receptors in paired tumor and paracancerous specimens were determined by immunohistochemical staining (n = 37) and western blot (n = 30), respectively. Serum adiponectin level was lower in patients than in controls (5.0 ± 2.4 vs 8.4 ± 3.5 μg/mL, P < 0.01), and was inversely associated with histological grade and lymph node metastasis but not tumor size. Local adiponectin levels in tumor tissue gradually decreased as tumor‐node‐metastasis stage increased, while the expression of adiponectin receptors was unchanged. In addition, serum adiponectin levels in the TSCC patients without metabolic and cardiovascular diseases, or without smoking and drinking habits, were still lower than in controls. Furthermore, adiponectin inhibited the migration, but not proliferation, of SCC15 cells in vitro. These results indicate that a decreased adiponectin level is associated with risk of TSCC. Hypoadiponectinemia might be used as a biomarker to predict an aggressive phenotype of TSCC.

CTRP3 promotes energy production by inducing mitochondrial ROS and up-expression of PGC-1α in vascular smooth muscle cells

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine with modulation effects on metabolism and inflammation. Adenosine triphosphate (ATP) exerts multiple biological effects in vascular smooth muscle cells (VSMCs) and energy imbalance is involved in vascular diseases. This study aimed to explore the effect of CTRP3 on energy production and its underlying mechanism in VSMCs. Our results indicated that exogenous CTRP3 increased ATP synthesis and the protein expression of oxidative phosphorylation (OXPHOS)-related molecules, including peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, sirtuin-3 (SIRT3), complex I, II, III, and V in cultured VSMCs. Depletion of endogenous CTRP3 by small interfering RNA (siRNA) reduced ATP synthesis and the expression of those molecules. PGC-1α knockdown abrogated CTRP3-induced ATP production and OXPHOS-related protein expression. Furthermore, CTRP3 increased mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential level. Pretreatment with N-acetyl-L-cysteine, a reactive oxygen species scavenger, and cyanidem-chlorophenylhydrazone, an uncoupler of OXPHOS, suppressed CTRP3-induced ROS production, PGC-1α expression and ATP synthesis. In conclusion, CTRP3 modulates mitochondrial energy production through targets of ROS and PGC-1α in VSMCs.

C1q/tumor necrosis factor-related protein-3 enhances the contractility of cardiomyocyte by increasing calcium sensitivity

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine that protects against myocardial infarction-induced cardiac dysfunction through its pro-angiogenic, anti-apoptotic, and anti-fibrotic effects. However, whether CTRP3 can directly affect the systolic and diastolic function of cardiomyocytes remains unknown. Adult rat cardiomyocytes were isolated and loaded with Fura-2AM. The contraction and Ca2+ transient data was collected and analyzed by IonOptix system. 1 and 2μg/ml CTRP3 significantly increased the contraction of cardiomyocytes. However, CTRP3 did not alter the diastolic Ca2+ content, systolic Ca2+ content, Ca2+ transient amplitude, and L-type Ca2+ channel current. To reveal whether CTRP3 affects the Ca2+ sensitivity of cardiomyocytes, the typical phase-plane diagrams of sarcomere length vs. Fura-2 ratio was performed. We observed a left-ward shifting of the late relaxation trajectory after CTRP3 perfusion, as quantified by decreased Ca2+ content at 50% sarcomere relaxation, and increased mean gradient (μm/Fura-2 ratio) during 500-600ms (-0.163 vs. -0.279), 500-700ms (-0.159 vs. -0.248), and 500-800ms (-0.148 vs. -0.243). Consistently, the phosphorylation level of cardiac troponin I at Ser23/24 was reduced by CTRP3, which could be eliminated by preincubation of okadaic acid, a type 2A protein phosphatase inhibitor. In summary, CTRP3 increases the contraction of cardiomyocytes by increasing the myofilament Ca2+ sensitivity. CTRP3 might be a potential endogenous Ca2+ sensitizer that modulates the contractility of cardiomyocytes.

Cartilage intermediate layer protein-1 alleviates pressure overload-induced cardiac fibrosis via interfering TGF-β1 signaling

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins in the myocardium and results in decreased ventricular compliance and diastolic dysfunction. Cartilage intermediate layer protein-1 (CILP-1), a novel identified cardiac matricellular protein, is upregulated in most conditions associated with cardiac remodeling, however, whether CILP-1 is involved in pressure overload-induced fibrotic response is unknown. Here, we investigated whether CILP-1 was critically involved in the fibrotic remodeling induced by pressure overload. Western blot analysis and immunofluorescence staining showed that CILP-1 was predominantly detected in cardiac myocytes and to a less extent in the interstitium. In isolated adult mouse ventricular myocytes and nonmyocytes, CILP-1 was found to be mainly synthesized by myocytes. CILP-1 expression in left ventricles was upregulated in C57BL/6 mice undergoing transverse aortic constriction (TAC). Myocardial CILP-1 knockdown aggravated whereas CILP-1 overexpression attenuated TAC-induced ventricular remodeling and dysfunction, as measured by echocardiography test, morphological examination, and gene expressions of fibrotic molecules. Incubation of cardiac fibroblasts with the conditioned medium containing full-length, N-terminal, or C-terminal CILP-1 inhibited transforming growth factor (TGF)-β1-induced Smad3 phosphorylation and the subsequent profibrotic events. We first demonstrated that C-terminal CILP-1 increased Akt phosphorylation, promoted the interaction between Akt and Smad3, and suppressed Smad3 phosphorylation. Blockade of PI3K-Akt pathway attenuated the inhibitory effect of C-CILP-1 on TGF-β1-induced Smad3 activation. We conclude that CILP-1 is a novel ECM protein possessing anti-fibrotic ability in pressure overload-induced fibrotic remodeling. This anti-fibrotic effect of CILP-1 attributes to interfering TGF-β1 signaling through its N- and C- terminal fragments.