QNZ – 2hydroxybutanoate

ERK1/2 mediates the lipopolysaccharide-induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac ﬁbroblasts

Liang-Chi Chena, Marthandam Asokan Shibub, Chung-Jung Liuc, Chien-Kuo Hand, Da-Tong Jue, Pei-Yu Chena, Vijaya Padma Viswanadhaf, Chao-Hung Laig, Wei-Wen Kuoh,1, Chih-Yang Huangd,i,j,1,*

A B S T R A C T

Myocardial ﬁbrosis is a critical event during septic shock. Upregulation in the ﬁbrosis signaling cascade proteins such as ﬁbroblast growth factor (FGF), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and activation of matriX metalloproteinases (MMPs) are widely associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac ﬁbrosis and heart failure. However, evidences suggest that the common upstream mediators of ﬁbrosis cascade play little role in cardiac ﬁbrosis induced by LPS; further, it is unknown if LPS directly triggers the expressions and/or activity of FGF-2, uPA, tPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts. In the present study, we treated primary cultures of cardiac ﬁbroblasts with LPS to explore whether LPS upregulates FGF-2, uPA, tPA, MMP-2, MMP-9 and enhance cellular migration. Further the precise molecular and cellular mechanisms behind these LPS induced responses were identiﬁed. Inhibition as- says on MAPKs using U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor) and QNZ (NFκB inhibitor) show that LPS-induced upregulation of FGF-2, uPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts was mediated through ERK1/2 signaling. Collectively, our results provide a link between LPS-induced cardiac dysfunction and ERK1/2 signaling pathway and thereby implies ERK1/2 as a possible target to regulate LPS induced upregulation of FGF-2, uPA, MMP-2, MMP-9 and cellular migration in cardiac ﬁbroblasts.

Keywords: Lipopolysaccharide Cardiac ﬁbroblast uPA
MMPs
Cellular migration

1. Introduction

Lipopolisaccharide (LPS) from gram negative bacteria is a strong stimulus for inﬂammatory response, inducing both upregulation and release of cytokines [1–3]. As cardiac cells including cardiac ﬁbroblasts express TLR4 receptors they are prone to LPS activity and many studies have suggested that LPS stimulation contributes to cardiovascular collapse [4]. Circulating LPS has been demonstrated to mediate vascular inﬂammation in atherosclerosis, insulin resistance and weight gain [5,6]. Further, increased plasma LPS was detected in chronic heart failure (CHF) patients with substantial immune activation [7]. Severe and potentially fatal hypotension and cardiac contractile dysfunction are the characteristics in patients with sepsis [8,9]. While, various cytokines such as TNF-α secreted in response to LPS can deteriorate cardiac function, studies have demonstrated that LPS directly decreases contractility in cardiomyocytes through toll-like receptor-4 (TLR-4) [10–12].
Circulating LPS also induces cardiac ﬁbrosis and alters left ventricular structure and function [13]. A large number of studies have been dedicated to exploring the molecular mechanisms involved in the cardiac dysfunction [14]. However speciﬁc molecular events involved in LPS induced cardiac dysfunction that trigger ﬁbrosis eﬀects are not clearly understood. Mitogen-activated protein kinases (MAPKs) include three major subfamilies such as the extracellular signal regulated ki- nases (ERKs), the c-Jun N-terminal kinases (JNKs) and the p38 MAPKs [15]. Studies have shown that ERK plays an important role in dauo- mycin-induced damages of cardiac myocytes [16], and that p38 MAPK and ERK1/2 mediate the interplay of TNFα and IL-10 in regulating oXidative stresses and cardiac myocyte apoptosis [17]. The p38 MAPK also mediates myocardial proinﬂammatory cytokine production, en- dotoXin-induced contractile suppression and TNFα-induced cardiac injury [18,19]. The JNK pathway is activated by oXidative stress and is implicated in the regulations of cardiac cell survival or apoptotic ac- tivity [20]. JNK is essential for MEKK1-mediated cardiac hypertrophy and dysfunction induced by Gq [21]. NFκB pathway is reported to play a key role in mediating the decreases in cardiomyoctes contractility, and initiating inﬂammatory response when toll-like receptor is acti- vated [10]. Another pathway that has received attention is mediated by Ca2+-calmodulin activated phosphatase calcineurin (PP2B). Calci- neurin plays an important mediator for cardiac hypertrophy, ischemia- induced cardiomyocytes apoptosis and heart dysfunction [22,23]. Transgenic mice overexpressing calcineurin in the heart indeed develop cardiac hypertrophy and heart failure that mimic human heart disease [24]. One of such signaling cascades may potentially mediate of LPS induced ﬁbrosis and it is essential to evaluate such possibilities in order to formulate eﬃcient strategies in treating LPS associated cardiac ef- fects.
MatriX metalloproteinases (MMPs) are a family of functionally re- lated zinc-containing enzymes that include interstitial collagenases, gelatinases, stromelysin, matrilysin, metalloelastase and membrane- type MMPs [25,26]. MMPs denature and degrade ﬁbrillar collagens and other components of the extracellular matriX (ECM). Studies show that dysregulation of MMPs is involved in the development of myocardial extracellular matriX remodeling and cardiac ﬁbrosis, thus contributing to the progression of heart failure. Signiﬁcant upregulation of MMP-2 and MMP-9 is observed during the progression of heart failure in the infarcted rat model [27]. Deﬁciency of MMP-9 and MMP-2 in mice prevents cardiac rupture and attenuates left ventricular enlargement, collagen accumulation, cardiac dilatation and dysfunction after acute myocardial infarction [28–30]. Another proteolytic plasminogen system with its plasminogen activators (PA), such as tissue-type plas- minogen activators (t-PA) and urokinase-type plasminogen activators (u-PA) is involved in cardiac dysfunction [28,31]. Loss or inhibition of uPA is shown to reduces left ventricular remodeling and dysfunction in uPA−/- mice with transverse aortic banding (TAB) [32]. Our previous studies in H9c2 cardiomyoblasts cells showed that 1 μg/mL of LPS can eﬀectively induce inﬂammation, hypertrophy and cell death [33–35].
In the present study, we have examined the eﬀects of LPS (1 μg/mL) on the levels of FGF-2, uPA, tPA, MMP-2 and MMP-9, and the migration eﬀects of cardiac ﬁbroblasts to understand and to further identify the possible targets involved in LPS associated cardiac remodeling process.

2. Materials and methods

2.1. Cells, antibodies, reagents and enzymes

Lipopolysaccharide (LPS) and gelatin were purchased from Sigma (Sigma Chemical Co., St. Louis, Missouri, USA). The U0126 (MEK1/2 inhibitor), SB203680 (p38 MAPK inhibitor), SP600125 (JNK inhibitor) and cyclosporine A (calcineurin inhibitor) were purchased from TOCRIS (Ellisville, Missouri, USA). 6-Amino-4-(4-phenoX-yphenylethylamino) quinazoline (QNZ), NFκB activation inhibitor was purchased from Peptides International (Louisville, Kentucky, USA). We utilized the following antibodies against FGF-2, ERK1/2, phospho- ERK1/2, uPA, tPA, MMP-2 and MMP-9 (Santa Cruz Biotechnology, Inc. Santa Cruz, California, USA); α-tubulin (Lab Vision Corporation, Fremont, California, USA) as loading control. Goat anti-mouse IgG antibody conjugated to horseradish peroXidase and goat anti-rabbit IgG antibody conjugated to horseradish peroXidase and rabbit anti-goat IgG horseradish peroXidase conjugate were purchased from Santa Cruz Biotechnology, Inc. in California, USA.

2.2. Cell culture

All protocols were reviewed and approved by the Institutional Review Board, and the animal care and use committee of the China Medical University, Taichung, Republic of China. Primary cardiac ﬁ- broblasts were isolated and cultured using commercially available ﬁ- broblast Isolation System Kit (Cellutron Life Technology, Highland Park, NJ, USA) according to the manufacturer’s protocol. Brieﬂy, hearts of 1–2 days old Sprague-Dawley rats were removed, and cardiac ﬁ- broblast cells and cardiomyocytes were dispersed in digestion solution at 37 °C. Cardiac ﬁbroblast cells and cardiomyocytes were separated and subsequently cultured in DMEM containing 10% fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine. After 3–4 days, cardiac ﬁbroblasts were incubated in serum-free essential medium overnight before treatment with indicated agents.

2.3. Immunoblotting

To isolate total proteins, cultured primary cardiac ﬁbroblasts were washed with cold PBS and resuspended in lysis buﬀer (50 mM Tris, pH 7.5, 0.5 M NaCl, 1.0 mM EDTA, pH 7.5, 10% glycerol, 1 mM BME, 1% IGEPAL-630 and a proteinase inhibitor cocktail (Roche Molecular Biochemicals)). After incubation for 30 min on ice, and centrifuged at 12000 g for 15 min at 4 °C, and the concentration of the proteins in the supernatant was determined by the Bradford method. Sample con- taining equal proteins (60 μg) were loaded and analyzed by Western blot analysis. Brieﬂy, proteins were separated by 12% SDS-PAGE and transferred onto PVDF membrane (Millipore, Belford, Massachusetts, USA). Membrane were blocked with blocking buﬀer (5% non-fat dry milk, 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for at least 1 h at room temperature. Membranes were incubated with primary antibodies in the above solution on an orbital shaker at 4 °C overnight. Following hybridization of primary antibody incubations, membranes were incubated with horseradish peroXidase-linked sec- ondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG) for one hour and the blots were appropriately documented as images using a Fujiﬁlm LAS-3000 chemiluminescence detection system (Tokyo, Japan).

2.4. Gelatin zymography for MMP-2 and MMP-9

Activity of MMP-2 and MMP-9 was assessed by gelatin zymography. After treatments with/without LPS in the presence or absence of in- hibitors such as MEK1/2 inhibitor U0126, p38 MAPK inhibitor SB203680, JNK inhibitor SP600125, calcineurin inhibitor cyclosporine A and NFκB inhibitor QNZ, we collected conditioned medium, and added suﬃcient 5X loading dye to conditioned medium for gelatin zymography assay. We performed gelatin zymography analysis by loading the miXture (conditioned medium and loading dye) onto 8% SDS-PAGE gels containing 0.1% gelatin and running the electrophoresis at 140 V for 2.5 h. The gels were washed in a 2.5% Triton X-100 solution with shaking for 30 min and then incubated in 50 mL reaction buﬀer (40 mM Tris-HCl, pH 8.0; 10 mM CaCl2, 0.01% NaN3) at 37 °C for 12 h before staining with 0.25% Coomassie brilliant blue R-250 in 50% methanol and 10% acetic acid for 1 h. Quantitative analysis was preformed after destaining in the destaining solution (10% acetic acid, 20% methanol) twice for 30 min.

2.5. Migration assay

Migration assay was performed using the 48-well Boyden chamber (Neuro Probe, Gaithersburg, MD, USA) plate with the 8-μm pore size polycarbonate membrane ﬁlters. The lower compartment was ﬁlled with DMEM containing 10% FCS. Cardiac ﬁbroblasts were placed in the upper part of the Boyden chamber containing serum-free medium and incubated for 48 h. After incubation, the cells on membrane ﬁlter were ﬁXed with methanol and stained with 0.05% Giemsa for 1 h. The cells on upper surface of the ﬁlter were removed with a cotton swab. The ﬁlters were then rinsed in double distilled water until additional stain was leached. The cells were then air-dried for 20 min. The migratory phenotypes were determined by counting the cells that migrated to the lower side of the ﬁlter with microscopy at 400X magniﬁcation, re- spectively.

2.6. Statistical analysis

Each experiment was duplicated at least three times. Results were presented as the mean ± SE, and statistical comparisons were made using the one-way analysis of variants. Signiﬁcance was deﬁned at the p < 0.05. 3. Results 3.1. LPS induces the expression of FGF-2, uPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts In the present study, we also explored the eﬀects of LPS on ex- pression of FGF-2, uPA, tPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts. After incubation with LPS (1 μg/mL) for various time periods (2, 6, 12, 18 and 24 h), we observed a notable increase in the expression of FGF-2, uPA, MMP-2 and MMP-9 within 2 h and the phenomenon was maintained for up to 24 h (Fig. 1). The results demonstrated that ad- ministration of LPS signiﬁcantly upregulated FGF-2, uPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts. 3.2. ERK1/2 mediates LPS-Upregulated FGF-2, uPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts Inhibition assays were performed to identify the signal transduction pathway(s) involved in the mechanism behind LPS-upregulated expression of FGF-2, uPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts. Cardiac ﬁbroblasts cells were pre-incubated for 1 h with 10−6 M U0126 (ERK1/2 activation inhibitor); 10−6 M SB203580 (p38 inhibitor); 10−6 M SP600125 (JNK 1/2 inhibitor); 10−6 M CsA (calcineurin in- hibitor) and 10−6 M QNZ (NFkB inhibitor) prior to treatment with 1 mg/mL LPS for 24 h. Protein extracts were then subsequently sub- jected to immunoblotting assay to assess the eﬀect of these inhibitors on LPS-induced expression of FGF-2, uPA, MMP-2 and MMP-9. The ﬁnding showed that only U0126, ERK1/2 inhibitor signiﬁcantly suppressed LPS-induced protein levels of FGF-2, uPA, MMP-2 and MMP-9 (Fig. 2A). 3.3. U0126 inhibits LPS-Induced expressions of FGF-2, uPA, MMP-2 and MMP-9 in cardiac ﬁbroblasts To check if the early ﬁbrosis responses induced by LPS includes ERK1/2 mediated ﬁbrosis eﬀects, cardiac ﬁbroblasts were preincubated with U0126 (1 μM) for 1 h and followed by LPS (1 μg/mL) challenge for either 12 h or 24 h; the results showed that U0126 signiﬁcantly inhibited LPS-induced protein levels of FGF-2, uPA, MMP-2 and MMP-9 at both 12 h and 24 h time point (Fig. 3A). Further to conﬁrm if ERK1/2 responds rapidly to LPS stimulation, serum-starved cardiac ﬁbroblasts were incubated with LPS (1 μg/mL) for 0, 30 and 60 min and were subjected to immunoblotting assay. Phosphorylation of ERK1/2 (T185, Y187) is signiﬁcantly induced in response to LPS stimulation (Fig. 3B). The results reveal ERK1/2 as an important factor in mediating LPS- induced increment in the levels of ﬁbrosis associated FGF-2, uPA, MMP- 2 and MMP-9 proteins in cardiac ﬁbroblasts. 3.4. ERK1/2 mediates LPS-Induced activation of MMP-2 and MMP-9 in cardiac ﬁbroblasts We further identiﬁed the role of ERK1/2 in activating MMP-2 and MMP-9 in cardiac ﬁbroblasts treated with LPS. After treatment of cul- tured primary cardiac ﬁbroblasts with U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), CsA (calcineurin inhibitor) or QNZ (NFκB inhibitor) in the presence of LPS, we observed that ERK1/2 inhibitor U0126 suppressed LPS-induced enhancement in MMP-2 and MMP-9 activity as seen from the results of gelatin zymorgraphy assay (Fig. 4). These results reveal that ERK1/2 plays an important factor in mediating LPS-induced cardiac re- modeling. 3.5. ERK1/2 mediates LPS-Induced cell migration in cardiac ﬁbroblasts It is well known that activated cardiac ﬁbroblasts or myoﬁbroblasts migrate to damaged tissue to elevate the ECM turnover [36], therefore in the present study we further determined the eﬀects of LPS on mi- gration ability in cardiac ﬁbroblasts by culturing with LPS (1 μg/mL) in the presence or absence of ERK1/2 activation inhibitor U0126 for 48 h, and subsequently observed the ability of migration in cardiac ﬁbro- blasts by migration assay. In migration assay (Fig. 5), we observed that LPS induced a dramatic increase in cellar migration in cardiac ﬁbro- blasts. However, U0126 signiﬁcantly blocked the eﬀects of LPS on cell migration of cardiac ﬁbroblasts. These ﬁndings proved that ERK1/2 mediates LPS-modulated the migration ability in cardiac ﬁbroblasts (see Fig. 6). 4. Discussion Circulating LPS is a possible result of factors such as high-fat diet, smoking and periodontal disease [5,6]. Although, acute subclinical level of LPS in circulation is often tolerated, persistence of even subclinical levels of LPS triggers cardiac ﬁbrosis [6,37]. Various studies have demonstrated that LPS acts through various mechanisms to trigger cardiac damages. In a much early study carried out by Yasuda et al. Low levels (1 ng/mL) of LPS for 6 h was shown to depresses contractility and β-adrenergic responses in cardiac myocytes by reducing the myoﬁlament response to Ca2+ in cardiac myocytes [11]. A relatively higher concentration of LPS (10 ng/mL) has been shown to initiate apoptosis in adult rat ventricular myocytes within 12 h in a caspase dependent manner mediated through angiotensin type 1 receptors (AT1-R) and activation of calcineurine [38,39]. In addition, LPS is also known to induce cardiac ﬁbrosis and promote left ventricle remodeling and re- duces the survival [37]. NOX2 has been previously identiﬁed as po- tential novel targets to attenuate subclinical level LPS induced cardiac ﬁbrosis [6]. NOX-2 is one of the ﬁve isoforms of NADPH oXidase (NOX) in phagocytes and in cells such as endothelial cells, cardiomyocytes and ﬁbroblasts. In monocytes, NOX-2 involved ERK activation mediates ROS production for interleukin-1β production and processing [40]. In diabetic rats NOX-2 promotes collagen accumulation thereby con- tributing to diabetic cardiomyopathy in rat neonatal cardiomyocytes [41]. NOX-2 involved ERK activation is also associated with the de- velopment of renal ﬁbrosis in obstructive nephropathy [42]. However the involvement of ERK1/2 in LPS induced cardiac ﬁbrosis and their role in cardiac ﬁbroblasts cells is not much clear yet. The results of the present study reveal that LPS treatment signiﬁcantly increased the ex- pression of FGF-2, uPA, MMP-2 and MMP-9, and upregulated the ac- tivity of MMP-2 and MMP-9 in cardiac ﬁbroblasts. The increase in these mediators of ﬁbrosis was found to be highly dependent on ERK/MAPK signaling pathway. Cardiac injury triggers cardiac ﬁbroblasts to be diﬀerentiated to myoﬁbroblasts which is more eﬃcient in secreting ECM proteins. Myoﬁbroblasts are highly responsive to cardiac injury and demonstrate elevated proliferation, migration and ECM secretion. Cardiac ﬁbroblasts also maintain the homeostasis of ECM proteins and support the in- tegrity of myocardial tissue [43,44]. In our results, the migration of cardiac ﬁbroblasts and the mediators of ﬁbroblast migration were sig- niﬁcantly promoted by LPS treatment the cell migration was reduced signiﬁcantly following U0126 treatment. These ﬁndings may suggest that ERK1/2 activation is involved in the activation cardiac ﬁbroblasts and highlights ERK1/2 as a critical factor in LPS-induced cardiac dys- function and heart failure, particularly in patients with sepsis. Activation of MMPs is reported to contribute to ECM remodeling, cardiac remodeling and ventricular dilation, thus leading to heart failure [45–47]. In ischemic cardiomyopathy model, myocardial MMPs are also activated to degrade ECM and causes ventricle dilation and dysfunction [48]. In the spontaneously hypertensive heart failure (SHHF) rats, MMP-2 and MMP-9 zymographic activities are upregu- lated in both compensatory hypertrophic stage and in decompensated heart failure stage [49]. In transgenic mice with cardiac-speciﬁc over- expression of TNFα show progressive cardiac hypertrophy and dilated cardiomyopathy occurs with accompanied by a signiﬁcant increase in MMP-2 and MMP-9, an increase in collagen synthesis, deposition and denaturation, and a decrease in undenatured soluble collagens [50,51]. Deactivation of MMP-9 and MMP-2 in mice prevented cardiac rupture, and attenuates collagen accumulation, left ventricular dilation, cardiac dysfunction after acute myocardial infarction [28–30]. In the present study, we observed the signiﬁcantly increased expression of MMP-2 and MMP-9, and the notably upregulated zymorgraphic activity of MMP-2 and MMP-9 in cardiac ﬁbroblasts in response to LPS challenge. In addition, study suggested that collagen accumulation in dilated cardi- omyopathy is co-regulated by MMPs and ﬁbroblast growth factor (FGF) [52]. FGF-2 is reported to mediate the development of pressure- or volume overload-induced cardiac hypertrophy and ECM proliferation as well [53,54]. Here, we found that FGF-2 was signiﬁcantly induced in cardiac ﬁbroblast in response to LPS stimulation. Another proteolytic plasminogen system involving u-PA is also in- volved in myocardial tissue degradation and congestive heart failure [28,31]. Loss or inhibition of uPA prevents cardiac rupture [28] and attenuates cardiac remodeling and dysfunction in uPA−/- mice with transverse aortic banding (TAB) [32]. Mice with deﬁciency in uPA were less able to form ﬁbrotic scars after myocardial infarction [28,31]. Absence of uPA in these mice abolishes the ability of ﬁbroblasts to migrate into infarcted tissue and synthesize collagen. In the present study, our results showed a dramatic increase in uPA protein level within 2 h after LPS challenge in cardiac ﬁbroblasts. However, LPS treatment showed no dramatic inﬂuence on expression of tPA. These ﬁndings suggested that upregulation of uPA is involved in LPS-medi- ated cardiac damages. Evidences have suggested that plasma concentrations of LPS are raised in patients with chronic heart failure (CHF) who possess the signiﬁcant activation of immune system [7]. LPS is a potent stimulus to induce the production of proinﬂammatory cytokines (PICs) in cardio- myocytes [55]. The elevation of circulating PICs such as IL-1 [56] and IL-6 [57] has been associated with the worsening of symptoms and even the mortality [58] in CHF patients. The PICs have been showed to cause cardiac dysfunction by suppressing myocardial contractility [59,60]. Studies on various pathological conditions show that cardiac ﬁbrosis in general is mediated through TGF-β and CTGF but the LPS associated ﬁbrosis pathogenesis is known to not inﬂuence the levels of TGF-β or CTGF [6]. Evidences show that the stimuli of cardiac damage like hypoXia, oXidative stress and ischemia/reperfusion damage results in the acti- vation of protein kinase cascades, which in turn activate MAPKs such as ERK1/2, p38 MAPK and JNK1/2 [14,61,62]. These serine-threonine kinases have been showed to phosphorylate the important downstream mediators that participate in cardiac pathologies such as heart failure, hypertensive cardiac hypertrophy and cardiomyopathy [14,61,62]. The ﬁndings of the present study suggest that ERK1/2 may play as a key mediator in LPS-induced cardiac dysfunction through modulating the expression and/or activation of FGF-2, uPA, MMP-2 and MMP-9. 5. Conclusion Evidences have revealed that LPS causes serious cardiovascular collapse and even death observed in patients with bacterial sepsis. Here our ﬁndings further suggest that LPS may participate in the develop- ment of cardiac damage and dysfunction, the harmful consequences in the heart, mainly through ERK1/2 signaling pathway. Therefore, in order to avoid the cardiac diseases such as dilated cardiomyopathy, cardiac ﬁbrosis and heart failure, and the poor cardiac prognosis in patients with sepsis, we propose that the blockage of ERK1/2 signaling pathway by inhibitors may be a good therapeutic approach to prevent LPS-induced cardiac damage, cardiac ﬁbrosis and heart failure. Of course, further more studies are required to identify the feasibility in clinical applications. References [1] R.B. Yang, M.R. Mark, A. Gray, A. Huang, M.H. Xie, M. Zhang, A. Goddard, W.I. Wood, A.L. Gurney, P.J. Godowski, Toll-like receptor-2 mediates lipopoly- saccharide-induced cellular signalling, Nature 395 (1998) 284–288. [2] J.H. Hwang, K.J. Kim, S.J. Ryu, B.Y. Lee, Caﬀeine prevents LPS-induced in- ﬂammatory responses in RAW264.7 cells and zebraﬁsh, Chem. Biol. Interact. 248 (2016) 1–7. [3] J.T. Leverence, M. Medhora, G.G. Konduri, V. Sampath, Lipopolysaccharide-induced cytokine expression in alveolar epithelial cells: role of PKCζ-mediated p47phoX phosphorylation, Chem. Biol. Interact. 189 (2011) 72–81. [4] P. Boza, P. Ayala, R. Vivar, C. Humeres, F.T. Caceres, C. Munoz, L. Garcia, M.A. Hermoso, G. Diaz-Araya, EXpression and function of toll-like receptor 4 and inﬂammasomes in cardiac ﬁbroblasts and myoﬁbroblasts: IL-1beta synthesis, se- cretion, and degradation, Mol. Immunol. 74 (2016) 96–105. [5] L.L. Stoll, G.M. Denning, N.L. Weintraub, Potential role of endotoXin as a proinﬂammatory mediator of atherosclerosis, Arterioscler. Thromb. Vasc. Biol. 24 (2004) 2227–2236. [6] W.Y.W. Lew, E. Bayna, E.D. Molle, R. Contu, G. Condorelli, T. Tang, Myocardial ﬁbrosis induced by exposure to subclinical lipopolysaccharide is associated with decreased miR-29c and enhanced NOX2 expression in mice, PLoS One 9 (2014). [7] J. Niebauer, H.D. Volk, M. Kemp, M. Dominguez, R.R. Schumann, M. Rauchhaus, P.A. Poole-Wilson, A.J. Coats, S.D. Anker, EndotoXin and immune activation in chronic heart failure: a prospective cohort study, Lancet 353 (1999) 1838–1842. [8] F. Jardin, D. Brun-Ney, B. Auvert, A. Beauchet, J.P. Bourdarias, Sepsis-related cardiogenic shock, Crit. Care Med. 18 (1990) 1055–1060. [9] M. Packer, Is tumor necrosis factor an important neurohormonal mechanism in chronic heart failure? Circulation 92 (1995) 1379–1382. [10] J.H. Boyd, S. Mathur, Y. Wang, R.M. Bateman, K.R. Walley, Toll-like receptor sti- mulation in cardiomyoctes decreases contractility and initiates an NF-kappaB de- pendent inﬂammatory response, Cardiovasc. Res. 72 (2006) 384–393. [11] S. Yasuda, W.Y. Lew, Lipopolysaccharide depresses cardiac contractility and beta- adrenergic contractile response by decreasing myoﬁlament response to Ca2+ in cardiac myocytes, Circ. Res. 81 (1997) 1011–1020. [12] S. Mohanty, Y. Gautam, A.K. Maurya, A.S. Negi, O. Prakash, F. Khan, D.U. Bawankule, Indenes and tetralenes analogues attenuates lipopolysaccharide- induced inﬂammation: an in-vitro and in-vivo study, Chem. Biol. Interact. 245 (2016) 12–19. [13] F. Asgharzadeh, R. Bargi, M. Hosseini, M. Farzadnia, M. Khazaei, Cardiac and renal ﬁbrosis and oXidative stress balance in lipopolysaccharide-induced inﬂammation in male rats, ARYA atherosclerosis 14 (2018) 71–77. [14] N.H. Bishopric, P. Andreka, T. Slepak, K.A. Webster, Molecular mechanisms of apoptosis in the cardiac myocyte, Curr. Opin. Pharmacol. 1 (2001) 141–150. [15] P.H. Sugden, A. Clerk, "Stress-responsive" mitogen-activated protein kinases (c-Jun N-terminal kinases and p38 mitogen-activated protein kinases) in the myocardium, Circ. Res. 83 (1998) 345–352. [16] W. Zhu, Y. Zou, R. Aikawa, K. Harada, S. Kudoh, H. Uozumi, D. Hayashi, Y. Gu, T. Yamazaki, R. Nagai, Y. Yazaki, I. Komuro, MAPK superfamily plays an important role in daunomycin-induced apoptosis of cardiac myocytes, Circulation 100 (1999) 2100–2107. [17] S. Dhingra, A.K. Sharma, D.K. Singla, P.K. Singal, p38 and ERK1/2 MAPKs mediate the interplay of TNF-alpha and IL-10 in regulating oXidative stress and cardiac myocyte apoptosis, Am. J. Physiol. Heart Circ. Physiol. 293 (2007) H3524–H3531. [18] M. Wang, R. Sankula, B.M. Tsai, K.K. Meldrum, M. Turrentine, K.L. March, J.W. Brown, C.A. Dinarello, D.R. Meldrum, P38 MAPK mediates myocardial proinﬂammatory cytokine production and endotoXin-induced QNZ contractile suppres- sion, Shock 21 (2004) 170–174.
[19] D.R. Meldrum, C.A. Dinarello, J.C. Cleveland Jr., B.S. Cain, B.D. Shames, X. Meng, A.H. Harken, Hydrogen peroXide induces tumor necrosis factor alpha-mediated cardiac injury by a P38 mitogen-activated protein kinase-dependent mechanism, Surgery 124 (1998) 291–296 discussion 297.
[20] C.J. Dougherty, L.A. Kubasiak, H. Prentice, P. Andreka, N.H. Bishopric, K.A. Webster, Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oXidative stress, Biochem. J. 362 (2002) 561–571.
[21] T. Minamino, T. Yujiri, N. Terada, G.E. Taﬀet, L.H. Michael, G.L. Johnson, M.D. Schneider, MEKK1 is essential for cardiac hypertrophy and dysfunction in- duced by Gq, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 3866–3871.
[22] B.J. Wilkins, J.D. Molkentin, Calcineurin and cardiac hypertrophy: where have we been? Where are we going? J. Physiol. 541 (2002) 1–8.
[23] A. Lakshmikuttyamma, P. Selvakumar, R. Kakkar, R. Kanthan, R. Wang, R.K. Sharma, Activation of calcineurin expression in ischemia-reperfused rat heart and in human ischemic myocardium, J. Cell. Biochem. 90 (2003) 987–997.
[24] J.D. Molkentin, J.R. Lu, C.L. Antos, B. Markham, J. Richardson, J. Robbins, S.R. Grant, E.N. Olson, A calcineurin-dependent transcriptional pathway for cardiac hypertrophy, Cell 93 (1998) 215–228.
[25] L.M. Coussens, B. Fingleton, L.M. Matrisian, MatriX metalloproteinase inhibitors and cancer: trials and tribulations, Science 295 (2002) 2387–2392.
[26] S.M. Ellerbroek, M.S. Stack, Membrane associated matriX metalloproteinases in metastasis, Bioessays 21 (1999) 940–949.
[27] J.T. Peterson, H. Li, L. Dillon, J.W. Bryant, Evolution of matriX metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat, Cardiovasc. Res. 46 (2000) 307–315.
[28] S. Heymans, A. Luttun, D. Nuyens, G. Theilmeier, E. Creemers, L. Moons, G.D. Dyspersin, J.P. Cleutjens, M. Shipley, A. Angellilo, M. Levi, O. Nube, A. Baker, E. Keshet, F. Lupu, J.M. Herbert, J.F. Smits, S.D. Shapiro, M. Baes, M. Borgers, D. Collen, M.J. Daemen, P. Carmeliet, Inhibition of plasminogen activators or matriX metalloproteinases prevents cardiac rupture but impairs therapeutic angio- genesis and causes cardiac failure, Nat. Med. 5 (1999) 1135–1142.
[29] A. Ducharme, S. Frantz, M. Aikawa, E. Rabkin, M. Lindsey, L.E. Rohde, F.J. Schoen, R.A. Kelly, Z. Werb, P. Libby, R.T. Lee, Targeted deletion of matriX metalloprotei- nase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction, J. Clin. Investig. 106 (2000) 55–62.
[30] S. Hayashidani, H. Tsutsui, M. Ikeuchi, T. Shiomi, H. Matsusaka, T. Kubota, K. Imanaka-Yoshida, T. Itoh, A. Takeshita, Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction, Am. J. Physiol. Heart Circ. Physiol. 285 (2003) H1229–H1235.
[31] E. Creemers, J. Cleutjens, J. Smits, S. Heymans, L. Moons, D. Collen, M. Daemen, P. Carmeliet, Disruption of the plasminogen gene in mice abolishes wound healing after myocardial infarction, Am. J. Pathol. 156 (2000) 1865–1873.
[32] S. Heymans, F. Lupu, S. Terclavers, B. Vanwetswinkel, J.M. Herbert, A. Baker, D. Collen, P. Carmeliet, L. Moons, Loss or inhibition of uPA or MMP-9 attenuates LV remodeling and dysfunction after acute pressure overload in mice, Am. J. Pathol. 166 (2005) 15–25.
[33] C.J. Liu, Y.C. Cheng, K.W. Lee, H.H. Hsu, C.H. Chu, F.J. Tsai, C.H. Tsai, C.Y. Chu, J.Y. Liu, W.W. Kuo, C.Y. Huang, Lipopolysaccharide induces cellular hypertrophy through calcineurin/NFAT-3 signaling pathway in H9c2 myocardiac cells, Mol. Cell. Biochem. 313 (2008) 167–178.
[34] M.J. Fan, R. Huang-Liu, C.Y. Shen, D.T. Ju, Y.M. Lin, P.Y. Pai, P.Y. Huang, T.J. Ho, F.J. Tsai, C.H. Tsai, C.Y. Huang, Reduction of TLR4 mRNA stability and protein expressions through inhibiting cytoplasmic translocation of HuR transcription factor by E-2 and/or ER alpha in LPS-treated H9c2 cardiomyoblast cells, Chin. J. Physiol. 57 (2014) 8–18.
[35] P.C. Li, Y.C. Tien, C.H. Day, P.Y. Pai, W.W. Kuo, T.S. Chen, C.H. Kuo, C.H. Tsai, D.T. Ju, C.Y. Huang, Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts, Cardiovasc. ToXicol. 15 (2015) 172–179.
[36] S. Kim, H. Iwao, Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases, Pharmacol. Rev. 52 (2000) 11–34.
[37] W.Y.W. Lew, E. Bayna, E.D. Molle, N.D. Dalton, N.C. Lai, V. Bhargava, V. Mendiola, P. Clopton, T. Tang, Recurrent exposure to subclinical lipopolysaccharide increases mortality and induces cardiac ﬁbrosis in mice, PLoS One 8 (2013).
[38] H.L. Li, J. Suzuki, E. Bayna, F.M. Zhang, E.D. Molle, A. Clark, R.L. Engler, W.Y.W. Lew, Lipopolysaccharide induces apoptosis in adult rat ventricular myo- cytes via cardiac AT(1) receptors, Am J Physiol-Heart C 283 (2002) H461–H467.
[39] J. Suzuki, E. Bayna, H.L. Li, E. Dalle Molle, W.Y.W. Lew, Lipopolysaccharide acti- vates calcineurin in ventricular myocytes, J. Am. Coll. Cardiol. 49 (2007) 491–499.
[40] A. Singh, V. Singh, R.L. Tiwari, T. Chandra, A. Kumar, M. Dikshit, M.K. Barthwal, The IRAK-ERK-p67phoX-NoX-2 axis mediates TLR4, 2-induced ROS production for IL-1beta transcription and processing in monocytes, Cell. Mol. Immunol. 13 (2016) 745–763.
[41] Y. Liu, J. Zhang, NoX2 contributes to cardiac ﬁbrosis in diabetic cardiomyopathy in a transforming growth factor-β dependent manner, Int. J. Clin. EXp. Pathol. 8 (2015) 10908–10914.
[42] J. Qin, W.J. Mei, Y.Y. Xie, L. Huang, Q.J. Yuan, G.Y. Hu, L.J. Tao, Z.Z. Peng, Fluorofenidone attenuates oXidative stress and renal ﬁbrosis in obstructive ne- phropathy via blocking NOX2 (gp91phoX) expression and inhibiting ERK/MAPK signaling pathway, Kidney Blood Pres. Res. 40 (2015) 89–99.
[43] K.E. Porter, N.A. Turner, Cardiac ﬁbroblasts: at the heart of myocardial remodeling, Pharmacol. Therapeut. 123 (2009) 255–278.
[44] J.G. Travers, F.A. Kamal, J. Robbins, K.E. Yutzey, B.C. Blaxall, Cardiac ﬁbrosis the ﬁbroblast awakens, Circ. Res. 118 (2016) 1021–1040.
[45] D.L. Mann, F.G. Spinale, Activation of matriX metalloproteinases in the failing human heart: breaking the tie that binds, Circulation 98 (1998) 1699–1702.
[46] Z. Gunja-Smith, A.R. Morales, R. Romanelli, J.F. Woessner Jr., Remodeling of human myocardial collagen in idiopathic dilated cardiomyopathy. Role of me- talloproteinases and pyridinoline cross-links, Am. J. Pathol. 148 (1996) 1639–1648.
[47] S.C. Tyagi, Proteinases and myocardial extracellular matriX turnover, Mol. Cell. Biochem. 168 (1997) 1–12.
[48] S.C. Tyagi, S.G. Kumar, S.R. Alla, H.K. Reddy, D.J. Voelker, J.S. Janicki, EXtracellular matriX regulation of metalloproteinase and antiproteinase in human heart ﬁbroblast cells, J. Cell. Physiol. 167 (1996) 137–147.
[49] H. Li, H. Simon, T.M. Bocan, J.T. Peterson, MMP/TIMP expression in spontaneously hypertensive heart failure rats: the eﬀect of ACE- and MMP-inhibition, Cardiovasc. Res. 46 (2000) 298–306.
[50] Y.Y. Li, Y.Q. Feng, T. Kadokami, C.F. McTiernan, R. Draviam, S.C. Watkins, A.M. Feldman, Myocardial extracellular matriX remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor ne- crosis factor alpha therapy, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 12746–12751.
[51] T. Kubota, C.F. McTiernan, C.S. Frye, S.E. Slawson, B.H. Lemster, A.P. Koretsky, A.J. Demetris, A.M. Feldman, Dilated cardiomyopathy in transgenic mice with cardiac-speciﬁc overexpression of tumor necrosis factor-alpha, Circ. Res. 81 (1997) 627–635.
[52] S. Aharinejad, K. Krenn, P. Paulus, R. Schafer, A. Zuckermann, M. Grimm, D. Abraham, Diﬀerential role of TGF-beta1/bFGF and ET-1 in graft ﬁbrosis in heart failure patients, Am. J. Transplant. 5 (2005) 2185–2192.
[53] E. Kardami, Z.S. Jiang, S.K. Jimenez, C.J. Hirst, F. Sheikh, P. Zahradka, P.A. Cattini, Fibroblast growth factor 2 isoforms and cardiac hypertrophy, Cardiovasc. Res. 63 (2004) 458–466.
[54] Z.Y. He, B. Feng, S.L. Yang, H.L. Luo, Intracardiac basic ﬁbroblast growth factor and transforming growth factor-beta 1 mRNA and their proteins expression level in patients with pressure or volume-overload right or left ventricular hypertrophy, Acta Cardiol. 60 (2005) 21–25.
[55] D.R. Wagner, A. Combes, C. McTiernan, V.J. Sanders, B. Lemster, A.M. Feldman, Adenosine inhibits lipopolysaccharide-induced cardiac expression of tumor necrosis factor-alpha, Circ. Res. 82 (1998) 47–56.
[56] A. Blum, S. Sclarovsky, E. Rehavia, B. Shohat, Levels of T-lymphocyte subpopula- tions, interleukin-1 beta, and soluble interleukin-2 receptor in acute myocardial infarction, Am. Heart J. 127 (1994) 1226–1230.
[57] S.D. Anker, S. von Haehling, Inﬂammatory mediators in chronic heart failure: an overview, Heart 90 (2004) 464–470.
[58] M. Rauchhaus, W. Doehner, D.P. Francis, C. Davos, M. Kemp, C. Liebenthal, J. Niebauer, J. Hooper, H.D. Volk, A.J. Coats, S.D. Anker, Plasma cytokine para- meters and mortality in patients with chronic heart failure, Circulation 102 (2000) 3060–3067.
[59] A. Kumar, C. Haery, J.E. Parrillo, Myocardial dysfunction in septic shock, Crit. Care = Clin. 16 (2000) 251–287.
[60] A. Kumar, R. Brar, P. Wang, L. Dee, G. Skorupa, F. Khadour, R. Schulz, J.E. Parrillo, Role of nitric oXide and cGMP in human septic serum-induced depression of cardiac myocyte contractility, Am. J. Physiol. 276 (1999) R265–R276.
[61] T. Ravingerova, M. Barancik, M. Strniskova, Mitogen-activated protein kinases: a new therapeutic target in cardiac pathology, Mol. Cell. Biochem. 247 (2003) 127–138.
[62] C.P. Baines, J.D. Molkentin, STRESS signaling pathways that modulate cardiac myocyte apoptosis, J. Mol. Cell. Cardiol. 38 (2005) 47–62.