Adult Rat Cardiac Fibroblasts Research Articles

Abstract 173: Expanded Role for Thrombin/PAR1 Signaling in Cardiac Fibroblast Activation

Cardiac fibrosis is a feature of numerous types of heart disease that lead to disability and death and is characterized by the transformation of cardiac fibroblasts (CFs) to myofibroblasts (myofibs) that synthesize and release large amounts of collagen-rich extracellular matrix (ECM), thereby resulting in reduced cardiac function and eventually heart failure. CFs are the most abundant cell type in the heart and regulate the production of ECM as a normal part of wound repair but disease can result if CFs become overactive, at least in part by increased transformation to myofibs. Cardiac fibrosis can occur spontaneously in advanced age, after acute injury (e.g., myocardial infarction) and in disease states (e.g., diabetes mellitus and heart failure). We sought to identify novel therapeutic targets for cardiac fibrosis by using an unbiased approach (RT-PCR array) to define the most abundantly expressed G protein-coupled receptors (GPCRs) predicted to regulate the CF/myofib transformation. Certain GPCRs, e.g. angiotensin and endothelin receptors, can promote cardiac fibrosis but few others are known to do this. We hypothesized that previously unrecognized GPCRs play an important role in regulating the function of CFs. Using the RT-PCR GPCR array, we found that the protease-activated receptor 1 (PAR1) is the most abundant GPCR in adult rat CFs (rCFs). Thrombin activation of PAR1 in rCFs elevates (with distinct kinetics) the expression of a variety of pro-fibrotic markers, induces morphological changes to the myofib phenotype and stimulates synthesis of collagen, the primary ECM component without affecting cell proliferation. The thrombin-induced initiation of a proliferation-independent pro-fibrotic response in adult rCFs contrasts with effects of thrombin in neonatal rCFs, suggesting a differential wound repair response in the adult versus the still-developing heart. By defining the level of expression of GPCRs in rCFs and revealing an expanded role for PAR1 in the wound repair response of the heart, the data provide a proof-of-principle that our approach can identify novel functional and disease-relevant GPCRs in CFs-- and likely in other cardiovascular cells as well.

Defining the cellular repertoire of GPCRs identifies a profibrotic role for the most highly expressed receptor, protease‐activated receptor 1, in cardiac fibroblasts

G-protein-coupled receptors (GPCRs) have many roles in cell regulation and are commonly used as drug targets, but the repertoire of GPCRs expressed by individual cell types has not been defined. Here we use an unbiased approach, GPCR RT-PCR array, to define the expression of nonchemosensory GPCRs by cardiac fibroblasts (CFs) isolated from Rattus norvegicus. CFs were selected because of their importance for cardiac structure and function and their contribution to cardiac fibrosis, which occurs with advanced age, after acute injury (e.g., myocardial infarction), and in disease states (e.g., diabetes mellitus, hypertension). We discovered that adult rat CFs express 190 GPCRs and that activation of protease-activated receptor 1 (PAR1), the most highly expressed receptor, raises the expression of profibrotic markers in rat CFs, resulting in a 60% increase in collagen synthesis and conversion to a profibrogenic myofibroblast phenotype. We use siRNA knockdown of PAR1 (90% decrease in mRNA) to show that the profibrotic effects of thrombin are PAR1-dependent. These findings, which define the expression of GPCRs in CFs, provide a proof of principle of an approach to discover previously unappreciated, functionally relevant GPCRs and reveal a potential role for thrombin and PAR1 in wound repair and pathophysiology of the adult heart.

Cyclic AMP induces necroptotic cell death in cardiac fibroblasts

Increased levels of the second messenger cyclic AMP (cAMP) can promote cell loss of a variety of cell types. The precise mechanism(s) for this loss is/are not clearly defined, although cAMP can promote apoptosis of certain types of cells (Insel et al, Acta Physiol in press). In the present study, we used adult rat cardiac fibroblasts (rCFs) to test the hypothesis that cAMP induces necroptosis. Our results from morphological and biochemical studies provide evidence that cAMP induces necroptosis in rCFs and is mediated by protein kinase A and not Epac, another cAMP effector. The findings include destruction of the plasma membrane of rCFs, which differs from apoptotic shrinking of the membrane, and a prominent increase in expression of HMGB1, a marker of necrosis, following treatment with cAMP‐elevating agents. In addition, such agents induce death in a caspase‐PARP independent manner. The pan‐caspase inhibitor, z‐VAD‐fmk increases cell death induced by CPT‐cAMP but the necroptosis inhibitors necrostatin‐1 and cyclosporine A reduce this effect. Taken together, these findings lead us to conclude that cAMP induces necroptosis in rCFs and that this occurs via a PKA‐mediated mechanism. cAMP/PKA‐promoted necroptosis may contribute to decrease in number of CFs in vivo and may be a mechanism of cAMP‐promoted cell death in other types of cells. (Supported by research and training grants from NIH).

Estrogen receptor dependence of lysyl oxidase expression and activity in cardiac fibroblasts

Lysyl oxidase (LOX) is a collagen crosslinking enzyme essential for the deposition of collagen. We investigated the mechanism of estrogen mediated increases of LOX expression in rat cardiac fibroblasts and the role of estrogen receptors (ER). Adult rat cardiac fibroblasts were treated with ER‐α (MPP) and ‐β (PHTPP) specific antagonists, and a general ER inhibitor (ICI 181780), then treated with estrogen for 48 hrs. LOX protein expression was analyzed by Western blot. LOX catalytic activity was measured by fluorometric assay of cell‐conditioned media. Estrogen (1 to 50 ng/ml) increased LOX protein expression (150–290% for active form and 210–310% for proLOX vs control; P<0.05). Estrogen also increased LOX activity in media (120–140% vs media from untreated). LOX activation by estrogen was prevented by ER‐α and ‐β blockade; however ER‐β antagonism had a greater effect, reducing LOX activity below that of control (75% versus control and ER‐α block; P<0.05). ER‐β blockade also had a greater effect on LOX protein expression (58% decrease for active LOX vs estrogen treated) than ER‐α inhibitor (25%). ICI also significantly decreased LOX protein expression. These data indicate that both ER‐α and ‐β are involved in estrogen induced upregulation of LOX expression and activity in rat cardiac fibroblasts, with ER‐β playing a more significant role (Louisiana Board of Regents LEQSF2009‐12‐RDA10 and AHA #11GRNT7700002).

Effects of interleukin-33 on cardiac fibroblast gene expression and activity

Interleukin-33 (IL-33) is a recently described member of the interleukin-1 (IL-1) family. It is produced by diverse cell types in response to a variety of stresses including hemorrhage and increased mechanical load. Though only relatively recently discovered, IL-33 has been shown to participate in several pathological processes including promoting type 2 T helper cell-associated autoimmune diseases. In contrast, IL-33 has been also found to have protective effects in cardiovascular diseases. Recent studies have illustrated that IL-33 attenuates cardiac fibrosis induced by increased cardiovascular load in mice (transaortic constriction). Since cardiac fibrosis is largely dependent on increased production of extracellular matrix by cardiac fibroblasts, we hypothesized that IL-33 directly inhibits pro-fibrotic activities of these cells. Experiments have been carried out with isolated rat cardiac fibroblasts to evaluate the effects of IL-33 on the modulation of cardiac fibroblast gene expression and function to test this hypothesis. The expression of the IL-33 receptor, interleukin-1 receptor-like 1 (ST2), was detected at the mRNA and protein levels in isolated adult rat cardiac fibroblasts. Subsequently, the effects of IL-33 treatment (0–100ng/ml) on the expression of extracellular matrix proteins and pro-inflammatory cytokines/chemokines were examined as well as the effects on rat cardiac fibroblast activities including proliferation, collagen gel contraction and migration. While IL-33 did not directly inhibit collagen I and collagen III production, it yielded a dose-dependent increase in the expression of interleukin-6 and monocyte chemotactic protein-1. Treatment of rat cardiac fibroblasts with IL-33 also impaired the migratory activity of these cells. Further experiments illustrated that IL-33 rapidly activated multiple signaling pathways including extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in a dose-dependent manner. Experiments were carried out with pharmacological inhibitors to determine the role of specific signaling pathways in the response of fibroblasts to IL-33. These experiments illustrated that the activation of p38 mitogen-activated protein kinase and extracellular signal-regulated kinases are critical to the increased production of interleukin-6 and monocyte chemotactic protein-1 in response to IL-33. These studies suggest that IL-33 has an important role in the modulation of fibroblast function and gene expression. Surprisingly, IL-33 had no effect on the expression of genes encoding extracellular matrix components or on proliferation, markers typical of fibrosis. The major effects of IL-33 detected in these studies included inhibition of cell migration and activation of cytokine/chemokine expression. The previously reported inhibition of cardiac fibrosis may include more complicated mechanisms that involve other cardiac cell types. Future studies aimed at determining the effects of IL-33 on other cardiac cell types are warranted.

Hypoxic Stress Induces Transient Receptor Potential Melastatin 2 (TRPM2) Channel Expression in Adult Rat Cardiac Fibroblasts

When cardiac tissue is exposed to hypoxia, myocytes are damaged, while fibroblasts are activated. However, it is unknown what changes are induced by hypoxia in cardiac fibroblasts. In this study, using the whole cell patch-clamp technique, we investigated the effect of hypoxia on membrane currents in fibroblasts primarily cultured from adult rat hearts. Cardiac fibroblasts were incubated for 24 h under normoxic or hypoxic conditions using Anaeropack. Hypoxia increased a current which reversed at around −20 mV in the cardiac fibroblasts. This current was inhibited by clotrimazole, which is an inhibitor of transient receptor potential melastatin 2 (TRPM2) channel and intermediate-conductance Ca2+-activated K+ channel (KCa3.1). ADP ribose in the pipette solution enhanced this current. Quantitative RT-PCR revealed that mRNA of TRPM2, but not that of KCa3.1, was increased by hypoxia. RNA interference of TRPM2 prevented the development of the hypoxia-induced current. H2O2, an activator of TRPM2 channel, induced a higher [Ca2+]i elevation in hypoxia-exposed cardiac fibroblasts than that in normoxia-exposed cells. We conclude that hypoxia induces TRPM2 channel expression in adult rat cardiac fibroblasts.

Abstract P129: G Protein--Coupled Receptor Kinase-2 (GRK2) Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

Cardiac fibroblasts (CF) make up 70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. The objective of this study was to investigate the potential role of GRK2 in modulating CF transformation to myofibroblasts and collagen synthesis in adult human CF isolated from normal and failing left ventricles. CF isolated from failing ventricles showed a significant increase in expression of collagen I, III and VI compared with controls. α-SMA was increased 2-fold over controls, consistent with CF transformation to myofibroblasts. Baseline collagen synthesis was elevated 2-fold in failing CF and was not inhibited by isoproterenol (ISO)-stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in failing CF as assessed by basal and ISO-stimulated cAMP production. The primary mechanism appears to be a 2.5-fold increase in GRK2 activity as GRK2 phosphorylates and uncouples agonist-occupied β-ARs. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following ISO stimulation. In contrast, siRNA-mediated knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity by adenoviral-mediated βARKct expression or GRK2 knockdown in failing CF led to a significant decline in collagen and α-SMA expression. GRK2 inhibition restored β-AR signaling and ISO-stimulated inhibition of collagen synthesis and also significantly decreased collagen synthesis in response to TGFβ stimulation. In conclusion, GRK2 appears to play a significant role in regulating CF transformation and collagen synthesis in adult human CF and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

A physiological concentration of glucocorticoid inhibits the pro-inflammatory cytokine-induced proliferation of adult rat cardiac fibroblasts: Roles of extracellular signal-regulated kinase 1/2 and nuclear factor-κB

1. Inflammation-induced proliferation of cardiac fibroblasts plays an important role in cardiac remodelling. Pharmacological doses of exogenous glucocorticoids (GC) are the most effective therapy for inflammatory diseases. Similarly, physiological concentrations of endogenous GC have recently been shown to have anti-inflammatory effects. Therefore, the aim of the present study was to determine whether a physiological concentration of GC could inhibit pro-inflammatory cytokine-stimulated proliferation of cardiac fibroblasts and to explore the mechanisms involved. 2. Cardiac fibroblasts were isolated from adult male Sprague-Dawley rats and cell proliferation was measured using a CCK-8 kit. Western blotting was used to detect protein expression of extracellular-regulated kinase (ERK) 1/2 and nuclear factor (NF)-κB. 3. Cardiac fibroblast proliferation was significantly increased by tumour necrosis factor-α, interleukin (IL)-1β and angiotensin II and was accompanied by upregulated protein expression of ERK1/2 and NF-κB. A physiological concentration of hydrocortisone (127 ng/mL) not only inhibited the proliferation of cardiac fibroblasts, but also suppressed activation of ERK1/2 and NF-κB. These effects of hydrocortisone were abrogated by the glucocorticoid receptor (GR) antagonist RU-486 (100 nmol/L). Furthermore, inflammation-induced cardiac fibroblast proliferation was also blocked by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (100 nmol/L) and the NF-κB inhibitor pyrrolidine dithiocarbamate (1 μmol/L). Cytokine-induced ERK1/2 phosphorylation and cyclin D1 expression were attenuated by U0126, suggesting that the ERK1/2 and NF-κB signalling pathways were involved in cardiac fibroblast proliferation. 4. In conclusion, the results of the present study indicate that a physiological concentration of hydrocortisone can inhibit inflammation-induced proliferation of cardiac fibroblasts by preventing the activation of ERK1/2 and NF-κB.

TNF-α increases cardiac fibroblast lysyl oxidase expression through TGF-β and PI3Kinase signaling pathways

TNF-α is a proinflammatory cytokine that is upregulated in many cardiac diseases. The increase of TNF-α expression affects both heart function and the structure of the extracellular matrix. Lysyl oxidase (LOX) is a key enzyme responsible for the maturation of extracellular matrix proteins, including collagens type I and III. In this study, we investigated the regulation of LOX expression and activity by TNF-α using adult rat cardiac fibroblasts. Our results indicate that TNF-α has a dichotomous effect on LOX expression by cardiac fibroblasts. Low dose TNF-α (1–5ng/ml) decreased LOX expression, whereas higher doses (10–30ng/ml) increased expression. The higher dose TNF-α effect on LOX expression was attenuated by the inhibition of PI3Kinase/Akt pathway. TGF-β1 signaling played a significant role in mediating the TNF-α effect. TNF-α increased the expression of TGF-β, and TGF-β receptors type I and II, and also stimulated Smad3 phosphorylation. Inhibition of TGF-β receptor I or Smad3 prevented increased LOX expression by TNF-α. These findings indicate that TNF-α stimulated LOX expression may play an important role in progressive cardiac fibrosis.

Age-Dependent Expression of Collagen Receptors and Deformation of Type I Collagen Substrates by Rat Cardiac Fibroblasts

Little is known about how age influences the ways in which cardiac fibroblasts interact with the extracellular matrix. We investigated the deformation of collagen substrates by neonatal and adult rat cardiac fibroblasts in monolayer and three-dimensional (3D) cultures, and quantified the expression of three collagen receptors [discoidin domain receptor (DDR)1, DDR2, and β1 integrin] and the contractile protein alpha smooth muscle actin (α-SMA) in these cells. We report that adult fibroblasts contracted 3D collagen substrates significantly less than their neonate counterparts, whereas no differences were observed in monolayer cultures. Adult cells had lower expression of β1 integrin and α-SMA than neonate cultures, and we detected significant correlations between the expression of α-SMA and each of the collagen receptors in neonate cells but not in adult cells. Consistent with recent work demonstrating age-dependent interactions with myocytes, our results indicate that interactions between cardiac fibroblasts and the extracellular matrix change with age.

Adiponectin induces interleukin-6 production and its underlying mechanism in adult rat cardiac fibroblasts

It has been reported that adiponectin enhances interleukin-6 (IL-6) production in cardiac fibroblasts derived from neonatal rats and adult mice, but the mechanisms involved remain unknown. In the present study, we explored the effect and mechanisms of adiponectin on IL-6 production in adult rat cardiac fibroblasts. Globular adiponectin (gAd) increased IL-6 mRNA expression and protein secretion in cultured adult rat cardiac fibroblasts. gAd-induced IL-6 release was attenuated after RNA interference inhibition of adiponectin receptor 1 (AdipoR1), but not AdipoR2 or an adaptor protein APPL1. gAd increased the phosphorylation of AMP-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun-N-terminal kinase (JNK). Inhibitors of AMPK (araA), p38MAPK (SB202190), and ERK1/2 (PD98059 and U0126) but not JNK (SP600125) suppressed gAd-induced IL-6 production. In transient transfection assays of IL-6 promoter/luciferase reporter plasmids, gAd increased the transcriptional activity of the full-length IL-6 promoter. Deletion analysis of the IL-6 promoter indicated that activator protein-1 (AP-1), nuclear factor for IL-6 (NF-IL-6) and nuclear factor κB (NF-κB) binding sites were important for gAd-induced IL-6 transcription. Our data suggest that gAd enhances IL-6 synthesis and release in adult rat cardiac fibroblasts through AdipoR1. Activation of AMPK, p38MAPK, and ERK1/2 mediates the intracellular signal transduction. AP-1, NF-IL-6, and NF-κB cis-elements are required for gAd-induced IL-6 transcription.

G Protein-coupled Receptor Kinase-2 Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

P38 MAPK regulates G1-S transition in hypoxic cardiac fibroblasts

Cardiac fibroblast hyperplasia associated with augmented matrix production is central to wound healing following myocardial injury. Regulation of the cardiac fibroblast cell cycle by factors in the diseased myocardium that can potentially modify the hyperplastic response of cardiac fibroblasts has, however, not been investigated. We examined the regulation of the cardiac fibroblast cell cycle by hypoxia, a major constituent of myocardial ischemia. Significant reductions in DNA synthesis and cell number, and flow cytometry indicated decreased G1/S progression in hypoxic adult rat cardiac fibroblasts. Western blot analysis showed reduced levels of cyclin D and cyclin A, induction of p27 and hypophosphorylation of Rb under hypoxia. Skp2, which targets p27 for degradation, was significantly lower and inversely related to p27 protein levels in hypoxic cells. Marked p38 MAPK activation was observed under hypoxia and its inhibition using SB203580 reversed the effects of hypoxia on DNA synthesis, cell cycle phase distribution, p27, and cyclin D1 but not cyclin A. Interestingly, a 2-fold increase in p27 mRNA in hypoxic cells, demonstrated by real-time PCR, was unaffected by SB203580, which, however, reversed the hypoxic inhibition of Skp2. In summary, p38 MAPK is an important determinant of hypoxia-induced G0/G1 block in cardiac fibroblasts. p27 induction in hypoxic cardiac fibroblasts may involve direct transcriptional regulation, independent of p38 MAPK, and post-translational regulation via p38 MAPK-dependent suppression of its degradation by Skp2. The study identifies Skp2 as a potential downstream target of p38 MAPK, suggesting a novel mechanism of G1-S regulation in cardiac fibroblasts exposed to stress conditions.

Regulation of MT1-MMP and MMP-2 by Leptin in Cardiac Fibroblasts Involves Rho/ROCK-Dependent Actin Cytoskeletal Reorganization and Leads to Enhanced Cell Migration

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response to leptin, again via a Rho-dependent signaling mechanism. Our results suggest that leptin regulates myocardial matrix remodeling by regulating the cell surface localization of MT1-MMP in adult cardiac fibroblasts via Rho/ROCK-dependent actin cytoskeleton reorganization. Subsequent pro-MMP-2 activation then contributes to stimulation of cell migration.

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.

Beta 2-adrenergic receptor regulates cardiac fibroblast autophagy and collagen degradation

Autophagy is a physiological degradative process key to cell survival during nutrient deprivation, cell differentiation and development. It plays a major role in the turnover of damaged macromolecules and organelles, and it has been involved in the pathogenesis of different cardiovascular diseases. Activation of the adrenergic system is commonly associated with cardiac fibrosis and remodeling, and cardiac fibroblasts are key players in these processes. Whether adrenergic stimulation modulates cardiac fibroblast autophagy remains unexplored. In the present study, we aimed at this question and evaluated the effects of b 2-adrenergic stimulation upon autophagy. Cultured adult rat cardiac fibroblasts were treated with agonists or antagonists of beta-adrenergic receptors (b-AR), and autophagy was assessed by electron microscopy, GFP-LC3 subcellular distribution, and immunowesternblot of endogenous LC3. The predominant expression of b 2-ARs was determined and characterized by radioligand binding assays using [ 3H]dihydroalprenolol. Both, isoproterenol and norepinephrine (non-selective b-AR agonists), as well as salbutamol (selective b 2-AR agonist) increased autophagic flux, and these effects were blocked by propanolol (b-AR antagonist), ICI-118,551 (selective b 2-AR antagonist), 3-methyladenine but not by atenolol (selective b 1-AR antagonist). The increase in autophagy was correlated with an enhanced degradation of collagen, and this effect was abrogated by the inhibition of autophagic flux. Overall, our data suggest that b 2-adrenergic stimulation triggers autophagy in cardiac fibroblasts, and that this response could contribute to reduce the deleterious effects of high adrenergic stimulation upon cardiac fibrosis.

Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: Expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts

In fibrosing hearts, myofibroblasts are associated with cardiac extracellular matrix remodeling. Expression of key genes in the transition of cardiac fibroblast to myofibroblast phenotype in post-myocardial infarction heart and in vitro has not been well addressed. Contractile, focal adhesion-associated, receptor proteins, fibroblast growth factor-2 (FGF-2) expression, and motility were compared to assess phenotype in adult and neonatal rat cardiac fibroblasts and myofibroblasts. Neonatal and adult fibroblasts undergo phenotypic transition to myofibroblastic cells, marked by increased alpha-smooth muscle actin (alphaSMA), smooth muscle myosin heavy chain (SMemb), extra domain-A (ED-A) fibronectin, paxillin, tensin, FGF-2, and TbetaRII receptor. Elevated ED-A fibronectin confirmed fibroblast to supermature myofibroblastic phenotype transition. Presence of myofibroblasts in vivo was noted in sections of healed infarct scar after myocardial infarction, and their expression is similar to that in culture. Thus, cultured neonatal and adult cardiac fibroblasts transition to myofibroblasts in vitro and share expression profiles of cardiac myofibroblasts in vivo. Reduced motility with in vitro passage reflects enhanced production of focal adhesions.

FAK mediates the activation of cardiac fibroblasts induced by mechanical stress through regulation of mTOR complex

We evaluated whether focal adhesion kinase (FAK) plays a role in the proliferation and differentiation of cardiac fibroblasts into myofibroblasts in response to cyclic stretch (CS). Neonatal (NF‐ 3rd passage, 80% confluence) and adult (AF– 1st passage, 80% confluence) rat cardiac fibroblasts were exposed to CS (biaxial, 1Hz), which enhanced phospho‐FAKY397. Proliferation (anti‐Ki67 nuclear), differentiation into myofibroblasts (expression of α‐smooth muscle actin –α‐SMA) and the activity of matrix metalloproteinase‐2 (MMP‐2) were equally enhanced in stretched NF‐P3/80 and AF‐P1/80. Assays with the integrin inhibitor RGD peptide increased apoptosis (TUNEL) in non‐stretched and stretched NF‐P3/80, while FAK knockdown induced by siRNA modestly enhanced apoptosis only in stretched cells. RGD peptide or FAK silencing suppressed the activation of NF‐P3/80 invoked by CS. NF‐P3/80 depleted of FAK were defective in phospho‐AKTS473, TSC‐2T1462, and S6 kinaseT389 induced by CS. The activation of NF‐P3/80 invoked by CS was prevented by pre‐treatment with rapamycin, the mammalian target of rapamycin (mTOR) inhibitor, whereas treatment with leucine, activated S6K and rescued the stretch‐induced activation of NF‐P3/80 depleted of FAK. These findings demonstrate a critical role for the mTOR complex, FAK downstream, in mediating the activation of cardiac fibroblasts in response to mechanical stress.

IL‐1β and TNF‐α Differentially Regulate Lysyl Oxidase Expression and Activity in Adult Cardiac Fibroblasts

Cardiac fibroblasts constitutively express lysyl oxidase (LOX), an enzyme that catalyzes the crosslinking of collagen and modulates extracellular matrix maturation. Pro‐inflammatory cytokines, IL‐1β and TNF‐α, are elevated during cardiac disease and are known to modulate LOX expression in non‐cardiac cells. However, little is known about the regulation of LOX expression or activity in the heart. We tested the hypothesis that IL‐1β and TNF‐α decrease LOX expression and activity in isolated adult rat cardiac fibroblasts. After incubation in minimal serum media, fibroblasts were treated with TNF‐α (20 ng/ml) or IL‐1β (10 ng/ml) for 48 hrs. Cells were harvested and LOX expression assayed by Western blot. LOX activity was measured in cell culture media using Amplex red assay. Treatment with TNF‐α led to a significant reduction in LOX protein expression. However, IL‐1β did not affect LOX expression. Despite these differences in expression, both TNF‐α and IL‐1β significantly decreased LOX activity (64% and 60% vs. control, respectively). In addition, the TNF‐α regulation of LOX protein occurs in part through a Rho‐associated protein kinase (ROCK) pathway, as incubation of fibroblasts with the ROCK inhibitor, Y‐27632, attenuated the reduction of LOX expression. These findings indicate that the expression and activation of LOX in cardiac fibroblasts is differentially regulated by TNF‐α and IL‐1β.

Angiotensin 11 Enhances Interleukin-1.BETA.-Induced MMP-9 Secretion in Adult Rat Cardiac Fibroblasts

Cardiac fibroblasts play important roles during the cardiac remodeling through the secretion of matrix metalloproteinase (MMP)-9. Inflammatory cytokine, interleukin (IL)-1beta induces MMP-9 secretion in cultured cardiac fibroblasts. Angiotensin II is well known to play pivotal roles in cardiac remodeling, but the effect of angiotensin II on MMP-9 secretion in cardiac fibroblasts has not been fully clarified. In the present study, we investigated the effect of angiotensin II on basal and IL-1beta-induced MMP-9 secretion in adult rat cardiac fibroblasts. MMP-9 protein secreted into culture medium, and phosphorylation of nuclear factor (NF)-kappaB, c-Jun NH(2)-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) in cell lysates were measured by Western blotting. Angiotensin II (1 nM, 24 hr) alone-treatment did not induce MMP-9 secretion. However, angiotensin II significantly enhanced IL-1beta (4 ng/ml, 24 hr)-induced MMP-9 secretion. Telmisartan (10 nM), an angiotensin II type 1 receptor (AT1R) antagonist, significantly suppressed the enhancement of IL-1beta-induced MMP-9 secretion by angiotensin II, whereas PD123319 (10 nM), an angiotensin II type 2 receptor antagonist, was ineffective. IL-1beta (4 ng/ml, 10 min) induced phosphorylation of NF-kappaB, JNK, and ERK. Angiotensin II augmented the IL-1beta-induced phosphorylation of ERK but not NF-kappaB and JNK. PD98059 (50 microM), a selective inhibitor of ERK pathway, inhibited the angiotensin II enhancement of IL-1beta-induced MMP-9 secretion. These results suggest that angiotensin II enhances IL-1beta-induced MMP-9 secretion through the augmentation of ERK phosphorylation via AT1R in adult rat cardiac fibroblasts.