Transformation of fibroblasts to myofibroblasts, characterized by expression of ␣-smooth muscle actin (␣-SMA) and production of extracellular matrix (ECM) components, is a key event in connective tissue remodeling. Approaches to inhibit this transformation are needed in tissues, such as the heart, where excessive ECM production by cardiac fibroblasts (CFs) causes fibrosis, myocardial stiffening, and cardiac dysfunction. We tested whether adenylyl cyclase (AC) activation (increased cAMP levels) modulates the transformation of adult rat CF to myofibroblasts, as assessed by immunofluorescent microscopy, immunoblotting, and collagen synthesis. A 24-h incubation of CF with TGF-␤ or angiotensin II increased ␣-SMA expression, which was inhibited by the AC agonist forskolin and a cAMP analog that activates protein kinase A. Treatment with forskolin blunted serum-, TGF-␤-, and angiotensin II-stimulated collagen synthesis. CFs engineered to overexpress type 6 AC had enhanced forskolin-promoted cAMP formation, greater inhibition by forskolin of TGF-␤-stimulated ␣-SMA expression, and a decrease in the EC 50 of forskolin to reduce serum-stimulated collagen synthesis. The AC stimulatory agonist adrenomedullin inhibited collagen synthesis in CF that overexpressed AC6 but not in controls. Thus, AC stimulation blunts collagen synthesis and, in parallel, the transformation of adult rat CF to myofibroblasts. AC overexpression enhances these effects, ''uncovering'' an inhibition by adrenomedullin. These findings implicate cAMP as an inhibitor of ECM formation by means of blockade of the transformation of CF to myofibroblasts and suggest that increasing AC expression, thereby enhancing cAMP generation through stimulation of receptors expressed on CF, could provide a means to attenuate and prevent cardiac fibrosis and its sequelae.cardiac fibroblast ͉ cyclic AMP ͉ extracellular matrix ͉ fibrosis ͉ heart failure

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Prolonged activation of CFs leads to cardiac fibrosis and reduced myocardial contractile function. Resveratrol (RES) exhibits a number of cardioprotective properties; however, the possibility that this compound affects CF function has not been considered. The current study tests whether RES directly influences the growth and proliferation of CFs and differentiation to the hypersecretory myofibroblast phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase activation. This inhibition by RES reduced basal proliferation and blocked ANG II-induced growth and proliferation of CFs in a concentration-dependent manner, as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively. RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with 0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK activation over basal was similar to the phosphorylation induced by 100 nM ANG II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II- and transforming growth factor-beta-induced CF differentiation to the myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin expression and stress fiber organization in CFs. This study identifies RES as an anti-fibrotic agent in the myocardium by limiting CF proliferation and differentiation, two critical steps in the pathogenesis of cardiac fibrosis.

Cardiac fibroblast (CF) proliferation and differentiation into hypersecretory myofibroblasts can lead to excessive extracellular matrix (ECM) production and cardiac fibrosis. In turn, the ECM produced can potentially activate CFs via distinct feedback mechanisms. To assess how specific ECM components influence CF activation, isolated CFs were plated on specific collagen substrates (type I, III, and VI collagens) before functional assays were carried out. The type VI collagen substrate potently induced myofibroblast differentiation but had little effect on CF proliferation. Conversely, the type I and III collagen substrates did not affect differentiation but caused significant induction of proliferation (type I, 240.7 +/- 10.3%, and type III, 271.7 +/- 21.8% of basal). Type I collagen activated ERK1/2, whereas type III collagen did not. Treatment of CFs with angiotensin II, a potent mitogen of CFs, enhanced the growth observed on types I and III collagen but not on the type VI collagen substrate. Using an in vivo model of myocardial infarction (MI), we measured changes in type VI collagen expression and myofibroblast differentiation after post-MI remodeling. Concurrent elevations in type VI collagen and myofibroblast content were evident in the infarcted myocardium 20-wk post-MI. Overall, types I and III collagen stimulate CF proliferation, whereas type VI collagen plays a potentially novel role in cardiac remodeling through facilitation of myofibroblast differentiation.

Abstract-Angiotensin II (Ang II)-induced proliferation of cardiac fibroblasts is a major contributing factor to the pathogenesis of cardiac fibrosis. Ang II activates extracellular signal-regulated kinase (ERK) 1/2 to induce cardiac fibroblast proliferation, but the signaling pathways leading to ERK 1/2 activation have not been elucidated in these cells.The goal of the current study was to identify the intracellular mediators of Ang II-induced ERK 1/2 activation in adult rat cardiac fibroblasts. We determined that 100 nmol/L of Ang II-induced ERK 1/2 phosphorylation is inhibited by simultaneous chelation of cytosolic calcium and downregulation of protein kinase C (PKC) by phorbol ester or by the specific PKC␦ inhibitor rottlerin, as well as PKC␦ small interfering RNA, but not by inhibition of 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetate, phorbol ester, rottlerin, or PKC␦ small interfering RNA alone. We also found that Ang II does not transactivate the epidermal growth factor receptor in adult cardiac fibroblasts, because pretreatment with 1 mol/L of AG 1478 did not significantly inhibit [ 3 H]-thymidine incorporation or ERK 1/2 activation. In addition, immunoprecipitation of the epidermal growth factor receptor demonstrated no significant Ang II-induced phosphorylation of tyrosine residues. Inhibition of phosphatidylinositide 3-kinase, PKC, and src tyrosine kinase had no effect on Ang II-induced ERK 1/2 activation. Collectively, these data demonstrate that Ang II does not transactivate the epidermal growth factor receptor in adult rat cardiac fibroblasts to activate ERK 1/2, a common pathway described in vascular smooth muscle and other cell types, but rather occurs via activation of distinct parallel signaling pathways mechanistically controlled by intracellular Ca

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between G q and G s signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by ␤-adrenergic receptors (␤-AR). The potentiation of ␤-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of G q . Ionomycin and thapsigargin increased intracellular Ca 2؉ levels and potentiated isoproterenoland forskolin-stimulated cAMP production, whereas chelation of Ca 2؉ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or G␤␥ did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca 2؉ -activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased ␤AR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that ␤AR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca 2؉ -dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of crosstalk between G q and G s signaling pathways in cardiac fibroblasts.

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Activated cardiac fibroblasts (CFs) proliferate, differentiate, and migrate to regulate cardiac remodeling. ANG II activates extracellular signal‐regulated kinase (ERK) 1/2 to induce proliferation, but the signaling pathways leading to ERK 1/2 activation have not been elucidated in CFs. The goal of the current study was to identify the intracellular mediators of ANG II‐induced ERK 1/2 activation in adult rat CFs. We determined that ANG II does not transactivate the epidermal growth factor receptor (EGFR) in adult CFs, since pretreatment with AG 1478 did not inhibit [3H]‐thymidine incorporation or ERK 1/2 activation. We found that ANG II‐induced ERK 1/2 phosphorylation is inhibited by simultaneous chelation of cytosolic Ca2+ and downregulation of PKC by phorbol ester or by the specific PKC inhibitor rottlerin as well as PKC siRNA, but not by inhibition of each agent alone. We next investigated the role of specific PKC isoforms in migration of the activated CFs using in vitro wound healing assays. Through pharmacological blockade we determined that inhibition of PKC slows migration of CFs and inhibition of PKC also decreases the rate migration but to a lesser extent than blockade of PKC. These data reveal that PKC is a key mediator in both ANG II‐induced proliferation with concurrent Ca2+chelation and migration of CFs.