Angiotensin II (A II ) is a critical factor in cardiac remodeling which involves hypertrophy, fibroblast proliferation, and extracellular matrix production. However, little is known about the mechanism by which A II accelerates these responses. Osteopontin is an acidic phosphoprotein with RGD (arginine-glycine-aspartate) sequences that are involved in the vascular smooth muscle cell remodeling process. We identified the presence of osteopontin mRNA and protein in cultured rat cardiac fibroblasts and its prominent regulation by A II (10 Ϫ 11 M). Osteopontin message levels were increased fourfold ( P Ͻ 0.01) and protein fivefold ( P Ͻ 0.05) at 24 h after addition of A II (10 Ϫ 7 M). This response was inhibited by the AT 1 receptor blocker, losartan. Osteopontin mRNA levels were increased in hypertrophied ventricles from animals with renovascular hypertension (1.6-fold, P Ͻ 0.05) and aortic banding (2.9-fold, P Ͻ 0.05). To examine the function of osteopontin, we determined its effects on ( a ) the ability of cardiac fibroblasts to contract three-dimensional collagen gels and ( b ) cardiac fibroblast growth. A monoclonal antibody against osteopontin partially blocked A II -induced three-dimensional collagen gel contraction by cardiac fibroblasts (64 Ϯ 4 vs. 86 Ϯ 5% in the presence of antibody, P Ͻ 0.05), while osteopontin itself promoted contraction of the gels by fibroblasts (71 Ϯ 5%, P Ͻ 0.05 compared with control). Either a monoclonal antibody against  3 integrin which is a ligand for osteopontin or the RGD peptide blocked both A II and osteopontin-induced collagen gel contraction. Thus, the osteopontin RGD sequence binds to  3 integrins on the fibroblast to promote fibroblast binding to collagen. A II induced a threefold increase in DNA synthesis of cardiac fibroblasts, which was completely blocked by antibodies against osteopontin and  3 integrin, or by RGD peptide, but not by controls. Thus, A II -induced growth of cardiac fibroblasts also requires osteopontin engagement of the  3 integrin. Taken together, these results provide the first evidence that osteopontin is a potentially important mediator of A II regulation of cardiac fibroblast behavior in the cardiac remodeling process. ( J. Clin. Invest. 1996. 98:2218-2227.)
The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.
Hypertrophic cardiomyopathy and dilated cardiomyopathy are two major clinical phenotypes of "idiopathic" cardiomyopathy. Recent molecular genetic analyses have now revealed that "idiopathic" cardiomyopathy is caused by mutations in genes for sarcomere components. We have recently reported several mutations in titin/connectin gene found in patients with hypertrophic cardiomyopathy or dilated cardiomyopathy. A hypertrophic cardiomyopathy-associated titin/connectin mutation (Arg740Leu) was found to increase the binding to actinin, while other dilated cardiomyopathy-associated titin/connectin mutations (Ala743Val and Val54Met) decreased the binding to actinin and Tcap/telethonin, respectively. We also reported several other mutations in the N2-B region of titin/connectin found in hypertrophic cardiomyopathy and dilated cardiomyopathy. Since the N2-B region expresses only in the heart, it was speculated that functional alterations due to the mutations cause cardiomyopathies. In this study, we investigated the functional changes caused by the N2-B region mutations by using yeast-two-hybrid assays. It was revealed that a hypertrophic cardiomyopathy-associated mutation (Ser3799Tyr) increased the binding to FHL2 protein, whereas a dilated cardiomyopathy-associated mutation (Gln4053ter) decreased the binding. In addition, another TTN mutation (Arg25618Gln) at the is2 region was found in familial DCM. Because FHL2 protein is known to tether metabolic enzymes to N2-B and is2 regions of titin/connectin, these observations suggest that altered recruitment of metabolic enzymes to the sarcomere may play a role in the pathogenesis of cardiomyopathies.
Abstract-Cardiac fibrosis results from proliferation of interstitial fibroblasts and concomitant increased biosynthesis of extracellular matrix (ECM) components and is often complicated by cardiac hypertrophy. This study was conducted to investigate whether norepinephrine (NE) potentiates transforming growth factor- (TGF-)-induced cardiac fibrosis. The expression of the cardiac ECM proteins, plasminogen activator inhibitor-1 (PAI-1), fibronectin, and collagen type I, was examined by Western blotting using extracts from neonatal rat primary cardiac fibroblasts. In cardiac fibroblasts, treatment with a combination of NE and TGF-1 increased cell proliferation and ECM expression. Luciferase assays were conducted to clarify the effect of NE on TGF- signaling. TGF-1 (1 ng/mL) increased the specific signaling activity 2-fold, whereas the combination of NE (10 mol/L) and TGF-1 (1 ng/mL) resulted in an approximate 10-fold increase in specific signaling activity. We confirmed that treatment with NE markedly enhances TGF--induced phosphorylation of activating transcription factor 2 (ATF-2). These results indicated that NE has a synergistic effect on TGF- signaling. To determine whether this activation by NE was mediated by the TGF-1 receptor, we used a dominant negative vector of the TGF-1 type II receptor, and the synergistic effects were inhibited. Furthermore, this synergistic effect was attenuated by a specific inhibitor of p38, SB203680. These data indicate that NE enhances cardiac fibrosis through TGF-1 post-receptor signaling, predominantly via the p38 MAP kinase pathway.
The purpose of the present investigation was to compare the effects of angiotensin II (ANG II) other growth factors implicated to play a role in ventricular hypertrophy on cardiac fibroblast changes associated with cardiac remodeling. These changes included induction of early growth response (Egr-1) gene and increases in message levels of extracellular matrix proteins. ANG II treatment (10(-10)-10(-6) M) of rat cardiac fibroblasts induced 1) Egr-1 and 2) a fourfold (P < 0.02) increase in fibronectin and a twofold (P = 0.05) increase in laminin mRNA levels but no increases in that of collagens I, III, or IV at 24-48 h, and 3) a decrease in AT1-receptor mRNA levels to 26% (P < 0.001) of basal at 4-6 h. These effects were all inhibited by the AT1-receptor blocker, losartan, but not AT2-receptor blockers. Immunostaining of cultured cells with antibody against rat fibronectin demonstrated positive staining of cells in serum-free medium; staining was more intense in cells treated with ANG II (10(-6) M, 48 h). Fluorescent-activated cell sorting using an antibody against rat AT1 receptor demonstrated a receptor signal in cells maintained in serum-free medium; however, the receptor signal was not detectable in ANG II-treated cells. Serum and epidermal growth factor (EGF) also induced Egr-1, but norepinephrine (NE) and endothelin (ET) had no effect. Serum increased fibronectin mRNA levels by twofold (P < 0.05). EGF, NE, and ET had no effect on matrix gene expression. Serum, EGF, and NE also transiently downregulated AT1-receptor mRNA levels at 4-6 h of treatment. These results demonstrate that 1) ANG II both induces protooncogene expression and enhances fibronectin mRNA levels in cultured cardiac fibroblasts, whereas EGF only induces Egr-1, and NE and ET have no effects on either function; 2) ANG II effects are primarily mediated by the AT1 receptor; and 3) growth factors can regulate AT1-receptor mRNA levels. Thus ANG II, relative to NE, ET, and EGF, appears to play a prominent and direct role in fibroblast changes associated with cardiac hypertrophy.
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