Comparison of ANG II with other growth factors on Egr-1 and matrix gene expression in cardiac fibroblasts

Abstract

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.

Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction.

“…Neonatal cardiac fibroblast cultures were prepared from Sprague-Dawley rats 1-3 d after birth and characterized as described previously (22). Briefly, neonatal hearts were dissected free of atria, minced, and subjected to trypsin and DNAse II digestion.…”

“…A II AT 1 receptor mRNA levels and protein are prominently detectable in rat cardiac fibroblasts (20)(21)(22). We and others demonstrated that they mediate A II effects to promote cardiac fibroblast proliferation, early growth response gene expression, and production of extracellular matrix proteins, particularly fibronectin which can bind to osteopontin (20)(21)(22). Regulation of extracellular matrix formation and composition may be one mechanism by which A II regulates remodeling events in the heart.…”

“…These events increase the volume of fibroblasts and matrix relative to that of myocytes in the hypertrophied heart compared with normal heart, which ultimately results in ventricular dysfunction and heart failure (19). A II AT 1 receptor mRNA levels and protein are prominently detectable in rat cardiac fibroblasts (20)(21)(22). We and others demonstrated that they mediate A II effects to promote cardiac fibroblast proliferation, early growth response gene expression, and production of extracellular matrix proteins, particularly fibronectin which can bind to osteopontin (20)(21)(22).…”

Norepinephrine Enhances Fibrosis Mediated by TGF-β in Cardiac Fibroblasts

“…20 Briefly, cardiac myocytes were cultured in DMEM and fibroblasts in a DMEM/F12 nutrient mixture (DMEM/F12) (1:1) containing 10% fetal bovine serum (FBS, pH 7.3). To reduce the number of nonmyocardial cells (NMCs), mainly cardiac fibroblasts, dissociated cells were preplated in DMEM/F12 with 10% FBS.…”

Angiotensin II–Induced MMP-2 Activity and MMP-14 and Basigin Protein Expression Are Mediated via the Angiotensin II Receptor Type 1–Mitogen-Activated Protein Kinase 1 Pathway in Retinal Pigment Epithelium

“…18,[22][23][24][25][26] It has been well established that action of Ang II regulates expression of ECM proteins in various tissues. [27][28][29][30] It has been recently demonstrated that Ang II induces AT1-mediated breakdown of the RPE basement membrane by increasing MMP-2 activity in RPE. 18,26 A concomitant increase in MMP-14 expression has also been demonstrated, which might account, at least in part, for the Ang II-induced increase in MMP-2 activity.…”