Angiotensin-converting enzyme (ACE) inhibition attenuates insulin-like growth factor-I (IGF-I) induced cardiac fibroblast proliferation

Abstract

The effects of angiotensin-converting enzyme (ACE) inhibition and angiotensin type 1 (AT(1)) receptor blockade on insulin-like growth factor-I (IGF-I) induced proliferation and immediate-early-gene expression of neonatal rat cardiac fibroblasts were investigated. Moreover the role of the IGF-I receptor (IGF-IR) in this process was evaluated. IGF-I (10(-9) - 10(-7) M) stimulated neonatal rat cardiac fibroblast growth in a dose-dependent fashion (maximum: 3.5+/-0.1 fold, 10(-7) M), as determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation. ACE inhibition or AT(1) receptor blockade attenuated the IGF-I (10(-7) M) induced neonatal rat cardiac fibroblast growth in a concentration-dependent fashion (moexiprilat: 50+/-2%, enalaprilat: 31+/-2%, CV11974; 58+/-1%, all: 10(-7) M). IGF-I stimulated cellular growth was accompanied by an upregulation of the immediate early genes c-Fos (2.4+/-0.3 fold), Egr-1 (4.7+/-1.1 fold) and Sp1 (6.2+/-0.7 fold). IGF-I induced expression was completely inhibited by ACE inhibition or AT(1) receptor blockade. Stimulation with IGF-I or Ang II (10(-7) M) increased IGF-IR expression 5.7+/-0. 5 fold and 3.6+/-0.5 fold respectively. The IGF-I induced overexpression of the IGF-IR was reduced by ACE inhibition with moexiprilat (10(-7) M) by 79+/-7% and by AT(1) receptor blockade with CV11974 (10(-7) M) by 79+/-5%. These data demonstrate that the mitogenic action of IGF-I in neonatal rat cardiac fibroblasts is in part mediated by activation of the renin-angiotensin system (RAS) with subsequent upregulation of IGF-IR expression. This observation has important implications for the treatment of cardiac diseases with ACE inhibitors alone and their combination with IGF-I or growth hormone.

Figure 1

IGF-I induced cardiac fibroblast growth can be attenuated by ACE inhibition and by AT₁ receptor antagonism. (a) Starved neonatal rat cardiac fibroblast were stimulated with IGF-I at the indicated concentrations or coincubated with IGF-I (10⁻⁷ M) and the IGF-I receptor antagonist H-1356 (H; 10⁻⁷ M), moexiprilat (Mox; 10⁻⁷ M) or CV11974 CV; 10⁻⁷ M). Control cells were treated with vehicle (C) and with the inhibitors (H, Mox, CV) alone. Bars represents the relative BrdU incorporation compared to control cells (C). ^*P<0.01 vs control; ^**P<0.01 vs control and vs IGF-I (10⁻⁷ M) (n=9). (b) Dose-response curves for moexiprilat (Mox) and enalaprilat (Ena) in neonatal rat cardiac fibroblasts stimulated with IGF-I (10⁻⁷ M) (n=9).

Figure 2

Immunoblot analysis revealed an increase of the immediate early genes Egr-1, c-Fos and Sp1 expression in starved neonatal rat cardiac fibroblasts stimulated with IGF-I (10⁻⁷ M, 60 min). This induction could be completely inhibited by a coincubation with the IGF-I receptor antagonist H-1356 (H; 10⁻⁷ M), moexiprilat (Mox; 10⁻⁷ M) or CV11974 (CV; 10⁻⁷ M). One of three similar experiments is shown.

Figure 3

Immunoblot analysis of the IGF-IR expression in neonatal rat cardiac fibroblasts. Starved neonatal rat cardiac fibroblasts were stimulated for 24 h with IGF-I (10⁻⁷ M) and coincubated with moexiprilat (Mox; 10⁻⁷ M) and CV11974 (CV; 10⁻⁷ M). Control cells were treated with vehicle (C) and with the inhibitors (Mox, CV) alone. Densitometric analysis of three immunoblots. The values were normalized to cells treated with vehicle alone (C). ^*P<0.01.

Figure 4

Immunoblot analysis of the IGF-IR expression in neonatal rat cardiac fibroblasts. Starved neonatal rat cardiac fibroblast were stimulated for 24 h with Ang II (10⁻⁷ M) and coincubated with CV11974 (CV; 10⁻⁷ M). Control cells were treated with vehicle (C) and with the inhibitor (CV alone). Densitometrical analysis of three immunoblots. The values were normalized to cells treated with vehicle alone (C). ^*P<0.01.