Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty

Abstract

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.

Figure 1.

A: Binding of ¹²⁵I-PDGF to immobilized decorin. Data are mean ± SD of triplicate assays and are expressed as % binding of ¹²⁵I-PDGF to immobilized decorin. In contrast to CS and BSA that did not reach half-maximal inhibition, unlabeled PDGF competed with ¹²⁵I-PDGF dose dependently and reached half-maximal concentration of about 0.37 μg/ml indicating the specificity of decorin binding to PDGF. BSA, bovine serum albumin; CS, chondroitin sulfate. Experiments were repeated a minimum of three times. *, P < 0.01 versus CS and BSA. B and C: Western blot analysis of affinity cross-linked decorin with PDGF confirmed decorin-PDGF complex formation using PDGF (B) or decorin (C) antibody. A higher molecular weight band (arrow) which was only detected in the lanes with cross-linked proteins (B and C, lanes 3, 4, and 5), represents decorin-PDGF complex. The lower molecular weight bands in lane 1 of B and lane 2 of C correspond to PDGF and decorin only, respectively. Results were similar in three independent experiments.

Figure 2.

A: Western blot analysis of PDGFR-β. PDGF inhibited PDGFR-β expression. Decorin did not affect PDGFR-β expression. B: Autoradiograph of a gel loaded with extracts containing 50 μg of protein from cells incubated with ¹²⁵I-PDGF in the presence or absence of unlabeled PDGF or decorin. Decorin did not affect binding of ¹²⁵I-PDGF to PDGF receptor. Unlabeled PDGF competed with ¹²⁵I-PDGF and prevented its binding to receptors indicating specificity of binding. C: Western blot analysis of phospho-PDGFR-β. Decorin inhibited PDGF-stimulated PDGFR-β phosphorylation (top). Immunoblotting with anti-PDGFR-β shows (bottom) that similar amounts of receptor were present in all samples. Results were similar in three independent experiments.

Figure 3.

Western blot analysis of caspase-3 after treating cells with decorin (30 μg/ml) or PDGF (10 ng/ml) for 24 hours. Each lane contains 50 μg of cellular proteins. Western blots were probed with anti-caspase-3 (top) or anti β-actin (bottom) to verify equal loading of protein in each lane. Positive control was from human Jurkat cells (tonsil). There was no evidence of cell apoptosis in decorin-treated cells as well as control non-treated or PDGF-treated cells. Results were similar in three independent experiments.

Figure 4.

PDGF-stimulated (A) DNA synthesis, (B) cell proliferation, (C) cell migration, and (D) collagen synthesis were significantly inhibited by decorin in a dose-dependent manner. PDGF-stimulated cell proliferation was significantly inhibited by decorin at 24 hours and 48 hours. Experiments were performed in triplicate and repeated a minimum of three times. ^†, P < 0.01 versus control non-treated cells; *, P ≤ 0.02 versus PDGF-treated cells.

Figure 5.

A: PCR of DNA from rabbit carotid arteries at 24 hours after transfection. Lanes 1 and 2: decorin-transfected arteries; lanes 3 and 4: β-gal-transfected arteries; lanes 5 and 6: injured non-transfected arteries; lanes 7 and 8: uninjured arteries and far left lane (ΦX174): DNA standards. In the decorin-transfected arteries, a 870-bp fragment was identified by using primers for the CMV promotor sequence and the rabbit decorin gene. B: Decorin (brown staining) demonstrated in medial layer by immunohistochemistry at 1 week after angioplasty and transfection. Arrowhead, external elastic lamina; arrow, internal elastic lamina; l, lumen; m, media; a, adventitia.

Figure 6.

Intimal cross-sectional area at 10 weeks after angioplasty. *, P < 0.04 versus β-gal and control.

Figure 7.

Movat-pentachrome-stained sections of injured carotid arteries at 10 weeks after angioplasty. A: Control (injured non-transfected). B: β-gal-transfected. C: Decorin-transfected. Marked inhibition of intimal hyperplasia seen in decorin-transfected arteries. Arrowhead, external elastic lamina; arrow, internal elastic lamina; l, lumen; i, intima; m, media; a, adventitia.