The proteoglycan osteoglycin/mimecan is correlated with arteriogenesis

Abstract

Arteriogenesis or collateral growth is able to compensate for the stenosis of major arteries. Using differential display RT-PCR on growing and quiescent collateral arteries in a rabbit femoral artery ligation model, we cloned the rabbit full-length cDNA of osteoglycin/mimecan. Osteoglycin was present in the adventitia of collateral arteries as a glycosylated protein without keratan sulfate side chains, mainly produced by smooth muscle cells (SMCs) and perivascular fibroblasts. Northern blot, Western blot, and immunohistochemistry confirmed a collateral artery-specific downregulation of osteoglycin from 6 h to 3 weeks after the onset of arteriogenesis. Treatment of primary SMCs with the arteriogenic protein fibroblast growth factor-2 (FGF-2) resulted in a similar reduction of osteoglycin expression as observed in vivo. Application of the FGF-2 inhibitor polyanethole sulfonic acid (PAS) blocked the downregulation of osteoglycin and interfered with arteriogenesis. From our study we conclude that downregulation of osteoglycin is a fundamental requirement for proper arteriogenesis.

Fig. 1

Sequence of rabbit osteoglycin. The rabbit osteoglycin cDNA consisted of 2,493 bp and contained an open reading frame coding for a protein of 298aa. The protein starts with a signal peptide of 19 amino acids (underlined). The three consensus sequences for N-linked glycosylation are printed in red. The leucine-rich repeats of the S-type are marked in blue, that of the T-type in green

Fig. 2

Osteoglycin mRNA expression at various time points after occlusion of the femoral artery. ABar graphs representing Northern blot results on osteoglycin expression in growing collateral arteries from 3 h to 3 weeks after femoral artery ligation (for 3–24 h: n = 4 for each time point; for 3d to 3w: n = 8 for each time point; control: n = 8). B Representative Northern blot showing osteoglycin expression (top) in quiescent (con) and growing collateral arteries at days 3, 7 as well as 3 weeks after femoral artery occlusion. The blot was rehybridized with an 18S rRNA specific probe (bottom). Results are expressed as mean ± SEM (* P < 05 versus con; con control)

Fig. 3

Western blot analyses of osteoglycin. A Representative Western blot showing the level of osteoglycin protein in growing collateral arteries at days 3, 7 and 3 weeks after femoral artery occlusion as well as in control vessels. B Quantification of the Western blot analyses. n = 7 for control and for d3, n = 6 for d7, and n = 5 for 3 weeks. Results are expressed as mean ± SEM (* P < 05 versus con). C Protein preparations before (−) and after (+) deglycosylation (n = 4 for each time point). Ponceau S staining was used in all Western blots to demonstrate equal loading of the protein samples. (M Cruz marker molecular weight standard, con control, 3d 3 days after occlusion, 7d 7 days after occlusion, 3w 3 weeks after occlusion)

Fig. 4

Localization of osteoglycin mRNA (A, C, D), protein (B, E, F), and the proliferation marker ki-67 (G) in collateral arteries and surrounding tissue. A, C, D In situ hybridization. A The hybridization with the sense probe was used as a negative control. C, D Positive signals for osteoglycin were detected in the SMCs of the collateral media (arrows) and in fibroblasts (arrowheads). B, E, F Immunohistochemistry. B Negative control for immunoperoxidase reaction. E Quiescent collateral arteries showed a thin media and continuous internal elastic lamina (arrow). Note the prominent staining for osteoglycin in the adventitia (black arrowheads). F, G (consecutive sections): After 7 days of femoral artery occlusion growing collateral arteries show fragmented elastic lamina (arrows in F), prominent neointima formation (NI in F), and SMC proliferation (arrows in G; ki67 staining). Note the strong reduction of osteoglycin protein in the adventitia of growing collateral arteries (compare black arrowheads in E and F), whereas in the fascia of the muscle the signal intensity remains the same (red arrowheads)

Fig. 5

Stimulation of SMCs with distinct cytokines and growth factors. Bar graphs representing Northern blot results on osteoglycin expression in primary rabbit SMCs after 24, 48, and 72 h of stimulation with distinct growth factors and cytokines. The factors and concentrations used are indicated in the “Material and Methods” section. Results are expressed as mean ± SEM (* P < 0.05 versus con; con control, OsM Oncostatin M). All experiments were performed in triplicate

Fig. 6

Application of PAS. Postmortem angiograms of rabbit hind limbs. A After 1 week of infusion of PAS without ligation of the femoral artery. B After 1 week of femoral artery ligation with concomitant infusion of PAS. C After 1 week of femoral artery ligation without infusion of PAS. Upon femoral artery ligation, only rabbits not treated with PAS (C) showed collateral arteries with corkscrew formation (arrows), a typical sign for growing collaterals. DBar graphs representing Northern blot results on osteoglycin expression after 1 week of infusion of PAS in resting collateral arteries (con + PAS) and in growing collateral arteries 7 days after ligation of the femoral artery (7d occ + PAS; n = 4). Results are expressed as mean ± SEM. No statistical difference was found