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Abstract
Tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS) activity, is known to play important roles in modulating both NO and superoxide production during vascular diseases such as atherosclerosis. However, the role of BH4 in functions of vascular smooth muscle cells is not fully known. In this study, we tested the effects of BH4 and dihydrobiopterin (BH2), a BH4 precursor, on migration and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) in rat aortic smooth muscle cells (RASMCs). Cell migration and proliferation were measured using a Boyden chamber and a 5-bromo-2′-deoxyuridine incorporation assay, respectively, and these results were confirmed with an ex vivo aortic sprout assay. Cell viability was examined by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assays. BH4 and BH2 decreased PDGF-BB-induced cell migration and proliferation in a dose-dependent manner. The inhibition of cell migration and proliferation by BH4 and BH2 was not affected by pretreatment with NG-nitro-L-arginine methyl ester, a NOS inhibitor. Moreover, the sprout outgrowth formation of aortic rings induced by PDGF-BB was inhibited by BH4 and BH2. Cell viability was not inhibited by BH4 and BH2 treatment. The present results suggest that BH4 and BH2 may inhibit PDGF-stimulated RASMC migration and proliferation via the NOS-independent pathway. Therefore, BH4 and its derivative could be useful for the development of a candidate molecule with an NO-independent anti-atherosclerotic function.
References
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Fig. 1.
Effects of BH4 and BH2 on PDGF-BB-induced migration of RASMCs. PDGF (10 ng/ml) and BH4 (0.1∼1,000 μM; A) or BH2 (0.1∼1,000 μM; B) were loaded in the upper chamber and incubated for 90 min (n=9). Migration was examined using a Boyden chamber assay. The migration of RASMCs in the quiescent state was expressed as 100% (n=9). ∗p<0.05 vs. the PDGF-BB-stimulated response in the absence of BH2 or BH4.
Fig. 2.
Effects of BH4 and BH2 on PDGF-BB-induced proliferation of RASMCs. RASMCs were treated with BH4 (0.1∼1,000 μM; A) or BH2 (0.1∼1,000 μM; B) and then stimulated with PDGF-BB (10 ng/ml) for 36 h. Cell proliferation was quantified using a BrdU incorporation assay. Proliferation in the quiescent state was expressed as 100% (n=8). ∗p<0.05 vs. the PDGF-BB-induced response in the absence of BH2 or BH4.
Fig. 3.
Effects of NOS inhibition on BH4- and BH2-induced inhibition of migration of RASMCs. After treatment with L-NAME (1 and 3 mM) and/or BH4 (100 μM; A) or BH2 (100 μM; B), the cells were loaded into the upper chamber containing PDGF-BB (10 ng/ml) and incubated for 90 min (n=8), as described in the Methods section. Cell migration in the quiescent state was expressed as 100% (n=8). ∗p<0.05 vs. the PDGF-BB-stimulated response in the absence of BH2 and BH4.
Fig. 4.
Effects of BH4 and BH2 on PDGF-BB-induced sprout formation of aortic rings. (A) Aortic rings (1-mm) embedded and cultured in Matrigel were treated with PDGF-BB, vehicle (0.1% DMSO), and BH4 or BH2. The effects were observed on day 5 of the stimulation. Upper panels: effects of vehicle and PDGF-BB (10 ng/ml) on the outgrowth of aortic-ring sprouts. Middle and lower panels: representative photographs showing the effects of BH4 (0.1∼1,000 μM) and BH2 (0.1∼1,000 μM), respectively. (B, C) The statistical data obtained from panel A. The sprout length level in vehicle-treated rings was expressed as 100% (n=4). ∗p<0.05 vs. the PDGF-BB-induced responses in the absence of BH2 and BH4.
Fig. 5.
Effects of BH4 and BH2 on RASMC viability. Cell viability was evaluated by the XTT assay as described in the Methods section. RASMCs were treated with BH4 (0.1∼1,000 μM; A) or BH2 (0.1∼1,000 μM; B) for 24 h. After addition of the XTT dye, cells were incubated for 4 h. Cell viability was quantified by measuring optical density values with an enzyme-linked immunosorbent assay reader at 450 nm. Cell viability in the quiescent state was considered 100% (n=4).
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