Abstract
Proliferation of smooth muscle cells (SMCs) in atherosclerosis may be modulated by several growth regulatory molecules. At least two mitogens for SMCs, platelet-derived growth factor (PDGF) A-chain and heparin-binding epidermal growth factor-like growth factor (HB-EGF), can be produced by SMCs themselves and may stimulate smooth muscle proliferation in an autocrine or paracrine fashion. We examined the effects of thrombin, which may be generated at the site of vascular injury during atherogenesis, and the potent anti-inflammatory glucocorticoid, dexamethasone (DEX), on the expression of the genes encoding these two growth factors. Since both PDGF A-chain and HB-EGF have affinity for heparin, we also examined the effect of thrombin and DEX on the release of heparin binding mitogenic activity from SMCs. Treatment of SMCs with thrombin resulted in increases both in the level of the PDGF-A and HB-EGF transcripts in the cells, as well as in released heparin-binding growth factor activity. DEX inhibits the thrombin-stimulated release of mitogenic activity in a dose-dependent manner. An enzyme-linked immunoadsorbent assay showed that DEX inhibits both constitutive and thrombin-stimulated release of PDGF-AA. DEX also decreases both constitutive and thrombin-stimulated mRNA levels for PDGF A-chain and HB-EGF and destabilizes the transcripts for both growth factors. A nuclear run-on assay revealed that DEX acts, in addition, to inhibit constitutive and thrombin-stimulated transcription of the PDGF A-chain and HB-EGF genes. Thus, these findings indicate that expression of PDGF A-chain and HB-EGF may be regulated by thrombin and glucocorticoid at the transcription level. Our results are consistent with the involvement of thrombin-induced growth factor expression in neointimal SMC proliferation and suggest the possibility that intimal proliferation may be attenuated by glucocorticoids.
Highlights
Proliferation of smooth muscle cells (SMCs) in ath- Intimal smooth muscle proliferation is a critical event in erosclerosis may be modulated by several growrtehg- the formation of the advanced lesions of atherosclerosis
Effect of Thrombin and DEX Ronelease of Heparin Binding Mitogenic Activity by Human SMCs-Since Platelet-derived growth factor (PDGF) (Raines and Ross, 1982) and Heparin-binding EGF-like growth factor (HB-EGF) (Besner et al, 1990; Higashiyama et al, 1991; Temizer et al, 1992) both bind to heparin and aresecreted by SMCs, we first examined whether thrombinandDEX could affect the release of heparin-binding mitogenic activity from human SMCs
Conditioned media from SMCs treated with thrombin or DEX were fractionated on heparin-Sepharose, and bound proteins were step-eluted under conditions that would elute PDGF and HBEGF, but other heparin-binding mitogens such as basic and acidic fibroblast growth factor (Shing et al, 1984) as well
Summary
Culture of Smooth Muscle Cells-Human aortic SMCswere isolated from newborn human thoracic aorta, as described previously (Ross and Kariya, 1980) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. Cells weregrown to confluence and changed to DMEM with 1%human plasma-derived serum (Raines and Ross, 1985) for 48 h prior to initiation of experiments. Northern Blotting-Total cellular RNA was extracted from cells as described previously (Chomoczynski and Sacchi, 1987). RNA samples were subjected to limited base hydrolysis (0.2 M NaOH on ice for 3 min), ethanol-precipitated, and suspended at 1 X 10' cpm/ml in hybridization buffer. The membranes were hybridized for 45 h with the radiolabeled run-on RNAs and washed as described for Northern blotting. The cells were stimulated with 2 units/ml human thrombin itnhe presence or absence of 1p~ DEX and labeled 16-48 h after sample addition with 2 mCi/ml [3H]thymidine inthe presence of 3 mM cold thymidine. The cells were stimulated with 2 units/ml human thrombin itnhe presence or absence of 1p~ DEX and labeled 16-48 h after sample addition with 2 mCi/ml [3H]thymidine inthe presence of 3 mM cold thymidine. [3H]Thymidineincorporation was measured as described above
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