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The triazolopyrimidine trapidil has been found in controlled clinical trials to prevent restenosis after vascular injury. Although trapidil is widely regarded as a platelet-derived growth factor receptor (PDGF) antagonist, its precise mode of action is still unknown. This study was designed to investigate the inhibition of mitogenesis by trapidil in cultured bovine coronary artery smooth muscle cells (SMC) and to identify major signal transduction pathways involved. Trapidil inhibited PDGF-BB-induced mitogenesis in SMC in a concentration-dependent manner. Comparable inhibitory effects were obtained after stimulation of smooth muscle cells by phorbol ester, which suggests that the action of trapidil was not restricted to PDGF receptor-mediated mechanisms. Trapidil also inhibited PDGF- and phorbol ester-induced mitogen-activated protein kinase as well as Raf-1 kinase activity. As a possible target of trapidil, stimulation of cellular protein kinase A (PKA) activity was detected. Trapidil also induced the phosphorylation of vasodilator-stimulated phosphoprotein in SMC. Antimitogenic effects of trapidil were completely abolished by PKA inhibitors. Neither a direct stimulation of cAMP formation nor a phosphodiesterase inhibition was observed at antimitogenic concentrations of trapidil. However, trapidil directly activated purified PKA holoenzyme in a cAMP-independent manner. In conclusion, trapidil exerts its antimitogenic effects on SMC by direct activation of PKA. Thus, PKA-mediated inhibition of the Raf-1/MAP kinase pathway may be involved in the antimitogenic actions of the compound.

This study compares the antimitogenic effects of iloprost and prostaglandin E1 on platelet-derived growth factor-BB stimulated DNA synthesis ([3H]thymidine incorporation) in bovine coronary artery smooth muscle cells. When added 20-24 h after stimulation with platelet-derived growth factor-BB (20 ng/ml), both iloprost and prostaglandin E1, concentration-dependently (IC50 3-5 nM) inhibited DNA synthesis. However, when added together with the growth factor (0-24 h), the inhibition of DNA synthesis by iloprost was markedly attenuated, indicating tolerance development. In contrast, no tolerance to antimitogenic effects of prostaglandin E1 or forskolin were observed. When added to iloprost-tolerant cells, both prostaglandin E1 and forskolin, still inhibited DNA synthesis. There was no evidence for transcriptional down-regulation of prostacyclin receptor gene by iloprost. The data demonstrate a tolerance development to antimitogenic actions of prostacyclin but not of prostaglandin E1 and suggest that the receptors, mediating the antiproliferative actions of these prostaglandins, may be different.

Background: Previous studies have shown that thrombin is a potent though slow-acting mitogen for vascular smooth muscle cells (SMC). Because thrombin generation in vivo is accompanied by platelet activation, it has been suggested that platelet-derived factors might enhance thrombin-induced SMC proliferation. No information is available so far on the possible role of thromboxane A2., Methods and Results: Thrombin (1 U/mL) caused a threefold to fourfold increase of DNA synthesis in cultured bovine coronary artery SMC as assessed from [3H]thymidine incorporation. U 46619, a stable thromboxane A2 mimetic, had only a minor stimulating effect on its own but potentiated the thrombin effect sixfold to sevenfold above control (P<.05). These findings were paralleled by a 52+/-5% (P<.05) increase in cell number at 48 hours after addition of both mitogens as compared with 24+/-5% with thrombin alone and no change with U 46619 alone. Thromboxane A2 receptor mRNA was found to be upregulated sixfold 20 minutes after thrombin stimulation. Pretreatment of SMC with thrombin for 4 hours markedly increased U 46619-induced mitogen-activated protein kinase activity, indicating thrombin-induced upregulation of functional thromboxane receptors in SMC., Conclusions: Thrombin-induced proliferation of SMC is markedly enhanced by thromboxane A2. This might result in an enhancement of SMC proliferation by platelet-derived thromboxane A2 in vivo.

Vasodilatory prostaglandins (PGI2, PGE2, PGE1) are known inhibitors of proliferation of vascular smooth muscle cells (SMC) after stimulation with mitogenic factors. However, endogenous prostaglandins do not prevent SMC proliferation subsequent to vessel injury in vivo. Since vascular cells produce large amounts of antiproliferative prostaglandins, especially subsequent to COX-2 expression, insufficient vascular PGI2 formation is not likely to explain the failure of endogenous prostaglandins to prevent excessive SMC growth. In this paper we demonstrate a rapid development of tolerance to PGI2 in SMC, resulting in diminished antiproliferative activity. These findings may not only be relevant for the control of SMC growth by endogenously synthesized prostaglandins but also for clinical use of PGI2 mimetics.

The activation of thrombin is the key event in clot formation after vascular injury. Thrombin itself, but also other clot-derived factors, such as thromboxane A2 (TXA2), are mitogenic for vascular smooth muscle cells. We have studied the possible interactions between thrombin and TXA2 in stimulation of coronary artery smooth muscle cell (SMC) proliferation. Thrombin (1 U/ml) caused a significant proliferatory response in SMC. U 46619, a stable TXA2 mimetic, had only a minor stimulating effect by its own but markedly potentiated the thrombin-induced mitogenesis. A possible mechanism for these potentiating effects is provided by the demonstration of a marked (6 fold) but transient (maximum after 20 min) increase in the expression of TXA2 receptor (TP receptor) mRNA in SMC by thrombin. Since a significant clot-related TXA2 generation was detected for at least 2 hours, the up-regulation of TP receptors by thrombin may represent a mechanism that is relevant for the in vivo situation of SMC proliferation after vessel injury.

In addition to inhibition of platelet function, prostacyclin and its stable analogues are reported to attenuate vascular smooth muscle cell proliferation. However, desensitization of prostacyclin responsiveness is a known phenomenon in platelets. In this study we investigated the time-dependent effects of the prostacyclin-mimetic iloprost and of PGE1, respectively, on PDGF-induced proliferation of cultured coronary artery smooth muscle cells. Proliferation, assessed by [3H]thymidine incorporation was markedly inhibited by coincubation with iloprost (100 nM) and PGE1 (100 nM) for 4 h. In contrast, addition of iloprost (100 nM) for 24 h did not decrease smooth muscle cell proliferation, whereas inhibition by PGE1 or by forskolin was not diminished. These results suggest a homologous desensitization of anti-mitogenic effects of iloprost in coronary artery smooth muscle cells, probably at receptor-level.