Hyperproliferation of vascular smooth muscle cells (VSMCs) is critically involved in the onset of atherosclerosis and restenosis. Although caffeic acid phenethyl ester (CAPE, 1), one of the main constituents of honeybee propolis, has been shown to exert a beneficial effect in models of vascular injury in vivo, detailed mechanistic investigations in vascular cells are scarce. This study has examined the antiproliferative activity of 1 in platelet-derived growth factor (PDGF)-stimulated primary rat aortic VSMCs and aimed to shed light on underlying molecular mechanisms. Compound 1 inhibited the proliferation of VSMCs upon exposure to PDGF in a dose-dependent manner by interfering with cell cycle progression from the G0/1- to the S-phase. Enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) as well as stabilization of hypoxia-inducible factor (HIF)-1α and subsequent induction of heme oxygenase-1 (HO-1) could be identified as molecular events contributing to the observed growth arrest in PDGF-activated VSMCs upon exposure to 1.

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to the pathogenesis of restenosis. Thus, drugs interfering with cell cycle progression in VSMC are promising candidates for an antirestenotic therapy. In this study, we pharmacologically characterize, a novel derivative of the cyclin-dependent kinase (CDK) inhibitor roscovitine (ROSC), in PDGF-BB-activated VSMC. Cell proliferation was quantified measuring DNA synthesis via 5-bromo-2Ј-deoxyuridine incorporation. Analysis of cell cycle distribution was done by flow cytometry using propidium iodide-stained nuclei. Key regulators of the cell cycle and relevant signaling pathways were dissected by Western blot analyses. In addition, in vitro kinase assays and in silico studies regarding the pharmacokinetic profile of both compounds were performed. LGR1406 shows a stronger (IC 50 ϭ 3.0 M) antiproliferative activity than ROSC (IC 50 ϭ 16.9 M), halting VSMCs in G 0 /G 1 phase of the cell cycle, whereas ROSC does not arrest but rather delays cell cycle progression. Neither of the compounds interferes with early PDGF-BB-induced signaling pathways (p38, extracellular signal-regulated kinase 1/2, c-Jun NH 2 -terminal kinase, Akt, signal transducer and activator of transcription 3), and both inhibit CDKs, with LGR1406 exerting a slightly higher potency against CDK1/2 and 4 than ROSC. Expression of cyclins A and E as well as hyperphosphorylation of the pocket proteins retinoblastoma protein and p107 are negatively affected by both compounds, although to a different extent. In silico calculations predicted a much higher metabolic stability for LGR1406 compared with ROSC. Altogether, ROSC derivatives, such as LGR1406 seem to be promising compounds for further development in antirestenotic therapy.Developing strategies against restenosis, the renarrowing of an artery after angioplastic interventions, remains an important goal of vascular biology and pharmacological research. Currently, drug-eluting stents have become the treatment of choice for patients undergoing percutaneous coronary revascularization (Kukreja et al., 2008). Current marketed first-generation drug eluting stents use drugs such as rapamycin and paclitaxel targeting mammalian target of rapamycin and tubulin, respectively (Windecker and Jü ni, 2008). Unfortunately, some concerns have been raised recently due to a potential increased risk of late-stent thrombosis (Steffel et al., 2008). Therefore, the identification of new drug candidates interfering with vascular smooth muscle cell (VSMC) proliferation using mechanisms other than rapamycin and paclitaxel is an important pharmacological topic.Roscovitine (ROSC) has been characterized as a selective inhibitor of cyclin dependent kinases (CDK) 1, 2, and 5 in enzyme-based assays (Meijer et al., 1997), inhibiting the proliferation of various cell types ranging from numerous cancer cell lines to keratinocytes and fibroblasts. The ob-

During the pathology of atherosclerosis and restenosis, hyperproliferation and migration of vascular smooth muscle cells (VSMC) contribute to lumen loss of the vessel and neointima formation [1]. Drugs interfering with both, proliferation and migration of VSMC are therefore promising candidates for the treatment of vasculoproliferative diseases. In one of our previous studies Indirubin-3-monoxime (I3MO) has been identified to inhibit VSMC proliferation in vitro as well as neointima formation in vivo [2]. I3MO specifically interfered with PDGF-BB-induced STAT3 phosphorylation without affecting other common early growth factor signalling pathways. In this work we now investigated the influence of I3MO on VSMC migration. In order to trigger migration we used PDGF-BB (10 ng/ ml), the major migratory stimulus for VSMC, and assessed migration by employing transwell-as well as wounding assays. With both methods we consistently observed significant and dose-dependent inhibition of migration by I3MO with complete abrogation at 5 µM. Employing phalloidin staining in order to visualise the actin cytoskeleton we show that I3MO already impairs early cytoskeletal rearrangements upon PDGF stimulation, such as formation of stress fibres, membrane ruffles or filopodia. We were able to exclude an inhibitory activity of I3MO on the activation of the small GTPase Rac-1, one of the key regulators of cell migration. However, in consistency with the inhibitory activity of I3MO on STAT3 phosphorylation we found that I3MO inhibits expression of cytosolic phospholipase A2 (cPLA2), a STAT3 target gene and mediator of VSMC migration [3].To summarize, we demonstrate that next to its antiproliferative activity against VSMC I3MO inhibits VSMC migration. Interference with the STAT3 -cPLA2 pathway is likely to contribute to the anti-migratory action of I3MO, but further studies are needed to elucidate the precise mechanism of action.[1]Dzau VJ, Braun-Dullaeus RC, Sedding DG. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med. 2002Med. : 8: 1249Med. -1256Med. . doi:10.1038Med. /nm1102-1249 Schwaiberger A. PhD Thesis, University of Vienna, Austria, 2008. [3] Neeli I, Liu Z, Dronadula N, Ma ZA, Rao GN. An essential role of the Jak-2/STAT-3/cytosolic phospholipase A(2) axis in platelet-derived growth factor BB-induced vascular smooth muscle cell motility.

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to neointima formation during the pathogenesis of atherosclerosis and restenosis. Drugs interfering with the cell cycle progression of VSMCs are therefore promising candidates for the treatment of vasculoproliferative diseases. Roscovitine (ROSC) has been characterised as a selective CDK1/2/5/7/9 inhibitor using enzyme-based assays, but the inhibition of other kinases (e.g. ERK, AuroraA) has been demonstrated as well. It has been shown that ROSC inhibits VSMC growth but the mode of action has not been solved satisfyingly and there are no data describing its influence on platelet-derived growth factor (PDGF) signalling, a major proliferative stimulus for VSMCs. We therefore investigated the effect of ROSC on PDGF-activated VSMCs. Using a BrdU incooporation assay, we show that ROSC inhibits PDGFinduced VSMC growth in a concentration dependent manner (IC 50 : ~15 μM). Detailed FACS analysis of the cell cycle revealed that ROSC delays S-phase entry upon PDGF stimulation without leading to enhanced apoptosis. By Western blot analysis we excluded that ROSC has any inhibitory effect on common early signaling events (phosphorylation of ERK, p38, Akt). Contrary to results published on colon carcinoma cells, where ROSC causes a loss of cyclinD1, we demonstrate that cyclinD1 levels are markedly increased even after 24 h of PDGF stimulation, confirming that there is no interference with early steps in cell cycle progression. Despite high levels of cyclinD1 the phosphorylation of the retinoblastoma protein at Ser807/ 811 is delayed and reduced, as is the induction of the early S-phase cyclin A. We conclude that ROSC limits VSMC growth by directly inhibiting CDK2 or 4 and constitutes a promising compound for further development as e.g. stent-coating agent.