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Local cyclin-dependent kinase inhibition by flavopiridol inhibits coronary artery smooth muscle cell proliferation and migration: Implications for the applicability on drug-eluting stents to prevent neointima formation following vascular injury

BIRGIT JASCHKE^*, STEFAN MILZ, MICHAEL VOGESER, CORNELIA MICHAELIS^*,, MARC VORPAHL^*, ALBERT SCHÖMIG^*, ADNAN KASTRATI^* and RAINER WESSELY^*,1

^* Deutsches Herzzentrum and 1. Medizinische Klinik, Technische Universität;
Anatomische Anstalt, Ludwig-Maximilians-Universität;
Institut für Experimentelle Onkologie und Therapieforschung, Technische Universität; and
Institut für Klinische Chemie, Ludwig-Maximilians-Universität, Klinikum Großhadern, Munich, Germany

¹Correspondence: Deutsches Herzzentrum, Lazarettstr. 36, München 80636, Germany. E-mail: rwessely{at}dhm.mhn.de

SPECIFIC AIMS

The synthetic CDK inhibitor flavopiridol has been shown to cause growth arrest in cultured aortic smooth muscle cells. Moreover, orally administered flavopiridol limited neointima formation after experimental vascular injury. This study was designed to assess growth inhibitory effects of flavopiridol on human coronary artery smooth muscle cells (CASMC) and to determine whether flavopiridol affects CASMC migration. Applicability and therapeutic efficacy of flavopiridol on drug-eluting stents was evaluated as a prevention to formation of in-stent stenosis by suppression of neointima formation.

PRINCIPAL FINDINGS

1. Flavopiridol efficiently abrogates mitogen-mediated proliferation and migration of human coronary artery smooth muscle cells
In-stent restenosis remains to be a major problem of interventional vascular procedures. Drug-eluting stents inhibiting vascular smooth muscle cell proliferation and migration within the injured vessel segment have shown to be effective inhibitors of neointima formation, the pathoanatomical correlate of in-stent restenosis. We now demonstrate that flavopiridol induces a potent, dose-dependent antiproliferative effect in cultured CASMC, shown by significant inhibition of BrdU incorporation starting at 0.05 µM. Flavopiridol induces both a G₁/S as well as a G₂/M block in CASMC as determined by flow cytometry based cell cycle analysis. In addition to smooth muscle cell proliferation, migration of this particular cell type into the neointima serves as another major mechanism of the restenotic disease process, representing another therapeutic target to abrogate neointima formation. We show that Flavopiridol effectively inhibits mitogen induced CASMC migration in a Boyden chamber assay, which is known to correspond with cellular invasiveness in vivo at doses beginning at 0.01 µM.

2. Flavopiridol leads to enhanced expression of cyclin dependent kinase inhibitors of the cip/kip family p21^cip1 and p27^kip1, downregulation of cyclins A and D and expeditious induction of p53 in CASMC
We next sought to further address the effects of flavopiridol on expression of important cell cycle regulatory proteins in CASMC. Members of the cip/kip class of CDK inhibitors such as p21^cip1 and p27^kip1, negative regulators of the cell cycle, play an important role in both pathogenesis as well as treatment of vasoproliferative diseases such as in-stent restenosis. Overexpression of either p21^cip1 or p27^kip1 leads to inhibition of neointima formation in experimental animal models. We demonstrate up-regulated protein expression of both p27^kip1 and p21^cip1 as early as 6 h and 12 h after initialization of flavopiridol treatment (Fig. 1 ). The tumor suppressor protein p53 mediates its activities through transcriptional activation of cell cycle regulatory genes. Absence of p53 may lead to enhanced neointima formation following vascular injury. Whereas p53 expression was very low or even undetectable in untreated control cells, p53 levels were induced early after incubation with flavopiridol (Fig. 1) . Protein levels of the major inhibitor and transcriptional target of p53, MDM2, went up starting 12 h after p53 induction and reached maximum levels at 18–24h, representing a feedback loop resulting in inhibition of p53 protein expression. Cyclins D, E, and A are fundamentally involved in regulation of G₁ and S phase. Flavopiridol led to a significant decrease of both cyclin A₁/A₂ and D₁ levels compared with non-flavopiridol-treated cells growing in fully supplemented medium. Whereas cyclin D₁ levels reconstituted gradually, cyclin A₁/A₂ levels were downregulated after 18h, presumably since cells subsequent to the G₁ checkpoint kept their cyclin A levels for the duration of one cell cycle. Cyclin E protein levels were not downregulated by flavopiridol (Fig. 1) . Retinoblastoma protein (Rb) phosphorylation was significantly and dose-dependently inhibited by flavopiridol as determinded by Rb phosphorylation ELISA.

View larger version (61K): [in this window] [in a new window]   Figure 1. Effect of flavopiridol treatment (0.1 µM) on various cell cycle regulatory proteins. Whereas cyclin E levels do not diminish with flavopiridol treatment, cyclin A1/A2 and D1 levels are downregulated by flavopiridol and recover at later time points. Flavopiridol leads to a rapid increase of transcription factor p53 levels, followed by an increase of both p21cip1 and p27kip1 CKI levels. p53 levels are inversely regulated to its main inhibitor and transcriptional target MDM2, but still detectable 48 h after beginning flavopiridol treatment in contrast to untreated cells.

3. No detectable cytotoxic and proapoptotic effect in coronary artery smooth muscle and endothelial cells (CAEC) treated with flavopiridol
Intravascular death of CASMC may elicit an inflammatory response which can lead to instability of the vessel wall, aggravated wound healing, and enhanced restenosis. Therefore, a cytopathic effect should be avoided during pharmacological prevention of vasoproliferative processes. Similarly, endothelial cell death may lead to prolonged and incomplete reendothelialization of the injured vascular wall and, if a stent was implanted, the stent struts, eventually leading to stent thrombosis. Therefore, we examined whether flavopiridol exhibits a cytotoxic effect in CASMC as well as CAEC and if there is accelerated apoptosis in a mitogenic environment. For cytotoxicity (necrosis), LDH release was measured in both cell lines 48 h after addition of flavopiridol. Whereas baseline LDH levels were very low in CASMC, they were higher in CAEC suggesting an increased susceptibility to cellular injury of these cells in culture, despite use of appropriate cell culture medium. Flavopiridol did not lead to an appreciable increase in LDH release in both cell lines at doses exhibiting a sustained effect on cellular proliferation and migration. Similarly the apoptosis rate in smooth muscle cells as well as in the endothelial cells (CAEC) did not increase significantly during flavopiridol treatment.

4. Drug-eluting stents coated with flavopiridol limit in-stent stenosis following vascular injury
To investigate the therapeutic effect of flavopiridol coated stents on the inhibition of neointima hyperplasia, phosphorylcholine coated drug delivery stents (BiodivYsio^TM DD stents, Biocompatibles, Ireland) were coated with flavopiridol. Both pharmacological and biological release kinetics showed almost complete release of the drug within 3 h (Fig. 2 A, B). However, this was sufficient to limit in-stent neointima formation in flavopiridol coated stents significantly (Fig. 2C, D ). Under single injection of heparin at the time of surgery, flavopiridol coated stents did not present a higher stent thrombosis rate compared with control stents as determined by serial duplex sonography at 24 h and 14 days after surgery and histological post mortem analysis (flavopiridol n=3/15, controls n=4/17, P=n.s.).

View larger version (21K): [in this window] [in a new window]   Figure 2. Flavopiridol eluting stents inhibit neointima formation in vivo. To verify therapeutic efficacy of flavopiridol coated stents, coated BiodivYsioTM DD stents were deployed in balloon injured rat carotid arteries. Both pharmacological (A) and biological (B) flavopiridol release kinetics from this particular stent were assayed in vitro indicating entire release of the compound within three hours. Pharmacological release was determined by HPLC measurement, biological release by assaying the growth inhibitory effect of stent-released flavopiridol at indicated time points on cultured CASMC. C) Histomorphometric analysis of stented rat carotid arteries 14 days post implantation revealed a significant decrease of neointima formation in animals receiving a flavopiridol coated compared with identical but uncoated stents. D) Representative images of a flavopiridol stented vs. a uncoated stented carotid artery. ** P< 0.01.

CONCLUSIONS AND SIGNIFICANCE

In-stent restenosis can be treated successfully with drug-eluting stents coated with compounds exhibiting cell cycle inhibitory properties (e.g., rapamycin and paclitaxel). Since a subset of patients still acquires in-stent restenosis even after receiving these currently available coated stents, a search for novel drug alternatives is warranted. Cyclin-dependent kinases are key regulators of the cell cycle and represent a group of serine/threonine kinases that form active heterodimeric complexes following binding to cyclins. Since cell cycle inhibition represents the major therapeutic target to prevent neointima formation, synthetic inhibition of cyclin-dependent kinases by flavopiridol offers a unique opportunity to inhibit several key regulators of distinct cell cycle phases. Results of this study indicate that flavopiridol induces a potent antiproliferative and antimigratory effect in CASMC. Important negative regulators of the smooth muscle cell cycle such as cip/kip CKIs and p53 are reliably induced, thereby avoiding an appreciable cytotoxic effect or accelerated apoptosis in both smooth muscle and endothelial cells of coronary artery origin. Despite the relatively short flavopiridol release time of 3 h from the stent platform used in this study, this was sufficient to inhibit neointima formation in the rat carotid injury and stent model significantly. In this model, stent thrombosis rate was not significantly different compared with identical but non-flavopiridol coated stents. This is the first study reporting successful application of a synthetic CDK inhibitor on a drug eluting stent platform to limit in-stent neointimal hyperplasia. Based on the findings of this study, flavopiridol, being the first CDK inhibitor to enter clinical trials in oncology, may be considered as an appropriate candidate to further elucidate the efficacy of this new class of specific and potent antiproliferative therapeutics in prevention and treatment of human in-stent restenosis on drug-eluting stents.

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the experimental design and analytical approach. CASMC,coronary artery smooth muscle cells; CAEC, coronary artery endothelial cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1646fje; doi: 10.1096/fj.04-1646fje

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