Local cyclin-dependent kinase inhibition by ... - The FASEB Journal

The FASEB Journal express article 10.1096/fj.04-1646fje. Published online June 4, 2004.

Local cyclin-dependent kinase inhibition by flavopiridol inhibits coronary artery smooth muscle cell proliferation and migration: Implications for the applicability on drugeluting 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* *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, LudwigMaximilians-Universität, Klinikum Großhadern, Munich, Germany. Corresponding author: Rainer Wessely, M.D., Deutsches Herzzentrum, Lazarettstr. 36, 80636 München, Germany; E-mail: [email protected] ABSTRACT In-stent restenosis is a hyperproliferative disease which can be successfully treated by drugeluting stents releasing compounds that exhibit cell-cycle inhibitory properties to inhibit coronary smooth muscle cell (CASMC) proliferation and migration, resembling the key pathomechanisms of in-stent restenosis. Cyclin-dependent kinases (CDK) are key regulators of the eukaryotic cell cycle. CDK activity may be blocked by novel compounds such as flavopiridol. Therefore, CDK inhibitors are attractive drugs to be used for the local prevention of in-stent restenosis. In this study, we demonstrate that flavopiridol leads to potent inhibition of CASMC proliferation and migration. Molecular effects on cell-cycle regulatory mechanisms and distribution were evaluated by post-transcriptional assessment of distinct cyclins and cyclindependent kinase inhibitor (CKI) levels and flow cytometry. Cellular necrosis and apoptosis was assessed in CASMC and coronary endothelial cells. Flavopiridol induced a potent antiproliferative effect by cell-cycle inhibition in G1 and G2/M and led to increased protein levels of CKIs p21cip1 and p27kip1 as well as p53 in CASMC. Hyperphosphorylation of retinoblastoma protein was abrogated and mitogen-mediated smooth muscle cell migration significantly reduced. No accelerated cytotoxicity or increased apoptosis was detectable. Flavopiridol-coated stents, implanted in rat carotid arteries, led to significant decrease of neointima formation. As proof of principle, our results demonstrate that stents eluting CDK inhibitors such as flavopiridol effectively inhibit neointima formation. Therefore, this new class of therapeutics may be suitable for further clinical investigations on drug-eluting stents to prevent in-stent restenosis. Key words: restenosis • cell cycle • drug-coated stents

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asoproliferative diseases, including bypass atherosclerosis, transplant vasculopathy and in-stent restenosis are among the most common diseases leading to significant morbidity and mortality worldwide (1). As indicated by the term, cellular proliferation and migration, in particular of vascular smooth muscle cells, represent the major pathophysiological processes in the pathogenesis of the disease. A number of distinct and partially redundant mitogenic disease pathways converge in the cell cycle as the ultimate interface involved in cellular proliferation. The cell-cycle machinery is regulated by oscillatory and locally coordinated expression of complexes consisting of cyclins and their associated cyclin-dependent kinases (CDK). Cyclin-dependent kinases are key regulators of the cell cycle and its transitions (2). For example, in mammalian cells, CDK2, CDK4, CDK6, and associated cyclins control G1/S transition, CDK2 governs S-phase progression, and CDK1 is involved in G2/M and M-phase regulation (3). Cyclin/CDK complexes can be kept inactive by cyclin-dependent kinase inhibitors (CKI) such as p21cip1, p27kip1 (4) or members of the ink-family, for example, p15ink4B (5). CDK inhibition leads to highly effective cell cycle arrest in most cells (2), suggesting a role for CDK inhibition as a potential therapeutic target to limit vasoproliferative disease processes such as neointima formation, the cause of in-stent restenosis (6). Flavopiridol is a synthetic, highly specific inhibitor of CDK kinase activity, leading to growth arrest at both, G1/S and G2/M transitions. It has already entered clinical trials and is currently evaluated in phase III trials in cancer patients. In a cancer cell line, the IC50 for the inhibition of CDK1/cyclin B, kinase activity was 0.3 µM, for CDK2/cyclin A, it was 0.1 µM and for CDK4/cyclin D, it was 0.4 µM (7). It has been shown previously that flavopiridol induces growth arrest in cultured human aortic smooth muscle cells and inhibits expression of cyclin D1 and phosphorylation of the retinoblastoma protein without interfering with cell viability as determined by cell counting (8). In this study, oral administration of flavopiridol significantly inhibited neointima formation after experimental vascular injury. Because early and highly effective inhibition of vascular smooth muscle cell proliferation and migration is believed to be a key therapeutic approach to limit in-stent restenosis (6), the aim of this study was to extend the characterization of the molecular and cellular effects of flavopiridol to coronary artery smooth muscle cell (CASMC) proliferation and determine its influence on mitogen-induced CASMC migration. In addition, we sought to examine its local therapeutic applicability and effectiveness to inhibit neointimal hyperplasia in an experimental drug-eluting stent model. METHODS Materials Flavopiridol (L86-8275), a novel synthetic alkaloid derivative from the plant Dysoxylum binectariferum (7), was kindly provided by Aventis, Inc. (Bridgewater, NJ). The water-soluble compound has a molecular weight of 438 g/mol. Cell culture Human coronary artery smooth muscle cells (CASMC; #CC-2583, Clonetics, Walkersville, MD) and human coronary artery endothelial cells (CAEC; #CC-2585, Clonetics) were obtained at passage 3 and used in passages not higher than 10. CASMC were grown in smooth muscle cell basal medium (SmBM®, Clonetics, #CC-3182, including 0.5 µg/ml hEGF, 5 mg/ml insulin, 1 µg/ml bFGF, 50 mg/ml gentamicin and 5% FBS; #CC-4149). CAEC were maintained in


endothelial cell basal medium (EBM-2®, #CC-3162) and 5% FBS (#CC-4176), both from Clonetics. Treatment with flavopiridol was performed in fully supplemented medium for the indicated time and concentration ranges. Proliferation assay 60,000 CASMC were seeded in 35 mm plates the day before incubation with various concentrations of flavopiridol (triplicates). Every 24 h, three 100× power fields per well were imaged and subsequently counted manually. BrdU-ELISA Cellular proliferation was analyzed by a colorimetric bromodeoxyuridine (BrdU) ELISA kit (Roche, Mannheim, Germany, #1647229) as described previously (9). Cells were seeded on 96well plates at a density of ~20%. Cells were exposed to a 10 µM BrdU pulse 15 h before analysis. FACS analysis Cell cycle distribution was monitored by flow cytometry (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ) using propidium iodine staining (Cycle TestTM Kit Plus DNA Reagent Kit, BD, #340242), according to the protocol of the manufacturer. Proliferating CASMC grown in fully supplemented medium, including 10 ng/ml PDGF, were incubated with flavopiridol for 24 h, respectively. Software-based cell cycle analysis was performed using ModFit 2.0 (Verity, Topsham, ME). Western blot Western blotting was performed as described previously (9). Membranes were probed with antibodies directed against p21cip1 (Becton Dickinson, Heidelberg, Germany, #610233), p27kip1 (BD, #610241), cyclin A (Santa Cruz, #sc-751), cyclin D1 (Santa Cruz, #sc-8396), cyclin E (Santa Cruz, #sc-198), MDM2 (BD, #556353) or p53 (Santa Cruz, #sc-126), respectively. LDH assay Cytotoxicity was measured using a colorimetric assay (Roche, Mannheim Germany, #1644793) for the quantification of cell injury based on the measurement of LDH activity released from the cytosol of damaged cells into the supernatant as described previously (10). Maximum LDH release was determined by 1% (vol/vol) TritonX-100 (Sigma, #X-100) treatment. Migration assay The QCMTM-FN Quantitative Cell Migration Assay (#ECM500) from Chemicon (Temecula, CA) was used to measure cell migration due to haptotaxis toward a fibronectin gradient in a Boyden chamber system. The kit was applied according to the protocol of the manufacturer using 15,000 CASMC per well. 24 h after flavopiridol incubation, cells on the bottom side of the membrane were fixed, stained with crystal violet and manually counted on an inverted microscope.


Assessment of apoptosis To determine the apoptotic effect of flavopiridol on CASMC and CAEC, a single-stranded DNA apoptosis ELISA kit from Chemicon (#APT225) was used according to the supplied protocol. As a control, apoptosis was induced by treatment with 100 mM FeSO4 and 100 mM H2O2 in HBSS for 2 h at 37°C. (p)Rb-ELISA To determine the phosphorylation state of the Retinoblastoma protein (Rb), a (p)Rb-ELISA (BioSource, Camarillo, CA, #KH-O0021 and #KH-O0011) was used, and the ratio of hyperphosphorylated and total Rb was calculated. This assay recognizes the phosphorylation of threonine 821, which is catalyzed by cyclin E/cdk2 and cyclin A/cdk2. Cells were growtharrested by serum withdrawal for 48 h, then stimulated with fully supplemented medium and incubated with flavopiridol simultaneously. Stent coating and flavopiridol release kinetics BiodivYsioTM (Biocompatibles, Galway, Ireland) Matrix LO DD stents (10 mm length, 2.0 mm diameter) were dip-coated in 25 mg/ml flavopiridol solution for 10 min and air-dried for 15 min. For determination of both pharmacological and biological release kinetics, stents were deployed and submersed in 2 ml SmBM medium. Every hour up to 24 h, medium was changed and flavopiridol concentrations were measured by HPLC, and the biological activity of released flavopiridol was assayed by BrdU ELISA on CASMC. For HPLC analysis, a validated HPLC method using protein precipitation with acetonitrile as the sample preparation, gradient elution, and ultraviolet detection was applied as described previously (11). Experimental model of stent implantation into the rat carotid artery To determine the effect of flavopiridol on neointimal hyperplasia, a small animal model was used to examine the therapeutic effect of a drug-eluting stent system. At first, the left common carotid artery was exposed and injured by withdrawal of an inflated 2 French Fogarty catheter as described previously (9, 12). Thereafter, the premounted drug-eluting stent system coated with flavopiridol was inserted through the identical access site via the external carotid artery and placed into the common carotid artery. Subsequently, the stent was deployed with 10 atmospheres for 10 s. During surgery, animals received 80 IE of heparin. 14 days after surgery, animals were killed and the common carotid artery embedded in methylmethacrylate as described previously (13). For histomorphometric analysis, 100 µm thick sections were cut with a Leitz saw microtome, stained according to the Gallamin-Giemsa technique and scanned with a Zeiss Axiovert 100 System. Morphometric analysis was performed by SigmaScan 5.0 software (SPSS Inc., Chicago, IL). Statistical analysis Results are expressed as mean plus/minus standard deviation. The significance of variability amongst the means of the experimental groups was determined by one- or two-way ANOVA, using SPSS for Windows V10.0 software. Differences among experimental groups were considered to be statistically significant when P < 0.05. Page 4 of 20 (page number not for citation purposes)

RESULTS Flavopiridol efficiently abrogates mitogen-mediated proliferation of human coronary artery smooth muscle cells Since proliferation of coronary artery smooth muscle cells is a key process in the pathogenesis of neointima formation, we first analyzed the effect of flavopiridol on CASMC proliferation. With respect to cell count at various time points, flavopiridol displayed a pronounced antiproliferative effect even at low dosages (Fig. 1). Cell number was consistently lower at flavopiridol concentrations of more than 0.05 µM compared with nontreated controls. Cellular proliferation was completely abrogated at 0.1 µM. Cells treated with flavopiridol did not maintain a typical dedifferentiated phenotype but changed to an elongated and thin phenotype, which is morphologically consistent with a contractile, (re-) differentiated phenotype (14) (Fig. 1). Sphase progression of mitogen-stimulated CASMC treated with flavopiridol was examined by BrdU ELISA. Flavopiridol induced a dose-dependent decrease of BrdU incorporation (Fig. 2A). Congruent with cell counting, doses up to 0.025 µM did not show any effect on cellular proliferation in this assay. The maximum growth inhibitory effect, which was not different from serum-starved, quiescent control cells, was achieved at doses higher than 0.1 µM. Cell cycle analysis by flow cytometry showed a dose-dependent accumulation of flavopiridol-treated CASMC cells in both, G1 and G2/M phase (Fig. 2B). Accordingly, hyperphosphorylation of the retinoblastoma protein was inhibited by flavopiridol in a dose-dependent manner (Fig. 2C). Interference of flavopiridol with mitogen mediated migration of human coronary artery smooth muscle cells Another key mechanism of neointima formation besides proliferation of vascular smooth muscle cells is mitogen-mediated migration of these particular cells into the neointima. Therefore, the inhibitory effect of flavopiridol on mitogen-mediated cellular migration was analyzed using a Boyden chamber assay. Migratory cellular activity in this model corresponds with invasive cellular capacity in vivo (15). Flavopiridol led to dose-dependent inhibition of CASMC migration toward a fibronectin gradient (Fig. 3). Interestingly, even at low concentrations, which did not induce a detectable effect on CASMC proliferation as determined by BrdU assay, there was a perceptible inhibitory effect of flavopiridol on CASMC migration. Flavopiridol leads to enhanced expression of cyclin-dependent kinase inhibitors of the cip/kip family p21cip1 and p27kip1, down-regulation of cyclins A and D and expeditious induction of p53 Cyclin-dependent kinase inhibitors (CKI) of the cip/kip family are important regulators of cyclin-dependent kinase activity. Pharmacological induction of this particular family of CKI has been shown to play a key role in the concept of successful therapeutic approaches to limit neointimal hyperplasia (16). Interestingly, members of the ink4 class of CKI do not appear to have comparable therapeutic efficacy (17). Therefore, the effect of flavopiridol on the expression of the major cip/kip family members p21cip1 and p27kip1 was examined by Western blotting (Fig. 4). Protein expression of p21cip1 was increased time dependently by flavopiridol as early as after 12 h and peaked at 18 h. p27kip1 levels were induced even at 6 h and continued to rise until 48 h. Thus, flavopiridol accomplished dose-dependent induction of both CKIs. The tumor suppressor Page 5 of 20 (page number not for citation purposes)

protein p53 is a nuclear phosphoprotein, which mediates its activities predominantly through transcriptional activation of cell cycle regulatory genes (18). Absence of p53 activity may lead to enhanced vascular smooth muscle cell proliferation and atherosclerosis (19). Additionally, lack of p53 expression leads to enhanced neointima formation following vascular injury (20). Therefore, we examined the expression pattern of p53 in flavopiridol-treated CASMC. Whereas p53 expression was very low or even undetectable in untreated control cells, p53 levels rose early after initiation of flavopiridol treatment and peaked after 12 h. Thereafter, levels decreased significantly but were still detectable even after 48 h. Protein levels of the major inhibitor and transcriptional target of p53, MDM2, rose beginning 12 h after p53 induction and reached maximum levels at 18–24 h. This may represent a feedback loop resulting in the inhibition of p53 protein expression. Cyclin D, E, and A are fundamentally involved in the regulation of G1 and S phase. Flavopiridol led to a significant decrease of both cyclin A1/A2 and D1 levels compared with nonflavopiridol-treated cells growing in fully supplemented medium. Whereas cyclin D1 levels reconstituted gradually, cyclin A1/A2 levels were down-regulated after 18 h, 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 down-regulated by flavopiridol (Fig. 4). No detectable cytotoxic and proapoptotic effect of coronary artery smooth muscle and endothelial cells 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 therefore enhanced restenosis (21). Therefore, cytotoxicity 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, thus 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 the flavopiridol. Whereas baseline LDH levels were very low in CASMC, they were higher in CAECs, suggesting a higher susceptibility to cellular injury of these cells in culture, despite the use of appropriate cell culture medium. However, flavopiridol did not lead to an appreciable increase in LDH release in both cell lines at doses that exhibited a sustained effect on cellular proliferation and migration (Fig. 5A and B). Similarly, the apoptosis rate in smooth muscle cells as well as in endothelial cells did not increase notably during flavopiridol treatment (Fig. 5C and D). Drug-eluting stents coated with flavopiridol limit in-stent stenosis following vascular injury To investigate the therapeutic effect of flavopiridol-coated stents on the vascular proliferative response following vessel injury, drug delivery stents were dip-coated as described. Both pharmacological and biological release kinetics showed almost complete release of the drug within 3 h (Fig. 6A, B). However, this was sufficient to limit in-stent neointima formation in flavopiridol-coated stents significantly (Fig. 6C, 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.).


DISCUSSION In-stent restenosis is predominantly due to neointimal hyperplasia (22). Cytokine and growth factor release following vascular injury lead to activation of cell proliferation, primarily of vascular smooth muscle cells (23). Complex, partially redundant signaling pathways converge into the cell cycle as the ultimate step of proliferative signaling cascades. The redundancy of signal transduction pathways is likely to prevent the success of targeting one or two signal transduction factors alone. Therefore, the final common pathway of cellular proliferation, the cell cycle, is regarded as the most promising therapeutic target to limit neointima formation. In human trials, systemic administration of a variety of drugs did not prove successful in limiting vascular restenosis. Drug-eluting stents offer diverse advantages over systemic pharmacotherapy, such as minimal or no systemic side effects, higher local drug concentration at the target location and no interference with patient compliance. In clinical trials, stents coated with cell cycle inhibitors, either the G1 inhibitor sirolimus (24) or the mitosis inhibitor paclitaxel (25) have proven to be effective in limiting human neointima formation and hence in-stent restenosis. Being additionally an efficient immunosuppressive drug, it has been found that the potent antiproliferative effect of sirolimus is predominantly due to its direct inhibitory effects on the cell cycle machinery and not on its immune modulatory functions (26, 27). However, even sirolimuscoated stents are not capable of solving the restenosis problem entirely. For example, there is still a considerable restenosis rate in diabetic patients (24). Thus, evaluation of 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 (28). Several CDKs, mainly CDK4, CDK6, and probably CDK3, are involved in G1 regulation (5), CDK2 is mainly involved in the regulation of G1/S transition (29), and CDK1 predominantly regulates G2/M transition. Since cell cycle inhibition represents the major therapeutic target to prevent neointima formation, synthetic inhibition of cyclin-dependent kinases offers a unique opportunity to inhibit several key regulators of distinct cell-cycle phases. In cancer therapy, at least three compounds, flavopiridol, UCN-01 and roscovitine have entered clinical trials up to phase 3. Flavopiridol is a highly selective inhibitor of most cyclin-dependent kinases. In different human tumor cell lines, the IC50 of kinase inhibition by flavopiridol for CDK1/cyclin B, CDK2/cyclin A, CDK2/cyclin E, CDK4/cyclin D, and for CDK7/cyclin H has already been determined (7, 30, 31). Protein kinases such as CDKs selectively transfer phosphate groups from ATP to protein substrates, thereby modulating their activity and/or their attachment sites to activate downstream signaling molecules (31). The mechanism by which flavopiridol inhibits CDK activity is not solely based on interactions with the ATP site of various CDKs (32). Recent studies demonstrated additional effects by which flavopiridol induces cell cycle inhibition, apparently by transcription-related mechanisms through inhibition of pTEFb (positive transcription elongation factor), thereby blocking RNA polymerase II transcription (33) leading to an early decrease of cyclin D levels in MCF-7 cancer cells (34). To our knowledge, this is the first report about local therapeutic application of a synthetic CDK inhibitor to prevent neointima formation following stent placement. A previous study by Ruef et al. (8) indicated that oral flavopiridol treatment in rats following carotid injury without stenting Page 7 of 20 (page number not for citation purposes)

attenuates the development of vascular stenosis. Because oral drug administration has never been shown to be therapeutically effective in the inhibition of in-stent restenosis in humans, we examined the effect of local flavopiridol administration to the injured vessel wall. In addition, the pathophysiology of restenosis after stenting is quite distinct from mere vascular injury. Therefore, the findings from the study by Ruef et al. cannot readily be extrapolated to the scenario of local stent-based drug delivery. In this report, we examine the molecular and cellular effects of flavopiridol in a noncancer cell line, coronary artery smooth muscle cells. We show that flavopiridol highly efficiently impedes CASMC proliferation by exhibiting both a G1 and a G2/M block in proliferating cells. This phenomenon may be advantageous to sirolimus or paclitaxel, which exclusively block in G1 or M phase, respectively. Up to relatively high doses of flavopiridol, which proved sufficient to fully block DNA replication, we were not able to detect an immediate appreciable cytotoxic effect in CASMC as determined by lack of LDH release from treated cells. In addition, there was no increase in the rate of cellular apoptosis. Surprisingly, just like CASMC, coronary artery endothelial cells neither exhibited a measurable LDH increase nor showed any evidence of accelerated apoptosis following flavopiridol treatment. In tumor cell lines, flavopiridol has been associated with the induction of apoptosis (35). However, this effect may vary considerably between cell lines and may be cell-cycle dependent (36). For example, HUVECs (human umbilical vein endothelial cells) express high levels of p27kip1, low or undetectable amounts of CDK1, cyclin A, B and cdc25C mRNA, as well as low CDK2 kinase activity. However, apoptosis can be reliably induced at doses higher than 100 nM flavopiridol (36), suggesting that flavopiridol may effect other pathways that mediate apoptotic responses. Another important fact might be that CAEC used for this study were at very low passages (≤5), whereas most other endothelial cell lines are used at considerably higher passages. Concordant with the lack of a cytotoxic or proapoptotic effect in endothelial cells at dosages evaluated in this study, animals receiving flavopiridol-coated stents did not exhibit an increased rate of stent thrombosis in this study as determined by duplex sonography and post mortem histology. Cyclin-dependent kinase inhibitors of the cip/kip class, in particular p21cip1 and p27kip1 are considered to play a prominent therapeutic role for the suppression of mitogen-induced vascular smooth muscle cell proliferation and migration (17, 37). Rapamycin, widely used on drugeluting stents, inhibits the kinase TOR (target of rapamycin) after binding to its intracellular receptor FK506 binding protein (FKBP12), ultimately leading to increased p27kip1 protein levels and G1 arrest (38). The importance of this specific mechanism for the inhibition of proliferation in vascular smooth muscle cells is illustrated by the observation that resistance to rapamycin treatment is associated with defective p27kip1 regulation (39). However, another study found that rapamycin may also exhibit a growth inhibitory effect in p27kip1-deficient cultured smooth muscle cells (40). In addition, there is evidence that p27kip1 may not be required for rapamycindependent attenuation of lesion formation induced by mechanical injury (40) or hypercholesterolemia (41) in distinct animal models. Thus, both p27kip1 dependent and independent mechanisms of rapamycin actions on smooth muscle cell growth arrest have been reported. In contrast, p27kip1 is essential for efficient rapamycin-dependent inhibition of smooth muscle cell migration (42). p21cip1 levels are increased even in later phases of neointima formation (43), suggesting that p21cip1 is part of an important feedback mechanism leading to cell cycle arrest in initially proliferating neointimal cells. Therefore, restoration of CKI function may be considered as crucial to abrogate the hyperproliferative response in vasoproliferative diseases. Page 8 of 20 (page number not for citation purposes)

Flavopiridol led to a prominent and sustained induction of both p21cip1 and p27kip1 protein levels in CASMC. The fact that p27kip1 is additionally involved in the inhibition of vascular smooth muscle cell migration (42) provides a possible explanation for the flavopiridol-mediated inhibition of CASMC migration. According to previous reports in cancer cell lines (34), flavopiridol lead to a decrease of cyclin A and D levels in CASMC, whereas cyclin E protein levels did not diminish. The fact that cyclin D-CDK4/6 kinase activity is essential for Rb hyperphosphorylation (44) explains the finding that Rb hyperphosphorylation was inhibited by flavopiridol. Endogenous expression levels of the transcription factor p53 are associated with attenuated neointima formation (20). We found a rapid up-regulation of p53 in flavopiridoltreated CASMC followed by decreasing protein levels after 12 h; however, p53 was maintained at lower levels up to 48 h following flavopiridol treatment. As expected, protein levels of the major inhibitor of p53, MDM2 were regulated inversely. Induction of p21cip1 might be in part due to p53, since p53 transcriptionally activates p21cip1 (45); however, the up-regulation of p21 might also be due to other transcription factors not addressed in this study, for example interferon regulatory factor-1 (IRF-1) (9). For the evaluation of drug-eluting stents coated with a CDK inhibitor, we used the BiodivYsio DD Matrix LO stent platform with phosphorylcholine coating in the rat carotid artery stent model. Evaluation of this commercially available stent system in human small coronary arteries has been proven to be safe and effective (46). Determination of the biological release kinetics revealed fast flavopiridol release from the stent, most of the drug was released within 3 h. As stated above, stent patency was not different in the verum and placebo group. However, the short release of the CDK inhibitor from the stent was sufficient to significantly reduce neointima formation 14 days after vascular injury. The observation that a short release is sufficient to limit neointima formation in the rat carotid stent model may be explained by the fact that the development of in-stent restenosis differs significantly between animals and humans (47). Given the fact that lesion formation in man takes ~8–10 times longer than in the rat, a shorter drug release may therefore be sufficient in the rat to inhibit neointimal processes. The question for optimal drug release remains open and may be highly dependent on the mode of drug action and pharmacogenetics as well as lesion characteristics. In preliminary reports, the Matrix LO stent system has proven to be effective in drug delivery into the vascular wall by limiting the rate of in-stent restenosis in de novo coronary lesions in human patients (48). However, in our study, we considered this particular stent system as a platform to prove the concept that the delivery of a novel CDK inhibitor such as flavopiridol can efficiently inhibit in-stent restenosis. In the human system, a prolonged release may be more effective and desirable. Limitation of the study We analyzed the therapeutic effect of flavopiridol-eluting stents in the rat carotid stent model. In a recently published report from a consensus group, it is stated that “the ideal animal model for drug-eluting stent evaluation is uncertain”, and “it is unclear that any single animal species is any more predictive of human response and for specific indications” (49). Thus, as any animal model, extrapolation of the data to the human clinical situation remains uncertain. However, there is no standard or proven better animal model than the rat vascular injury model for an experimental therapeutic approach to inhibit in-stent neointimal growth. In addition, we showed that flavopiridol does not induce an increased cytotoxic effect or apoptosis in human coronary endothelial cells and there was no accelerated stent thrombosis rate in the animal model. Page 9 of 20 (page number not for citation purposes)

However, this data may not be sufficient to rule out an effect on the stent thrombosis rate in the human system. Despite these limitations, considering the prominent effect of flavopiridol on CASMC proliferation and migration as well as the favorable profile concerning cytotoxicity and apoptosis, even in human coronary endothelial cells, synthetic CDK inhibitors deserve further evaluation of their antirestenotic effect. Because flavopiridol is the first CDK inhibitor to enter clinical trials (50), it may be considered as an appropriate candidate to further elucidate the efficacy of this new class of specific and potent antiproliferative therapeutics in the prevention and treatment of human in-stent restenosis on drug-eluting stents. ACKNOWLEDGMENT This study was supported in part by a grant awarded from the Kommission für Klinische Forschung, Deutsches Herzzentrum, Munich (to R.W.) and by the Bayerische Forschungsstiftung (to R.W., A.S., A.K.). We wish to thank Dr. Franziska Wegener for expert technical assistance and Dr. Ludger Hengst for critically reading the manuscript. REFERENCES 1.

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