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Cardiovascular Research 2004 63(1):11-21; doi:10.1016/j.cardiores.2004.02.009
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Copyright © 2004, European Society of Cardiology

Control of vascular cell proliferation and migration by cyclin-dependent kinase signalling: new perspectives and therapeutic potential

Vicente Andrés*

Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Cientificas, C/Jaime Roig 11, Valencia, 46010 Spain

*Tel.: +34-96-3391752; fax: +34-96-3391750. Email address: vandres{at}ibv.csic.es

Received 21 December 2003; revised 28 January 2004; accepted 18 February 2004


   Abstract
Top
 Abstract
1. Introduction
 Acknowledgments
 References
 
Neointimal lesion development is a chronic inflammatory process that involves excessive cell proliferation and migration within the artery wall. Progression through the mammalian cell cycle requires the sequential activation of holoenzymes composed of a catalytic cyclin-dependent protein kinase (CDK) and a regulatory subunit named cyclin. Members of the family of CDK inhibitory proteins (CKIs) interact with and inhibit the activity of CDK/cyclins. Cell migration occurs predominantly at the G1/S phase of the cell cycle, and both CDKs and CKIs are among the molecular machines that coordinately regulate the cycling events that control cell proliferation and locomotion. The purpose of this review is to discuss the role of CDK/cyclins and CKIs in the regulation of vascular cell proliferation and migration and in the control of neointimal thickening. Pharmacological and gene therapy strategies targeting these cell cycle regulators for the treatment of cardiovascular disease will also be discussed.

KEYWORDS Vascular cell proliferation; Migration; CDK


   1. Introduction
Top
 Abstract
 1. Introduction
Acknowledgments
 References
 
Atherosclerosis is a multifactorial process that involves adaptative and innate immune mechanisms [1–4] (Fig. 1). Endothelial cell (EC) dysfunction induced by atherogenic stimuli is one of the earliest alterations at sites of predisposition to atherosclerosis. The damaged endothelium promotes the adhesion and transendothelial migration of circulating leukocytes. Early fatty streaks contain mostly highly proliferative macrophages that avidly uptake lipoproteins to become lipid-laden foam cells. Activated intimal leukocytes produce a plethora of inflammatory mediators that promote vascular smooth muscle cell (VSMC) proliferation and migration towards the atherosclerotic lesion, thus further contributing to atheroma growth [1,2,5,6]. Excessive cell proliferation and migration are also involved in the growth of vascular obstructive lesions during restenosis post-angioplasty, transplant vasculopathy, and graft atherosclerosis. Plaque rupture or erosion at advanced disease stages can lead to acute occlusion due to thrombus formation, resulting in myocardial infarction or stroke.


Figure 1
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  		Fig. 1 Mechanisms involved in atherothrombosis. Based on the response-to-injury hypothesis proposed by Ross [1], the cartoon illustrates different stages of atheroma progression, from a "healthy" artery wall (far left) to an advanced plaque (far right). Several atherogenic stimuli (i.e., hypercholesterolemia, hypertension, diabetes, smoking) trigger the recruitment and activation of circulating leukocytes, which in turn promote the accumulation of SMCs within the growing atheroma. Plaque rupture or erosion can lead to thrombus formation and acute ischemic events.

 
While several proliferation markers are expressed in human neointimal lesions [7–16], the magnitude of proliferation during human atherosclerosis and restenosis has been controversial, with some studies reporting very low proliferative rates [8,9,11,13,15] and others reporting abundance of proliferation [10,17]. Factors that may contribute to these differences include technical issues (e.g., differences in tissue fixatives, antigen accessibility, analysis of different proliferation markers), differences in the arteries being analyzed (e.g., peripheral versus coronary and carotid arteries, primary atheroma versus restenotic lesion), and variance in lesion stage at the time of tissue harvesting. Indeed, cell proliferation appeared more pronounced in restenotic versus primary lesions [11,17,18], and primary VSMCs obtained from human advanced primary stenosing displayed fewer proliferation than cells from fresh restenosing lesions [19], suggesting that cell proliferation is maximal at early stages of neointimal thickening. Consistent with this interpretation, atheroma size and cellular proliferation within the atheromatous plaque of hyperlipidemic rabbits are inversely correlated [20–22], and experimental angioplasty is characterized by the reestablishment of the quiescent phenotype after the initial proliferative burst [5,23].

The mammalian cell cycle is controlled by holoenzymes composed of a catalytic cyclin-dependent protein kinase (CDK) and a regulatory cyclin [24]. Different CDK/cyclin complexes are orderly activated at specific phases of the cell cycle (Fig. 2). CDK/cyclin-dependent hyperphosphorylation of the retinoblastoma protein (pRb) and the related pocket proteins p107 and p130 from mid G1 to mitosis contributes to the transactivation of genes with functional E2F-binding sites, including growth and cell-cycle regulators (i.e., c-myc, pRb, cdc2, cyclin E, cyclin A), and genes encoding proteins required for nucleotide and DNA biosynthesis (i.e., DNA polymerase {alpha}, histone H2A, proliferating cell nuclear antigen, thymidine kinase) [25]. The identities of substrates of the yeast CDK1 (CDC2) have revealed that this enzyme employs a global regulatory strategy involving phosphorylation of other regulatory molecules as well as phosphorylation of the molecular machines that drive cell-cycle events [26].


Figure 2
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  		Fig. 2 Cell cycle control in mammalian cells. Activation of specific CDK/cyclin complexes drives progression through the cell cycle (CDK1=CDC2). CKIs interact with and inactivate CDK/cyclin holoenzymes.

 
Cyclin availability and phospo/dephosphorylation of CDKs and cyclins regulate CDK/cyclin activity. CDK activity is attenuated by the interaction with CDK inhibitory proteins (CKIs) of the Cip/Kip (for CDK interacting protein/Kinase inhibitory protein) and Ink4 (for inhibitor of CDK4) families [24] (Fig. 2). Cip/Kip proteins (p21Cip1, p27Kip1, p57Kip2) bind to and inhibit a wide spectrum of CDK/cyclin holoenzymes, while Ink4 proteins (p16Ink4a, p15Ink4b, p18Ink4c, p19Ink4d) are specific for cyclin D-associated CDKs. Mitogenic and antimitogenic stimuli affect the rates of CKI synthesis and degradation, as well as their redistribution among different CDK/cyclin heterodimers.

2. Control of vascular cell proliferation and neointimal lesion growth by CDKs and CKIs
VSMC proliferation in the balloon-injured rat carotid artery is associated with a temporally and spatially coordinated expression and activation of CDKs and cyclins [16,27,28]. CDKs and cyclins are also expressed in human VSMCs within atherosclerotic and restenotic tissue [10,16,29], suggesting that assembly of functional CDK/cyclin holoenzymes in the injured arterial wall is a hallmark of vascular proliferative disease.

Numerous studies have implicated p27Kip1 and p21Cip1 in the control of vascular cell proliferation in vitro and neointimal thickening in vivo. For example, interleukin-1β-induced p27Kip1 and p21Cip1 downregulation may stimulate CDKs and promote neointimal VSMC proliferation [30]. In contrast, salicylate-dependent inhibition of PDGF-induced VSMC growth correlated with p27Kip1 and p21Cip1 accumulation [31]. Likewise, beraprost sodium-dependent VSMC growth arrest and reduction of intimal thickening in the balloon-injured dog coronary artery correlated with high p27Kip1 expression [32], and p21Cip1 induction was associated with tranilast-dependent inhibition of CDK2 and CDK4 activities, VSMC growth arrest in vitro and reduced intimal hyperplasia in the rat balloon-injured carotid artery [33]. p27Kip1 and p21Cip1 upregulation may contribute to VSMC growth arrest induced by non-steroidal anti-inflammatory drugs [34], nitric oxide donors [35] and gene transfer of endothelial nitric oxide synthase [36]. Simvastatin-dependent inhibition of CDK2 activity and VSMC proliferation correlates with prevention of Rho GTPase-induced downregulation of p27Kip1 without changes in p21Cip1, p16Ink4a, or p53 levels [37]. p27Kip1 may also mediate protein kinase C{delta}-mediated S-phase arrest in capillary ECs [38], and CDK2 inhibition and growth arrest in contact-inhibited ECs [39]. In vitro exposure of VSMCs to carbon monoxide (CO) transiently increases p21Cip1 expression and induces growth arrest, and treatment of rats with CO suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury [40]. However, although p21Cip1 was essential for CO-dependent VSMC growth arrest in vitro, the therapeutic effect of CO in a mouse model of mechanical arterial injury was not impaired in p21Cip1-null mice [40].

Hemodynamic forces appear to play an important role in atheroma development [1,2]. EC growth arrest induced by steady laminar stress correlated with p21Cip1 upregulation without changes in p27Kip1 protein levels [41]. Mattiussi et al. have shown that p21Cip1 gene transfer protects ECs from apoptosis induced by laminar shear stress in vitro, and significantly reduced in vivo apoptosis in ECs of ischemic hindlimbs [42]. On the other hand, stretch-mediated inactivation of forkhead transcription factors and p27Kip1 downregulation in VSMCs was accompanied by activation of CDK2, pRb hyperphosphorylation and proliferation, demonstrating that the earliest cell cycle events in VSMCs can occur in a solely mechanosensitive fashion [43]. RhoA-dependent p27Kip1 downregulation, mediated in part via phosphatidylinositol-3-kinase, induces VSMC proliferation and may contribute to the enhanced vascular responsiveness associated to hypertension [44].

p21Cip1 and p27Kip1 accumulation at late time points after balloon angioplasty in rat and porcine arteries may limit neointimal hyperplasia after the initial proliferative wave [14,45,46]. Transluminal injury in the murine femoral artery induces a rapid apoptotic response and p27Kip1 downregulation in medial VSMCs followed by a gradual increase in cell proliferation that peaked at 2 weeks in both the media and neointima and decreased thereafter coinciding with p27Kip1 upregulation [47]. The following observations suggest a role for CKIs as regulators of human neointimal hyperplasia: (a) reduced p27Kip1 expression in primary atherosclerotic and restenotic lesions compared with aorta, internal mammary artery, and carotid artery thrombendarterectomy specimens [48]; (b) p21Cip1 upregulation in restenosis compared with primary lesions and other vascular regions [48]; (c) more frequent p27Kip1 and p21Cip1 expression within regions of coronary atheromas not undergoing proliferation [14]; (d) concordant expression of TGF-β receptors in virtually all cells positive for p27Kip1 within atherosclerotic plaques, suggesting that the anti-mitogenic action of TGF-β1 in these lesions may be mediated by p27Kip1 [29]; and (e) p53 and p21Cip1 coexpression in carotid atheromatous plaque cells that revealed lack of proliferation markers, suggesting that p21Cip1 induction may occur via p53-dependent transcriptional activation [49].

We have established a causal link between decreased p27Kip1 protein expression and atherogenesis in hypercholesterolemic apolipoprotein E (apoE)-null mice by demonstrating that whole-body genetic inactivation of p27Kip1 increases arterial VSMC and macrophage proliferation and accelerates atherosclerosis [50]. We have also shown that selective inactivation of p27Kip1 in hematopoietic progenitor cells increases neointimal macrophage proliferation and is sufficient to accelerate atherosclerosis in fat-fed apoE-deficient mice [51], consistent with previous studies demonstrating enhanced haematopoietic progenitor cell proliferation upon p27Kip1 inactivation [52], and implicating p27Kip1 as a critical macrophage growth suppressor [53,54]. Because macrophages were the most abundant neointimal cells in our study [51], it seems reasonable to suggest that macrophage p27Kip1 safeguards against the inflammatory/proliferative response induced by dietary cholesterol in apoE-null mice. Regarding the consequences of CKI inactivation on neointimal thickening induced by vascular mechanical injury, a threefold increase in lesion size was observed in p21Cip1-null versus wild-type mice [40], but p27Kip1 genetic ablation did not affect lesion size [55]. Redundant roles between p21Cip1 and p27Kip1, or a compensatory increase in p21Cip1 expression (or other CKIs) might account for the lack of phenotype of p27Kip1-null mice in the setting of mechanical arterial injury.

Regional differences in the proliferative response of VSMCs have been established, both when comparing cells from different compartments of the same vessel and cells isolated from vessels from different vascular beds [56–62], and several studies suggest that distinct CKI regulation may contribute to establishing these regional dissimilarities. Sustained p27Kip1 expression in spite of growth stimuli may contribute to the resistance to growth of human VSMCs isolated from internal mammary artery compared with saphenous vein VSMCs, and to the longer patency of arterial versus venous grafts [61]. Likewise, distinct p15Ink4b and p27Kip1 expression correlated with different proliferative potential of intimal and medial VSMCs stimulated with basic fibroblast growth factor [62]. We have recently shown intrinsic differences in the regulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and p27Kip1 that may contribute to creating regional variability in the proliferative and migratory capacity of VSMCs from vascular beds with different atherogenicity [63]. Developmental differences in ERK activity also correlate with distinct proliferative and migratory capacity of VSMCs derived from the aortae of newborn versus adult rats [64].

Using primary cultures of human VSMCs, mitogen-induced proliferation was higher in cells derived from normal media (M-VSMCs), intermediate in cells from in-stent stenosis sites (ISS-VSMC), and lower in cells from primary atherosclerotic plaques (P-VSMC) [65]. Compared with M-VSMCs, P-VSMCs expressed high levels of p16Ink4a and p21Cip1, reduced p27Kip1 and cyclin E expression, and diminished pRb phosphorylation. ISS-VSMCs also displayed high levels of p21Cip1 and reduced p27Kip1 expression compared with M-VSMCs, although cyclin A and E expression was highest in ISS-VSMCs. Based on these findings, the authors have suggested that ISS-VSMCs derive from P-VSMCs driven to proliferate through cyclin E overexpression [65].

3. CDK inhibitory approaches to reduce neointimal thickening
Pharmacological (Table 1) and gene therapy (Table 2) CDK inhibitory strategies can limit experimental neointimal lesion growth. The relative inhibitory potency of the purine derivative CVT-313 varies from very high for CDK2, moderate for CDK1, and very low for CDK4 [66]. Flavopiridol (L86-8275) is a more potent CDK inhibitor that displays higher specificity towards CDK4 than towards CDK1 and CDK2 [67,68]. Flavopiridol- and CVT-313-dependent VSMC growth arrest correlates with decreased pRb hyperphosphorylation [66,69]. In the rat carotid model of balloon angioplasty, a brief intraluminal exposure to CVT-313 [66] or oral administration of flavopiridol for 5 days beginning at the day of injury [69] reduced neointima formation by 80% and 39%, respectively.


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  		Table 1 Pharmacological CDK inhibitors that limit neointimal hyperplasia in the rat carotid artery model of balloon angioplasty

 

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  		Table 2 CDK/cyclin inhibitory gene therapy strategies that limited experimental neointimal thickening

 
Antisense oligodeoxynucleotide (ODN) against CDK2 [27,70], CDC2 [27,71,72], and cyclin B1 [72] reduced neointimal thickening after balloon angioplasty. Likewise, cyclin G1 inactivation by retrovirus-mediated antisense gene transfer inhibited VSMC proliferation and neointima formation after balloon angioplasty [73], and antisense ODN to CDC2/PCNA [74] and CDK2 [75] attenuated graft atherosclerosis. Additional approaches based on the inactivation of positive cell cycle regulators that do not directly target CDK/cyclin activity (i.e., E2F, c-myc, etc.) have been reviewed elsewhere [6,76].

Gene transfer of p21Cip1 [77–79], p27Kip1 [45,80], and p57Kip2 [81] reduced neointima formation after angioplasty in normocholesterolemic animals. Likewise, p21Cip1 overexpression attenuated neointimal thickening after balloon injury in hypercholesterolemic mice [82] and following vein grafting [83]. Lamphere et al. [84] generated chimeric p16Ink4a and p27Kip1 molecules, which were of comparable potency to the parental p27Kip1 in inhibiting the activities of several CDKs in vitro. Among these chimeras, W9 was the most potent growth suppressor of human coronary artery VSMCs and ECs when compared to the parental p16Ink4a and p27Kip1, p27Kip1 derivatives, or several alternative p27Kip1–p16Ink4a chimeras. Importantly, W9 inhibited neointimal hyperplasia and artery occlusion when delivered to balloon-injured porcine arteries [85], and was more effective than the p27Kip1 and p16Ink4a parental genes in inhibiting neointimal thickening after balloon angioplasty in cholesterol-fed rabbits [86]. Further approaches based on the overexpression of growth suppressor that do not directly target CDK/cyclin activity (i.e., pRb, p53, Gax, etc.) are reviewed elsewhere [6,76].

4. Control of cell migration by CDKs and CKIs
Several cytostatic agents (e.g., quercetin, mimosine, suramin, rapamycin, and troglitazone) can reduce the migratory potential of VSMCs and tumour cells [87–92]. Likewise, 17β-estradiol and the transcription factors p53, AP-1 and c-myc regulate in a coordinated manner the proliferative and migratory potential of ECs and VSMCs [93–96]. NBT-II rat bladder carcinoma cells synchronized in G1 migrated simultaneously upon FGF-1 stimulation, and cells arrested in G2/M did not respond to stimulation by this mitogen [97]. Moreover, maximal migration of PDGF-BB-stimulated VSMCs occurred in late G1 [98]. These studies indicate that the position in the cell cycle is a key determinant of a cell's competence for migration. Consistent with this notion, many genes involved in cell motility (including cytoskeletal reorganization genes and extracellular matrix remodelling genes) exhibit cell cycle-dependent regulation in human fibroblasts [99]. Moreover, disruption of the actin cytoskeleton in murine Swiss 3T3 fibroblasts leads to inhibition of mitogen-induced cyclin E expression, CDK2 phosphorylation, and nuclear accumulation of the pRb-related p107 protein, further establishing a link between the cell cycle and locomotion machineries [100].

Overexpression of p27Kip1, p16INK4a and p21Cip1 inhibits cell spreading and migration in human umbilical vein ECs, CS-1 β3 melanoma cells, VSMCs and NIH-3T3 fibroblasts [63,101–103]. Moreover, p27Kip1-null VSMCs were more resistant than wild-type cells to the antimigratory properties of rapamycin [104]. Regarding the role of CDKs on cell locomotion, CDK6 localized to the ruffling edge of spreading cells and suppressed p16INK4a-mediated inhibition of cell spreading [102]. By analyzing chimeric mice obtained by injection of CDK5-null embryonic stem cells into host blastocysts, Ohshima et al. [105] suggested that CDK5 is required for specific components of neuronal migration that are differentially required by different neuronal cell types and by even a single neuronal cell type at different developmental stages. Moreover, it has been suggested that CDK5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells [106]. Mechanisms that may contribute to CDK5-dependent control of cell migration include the regulation of cytoskeletal dynamics by p39/CDK5 complexes [107], and CDK5 phosphorylation of serine 732 of focal adhesion kinase (FAK) through regulation of a microtubule fork important for nuclear translocation [108,109].

In addition to their well-established role in regulating cell shape and locomotion, specific components of the extracellular matrix and integrins play an important role in the control of vascular and nonvascular cell proliferation [110,111]. Significant changes in matrix collagen content occur during neointimal lesion development [112–114]. Because polymerized collagen may mimic the scenario of a normal artery composed of quiescent VSMCs, and monomer collagen might resemble the extracellular matrix surrounding proliferating neointimal VSMCs, Koyama et al. [115] studied the growth properties of VSMCs cultured on monomer collagen fibers and on polymerized collagen. Mitogen-stimulated VSMCs grown on monomer collagen disclosed high proliferative activity, but underwent G1 arrest when seeded on polymerized collagen. This inhibitory effect of polymerized collagen appeared to be mediated by {alpha}2 integrins, and correlated with suppression of p70S6K and p27Kip1 upregulation (and to a lesser extent p21Cip1). Interestingly, the quiescent phenotype of nonadherent NRK fibroblasts correlated with an increased association of p27Kip1 and p21Cip1 to cyclin E-containing holoenzymes [116], and E-cadherin-dependent inhibition of nonadherent EMT/6 mouse mammary carcinoma cells involves upregulation of p27Kip1 [117]. Moreover, evidence has been presented suggesting that tension-dependent changes in cell shape and cytoskeletal structure play an important role in the control of EC proliferation via modulation of cell cycle regulatory factors, including p27Kip1 [118]. It is also noteworthy that antisense ODN-mediated p21Cip1 inactivation in VSMCs attenuated the production and secretion of the ECM proteins fibronectin and laminin [119]. Further in vivo studies are required to elucidate possible links between various CKIs, the maintenance of cell shape, and dynamic modulation of cell–substrate interactions within the artery wall.

In view of the molecular connections between cell proliferation and migration, we have investigated whether the dual function of p27Kip1 as a cell-cycle and migration inhibitor is achieved via common or independent molecular pathways [120]. Physiologically high level of p27Kip1 expression inhibited CDK activity and attenuated both proliferation and migration in VSMC and fibroblast cultures, and mutations that rendered p27Kip1 unable to abrogate CDK activity also prevented p27Kip1-induced growth arrest and migration blockade. Moreover, a constitutively active mutant of pRb insensitive to CDK-dependent hyperphosphorylation inhibited both cell proliferation and migration, and pRb inactivation by forced expression of the adenoviral oncogene E1A correlated with high proliferative and migratory activity. Thus, cellular proliferation and migration appear to be coordinately regulated by the p27Kip1/CDK/pRb/E2F pathway (Fig. 3). Consistent with this notion, E2F-1-null keratinocytes exhibited delay in transit through both G1 and S phases of the cell cycle and substantially impaired migration [121]. Future studies are necessary to identify E2F-regulated genes implicated in cell locomotion.


Figure 3
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  		Fig. 3 Coordinate control of cell proliferation and migration. In the presence of low level of CKIs, active CDK/cyclin holoenzymes trigger the hyperphosphorylation of pRb, release of E2F and high proliferative and migratory activity. In contrast, CDK/cyclin inactivation by high level of CKIs leads to the accumulation of hypophosphorylated pRb, sequestration of E2F and low proliferative and migratory activity. The schematic shows several CDK/cyclin inhibitory strategies that inhibited experimental neointimal thickening (see Tables 1 and 2Go).

 
Regarding the signal transduction pathways that contribute to coordinate regulation of cell growth and locomotion, higher proliferative and migratory capacities of phenotypically distinct VSMC subtypes (aorta versus femoral artery VSMC, or aorta VSMCs from newborn versus old rats) correlate with greater ERK activity [63,64]. We have shown that retrovirus-mediated constitutive activation of ERK diminished p27Kip1 expression and markedly augmented PDGF-BB-mediated VSMC proliferation and migration, and pharmacological ERK inhibition increased p27Kip1 expression and attenuated PDGF-BB-induced VSMC proliferation and migration [63]. Likewise, Zhan et al. [122] found that adenovirus-mediated overexpression of dominant negative mutated variants of ERK, p38 and c-Jun N terminal kinase (JNK) inhibited PDGF-BB-stimulated VSMC proliferation and migration. These authors also showed that ERK and JNK inactivation suppressed PDGF-BB-induced downregulation of p27Kip1, whereas p38 blockade decreased PDGF-BB-dependent upregulation of p21Cip1.

5. Concluding remarks
Excessive cell proliferation and migration contribute to neointimal thickening, and CDKs, cyclins and CKIs are key regulators of these processes in vitro. Moreover, changes in the expression and/or activity of these cell cycle regulators have been documented in several animal models of vascular proliferative disease and in human atherosclerotic and restenotic tissue, and CDK/cyclin inhibition limited experimental neointimal thickening. Regarding the potential therapeutic application of these strategies to prevent human vascular obstructive disease, several issues need to be considered in the risk–benefit analysis of this approach. For instance, the prevention of cell proliferation and migration can be accompanied by VSMC an EC apoptosis leading to increased dysfunction of the vessel and possible predisposition to rupture of advanced atherosclerotic plaques. Of note in this regard, the anti-proliferative effect of the CDK inhibitor roscovitine is more potent in cells derived from human in-stent stenosis sites than in medial VSMCs [65]. Thus, studies to assess the therapeutic efficacy of roscovitine to limit in-stent stenosis are warranted. Given the emerging evidence suggesting the contribution of progenitor cells to vascular lesion formation (reviewed in Refs. [123,124]), potential effects of cell cycle inhibition on the behaviour of circulating progenitor cells warrant investigation. For example, it appears important to ascertain whether cell cycle inhibiting strategies can effectively prevent the accumulation, proliferation and differentiation of progenitor cells to VSMC, thereby preventing increased neointimal formation, or whether such strategies will rather be detrimental by inhibiting endothelial progenitor cell adhesion, proliferation, neoangiogenesis and restoration of damaged tissue.

Because patients frequently exhibit advanced atherosclerotic plaques when first diagnosed and cell proliferation is likely to peak at the early stages of human atheroma development, the potential benefit of antiproliferative strategies for the treatment of human atherosclerosis is uncertain. Indeed, the antiproliferative approaches used so far in the setting of vascular obstructive disease have focused on restenosis and graft atherosclerosis, during which neointimal hyperplasia is spatially localized and develops over a short period of time (typically 2–12 months). Synthetic CDK inhibitors (CVT-313, flavopiridol), antisense strategies targeting CDK/cyclins, and CKI overexpression reduced neointimal hyperplasia after experimental graft atherosclerosis and angioplasty, although the therapeutic efficacy of these CDK inhibitory strategies has not been yet assessed in clinic. In this regard, it is notable that several promising in vitro and animal study cell cycle inhibition approaches have not been successful to date in clinical trials. Factors that may contribute to these differences include inadequate study designs (i.e., incorrect doses, duration of treatment, or mode of application), inter-species heterogeneity, and phenotypic variability of VSMCs in different vascular beds.

Despite all the above drawbacks and potential detrimental side effects, antiproliferative approaches have shown promising results in preventing human neointimal thickening. The bacterial macrolide rapamycin (sirolimus, rapamune) is the pharmacological agent with which most experience has been gathered so far for the prevention of in-stent restenosis. Rapamycin is a potent immunosuppressant that strongly inhibits VSMC proliferation and migration via both p27Kip1-dependent and p27Kip1-independent mechanisms (reviewed in Refs. [125]). Rapamycin potently inhibited neointimal thickening in animal models of angioplasty, graft atherosclerosis, and diet-induced atherosclerosis [55,126–134], and recent clinical trials using rapamycin-impregnated stents have shown promising results for the prevention of neointimal proliferation, restenosis, and associated clinical events in patients undergoing coronary angioplasty [135–137]. Activation of E2F is triggered by CDK-dependent phosphorylation of pRb and related pocket proteins, therefore E2F inhibition may be a common mechanism by which different CDK/cyclin inhibitory strategies reduce neointimal thickening. E2F inactivation via transfection of synthetic ODN containing an E2F consensus binding site reduced hyperplasia after experimental balloon angioplasty and vein grafting [138,139], and application of this E2F ‘decoy’ strategy is safe and can achieve sequence-specific inhibition of cell-cycle gene expression and DNA replication in patients receiving ortocoronary bypass [140,141]. Despite these encouraging results, significant effort in basic research is warranted to identify additional target genes and strategies for the treatment of cardiovascular disease.


   Acknowledgments
Top
 Abstract
 1. Introduction
 Acknowledgments
References
 
I apologize to colleagues whose work has not been directly cited due to space limitations. I thank María J. Andrés-Manzano for preparing the figures. Work supported by grants from the Spanish Ministry of Science and Technology and Fondo Europeo de Desarrollo Regional (SAF2001-2358, SAF2002-1443), and from Instituto de Salud Carlos III (Red de Centros C03/01).


   Notes
 
Time for primary review 34 days


   References
Top
 Abstract
 1. Introduction
 Acknowledgments
 References
 

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