June 2010 | Vol 7 | N.º 6 | CNIC-21 [PDF (687KB)]

Targeting cell cycle regulators in the diagnosis and treatment of atherosclerosis and restenosis: Emerging topics, current limitations and future directions

Carlos Silvestre¹, José Javier Fuster¹, Pedro Molina and Vicente Andrés. About the authors
Correspondence *Vicente Andrés, Centro Nacional de Investigaciones Cardiovasculares (CNIC), C/ Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Email vandres@cnic.es

Abstract
The recognition that abnormal cell proliferation is a key factor in the development of atherosclerosis and restenosis has provided proof-of-principle for the potential of cytostatic interventions in the treatment of these disorders. Indeed, drug-eluting stents (DES) that deliver anti-proliferative drugs locally have revolutionised interventional cardiology by significantly reducing in-stent restenosis and the need for target-vessel revascularisation. However, their use is limited by late thrombosis, which may be caused by delayed re-endothelialisation as a consequence of reduced endothelial cell proliferation, an unwanted side effect of DES. Strategies involving the use of cytostatic drugs to treat native atherosclerosis must also take into account the possible clinical complications associated with indiscriminate growth suppression within the artery wall. More work is therefore required both to improve existing therapeutic strategies and to develop new cytostatic approaches to achieve precise manipulation of the cell cycle in the vessel wall. Aside from its therapeutic applications, research on cell cycle regulatory factors may also give rise to genetic tools that could be used to identify patients who are at high risk for atherosclerosis and restenosis.

Introduction
Atherosclerosis and associated ischemic events are the leading cause of morbidity and mortality in industrialised countries. Human and animal studies have demonstrated that atherosclerosis is caused by a complex inflammatory process that is triggered by cardiovascular risk factors such as hyperlipidemia, diabetes, smoking and hypertension. Abnormal cell proliferation is a hallmark of the atherosclerotic plaque and plays a central role in the development of the disease. Percutaneous coronary intervention (PCI) to deploy stents is the procedure used most often for revascularising atherosclerotic vessels in patients who are at high risk of acute cardiovascular accidents owing to limited blood flow in the coronary arteries. However, a significant percentage of patients undergo recurrent arterial narrowing after stent implantation, known as in-stent restenosis (ISR). The main pathophysiological mechanism underlying ISR is the proliferation and migration of medial vascular smooth muscle cells (VSMCs) into the intima, which occurs in response to inflammation provoked by the mechanical damage due to stent implantation. Therefore, native atherosclerosis and ISR have many features in common, such as their obstructive and inflammatory nature and the central role of cell proliferation in both disorders.

In human atherosclerosis, excessive cell proliferation within the atheroma has been demonstrated by several studies that reported the expression of proliferation markers in atherosclerotic specimens.¹ However, the results regarding the degree of cell proliferation within the human atherosclerotic plaque are conflicting, with some studies finding very low proliferation rates^2-7 and others reporting high proliferative activity^8-10. This probably reflects the fact that cell proliferation is primarily a characteristic of the early stages of atherosclerosis and is strongly diminished in advanced plaques. In the case of restenosis, cell proliferation is clearly established as the main causative mechanism. Indeed, the recent introduction of drug-eluting stents (DES) that deliver cytostatic agents at the site of PCI have led to a significant reduction (as much as 80%) in ISR rates compared with traditional bare-metal stents (BMS).¹¹

Cell proliferation in mammals is tightly regulated by a large number of proteins that modulate the mitotic cell cycle. Progression through the cell cycle requires the sequential activation of holoenzymes composed of a catalytic cyclin-dependent kinase (CDK) and a regulatory subunit, the cyclin. The transition through the cell cycle is also controlled by CDK inhibitors (CKIs), which block CDK activity and negatively regulate cell cycle progression. Known CKIs include members of the Cip/Kip family (p21^Cip1, p27^Kip1 and p57^Kip2) and the Ink4 family (p15^Ink4b, p16^Ink4a, p18^Ink4c and p19^Ink4d). Cell cycle progression is also modulated by various transcriptional regulators, such as p53, members of the E2F/DP family, and the retinoblastoma protein (Rb). The central role of cell-cycle regulators in vascular occlusive diseases is supported by a number of animal studies (summarised in Table 1).

This review examines the potential of cell-cycle regulators as diagnostic markers or therapeutic targets for the prevention and treatment of atherosclerosis and ISR. We will discuss emerging topics, current limitations and future directions. The relevance of cell proliferation to graft atherosclerosis, a related vascular occlusive disease, has been discussed extensively elsewhere1 and will thus not be addressed here.have revolutionised interventional cardiology by significantly reducing in-stent restenosis and the need for target-vessel revascularisation. However, their use is limited by late thrombosis, which may be caused by delayed re-endothelialisation as a consequence of reduced endothelial cell proliferation, an unwanted side effect of DES. Strategies involving the use of cytostatic drugs to treat native atherosclerosis must also take into account the possible clinical complications associated with indiscriminate growth suppression within the artery wall. More work is therefore required both to improve existing therapeutic strategies and to develop new cytostatic approaches to achieve precise manipulation of the cell cycle in the vessel wall. Aside from its therapeutic applications, research on cell cycle regulatory factors may also give rise to genetic tools that could be used to identify patients who are at high risk for atherosclerosis and restenosis.

Cell proliferation regulators as genetic markers of vascular occlusive diseases
It is widely accepted that classic environmental risk factors only partly explain the development of atherosclerosis, and that genetic risk factors are critically involved in this pathology and its clinical manifestations. Recent studies have established links between genetic polymorphisms in negative regulators of cell proliferation and an increased risk of cardiovascular disease. The strongest susceptibility locus for coronary artery disease (CAD) and myocardial infarction (MI) in humans, which has been replicated by different groups, is located in an intergenic non-coding region on chromosome 9p21 next to the INK4/ARF locus (reviewed in ¹²), which plays important roles in cell proliferation, apoptosis and senescence.^13,14 Although some conflicting results have recently been reported,¹⁵ several studies have found altered expression of certain INK4/ARF transcripts in individuals carrying certain single nucleotide polymorphisms (SNPs) at 9p21 that are associated with an increased risk of atherosclerosis^16,17. These results provide a direct link between these atherosclerosis-associated SNPs and the INK4/ARF locus and suggest a key role for these genes in protection against the development of atherosclerosis. Consistent with this notion, cardiac expression of INK4/ARF transcripts is severely reduced upon deletion of the orthologous 70 kb non-coding interval on mouse chromosome 4. Tissue culture experiments reveal that this deletion increases proliferation and diminishes senescence in VSMCs, cellular phenotypes that might accelerate the progression of CAD¹⁹. Our studies in atherosclerosis-prone apolipoprotein E-null mice (apoE-KO) reveal that ARF deficiency reduces apoptosis in macrophages and VSMCs and aggravates atherogenesis, but does not affect plaque proliferative activity and senescence, possibly due to a compensatory upregulation of p16^INK4a.18 Additional work is therefore required to unravel the molecular mechanisms that link polymorphisms at chromosome 9p21 with INK4/ARF expression and to further delineate the role of this locus in CAD. Further underlining the importance of cell proliferation in atherogenesis, genetic studies in small cohorts of patients have identified MI-associated variants in other genes encoding cell-cycle inhibitors, such as CDKN1C (encoding p57^Kip2)20 and CKDKN1B (encoding p27^Kip1)21. Although additional studies with larger cohorts are needed to validate these genotype-disease associations, these findings raise the possibility of determining atherosclerotic risk in humans through genetic profiling.

Susceptibility to ISR is strongly influenced by several factors, mostly related to lesion characteristics and clinical conditions such as diabetes or previous ISR.^22,23 As in atherosclerosis, there is increasing evidence that genetic factors play an important role in this disease^24,25, and genetic studies could contribute to the assessment of ISR risk in the future. Because excessive cell proliferation is central to the aetiopathogenesis of restenosis, genetic analysis of cell cycle regulators may be a good strategy for identifying new markers of ISR. Remarkably, van Tiel and colleagues have recently identified an association between the same polymorphism in CDKN1B that is associated with atherosclerotic risk (-838A>C) and a decreased risk of ISR in patients receiving BMS²⁶. In contrast, two studies in patients treated with DES found no association between the risk of ISR and SNPs in CDKN1B, TP53 (encoding p53) or at the 9p21 locus^27,28. However, caution must be taken in the interpretation of these results because the use of DES that reduce neointimal lesion progression by inhibiting cell proliferation may affect the function of cell-cycle regulators, and therefore mask any association between these genetic variants and the risk of ISR. Our research group is actively engaged in studies aimed at identifying SNPs in cell-cycle regulatory genes that might affect an individual’s risk of ISR. We also plan to complement our genetic studies with functional studies into the molecular basis of genotype-phenotype associations. Our aim is to discover novel genetic markers that will allow individual stratification of ISR risk and therefore aid the interventional cardiologist in deciding on the optimal treatment before the intervention (e.g., to prescribe BMS and DES to patients at low and high risk of ISR, respectively). These studies may also identify novel therapeutic targets.

Cell proliferation regulators as therapeutic targets in vascular occlusive diseases
The important role of excessive cell proliferation and cell cycle regulatory genes in atherosclerosis suggests that antiproliferative therapies would be a useful strategy in the treatment of this disease^1,29. However, the clinical potential of this strategy has two important potential limitations. First, atherosclerotic disease usually remains asymptomatic for decades and is diagnosed only at an advanced stage, when there is little or no cell proliferation within the atheroma. This problem might be overcome in the near future, given the rapid development of noninvasive imaging techniques to identify subclinical (asymptomatic) atherosclerosis (reviewed in ³⁰). However, even where applicable, the indiscriminate blockade of cell proliferation in the arterial wall may lead to unwanted side-effects due to the complex cell biology of the atherogenic process. For instance, although limiting the proliferation of monocytes/macrophages within the atherosclerotic plaque will likely reduce plaque size, the inhibition of VSMC proliferation might result in a thinner fibrous cap and therefore in a more vulnerable plaque, thus increasing the risk of thrombosis and subsequent ischemic events. Moreover, many cell-cycle regulators also modulate other important cellular processes such as apoptotic cell death and cell senescence, which also influence the development of the atherosclerotic plaque. Therefore, special care must be taken when devising strategies to target these genes, and carefully designed, genetically-engineered models are required to identify suitable therapeutic targets and to assess the benefits and safety of anti-proliferative therapies.

In our laboratory, we are generating mouse models of experimental atherosclerosis that will allow the inactivation of cell-cycle regulatory genes of interest in a cell-type-specific manner. This approach takes advantage of the Cre recombinase of the P1 bacteriophage, which catalyses site-specific recombination of DNA between specific sequences (called LoxP sites) and is a widely-used tool in genetic engineering. Cre is especially useful for the generation of knockout animals because it allows the deletion of specific DNA segments flanked by LoxP sites (“floxed” sites)³¹ (Figure 1A).

We have interbred atherosclerosis-prone apoE-KO mice with various mouse strains that express Cre under the control of gene promoters whose activity is mostly restricted to myeloid cells (LysM-Cre mice), VSMCs (SM22-Cre mice) or endothelial cells (ECs) (Tie2-Cre mice); pilot experiments to confirm the cell-type-specificity of these promoters in atherosclerotic plaques in mice are underway. Our ultimate goal is to interbreed these apoE-KO/Cre lines with mice harbouring floxed versions of specific genes of interest (Figure 1B). We previously showed that the tumour suppressor p27 limits neointimal cell proliferation and protects against atherosclerosis in apoE-KO mice^33,34, and our initial goal is therefore to cross the apoE-KO/Cre lines with p27-floxed and p27-stop-floxed mice.³² The cross with p27-floxed mice should permit the generation of apoE-KO mice with p27 ablated specifically in VSMCs, ECs or macrophages, thereby promoting the proliferation of these cell types. In addition, p27-stop-floxed mice exhibit global inactivation of p27 due to the insertion of a floxed stop sequence within the p27 coding region. In these mice, restricted expression of Cre in VSMCs, ECs or macrophages will restore p27 expression exclusively in these cell types, thereby limiting their proliferative potential within the atheroma. This Cre/loxP-based approach can be complemented with studies using transgenic mice that overexpress cell cycle genes in a cell-type-specific manner. We expect that these mouse models will shed light on the molecular mechanisms by which deregulated cell cycle activity affects the size and composition of the atherosclerotic plaque and to identify genes that may differentially affect the proliferation of macrophages, VSMCs and ECs. This knowledge will provide insight into the potential therapeutic benefits and risks of cell type-specific changes in proliferation within the atheroma. In the future, this strategy will be extended to the analysis of other cell proliferation regulators and factors involved in the regulation of apoptosis.

Whereas antiproliferative therapies for atherosclerosis are a goal for the future, they are already one of the main treatments for the neointimal thickening associated with ISR. Thanks to its capacity to inhibit ISR, the introduction of DES has revolutionised interventional cardiology, replacing BMS as the main revascularisation procedure^22,23, and DES now accounts for 2 out of 3 implanted stents in Europe³⁵. However, despite initially promising results, several limitations of DES have become apparent. The most important shortcoming is an increased risk of late in-stent thrombosis, which is associated with high rates of MI and death thus enforcing the prolonged administration of anti-platelet drugs. Other limitations are its deleterious effects on endothelial function and suboptimal anti-restenotic action in high risk patients (individuals with complex lesions or diabetes)³⁶. Although the cause of in-stent thrombosis is not entirely clear, the main contributing factors appear to be a hypersensitive reaction to stent polymers and delayed vascular healing³⁷. This delay is thought to be caused by the non-selective action of the drugs delivered by DES, which block the proliferation and migration of not only VSMCs but also ECs. Inhibition of EC proliferation is thought to retard the re-endothelisation of the affected vessel and thereby increase the risk of thrombotic events.^36,37 Therefore, the development of cell-type specific therapies that inhibit VSMC proliferation without affecting endothelial integrity appears to be critical for the improvement of DES-based therapies. The design of such new antiproliferative therapies requires pre-clinical studies based on genetically-modified animals or gene therapy strategies aimed at cell-specific gene ablation or overexpression to evade the adverse side-effects associated with reduced EC proliferation. Our mouse models for Cre-mediated cell-specific gene ablation, depicted in Fig. 1, will allow us to study the effect of cell-type specific growth arrest on neointimal lesion formation in response to mechanical injury of the vessel wall. This will provide important insights into the potential adverse consequences of anti-proliferative therapies in the context of ISR. Similar approaches have been developed using VSMC- or EC-specific promoters to inactivate or overexpress proteins of interest in specific cell populations.^38-41 An alternative strategy is local gene delivery via viral vectors carrying target genes whose expression can be driven by a cell-specific promoter.^42,43 In conclusion, we expect to contribute to the understanding of the mechanisms underlying the side-effects of DES and to the discovery of new pharmacological agents that limit VSMC proliferation without affecting endothelial function, leading to safer methods for the reduction of restenosis.

CONCLUSIONS
Although the anti-restenosis benefits of DES that deliver cytostatic drugs are unquestionable, their use has been linked to late thrombosis, possibly triggered by reduced EC proliferation and delayed re-endothelialisation. Similarly, problems associated with indiscriminate growth suppression within the artery wall may limit the use of cytostatic approaches to treat native atherosclerosis. In order to improve the safety and efficacy of anti-proliferative treatments, additional work is needed to develop new strategies to precisely manipulate the cell cycle in the cell types that participate in vascular remodelling during atherosclerosis and restenosis (VSMCs, ECs and macrophages). In addition, recent evidence that SNPs in cell cycle genes or adjacent intergenic regions are associated with the risk of cardiovascular disease warrants further genetic studies to identify additional polymorphic variants in this family of genes as potential biomarkers of atherosclerosis and ISR. More work is also required to elucidate the molecular mechanisms that underlie genotype-phenotype associations in these diseases.

Acknowledgements
We thank Simon Bartlett for English editing and María J. Andrés-Manzano for art work. VA’s lab is funded by the Spanish Ministry of Science and Innovation (MICINN) and Fondo Europeo de Desarrollo Regional (FEDER) (grant SAF2007-62110), Instituto de Salud Carlos III (RECAVA grant RD06/0014/0021), Fundación Ramón Areces and Fina Biotech. J.J.F. and C.S. were supported by the CSIC-I3P predoctoral fellowship programme (cosponsored by the ERDF) and the Fundación Mario Losantos del Campo, respectively. The CNIC is supported by the MICINN and the Pro-CNIC Foundation.