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Marisol Gascón-Irún, Silvia M. Sanz-González and Vicente Andrés
Laboratory of Vascular Biology, Department of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Spanish Council for Scientific Research (CSIC), Valencia, Spain.
Excessive cellular proliferation is thought to contribute to the pathogenesis of several forms of cardiovascular disease (e. g., atherosclerosis, restenosis after angioplasty, and vessel bypass graft failure). Therefore, candidate targets for the treatment of these disorders include cell cycle regulatory factors, such as cyclin-dependent kinases (CDKs), cyclins, CDK inhibitory proteins (CKIs), tumor suppressors, growth factors and their receptors, and transcription factors. Importantly, animal models of atherosclerosis have demonstrated an inverse correlation between neointimal cell proliferation and atheroma size, suggesting that excessive cell growth prevails at the onset of atherogenesis. Cell growth may also predominate at the onset of human atherosclerosis. Thus, given that affected humans often exhibit advanced atherosclerotic plaques when first diagnosed, the potential benefit of antiproliferative strategies for the treatment of atherosclerosis in clinic is doubtful.
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). Vascular interventions, both endovascular and open surgical, allow minimally invasive, easily monitored gene delivery. Thus, gene therapy strategy are emerging as an attractive approach for the treatment of vascular proliferative disease. In this review, we will discuss the use of gene therapy strategies against cellular proliferation in animal models and clinical trials of cardiovascular disease.
KEY WORDS: atherosclerosis, restenosis, bypass graft failure, cell cycle, gene therapy
LIST OF ABBREVIATIONS: apoE, apolipoprotein E; AP-1, activator protein-1; BrdU, 5-bromodeoxyuridine; CDK, cyclin-dependent kinase; CKI, CDK inhibitory protein; EC, endothelial cell; ERK, extracellular signal-regulated kinase; IVUS, intravascular ultrasound; JNK, c-jun NH2-terminal protein kinase; MAPK, mitogen-activated protein kinase; ODN, oligodeoxynucleotide; PCNA, proliferating cell nuclear antigen; PDGF, platelet-derived growth factor; pRb, retinoblastoma protein; PTCA, percutaneous transluminal angioplasty; SAPK, stress-activated protein kinase; TGF-transforming growth factor-; VSMC, vascular smooth muscle cell.
II. Preclinical studies
IIA. Antisense approach
IIA1. CDKs and cyclins
IIA2. Mitogen-responsive nuclear factors that promote cell growth
IIC. Transcription factor ‘decoy’ strategies
IID. Overexpression of growth suppressors
IIE. Overexpression of transdominant negative mutants of positive cell cycle regulators.
III. Clinical studies
IIIA. E2F ‘decoy’
IIIB. c-myc antisense ODN
Large-scale clinical trials conducted over the last decades have allowed the identification of independent risk factors that increase the prevalence and severity of atherosclerosis (e. g., hypercholesterolemia, hypertension, smoking). Cardiovascular risk factors initiate and perpetuate an inflammatory response within the injured arterial wall that promotes the development of atherosclerotic plaques (Dzau et al., 2002; Lusis, 2000; Ross, 1999; Steinberg, 2002) (Fig. 1). Chemokines and cytokines secreted by leukocytes that accumulate within the injured arterial wall promote their own proliferation, as well as the growth and migration of the underlying vascular smooth muscle cells (VSMCs) (Fig. 2). This inflammatory response also plays a critical role during restenosis after angioplasty and graft atherosclerosis. Thus, understanding the molecular mechanisms that control hyperplastic growth of vascular cells should help develop novel therapeutic strategies for the treatment of vascular obstructive disease.
Although arterial cell proliferation occurs in animal models during all phases of atherogenesis (Cortés et al., 2002; Díez-Juan and Andrés, 2001; Ross, 1999), studies with hyperlipidemic rabbits have shown an inverse correlation between atheroma size and cellular proliferation within the atheromatous plaque (McMillan and Stary, 1968; Rosenfeld and Ross, 1990; Spraragen et al., 1962). Experimental angioplasty is also characterized by abundant proliferation of VSMCs, followed by the reestablishment of the quiescent phenotype, typically within 2-4 weeks (Andrés, 1998; Bauters and Isner, 1997; Libby and Tanaka, 1997). These animal studies suggest that vascular cell proliferation prevails at the onset of atherogenesis and restenosis.
Expression of proliferation markers in human primary atheromatous plaques and restenotic lesions has been well documented (Burrig, 1991; Essed et al., 1983; Gordon et al., 1990; Katsuda et al., 1993; Kearney et al., 1997; Nobuyoshi et al., 1991; O'Brien et al., 1993; O'Brien et al., 2000; Orekhov et al., 1998; Rekhter and Gordon, 1995; Tanner et al., 1998; Veinot et al., 1998; Wei et al., 1997). However, controversy exists regarding the magnitude of the proliferative response, ranging from a very low index of cell proliferation (Gordon et al., 1990; Katsuda et al., 1993; O'Brien et al., 1993; O'Brien et al., 2000; Rekhter and Gordon, 1995; Veinot et al., 1998) to abundance of dividing cells (Essed et al., 1983; Kearney et al., 1997; Nobuyoshi et al., 1991; Pickering et al., 1993). Aside from methodological issues (e. g., differences in the fixatives used for tissue preservation, antigen accessibility, diversity of proliferation markers analyzed in these studies), some of the reported variance with regard to the issue of cell proliferation might relate to differences in the arteries being analyzed (i. e., peripheral, coronary and carotid arteries) and variance in the stage of atherogenesis at the time of tissue harvesting (Isner, 1994).
The cell types that undergo cell proliferation within human atherosclerotic tissue include VSMCs, leukocytes and endothelial cells (ECs) (Burrig, 1991; Gordon et al., 1990; Katsuda et al., 1993; O'Brien et al., 1993; Orekhov et al., 1998; Rekhter and Gordon, 1995; Veinot et al., 1998). Histological examination in 20 patients undergoing antemorten coronary angioplasty revealed that the extent of intimal proliferation was significantly greater in lesions with evidence of medial or adventitial tears than in lesions with no or only intimal tears (Nobuyoshi et al., 1991). Human carotid artery primary atherosclerotic tissue retrieved by endarterectomy surgery displayed greater proliferative activity in the intimal lesion versus the underlying media (Rekhter and Gordon, 1995). Moreover, monocyte/macrophage proliferation predominated in the intima (46% versus 9.7% -actin immunoreactive VSMCs, 14.3% ECs, 13.1% T lymphocytes), whereas VSMC proliferation prevailed in the media (44.4% versus 20% ECs, 13.0% monocyte/macrophages, and 14.3% T lymphocytes). It is also noteworthy that cell proliferation in human peripheral and coronary ateries is greater in restenotic versus primary lesions (O'Brien et al., 1993; O'Brien et al., 2000; Pickering et al., 1993). Furthermore, cultured VSMCs from human advanced primary stenosing disclosed lower proliferative capacity than cells from fresh restenosing lesions (Dartsch et al., 1990). Thus, similar to the situation in animal models, proliferation during human atherosclerosis and restenosis might peak at the onset of these pathologies and then progressively decline.
Cell cycle progression is controlled by several cyclin-dependent kinases (CDKs) that associate with regulatory cyclins (Morgan, 1995) (Fig. 3). Active CDK/cyclin holoenzymes hyperphosphorylate the retinoblastoma protein (pRb) and the related pocket proteins p107 and p130 from mid G1 to mitosis. Phosphorylation of pRb and related pocket proteins contributes to the transactivation of genes with functional E2F-binding sites, including several growth and cell-cycle regulators (i.e., c-myc, pRb, cdc2, cyclin E, cyclin A), and genes encoding proteins that are required for nucleotide and DNA biosynthesis (i. e., DNA polymerase , histone H2A, proliferating cell nuclear antigen, thymidine kinase) (Dyson, 1998; Lavia and Jansen-Durr, 1999; Stevaux and Dyson, 2002). Interaction of CDK/cyclins with CDK inhibitory proteins (CKIs) attenuates CDK activity and promotes growth arrest (Philipp-Staheli et al., 2001). CKIs of the Cip/Kip family (p21Cip1, p27Kip1 and p57Kip2) bind to and inhibit a wide spectrum of CDK/cyclin holoenzymes, while members of the Ink4 family (p16Ink4a, p15Ink4b, p18Ink4c, p19Ink4d) are specific for cyclin D-associated CDKs. Mitogenic and antimitogenic stimuli affect the rates of synthesis and degradation of CKIs, as well as their redistribution among different CDK/cyclin pairs (Philipp-Staheli et al., 2001). For example, p27Kip1 promotes the assembly of CDK4/cyclin D complexes by binding to them, thus facilitating CDK2/cyclin E activation through G1/S phase.
VSMC proliferation in the balloon-injured rat carotid artery is associated with a temporally and spatially coordinated expression of CDKs and cyclins (Braun-Dullaeus et al., 2001; Wei et al., 1997). Importantly, augmented expression of these factors is associated with an increase in their kinase activity (Abe et al., 1994; Wei et al., 1997), demonstrating the assembly of functional CDK/cyclin holoenzymes in the injured arterial wall. Expression of CDK2 and cyclin E was also detected in human VSMCs within atherosclerotic and restenotic tissue (Ihling et al., 1999; Kearney et al., 1997; Wei et al., 1997), suggesting that induction of positive cell-cycle control genes is a hallmark of vascular proliferative disease in human patients.
In the following sections, we will discuss the use of gene therapy strategies targeting cellular proliferation in preclinical (Table 1) and clinical studies (Table 2) related to cardiovascular disease.
II. Preclinical studies
Antiproliferative gene therapy strategies designed for the treatment of experimental cardiovascular disease include the following: 1) inactivation of positive cell cycle regulators (e. g., CDK/cyclins, protooncogenes, E2F, growth factors) by antisense approaches, ribozymes, and transcription factor ‘decoy’ strategies (Fig. 4), 2) overexpression of negative regulators of cell growth (e. g., CKIs, p53, pRb, GAX, and GATA-6), and 3) overexpression of transdominant negative mutants of positive cell cycle regulators (e. g., Ras, mitogen-activated protein kinases).
IIA. Antisense approach
The gene of interest is inactivated by using a synthetic antisense oligodeoxynucleotide (ODN) that hybridizes in a complementary fashion and stoicheometrically with the target mRNA.
IIA1. CDKs and cyclins
The efficacy of antisense ODN strategies targeting CDKs and cyclins to reduce neointimal lesion formation has been demonstrated in several animal models of balloon angioplasty. These studies include antisense oligodeoxynucleotides against CDK2 (Abe et al., 1994; Morishita et al., 1994a), CDC2 (Abe et al., 1994; Morishita et al., 1993; Morishita et al., 1994b) and cyclin B1 (Morishita et al., 1994b). Interestingly, cotransfection of antisense ODN against CDC2 kinase and cyclin B1 resulted in further inhibition of neointima formation, as compared to blockade of either gene target alone (Morishita et al., 1994b). Of note, Morishita et al. reported sustained inhibition of neointima formation in the rat carotid balloon-injury model after a single intraluminal molecular delivery of combined CDC2 and proliferating cell nuclear antigen (PCNA) antisense ODNs (Morishita et al., 1993), whereas this approach had no effect in the coronary arteries of pigs after balloon angioplasty (Robinson et al., 1997). Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibited VSMC proliferation and neointima formation after balloon angioplasty (Zhu et al., 1997). Attenuated graft atherosclerosis has been also observed upon inactivation of CDC2/PCNA (Mann et al., 1995; Miniati et al., 2000 2000) and CDK2 (Suzuki et al., 1997) with antisense ODN.
IIA2. Mitogen-responsive nuclear factors that promote cell growth
Several “immediate-early” genes (e. g., c-fos, c-jun, c-myc, c-myb, egr-1) are induced in serum-stimulated VSMCs, and their overexpression can promote VSMC proliferation in vitro (Bennett et al., 1994b; Brown et al., 1992; Campan et al., 1992; Castellot et al., 1985; Gorski and Walsh, 1995; Kindy and Sonenshein, 1986; Reilly et al., 1989; Rothman et al., 1994). VSMCs cultured from atheromatous plaques present higher levels of c-myc mRNA than VSMCs from normal arteries (Parkes et al., 1991), and arterial injury induced the expression of several “immediate-early” genes (Lambert et al., 2001; Miano et al., 1990; Miano et al., 1993; Sylvester et al., 1998). Antisense ODNs against c-myc and c-myb reportedly inhibited in a sequence-specific manner both VSMC proliferation in vitro (Bennett et al., 1994a; Biro et al., 1993; Brown et al., 1992; Ebbecke et al., 1992; Gunn et al., 1997; Pukac et al., 1990; Shi et al., 1994a; Shi et al., 1993; Simons and Rosenberg, 1992), and neointima formation after angioplasty (Bennett et al., 1994a; Gunn et al., 1997; Kipshidze et al., 2001; Kipshidze et al., 2002; Shi et al., 1994b; Simons et al., 1992) and vein grafting (Mannion et al., 1998) in vivo. However, these inhibitory effects may be mediated by a nonantisense mechanism (Burgess et al., 1995; Chavany et al., 1995; Guvakova et al., 1995; Villa et al., 1995; Wang et al., 1996).
It has been recently shown that nanospheres containing antisense ODN against PDGF receptor inhibit neointimal thickening in the rat carotid model of balloon angioplasty (Cohen-Sacks et al., 2002).
Ribozymes represent a unique class of RNA molecules that catalytically cleave the specific target RNA, thus resulting in targeted gene inactivation. Su et al. designed a DNA-RNA chimeric hammerhead ribozyme targeted to human transforming growth factor-1 (TGF-1) that significantly inhibited angiotensin II-stimulated TGF-1 mRNA and protein expression in human VSMCs, and efficiently inhibited the growth of these cells (Su et al., 2000). Likewise, cleavage of the platelet-derived growth factor (PDGF) A-chain mRNA by hammerhead ribozyme attenuated human and rat VSMC growth in vitro (Hu et al., 2001a; Hu et al., 2001b) and inhibited neointima formation in the rat carotid artery model of balloon injury (Kotani et al., 2003).
Studies using experimental models of angioplasty provided the first evidence that ribozymes might represent useful tools in cardiovascular therapy. Frimerman et al. reported the efficacy of chimeric hammerhead ribozyme to PCNA in reducing stent-induced stenosis in a porcine coronary model (Frimerman et al., 1999), and ribozyme strategy against TGF-1 inhibited neointimal formation after balloon injury in the rat carotid artery model (Yamamoto et al., 2000). 12-Lipoxygenase products of arachidonate metabolism have growth and chemotactic effects in vascular smooth muscle cells, and ribozyme against this enzyme prevents intimal hyperplasia in balloon-injured rat carotid arteries (Gu et al., 2001).
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