GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 81‘decoy’ ODN with a circular dumbbell structure (CD-E2F)and compared its properties with those of conventionalphosphorothioated E2F ‘decoy’ ODN (PS-E2F). CD-E2Fdisplayed more stability and stronger antiproliferativeactivity than PS-E2F when assayed in cultured VSMCs,and was more effective in inhibiting neointimal formationin vivo.2. Activator protein-1 (AP-1)Cell proliferation in the rat carotid artery model ofangioplasty correlated with elevated expression and highDNA-binding activity of transcription factors of the AP-1family (Miano et al, 1990; Miano et al, 1993; Hu et al,1997; Sylvester et al, 1998; Andrés et al, 2001). Underconditions of PDGF stimulation, AP-1 ‘decoy’ ODNdelivery into cultured human VSMCs significantlyreduced cell number and TGF-β1 production (Kume et al,2002), and attenuated neointimal thickening when appliedat the site of balloon angioplasty in rabbit carotid artery(Kume et al, 2002) and minipig coronary arteries(Buchwald et al, 2002). Circular dumbbell AP-1 ‘decoy’ODN was more effective in inhibiting the proliferation ofVSMCs in vitro and neointimal hyperplasia in vivocompared to conventional phosphorothioated AP-1 decoyODN, (Ahn et al, 2002b).D. Overexpression of growth suppressors1. CKIsThe efficacy of CKIs in inhibiting CDK activity andcell cycle progression has been widely documented in avariety of normal and tumour cells in vitro. The firstevidence that p21 Cip1 and p27 Kip1 may function as negativeregulators of neointimal hyperplasia was suggested inanimal studies showing the upregulation of these CKIs atlate time points following balloon angioplasty, coincidingwith the restoration of the quiescent phenotype after theinitial proliferative wave (Chen et al, 1997; Tanner et al,1998). The protective role of p27 Kip1 against neointimalthickening has been rigorously demonstrated inhypercholesterolemic apolipoprotein E (apoE)-deficientmice, in which genetic inactivation of p27 Kip1 acceleratedatherogenesis in a dose-dependent manner (Díez-Juan andAndrés, 2001). However, neointimal hyperplasia aftermechanical damage of the arterial wall was similar inwild-type and p27 Kip1 -null mice (Roque et al, 2001b).Redundant roles between p21 Cip1 and p27 Kip1 , orcompensatory increase in p21 Cip1 expression (or otherCKIs) might account for the lack of phenotype of p27 Kip1 -null mice in the setting of mechanical arterial injury.Several studies have suggested a role of CKIs inestablishing regional phenotypic variance in VSMCs fromdifferent vascular beds. Using human VSMCs isolatedfrom internal mammary artery and saphenous vein, Yanget al. (1998) suggested that sustained p27 Kip1 expression inspite of growth stimuli may contribute to the resistance togrowth of VSMCs from internal mammary artery and tothe longer patency of arterial versus venous grafts (Yanget al, 1998). Likewise, different expression of p15 Ink4b andp27 Kip1 has been correlated with distinct proliferativeresponse of intimal and medial VSMCs towards basicfibroblast growth factor (bFGF or FGF2) (Olson et al,2000). Intrinsic differences in the regulation of p27 Kip1might also play an important role in creating variance inthe proliferative and migratory capacity of VSMCsisolated from different vascular beds, which might in turncontribute to establishing regional variability inatherogenicity (Castro et al, 2003).Tanner et al (1998) have reported more frequentexpression of p27 Kip1 and p21 Cip1 within regions of humancoronary atheromas not undergoing proliferation.Concordant expression of TGF-β receptors I and II invirtually all cells positive for p27 Kip1 within humanatherosclerotic plaques indicates that TGF-β1 present inthese lesions may contribute to p27 Kip1 upregulation (Ihlinget al, 1999). Moreover, coexpression of p53 and p21 Cip1 inhuman carotid atheromatous plaque cells that revealedlack of proliferation markers suggests that induction ofp21 Cip1 may occur via transcriptional activation by p53(Ihling et al, 1997).Ectopic expression of p21 Cip1 and p27 Kip1 , but notp16 Ink4a , significantly reduced neointimal thickening inseveral animal models of angioplasty (Chang et al, 1995a;Yang et al, 1996; Chen et al, 1997; Ueno et al, 1997a;Tanner et al, 2000; Condorelli et al, 2001). Overexpressionof p21 Cip1 also attenuated neointimal lesion formation in arabbit model of vein grafting (Bai et al, 1998).2. p53p53 is a transcription factor that functions as a tumorsuppressor displaying both antiproliferative andproapoptotic actions. These effects result from complexregulatory networks, including transcriptional activation ofantiproliferative and proapoptotic genes (e. g., p21 Cip1 andBax, respectively), transcriptional repression ofproproliferative and antiapoptotic genes (e. g., IGF-II andbcl-2, respectively), and direct protein-protein interactions(e. g., with helicases and caspases). Increased VSMCproliferation has been shown as a result of antisense p53ODN transfection (Aoki et al, 1999; Matsushita et al,2000), and p53 gene transfer has the opposite effect(Yonemitsu et al, 1998). Mayr et al (2002) showed ahigher rate of proliferation and migration of VSMCsisolated from p53-deficient mice than its wild-typecounterparts. Consistent with these findings, earlymigration and proliferation of VSMCs happened inexplanted porcine tunica media tissue after mitogeninduceddownregulation of p53 (Rodriguez-Campos et al,2001).p53 deficiency has been demonstrated to have aproatherogenic effect in studies of genetic inactivation inhypercholesterolemic apoE and apoE*3-Leiden mice,although the relative contribution of increased cellularproliferation and decreased apoptosis in these animalmodels remains obscure (Guevara et al, 1999; van Vlijmenet al, 2001). Mice deficient for p53 also disclosedaccelerated vein graft atherosclerosis (Mayr et al, 2002).Regarding human atherosclerosis, p53 is overexpressedbut not mutated in human atherosclerotic tissue (Iacopetta81