| BACKGROUNDPostinjury intimal hyperplasia and vascular restenosis remain unsolved problems in the treatment of vascular disorders. A large body of evidence has demonstrated that the pathogenesis of intimal hyperplasia involves diverse signaling cascades that ultimately converge on vascular smooth muscle cells (SMCs), stimulating their phenotype modulation whereby they lose their differentiation markers and gain the abilities to proliferate, migrate and secret extracellular matrix (ECM). In recent years, many anti-proliferative agents, such as rapamycin, have been found to inhibit SMC proliferation and migration. The widely clinical utilization of these agents via drug-eluting stents has dramatically reduced restenosis rate. These agents, however, also inhibit the healing and reendothelialization of the injured artery. In-stent subacute and late thrombosis become major concerns after percutaneous transluminal intervention using drug-eluting stents. Therefore, it is desirable to develop new therapeutic agents that selectively inhibit SMCs proliferation and accelerate, at least do not delay reendothelialization.The cellular repressor of E1A-stimulated genes (CREG) associated with the mature SMC phenotype might be a potential candidate in this regard. CREG is a secreted glycoprotein which has been shown to antagonize transcription activation and cellular transformation induced by the adenovirus E1A oncoprotein. The human homolog of CREG contains 220 amino acid residues and 3 consensus N-glycosylation sites. When overexpressed in human embryonic carcinoma cells, CREG enhances cellular differentiation and inhibits cell cycle progression. Our previous studies have shown that CREG is upregulated at both mRNA and protein levels during the phenotypic conversion of cultured SMCs from a proliferative and synthetic state to a non-proliferative and differentiated state. Since SMC dedifferentiation and proliferation play a key role in the neointimal hyperplasia after acute arterial injury and since CREG has been shown to inhibit proliferation in cultured cells, we hypothesized that arterial wall injury might acutely downregulate CREG expression and that local CREG overexpression might attenuate neointimal hyperplasia.MATERIALS AND METHODSThe human full-length CREG cDNA was subcloned into the shuttle plasmid pshuttle-CMV at KpnI and XhoI restriction sites. Recombinant adenovirus was generated by bacterial homologous recombination between pshuttle-CMV containing CREG cDNAs and the viral backbone vector pAdEasy-1. Viral packaging was carried out in 293 cells as described.Human VSMC was infected by Ad-CREG and Ad-GFP, and then the expression of CREG and differentiation markers of VSMC such as SMα- actin and SM MHC was detected by immunohistochemistry and Western blot, proliferation marker Brud labeled cells was detected by immunohistochemistry.The left external carotid arteries of 60 Male New Zealand white rabbits were injured by a balloon catheter and inbubated with Ad-CREG, Ad-GFP or saline for 30 min. The rabbits were sacrificed with an overdose of pentobarbital sodium at 3, 7, 14, and 28 days (5 rabbits for each time point) after balloon injury. Angiography of bilateral CCA was performed before sacrifice. Injured and control arteries were harvested and immunorstained with anti-CREG, SM MHC,α-actin, BrdU, and conterstained with hematoxylin Homogenates of rabbit CCA were analyzed by Western blot analysisfor CREG, SMα-actin, SM myosin heavy chain (SM MHC), GFP, andβ-actin expression.RESULTSThe recombinant adenovirus containing CREG was successfully constructed. The extro-gene was found to be effectively transferred and expressed in the target cells. CREG transfection promotes cell differentiation and inhibits cell proliferation. CREG expression positively correlates with SMC differentiation status in normal and balloon injured rabbit arteries. Immunohistochemical and Western Blot detected high levels of CREG expression in the artery. However, the expression was greatly reduced in the media at 3 days after injury, which was accompanied by a reduction in SMα-actin and SM MHC, and by an increase in BrdU-labeled cells. After 7 days, the CREG expression gradually restored as SMCs began to redifferentiate. Four weeks after injury, high levels of CREG expression was observed not only in the vascular media but also in the endothelium and the newly formed neointima. These data strongly suggest that CREG expression positively correlates with the maturation state of SMCs in normal and injured arterial walls.Morphometric analysis of the injured artery revealed that Ad-CREG transduction reduced the neointima area by 50% as compared to Ad-GFP and saline controls (Ad-CREG vs saline or Ad-GFP controls, P<.01, n=5). The intima/media ratio of the Ad-CREG-transduced arteries was also decreased by more than 50% (Ad-CREG vs saline or Ad-GFP controls, P<.01, n=5). Quantitative angiography performed at 28 days after injury showed larger minimal lumen diameters in CREG-transduced arteries as compared to the GFP transduced or the saline control. In contrast, the diameter stenosis rate, which inversely correlates with the minimal lumen diameter, was lower in Ad-CREG transduced arteries (Ad-CREG vs saline or Ad-GFP, P <.01, n=5). These data indicate that local transfer of CREG gene significantly inhibits neointimal hyperplasia and prevents restenosis after balloon injury to the carotid artery.A significant reduction in BrdU-positive neointimal cells was noted in CREG transduced arteries at 3 and 7 days after balloon as compared to the GFP transduced and saline control arteries. Furthermore, CREG transfer was also observed to inhibit injury-induced reduction of SMα-actin and SM MHC. These results support the notion that CREG promotes a quiescent, differentiated SMC phenotype in the arterial wall.Immunohistochemistry for the endothelial marker CD31 often revealed a nonconsecutive linear staining on the surface of neointima at 14 days after balloon injury). There was no significant difference in the endothelialization rate, defined as percentage of the luminal surface covered by CD31-positive cells, between the Ad-CREG-transduced arteries and Ad-GFP/saline controls (58.4±5.2% vs62.3±6.7%/57.8±6.3%, p>.05). After 4 weeks, the luminal surface of injured artery was completely covered by CD31-positive endothelial cells in all three groups, suggesting that Ad-CREG transduction does not affect endothelial repair after vascular injury.CONCLUSIONThese data suggest that forced expression of CREG in the artery wall after acute vascular injury inhibits SMCs proliferation, induces cellular differentiation and attenuates neointimal hyperplasia. CREG delivery may have therapeutic potential for the prevention of restenosis after vascular angioplasty.
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