Expression of apolipoprotein A-I in rabbit carotid endothelium protects against atherosclerosis.

Expression of atheroprotective genes in the blood vessel wall is potentially an effective means of preventing or reversing atherosclerosis. Development of this approach has been hampered by lack of a suitable gene-transfer vector. We used a helper-dependent adenoviral (HDAd) vector to test whether expression of apolipoprotein A-I (apoA-I) in the artery wall could retard the development of atherosclerosis in hyperlipidemic rabbits. Carotid arteries were infused with an HDAd expressing rabbit apoA-I or a "null" HDAd and harvested 2 and 4 weeks later. ApoA-I mRNA and protein were detected only in HDAdApoAI arteries. Lesion size, lipid and macrophage content, and adhesion molecule expression were similar in both groups at 2 weeks. Between 2 and 4 weeks, most of these measures of atherosclerosis increased in HDAdNull arteries, but were stable or decreased in HDAdApoAI arteries (P ≤ 0.04 for all end points in 4-week HDAdApoAI versus HDAdNull arteries). A longer-term study in chow-fed rabbits revealed persistence of HDAd vector DNA and apoA-I expression for ≥48 weeks, with stable vector DNA content and apoA-I expression from 4 to 48 weeks. Expression of apoA-I in arterial endothelium significantly retards atherosclerosis. HDAd provides prolonged, stable expression of a therapeutic transgene in the artery wall.

Helper-dependent adenoviral vectors are superior in vitro to first-generation vectors for endothelial cell-targeted gene therapy.

Arterial endothelial cells (EC) are attractive targets for gene therapy of atherosclerosis because they are accessible to hematogenous and catheter-based vector delivery and overlie atherosclerotic plaques. Vector-mediated expression-in EC-of proteins that mediate cholesterol transfer out of the artery wall and decrease inflammation could prevent and reverse atherosclerosis. However, clinical application of this strategy is limited by lack of a suitable gene-transfer vector. First-generation adenovirus (FGAd) is useful for EC gene transfer in proof-of-concept studies, but is unsuitable for atheroprotective human gene therapy because of limited duration of expression and proinflammatory effects. Moreover, others have reported detrimental effects of FGAd on critical aspects of EC physiology including proliferation, migration, and apoptosis. Here, we investigated whether helper-dependent adenovirus (HDAd) either alone or expressing an atheroprotective gene [apolipoprotein A-I (apoA-I)] could circumvent these limitations. In contrast to control FGAd, HDAd did not alter any of several critical EC physiologic functions (including proliferation, migration, apoptosis, metabolic activity, and nitric oxide (NO) production) and did not stimulate proinflammatory pathways [including expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and interleukin-6 (IL-6)]. Expression of apoA-I by HDAd reduced EC VCAM-1 expression. HDAd is a promising vector and apoA-I is a promising gene for atheroprotective human gene therapy delivered via EC.

Recombinant leptin promotes atherosclerosis and thrombosis in apolipoprotein E-deficient mice.

OBJECTIVE: The direct role of leptin in vascular disease remains controversial. The objective of this study was to examine the effects of leptin treatment on atherosclerosis and thrombosis in atherosclerotic-prone mice. METHODS AND RESULTS: Sixteen-week-old, male apolipoprotein E-deficient mice were treated with injections of recombinant leptin (125 microg per day IP; n=10) or vehicle (n=10) for 4 weeks. Leptin treatment resulted in reduced epididymal fat (352+/-30.7 versus 621+/-61.5 mg; P=0.005) and fasting insulin (0.57+/-0.25 versus 1.7+/-0.22 ng/mL; P=0.014). Despite these metabolic benefits, leptin treatment resulted in an increase in atherosclerosis (8.0+/-0.95% versus 5.4+/-0.59% lesion surface coverage; P<0.05). Leptin treatment also resulted in a shortened time to occlusive thrombosis after vascular injury (21+/-2.1 versus 34.6+/-5.4 minutes; P=0.045). CONCLUSIONS: These studies indicate that exogenous leptin promotes atherosclerosis and thrombosis and support the concept that elevations of leptin may increase the risk for cardiovascular disease.

Constriction of carotid arteries by urokinase-type plasminogen activator requires catalytic activity and is independent of NH(2)-terminal domains.

Urokinase-type plasminogen activator (uPA) is expressed at increased levels in stenotic, atherosclerotic human arteries. However, the biological roles of uPA in the artery wall are poorly understood. Previous studies associate uPA with both acute vasoconstriction and chronic vascular remodeling and attribute uPA-mediated vasoconstriction to the kringle - not the catalytic - domain of uPA. We used an in-vivo uPA overexpression model to test the hypothesis that uPA-induced vasoconstriction is a reversible vasomotor process that can be prevented - and uPA fibrinolytic activity preserved - by: 1) removing the growth factor and kringle domains; or 2) anchoring uPA to the endothelial surface. To test this hypothesis we constructed adenoviral vectors that express: wild-type rabbit uPA (AduPA); a uPA mutant lacking the NH(2)-terminal growth-factor and kringle domains (AduPAdel); a mutant lacking catalytic activity (AduPAS-->A), and a cell-surface anchored mutant (AdTMuPA). uPA mutants were expressed and characterised in vitro and in carotid arteries in vivo. uPAS-->A had no plasminogen activator activity. Activity was similar for uPA and uPAdel, whereas AdTMuPA had only cell-associated activity. AduPAS-->A arteries were not constricted. AduPA, AduPAdel, and AdTM-uPA arteries were constricted (approximately 30% smaller lumens; p< or =0.008 vs. AdNull arteries). Papaverine reversed constriction of AduPA arteries. uPA-mediated arterial constriction is a vasomotor process that is mediated by uPA catalytic activity, not by the NH(2)-terminal domains. Anchoring uPA to the endothelial surface does not prevent vasoconstriction. uPA catalytic activity, generated by artery wall cells, may contribute to lumen loss in human arteries. Elimination of uPA vasoconstrictor activity requires concomitant loss of fibrinolytic activity.