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Abstract: Upregulation of plasminogen activator inhibitor type 1 (PAI-1) expression is a critical mechanism through which transforming growth factor-β1 (TGF-β1) accelerates intimal growth. The aim of this study was to identify signaling pathways through which TGF-β1 upregulates PAI-1 expression in endothelial cells (EC) and test interventions for blocking these pathways. We transduced cultured bovine EC with an adenoviral vector containing the PAI-1 promoter fused to a β-galactosidase reporter gene. We used these cells, along with vectors expressing potential modifiers of TGF-β1 signaling and pharmacologic antagonists of mitogen-activated protein kinase (MAPK) pathways to identify key mediators of basal and TGF-β1-regulated PAI-1 expression. Basal activity of the PAI-1 promoter was directly correlated with Ras activation and was blocked by a dominant negative (DN) type I TGF-β receptor. TGF-β1-stimulated activity of the PAI-1 promoter did not require Ras activation, and was lessened or eliminated by expression of either DN type I or type II TGF-β receptors and by inhibition of either of two MAPKs: MEK and p38. Our results suggest unanticipated pathways of TGF-β1 signaling in EC and point to new strategies to limit TGF-β1-induced vascular disease. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Data from clinical studies, cell culture, and animal models implicate the urokinase plasminogen activator (uPA)/uPA receptor (uPAR)/plasminogen system in the development of atherosclerosis and aneurysms. However, the mechanisms through which uPA/uPAR/plasminogen stimulate these diseases are not yet defined. We used genetically modified, atherosclerosis -prone mice, including mice with macrophage-specific uPA overexpression and mice genetically deficient in uPAR to elucidate mechanisms of uPA/uPAR/plasminogen-accelerated atherosclerosis and aneurysm formation. We found that macrophage-specific uPA overexpression accelerates atherosclerosis and causes aortic root dilation in fat-fed Ldlr-/- mice (as we previously reported in Apoe-/- mice). Macrophage-expressed uPA accelerates atherosclerosis by stimulation of lesion progression rather than initiation and causes disproportionate lipid accumulation in early lesions. uPA-accelerated atherosclerosis and aortic dilation are largely, if not completely, independent of uPAR. In the absence of uPA overexpression, however, uPAR contributes modestly to both atherosclerosis and aortic dilation. Microarray studies identified S100A8 and S100A9 mRNA as the most highly up-regulated transcripts in uPA-overexpressing macrophages; up- regulation of S100A9 protein in uPA-overexpressing macrophages was confirmed by Western blotting. S100A8/A9, which are atherogenic in mice and are expressed in human atherosclerotic plaques, are also up-regulated in the aortae of mice with uPA- overexpressing macrophages, and macrophage S100A9 mRNA is up-regulated by exposure of wild-type macrophages to medium from uPA-overexpressing macrophages. Macrophage microarray data suggest significant effects of uPA overexpression on cell migration and cell-matrix interactions. Our results confirm in a second animal model that macrophage- expressed uPA stimulates atherosclerosis and aortic dilation. They also reveal uPAR independence of these actions and implicate specific pathways in uPA/Plg-accelerated atherosclerosis and aneurysmal disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-β1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-β1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation. [ABSTRACT FROM AUTHOR]