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 g per day IP; nϭ10) or vehicle (nϭ10) 4 and platelets. 5 Plasma leptin levels have also been correlated with cardiovascular complications in humans, an effect independent of body mass index and traditional risk factors. 6 -8 Nevertheless, the direct role of leptin in vascular disease remains controversial. Atherosclerotic mouse models with complete deficiency of leptin suggest that the absence of leptin might promote atherosclerosis. 9,10 However, these studies have been confounded by the extreme obesity and dyslipidemia that result from the loss of leptin-mediated central effects. 9,10 As a result, the direct effect of leptin on atherosclerosis has not yet been addressed. Therefore, to examine the direct role of leptin in atherosclerosis, we analyzed the effect of exogenous recombinant murine leptin on atherosclerosis using apolipoprotein E (apoE)-deficient mice.In addition, we tested the effects of chronic leptin therapy on thrombosis in these atherosclerotic-prone mice to determine whether potential metabolic improvements achieved with leptin therapy would outweigh the acute prothrombotic effect of leptin described previously. 11,12 Methods MiceMale apoE-deficient mice were purchased from the Jackson Laboratory (stock No. 002052; Bar Harbor, Maine) and provided Western chow (TD88137; Harlan Teklad) from 14 to 20 weeks of age. All procedures complied with the principles of laboratory and animal care established by the National Society for Medical Research and were approved by the University of Michigan committee on use and care of animals. Leptin TreatmentBeginning at 16 weeks of age, mice received 200 L intraperitoneal injections daily of either 125 g of recombinant murine leptin (R & D Systems) or vehicle control (nϭ10 per group). The dose of leptin chosen was based on a protocol used to achieve weight loss and fertility in leptin-deficient mice. 13 We determined that a 4-week duration of injections at this dose would be adequate to test the hypothesis that leptin promotes atherosclerosis in this particular model of hyperlipidemia. 14 http://atvb.ahajournals.org/ Downloaded from esthesia (100 mg/kg). Mice were perfused with saline and fixed using formalin with a 25-gauge needle inserted into the left ventricle at a rate of 1 mL/min. After formalin fixation, the arterial tree was meticulously dissected from the carcass and placed in 70% ethanol for Ն72 hours. The surface area occupied by atherosclerosis was then quantitated at the thoracic aorta and major branches, including the brachiocephalic, carotid, and subclavian arteries, via Oil Red O staining and quantitative morphometry, as described previously. 15 The lesion area was calculated for the control and treat...

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.

The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity, and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin-10) in large vessel endothelial cells (EC) at levels as high as obtained with the CMV immediate-early promoter, while retaining EC-specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC-specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely 4 copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large vessel EC, while minimizing systemic effects.