Search

Apolipoprotein E (apoE) is a 34 kDa glycoprotein with multiple actions that help protect against the development of atherosclerosis. Here, we have assessed the atheroprotective potential of an [E1(-), E3(-), polymerase(-)] adenovirus vector expressing human apoE, comparing intramuscular and intravenous (liver-directed) injections in hypercholesterolaemic apoE-deficient mice (apoE(-/-)). Intramuscular injections resulted in low expression of apoE and afforded no protection against atherogenesis. In contrast, 3 and 7 days after intravenous injections into young (6-8-week-old) apoE(-/-) mice, plasma levels of apoE were elevated and were accompanied by reductions in plasma cholesterol and normalization of lipoprotein profiles. Thereafter, plasma apoE was still detectable up to day 70, but gradually declined, although no humoral immune response was evoked, and there was a return to dyslipoproteinaemia. High levels of the vector genome were still present in livers of treated animals at 70 days, implying that decrease in apoE expression was due to cellular shutdown of the cytomegalovirus promoter. Importantly, liver-directed apoE gene transfer to these young mice retarded progression of atherosclerosis by 38% (treated, 8.21 +/- 1.05%; untreated, 13.26 +/- 0.98%, P < 0.05), during the 70 day study period. Moreover, when 10-month-old apoE(-/-) mice with advanced atherosclerosis were treated with the adenovirus vector, there was clear regression of aortic lesion area by 1 month [24.3 +/- 1.7% compared to 40.7 +/- 2.6% in baseline controls (P < 0.002)]. We conclude that the stability of the adenovirus vector genome in the livers of intravenously treated animals provides an ideal platform to evaluate liver-specific promoters for sustained transgene expression and control of atherosclerotic lesion pathology.

Apolipoprotein E (apoE) is a multifunctional plasma glycoprotein involved in lipoprotein metabolism and a range of cell signalling phenomena. ApoE-deficient (apoE(-/-)) mice exhibit severe hypercholesterolaemia and are an excellent model of human atherosclerosis. ApoE somatic gene transfer and bone marrow transplantation in apoE(-/-) mice results in reversal of hypercholesterolaemia, inhibition of atherogenesis and regression of atherosclerotic plaque density. Replication defective adeno-associated virus vectors (rAAVs) are an attractive system currently in clinical trial for muscle-based heterologous gene therapy to express secreted recombinant plasma proteins. Here we have applied rAAV transduction of skeletal muscle to express wild-type (epsilon3) and a defective receptor-binding mutant (epsilon2) human apoE transgene in apoE(-/-) mice. In treated animals, apoE mRNA was present in transduced muscles and, although plasma levels of recombinant apoE fell below the detection levels of our ELISA (ie <10 ng/ml), circulating antibodies to human apoE and rAAV were induced. Up to 3 months after a single administration of rAAV/apoE3, a significant reduction in atherosclerotic plaque density in aortas of treated animals was observed (approximately 30%), indicating that low-level rAAV-mediated apoE3 expression from skeletal muscle can retard atherosclerotic progression in this well-defined genetic model.

A human cell line THP-1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low-density lipoprotein (n-LDL), acetylated-low-density lipoprotein (Ac-LDL), oxidised-LDL, or 25-OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: 1) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labelled LDL to study lipoprotein uptake; 2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and 3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP-1 macrophages incubated with diI-labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam-cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foam-cells in vitro offers a way of studying their behaviour at the single cell level.

A new rabbit model of atherosclerosis is described in which several of the features seen in early human atherosclerosis are generated within a period of 7 days. The positioning of a hollow silastic collar around the carotid artery of a cholesterol-fed rabbit results in macrophage and smooth muscle cell infiltration into the arterial subendothelium, foam cell formation and the deposition of extracellular lipid. A time-dependent accumulation of extracellular cholesteryl ester occurs within the arterial wall. Each of these changes occurs in the presence of a morphologically intact endothelium as assessed using light microscopy, scanning and transmission electron microscopy. A high cholesterol diet did not affect the extent of proliferation but exacerbated cholesteryl ester accumulation. It is proposed that the changes induced by the collar may be mediated by obstruction of the adventitial vasa vasorum with the creation of a localised ischaemic region.