Rapid Regression of Atherosclerosis Induced by Liver-Directed Gene Transfer of ApoE in ApoE-Deficient Mice

Abstract

Abstract-Apolipoprotein E (apoE) is a multifunctional protein synthesized by the liver and tissue macrophages.ApoE-deficient mice have severe hyperlipidemia and develop accelerated atherosclerosis on a chow diet. Both liver-derived and macrophage-derived apoEs have been shown to reduce plasma lipoprotein levels and slow the progression of atherosclerosis in apoE-deficient mice, but regression of atherosclerosis has not been demonstrated in this model. We utilized second-generation adenoviruses to achieve hepatic expression of human apoE in chow-fed, apoE-deficient mice with established atherosclerotic lesions of different stages. As expected, hepatic expression of human apoE3 significantly reduced plasma cholesterol levels. Liver-derived apoE also accumulated substantially within preexisting atherosclerotic lesions, indicating that plasma apoE gained access to the arterial intima. Hepatic expression of human apoE3 for 6 weeks resulted in significant quantitative regression of both early fatty streak lesions as well as advanced, complex lesions in both the aortic root and the aortic arch. In addition, hepatic expression of apoE induced substantial morphological changes in lesions, including decreased foam cells and increased smooth muscle cells and extracellular matrix content. In parallel, human apoE4 and apoE2 were also expressed in the liver by using recombinant adenoviruses. ApoE4 reduced cholesterol levels to the same extent as did apoE3 and also prevented progression but did not induce significant regression of preexisting lesions. ApoE2 reduced cholesterol levels to a lesser degree than did apoE3 and apoE4 and lesion progression was reduced, but regression was not induced. In summary, (1) regression of preexisting atherosclerotic lesions in apoE-deficient mice can be rapidly induced by hepatic expression of apoE, despite the absence of macrophage-derived apoE; (2) the morphological changes seen in this model of regression resemble those in other animal models, induced over longer periods of time; (3) liver-derived apoE gained access to and was retained by intimal atherosclerotic lesions; and (4) apoE4 was less effective in inducing regression, despite its effects on plasma lipoproteins that were similar to those of apoE3. The rapid regression of preexisiting atherosclerotic lesions induced by apoE gene transfer in apoE-deficient mice could provide a convenient murine model for investigation of the molecular events associated with atherosclerosis regression.

Inhibition of atherosclerosis in apolipoprotein-E-deficient mice following muscle transduction with adeno-associated virus vectors encoding human apolipoprotein-E

“…17,37,38 The restoration of normal cholesterolaemic levels and protection against atherosclerosis has been achieved in apoE -/-animal models by the gene transfer of human apoE, using retroviral, adenoviral and plasmid expression vectors. 22,23,26,[39][40][41] As a platform for secretion of therapeutic proteins into the circulation, muscle has been used extensively in rAAVmediated gene transfer yielding sustained transgene expression in the absence of immune clearance of transduced cells. 29,[31][32][33][34][35] We have examined the feasibility of targeting muscle as a platform for the secretion of anti-atherogenic proteins by the transfection of mouse myoblast cultures with rAAV-based plasmid vectors containing apoA1 and LCAT cDNAs.…”

“…20,21 ApoE2 is less efficient at correcting the hyperlipidaemic phenotype compared with apoE3, due to reduced efficiency in mediating lipoprotein plasma clearance and liver uptake, but nevertheless has been shown to exhibit residual anti-atherogenic activity and retardation of atherosclerotic lesion development in apoE -/-mice, following liver-and muscle-directed gene transfer. 22,23 A major limitation of studies utilising rAd vectors to mediate apoE gene transfer has been the induction of adverse host immune and inflammatory responses, 24,25 which lead to only transient transduction and to rapid target cell elimination. For studies showing quantitative regression of atherosclerotic plaques, recourse to immunodeficient athymic apoE -/-mice was required.…”

“…In this respect, local effects of apoE in situ, at the atherosclerotic lesion site may be an important parameter in controlling atherosclerotic lesion progression, secondary to the lowering of plasma cholesterol levels. Notably, deposition of recombinant apoE3 within atherosclerotic lesions has been demonstrated following rAd-mediated liver transduction, 22 and associated with macrophages following bone marrow transplantation. 39 A defining stage in the development of atherosclerotic lesions that precedes the attachment of monocytes to the vessel wall, is the accumulation of remnant lipoproteins on the intimal extracellular matrix which occurs as early as 3 weeks of age in apoE -/-mice.…”

“…[10][11][12] The efficiency of this process is isoform-dependent with apoE3 being more efficient in forming ␥LpE particles, reflected by the much enhanced ability of ⑀3/⑀3 plasma samples to mobilise cell-derived cholesterol compared with ⑀2/⑀2 subjects. 11 A direct comparison of apoE2, apoE3 and apoE4 isoforms by adenoviral gene transfer into apoE -/-mice, demonstrated the impaired ability of apoE2 to clear VLDL, IDL and remnant lipoproteins from the circulation compared with apoE3 and apoE4, 22,40 due to reduced affinity for the LDL-R and LRP. 6,52,53 In addition to a humoral immune response against apoE2 and apoE3, antibodies directed against the virus capsid proteins VP1 and VP2 were present 4-6 weeks after vector administration.…”

“…The dissected aortas were stained with Oil-Red-O stain (Sigma) modified from previously described methods. 22,23 Briefly, the aortas were fixed in PBS/4% formaldehyde for 3 days, washed in PBS followed by staining with 1.8% OilRed-O in 60% isopropanol for 15 min at room temperature, then destained in 60% isopropanol for 5 min. The stained aortas were transferred to PBS for storage at 4°C.…”