Hepatocyte-specific ABCA1 transfer increases HDL cholesterol but impairs HDL function and accelerates atherosclerosis.

AIMS: The ATP-binding cassette transporter A1 (ABCA1) lipidates apolipoprotein (apo) A-I. The hypothesis that hepatocyte-specific ABCA1 overexpression results in high-density lipoprotein (HDL) dysfunction was evaluated by comparing the effects of murine ABCA1 (AdABCA1) and human apo A-I (AdA-I) transfer on lipoprotein profile, HDL function, and progression of atherosclerosis. METHODS AND RESULTS: Gene transfer in male and female C57BL/6 apo E(-/-) mice was performed at the age of 3 months with E1E3E4-deleted adenoviral vectors containing hepatocyte-specific expression cassettes. Atherosclerosis was quantified at baseline and 56 days later in AdABCA1, AdA-I, and control mice. HDL cholesterol after AdA-I transfer was 1.7-fold (P < 0.001) and 1.8-fold (P < 0.001) higher in male and female mice, respectively, and potently inhibited atherosclerosis progression compared with respective controls. Notwithstanding a 1.4-fold (P < 0.01) and a 1.7-fold (P < 0.01) increase of HDL cholesterol in male and female mice, respectively, after AdABCA1 transfer, the intima was 2.2-fold (P < 0.001) larger in male and 1.3-fold (P = NS) larger in female mice compared with respective controls. HDL isolated from control and AdA-I mice but not from AdABCA1 mice enhanced endothelial progenitor cell (EPC) migration in vitro and reduced endothelial cell death in vitro after serum and growth factor withdrawal. Scavenger receptor class B type I (SR-BI) protein level in the liver was significantly lower in AdABCA1 mice than in control and AdA-I mice. CONCLUSION: Hepatocyte-specific ABCA1 transfer decreases SR-BI protein level in the liver and abrogates beneficial effects of HDL on EPCs and endothelial cells. Decreased HDL function may underlie accelerated atherosclerosis in AdABCA1 apo E(-/-)mice.

The relative atherogenicity of VLDL and LDL is dependent on the topographic site.

To evaluate whether the relative atherogenicity of VLDL and LDL is dependent on the topographic site, atherosclerosis was compared at four topographic sites in homozygous LDL receptor (LDLr)-deficient rabbits fed normal chow and in heterozygous LDLr-deficient rabbits with the same genetic background fed a 0.15% cholesterol diet to match cholesterol levels. VLDL cholesterol was significantly higher and LDL cholesterol significantly lower in LDLr(+/-) diet rabbits compared with LDLr(-/-) rabbits. Intimal area in the ascending thoracic aorta and in the abdominal aorta at the level of the renal arteries was 1.4-fold (P < 0.05) and 1.5-fold (P < 0.05) higher, respectively, in LDLr(-/-) rabbits than in LDLr(+/-) diet rabbits, whereas no significant difference occurred in the descending thoracic aorta and in the abdominal aorta just above the bifurcation. Differences remained statistically significant after adjustment for plasma cholesterol, triglycerides, and sex. Compared with LDLr(+/-) diet rabbits, higher intimal lipoprotein lipase (LPL) and apolipoprotein (apo) B levels were observed in LDLr(-/-) rabbits only at the level of the descending thoracic aorta. Intimal apo E levels in LDLr(-/-) rabbits were significantly lower in sites with a larger intima than in LDLr(+/-) diet rabbits. In conclusion, the relative atherogenicity of VLDL and LDL is dependent on the topographic site.

Species differences in hepatocyte-directed gene transfer: implications for clinical translation.

The liver is a key organ in numerous metabolic pathways, in cholesterol metabolism, and in production of coagulation factors. Therefore, gene transfer to hepatocytes has been extensively pursued. There are numerous biological parameters that may affect the outcome of hepatocyte-directed gene transfer. Species or strain variation of any of these multiple determinants hinders the process of clinical translation. This review specifically focuses on functional aspects of liver histology that are pertinent for gene transfer to parenchymal liver cells. We discuss the reticulo-endothelial cells of the liver and the spleen, and their impact on innate immune responses after adenoviral transfer and on vector clearance. Liver sinusoidal endothelial cells contain pores, called fenestrae, and have no basal lamina. Fenestrae are clustered in sieve plates and may provide direct access for circulating gene transfer vectors to the space of Disse, in which microvilli of parenchymal liver cells protrude. We present multiple lines of evidence that the species differences in the diameter of sinusoidal fenestrae are a critical determinant of transgene expression after adenoviral transfer. The small diameter of fenestrae in humans should be considered in any rational design of gene transfer technologies for hepatocyte-directed transfer. Hydrodynamic gene transfer is highly successful in rodents. The significantly lower efficacy in higher species may also partially be due to species differences in liver architecture. Finally, we discuss species differences in adaptive immune responses against the transgene product that may constitute one of the most significant hurdles for clinical translation.

Wild-type apo A-I and apo A-I(Milano) gene transfer reduce native and transplant arteriosclerosis to a similar extent.

Apolipoprotein (apo) A-I(Milano) is an apo A-I mutant characterized by a cysteine for arginine substitution at position 173. Apo A-I(Milano) carriers have much less atherosclerosis than expected from their low plasma high-density lipoprotein cholesterol levels, suggesting that this mutant may have superior atheroprotective properties. Here, we compare the effect of hepatocyte-directed gene transfer of wild-type human apo A-I and human apo A-I(Milano) on endothelial progenitor cell (EPC) biology and on the progression of native atherosclerosis and allograft vasculopathy in C57BL/6 apo E(-/-) mice. Human apo A-I and apo A-I(Milano) transfer resulted in an equivalent increase of EPC number and function as well as EPC incorporation and endothelial regeneration in allografts and inhibited the progression of native atherosclerosis and allograft vasculopathy to a similar extent. In conclusion, the current head-to-head comparison indicates that human apo A-I(Milano) transfer is not superior compared to wild-type human apo A-I transfer.

Human apolipoprotein A-I gene transfer reduces the development of experimental diabetic cardiomyopathy.

BACKGROUND: The hallmarks of diabetic cardiomyopathy are cardiac oxidative stress, intramyocardial inflammation, cardiac fibrosis, and cardiac apoptosis. Given the antioxidative, antiinflammatory, and antiapoptotic potential of high-density lipoprotein (HDL), we evaluated the hypothesis that increased HDL via gene transfer (GT) with human apolipoprotein (apo) A-I, the principal apolipoprotein of HDL, may reduce the development of diabetic cardiomyopathy. METHODS AND RESULTS: Intravenous GT with 3x10(12) particles/kg of the E1E3E4-deleted vector Ad.hapoA-I, expressing human apoA-I, or Ad.Null, containing no expression cassette, was performed 5 days after streptozotocin (STZ) injection. Six weeks after apoA-I GT, HDL cholesterol levels were increased by 1.6-fold (P<0.001) compared with diabetic controls injected with the Ad.Null vector (STZ-Ad.Null). ApoA-I GT and HDL improved LV contractility in vivo and cardiomyocyte contractility ex vivo, respectively. Moreover, apoA-I GT was associated with decreased cardiac oxidative stress and reduced intramyocardial inflammation. In addition, compared with STZ-Ad.Null rats, cardiac fibrosis and glycogen accumulation were reduced by 1.7-fold and 3.1-fold, respectively (P<0.05). Caspase 3/7 activity was decreased 1.2-fold (P<0.05), and the ratio of Bcl-2 to Bax was upregulated 1.9-fold (P<0.005), translating to 2.1-fold (P<0.05) reduced total number of cardiomyocytes with apoptotic characteristics and 3.0-fold (P<0.005) reduced damaged endothelial cells compared with STZ-Ad.Null rats. HDL supplementation ex vivo reduced hyperglycemia-induced cardiomyocyte apoptosis by 3.4-fold (P<0.005). The apoA-I GT-mediated protection was associated with a 1.6-, 1.6-, and 2.4-fold induction of diabetes-downregulated phospho to Akt, endothelial nitric oxide synthase, and glycogen synthase kinase ratio, respectively (P<0.005). CONCLUSIONS: ApoA-I GT reduced the development of streptozotocin-induced diabetic cardiomyopathy.

Lipid emulsions potently increase transgene expression in hepatocytes after adenoviral transfer.

Elimination of Kupffer cells by cytotoxic clodronate liposomes increases transgene expression in the liver after adenoviral transfer. Here, we demonstrate that empty l-alpha-phosphatidylcholine liposomes block uptake of vectors in the reticuloendothelial cells of the liver and increase human apolipoprotein (apo) A-I (approved gene symbol apo A-I) expression in C57BL/6 (1.3-fold) and Balb/c mice (3.1-fold) to the same extent as clodronate liposomes (1.5- and 3.4-fold, respectively). A similar elevation of human apo A-I levels was induced by the lipid emulsion Intralipid (1.5- and 2.8-fold in C57BL/6 and Balb/c mice, respectively). Not only Kupffer cells but also hepatic sinusoidal endothelial cells (HSEC) constitute the reticuloendothelial cells of the liver. The uptake of adenoviral vectors 1 h after transfer in C57BL/6 mice was 2.9-fold lower in Kupffer cells than in HSEC. In contrast, Kupffer cell uptake in Balb/c mice was 2.6-fold higher than in HSEC. Vector uptake in reticuloendothelial cells of the liver was reduced and transgene expression was increased in splenectomized and Rag2-deficient Balb/c mice but not in splenectomized and Rag1-deficient C57BL/6 mice. In conclusion, lipid emulsions for parenteral clinical use block uptake of adenoviral vectors by the reticuloendothelial cells of the liver and potently increase transgene expression.

Elimination of innate immune responses and liver inflammation by PEGylation of adenoviral vectors and methylprednisolone.

Improvement of the therapeutic index of adenoviral gene transfer requires the development of strategies to abrogate adenoviral capsid-induced inflammation and cytokine production. The effect of monomethoxypolyethylene glycol (MPEG) conjugation to adenoviral vectors and of methylprednisolone (MP) on innate immunity, liver inflammation, and thrombocyte counts was evaluated after transfer of 1011 particles of E1/E3/E4- deleted adenoviral vector expressing human apolipoprotein A-I (apoA-I). Gene transfer with unPEGylated vectors induced peak interleukin-6 (IL-6) plasma levels that were 66-fold above baseline levels in C57BL/6 mice. PEGylation combined with 4 mg of MP 6 hr before and at the time of gene transfer suppressed IL-6 plasma levels to baseline values at all time points. This combination resulted in 24-, 28-, 5.9-, 42-, 26-, and 2.5- fold reduced mRNA expression in the liver of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, interferon-inducible protein-10, macrophage inflammatory protein-1 beta, lipopolysaccharide-induced CXC chemokine, and keratinocyte-derived chemokine, respectively; abrogated neutrophil infiltration in the liver; and reduced alanine aminotransferase levels. PEGylation reduced vector uptake in the spleen and in nonparenchymal liver cells. PEGylation also inhibited the development of thrombocytopenia. In conclusion, PEGylation of adenoviral vectors combined with MP administration improves the therapeutic index of adenoviral gene transfer.