Fenestrae allow the passage of gene transfer vectors from the sinusoidal lumen to the surface of hepatocytes. We have previously shown that the diameter of fenestrae correlates with species and strain differences of transgene expression following intravenous adenoviral transfer. In the current study, we demonstrate that the diameter of fenestrae in humans without liver pathology is 107 ± 1.5 nm. This is similar to the previously reported diameter in New Zealand White (NZW) rabbits (103 ± 1.3 nm) and is significantly smaller than in C57BL/6 mice (141±5.4 nm) and SpragueDawley rats (161 ± 2.7 nm). We show that the diameter of fenestrae in one male NZW rabbit and its offspring characterized by a more than 50-fold increase of transgene expression after adenoviral gene transfer is significantly (113 ± 1.5 nm; Po0.001) larger than in control NZW rabbits. In vitro filtration experiments using polycarbonate filters with increasing pore sizes demonstrate that a relatively small increment of the diameter of pores potently enhances passage of adenoviral vectors, consistent with in vivo data. In conclusion, the small diameter of fenestrae in humans is likely to be a major obstacle for hepatocyte transduction by adenoviral vectors.

Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E-deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.

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 3×10 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). Conclusion— ApoA-I GT reduced the development of streptozotocin-induced diabetic cardiomyopathy.

Macrophage infiltration into the subendothelial space at lesion prone sites is the primary event in atherogenesis. Inhibition of macrophage homing might therefore prevent atherosclerosis. Since HDL levels are inversely correlated with cardiovascular risk, their effect on macrophage homing was assessed in apoE-deficient (apoE-/-) mice. Overexpression of human apolipoprotein AI in apoE-/- mice increased HDL levels 3-fold and reduced macrophage accumulation in an established assay of leukocyte homing to aortic root endothelium 3.2-fold (P<0.005). This was due to reduced in vivo betaVLDL oxidation, reduced betaVLDL triggered endothelial cytosolic Ca2+ signaling through PAF-like bioactivity, lower ICAM-1 and VCAM-1 expression, and diminished ex vivo leukocyte adhesion. Adenoviral gene transfer of human PAF-acetylhydrolase (PAF-AH) in apoE-/- mice increased PAF-AH activity 1.5-fold (P<0.001), reduced betaVLDL-induced ex vivo macrophage adhesion 3.5-fold (P<0.01), and reduced in vivo macrophage homing 2.6-fold (P<0.02). These inhibitory effects were observed in the absence of increased HDL cholesterol levels. In conclusion, HDL reduces macrophage homing to endothelium by reducing oxidative stress via its associated PAF-AH activity. This protective mechanism is independent of the function of HDL as cholesterol acceptor. Modulation of lipoprotein oxidation by PAF-AH may prevent leukocyte recruitment to the vessel wall, a key feature in atherogenesis.

No abstract

Objective— Transplant arteriosclerosis is the leading cause of graft failure and death in patients with heart transplantation. Endothelial progenitor cells (EPCs) contribute to endothelial regeneration in allografts. We investigated whether increased HDL cholesterol induced by adenoviral human apoA-I (AdA-I) transfer increases number and function of EPCs, promotes incorporation of EPCs in Balb/c allografts transplanted paratopically in C57BL/6 ApoE −/− mice, and attenuates transplant arteriosclerosis. Methods and Results— EPC number in ApoE −/− mice was increased after AdA-I transfer as evidenced by 1.5-fold ( P <0.01) higher Flk-1 Sca-1–positive cells and 1.4-fold ( P <0.01) higher DiI-acLDL isolectin-positive spleen cells. In addition, HDL enhanced EPC function in vitro. Incorporation of bone marrow–derived EPCs was 5.8-fold ( P <0.01) higher at day 21 after transplantation in AdA-I-treated apoE −/− mice compared with control mice. Enhanced endothelial regeneration in AdA-I-treated apoE −/− mice as evidenced by a 2.6-fold ( P <0.01) increase of CD31-positive endothelial cells resulted in a 1.4-fold ( P =0.059) reduction of neointima and a 3.9-fold ( P <0.01) increase of luminal area. Conclusion— Human apoA-I transfer increases the number of circulating EPCs, enhances their incorporation into allografts, promotes endothelial regeneration, and attenuates neointima formation in a murine model of transplant arteriosclerosis.

No abstract

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.