Bart De Geest

Adenovirus-Mediated Gene Transfer of Human Platelet-Activating Factor–Acetylhydrolase Prevents Injury-Induced Neointima Formation and Reduces Spontaneous Atherosclerosis in Apolipoprotein E–Deficient Mice

Background—Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E–deficient mice. Methods and Results—Intravenous administration of an adenovirus (5×108 plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P <0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P <0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P <0.001), macrophages by 69% (P =0.006), and smooth muscle cells by 84% (P =0.002) in the arterial wall. This resulted in a 77% reduction (P <0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P =0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P =0.01 and P =0.04, respectively). Conclusions—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.

HDL-associated PAF-AH reduces endothelial adhesiveness in apoE−/− mice

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 βVLDL oxidation, reduced βVLDL 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 β VLDL‐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.—Theilmeier, G., De Geest, B., Van Veldhoven, P. P., Stengel, D., Michiels, C., Lox, M., Landeloos, M., Chapman, M. J., Ninio, E., Collen, D., Himpens, B., Holvoet, P. HDL‐associated PAF‐AH reduces endothelial adhesiveness in apoE−/− mice. FASEB J. 14, 2032–2039 (2000)

Effects of Adenovirus-Mediated Human Apo A-I Gene Transfer on Neointima Formation After Endothelial Denudation in Apo E–Deficient Mice

BACKGROUND Inactivation of apolipoprotein (apo) E genes in mice markedly increases beta-VLDL levels and accelerates progression of complex atherosclerotic lesions. The present study investigated (1) the effect of apo E deficiency (apo E-/-) on neointima formation after endothelial denudation; and (2) the effect of increased HDL, induced by adenovirus-mediated transfer of a human apo A-I gene, on neointima formation. METHODS AND RESULTS Guidewire-induced abrasion of the endothelium of the common carotid artery did not produce neointima formation within 18 days after injury in C57BL/6J mice (n=12) but was associated with an intima/media ratio of 0.82+/-0.25 in age-matched C57BL/6J apo E-/- mice (n=12). Neointima consisted primarily of smooth muscle alpha-actin positive cells. Injection in C57BL/6J apo E-/- mice of 2x10(9) (n=5) or 4x10(9) (n=7) plaque forming units (p.f.u.) of a recombinant human apo A-I adenovirus 3 days before injury resulted in an increase of HDL cholesterol from 36+/-5 to 75+/-3 mg/dL (P<.05) and to 96+/-13 mg/dL (P<.05), respectively, and of the HDL cholesterol/non-HDL cholesterol ratio from 0.063+/-0.003 to 0.15+/-0.01 (P<.05) and to 0.16+/-0.015 (P<.05), respectively. Intima/media ratio decreased to 0.28+/-0.06 (P=NS versus C57BL/6J apo E-/- mice) with 2x10(9) p.f.u. of apo A-I virus and to 0.03+/-0.01 with 4x10(9) p.f.u. (P<.01 versus C57BL/6J apo E-/- mice). Injection of 4x10(9) p.f.u. of RR5 (n=7) or tissue plasminogen activator (t-PA) control virus (n=6) did not result in a significant alteration of HDL cholesterol (44+/-11 and 26+/-4 mg/dL, respectively) nor in a reduction of intima/media ratio (0.81+/-0.35 and 0.86+/-0.23, respectively). CONCLUSIONS Apo E deficiency is associated with increased neointima formation after endothelial denudation. Gene transfer of apo A-I increases HDL cholesterol and significantly reduces neointima formation, which suggests a direct vascular protective effect of HDL.

The Role of Liver Sinusoidal Cells in Hepatocyte-Directed Gene Transfer

Hepatocytes are a key target for gene therapy of inborn errors of metabolism as well as of acquired diseases such as liver cancer and hepatitis. Gene transfer efficiency into hepatocytes is significantly determined by histological and functional aspects of liver sinusoidal cells. On the one hand, uptake of vectors by Kupffer cells and liver sinusoidal endothelial cells may limit hepatocyte transduction. On the other hand, the presence of fenestrae in liver sinusoidal endothelial cells provides direct access to the space of Disse and allows vectors to bind to receptors on the microvillous surface of hepatocytes. Nevertheless, the diameter of fenestrae may restrict the passage of vectors according to their size. On the basis of lege artis measurements of the diameter of fenestrae in different species, we show that the diameter of fenestrae affects the distribution of transgene DNA between sinusoidal and parenchymal liver cells after adenoviral transfer. The small diameter of fenestrae in humans may underlie low efficiency of adenoviral transfer into hepatocytes in men. The disappearance of the unique morphological features of liver sinusoidal endothelial cells in pathological conditions like liver cirrhosis and liver cancer may further affect gene transfer efficiency. Preclinical gene transfer studies should consider species differences in the structure and function of liver sinusoidal cells as important determinants of gene transfer efficiency into hepatocytes.

Human ApoA-I Transfer Attenuates Transplant Arteriosclerosis via Enhanced Incorporation of Bone marrow–derived Endothelial Progenitor Cells

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.

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 3×1012 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.

Critical role of scavenger receptor-BI-expressing bone marrow-derived endothelial progenitor cells in the attenuation of allograft vasculopathy after human apo A-I transfer.

Allograft vasculopathy is the leading cause of death in patients with heart transplantation. Accelerated endothelial regeneration mediated by enhanced endothelial progenitor cell (EPC) incorporation may attenuate the development of allograft vasculopathy. We investigated the hypothesis that modulation of EPC biology and attenuation of allograft vasculopathy by increased high-density lipoprotein cholesterol after human apo A-I (AdA-I) transfer requires scavenger receptor (SR)-BI expression in bone marrow-derived EPCs. After AdA-I transfer, the number of circulating EPCs increased 2.0-fold (P < .001) at different time points in C57BL/6 mice transplanted with SR-BI(+/+) bone marrow but remained unaltered in mice with SR-BI(-/-) bone marrow. The effect of high-density lipoprotein on EPC migration in vitro requires signaling via SR-BI and extracellular signal-regulated kinases and is dependent on increased nitric oxide (NO) production in EPCs. Human apo A-I transfer 2 weeks before paratopic artery transplantation reduced intimal area at day 21 3.7-fold (P < .001) in mice with SR-BI(+/+) bone marrow but had no effect in mice with SR-BI(-/-) bone marrow. AdA-I transfer potently stimulated EPC incorporation and accelerated endothelial regeneration in chimeric SR-BI(+/+) mice but not in chimeric SR-BI(-/-) mice. In conclusion, human apo A-I transfer accelerates endothelial regeneration mediated via SR-BI expressing bone marrow-derived EPCs, thereby preventing allograft vasculopathy.

Vascular-Protective Effects of High-Density Lipoprotein Include the Downregulation of the Angiotensin II Type 1 Receptor

There is growing evidence that a cross-talk exists between the renin-angiotensin (Ang) system and lipoproteins. We investigated the role of high-density lipoprotein (HDL) on Ang II type 1 receptor (AT1R) regulation and subsequent Ang II–mediated signaling under diabetic conditions. To investigate the effect of HDL on AT1R expression in vivo, apolipoprotein A-I gene transfer was performed 5 days after streptozotocin injection. Six weeks after apolipoprotein A-I gene transfer, the 1.9-fold (P=0.001) increase of HDL cholesterol was associated with a 4.7-fold (P<0.05) reduction in diabetes mellitus–induced aortic AT1R expression. Concomitantly, NAD(P)H oxidase activity, Nox 4, and p22phox mRNA expression were reduced 2.6-fold, 2.0-fold, and 1.5-fold (P<0.05), respectively, whereas endothelial NO synthase dimerization was increased 3.3-fold (P<0.005). Apolipoprotein A-I transfer improved NO bioavailability as indicated by ameliorated acetylcholine-dependent vasodilation in the streptozotocin-Ad.hapoA-I group compared with streptozotocin-induced diabetes mellitus. In vitro, HDL reduced the hyperglycemia-induced upregulation of the AT1R in human aortic endothelial cells. This was associated with a 1.3-fold and 2.2-fold decreases in reactive oxygen species and NAD(P)H oxidase activity, respectively (P<0.05). Finally, HDL reduced the responsiveness to Ang II, as indicated by decreased oxidative stress in the hyperglycemia+HDL+Ang II group compared with the hyperglycemia+Ang II group. In conclusion, vascular-protective effects of HDL include the downregulation of the AT1R.

Impact of HDL on adipose tissue metabolism and adiponectin expression

OBJECTIVE The objective of the current study was to investigate the hypothesis that high-density lipoprotein (HDL) influences adipocyte metabolism and adiponectin expression. Therefore, HDL was increased in vivo via apolipoprotein (apo) A-I gene transfer and in vitro via supplementation of HDL to partly differentiated adipocytes, in the presence or absence of lipopolysaccharide (LPS), known to decrease HDL cholesterol and adiponectin levels in vivo. METHODS AND RESULTS Apo A-I transfer resulted in a significant increase of HDL cholesterol in control and LPS-injected C57BL/6 mice, which was paralleled by an increase in plasma adiponectin levels and adiponectin expression in abdominal fat. Triglyceride and free fatty acids levels after LPS administration were 2.2-fold (p<0.05) and 1.3-fold (p<0.05) lower, respectively, in Ad.hapoA-I-LPS than in Ad.Null-LPS mice. In parallel, the LPS-induced mRNA expression of hormone sensitive lipase was 3.5-fold (p=0.05) decreased in the Ad.hapoA-I-LPS group. On the other hand, apo A-I transfer abrogated the LPS-mediated reduction in lipin-1 and CD36 mRNA expression by 8.2-fold (p<0.05) and 18-fold (p<0.05), respectively. Concomitantly, the phosphorylation state of Akt was 2.0-fold (p<0.05) increased in the Ad.hapoA-I-LPS compared to the Ad.Null-LPS group. Pre-incubation of partly differentiated adipocytes with HDL (50 microg protein/ml) increased adiponectin expression by 1.5-fold under basal conditions (p<0.05) and could abrogate LPS-induced down-regulation of adiponectin, both in a phosphatidylinositol-3-kinase-dependent manner. CONCLUSIONS HDL affects adipocyte metabolism and adiponectin expression.

Sustained Expression of Human Apolipoprotein A-I after Adenoviral Gene Transfer in C57BL/6 Mice: Role of Apolipoprotein A-I Promoter, Apolipoprotein A-I Introns, and Human Apolipoprotein E Enhancer

Elevation of HDL cholesterol, after adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. Previously, we observed transient human apo A-I expression after adenoviral gene transfer with a cytomegalovirus (CMV)-driven construct containing the human apo A-I cDNA. Therefore, the effects of promoters (CMV or 256 base pairs of the human apo A-I promoter), introns of the human apo A-I gene, and the liver-specific human apolipoprotein E (apo E) enhancer on adenovirus-mediated human apo A-I expression were evaluated in C57BL/6 mice. In the presence of the CMV promoter, human apo A-I introns prolonged expression above 20 mg/dl from 14 to 35 days. Addition of one, two, or four copies of the human apo E enhancer in these constructs resulted in a copy-dependent but transient increase in expression for 14 days. The apo A-I promoter induced 3.2-fold lower peak levels of human apo A-I than did the CMV promoter, but insertion of four apo E enhancers in the apo A-I promoter-driven construct resulted in human apo A-I levels above 20 mg/dl for 6 months. The decline between day 6 and day 35 of human apo A-I expression driven by the CMV promoter was due to (1) a 2.5-fold decline in transgene DNA levels that is not observed with apo A-I promoter-driven constructs, and (2) CMV promoter attenuation as evidenced by a 7.6-fold decline in the human apo A-I mRNA/human apo A-I DNA copy number ratio between day 6 and day 35. Hepatotoxicity, as evidenced by up to 10-fold higher serum levels of transaminases on day 6 after gene transfer with CMV promoter-driven constructs than with apo A-I promoter-driven constructs, probably caused the accelerated decline of transgene DNA. In conclusion, gene transfer with an adenovirus comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA, and four apo E enhancers, all of human origin, is associated with low hepatotoxicity and with the absence of promoter shutoff resulting in human apo A-I expression above 20 mg/dl for up to 6 months.