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Circulation. 1997;96:4349-4356

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(Circulation. 1997;96:4349-4356.)
© 1997 American Heart Association, Inc.


Articles

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

Bart De Geest, MD; Zhian Zhao, MSc; Désiré Collen, MD, PhD; ; Paul Holvoet, PhD

From the Center for Molecular and Vascular Biology, University of Leuven (Belgium).

Correspondence to D. Collen, MD, PhD, Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium. E-mail desire.collen{at}med.kuleuven.ac.be


*    Abstract
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*Abstract
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down arrowResults
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Background Inactivation of apolipoprotein (apo) E genes in mice markedly increases ß-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 2x109 (n=5) or 4x109 (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 2x109 p.f.u. of apo A-I virus and to 0.03±0.01 with 4x109 p.f.u. (P<.01 versus C57BL/6J apo E-/- mice). Injection of 4x109 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.


Key Words: atherosclerosis • endothelial injury • lipoproteins • apolipoproteins


*    Introduction
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up arrowAbstract
*Introduction
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down arrowResults
down arrowDiscussion
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Apolipoprotein A-I (apo A-I), a single-chain polypeptide of 243 amino acids, is the principal apolipoprotein of high-density lipoproteins (HDL). Epidemiological studies have demonstrated an inverse relationship between plasma levels of HDL and risk for ischemic cardiovascular disease.1–3 Studies in transgenic mice have demonstrated that apo A-I has an antiatherogenic effect4–6 and that the protein composition of HDL significantly affects its antiatherogenic potential.7,8 The antiatherogenic effect of HDL has been ascribed to reverse cholesterol transport, the process by which cholesterol is transported from peripheral tissues to the liver for excretion and to endocrine organs for steroid hormone synthesis.9,10

High HDL is associated with decreased risk for restenosis after percutaneous transluminal coronary angioplasty (PTCA) in humans.11,12 Infusion of apo A-I Milano, a natural apo A-I mutant with a cysteine for arginine mutation at position 173, reduces neointima formation in cholesterol fed New Zealand White rabbits.13 The protective effect of HDL on restenosis and on neointima formation may be independent of reverse cholesterol transport. Indeed, HDL inhibits the oxidation of low-density lipoproteins (LDL)14,15 and reverses the inhibitory effect of oxidized LDL on endothelium-dependent arterial relaxation in vivo.16

Mice with inactivation of both apo E alleles are characterized by very high levels of ß-VLDL and accelerated progression of complex atherosclerotic lesions.17–20 Because introduction of a human apo A-I transgene in C57BL/6J apo E-/- mice has been shown to be a potent suppressor of atherosclerosis,5,6 we speculated that adenovirus-mediated transfer of a recombinant human apo A-I transgene might suppress neointima formation in these mice. This study demonstrates that endothelial denudation caused neointima formation in C57BL/6J apo E-/- but not in C57BL/6J control mice. Gene transfer with a recombinant adenovirus that induced a transient production of human apo A-I resulted in a 2.7-fold increase in HDL cholesterol levels and in a significant suppression of neointima formation. The present study suggests a direct vascular protective effect of HDL.


*    Methods
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*Methods
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Preparation of Recombinant Adenovirus and Viral Stocks
The entire human apo A-I cDNA, including presequences and prosequences,21 was cloned into the pACCMVpLpA vector.22 Five micrograms of this plasmid (pACCMVapo A-I) was cotransfected into 293 cells together with 2.5 µg of the pJM17 plasmid that contains the full-length adenovirus serotype 5 genome.23 Transfection efficiency was boosted after 6 hours by glycerol shock with 15% glycerol in Dulbecco's modified Eagle medium (DMEM, Gibco). Homologous recombination between pJM17 and pACCMVapo A-I leads to the formation of a recombinant adenovirus (AdCMVapo A-I) in which most of the early region 1 (E1) of the adenoviral genome is replaced by the expression cassette containing the apo A-I cDNA under control of the cytomegalovirus (CMV) promoter. The recombinant adenovirus is defective because of the absence of the E1 region, which is provided in trans by the 293 cells.24 The cells were overlaid with 0.65% noble agar in modified Eagle medium (MEM, Gibco) supplemented with 4% fetal bovine serum (Gibco) 24 hours after transfection. Viral plaques, appearing within 7 to 10 days, were picked and used to infect 293 cells. Recombinant adenovirus was identified by measuring human apo A-I concentration in the conditioned medium by ELISA.21 Large-scale production of recombinant adenovirus was performed by infecting confluent monolayers of 293 cells in 15-cm dishes. When 90% of the cells showed a cytopathic effect, Igepal CA-630 (Sigma) was added to a final concentration of 0.1% to lyse the cells. Cellular debris was removed by centrifugation at 12 000g for 10 minutes. After addition of 0.5 volume of 20% polyethylene glycol in 2.5 mol/L NaCl, virus-containing extracts were incubated on ice for 1 hour, and virus was precipitated by centrifugation at 12 000g for 20 minutes. The pellet was resuspended in 20 mmol/L Tris-HCl, pH 7.8, containing CsCl to obtain a relative density of 1.1. After CsCl step gradient, ultracentrifugation at 22 000 rpm for 2 hours, the virus was harvested from the 1.3 and 1.4 interface. The recombinant virus was desalted by chromatography on Sepharose CL4B in an isotonic saline buffer (10 mmol/L Tris-HCl, pH 7.4, 137 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2). The virus was stored at -80°C until use. Viral titers were determined by plaque assay, and doses were expressed in plaque forming units (p.f.u.). The experiments described in this report were performed with one batch of virus, although spot checks with other batches confirm the generality and reproducibility of the observations. The generation of the recombinant adenovirus AdRR5, which does not contain an insert after the CMV promoter, has been described earlier.25 The human tissue plasminogen activator (t-PA) virus was a kind gift of Dr R.D. Gerard (Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute of Biotechnology, Leuven). t-PA plasma levels were determined by ELISA as described previously.26

Animal Experiments
All experimental procedures in animals were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee. Apo E-/- mice,27 backcrossed for six generations into the C57BL/6J background, were purchased from Jackson Laboratory (Bar Harbor, Maine). These mice had 98.4% C57BL/6J background. Mice were fed normal chow ad libitum. All mice used in this study were approximately 4 months of age and weighed between 22 and 30 g.

Viral Injections
Mice were anesthestized by intraperitoneal injection of 60 mg/kg pentobarbital (Abbott Laboratories). The jugular vein was prepared by blunt-end dissection and a 2F gauge catheter was introduced in the vein. Each viral dose was given in a final volume of 300 µL.

Guidewire Injury Model
Endothelial denudation of the common carotid artery of mice was induced with a guidewire essentially as described by Lindner et al.28 The right common carotid artery and the right external carotid artery were exposed by blunt-end dissection. A guidewire (epidural spinal anesthesia guidewire; Portex; diameter of 320 µm) was introduced through the external carotid artery and moved proximally into the common carotid artery. The common carotid artery was abraded three times over its entire length. Eighteen days after injury, mice were anesthestized with 60 mg/kg Nembutal and a maximal blood volume was collected by puncture of the inferior vena cava. Perfusion fixation under physiological pressure was performed for 10 minutes with 4% formol in phosphate-buffered saline (PBS, pH 7.0) after intracardiac puncture. The common carotid artery was dissected, fixed in 4% formol in PBS for 5 hours and transferred to PBS containing 20% sucrose. Arteries were embedded in OCT compound (Tissue-Tek, Miles Inc), snap-frozen in precooled 2-methyl butane, and stored at -80°C until further use. Seven-micron-thick cryosections were made through the whole injured artery and stained with hematoxylin-eosin.

Morphometric Analysis
Morphometric analysis was performed in a blinded manner with the Leica 2 Quantimet system. The area within the external elastic lamina, the area within the internal elastic lamina, and the lumen size were determined in the injured segment of the artery at distances of 84 µm. The length of the injured segment was between 5 and 7 mm, and approximately 80 sections were analyzed per artery. Media was defined as the area between the internal and external elastic lamina. Intima was defined as the area within the internal elastic lamina not occupied by vessel lumen, thrombus, and organized thrombus. Intima/media ratio was defined as the ratio between the area occupied by neointima and the area occupied by media.

Immunohistochemistry
Endothelial cells were immunostained with rabbit anti–von Willebrand Factor antibodies (Dakopatts; diluted 1:100), biotinylated goat anti-rabbit antibodies (Dakopatts), and peroxidase-labeled avidin (Dakopatts; diluted 1:100). Peroxidase reaction was performed in 0.05 mol/L Tris:HCl (pH 7.0) containing 0.06% 3,3'-diaminobenzidine and 0.01% H2O2. Tissue sections were counterstained with hematoxylin. Smooth muscle cells and inflammatory cells were detected in an indirect staining procedure using respectively a cross-reacting murine monoclonal biotinylated antibody against human smooth muscle cell {alpha}-actin (clone1A4; Sigma; diluted 1:500) or a rat biotinylated monoclonal antibody against murine common leukocyte antigen/CD45 (clone 30F11.1; Pharmingen; diluted 1:100).

RNA Extraction and Northern Blotting
Total RNA was extracted from mouse liver by a single step method (Ultraspec RNA isolation system, Biotecx) based on the guanidinium isothiocyanate acid phenol method of Chomczynski and Sacchi.29 Approximately 10 µg of RNA was separated on a 1.25% agarose gel containing 0.21 mol/L formaldehyde and 3.8 vol% formamide and was blotted overnight on a nylon membrane (Hybond-N, Amersham Life Sciences). After UV cross-linking, the membrane was prehybridized in Quikhyb (Stratagene) for 4 hours. A human genomic 2.2 kb apo A-I fragment was labeled with a random primer labeling kit (Prime-Random II Primer Labeling Kit, Stratagene) and 15 million cpm were added to the prehybridization solution. After hybridization for 16 hours, the blot was washed for 20 minutes in a solution containing 2xSSC (1xSSC is 0.15 mol/L NaCl, 0.015 mol/L trisodiumcitrate 2H2O, pH 7.0) and 0.05% SDS and then washed twice for 20 minutes in a buffer containing 0.1% SSC and 0.1% SDS. The membrane was exposed overnight at -80°C to a Hyperfilm-MP (Amersham Life Sciences).

Plasma Lipid and Lipoprotein Analyses
Blood was obtained after an overnight fast by puncture of the retro-orbital plexus or by puncture of the vena cava and anticoagulated with 0.1 volume of 4% trisodiumcitrate. Plasma was obtained by centrifugation and lipoprotein fractions were separated by gel filtration of 200 µL of plasma on a Superdex 200HR column equilibrated with 20 mmol/L Tris:HCl buffer, pH 8.1, containing 0.15 mol/L NaCl, 1 mmol/L EDTA, and 0.02 mg/mL sodium azide in a FPLC system (Waters Associates). Phospholipid and triglyceride levels were determined by standard enzymatic assays (Biomérieux and Sigma, respectively). Cholesterol of fractions obtained after gel filtration was extracted with methanol/chloroform 2:1 (vol:vol). Esterified and unesterified cholesterol was quantitated by high-performance liquid chromatography on a reversed-phase column (Zorbax ODS; Du Pont de Nemours) essentially as descibed by Vercaemst et al.30 Samples were eluted isocratically at 45°C with a mixture of acetonitrile/isopropanol 50:50 (vol:vol).

Statistical Analysis
Intima/media ratio of control apo E–deficient mice, mice treated with 4x109 p.f.u. of RR5 control virus and 4x109 p.f.u. of t-PA adenovirus, were compared by Kruskal-Wallis nonparametric ANOVA test on logarithmically transformed values in the INSTAT V2.05a statistical program (Graph Pad Software), which revealed the absence of a statistically significant difference between the three control groups. Therefore, these three control groups were pooled and compared with mice treated with 2x109 p.f.u. and 4x109 human apo A-I adenovirus with a Kruskal-Wallis nonparametric ANOVA test on logarithmically transformed values followed by Dunn's multiple comparisons test. Significance of differences in lipid values was assessed by a two-tailed unpaired alternate Welch t test.31 A probability value of <.05 was considered statistically significant.


*    Results
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up arrowMethods
*Results
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Expression of Human Apo A-I in C57BL/6J and C57BL/6J Apo E-/- Mice
Fig 1ADown illustrates the time course of apo A-I expression in C57BL/6J mice after injection of 10,9 2x10,9 4x109, and 1010 p.f.u. of human apo A-I adenovirus. Injection of 2x109 p.f.u. resulted in 36-fold increased human apo A-I levels at day 3 and 6.5-fold higher human apo A-I levels at day 6 compared with injection of 109 p.f.u. A dose of 4x109 p.f.u. did not produce a significant further increase of the apo A-I levels. In C57BL/6J mice treated with 2x10,9 4x109, or 1010 p.f.u., human apo A-I levels decreased 3-fold between day 6 and day 9, whereas in mice treated with 109 p.f.u. maximal human apo A-I levels were obtained at day 9. Fig 1BDown shows that the time course of human apo A-I expression after injection of a dose of 2x109 p.f.u. of recombinant virus in C57BL/6J apo E-/- mice was very similar to that in C57BL/6J mice. Northern blot analysis of total liver RNA of C57BL/6J apo E-/- mice at days 6, 9, 14, and 21 after virus injection demonstrated a strong signal of human apo A-I RNA at days 6 and 9, which was markedly reduced at day 14 (data not shown).



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Figure 1. Human apolipoprotein (apo) A-I expression: (A) after injection of a dose of 109 ({blacksquare}), 2x109 ({circ}), 4x109 ({bullet}) or of 1010 ({blacktriangleup}) p.f.u. of human apo A-I adenovirus in C57BL/6J mice and (B) after injection of a dose of 2x109 p.f.u. of apo A-I adenovirus in C57BL/6J apo E-/- mice. Data represent mean±SEM of four independent experiments.

Effect of Human Apo A-I Adenovirus on Lipoprotein Profiles and Lipid Values in C57BL/6J and C57BL/6J Apo E-/- Mice
The cholesterol and human apo A-I distribution profiles in C57BL/6J mice at day 3 after gene transfer with 4x109 p.f.u. of human apo A-I adenovirus and the cholesterol profile in C57BL/6J control mice, C57BL/6J mice 3 days after injection with 4x109 p.f.u. of RR5 or t-PA control virus, are illustrated in Fig 2ADown. The corresponding profiles in C57BL/6J apo E-/- mice are shown in Fig 2BDown. Ninety percent of the human apo A-I was associated with HDL fractions both in C57BL/6J and C57BL/6J apo E-/- mice. The TableDown summarizes cholesterol and phospholipid levels of lipoprotein fractions after gel filtration and triglyceride plasma levels in both C57BL/6J mice and C57BL/6J apo E-/- control mice and in C57BL/6J mice and C57BL/6J apo E-/- mice 3 days after injection with 4x109 p.f.u. of RR5 control virus, t-PA control virus, and human apo A-I adenovirus. HDL cholesterol levels increased 2.8-fold in C57BL/6J mice (P<.05) and 2.7-fold in C57BL/6J apo E-/- mice (P<.05) 3 days after treatment with 4x109 p.f.u. of human apo A-I adenovirus (TableDown). Injection in C57BL/6J apo E-/- mice of 2x109 p.f.u. of human apo A-I adenovirus resulted in a 2.1- fold increase of HDL cholesterol (75±3 mg/dL; P<.05). Non–HDL cholesterol levels after treatment with 4x109 p.f.u. of human apo A-I adenovirus increased 3.1-fold in C57BL/6J mice (P<.05) and were not significantly altered in C57BL/6J apo E-/- mice treated with either 2x109 p.f.u. (490±29 mg/dL) or with 4x109 p.f.u. (TableDown). The HDL cholesterol/non–HDL cholesterol ratio increased 2.5-fold from 0.063±0.003 in untreated C57BL/6J apo E-/- mice to 0.15±0.01 (P<.05) and 0.16±0.015 (P<.05) in C57BL/6J apo E-/- mice treated with 2x109 p.f.u. and 4x109 p.f.u. of human apo A-I adenovirus, respectively. No significant alteration of HDL cholesterol was seen after treatment with 4x109 p.f.u. of RR5 or of t-PA control virus in either C57BL/6J or C57BL/6J apo E-/- mice. Injection of 4x109 human t-PA adenovirus resulted in human t-PA plasma levels of 9.7±0.76 µg/mL in C57BL/6J mice (n=4) and 8.8±1.0 µg/mL in C57BL/6J apo E-/- mice (n=6), whereas baseline murine t-PA levels are 2.5±0.65 ng/mL (mean±1.96 SEM).32 Non–HDL cholesterol levels in C57Bl/6J mice increased 1.8-fold after treatment with RR5 control virus (P<.05) and 3.1-fold after treatment with t-PA control virus (P<.01). No significant alteration of non–HDL cholesterol occurred in C57BL/6J apo E-/- mice after treatment with of 4x109 p.f.u. of both control viruses.



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Figure 2. A, Cholesterol distribution profile in untreated C57BL/6J mice ({circ}) and in C57BL/6J mice treated with 4x109 p.f.u. of RR5 control virus ({bullet}), tissue plasminogen activator (t-PA) control virus ({square}), and human apolipoprotein (apo) A-I adenovirus ({blacksquare}). B, Cholesterol distribution profile in untreated C57BL/6J apo E-/- mice ({circ}) and in C57BL/6J apo E-/- mice treated with 4x109 p.f.u. of RR5 control virus ({bullet}), t-PA control virus ({square}), and human apo A-I adenovirus ({blacksquare}). Plasma samples were fractionated on a Superdex 200 HR column and cholesterol levels were determined as described in "Methods." Each profile is the average of the profiles of three different mice. Human apo A-I ({bigtriangleup}) in C57BL/6J (A) and C57BL/6J apo E-/- (B) mice was predominantly associated with HDL fractions (fractions 19 to 25).


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Table 1. Cholesterol and Phospholipid Levels of Lipoprotein Fractions Isolated by Gel Filtration and Plasma Triglyceride Levels in C57BL/6J Mice and C57BL/6J ApoE-/- Mice 3 Days After Injection With 4x109 p.f.u. RR5, t-PA, or Human Apo A-I Adenovirus

Fig 3Down illustrates phospholipid distribution profiles in C57BL/6J (Fig 3ADown) and C57BL/6J apo E-/- mice (Fig 3BDown) for control mice and for mice 3 days after gene transfer with 4x109 p.f.u. of RR5 control virus, t-PA control virus, and human apo A-I adenovirus. Phospholipid levels increased 2.9-fold in apo A-I adenovirus–treated C57BL/6J mice (P<.05) and 5.6-fold in apo A-I adenovirus–treated C57BL/6J apo E-/- mice (P<.01); non–HDL phospholipids increased 4.2-fold in (P<.05) and 3.0-fold (P<.05), respectively. There was no statistically significant alteration of HDL phospholipids in either C57BL/6J or C57BL/6J apo E-/- mice after treatment with 4x109 p.f.u. of RR5 or t-PA control virus. Non–HDL phospholipids increased 2.0-fold (P=NS) and 1.3-fold (P=NS) in C57BL/6J and C57BL/6J apo E-/- mice, respectively, after treatment with 4x109 p.f.u. of RR5 control virus and increased 3.6- fold (P<.01) and 1.8- fold (P<.05) after treatment with 4x109 p.f.u. of human t-PA virus.



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Figure 3. Phospholipid distribution profile in C57BL/6J mice, untreated ({circ}) or treated with 4x109 p.f.u. of RR5 control virus ({bullet}), t-PA control virus ({square}), and human apo A-I adenovirus ({blacksquare}) (B). Phospholipid distribution profile in C57BL/6J apo E-/- mice, untreated ({circ}) or treated with either 4x109 p.f.u. of RR5 control virus ({bullet}), t-PA control virus ({square}), and human apo A-I adenovirus ({blacksquare}). Plasma samples were fractionated on a Superdex 200 HR column and phospholipid levels were determined as described in "Methods." See abbreviations in Fig 2Up.

Treatment with 4x109 p.f.u. of human apo A-I adenovirus induced a 2.1-fold (P<.05) and a 3.9-fold (P<.05) increase of triglycerides in C57BL/6J and C57BL/6J apo E-/- mice, respectively. No significant alteration of triglycerides was seen after treatment with 4x109 p.f.u. of RR5 or t-PA control virus in either C57BL/6J or C57BL/6J apo E-/- mice.

Endothelial Denudation and Neointima Formation in C57BL/6J and C57BL/6J Apo E-/- mice
Endothelial denudation of the common carotid artery of mice was induced with a guidewire essentially as described by Lindner et al.28 To confirm the reproducibility of the model, neointima formation was measured in Swiss Webster mice, which were previously used by Lindner et al. Mean intima/media ratio was 0.44±0.29 (n=7) 18 days after injury (data not shown). However, no detectable neointima formation was obtained in 12 C57BL/6J mice notwithstanding the fact that endothelial denudation was confirmed by the loss of immunostaining for von Willebrand factor within the internal elastic membrane 2 days after injury. Representative photographs illustrating the extent of neointima formation in injured arteries of nontreated and human apo A-I adenovirus treated C57BL/6J apo E-/- mice are shown in Fig 4Down. Recombinant adenoviruses were injected 3 days before injury and the extent of neointima formation was assessed 18 days after injury. Thrombus was detected 18 days after injury in 4 out of 16 C57BL/6J apo E-/- control mice, in 2 out of 9 RR5-treated animals (P=NS), 1 out of 8 apo A-I adenovirus–treated mice (P=NS), and 1 out of 7 t-PA–treated mice (P=NS). These arteries were not included in the analysis. Mean intima/media ratio was 0.82±0.25 in C57BL/6J apo E-/- mice (n=12), 0.81±0.35 in C57BL/6J apo E-/- mice treated with 4x109 p.f.u. of RR5 adenovirus (n=7), 0.86±0.23 in C57BL/6J apo E-/- mice treated with 4x109 p.f.u. of human t-PA adenovirus (n=6), 0.28±0.06 in C57BL/6J apo E-/- mice treated with 2x109 p.f.u. of human apo A-I adenovirus (n=5), and 0.03±0.01 in C57BL/6J apo E-/- mice treated with 4x109 p.f.u. of human apo A-I adenovirus (n=7) (Fig 5Down). Comparison of C57BL/6J apo E-/- control mice, mice treated with 4x109 p.f.u. of RR5 control virus, and mice treated with 4x109 t-PA adenovirus by Kruskal-Wallis nonparametric ANOVA test on logarithmically transformed values demonstrated the absence of a statistically significant difference between these three control groups. Subsequently, C57BL/6J apo E-/- control mice, mice treated with 4x109 p.f.u. of RR5 control virus, and mice treated with 4x109 t-PA adenovirus were grouped and comparison with mice treated with 2x109 and 4x109 apo A-I adenovirus by Kruskal-Wallis nonparametric ANOVA test on logarithmically transformed values showed a probability value of .0029. Comparison of all grouped control mice with mice treated with 4x109 apo A-I adenovirus was statistically significant at P<.01.



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Figure 4. Representative light micrographs (x200) of cross sections of the common carotid artery 18 days after endothelial denudation in C57BL/6J mice (a), C57BL/6J apo E-/- mice (b), and C57BL/6J apo E-/- mice treated with 4x109 p.f.u. of human apo A-I adenovirus (c).



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Figure 5. Intima/media ratio in C57BL/6J mice (n=12), C57BL/6J apo E-/- mice (n=12), C57BL/6J apo E-/- treated with 4x109 p.f.u. of RR5 adenovirus (n=7), C57BL/6J apo E-/- treated with 4x109 p.f.u. of human t-PA adenovirus (n=6), C57BL/6J apo E-/- treated with 2x109 p.f.u. of human apo A-I adenovirus (n=5), and in C57BL/6J apo E-/- treated with 4x109 p.f.u. of human apo A-I adenovirus (n=7). Data represent mean±SEM of n independent experiments. See Fig 2Up abbreviations.

Endothelial denudation was confirmed at day 2 by the loss of immunostaining for von Willebrand factor within the internal elastica membrane (Fig 6ADown). At day 18 after endothelial abrasion, reendothelialization had occurred (Fig 6BDown). Neointima was characterized by a dense population of smooth muscle {alpha}-actin immunoreactive cells (Fig 6CDown), whereas CD-45 immunoreactive cells were only occasionally observed (Fig 6DDown).



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Figure 6. Phase contrast micrographs (a and b) of cross sections of the common carotid artery of C57BL/6J apo E-/- mice 2 days (a, x400) and 18 days (b, x200) after endothelial denudation. The absence or presence of the endothelium was revealed by immunostaining for von Willebrand factor. Light micrographs (c and d, x200) of cross sections of C57BL/6J apo E-/- mice immunostained for smooth muscle cell {alpha}-actin (c) and for the common leukocyte antigen CD45 (d).


*    Discussion
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up arrowAbstract
up arrowIntroduction
up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
The present study demonstrates (1) that apo E deficiency was associated with increased neointima formation after endothelial denudation in the carotid arteries; and (2) that adenovirus-mediated transfer of human apo A-I, resulting in a significant increase of HDL in the absence of a decrease of VLDL, was associated with reduced neointima formation. These data suggest that HDL reduce neointima formation associated with endothelial injury, even in the presence of elevated levels of atherogenic ß-VLDL.

Previously it has been demonstrated that apo E-/- mice with elevated plasma levels of ß-VLDL due to delayed clearance of large atherogenic particles from the circulation showed accelerated progression of complex atherosclerotic lesions19,20 and that introduction of a human apo A-I transgene in apo E-/- mice significantly reduced the progression rate.5,6 Elevated HDL cholesterol levels in these mice accounted for 78% of the observed variance of mean lesion area.6 An approximately 50% increase in HDL cholesterol levels has previously been reported after gene transfer with 109 p.f.u. of a human apo A-I adenovirus.33

In the present study, adenovirus-mediated transfer of human apo A-I in C57BL/6J apo E-/- mice resulted in a 2.7-fold increase of HDL cholesterol that was similar to that observed after transfer of a human apo A-I transgene in apo E-/- mice.5,6 This increase was associated with a significant reduction of neointima formation after endothelial denudation. Human apo A-I gene transfer was, however, also associated with an increase of non–HDL phospholipids and triglycerides both in C57BL/6J and in C57BL/6J apo E-/- mice and an increase in non–HDL cholesterol in C57BL/6J mice. The increases of non–HDL cholesterol and phospholipids can be at least partially explained by an adenovirus-induced acute phase response, which is known to be associated with increases of phospholipids and ß- and pre–ß-lipoproteins.34,35 Although gene transfer with the RR5 control virus did not significantly alter non–HDL phospholipids, administration of t-PA control virus led to a significant increase in non–HDL phospholipids, both in C57BL/6J mice and C57BL/6J apo E-/- mice, probably due to the inflammatory response directed against viral gene products and the transgene. This response was more pronounced in mice treated with apo A-I or t-PA virus in comparison with the RR5 virus, which does not produce a transgene. The increase in non–HDL cholesterol seen after gene transfer with 4x109 p.f.u. of human apo A-I adenovirus was also observed after treatment with the same dose of RR5 and t-PA control virus but again was more pronounced after treatment with t-PA virus compared with the RR5 virus. A smaller but statistically significant increase in non–HDL cholesterol has also been observed in human apo A-I transgenic mice and rabbits.4,36

Apo E production by monocytes and macrophages in the vessel wall may reduce atherogenesis independent of circulating lipoproteins in the blood37,38 by redistributing excess cholesterol39 and enhancing reverse cholesterol transport.40 Overexpression of human apo E in the arterial wall of transgenic mice did not alter lipoprotein profiles but decreased lesion area with 70%,37 whereas macrophage-specific expression of human apo E significantly reduced atherosclerosis in the aortic sinus and proximal aorta compared with apo E-/- mice matched for plasma cholesterol.38 It is possible that local vascular production of apo E plays a protective role in neointima formation after endothelial denudation and that lack of apo E containing HDL in the vessel wall is associated with increased intimal hyperplasia. This has however to be tested in apo E-/- mice selectively expressing apo E in macrophages and backcrossed to the C57BL/6J background.

Alternatively, increased neointima formation may be due to a direct effect of atherogenic ß-VLDL on smooth muscle cells exposed after endothelial denudation. It has indeed been shown that lysophosphatidylcholine, a major phospholipid component of atherogenic lipoproteins such as ß-VLDL and oxidized LDL, that may be generated by the action of leukocyte-secreted phospholipase A2 at sites of inflammation, may induce growth factor gene expression that may contribute to the migration and proliferation of smooth muscle cells.41 The stimulation of smooth muscle cell proliferation by VLDL equalled that obtained by direct stimulation with platelet derived growth factor.42 Injury of the endothelium may have been associated with the release of radicals that induced oxidation of ß-VLDL and LDL that infiltrated the arterial wall.

There are different possible mechanisms by which HDL may inhibit neointima formation. Increase of HDL levels may result in a direct inhibition of smooth muscle cell proliferation by inhibition of growth factor synthesis43 or in an indirect inhibition by increasing the degradation of lysophosphatidylcholine by plasma lysolecithin acyltransferase44 or by neutralizing the effect of lysophosphatidylcholine.16

Residual thrombus 18 days after injury was seen in 4 of 16 C57BL/6J apo E-/- mice, 2 of 9 RR5 control virus–treated C57BL/6J apo E-/- mice, 1 of 7 t-PA control virus–treated mice, and 1 of 8 human apo A-I adenovirus–treated mice. The arteries with thrombus were discarded from the final analysis because in the presence of thrombus it is difficult to discern between clot colonization and neointima formation unrelated to presence of thrombus. Furthermore, neointima formation and clot colonization may represent distinct biological processes. The inclusion or exclusion of arteries with thrombus, however, did not affect the overall conclusions of this study.

The absence of detectable neointima formation in C57BL/6J mice was surprising and points to the importance of a fixed genetic background in these studies. Indeed, we confirmed that Swiss Webster mice, which were used in the original guidewire injury model described by Lindner et al,28 developed neointima after endothelial denudation. Our data obtained in C57BL/6J mice are also in agreement with the previous observation of Sullivan et al45 that 17ß estradiol replacement in ovariectomized C57BL/6J mice potently suppressed carotid response to injury.

In conclusion, the present study demonstrates that apo E deficiency induces substantial neointima formation after endothelial denudation in C57BL/6J mice that can be markedly reduced by a transient increase in HDL induced by adenovirus-mediated transfer of human apo A-I.


*    Acknowledgments
 
Bart De Geest is a research assistant of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. This study was supported in part by the Interuniversitaire Attractiepolen (Program 4/34) and by the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (Program G 3063.94). The authors are grateful to H. Bernar, A. Bouché, E. Brouwers, E. Deridder, M. Landeloos, and M. Lox for technical assistance.


*    Footnotes
 
Presented in part during the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 9 to 13, 1996.

Received January 22, 1997; revision received September 8, 1997; accepted September 11, 1997.


*    References
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*References
 
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P. Carmeliet, L. Moons, and D. Collen
Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis
Cardiovasc Res, July 1, 1998; 39(1): 8 - 33.
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