Endothelial expression of human ABCA1 in mice increases plasma HDL cholesterol and reduces diet-induced atherosclerosis.

The role of endothelial ABCA1 expression in reverse cholesterol transport (RCT) was examined in transgenic mice, using the endothelial-specific Tie2 promoter. Human ABCA1 (hABCA1) was significantly expressed in endothelial cells (EC) of most tissues except the liver. Increased expression of ABCA1 was not observed in resident peritoneal macrophages. ApoA-I-mediated cholesterol efflux from aortic EC was 2.6-fold higher (P < 0.0001) for cells from transgenic versus control mice. On normal chow diet, Tie2 hABCA1 transgenic mice had a 25% (P < 0.0001) increase in HDL-cholesterol (HDL-C) and more than a 2-fold increase of eNOS mRNA in the aorta (P < 0.04). After 6 months on a high-fat, high-cholesterol (HFHC) diet, transgenic mice compared with controls had a 40% increase in plasma HDL-C (P < 0.003) and close to 40% decrease in aortic lesions (P < 0.02). Aortas from HFHC-fed transgenic mice also showed gene expression changes consistent with decreased inflammation and apoptosis. Beneficial effects of the ABCA1 transgene on HDL-C levels or on atherosclerosis were absent when the transgene was transferred onto ApoE or Abca1 knockout mice. In summary, expression of hABCA1 in EC appears to play a role in decreasing diet-induced atherosclerosis in mice and is associated with increased plasma HDL-C levels and beneficial gene expression changes in EC.

RNA (2 µg) was reverse-transcribed using ABI TaqMan Reverse Transcriptase Reagents Kit. Real-time qPCR assays were performed with an ABI 7900HT, with 40 ng of cDNA per reaction. A list of ABI TaqMan assays used in the study is shown in supplementary Table I. For expression analysis of mouse 28S rRNA, the following primers and TaqMan MGB probe were custom designed, using ABI Primer Express 3.0 software: forward primer, TCTGCCCAGTGCTCTGAATG; reverse primer, CGTT-TACCCGCGCTTCAT; fl uorogenic probe with FAM and quencher, CAAAGTGAAGAAATTC.
A calibration curve based on the expression of mouse Abca1 gene in the kidney was constructed and used to determine the relative expression of hABCA1, as well as other genes. Normalization of expression data was done by using results for expression of mouse ␤ -actin and 28S rRNA genes. In the latter case, concentration of cDNA in PCR wells was decreased by 512-fold in comparison with test samples to match the level of expression. The presence of contaminating DNA was excluded by performing no reverse transcription controls. SDS 2.2.1 software from ABI7900HT was used for calculations. The relative level of expression was also measured by the comparative C T ( ⌬ ⌬ C T ) method ( 24 ) with mouse ␤ -actin, 18S, or 28S rRNA genes as reference genes.
Gene expression was also determined with Endothelial Cell Biology PCR Array PAMM-015A from SA Biosciences. A complete list of the genes included in the array can be found in supplementary Table II.
Western blot analysis was done as previously described ( 25 ) with an anti-ABCA1 antibody from Abcam (ab18180), which reacts with ABCA1 from both human and mouse tissue. Anti-human/mouse ␤ -actin was used as a control.

Animal studies
Mice were housed under controlled conditions, with a 12/12 h light/dark cycle, and they were fed either a standard rodent autoclaved chow diet, containing 4.0% fat (NIH31 chow diet; Zeigler Brothers Inc., Gardners, PA), or the Paigen diet (a cocoa butter diet TD90221, Harlan Teklad, Madison, WI), containing 1.25% cholesterol and 0.5% cholic acid (HFHC diet). For evaluation of atherosclerosis, en face measurements of the surface of mouse aortas covered by lipid deposits were performed as previously described ( 26 ). Quantifi cation of aortic plaques was done in a blinded fashion using the Image-Pro Plus version 4.1 software (Media Cybernetics, Inc., MD). All animal experiments were approved by the Animal Care and Use Committee of the NHLBI (#H-0050R2).

Analyses of plasma lipids and lipoproteins
Total cholesterol, triglycerides, phospholipids, and free cholesterol in EDTA-plasma were quantifi ed after a 4 h fast using enzymatic kits, as described elsewhere ( 27 ). HDL-C was determined after precipitation of apoB-containing lipoproteins with Raichem HDL-Cholesterol reagent.

Cell culture studies
Resident mouse peritoneal macrophages were obtained after peritoneal lavage ( 28 ) with 10 ml of sterile PBS, without Ca 2+ and Mg 2+ , containing 50 µg/ml of heparin. Cells from 10 mice were placed into 6-well tissue culture plates and allowed to adhere for 1.5 h in serum-free DMEM or RPMI-1640, after which nonadherent cells were removed by rinsing with PBS. Adhered cells were directly used for RNA isolation. Mouse aortic EC were isolated the maintenance of plasma HDL-cholesterol (HDL-C) ( 15 ).
EC express several genes involved in RCT, such as ABCA1, ABCG1, and SR-B1 (16)(17)(18), which are key transporters and receptors that are known to promote either the cellular effl ux or uptake of cholesterol to and from HDL. Endothelial expression of ABCG1 was recently shown to protect against endothelial dysfunction in mice fed a high-fat, high-cholesterol (HFHC) diet ( 19 ). It is also has been shown that ABCA1 participates in the transport of lipid-poor apoA-I across EC, whereas ABCG1 and SR-B1 facilitate the transcytosis of HDL across endothelium ( 18,20,21 ). To further examine the role of ABCA1 in EC, we describe the development of transgenic mice overexpressing human ABCA1 (hABCA1), using the endothelial-specifi c Tie2 promoter ( 22 ). Endothelial-specifi c expression of hABCA1 was found to raise plasma HDL-C and reduce diet-induced atherosclerosis.

Generation of Tie2 hABCA1 transgenic mice
Full-length (6.88 kb) hABCA1 cDNA was fl anked by Not I linkers and inserted into a unique Not I cloning site of the expression vector pSPTg.T2FpAXK ( 22,23 ). The vector has a 2.1 kb mouse Tie2 promoter, a SV40 3 ′ terminal untranslated sequence with a polyA signal, and a 1.6 kb Tie2 enhancer, which results in specifi c transgene expression in EC of mice ( 22,23 ). The plasmid was digested with Sal I, and a 10.7 kb DNA fragment, containing the complete expression cassette, including the Tie2 promoter, was isolated from a 0.8% agarose gel and purifi ed by CsCl density gradient ultracentrifugation (Beckman TL-100 table  top ultracentrifuge at 95,000 rpm for 24 h at 20°C). After dialysis in 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, the DNA fragment was microinjected into pronuclei of fertilized eggs from C57Bl/6J females (Jackson Laboratory, ME).
For genotyping, DNA was isolated from tail clips or blood with the Maxwell16 System (Promega), and then analyzed by real-time quantitative PCR with ABI 7900HT and TaqMan expression assays (see supplementary Table I). Tie2 hABCA1 DNA was used for the calibration curve. A strain of mice containing approximately 60 copies of the transgene per genome in the heterozygous state was produced. Unless otherwise indicated, homozygous transgenic mice were used in all studies. The integrity of the transgene in the genomic DNA was confi rmed by Southern blot hybridization, after digestion with Not I. All breeding was done with C57Bl/6N mice (Taconic, NY). The Tie2 hABCA1 transgene was transferred after fi ve generations onto Abca1 knockout (AbcA1-Ko) mice (Jackson Laboratory, Stock Number 003897) and ApoE-Ko mice (Jackson Laboratory, Stock Number 002052). Sibling controls were used in the analysis of the effect of the hABCA1 transgene in AbcA1-Ko and ApoE-Ko mice. In all other cases, C57Bl/6N mice were used as controls.

Gene expression analysis
For RNA isolation, tissue samples preserved in RNAlater (Invitrogen, Cat. No. AM7020) were homogenized in TRIzol Reagent (Invitrogen, Cat. No. 15596026), in a Precellys24 (Bertin Technologies, France). RNA was isolated with the Invitrogen PureLink RNA Mini Kit (Cat. No. 12183018A) after DNase treatment. RNA concentration and integrity were evaluated with a NanoDrop and an Agilent 2100 analyzer using RNA 6000 Pico from aorta tissue explants, following published protocols ( 29,30 ). Mouse lung EC for effl ux studies were isolated by an immunomagnetic method ( 31 ) using two rounds of selection (see supplementary data for detailed protocols). Cultured mouse lung and aortic EC, which were approximately 90% pure as determined by staining with Dil-acetylated LDL ( 32 ), had a characteristic cobblestone appearance when confl uent and formed tube-like structures when preconfl uent on dishes covered by Poly-D-lysine. Both aortic and lung EC were used at passage numbers less than fi ve. Normal human primary aortic EC (ATCC number PCS-100-011) and human endothelium-derived EA.hy926 cells (ATCC number CRL-2922) were grown according to supplier's recommendations.
To minimize possible effects of cultivation of EC on gene expression, EC were also isolated for gene expression studies after only one round of immunomagnetic selection, using the approach described above or by a rapid method shown below to yield highly purifi ed EC. Minced mouse lungs were incubated with collagenase (2 mg/ml) in 25 ml of DMEM (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C, with gentle agitation every 5 to 8 min. Lung cells were then separated by 12 passes through a 14 gauge blunt-ended needle and then passed through a 70 micron disposable cell strainer into 15 ml of high-glucose DMEM containing 20% fetal calf serum. Cells were pelleted by centrifugation at 400 g for 8 min at 4°C and resuspended in 1 ml of cold PBS with 0.1% BSA. Cells were incubated with anti-ICAM-2 antibody-conjugated magnetic beads for 12 min at RT on a rotating platform. The anti-ICAM-2 beads were prepared by mixing a rat, anti-mouse, FITC-conjugated CD102 antibody (BD Pharmingen, Franklin Lakes, NJ) with sheep anti-rat IgG magnetic beads (Invitrogen, Carlsbad, CA). Vascular EC were then isolated with a Magnetic Particle Concentrator (DynaMag™-2, Invitrogen) and resuspended in 1 ml of PBS with 0.1% BSA. Five rounds of cell purifi cation were performed, and EC were resuspended in 500 µl of PBS with 0.1% BSA. Isolated vascular EC were shown by fl ow cytometry to have 95% purity and 85% viability, using the FITCconjugated anti-CD102 antibody and 7-AAD.
Cholesterol effl ux to apoA-I and HDL was measured in quadruplicate, in at least two independent experiments, as previously described ( 33 ). Cells were labeled by incubation for 24 h in endothelial growth media (see supplementary data), with 10% FCS and 1 µCi/ml of [1,[2][3] H]cholesterol (PerkinElmer). Cells were washed two times with Dulbecco's PBS, without Ca 2+ and Mg 2+ , and cholesterol effl ux to apoA-I or HDL diluted in serum-free growth media, supplemented with 0.1% BSA, was monitored after 18 h.

Statistical analysis
Unless otherwise indicated, all data were analyzed by Student's t -test with GraphPad PRISM version 5.03 software. Statistically signifi cant differences were defi ned as a two-tailed probability of less than 0.05. Values for effl ux studies are expressed as mean ± SD. In all other cases, results are presented as mean ± SEM.

Expression of hABCA1 transgene and endogenous genes
Except for the liver, hABCA1 expression was observed in all tissues tested. Using total tissue homogenates, the level of hABCA1 mRNA expression on a normal chow diet compared with the endogenous murine Abca1 gene ranged between 1% and 12%, depending on the tissue ( Fig. 1A ). Because EC typically composes less than 1-2% of all cells in fl ux to apoA-I ( P < 0.00001) ( Fig. 3A ). No difference in the amount of cholesterol effl ux between control and transgenic aortic EC was observed when HDL was used as an acceptor. most tissues ( 1,2 ), the ABCA1 expression values from total tissue homogenates is an underestimate of the level of hABCA1 expression in endothelial cells. To better examine this issue, we compared the level of hABCA1 in EC isolated from the lung of transgenic mice, human primary aortic EC, and the human EA.hy926 endothelial cell line and found a 4.5-to 11-fold higher level of mRNA for hABCA1 in the cells from the transgenic mice ( Fig. 1B ). When we compared the level of hABCA1 to mAbcA1 in isolated lung EC from transgenic mice, we observed an approximate 2-fold increase in the enrichment of hABCA1 compared with what was observed for total lung tissue homogenates ( Fig. 1A ). The level hABCA1 mRNA, however, was only 10.3 ± 3% relative to that of mAbcA1 in isolated lung EC. By Western blot analysis of primary cultures of aortic EC, the total level of ABCA1 protein was 1.7-fold higher ( P < 0.05) in transgenic mice versus control mice ( Fig. 1C ).
Tie2 promoter constructs, containing a longer promoter segment than used in this study, have also been reported to be active in macrophages of transgenic mice ( 34 ). To confi rm the endothelial specifi city of hABCA1 expression, we compared the level of hABCA1 in lung EC and peritoneal macrophages ( Fig. 2A ). The relative expression of hABCA1 in lung EC was more than 80-fold higher than in peritoneal macrophages, which only showed trace expression of hABCA1. These results are consistent with the known endothelial specifi city of the 2.1 kb Tie2 promoter coupled with the 1.6 kb Tie2 enhancer used in this study ( 22,23 ).
The expression of the hABCA1 transgene in EC of mice did not signifi cantly alter the expression of endogenous mouse Abca1 , Abcg1 , or Sr-b1 genes in most tissues tested, nor in isolated lung EC and peritoneal macrophages ( Table 1 , compare also Fig. 2B, C ). Only in the kidney of transgenic animals was the expression of mouse Abca1 signifi cantly altered (1.8-fold increase; P =0.01, Table 1 ). Note that the level of Abca1 expression in macrophages of normal mice was relatively high compared with lung EC ( Fig. 2B ) (7.7 ± 0.9-fold increase; P < 0.002). In contrast, Sr-b1 was more abundantly expressed in EC compared with macrophages (17.6 ± 3.4-fold increase; P < 0.03). Lung EC and macrophages, however, had a similar high level of Abcg1 expression in both normal and transgenic mice, which was 3 ± 0.7-fold higher than in liver ( P < 0.001) ( Fig. 2D ). We also examined the level of apoA-I gene expression in isolated lung EC, peritoneal macrophages, and aorta, because apoA-I has been reported to be produced by endothelial cells lining the blood-brain barrier ( 35,36 ), but only trace amounts of apoA-I mRNA were detected in these tissues and cells (data not shown).

Effect of hABCA1 transgene on cholesterol effl ux
The functional impact of hABCA1 expression in EC was evaluated by cholesterol effl ux studies ( Fig. 3 ). Experiments with primary cultures of aortic EC isolated from Tie2 hABCA1 transgenic and C57Bl/6N control mice showed that overexpression of hABCA1 transgene in EC was accompanied by a 2.6 ± 0.6-fold increase in cholesterol ef- surface lesions compared with C57Bl/6N control mice ( Fig. 4A ). The effect of the hABCA1 transgene on atherosclerosis was also confi rmed in a second study, in which Tie2 hABCA1 transgenic mice and their nontransgenic sibling controls were placed on HFHC diet for 3 months. Because of relatively short time of this study, only a limited amount of atherosclerosis, however, was observed (supplementary Fig. I). When the Tie2 hABCA1 transgene was transferred onto an AbcA1-Ko background, however, no signifi cant atheroprotection from the transgene or change in plasma lipids was observed ( Fig. 4B ). Similarly, no effect To examine just the role of the transgene in cholesterol effl ux, EC were isolated from Tie2 hABCA1×AbcA1-Ko mice, as well as from AbcA1-Ko mice and wild-type C57Bl/6N control mice ( Fig. 3B ). Because we were unable to obtain aortic EC from tissue explants from AbcA1-Ko mice, only lung EC were used for this part of the study. As expected, cholesterol effl ux to apoA-I was not observed from lung EC isolated from AbcA1-Ko mice. Cholesterol effl ux, however, was approximately 30% higher ( P < 0.03) for cells from Tie2 hABCA1×AbcA1-Ko versus control mice. No signifi cant differences were observed when HDL was used as an acceptor, except for a modest decrease in cholesterol effl ux from cells isolated from AbcA1-Ko mice.

Effect of hABCA1 transgene on plasma lipids
Tie2 hABCA1 transgenic mice had approximately a 25% increase ( P < 0.0001) in plasma HDL-C compared with control mice on a normal chow diet ( Table 2 ). They also had slightly higher plasma phospholipids, total cholesterol, and lower triglycerides. On apoE-Ko or AbcAI-Ko background, the hABCA1 transgene did not, however, signifi cantly alter plasma HDL-C ( Table 2 ). In transgenic mice on AbcA1-Ko background, the level of plasma triglycerides and phospholipids was also increased in comparison with nontransgenic sibling control mice, but no effect from the transgene was observed on HDL-C, which was undetectable in both the transgenic Tie2 hABCA1×AbcA1-Ko mice and the AbcA1-Ko mice ( Table 2 ). When placed on a HFHC diet for 6 months, Tie2 hABCA1 transgenic mice had 40% higher ( P < 0.003) HDL-C than control mice ( Table 2 ), but no other signifi cant lipid changes were observed. In the absence of endogenous AbcA1, lipid profi les of plasma taken from the transgenic mice after 1.5 months on HFHC diet did not signifi cantly differ from nontransgenic sibling control mice (data not shown).

Effect of hABCA1 transgene on atherosclerosis
After 6 months on a HFHC diet, Tie2 hABCA1 transgenic mice had approximately 40% less ( P < 0.02) aortic Tissues from normal C57Bl/6N females (N = 3) or transgenic females (N = 3) or peritoneal macrophages from C57Bl/6N males (N = 10) and transgenic males (N = 11) were analyzed in duplicate in two independent experiments. All results are normalized to expression of mouse AbcA1 in normal kidneys, which was assigned a value of 100%. In two independent experiments (N = 6 in each case), no signifi cant difference was found in the expression of the hABCA1 transgene, AbcG1, or Sr-B1 between females and males for peritoneal macrophages.

DISCUSSION
The two main fi ndings of this study are that increased endothelial-specifi c expression of hABCA1 was associated with increased HDL-C and protected against diet-induced atherosclerosis in C57Bl/6N mice. These changes were observed despite the fact that the level of expression of hABCA1 in aortic EC of transgenic mice was relatively modest ( Fig. 1 ). Based on Western blot analysis, there was a 1.7-fold increase in total ABCA1 protein in aortic EC from transgenic mice compared with control mice ( Fig. 1C ), which is similar to the induction observed for endogenous mouse AbcA1 mRNA in the aorta of mice placed on a HFHC diet ( Fig. 5 ). Level of hABCA1 mRNA in lung EC from transgenic mice was significantly higher (4.5-to 11-fold increase) compared with cultured human primary aortic cells or the EA.hy926 human endothelial cell line ( Fig. 1B ). In contrast, hABCA1 mRNA was present at approximately 10% of the level of mouse AbcA1 in lung EC isolated from transgenic mice. Some of these apparent differences in the level of hABCA1 are likely due to rapid gene expression changes in cells once isolated from tissues and or perhaps differences in the expression of genes in endothelial cells from different tissues ( 2,43 ). Besides having a different promoter, the transgene also had a heterologous SV40 3 ′ UTR, which could have potentially improved the translation of the transgene transcript compared with the endogenous gene. On the basis of the cholesterol effl ux results, the level of increased expression of hABCA1 in the aortic EC from transgenic mice was enough to increase cholesterol effl ux to apoA-I by approximately 2.6-fold ( Fig.  3A ) and to restore cholesterol effl ux to normal levels in lung EC when on the AbcA1-Ko background ( Fig. 3B ). Overall, these results are consistent with only a relatively low level of hABCA1 transgene expression in EC but within a physiologic range and suffi cient to alter cholesterol effl ux. of the hABCA1 transgene was observed in plasma lipids ( Table 2 ) or in the level of atherosclerosis when expressed in mice on an apoE-Ko background ( Fig. 4C ).

Effect of HFHC diet on expression of hABCA1 and endogenous genes
The HFHC diet for 3 months signifi cantly induced in control mice the expression of mouse Abca1 and Abcg1 in several different tissues, particularly the liver and kidney ( Fig. 5A ). In contrast, endogenous Sr-b1 did not show major gene expression changes in response to the diet. Overall, the gene expression changes observed from the HFHC diet treatment in the Tie2 hABCA1 transgenic mice were more blunted compared with control mice, particularly for mAbcA1 ( Fig. 5B ). Only changes for increased expression of Abcg1 in heart and liver and Sr-b1 in the liver were statistically signifi cant. As expected, the HFHC diet did not cause major changes in the tissue expression of the hABCA1 transgene when under the control of the heterologous Tie2 promoter ( Fig. 1A ).
Aortic gene expression was analyzed after 3 months on a HFHC diet, using RT 2 Profi ler PCR array system for genes related to endothelial cell function (see supplementary Tables II and III). The expression of 9 out of the 84 examined genes was found to be changed in Tie2 hABCA1 transgenic mice compared with control mice ( Fig. 6A ). Two of the 3 genes with increased expression in transgenic mice, namely, the Ras homolog gene family member B (Rhob), which is involved in angiogenesis and EC survival ( 37 ), and tissue factor pathway inhibitor (Tfpi), an anticoagulant factor protein ( 38 ), are known to be antiatherogenic. No difference was observed after HFHC diet in the expression of the atheroprotective eNOS gene ( 39 ), but it was 2.2-fold more expressed in Tie2 hABCA1 transgenic mice compared with control mice on a normal chow diet ( Fig. 6B ). Several of the remaining 6 genes with decreased expression in the aortas of transgenic mice, namely, Casp6, Cxc11, Pecam1, fl ects their role in cholesterol traffi cking of EC. As can be observed in Fig. 5 , the expression of the hABCA1 transgene appeared to blunt the induction of the endogenous Abca1 gene in the aorta of the transgenic mice placed on a HFHC diet, but it did not affect the expression of the other endogenous genes that can modulate cholesterol effl ux, namely, Abcg1 and Sr-b1. Tie2 hABCA1 transgenic mice were observed to have a 25% increase in HDL-C on a normal chow diet compared with control mice ( Table 2 ). Interestingly, the transgene in EC was only able to infl uence the level of HDL-C in the presence of the endogenous mouse AbcA1 gene or, in other words, only when the liver and intestine were able to produce nascent HDL, which then interacts with ABCA1 in peripheral cells ( 14,15,44 ). When the Tie2 hABCA1 transgene was transferred onto AbcA1-Ko mice, HDL-C was still very low ( Table 2 ), despite the fact that, in vitro, isolated EC from these mice were able to effl ux cholesterol to apoA-I Besides AbcA1, mouse EC were also found to express AbcG1 and Sr-b1 ( Fig. 2B, C ), which can also contribute to cholesterol effl ux. From data presented in Fig. 2D and Table 1 , it is evident that lung EC had relatively high level of expression of AbcG1, which was close to the level found in peritoneal macrophages and exceeded that found in the liver by approximately 3-fold. Lung EC had especially high level of Sr-b1 expression, which was 18-fold higher than in macrophages and comparable to the level observed in the liver ( Fig. 2B-D ). This observed pattern of relatively high expression of these other transporters probably re- promoter is known, however, to be expressed in most EC ( 22,23 ), but whether increased ABCA1 expression in a particular tissue or site in the vasculature was more critical to the observed changes in HDL metabolism or atheroprotection was not assessed in this study.
Tie2 hABCA1 transgenic mice were also observed to have decreased atherosclerosis when placed on a HFHC diet compared with control mice ( Fig. 4A ). This may be related to the 40% increase in HDL-C in the transgenic mice on the HFHC diet ( Table 2 ). When the Tie2 hABCA1 transgene was transferred to the AbcA1-Ko or apoE-Ko mice and no changes were found in HDL-C, there was no apparent protection from the transgene on atherosclerosis ( Fig. 4B, C ). Besides the effect of HDL in promoting cholesterol effl ux, the increased HDL in the transgenic mice could also have reduced atherosclerosis by some of the other benefi cial properties of HDL ( 48 ). For example, HDL has been shown to reduce infl ammation by suppressing activation of NF-kappa B and also by decreasing the expression of adhesion proteins (48)(49)(50). We observed that in the aortas of theTie2 hABCA1 transgenic mice on a normal chow diet, but not the HFHC diet, there was increased expression of eNOS ( Fig. 6B ), which is known to have several anti-atherogenic effects ( 49,51 ). Caspase 6, Ripk1 [receptor (TNFRSF)-interacting serinethreoninekinase 1] and Tnfsf10 [tumor necrosis factor (ligand) superfamily, member 10] were found to be downregulated in the Tie2 hABCA1 transgenic mice ( Fig. 6A ) and are known to play important roles in programmed cell death or apoptosis (40)(41)(42). The expression of Cxcl1 [chemokine (C-X-C motif) ligand 1] and Pecam1 (platelet/EC adhesion molecule 1), which promote transendothelial migration of leukocytes and vascular infl ammation ( 52,53 ) were also downregulated. In contrast, the expression of the antiatherogenic genes Rhob, which is involved in angiogenesis and EC survival ( 37 ) and Tfpi, an anticoagulant factor protein ( 38 ), were increased in Tie2 hABCA1 transgenic mice compared with control mice on a HFHC diet ( Fig. 6A ).
It is known that a number of infl ammatory disorders, such as systemic vasculitis, Kawasaki disease, and Behcet's disease, are associated with low HDL, endothelial dysfunction, and pro-atherogenic gene expression (54)(55)(56)(57). Endothelial dysfunction can also occur in diabetes ( 58 ) and hypertension ( 59 ), and it is often also associated with decreased HDL-C ( 60,61 ). The mechanistic link between endothelial dysfunction and HDL-C is not clear, but it is likely related to increased production of infl ammatory cytokines and their effect in suppressing HDL production and accelerating its catabolism ( 62 ). Low HDL can then further negatively affect endothelial function, leading to the rapid progression of atherosclerosis ( 63,64 ). Results from this study, therefore, suggest that endothelial expression of ABCA1 can perhaps play an important role in these diseases and in this complex interrelationship between endothelial dysfunction, low HDL, and atherosclerosis.
The authors thank Dr. Thomas N. Sato for providing the pSPTg. T2FpAXK plasmid, Dr. Yoshitaka Sekine for help with Western blot experiments, and Dr. Chengyu Liu for help in creating the transgenic mice. ( Fig. 3B ). Unlike brain capillary EC, which are able to express and secrete apoA-I ( 35,36 ), the expression of apoA-I in EC from other sites was undetectable, which could account for the dependence of EC for nascent HDL produced by the liver and intestine to act as an acceptor for cholesterol effl ux stimulated by ABCA1 in EC. Endothelial expression of hABCA1 transgene was also unable to affect the level of plasma HDL-C in apoE-defi cient mice ( Table 2 ). It was previously shown that the expression of the hABCA1 transgene in liver of apoE-Ko mice also had a minimal effect on plasma lipids, and in fact, increased aortic lesion area compared with apoE-Ko animals ( 45 ). The mechanism for the low levels of HDL-C in apoE-Ko mice is not known, but apoE has been proposed to play an important role in the biogenesis and metabolism of HDL, at least in mice ( 46,47 ).
In this study, the effect of Tie2 hABCA1 transgene was specifi cally determined in lung and aortic EC, where the transgene increased level of expression of ABCA1 and cholesterol effl ux to apoA1 (Figs. 1C and 3). EC from different areas of the vasculature can display remarkable heterogeneity in structure and function ( 2,43 ). The Tie2 In cases when this ratio was <1, it was presented as negative ratio transgenic/control (downregulation). * P < 0.05; ** P р 0.02. (B) RNA from aortas of Tie2 hABCA1 transgenic (N = 3) and control C57Bl/6N (N = 3) females on a normal chow diet or after 3 months on HFHC diet were analyzed for eNOS gene expression. Comparative Ct method was used in calculations. * P < 0.04.