Regulation of Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT) Synthesis, Degradation, and Translocation by High-Density Lipoprotein2 at a Low Concentration
Abstract—Although plasma HDL2 cholesterol concentration stands in inverse relation to risk for atherosclerotic disease, little is known about the mechanism of the apparent cardioprotection. In mouse P388D1 macrophages, HDL2 at a low concentration (≤40 μg/mL) inhibits macrophage acyl-coenzyme A:cholesterol acyltransferase (ACAT), the enzyme that catalyzes esterification of intracellular cholesterol. The effects of HDL2 on ACAT synthesis, degradation, and intracellular translocation were investigated in mouse P388D1 macrophages. HDL2 at a low concentration enhanced ACAT synthesis but not total ACAT mass. Immunocytochemical studies showed that in the absence of lipoproteins, ACAT associated primarily with the perinuclear region of the cell. The addition of HDL2, however, induced the transfer of ACAT to vesicular structures and the cell periphery adjacent to the plasma membrane. Subfractionation combined with immunoprecipitation complemented these observations and showed that HDL2 promoted the transfer of ACAT to the plasma membrane fraction. Brefeldin A, which inhibits vesicular protein transport from the endoplasmic reticulum to the Golgi compartment in mammalian cells, blocked ACAT translocation and partially restored ACAT activity. These results suggest that HDL2 is an initiating factor in a signal transduction pathway that leads to intracellular ACAT translocation and inactivation.
- Received March 15, 2000.
- Accepted September 22, 2000.
Plasma HDL cholesterol concentration has a strong inverse relation to coronary artery disease incidence.1 2 HDL is an acceptor of macrophage cholesterol,3A and our recent studies of macrophages suggest that low-concentration HDL2 (40 μg/mL) inhibits acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity.3B Recruitment into the artery wall of blood-borne monocytes, which can differentiate into macrophage foam cells, is an early step in atherogenesis.4 5 6 7 8 Lipoprotein-derived free cholesterol (FC) can transfer to the plasma membrane9 and then to the rough endoplasmic reticulum (ER10 ), where it is converted to CE by ACAT.11 12
Human ACAT, which was first cloned by Chang et al,13 is located in the ER.14 15 In ACAT knockout mice, there was no detectable CE in the adrenal gland and no ACAT activity within peritoneal macrophages.16 In C57BL/6J mice fed an atherogenic diet15 and in cholesterol-loaded macrophages in vitro,17 ACAT activity was upregulated. ACAT mRNA levels did not appear to be altered under these conditions, however. Thus, ACAT activity appears to be regulated posttranscriptionally. ACAT contains 5 leucine heptad domains that mediate multimer formation and 2 myristoylation sites that could control attachment to membranes.13
ACAT inhibition by HDL2 at a low concentration could be due to a direct effect on the enzyme, a change in substrate availability, or an altered distribution within the cell. In the present study, we determined whether ACAT synthesis, degradation, and intracellular localization are controlled by HDL2 at a low concentration.
RPMI-1640, gentamicin, and glutamine were from Gibco Laboratories. Defined FBS and other chemicals were purchased from Sigma Chemical Co unless otherwise indicated. [3H]Acetate and [14C]oleate were from Amersham Pharmacia Biotech, Inc, and [35S]methionine was from ICN Biomedicals Inc. Vectors were from Invitrogen. Murine P388D1 cells and ACAT c1 gene sequences were from Drs David P. Via and Ta-Yuan Chang, respectively. HDL2 was isolated from normal human plasma through flotation between d=1.063 g/mL and d=1.12 g/mL, concentrated, sterilized, divided into aliquots, sealed under argon, stored at 4°C, and used within 1 month.
P388D1 cells were maintained in RPMI-1640 medium that contained 10% FBS and 0.1 g/L gentamicin. Before each experiment, cells were pretreated for 24 hours with serum-free RPMI-1640 containing 5% BSA. Cells were incubated with HDL2 for 4 hours in an atmosphere of 95% air and 5% CO2.
ACAT Fusion Antigen Preparation
ACAT cDNA was subcloned from pcDNA/neo/ACAT c1 (8.0 kb) into the expression vector PRsetB (2.9 kb), by which the ACAT gene (bp 1486 to 2455, corresponding to ACAT amino acid residues 31 to 353) was engineered downstream of the T7 promoter and ATG His6 track, using BamHI and EcoRI sites. In-frame insertion of the ACAT C1 gene into the new construct was verified by DNA sequencing. The PRsetB/ACAT vector was transfected into fresh, competent JM109 cells,18 and protein expression was induced by the addition of M13/T7 phages (5 pfu/cell) and isopropyl α-d-thiogalactopyranoside (1 mmol/L). The ACAT fusion protein was isolated from the inclusion bodies through SDS-PAGE (15% acrylamide) and collected via electroelution.
ACAT IgG Purification by Affinity Chromatography
After 4 inoculations with the fusion protein, rabbit serum proteins were precipitated with saturated ammonium sulfate (pH 7.4) and purified with Econo-Pac Serum IgG columns (Bio-Rad). An ACAT affinity gel was prepared with Affi-Gel 10 (Bio-Rad) and the ACAT fusion protein. Antisera were applied to the column, and nonspecific rabbit IgGs were eluted with 1.2 mol/L NaCl and 50 mmol/L phosphate buffer (pH 7.4). ACAT IgGs were eluted in 4.5 mol/L guanidine hydrochloride, dialyzed against PBS at 4°C, sterilized, and stored in PBS.
Cells were incubated for 18 hours with [35S]methionine with or without 40 μg/mL HDL2 (50 μCi/60-mm dish) in methionine- and cysteine-free medium. At various times, cells were harvested and washed in PBS and then lysed in 0.5 mL/dish of extraction buffer (1 mmol/L PMSF, 50 mmol/L Tris, 150 mmol/L NaCl, 1% Nonidet P-40, pH 9.0); 500 μg of each sample was used for immunoprecipitation. The lysate was precleaned with 2 μg/mL preimmune rabbit IgG (Sigma Chemical Co) at 4°C for 4 hours. Protein–preimmune IgG complexes were removed by the addition of 5% by volume 20% protein A–agarose beads (Sigma Chemical Co). The supernatant was continuously incubated with 2 μg/mL ACAT IgG at 4°C overnight. Immune complexes were removed; centrifuged into a pellet; washed 3 times in 50 mmol/L Tris, 100 mmol/L NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS, pH 8.0; washed in PBS; dissolved in SDS-PAGE sample buffer; and separated on 12% SDS-PAGE. Proteins were transferred to a supported nitrocellulose membrane (Bio-Rad), which was exposed to a CS screen (Bio-Rad) for 48 hours. Images were scanned and analyzed with a Molecular Imager (Bio-Rad).
To study ACAT turnover, cells were pretreated for 18 hours with [35S]methionine (50 μCi/60-mm dish) in a methionine- and cysteine-free medium. Cells were washed and incubated in serum-free medium; protein synthesis was terminated with 5 μg/mL cycloheximide for 2 hours. Incubation was continued with 5 μg/mL cycloheximide with or without 40 μg/mL HDL2 for various times. To quantify ACAT mass, cells were labeled with [35S]methionine (25 μCi/100-mm dish) in serum-free medium for 18 hours. Cells were then washed and incubated for 4 hours with 40 μg/mL HDL2 in the fresh labeling medium (serum free, 5% BSA, and 25 μCi [35S]methionine). ACAT turnover and total mass were determined by measuring the amount of immunoprecipitatable ACAT-associated radioactivity.
Immunocytochemistry and Immunofluorescence
Quiescent P388D1 cells were cultured overnight on coverslips in serum-free RPMI supplemented with 5 μg/mL BSA. Cells were incubated for 4 hours with or without 40 μg/mL HDL2. Some cultures also contained 2 μg/mL brefeldin A. After a PBS wash, cell monolayers were fixed in 4% paraformaldehyde at room temperature for 30 minutes. Cells were then permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes, washed in Tris-buffered saline (TBS), and blocked with 1% BSA-TBS for 4 hours.
For immunocytochemistry, cells were incubated overnight at 4°C with ACAT IgG (1 μg/mL). The goat anti-rabbit IgGs (biotinylated, 1:1000 dilution; Elite ABC Kit; Vector Laboratories Intl Corp) were used as the second antibody to blot the cell monolayer at room temperature for 4 hours. After 3 washes with 0.25% Tween 20-TBS, cells were incubated overnight with a 1:2000 dilution of avidin-peroxidase conjugate; 3-amino-9-ethylcarbazole was used to detect the peroxidase activity after a 30-minute incubation. Cells were counterstained in 50% hematoxylin for 30 to 60 seconds. After washing in water and drying, the coverslip was mounted on a slide (Gel/Mount; Biomeda Corp).
For immunofluorescence studies, cells were incubated overnight at 4°C with rabbit anti-ACAT IgG (0.5 μg/mL) and with mouse anti-KDEL IgG (1:400 dilution; StressGen Biotechnologies Corp), which was used to label the ER. The cell monolayer was incubated for 2 hours with Texas Red–conjugated goat anti-rabbit IgG (Vector Laboratories Inc) and fluorescein-conjugated goat anti-mouse F(ab)′2 (Immunotech), respectively, at 1:1000 and 1:50 dilutions. After a PBS wash and drying, the coverslip was mounted on the slide (Gel/Mount; Biomeda Corp) and examined by deconvolution fluorescence microscopy with a Zeiss Axiovert S100 TV microscope and DeltaVision Restoration Microscopy System (Applied Precision, Inc). A Z series of focal planes were digitally imaged and deconvolved with the DeltaVision constrained iterative algorithm to generate high-resolution images.
Common and distinct immunolocalization of ACAT with KDEL sequences was determined on images obtained by fluorescence microscopy. ACAT P388D1 cells were plated on coverslips, treated with or with HDL2 and brefeldin A, and immunostained with either ACAT or KDEL antibodies. Specimens were examined with a Nikon OPTIPHOT microscope with a UFX-IIA photomicrographic attachment. Images were captured and recorded with the Image-Pro Plus Software Package (Media Cybernetics), and fields at 40× were used for quantitative analysis with IP Laboratory Spectrum (Signal Analytic Corporation). Signals derived from ACAT and KDEL stains were separately segmented, and the sizes of areas that covered qualified segments were measured with this program. Ratios (R) of ACAT to KDEL in 50 cells were expressed as mean±SD values, and statistical significance among groups was evaluated with ANOVA; complete superposition of ACAT and KDEL localization corresponds to R=1.
Effect of Brefeldin A on ACAT Activity
Cellular ACAT activity was determined by measuring the rate of cholesteryl [14C]oleate formation from [14C]oleate.19 The serum-free RPMI-1640 medium used for pulsing consisted of 40 μg/mL HDL2, 100 μmol/L [1-14C]oleate–BSA complex, and 2 μg/mL brefeldin A. Cells were incubated in the pulsing medium for 4 hours at either 37°C or 4°C, followed by a 30-minute chase in the serum-free medium at 37°C. Cellular lipids were separated by thin-layer chromatography and quantified by liquid scintillation counting.
Subfractionation and Analysis of Cellular Organelles
P388D1 cells were pretreated for 18 hours with [35S]methionine (50 μCi/60-mm dish); harvested in 5 mmol/L Tris·HCl, 10 mmol/L KCl, 2 mmol/L CaCl2, and 1 mmol/L PMSF, pH 7.4; and lysed via sonication. Sucrose was added to a final concentration of 0.25 mol/L. The homogenate was subjected to sequential centrifugation. Nuclei, mitochondria, plasma membranes, and microsomes, respectively, were collected at 1200g (10 minutes), 3000g (10 minutes), 10 000g (20 minutes), and 100 000g (18 hours). Each fraction was washed in PBS, and 500 μg protein from each fraction was used for the immunoprecipitation. Aliquots were used to identify fractions corresponding to the nucleus, ER, and plasma membrane. The respective markers for these organelles were DNA, RNA, and 5′ nucleotidase activity.
Specificity of Polyclonal ACAT Antibody
To confirm the specificity of ACAT IgG, preimmune IgG and ACAT IgG were used to immunoprecipitate the P388D1 cell lysate. Preimmune IgG nonspecifically precipitated 43-, 40-, 32-, 17-, and 11-kDa proteins. Anti-ACAT IgG precipitated 65-, 46-, and 17-kDa proteins. The 46-kDa protein represents ≈90% of the intensity and corresponds to the mouse ACAT-1 protein, which migrates abnormally in SDS-PAGE.16
Effect of HDL2 on Cellular ACAT Turnover
The rates of ACAT synthesis in the presence and absence of HDL2 at a low concentration (40 μg/mL) were estimated from the rates of incorporation of [35S]methionine into immunoprecipitatable ACAT that was subjected to SDS-PAGE and analyzed for radioactivity (Figure 1A⇓). At 15, 30, and 240 minutes, HDL2 (40 μg/mL) increased synthesis of the 46-kDa ACAT by 80%, 35%, and 20%, respectively. Synthesis of the 65-kDa ACAT was also stimulated by HDL2 but accounted for <10% of newly synthesized ACAT. Synthesis of a nonspecific 32-kDa protein that was associated with preimmune IgG was unchanged.
Studies of ACAT degradation showed that ≈20% of the 65- and 46-kDa ACAT proteins were degraded in serum-free medium after a 30-minute incubation (Figure 1B⇑). No significant additional ACAT degradation (46 and 65 kDa) was observed during the next 210 minutes. The nonspecific 32-kDa protein, however, was degraded by 17% and 90% of the initial amount after 30-minute and 4-hour incubations, respectively. Incubations with HDL2 (40 μg/mL) did not affect the rates of degradation of the 46- and 65-kDa ACAT proteins and the nonspecific 32-kDa protein. These results suggest that ACAT proteins are more resistant to intracellular degradation than the nonspecific 32-kDa protein. HDL2 did not significantly change either the 46-kDa or the 65-kDa ACAT protein after a 4-hour incubation (Figure 1C⇑).
Effect of HDL2 and Brefeldin A on Subcellular ACAT Location
Staining of proteins in P388D1 macrophages with preimmune serum was negative (Figure 2A⇓). Cells exposed to an ACAT-specific antibody showed prominent ACAT-positive regions, which were distinguished by a pinkish brown color. In the absence of HDL2, the ACAT-positive regions were perinuclear (Figure 2B⇓). In the presence of HDL2, ACAT-positive regions were diffuse and shifted from the perinuclear region to the cell periphery (Figure 2D⇓). Some ACAT-positive staining within punctate rings, whose size and shape were consistent with vesicles (Figure 2E⇓), was observed only in the presence of low-concentration HDL2. The translocation of ACAT from 1 part of the cell to another in response to HDL2 was supported by tests with brefeldin A, which inhibits vesicular protein transport from the ER to the Golgi compartment.20 21 22 23 In the absence of HDL2, the specific brown stain denoting the location of ACAT was more concentrated near the nucleus in brefeldin A–treated cells (Figure 2C⇓) than in untreated cells (Figure 2B⇓). When cultures were treated simultaneously with brefeldin A and HDL2, however, the ACAT was confined to the perinuclear region (Figure 2F⇓). Thus, brefeldin A blocked the HDL2-mediated translocation of ACAT away from the perinuclear region.
Subcellular ACAT distribution was also investigated with dual immunofluorescence microscopy (Figure 3⇓). In control cells, regions of green and red fluorescence due to labeling of KDEL and ACAT were confined to a common perinuclear region (Figures 3A⇓ and 3B⇓). The superposition of the fluorescence from KDEL and ACAT labels, seen as a yellow image in control cells (Figure 3C⇓), confirms that both labels were confined to the same region of the cell (R=1.11±0.06).
After incubation with low-concentration HDL2, the fluorescent label associated with KDEL remained associated with the perinuclear region (Figure 3D⇑). In contrast, the fluorescent label associated with ACAT was more diffusely distributed and extended beyond the ER network (Figures 3E⇑ and 3F⇑). Quantitative analysis showed that the ratio of the ACAT-positive to KDEL-positive areas was higher in the presence of HDL2 (R=1.51±0.06; +36%; P<0.0004). Thus, the incubation of low-concentration HDL2 with P388D1 macrophages stimulates the transport of a fraction of the ACAT from the ER to the cell periphery. However, in the presence of brefeldin A and HDL2 (Figures 3G⇑–3I), ACAT and KDEL were confined to the perinuclear region (R=0.99±0.01). Thus, brefeldin A blocks the HDL2-mediated transport of ACAT to the cell periphery.
Effect of HDL2 and Brefeldin A on ACAT Activity
Effect of HDL2 on ACAT Distribution Among Organelles
Cells were incubated with and without HDL2 for 4 hours. The subcellular organelles were isolated from cell lysate via centrifugation and identified (Figures 4A⇓ and 4B⇓). The ACAT associated with each fraction was identified via immunoprecipitation (Figure 4C⇓). In the absence of HDL2, the 46-kDa ACAT was confined exclusively to the fractions containing ER and nuclei. After a 4-hour incubation with HDL2, however, a band corresponding to the 46-kDa ACAT appeared in the plasma membrane fraction but not in the nuclear fraction. Like the 46-kDa ACAT, the 65-kDa ACAT was concentrated in the ER with a small amount being translocated to the plasma membrane fraction in response to incubation with HDL2. The ACAT antibody also precipitated proteins of 86 and 35 kDa from all fractions.
HDL may be cardioprotective through reverse cholesterol transport, whereby HDL accepts FC from peripheral tissue and transports it to the liver for degradation or recycling.24 25 Our previous studies in murine P388D1 macrophages showed that incubation with HDL2 at low concentrations was associated with reduced esterification of cellular cholesterol and enhanced FC efflux.3B The present study was designed to identify the mechanism(s) by which low-concentration HDL2 inhibits ACAT activity.
Specificity of Polyclonal ACAT Antibody
ACAT is highly polymorphic. According to radiation inactivation experiments,26 27 the functional size of ACAT is ≈170 to 200 kDa. Western blot analysis with our polyclonal ACAT antibody raised against an ACAT fusion protein (≈57% of full-length ACAT) revealed 46- and 65-kDa ACAT-positive proteins in murine P388D1 macrophages. The 65-kDa ACAT was a minor component and represented only ≈10% of total ACAT. The appearance of 46-kDa ACAT protein was independent of the time (30 minutes to 48 hours) between protein electrophoresis and of the addition of the protease inhibitor PMSF to the assay solution (data not shown). Thus, it is also probable that the 46-kDa species, which is the dominant form of ACAT in P388D1 macrophages, was not formed through postculture proteolysis. This conclusion is consistent with the data of others. A polyclonal ACAT antibody (DM10) reacted with a 46-kDa mouse ACAT.14 16 Quantitative disappearance of 46-kDa ACAT protein was observed in heterozygotic and homozygotic ACAT knockout mice.16 The discrepancy between the calculated and observed sizes of ACAT proteins may be due to the unusually hydrophobicity of ACAT or to limitations of the antibody.13 14 In ACAT-transfected Sf9 cells, DM9 antibodies recognized a 56-kDa (minor) and a 50-kDa (major) ACAT protein,28 whereas DM10 antibody detected only the 50-kDa ACAT.14 Although ACAT gene expression products exist in multiple isoforms, the 46- and 65-kDa ACAT species are nearly always found, with the former being more prominent in Western blots.
Chemical modification of rabbit ACAT revealed 2 species distinguished by different apparent Ki values toward diethylpyrocarbonate.29 30 The human ACAT gene has 2 promoters that could produce different ACAT mRNAs.31 Multiple ACAT transcripts occur in a variety of mouse15 and rabbit32 tissues, indicating that the ACAT gene may encode different isoforms. According to our data, P388D1 macrophages express ACAT species that are either different isoforms or the same protein with a structural modification. Most of our conclusions are based on changes in the more abundant 46-kDa ACAT.
Translational Regulation of ACAT by HDL2 at Low Concentrations
HDL2 at a low concentration induced synthesis of 46-kDa ACAT protein synthesis (80% more than control) in the first 15 minutes (Figure 1A⇑). After a 4-hour incubation, the rate of ACAT synthesis was only 20% higher in the HDL2-treated macrophages than in control. Similar results were observed with the 65-kDa ACAT, suggesting that the expression of both species is regulated by the same mechanism. These results suggest that the induction of ACAT synthesis is an early cellular response to low-concentration HDL2. During a 4-hour incubation, low-concentration HDL2 did not affect the rate of degradation of either 46- or 65-kDa ACAT (Figure 1B⇑) or the total ACAT pool size (Figure 1C⇑). Taken together, the data in Figure 1⇑ suggest that the amount of new ACAT formed during the 4-hour incubation is small compared with the size of a large, and apparently quite stable, existing ACAT pool. Therefore, even the 80% and 20% increases in ACAT synthesis observed at 15 and 240 minutes, respectively, in the presence of low-concentration HDL2 do not contribute significantly to the overall ACAT pool size. Differences in ACAT activity and cellular CE content are probably not due to differences in cellular ACAT content. This conclusion is not unprecedented: macrophages17 and hepatoblastoma cells33 possess higher ACAT activities than controls without upregulation of ACAT mRNA and protein mass.
Intracellular Translocation of ACAT in Response to Low-Concentration HDL2
HDL2 at low concentrations inhibited ACAT activity and cellular CE.3B Our biochemical evidence supports the hypothesis that low-concentration HDL2 induces the vesicular transfer of ACAT from the ER to the cell periphery. Brefeldin A, which blocks vesicular protein transport, reversed the HDL2-mediated ACAT inhibition (Table⇑). Because our observations were made with intact cells, the HDL2-mediated decrease in ACAT activity could be due to a decrease in the availability of the substrate cholesterol or an increase in cellular phospholipid, which is the major cell membrane component that binds cholesterol. However, cholesterol and phospholipid syntheses in cells incubated with HDL2 alone or with HDL2 plus brefeldin A were the same. Most evidence suggests that brefeldin A does not affect intracellular transport of cholesterol34 35 or phosphatidylethanolamine.36 Thus, brefeldin A must directly affect ACAT translocation and not the translocation or cellular concentration of its substrates.
Using antibody DM10 and immunofluorescence methodology, Chang et al14 found ACAT mainly in the nuclear envelope and the ER. Because of the contiguity between the nuclear membrane and the ER, and the similar pattern of ACAT distribution between the nucleus and ER, some ACAT must be associated with the nuclear membrane or 1 or more perinuclear sites.37 We found essentially the same subcellular distribution of ACAT in the absence of HDL2 (Figures 2B⇑ and 3A⇑–3C). When incubated with low-concentration HDL2, however, ACAT occurred within punctate rings consistent with vesicles (Figure 2E⇑) and at the cell periphery beyond the ER network (Figures 2D⇑ and 3F⇑). A similar effect has been observed in cultured human macrophages in which ACAT was confined to tubular rough ER, but in response to acetylated-LDL, it appeared in small vesicles that were positive for a marker for ER.38 ACAT translocation to the cell periphery in response to low-concentration HDL2, however, was blocked by brefeldin A (Figures 2F⇑ and 3I⇑).
Subcellular fractionation studies showed an accumulation of ACAT in the fraction that contains the ER even after incubation with HDL2. However, the subcellular fractions that contained the ER also contained vesicular bodies, which according to the immunocytochemical findings are ACAT rich (Figure 2D⇑). In the presence of low-concentration HDL2, some ACAT was associated with the plasma membrane fraction. The amount was small, however, and the limitations of this technique preclude a definitive assignment.
We propose that the incubation of P388D1 macrophages with low-concentration HDL2 induces vesicular transport of ACAT from the ER and nuclear envelope to the cell periphery. The attendant inhibition of ACAT, which increases the intracellular cholesterol pool size, would complement the role of HDL2 as a cholesterol acceptor in the reverse cholesterol transport pathway. The HDL2-mediated translocation of ACAT from the ER to the cell periphery is temporally associated with and may be mechanistically linked to reduced ACAT activity, reduced cellular CE content, and enhanced FC efflux. The initiating event could be the binding of HDL2 to a specific cell surface receptor that is part of a signal transduction pathway that leads to intracellular ACAT translocation and inactivation. Translocation could lead to inactivation via several mechanisms. ACAT substrates acyl-CoA and cholesterol may be less accessible to the enzyme. This seems unlikely. Most intracellular membranes contain cholesterol, with the plasma membranes, in particular, being cholesterol rich.39 Although not all acyl-CoA is concentrated in membranes, it can be rapidly transported to membranes via spontaneous transfer.40 Second, ACAT may require a cofactor that resides exclusively in the ER. Third, ACAT may exist in active and inactive structures according to its location in the ER or cell periphery, respectively. Future studies should focus on the latter possibilities and on the structure and function of the 65- and 46-kDa ACAT proteins.
This work was supported by National Institutes of Health grants HL-30914 and HL-56865. Dr Li was a student in the Cardiovascular Sciences Graduate Program of The DeBakey Heart Center.
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