All reagents were obtained from Sigma (St. Louis, MO) unless otherwise indicated. Cholesterol was from Steraloids Inc. (Cat. C6760; Newport, RI). [³H]cholesterol (40 Ci/mmol) was from PerkinElmer Life Sciences (Cat. NET139001MC; Waltham, MA). The LCAT activity assay kit was from EMD Biosciences (Cat. 428900; San Diego, CA). The Total Cholesterol E and HDL Cholesterol Kits were from Wako Chemical USA Inc. (Cat. 439-17501; Richmond, VA). The 4-20% Tris-Glycine native gradient gel and running buffer were from Invitrogen (Calsbad, CA) and the high molecular weight marker was from GE healthcare (Cat. 17-0445-01; Piscataway, NJ). Purified human apoA-I, rabbit anti-mouse apoA-I antibody, and purified mouse apoA-I were from Meridian Life Science Inc. (Cincinnati, OH). Goat anti-mouse apoA-I polyclonal antibody was from Rockland Immunochemicals Inc. (600-101-196; Gilbertsville, PA). DELFIA Eu-N1-labeled anti-rabbit antibody and Enhancement solution were from PerkinElmer Life Sciences (Waltham, MA). The BCA Protein Assay Kit was from Pierce Biotechnology (Cat. 23227; Rockford, IL).

ApoE-null mice were purchased from Jackson Laboratories (Bal Harbor, Maine). All animal experimental procedures followed the NIH guideline and were approved by the Merck Research Laboratories Instututional Animal Care and Use Committee. Mice were enrolled at 24 weeks of age after feeding with a high fat Western diet (HF; Harlan Teklad TD#88137) for 20 weeks. The D-4F peptide was prepared in PBS solution containing 50 mM mannitol and dosed at 20 mg/kg body weight subcutaneously once daily for 3 days except where indicated. Samples were collected at each designated time point and were added to tubes containing EDTA. Plasma was isolated by centrifugation and kept frozen at -80?C for further analysis.

Mouse plasma LCAT activity was determined using the LCAT Activity Assay Kit according to the manufacturer's instructions with some modifications. Briefly, 5 ?l aliquots of mouse plasma were incubated at 37?C with the fluorescently labeled cholesterol in assay buffer containing 150 mM NaCl, 10 mM Tris-HCl, 4 mM ?-mercaptoethanol and 1 mM EDTA at pH 7.4. The total assay volume was 200 ?l. After 0, 20 and 40 min., 45 ?l of the reaction mixture was added to 135 ?l of the READ reagent (150 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA at pH 7.4). The conversion of cholesterol (Em. 470 nm) to cholesteryl ester (Em. 390 nm) at 340 nm excitation was determined in a fluorescence microplate reader (SPECTRAmax GEMINI, Molecular Devices Co., Sunnyvale, CA). The change of ratio of the two intensities (470/390) was calculated.

Fresh human or mouse blood was drawn into tubes containing potassium EDTA (Becton Dickinson, Franklin lakes, NJ), and plasma was separated immediately by low-speed centrifugation at 2500 rpm (1430g ) for 30 min at 4?C. Size exclusion chromatography was performed at ambient temperature using a Superose-6 10/300 column on a Bio-Rad FPLC system (Hercules, CA). A 200 ?l aliquot of plasma was injected per run, and eluted in PBS with 1 mM EDTA at a flow rate 0.2 ml/min. A total of 72 fractions with 0.27 ml per fraction were directly collected into microtiter plate for further analysis. Total cholesterol in each fraction was measured using the Cholesterol E Kit, according to the manufacturer's instructions. LCAT activity was measured using the LCAT Activity Assay Kit as described above (Fig. 1). HDL fractions containing LCAT activity were pooled and stored at -80?C. Protein concentration was determined by BCA.

The substrate particles composed of L-?-Phosphatidylcholine derived from egg yolk phosphatidylcholine (EPC), cholesterol with 1% of [³H]cholesterol, and human apoA-I with a molar ratio of 100:10:1 as described ¹⁷, ¹⁸ with some modification. The molar ratio was 100:10:10 if D-4F peptide was used. A control EPC-cholesterol vesicle was also made at a molar ratio of 100:10. After lyophilizing EPC and cholesterol, apoA-I or D-4F and assay buffer (10 mM Tris, 140 mM NaCl, and 1 mM EDTA, pH 7.4) were added to the dry powder. The particles were created by sonication on ice with a microtip at power 23 for 5 min x 5 times with a 2 min pause between each cycle followed by centrifugation at 1000 rpm (228g) for 5 min at 4?C. The supernatant was kept at 4?C for further characterization. The particles were confirmed by native gradient gel electrophoresis and the size of particles was determined against high molecular weight markers (data not shown).

In the LCAT activation assay, the synthetic particles were used as substrates. After a 15 min incubation of 8 ?l of 10-fold diluted substrates with 0.6% bovine serum albumin (fatty acid free) in assay buffer mentioned above, ?-mercaptoethanol was added to a final concentration of 2 mM. The LCAT reaction was initiated by addition of 2 ?l HDL associated-human LCAT and total volume was brought to 40 ?l. After 5 hours at 37 ?C the reaction was quenched by freezing the samples in a -80?C freezer. After the samples were thawed, 20 ?l aliquots were spotted on silica TLC plates. The cholesterol and cholesteryl esters were then separated by thin-layer chromatography in petroleum ether/diethyl ether/acetic acid (80:30:1, v/v/v) mixture. The cholesterol and cholesteryl ester bands were scraped and counted in scintillation cocktail. The percentage conversion of cholesterol to cholesteryl ester was quantified by their respective associated radioactivity.

Mouse macrophage cells (RAW 264.7, ATCC) were cultured in Dulbecco's Modified Eagle Medium (Invitrogen 12491) with 10% FCS and 1% Pen Strep Glutamine (GIBCO 10378) in T225 flasks to a confluency of ~80%. The cells were then seeded into 48 well plates at a concentration of 1 x 10⁵ cells/0.2 ml /well in the same medium now supplemented with ³[H] cholesterol (Perkin-Elmer) to a final 5 ?Ci/ml. After 24 hours, the cells were washed once and starved overnight with serum-free medium supplemented with 0.1% lipid-free BSA. Finally, apoA1 and D-4F with or without cAMP in serum-free medium were added and incubated for another 24 hours. Subsequently, medium and cell lysate (0.5% Triton X-100 in 1 mM HEPES, pH 7.5) were collected and aliquots were mixed separately with scintillation cocktail and counted. Cholesterol efflux was calculated as the percentage of radioactivity associated with medium over the sum of radioactivity of both medium and lysate. The cAMP-dependent cholesterol efflux was determined by the difference between cholesterol measured in the presence and the absence of cAMP.

Plasma total cholesterol and HDL were quantified by Wako cholesterol kits. Apolipoprotein A-I was determined by ELISA and Western blot using rabbit anti-mouse apoA-I and quantified against purified mouse apoA-I. In the ELISA assay, the plates were coated with goat anti-mouse apoA-I polyclonal antibody at a concentration of 0.5 ?g/ml overnight at 4?C. After blocking, the purified mouse apoA-I standard was diluted to 50 ng/ml, and then 2 fold serial dilution, and mouse plasma samples were diluted 50,000 times in assay buffer, 100 ?l diluted samples were added to the wells and incubated at 37?C for 2 hours. Subsequently, rabbit anti-mouse apoA-I antibody was added and incubated at 37?C for 1 hour. Finally, DELFIA Eu-N1-labeled anti-rabbit antibody was added and incubated at room temperature for 30 minutes. The signal was detected by adding the enhancement solution and measured in an EnVision multilabel plate reader.

In addition of apoA-I, apoE is an important protein in the lipoprotein transport system. The atheroprotective properties of apoE has been demonstrated in numerous models. ApoE-null mice on a high fat/high cholesterol diet has also been well documented as an animal model for atherosclerosis ¹⁹. Wild type (WT) and apoE-null mice have different lipoprotein profiles, in that WT mice have high HDL and very low LDL and VLDL while apoE-null mice have high VLDL and very low HDL, with total cholesterol levels approximately 6 times higher than that of WT mice. In order to determine the role of LCAT in atherogenesis, we purified HDL-associated LCAT enzymes and measured their activities in a time course study at 37?C. We used fluorescence-labeled cholesterol as substrate and found that cholesteryl ester is formed in a dose- and time-dependent manner in the presence of LCAT (Fig. 2A). Addition of the HDL-associated LCAT increased the rate of cholesteryl ester formation dramatically. Further, we analyzed the enzyme kinetics. LCAT reaction is a first-order reaction, and rate constants increased linearly with increasing amounts of LCAT enzyme (Fig. 2B), therefore the increase in the rate constant is a measure of LCAT activity. We define a unit of LCAT activity as an increment in the rate constant k_app of 1.0 min^-1. Here, we observed that the specific LCAT activities of WT and apoE-null mice are 0.00199 ? 0.0001058 and 0.001322 ? 0.000124 min^-1?g^-1, respectively, indicating that the specific LCAT activity of HDL-associated LCAT enzyme from apoE-null mice is 1.5-fold lower than that from WT mice.

To optimize the assay condition and understand physiological regulation by metals, the effects of divalent cations and reducing agent ?-mecaptoethanol on LCAT activity were examined. We used EPC-cholesterol vesicles as substrate and added human apoA-I at a final concentration of 0.5 ?M which mimics the condition of the pre-formed particles of EPC/Ch/apoA-I (100:10:1, mol/mol/mol). The reaction was initiated by adding HDL-associated human LCAT enzyme as described in Materials and Methods. With the addition of 1 mM EDTA, an inhibition hierarchy of Zn²⁺>Mg²⁺>Ca²⁺ was observed, providing IC₅₀ values of 1.1, 7.3, and 21.7 mM for Zn²⁺, Mg²⁺ and Ca²⁺, respectively (Fig. 3A, 3B, 3C). The IC₅₀ observed for Zn²⁺ is close to the EDTA concentration added, therefore the IC₅₀ for free (unchelated) Zn²⁺ might be very low and would need to be carefully determined as described for greater accuracy ²⁰. No inhibition was observed with ?-mercaptoethanol at concentrations up to 10 mM (Fig. 3D).

To investigate the ability of human apoA-I and the mimetic peptide D-4F to activate LCAT, apoA-I and D-4F particles were synthesized with EPC and cholesterol as described in Materials and Methods. The particle sizes showed diameters of 8.6-12.2 nm on a native gel separated by electrophoresis (data not shown). The bands on the native gel were broader than particles made with Dipalmitoylphosphatidylcholine (DPPC) which had both size and discoidal shape confirmed by electron microscope (EM) (Fig. 4F).

ABCA1-dependent cholesterol efflux from peripheral tissues is the first step in the RCT process. In the mouse macrophage cell line, RAW 264.7, the basal expression level of ABCA1 is very low, but can be significantly increased by adding cAMP (Fig. 4A). This characteristic is useful for determining the ABCA1-dependent cholesterol efflux in vitro. ApoA-I added to RAW cells without cAMP produced almost no efflux activity, but in the presence of cAMP efflux levels increased dramatically (Fig. 4B). The activities of free apoA-I, apoA-I particles, free D-4F and D-4F particles in the stimulation of ABCA1-dependent cholesterol efflux from macrophages were assayed, resulting in EC₅₀ values of 101 nM, 103 nM, 2.2 ?M and 2.8 ?M, respectively (Fig. 4C, 4D). ApoA-I or D-4F particles or EPC-cholesterol vesicles were used as substrates in the LCAT activation assay. As shown in Fig. 5A and 5B, human HDL-associated LCAT had a basal LCAT activity converting 4.6% to cholesteryl ester; proteoliposome containing ApoA-I increased LCAT activity by 5.9 fold while peptidosome containing D-4F showed no LCAT activation in the assay.

The ability of apoA-I and D-4F to activate LCAT activity was also determined using the method that Datta et al. used to demonstrate LCAT activation by the 5F and 6F, analogs of D-4F ²¹. Following that method, our EPC-cholesterol vesicles were used as substrates and apoA-I and D-4F were titrated (Fig. 5C). Again, apoA-I showed a dose-dependent activation of LCAT with an EC₅₀ of 72.8 nM, while D-4F was unable to activate LCAT up to 28 ?g/ml (12.1 ?M).

In light of the effect of D-4F on LCAT activity in vitro, we performed additional experiments to measure plasma LCAT activity after treatment with D-4F in vivo. 20 mg/kg of D-4F peptide or 50 mM mannitol were injected into apoE-null mice as a single dose or once daily for 3 days. Twenty-four hours after a single dose (24 h) or after last injection of three doses (72 h), mice were sacrificed and plasma samples were analyzed for different parameters. A significant increase of LCAT activity was observed in the 72 h D-4F treated group but not in the 24 h group (Fig. 6A). The plasma samples were analyzed by FPLC and each fraction was assayed for total cholesterol levels. Total cholesterol levels increased slightly in the 24 h group (1,512 ? 54 mg/dl) and slightly decreased in 72 h group (1,218 ? 79 mg/dl) compared to the control group (1,301 ? 124 mg/dl), but none reached statistical significance. Surprisingly, HDL levels did not change 24 h after first dose but increased significantly 24 h after the third dose (from 14.3 ? 3.8 to 28.1 ? 3.5 mg/dl). When HDL cholesterol concentration was plotted against LCAT activity, the treated group (72h) had a parallel increase in both LCAT activity and HDL level, which demonstrated that LCAT activity is positively correlated with plasma HDL cholesterol concentration (Fig. 6B). Plasma taken 1 h and 4 h after the first dosing of D-4F showed no differences from the vehicle controls in LCAT activity or HDL levels, even though D-4F reached its highest concentration in plasma at 4 h. (data not shown). To understand whether the HDL increase is accompanied by an increase in apoA-I, apoA-I ELISAs with rabbit anti-mouse apoA-I antibody were done and confirmed by western blots. The apoA-I protein in the 72 h D-4F group significantly increased 1.27-fold compared to the vehicle group (Fig. 6C).