Recombinant Apolipoprotein A-I Cysteine Mutant N74C Inhibits The Vascular Remodeling And Carotid Atherosclerosis Induced By A Periarterial Collar In Apolipoprotein E-Deficient Mice

Comprehensive experimental and epidemiological studies have shown an inverse relationship between high density lipoprotein (HDL) or its major protein component, apolipoprotein A-I (apoA-I), and atherosclerosis. Mature apoA-I contains a series of 10 repeating units that comprise approximately 80% of the protein separated by helix-breaking amino acids such as proline. Eight of these repeats contain 22 amino acids while two have 11 amino acids. Apolipoprotein A-IMilano (apoA-IM) and apoA-Iparis, two natural cysteine mutants of apoA-I with dimers as their effective forms, have been shown to have enhanced cardiovascular protective activities. Structural analysis indicated that both mutants positioned at the polar-nonpolar interface on the a helix. According to the Edmundson wheel, the construction features of apoA-I and the unique cysteine mutation positioning of apoA-IM and apoA-Iparis at the polar-nonpolar interface on the helix, seven cysteine mutants of apoA-I were designed and constructed in our laboratory with the similar mutant positions to apoA-IM on the a-helices. We have shown that monomeric cysteine mutant proteins in lipid-free state exhibited different structural features as well as biological functions of lipid binding, anti-oxidation and promotion of cholesterol efflux from THP-1 derived macrophages. Furthermore, our in vivo study documented that A-I(N74C), had an increased anti-inflammatory capability in a LPS-induced endotoxemia mouse model, compared with wild-type apoA-I (apoA-Iwt) and apoA-IM.Aim:In this study, we expressed and purified recombinant proteins including apoA-Iwt, A-I(N74C) and apoA-IM in E. coli. In comparison with apoA-Iwt and apoA-IM, our aim is to investigate the anti-inflammatory, anti-oxidation and antiatherosclerotic functions of A-I(N74C) in vitro and in a rapid atherosclerosis model induced by perivascular carotid collar placement in apoE-/- mice, and to further explore its protection effects and possible mechanism against atherosclerosis. We hope that our findings could shed some lights on the relationship between structure and function of apoA-I.Methods:Recombinant apolipoproteins including apoA-Iwt, A-I(N74C) and apoA-IM were prokaryotic expressed and purified with AKTA FPLC system. After endotoxin removing with Pierce Detoxi-Gel Endotoxin Removing Gel and Columns, purified apoA-Iwt, A-I(N74C) and apoA-IM apolipoproteins were packaged to form recombinant HDL (rHDL) with soybean lecithin, named as rHDLwt, rHDL74 and rHDLmilano respectively, after endotoxin checkout with tachypleus amebocyte lysate. To evaluate the antioxidation ability of lipid-free cysteine mutant proteins, we used copper-mediated LDL oxidation system and thiobarbituric acid-reacting substance (TBARS) assay to measure the oxidation extent of LDL in vitro. Cellular uptake of oxLDL and intracellular lipid accumulation in vitro were performed with THP-1 derived macrophages using red-oil O staining and lipid extraction method.28 male apoE-/- mice fed on HF/HC cholesterol for 2 weeks were divided randomly into 5 groups and received via tail veins with:(1) Saline (n=5,0.3 ml); (2) Soybean lecithin (n=5,134 mg/kg body weight (bw) in 0.3ml); (3) rHDLwt (n=6,40 mg/kg bw in 0.3ml) (4) rHDL74 (n=6,40 mg/kg bw in 0.3ml) (5) rHDLmilano (n=6,40 mg/kg bw in 0.3ml). Five days after surgery, mice were injected through tail vein with indicated rHDLs every ten days for a total of three injections per mouse before mice were terminated. Twenty-four hours after the third tail vein injection of rHDLs, blood samples were obtained from the retro-orbital plexus and serum was isolated and stored at-80℃until analysis. Serum total cholesterol (TC) and triglyceride (TG) concentrations were determined by standard enzymatic methods. Serum HDL levels were measured using a direct HDL-cholesterol reagent kit. Serum interleukin-6 (IL-6) level was measured using a commercial available enzyme-linked immunosorbent assay (ELISA) kit. The ferric reducing ability of serum (FRAS) assay was used to determine the antioxidant activity of rHDLs in vivo. Both carotid arteries were removed and fixed in 10%formaldehyde solution and embedded in paraffin. Carotid artery sections were routinely stained with hematoxylin and eosin to observe the neointima formation and vascular remodeling. To detect macrophage content in atherosclerotic plaques, sections were stained immunohistochemically with rat monoclonal anti-mouse F4/80 antibody.Results:Antioxidant Activity against LDL in vitro:A-I(N74C) and apoA-IM significantly inhibited the malondialdehyde (MDA) formation in a dose-dependent pattern in the presence of 5μM CUSO4 relative to apoA-Iwt. Under Cu2+-mediated oxidation, apoA-Iwt, A-I(N74C) and apoA-IM showed antioxidant activities with IC50 values of 264,72 and 67 mg/L, respectively. Adding the same molar concentration of GSH to the apoA-Iwt to counteract the role of free thiols induced by cysteine mutant of apoA-I resulted in a decrease in IC50 value from 264 to 238 mg/L.Intracellular Lipid Accumulation in THP-1 Derived Macrophages: A-I(N74C) and apoA-IM significantly inhibited the intracellular lipid accumulation, with a 56.78% and 61.08% decrease respectively in the oil-red O staining area, whereas apoA-Iwt decreased only 30.65%. Consistent with the oil-red O staining results, apoA-I(N74C) and apoA-IM treated macrophages also showed a significant lower level of intracellular cholesterol accumulation relative to PBS and apoA-Iwt control groups (P<0.05).Antiatherosclerosis in vivo:In vivo injection of rHDL74 significantly reduced the serum TC and Non-HDL-C concentration, which is as effective as rHDLwt and lower than that of rHDLmilano. rHDL74 injection group showed a dramatic increase in absorption at 593 nm by 42% compared to the initial values, which was significantly higher compared to other groups. 24 hours post the last injection, rHDLwt treatment mice exhibited an acute increase in serum IL-6 (315.0±32.5 pg/ml) compared to Saline or SPC treated mice. Administration of rHDLmilano resulted in a 17% decrease (261.5±35.2 pg/ml), whereas rHDL74 resulted in a 32% decrease (215.6±19.4 pg/ml) in mouse serum IL-6 when compared to mice received with rHDLwt. Compared to rHDLwt, rHDL74 in vivo injection resulted in a significant decrease in the plaque size, the ratio of aorta intima to media, arterial remodeling and macrophages content in the lesions.Conclusion:1. Recombinant A-I(N74C) mutant significantly enhanced the antioxidant capacity against LDL in vitro, which is as effective as apoA-IM. The antioxidation of cysteine mutants did not only result from the free thiols induced by cysteine mutation, the changes of structure and interactions of cysteine mutants with other substrates may play a role in the enhancement of antioxidant activity.2. Recombinant A-I(N74C) mutant significantly inhibited the cellular uptake of oxLDL and intracellular lipid accumulation in THP-1 derived macrophages, which is also as effective as apoA-IM.The intracellular lipid accumulation assay in vitro and lipid profile in vivo together with our previous study on cholesterol efflux, suggesting that the reduction of the intracellular lipid accumulation treated with A-I(N74C) might result from the inhibition of LDL oxidation and cholesterol uptake instead of promoting cholesterol efflux.3. In comparison with rHDLwt and rHDLmilano groups, rHDL74 injection significantly decreased the serum IL-6 level and enhanced the total antioxidant capacity in apoE-/-mice, indicating a higher anti-inflammatory and antioxidant capacity of rHDL74 than rHDLwt and rHDLmilano. 4. In vivo injection of rHDL74 after carotid artery collar placement resulted in significant reduction of the development of carotid atherosclerosis, improvement of atheroma extent and I/M ratio, and reduction of lipid and macrophages content, which is as effective as rHDLmilano.5. The inhibition of rHDL74 in vascular remodeling and progression of atherosclerosis may be due to its enhanced anti-inflammatory and antioxidant activities instead of its ability in reverse cholesterol transport. Cancer (malignant tumor) is a major public health problem in Chinese and many other parts of the world. Most of the chemotherapeutic agent can't maintain an efficient concentration in targeted tissues and cells because of their poor selectivity to target cell and water-solubility, low cell availability and more side effects. HDL is the smallest and highest density lipoprotein in human being, which is reported to be an effective drug delivery model because of its specific structural characteristics and the targeted receptor-mediated uptake mechanism. The major protein component of HDL, apoA-I, plays a critical role in the process of receptor-mediated uptake of core lipid or encapsulated drugs. Mature apoA-I contains a series of 10 repeating units separated by helix-breaking amino acids such as proline. Eight of these repeats contain 22 amino acids while two have 11 amino acids. ApoA-IMilano (apoA-IM) and apoA-Iparis, two natural cysteine mutants of apoA-I, have been shown to have enhanced cardiovascular protective activities. Structural analysis indicated that both mutants positioned at the polar-nonpolar interface on a helix. According to the Edmundson wheel, the construction features of apoA-I and the unique cysteine mutation positioning of apoA-IM and apoA-Iparis at the polar-nonpolar interface on the helix, seven cysteine mutants of apoA-I were designed and constructed in our laboratory with the similar mutant positions to apoA-IM on the a-helices.Aim:In this study, we expressed and purified recombinant apolipoproteins including apoA-Iwt and its seven cysteine substitution mutants in E. coli. The apoA-IM mutant with higher receptor affinity was chosen as a potential delivery vehicle from seven cysteine mutants of apoA-I constructed by our laboratory. In comparison with free HCPT, conventional liposome and rHDLwt-HCPT nanoparticles, our aim is to investigate the effects of rHDLM-HCPT nanoparticles on drug release profiles, tissue distribution and cytotoxicity, and to further explore its feasibility in improvement of cytotoxicity, selectivity, poor solubility and side effect of anticancer drugs using apoA-I mutants or mimic peptides as delivery vehicles.Methods:ApoA-Iwt and its seven cysteine mutants were prokaryotic expressed and purified. The reconstituted apolipoproteins were labeled with FITC. The apoA-IM mutant with higher receptor affinity was chosen as a potential delivery vehicle from seven cysteine mutants of apoA-I constructed by our laboratory using flow cytometry. rHDL-HCPT nanoparticles were prepared by thin-film method with various drug/phospholipid ratios. The phospholipids, apolipoprotein and HCPT component were measured by Phosphalipids, BCA protein assay kits and spectrophotometry respectively. Drug-loading capacity and encapsulation efficiency were determined according to the component detected in rHDL-HCPT nanoparticles. The size and morphology of rHDL-HCPT nanoparticles were observed under transmission electron microscope. The rHDL-HCPT nanoparticles were freeze dried with various amount of sucrose and trehalose as cryopretectant to protect the leakage of encapsulated drug and lipid peroxidation. The drug release profiles of free HCPT, conventional liposome, rHDLwt-HCPT and rHDLM-HCPT nanoparticles were determined in vitro. BALB/c mice were injected via the tail vein with either 10 mg/kg free HCPT or rHDLM-HCPT in 0.05M PBS. At the indicated time intervals (0.5,1,3,5,24 h), five mice were bled until death and the serum, heart, liver, spleen, lung, kidneys and brain were collected and weighted. Tissues collected were homogenized and the amount of HCPT was measured by fluorescence analysis. In comparison with free HCPT, conventional liposome and rHDLwt-HCPT nanoparticles, the enhancement of cytotoxicity of rHDLM-HCPT with rHDLM as delivery vehicle was determined in SKOV-3 and HCT-116 cells in vitro by MTT assay.Results:Relative receptor affinity assay:In comparison with apoA-Iwt, the relative receptor affinity of apoA-IM increased significantly, whereas the receptor affinity of A-I(S52C), A-I(K107C) and A-I(G129C) mutants dramatically reduced. However, the cysteine mutants A-I(N74C), A-I(K195C) and A-I(S228C) kept the similar receptor affinity activity relative to apoA-Iwt.Preparation of rHDL-HCPT nanoparticles:The highest encapsulation efficiency was obtained at the ratio of 1:20 for HCPT and SPC. The drug-loading capacity was calculated to be 4.31%, according to the amount of HCPT, phospholipids and protein components in the detected solution of rHDLM-HCPT nanoparticles. The electron micrographs of the negatively stained rHDL-HCPT nanoparticles showed that, although some of the rHDLwt-HCPT and rHDLM-HCPT nanoparticles represented discoidal particles, the majority of the particles was spherical with a mean diameter of 25.36±10.47 nm and 22.39±10.25 nm respectively, which is similar with the diameter of native HDL.In vitro release study:In contrast to a significant burst release of free HCPT and conventional liposomes, rHDLwt-HCPT and rHDLM-HCPT nanoparticles exhibited a very steady sustained release pattern. After dialyzed for 24 h, the accumulative release fraction of free HCPT, conventional liposomes and rHDLwt-HCPT achieved 90.7%, 65.4% and 41.3%, respectively. However, only 26.9% of HCPT in rHDLM-HCPT released to medium 24h after dialysis.Tissue distribution in vivo:When free HCPT was administered intravenously to mice, none of the HCPT concentration in tissues exceeded 7μg/g tissue. However, when rHDLM was used as delivery vehicle, relative to free HCPT, the concentrations of HCPT in liver, kidneys and spleen increased by 5.5,15.4 and 32.3 times half an hour post injection, and that in lung and plasma increased by 1.8 and 1.3 times respectively. No HCPT was detected in the plasma of free HCPT injection group 5 hours post injection, whereas the plasma concentration of HCPT in rHDLM-HCPT group still kept at 0.421μg HCPT/ml at 24 hours. Compared with free HCPT injection group, the HCPT concentrations in liver, kidneys, spleen and lung increased by 2.2 to 5.7 times in rHDLM-HCPT group 24 hours post injection.Cytotoxicity of rHDL-HCPT nanoparticles in vitro:Compared with free HCPT, IC50 of rHDLM-HCPT for SKOV-3 cells decreased by 70 times, whereas that of conventional liposome and rHDLwt-HCPT decreased by 27 and 58 times respectively. In accordance with the cytotoxicity for SKOV-3 cells, IC50 of rHDLM-HCPT for HCT-116 cells lowered to one-fiftieth and one-tenth relative to free HCPT and conventional liposome. In comparison with rHDLwt-HCPT, IC50 of rHDLM-HCPT for HCT-116 cells decreased by thirty percent.Conclusion:1. Cysteine mutations occurring on the different a helix fragments exerted different influences on the relative receptor affinity of apoA-I. ApoA-IMwith higher receptor affinity than apoA-Iwt is suitable to serve as delivery vehicle.2. The rHDLM-HCPT nanoparticles were prepared by thin film method with apoA-IM as delivery vehicle. The highest encapsulation efficiency was obtained at the ratio of 1:20 for HCPT and SPC and the drug-loading capacity was calculated to be 4.31%. The majority of the rHDL-HCPT particles was spherical with similar mean diameter with the diameter of native HDL.3. Freeze drying with 3% sucrose as cryoprotectant could inhibit the leakage of encapsulated drug and lipid peroxidation.4. rHDLM-HCPT nanoparticles prepared with rHDLM as delivery vehicle exhibited a very steady sustained release pattern in vitro, which is superior to free HCPT, conventional liposomes and rHDLwt-HCPT nanoparticles.5. rHDLM-HCPT nanoparticles prepared with rHDLM as delivery vehicle significantly increased the concentration of HCPT in plasma, liver, spleen, lung and kidneys and Apolipoprotein A-V (apoA-V) was discovered in 2001 by comparative sequencing. Studies of human apoA-V transgenic and apoA-V-deficient mouse models revealed that apoA-V influences plasma triglyceride levels in humans. The major protein component of HDL, apoA-I, was found to play a critical role in cholesterol metabolism and antiatherosclerosis. Both apoA-I and apoA-V gene are located at the APOA-I/CIII/AIV/AVgene cluster on chromosome Ilq23, a locus well known for playing a major role in regulating plasma cholesterol and triglyceride (TG) levels. Compared to the high abundance apolipoproteins such as apoA-I and apoC-III, the plasma levels of apoA-V are up to 2000-fold lower with that of 0.1-0.4 ug/ml. However, apoA-V was highly enriched and most abundant in HDL particles but was rapidly and efficiently redistributed to apoA-V-deficient VLDL upon incubation. Moreover, apoA-V at high levels was shown to promote HDL formation, accounting at least in part for skewed cholesterol redistribution to large HDL particles. Although apoA-V and HDL appear to be related, however, little is known about the effects that apoA-V exerts on the structure and functions of HDL.Aim:In the present study, we expressed and purified recombinant proteins including apoA-Iwt and apoA-V in E. coli. A series of recombinant HDL (rHDLs) were prepared with various amount of apoA-I and apoA-V by sodium cholate dialysis. Our aim is to explore the structural and functional changes in response to increasing the apoA-V content in rHDL on the basis of the determination on rHDL structure and related functions of apoA-I and apoA-V. We hope that our findings could shed some lights on the potential function and precise mechanisms of apoA-V.Methods:Recombinant apoA-Iwt was prokaryotic expressed and purified with AKTA FPLC system. ApoA-V protein was prokaryotic expressed and purified, and renatured in 725 mM sodium cholate or 50mM sodium citrate (pH 3.0) for further application. A series of recombinant HDL (rHDLs) were prepared with various amount of apoA-I and apoA-V by sodium cholate dialysis. The concentrations of phospholipids and apolipoproteins in rHDLs were determined by the Phospholipid and BCA assay kits to compare the phospholipid/apolipoprotein ratios. The kinetics of association with lipid of apoA-I and apoA-V mixtures were determined by DMPC turbidity clearance assay. The size and morphology of rHDL were observed under transmission electron microscope. To evaluate the antioxidation ability of rHDL reconstituted with apoA-I and apoA-V, we used copper-mediated LDL oxidation system and TBARS assay to measure the oxidation extent of LDL in vitro. Cellular uptake of oxLDL and intracellular lipid accumulation in vitro were performed in THP-1 derived macrophages. Two distinct assay systems were employed to analyze the effect of rHDLs on LPL activity with VLDL and commercially obtained TG emulsion (Intralipid) as the substrate. HepG2 cells and BALB/c mice were used to investigate the effects of apoA-V in rHDL on the clearance of VLDL in vitro and in vivo. rHDLs reconstituted with apoA-V alone or with apoA-V and apoA-I were incubated with VLDL at 37°C for 30 min and the transfer of apoA-V from rHDL to VLDL was observed.Results:Preparation of rHDLs: Six rHDLs including AI-rHDL, AV-rHDL, (l:l)-rHDL, (10:l)-rHDL, (100:l)-rHDL and (1000:l)-rHDL, were prepared by cholate dialysis method. The relative ratios of apoA-I and apoA-V in rHDLs detected by SDS-PAGE electrophoresis was in agreement with the initial mass ratios. The final phospholipid/protein mass and molar ratios increased in response to increasing the content of apoA-V of rHDL. DMPC turbidity clearance assay showed an increased DMPC-cleaning speed when more apoA-V included in the reaction mixtures. Electron micrograph of the negatively stained rHDLs observed under transmission electron microscope revealed that the addition of apoA-V to rHDL resulted in the formation of larger particle sizes, which maybe because of enhanced lipid-binding capacity of apoA-V.Antioxidant assay against LDL in vitro: The anti-LDL oxidation of rHDL increased in response to increasing the content of apoA-V of rHDL. In order to verify the antioxidant activity of apoA-V, increased AV-rHDL was added to the same amount of AI-rHDL and found increased antioxidant capacity, suggesting that reconstituted apoA-V may have higher antioxidant activity against LDL in vitro. Lipid-free apoA-V also showed enhanced antioxidant capacity in vitro, which was lower than lipid-bound apoA-V.Intracellular lipid accumulation: AI-rHDL treatment significantly inhibited the uptake of oxLDL and the cellular lipid accumulation in THP-1 derived macrophages. By contrast, the addition of apoA-V in rHDL resulted in the increasing of cellular lipid accumulation.Assay of lipoprotein lipase activity: ApoA-V encapsulated into the rHDL complex could not activate the LPL activity in vitro with VLDL or intralipid as substrate. contrastly, the LPL activity decreased followed the increase of apoA-V component in rHDLs. Pre-incubation of rHDL with VLDL did not promote LPL activation in vitro.VLDL clearance assay:The addition of apoA-V in rHDLs resulted in significantly decreased VLDL clearance by HepG2 cells in vitro and in vivo. Pre-treatment with protamine sulphate completely inhibited the clearance of DiI-VLDL mediated by AV-rHDL, indicating that LPL is critical for VLDL clearance in vivo.Transfer of apoA-V:None apoA-V redistributed from rHDL to VLDL after 30 min incubation.Conclusion:1. A series of rHDLs were prepared with various amount of apoA-V and apoA-I by sodium cholate dialysis method. An increased DMPC-cleaning speed was found when more apoA-V included in the reaction mixtures, which may thus result in more phospholipids component and larger particle sizes of rHDL.2. Reconstituted apoA-V may have higher antioxidant activity against LDL in vitro. The enhanced antioxidant activity of apoA-V in rHDL maybe attribute to the antioxidation of apoA-V itself and the cooperation of apoA-V with apoA-I or phospholipid component in rHDLs.3. ApoA-V appears to have little contribution to the uptake of oxLDL and the intracellular lipid accumulation in THP-1 derived macrophages.4. The increasing of apoA-V in rHDLs did not show significant effects on LPL activation in vitro. Contrastly, the hydrolysis of TG decreased in response to the addition of apoA-V.5. The increasing of apoA-V in rHDLs did not show significant effects on VLDL clearance in vitro and in vivo. Plasma LPL is important for VLDL clearance in vivo.6. After incubation at 37℃for 30 min with VLDL, the apoA-V component of rHDL did not transfer from rHDL to VLDL.7. Inhibited LPL activation and decreased VLDL clearance induced by apoA-V in rHDL in vitro and in vivo maybe attribute to the lost of redistribution of apoA-Vfrom rHDL to VLDL, and the binding of LPL to apoA-V of rHDL.