A reconstituted HDL containing V156K or R173C apoA-I exhibited anti-inflammatory activity in apo-E deficient mice and showed resistance to myeloperoxidase-mediated oxidation.
ABSTRACT: It has been hypothesized that blood infusion of reconstituted HDL (rHDL) is a possible therapeutic strategy for the treatment of coronary artery disese. To compare short-term anti-inflammatory activity of wildtype (WT) apoA-I and point mutants, each rHDL containing WT, V156K, or R173C was infused into apo-E deficient atherosclerotic mice. Each rHDL was injected via the tail vein at a dosage of 120 mg/kg of body weight in 0.4 ml of tris-buffered saline (TBS), and blood was then collected at 24 and 48 h post-injection. Although regression activity was observed in each of the rHDL infused groups, a 30% reduction in the lipid-stained area of the aortic sinus was observed in the V156K and R173C-rHDL groups when compared to that of the WT-rHDL group, and this reduction was well correlated with an approximately 60% reduction in the accumulation of macrophages in the lesion area. Additionally, the groups that received the V156K and R173C-rHDL treatments showed smaller increases in the GOT, GPT, interleukin-6, myeloperoxidase (MPO) and lipid hydroperoxide (LPO) serum levels than those that received the WT-rHDL treatment. In addition, the strongest serum paraoxonase and ferric reducing ability was observed in the V156K and R173C-rHDL groups. In vitro nitration and chlorination of apoA-I by MPO treatment revealed that V156K-rHDL and R173C-rHDL were less susceptible to chlorination. Furthermore, rHDL treatment inhibited cellular uptake of oxidized LDL by macrophage cells and the production of proatherogenic species in culture media. In conclusion, blood infusions of the rHDLs exerted in vivo regression activity with anti-inflammatory and antioxidant activity in apo-E deficient mice and THP-1 cells, especially in those that were treated with V156K and R173C apoA-I.
Project description:The goal of this study was to investigate the effects of nonenzymatic glycation on the antiinflammatory properties of apolipoprotein (apo) A-I.Rabbits were infused with saline, lipid-free apoA-I from normal subjects (apoA-I(N)), lipid-free apoA-I nonenzymatically glycated by incubation with methylglyoxal (apoA-I(Glyc in vitro)), nonenzymatically glycated lipid-free apoA-I from subjects with diabetes (apoA-I(Glyc in vivo)), discoidal reconstituted high-density lipoproteins (rHDL) containing phosphatidylcholine and apoA-I(N), (A-I(N))rHDL, or apoA-I(Glyc in vitro), (A-I(Glyc in vitro))rHDL. At 24 hours postinfusion, acute vascular inflammation was induced by inserting a nonocclusive, periarterial carotid collar. The animals were euthanized 24 hours after the insertion of the collar. The collars caused intima/media neutrophil infiltration and increased endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). ApoA-I(N) infusion decreased neutrophil infiltration and VCAM-1 and ICAM-1 expression by 89%, 90%, and 66%, respectively. The apoA-I(Glyc in vitro) infusion decreased neutrophil infiltration by 53% but did not reduce VCAM-1 or ICAM-1 expression. ApoA-I(Glyc in vivo) did not inhibit neutrophil infiltration or adhesion molecule expression. (A-I(Glyc in vitro))rHDL also inhibited vascular inflammation less effectively than (A-I(N))rHDL. The reduced antiinflammatory properties of nonenzymatically glycated apoA-I were attributed to a reduced ability to inhibit nuclear factor-kappaB activation and reactive oxygen species formation.Nonenzymatic glycation impairs the antiinflammatory properties of apoA-I.
Project description:The antiatherogenic properties of apolipoprotein A-I (apoA-I) are derived, in part, from lipidation-state-dependent structural elements that manifest at different stages of apoA-I's progression from lipid-free protein to spherical high-density lipoprotein (HDL). Previously, we reported the structure of apoA-I's N-terminus on reconstituted HDLs (rHDLs) of different sizes. We have now investigated at the single-residue level the conformational adaptations of three regions in the central domain of apoA-I (residues 119-124, 139-144, and 164-170) upon apoA-I lipid binding and HDL formation. An important function associated with these residues of apoA-I is the activation of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for catalyzing HDL maturation. Structural examination was performed by site-directed tryptophan fluorescence and spin-label electron paramagnetic resonance spectroscopies for both the lipid-free protein and rHDL particles 7.8, 8.4, and 9.6 nm in diameter. The two methods provide complementary information about residue side chain mobility and molecular accessibility, as well as the polarity of the local environment at the targeted positions. The modulation of these biophysical parameters yielded new insight into the importance of structural elements in the central domain of apoA-I. In particular, we determined that the loosely lipid-associated structure of residues 134-145 is conserved in all rHDL particles. Truncation of this region completely abolished LCAT activation but did not significantly affect rHDL size, reaffirming the important role of this structural element in HDL function.
Project description:A key cardioprotective effect of high-density lipoprotein involves the interaction of its major protein, apolipoprotein A-I (apoA-I) with ATP-binding cassette transporter A1 (ABCA1), a macrophage cholesterol exporter. ApoA-I is thought to remove cholesterol from macrophages by a cascade of events. First it binds directly to ABCA1, activating signaling pathways, and then it binds to and solubilizes lipid domains generated by ABCA1. HDL isolated from human atherosclerotic lesions and blood of subjects with established coronary artery disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of myeloperoxidase (MPO), a heme protein secreted by macrophages. Here we show that chlorination (but not nitration) of apoA-I by the MPO pathway impairs its ability to interact directly with ABCA1, to activate the Janus kinase 2 signaling pathway, and to promote efflux of cellular cholesterol. In contrast, oxidation of apoA-I has little effect on its ability to stabilize ABCA1 protein or to solubilize phospholipids. Our results indicate that chlorination of apoA-I by the MPO pathway selectively inhibits two critical early events in cholesterol efflux: (1) the binding of apoA-I to ABCA1 and (2) the activation of a key signaling pathway. Therefore, oxidation of apoA-I in the artery wall by MPO-generated chlorinating intermediates may contribute to atherogenesis by impairing cholesterol efflux from macrophages.
Project description:The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of pre? HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of pre? formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-?, IL-6, and IL-1? release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE(-/-) mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL's anti-inflammatory and anti-atherosclerosis properties.
Project description:Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.
Project description:Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed ?-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.
Project description:<h4>Background</h4> Apolipoprotein (apo) A-IV, the third most abundant apolipoprotein in human high density lipoproteins (HDLs), inhibits intestinal and systemic inflammation. This study asks if apoA-IV also inhibits acute vascular inflammation. <h4>Methods</h4> Inflammation was induced in New Zealand White rabbits by placing a non-occlusive silastic collar around the common carotid artery. A single 1 mg/kg intravenous infusion of lipid-free apoA-IV or saline (control) was administered to the animals 24 h before collar insertion. The animals were euthanised 24 h post-collar insertion. Human coronary artery cells (HCAECs) were pre-incubated with reconstituted HDLs containing apoA-IV complexed with phosphatidylcholine, (A-IV)rHDLs, then activated by incubation with tumour necrosis factor (TNF)-α. Cell surface vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the TNF-α-activated HCAECs was quantified by flow cytometry. VCAM-1, ICAM-1 and 3β-hydroxysteroid-Δ24 reductase (DHCR24) mRNA levels were quantified by real time PCR. <h4>Results</h4> Apolipoprotein ApoA-IV treatment significantly decreased collar-induced endothelial expression of VCAM-1, ICAM-1 and neutrophil infiltration into the arterial intima by 67.6 ± 9.9% (p < 0.01), 75.4 ± 6.9% (p < 0.01) and 74.4 ± 8.5% (p < 0.05), respectively. It also increased endothelial expression of DHCR24 by 2.6-fold (p < 0.05). Pre-incubation of HCAECs with (A-IV)rHDLs prior to stimulation with TNF-α inhibited VCAM-1 and ICAM-1 protein levels by 62.2 ± 12.1% and 33.7 ± 5.7%, respectively. VCAM-1 and ICAM-1 mRNA levels were decreased by 55.8 ± 7.2% and 49.6 ± 7.9%, respectively, while DHCR24 mRNA expression increased by threefold. Transfection of HCAECs with DHCR24 siRNA attenuated the anti-inflammatory effects of (A-IV)rHDLs. Pre-incubation of TNF-α-activated HCAECs with (A-IV)rHDLs also inhibited nuclear translocation of the p65 subunit of nuclear factor-κB (NF-κB), and decreased IκBα phosphorylation. <h4>Conclusion</h4> These results indicate that apoA-IV inhibits vascular inflammation in vitro and in vivo by inhibiting NF-κB activation in a DHCR24-dependent manner.
Project description:Human serum amyloid A (SAA) is an exchangeable apolipoprotein (apo) in high-density lipoprotein (HDL) that influences HDL quality and functionality, particularly in the acute phase of inflammation. On the other hand, the structural and functional correlations of HDL containing SAA and apoA-I have not been reported. The current study was designed to compare the change in HDL quality with increasing SAA content in the lipid-free and lipid-bound states in reconstituted HDL (rHDL). The expressed recombinant human SAA1 (13 kDa) was purified to at least 98% and characterized in the lipid-free and lipid-bound states with apoA-I. The dimyristoyl phosphatidylcholine (DMPC) binding ability of apoA-I was impaired severely by the addition of SAA, while SAA alone could not bind with DMPC. The recombinant human SAA1 was incorporated into the rHDL (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC): cholesterol: apoA-I) with various apoA-I:SAA molar ratios from 1:0 to 1:0.5, 1:1 and 1:2. With increasing SAA1 content, the rHDL particle size was reduced from 98 Å to 93 Å, and the α-helicity of apoA-I:SAA was decreased from 73% to 40% for (1:0) and (1:2), respectively. The wavelength maximum fluorescence (WMF) of tryptophan in rHDL was red-shifted from 339 nm to 345 nm for (1:0) and (1:2) of apoA-I:SAA, respectively, indicating that the addition of SAA to rHDL destabilized the secondary structure of apoA-I. Upon denaturation by urea treatment from 0 M to 8 M, SAA showed only a 3 nm red-shift in WMF, while apoA-I showed a 16 nm red-shift in WMF, indicating that SAA is resistant to denaturation and apoA-I had higher conformational flexibility than SAA. The glycation reaction of apoA-I in the presence of fructose was accelerated up to 1.8-fold by adding SAA in a dose-dependent manner than that of apoA-I alone. In conclusion, the incorporation of SAA in rHDL impaired the structural stability of apoA-I and exacerbated glycation of HDL and apoA-I.
Project description:Phagocyte-derived myeloperoxidase (MPO) and pro-inflammatory high density lipoprotein (HDL) associate with rheumatoid arthritis (RA), but the link between MPO and HDL has not been systematically examined. In this study, we investigated whether MPO can oxidise HDL and determined MPO-specific oxidative signature by apoA-1 by peptide mapping in RA subjects with and without known cardiovascular disease (CVD).Two MPO oxidation products, 3-chlorotyrosine and 3-nitrotyrosine, were quantified by tandem mass spectrometry (MS/MS) in in vitro model system studies and in plasma and HDL derived from healthy controls and RA subjects. MPO levels and cholesterol efflux were determined. Site-specific nitration and chlorination of apoA-1 peptides were quantified by MS/MS.RA subjects demonstrated higher levels of MPO, MPO-oxidised HDL and diminished cholesterol efflux. There was marked increase in MPO-specific 3-chlorotyrosine and 3-nitrotyrosine content in HDL in RA subjects consistent with specific targeting of HDL, with increased nitration in RA subjects with CVD. Cholesterol efflux capacity was diminished in RA subjects and correlated inversely with HDL 3-chlorotyrosine suggesting a mechanistic role for MPO. Nitrated HDL was elevated in RACVD subjects compared with RA subjects without CVD. Oxidative peptide mapping revealed site-specific unique oxidation signatures on apoA-1 for RA subjects with and without CVD.We report an increase in MPO-mediated HDL oxidation that is regiospecific in RA and accentuated in those with CVD. Decreased cholesterol efflux capacity due to MPO-mediated chlorination is a potential mechanism for atherosclerosis in RA and raises the possibility that oxidant resistant forms of HDL may attenuate this increased risk.
Project description:Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) provides cardiovascular protection. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to analyze the dynamic solution structure of the apoA-I protein in the apo- and HDL-states and the protein structure conversion in HDL formation. Wild-type apoA-I protein was compared to human variants that either are protective (R173C, Milano) or lead to increased risk for ischaemic heart disease (A164S). Comparable secondary structure distributions in the HDL particles, including significant levels of beta strand/turn, were observed. ApoA-I Milano in HDL displayed larger size heterogeneity, increased protein flexibility, and an altered lipid-binding profile, whereas the apoA-I A164S in HDL showed decrease thermal stability, potentially linking the intrinsic HDL propensities of the variants to disease risk.