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An abundant dysfunctional apolipoprotein A1 in human atheroma

Abstract

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall.

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Figure 1: Phage display affinity maturation to form a high-affinity mAb specific for apoA1 oxidized by the MPO-H2O2-halide system.
Figure 2: Epitope mapping of affinity-matured mAb r8B5.2
Figure 3: Characterization of oxTrp72-apoA1 recovered from human atherosclerotic plaque.
Figure 4: Characterization of oxTrp72-apoA1 recovered from human plasma.
Figure 5: oxTrp72-apoA1 has impaired function in vivo and is associated with CVD.
Figure 6: Formation of a dysfunctional apoA1 form, oxTrp72-apoA1, within human atherosclerotic lesions.

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Acknowledgements

We thank M. Liang (Chinese Center for Disease Control and Prevention) for the gift of the dicistronic baculoviral shuttle vector used to subclone the scFv gene. This study was supported by US National Institutes of Health (NIH) grants P01HL098055 and HL119962. BioBank, the clinical study from which samples were analyzed, was supported in part from NIH grants P01HL098055, P01HL076491, R01HL103866, P20HL113452 and R01HL103931. This work was also supported in part by a grant from the LeDucq Fondation. S.L.H. is also partially supported by a gift from the Leonard Krieger Fund. Mass spectrometry instrumentation used was housed within the Cleveland Clinic Mass Spectrometry Facility, which is partially supported through a Center of Innovation Award by AB SCIEX.

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Y.H. participated in all laboratory, animal and human studies, assisted in statistical analyses, helped design the experiments and drafted the manuscript. B.S.L., G.S.G., V.G., C.S.K., Z.W. and X.F. assisted with various laboratory and mass spectrometry studies. D.S., J.B., M.K.C., S.Z.B. and C.-C.C. helped perform various animal experiments. J.A.D., D.S., T.K., X.G., M.K.C., J.E.H., A.J.D. and D.P. helped make various bacterial expression clones and produce and purify recombinant proteins used. J.A.D. and S.L. helped with mAb generation and screening. T.K. and T.T.N. helped with ELISA assays. L.L. and Y.W. provided statistical analyses of clinical data. J.A.D., L.C., E.F.P., P.L.F., V.G., W.H.W.T., J.S.P., E.A.F., J.D.S. and S.L.H. provided experimental analysis and expertise. All authors took part in critical review of the manuscript. The project was scientifically conceived and directed by S.L.H.

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Correspondence to Stanley L Hazen.

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W.H.W.T. has previously received research grant support from Abbott Laboratories. S.L.H., Z.W., B.S.L. and J.D.S. report being listed as co-inventors on pending and issued patents held by the Cleveland Clinic relating to cardiovascular diagnostics or therapeutics. S.L.H. reports having been paid as a consultant for the following companies: AstraZeneca Pharmaceuticals LP, Cleveland Heart Lab, Esperion, Lilly, Liposcience Inc., Merck & Co., Inc., Pfizer Inc., Procter & Gamble and Takeda. S.L.H. reports receiving research funds from Cleveland Heart Lab, Liposcience Inc., Procter & Gamble and Takeda. J.D.S. reports having the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from Cleveland Heart Lab and Esperion and being paid as a consultant for Esperion. S.L.H. reports having the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics or therapeutics from the following companies: Cleveland Heart Lab, Esperion, Frantz Biomarkers, LLC and Liposcience Inc. B.S.L. and Z.W. report having the right to receive royalty payments for inventions or discoveries related to cardiovascular diagnostics from Liposcience Inc.

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Huang, Y., DiDonato, J., Levison, B. et al. An abundant dysfunctional apolipoprotein A1 in human atheroma. Nat Med 20, 193–203 (2014). https://doi.org/10.1038/nm.3459

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