Cross-linking modifications of HDL apoproteins by oxidized phospholipids: structural characterization, in vivo detection, and functional implications.

Apolipoprotein A-I (apoA-I) is cross-linked and dysfunctional in human atheroma. Although multiple mechanisms of apoA-I cross-linking have been demonstrated in vitro, the in vivo mechanisms of cross-linking are not well-established. We have recently demonstrated the highly selective and efficient modification of high-density lipoprotein (HDL) apoproteins by endogenous oxidized phospholipids (oxPLs), including γ-ketoalkenal phospholipids. In the current study, we report that γ-ketoalkenal phospholipids effectively cross-link apoproteins in HDL. We further demonstrate that cross-linking impairs the cholesterol efflux mediated by apoA-I or HDL3 in vitro and in vivo Using LC-MS/MS analysis, we analyzed the pattern of apoprotein cross-linking in isolated human HDL either by synthetic γ-ketoalkenal phospholipids or by oxPLs generated during HDL oxidation in plasma by the physiologically relevant MPO-H2O2-NO2- system. We found that five histidine residues in helices 5-8 of apoA-I are preferably cross-linked by oxPLs, forming stable pyrrole adducts with lysine residues in the helices 3-4 of another apoA-I or in the central domain of apoA-II. We also identified cross-links of apoA-I and apoA-II with two minor HDL apoproteins, apoA-IV and apoE. We detected a similar pattern of apoprotein cross-linking in oxidized murine HDL. We further detected oxPL cross-link adducts of HDL apoproteins in plasma and aorta of hyperlipidemic LDLR-/- mice, including cross-link adducts of apoA-I His-165-apoA-I Lys-93, apoA-I His-154-apoA-I Lys-105, apoA-I His-154-apoA-IV Lys-149, and apoA-II Lys-30-apoE His-227. These findings suggest an important mechanism that contributes to the loss of HDL's atheroprotective function in vivo.

Differential expression of osteopontin, and osteoprotegerin mRNA in epicardial adipose tissue between patients with severe coronary artery disease and aortic valvular stenosis: association with HDL subclasses.

BACKGROUND: Previous studies suggest a relationship of the epicardial adipose tissue (EAT) with progression and calcification of the atherosclerotic plaque; however, it is unknown if this tissue expresses genes that may participate on these processes and if the expression of these genes is regulated by high-density lipoprotein (HDL) subclasses. METHODS: To explore this possibility, we determined the mRNA expression by qPCR of a pro-calcifying gene (osteopontin (OPN)), and two anti-calcifying genes (osteoprotegerin (OPG) and osteonectin (ON)), in biopsies of EAT obtained from 15 patients with coronary artery disease (CAD) determined by angiography, and 15 patients with diagnostic of aortic valve stenosis but without CAD as control group. We determined the distribution and composition of HDL subclasses by electrophoresis and their statistical relationship with the gene expression in EAT. RESULTS: EAT from CAD patients showed a higher expression level of OPN and OPG than control group, whereas ON expression was similar between groups. Large HDL subclasses were cholesterol-poor in CAD patients as estimated by the cholesterol-to-phospholipid ratio. A linear regression model showed an independent association of OPN expression with HDL3a-cholesterol, and OPG expression with the relative proportion of HDL3b protein. Logistic analysis determined that OPN expression was positively associated with the presence of atherosclerotic plaque CONCLUSION: OPN, ON, and OPG genes are transcribed in EAT; to the exception of ON, the level of expression was different in CAD patients and control group, and correlated with some HDL subclasses, suggesting a new role of these lipoproteins.

Cubilin maintains blood levels of HDL and albumin.

Cubilin is an endocytic receptor highly expressed in renal proximal tubules, where it mediates uptake of albumin and filtered forms of apoA-I/HDL. Cubilin deficiency leads to urinary loss of albumin and apoA-I; however, the consequences of cubilin loss on the homeostasis of blood albumin and apoA-I/HDL have not been studied. Using mice heterozygous for cubilin gene deletion (cubilin HT mice), we show that cubilin haploinsufficiency leads to reduced renal proximal tubular uptake of albumin and apoA-I and significantly increased urinary loss of albumin and apoA-I. Moreover, cubilin HT mice displayed significantly decreased blood levels of albumin, apoA-I, and HDL. The levels of albumin and apoA-I protein or mRNA expressed in the liver, kidney, or intestine of cubilin HT mice did not change significantly. The clearance rate of small HDL3 particles (density>1.13 g/ml) from the blood increased significantly in cubilin HT mice. In contrast, the rate of clearance of larger HDL2 particles from the blood did not change significantly, indicating a decreased half-life for HDL particles capable of filtering through the glomerulus. On the basis of these findings, we conclude that cubilin deficiency reduces renal salvage and delivery back to the blood of albumin and apoA-I, which decreases blood levels of albumin and apoA-I/HDL. These findings raise the possibility that therapeutic increase of renal cubilin expression might reduce proteinuria and increase blood levels of albumin and HDL.

The specific amino acid sequence between helices 7 and 8 influences the binding specificity of human apolipoprotein A-I for high density lipoprotein (HDL) subclasses: a potential for HDL preferential generation.

Humans have two major high density lipoprotein (HDL) sub-fractions, HDL(2) and HDL(3), whereas mice have a monodisperse HDL profile. Epidemiological evidence has suggested that HDL(2) is more atheroprotective; however, currently there is no direct experimental evidence to support this postulate. The amino acid sequence of apoA-I is a primary determinant of HDL subclass formation. The majority of the alpha-helical repeats in human apoA-I are proline-punctuated. A notable exception is the boundary between helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain and the C-terminal hydrophobic domain. In this study we ask whether the substitution of a proline-containing sequence (PCS) separating other helices in human apoA-I for the non-proline-containing sequence (NPCS) between helices 7 and 8 (residues 184-190) influences HDL subclass association. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL(2). In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL(3). Thus, the specific amino acid sequence between helices 7 and 8 influences HDL subclass association. The wild-type and mutant proteins exhibited similar physicochemical properties except that the two mutants displayed greater lipid-associated stability versus wild-type human apoA-I. These results focus new attention on the influence of the boundary between helices 7 and 8 on the properties of apoA-I. The expression of these mutants in mice may result in the preferential generation of HDL(2) or HDL(3) and allow us to examine experimentally the anti-atherogenicity of the HDL subclasses.