Oxidative Modifications of Apolipoprotein(a): Implications for Proinflammatory and Prothrombotic Roles of Lipoprotein(a) in the Vasculature

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) have been identified as a causal risk factor for calcific aortic valve disease (CAVD) and coronary heart disease (CHD). Relationships have recently been identified for genetic factors, such as single nucleotide polymorphisms (SNPs) in the LPA gene, specifically r10455872 and rs3798220, that have been correlated with increased Lp(a) plasma levels and risk of cardiovascular disease (CVD). Apo(a) bears striking homology with the zymogen plasminogen and possesses several similar structural features. A key feature shared between these proteins is the presence of multiple repeats of a kringle domain, which possesses the ability to bind to exposed lysine residues with high affinity. Apo(a) contains several copies of a plasminogen like KIV domain, one of which, KIV10, has been implicated in many proinflammatory processes in vitro. It has been hypothesized that the proinflammatory potential of Lp(a)/apo(a) is derived from the ability to be covalently modified by an oxidized phosphatidylcholine (oxPC) moiety. The work in this dissertation assesses the mechanism by which the oxPC moiety on apo(a) stimulates interleukin-8 (IL-8) production in macrophages. Targeted mutagenesis was used to determine a role for the KIV10 strong lysine binding site (sLBS) in the covalent addition of the oxPC moiety on apo(a) and identified the site of covalent oxPC modification at the amino acid level. Furthermore, characterization of the I4399M variant of apo(a), resulting from the rs3798220 SNP, from a perspective of its distinct structural and functional properties, revealed roles for the polymorphism on the structure and permeability of purified fibrin and plasma clots. The enhanced prothrombotic potential of this variant may be a result of an oxidized methionine residue, as identified by mass spectrometry. The identification of distinct functional properties associated with the oxidative modification of Lp(a)/ apo(a) offers insights into its proatherosclerotic and prothrombotic potentials

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a).

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp(55/57)→Ala(55/57) substitution of kringle (K)IV10)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV10 LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp(57)→Ala(57) substitution in KIV10 that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV10 LBS and is unique to human apo(a), which may explain Lp(a)'s pro-atherogenic potential

Roles of the low density lipoprotein receptor and related receptors in inhibition of lipoprotein(a) internalization by proprotein convertase subtilisin/kexin type 9

<div><p>Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are a causal risk factor for cardiovascular disease. The mechanisms underlying Lp(a) clearance from plasma remain unclear, which is an obvious barrier to the development of therapies to specifically lower levels of this lipoprotein. Recently, it has been documented that monoclonal antibody inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9) can lower plasma Lp(a) levels by 30%. Since PCSK9 acts primarily through the low density lipoprotein receptor (LDLR), this result is in conflict with the prevailing view that the LDLR does not participate in Lp(a) clearance. To support our recent findings in HepG2 cells that the LDLR can act as a <i>bona fide</i> receptor for Lp(a) whose effects are sensitive to PCSK9, we undertook a series of Lp(a) internalization experiments using different hepatic cells, with different variants of PCSK9, and with different members of the LDLR family. We found that PCSK9 decreased Lp(a) and/or apo(a) internalization by Huh7 human hepatoma cells and by primary mouse and human hepatocytes. Overexpression of human LDLR appeared to enhance apo(a)/Lp(a) internalization in both types of primary cells. Importantly, internalization of Lp(a) by LDLR-deficient mouse hepatocytes was not affected by PCSK9, but the effect of PCSK9 was restored upon overexpression of human LDLR. In HepG2 cells, Lp(a) internalization was decreased by gain-of-function mutants of PCSK9 more than by wild-type PCSK9, and a loss-of function variant had a reduced ability to influence Lp(a) internalization. Apo(a) internalization by HepG2 cells was not affected by apo(a) isoform size. Finally, we showed that very low density lipoprotein receptor (VLDLR), LDR-related protein (LRP)-8, and LRP-1 do not play a role in Lp(a) internalization or the effect of PCSK9 on Lp(a) internalization. Our findings are consistent with the idea that PCSK9 inhibits Lp(a) clearance through the LDLR, but do not exclude other effects of PCSK9 such as on Lp(a) biosynthesis.</p></div

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a).

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp(55/57)→Ala(55/57) substitution of kringle (K)IV10)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV10 LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp(57)→Ala(57) substitution in KIV10 that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV10 LBS and is unique to human apo(a), which may explain Lp(a)'s pro-atherogenic potential

Role of LDLR-related receptors in Lp(a) internalization.

<p>(A) HepG2 cells were transiently transfected with the indicated expression vectors or the empty parental pCMV6 vector. Cells were incubated with 10 μg/mL purified human Lp(a) for 4 hours. Cells were extensively washed to remove any bound Lp(a) and lysed to determine the relative amount of internalized Lp(a) compared to β-actin using western blot analysis. Representative western blots for Lp(a), β-actin, and the respective ectopically-expressed receptors are shown. (B) Lp(a) internalization assays were performed as in Panel A, except in the LRP-1-expressing CHO cell line K1 or the LRP-deficient CHO cell line 13-5-1. Also shown is a western blot confirming the absence of LRP-1 in the 13-5-1 cell line. The data represent the means ± s.e.m. of at least 3 independent experiments. *: <i>p</i> < 0.05 vs absence of PCSK9 by one-sample t-test; †: <i>p</i> < 0.05 versus absence of PCSK9 by Student’s t-test.</p

Effect of apo(a) isoform size on the ability of PCSK9 to regulate internalization.

<p>(A) HepG2 cells were treated with the indicated recombinant apo(a) variants (200 nM) in the presence or absence of 10 μg/mL purified PCSK9 for 4 hours. Cells were extensively washed to remove any bound apo(a) and lysed to determine the relative amount of internalized apo(a) compared to β-actin using western blot analysis. The internalization values in the presence of PCSK9 are expressed relative to the values obtained for that particular isoform in the absence of PCSK9. Representative blots are shown. The data represent the means ± s.e.m. of at least 7 independent experiments. *: <i>p</i> < 0.05 vs absence of PCSK9 by Student’s t-test. (B) The percent decrease in apo(a) internalization evoked by PCSK9 was calculated from the data in (A) and is plotted for each apo(a) isoform. No significant differences were observed (by one-way ANOVA).</p