Analysis of Lipoprotein(a) Catabolism

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been identified as an independent risk factor for vascular diseases including coronary heart disease and stroke. In the current study, we have examined the binding and degradation of recombinant forms of apolipoprotein(a) [r-apo(a)], the unique kringle-containing moiety of Lp(a), using a cultured cell model. We found that the incubation of human hepatoma (HepG2) cells with an iodinated 17 kringle-containing (17K) recombinant form of apo(a) resulted in a two-component binding system characterized by a high affinity (Kd = 12 nM), low capacity binding site, and a low affinity (Kd = 249 nM), high capacity binding site. We subsequently determined that the high affinity binding site on HepG2 cells corresponds to the LDL receptor. In the HepG2 cell model, association of apo(a) with the LDL receptor was shown to be dependent on the formation of Lp(a) particles from endogenous LDL. Using an apo(a) mutant incapable of binding to the high affinity site through its inability to form Lp(a) particles (17KΔLBS7,8), we further demonstrated that the LDL receptor does not participate in Lp(a) catabolism. The low affinity binding component observed on HepG2 cells, familial hypercholesterolemia (FH) fibroblasts and human embryonic kidney (HEK) 293 cells may correspond to a member(s) of the plasminogen receptor family, as binding to this site(s) was decreased by the addition of the lysine analogue epsilon-aminocaproic acid. The lysine-dependent nature of the low affinity binding site was further confirmed in HepG2 binding studies utilizing r-apo(a) species with impaired lysine binding ability. We observed a reduction maximum binding capacity for 17K r-apo(a) variants lacking the strong lysine binding site (LBS) in kringle IV type 10 (17KΔAsp) and the very weak LBS in kringle V (17KΔV). Degradation of Lp(a)/apo(a) was found to be mediated exclusively by the low affinity component on both HepG2 cells and FH fibroblasts. Fluorescence confocal microscopy, using the 17K r-apo(a) variant fused to green fluorescent protein, further confirmed that degradation by the low affinity component on HepG2 cells does not proceed by the activity of cellular lysosomes. Taken together, these data suggest a potentially significant route for Lp(a)/apo(a) clearance in vivo.

THE ROLE OF LIPOPROTEIN(a)/APOLIPOPROTEIN(a) IN ENDOTHELIAL DYSFUNCTION: MECHANISTIC STUDIES IN VASCULAR ENDOTHELIUM

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis.

Interactions of Lipoprotein(a) with the Plasminogen System: Mechanisms and Pathophysiological Consequences

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) are associated with increased risk of atherothrombotic disease. Lp(a) is a unique lipoprotein consisting of a low density lipoprotein-like moiety covalently linked to apolipoprotein(a) (apo(a)), a homologue of the fibrinolytic proenzyme plasminogen. Apo(a) is extremely heterogeneous in size with small isoforms being independently associated with increased cardiovascular risk. Several in vitro and in vivo studies have shown that Lp(a)/apo(a) can inhibit tissue-type plasminogen activator (tPA)-mediated plasminogen activation on fibrin surfaces, although the mechanism of inhibition by apo(a) remains controversial. Essential to fibrin clot lysis are a number of plasmin-dependent positive feedback reactions that enhance the efficiency of plasminogen activation, including the plasmin-mediated conversion of Glu1-plasminogen to Lys78-plasminogen. Additionally, abnormal fibrin clot structures have been associated with both an increased risk of cardiovascular disease and elevated Lp(a) levels. Similarly, oxidized phospholipids have been implicated in the development of cardiovascular disease, and are not only preferentially carried by Lp(a) in the plasma but have also been shown to covalently-modify both apo(a) and plasminogen. In this thesis, we built upon the understanding of the role of apo(a) in plasminogen activation on the fibrin/degraded fibrin surface by determining that: (i) apo(a) inhibits plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion and identifying the critical domains in apo(a) responsible for this effect, (ii) apo(a) isoform size does not affect either the inhibition of tPA-mediated plasminogen activation or the inhibition of plasmin-mediated Glu1-plasminogen to Lys78-plasminogen conversion, (iii) apo(a) modifies fibrin clot structure to form more dense clots with thinner fibers and reduced permeability, modifications that enhance the ability of apo(a) to inhibit tPA-mediated plasminogen activation and (iv) the phosphorus content of apo(a) affects its ability to inhibit tPA-mediated plasminogen activation and the phosphorus content of plasminogen affects its ability to be activated by tPA. By understanding these individual reactions, each of which has the potential to affect the broader fibrin clot lysis process, we have expanded our understanding of the overall effect of Lp(a)/apo(a) in the inhibition of plasminogen activation on the fibrin/degraded fibrin surface and thus broadened our understanding of how Lp(a)/apo(a) may mediate the inhibition of thrombolysis in vivo.