Analysis of Lipoprotein(a) Receptors: Roles in Catabolism and Pericellular Plasminogen Activation

Elevated plasma concentrations of lipoprotein(a) (Lp(a)) have been identified as an independent, causal risk factor for coronary heart disease. Lp(a) resembles low-density lipoprotein (LDL), but is distinguished by the covalent addition of apolipoprotein(a) (apo(a)) to the apolipoproteinB-100 (apoB-100) moiety of LDL. Apo(a) shares homology with the zymogen plasminogen and Lp(a)/apo(a) can inhibit plasminogen activation to the serine protease plasmin on the fibrin surface. In addition, apo(a)/Lp(a) can inhibit fibrinolysis through attenuation of Glu1-plasminogen to Lys78-plasminogen conversion: a key positive feedback mechanism in accelerating plasmin formation. Cellular receptors for Lp(a)/apo(a) are suggested to contribute to the pathophysiological mechanisms of Lp(a) such as inhibition of pericellular plasminogen activation as well as Lp(a) clearance from plasma. The roles and identities of these receptors, however, remain elusive. The work in this dissertation evaluates the roles of receptors implicated in the ability of apo(a)/Lp(a) to inhibit pericellular plasminogen activation as well as in Lp(a) clearance by hepatocytes. Apo(a)/Lp(a) was found to potently inhibit pericellular plasminogen activation on vascular and blood cells through attenuating Glu1- to Lys78-plasminogen conversion. For both these effects, critical roles for the strong lysine binding site in kringle IV type 10 as well as the kringle V domain within apo(a) were identified; there was also no dependency on apo(a) isoform size. We found that the urokinase receptor and integrins αMβ2 and αVβ3 all contribute to plasminogen activation and apo(a) mediated inhibition of plasminogen activation on the cell surfaces of vascular and blood cells, with only a minor role for receptors containing carboxyl-terminal lysines. In vivo evidence suggests a potential role for proprotein convertase subtilisin/kexin type 9 (PCSK9) in Lp(a) clearance through an unidentified receptor. Effects on the number of LDL-receptor (LDLR) molecules were found to underlie the ability of PCSK9 to modulate Lp(a) catabolism in hepatic cells and fibroblasts. This process was dependent on the apoB-100 component of Lp(a) and on clathrin-mediated endocytosis. Taken together, our data contribute importantly to our understanding of the mechanisms of Lp(a) pathogenicity with regard to inhibition of pericellular plasminogen activation and highlight a novel role for PCSK9 activity and the LDLR in modulating Lp(a) catabolism

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis

Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis

Omnipolarity applied to equi-spaced electrode array for ventricular tachycardia substrate mapping

Aims : Bipolar electrogram (BiEGM)-based substrate maps are heavily influenced by direction of a wavefront to the mapping bipole. In this study, we evaluate high-resolution, orientation-independent peak-to-peak voltage (Vpp) maps obtained with an equi-spaced electrode array and omnipolar EGMs (OTEGMs), measure its beat-to-beat consistency, and assess its ability to delineate diseased areas within the myocardium compared against traditional BiEGMs on two orientations: along (AL) and across (AC) array splines. Methods and results: The endocardium of the left ventricle of 10 pigs (three healthy and seven infarcted) were each mapped using an Advisor™ HD grid with a research EnSite Precision™ system. Cardiac magnetic resonance images with late gadolinium enhancement were registered with electroanatomical maps and were used for gross scar delineation. Over healthy areas, OTEGM Vpp values are larger than AL bipoles by 27% and AC bipoles by 26%, and over infarcted areas OTEGM Vpp values are 23% larger than AL bipoles and 27% larger than AC bipoles (P < 0.05). Omnipolar EGM voltage maps were 37% denser than BiEGM maps. In addition, OTEGM Vpp values are more consistent than bipolar Vpps showing less beat-by-beat variation than BiEGM by 39% and 47% over both infarcted and healthy areas, respectively (P < 0.01). Omnipolar EGM better delineate infarcted areas than traditional BiEGMs from both orientations. Conclusion: An equi-spaced electrode grid when combined with omnipolar methodology yielded the largest detectable bipolar-like voltage and is void of directional influences, providing reliable voltage assessment within infarcted and non-infarcted regions of the heart.This work was funded by Abbott Laboratories, St. Paul, MN, USA.S

Role of the Cofilin Activity Cycle in Astrocytoma Migration and Invasion

The cofilin pathway plays a central role in the regulation of actin polymerization and the formation of cell membrane protrusions that are essential for cell migration. Overexpression of cofilin has been linked to the aggressiveness of a variety of different cancers. In these cancers, the phosphorylation of cofilin at Ser3 is a key regulatory mechanism modulating cofilin activity. The activation status of cofilin has been directly linked to tumor invasion. Accordingly, in this study, we examined the expression of cofilin and its activation status in astrocytoma cell lines and astrocytic tumors. We show that cofilin expression was increased and correlated with increasing grade malignant astrocytoma. In addition, both cofilin and LIMK had elevated expression in astrocytoma cell lines. Knockdown of cofilin by siRNA altered astrocytoma cell morphology and inhibited astrocytoma migration and invasion. Conversely, overexpression of a cofilin phosphorylation mutant in an in vivo intracranial xenograft model resulted in a more highly invasive phenotype than those xenographs expressing wild-type cofilin. Animals harboring astrocytomas stably expressing the cofilin phosphorylation mutant (cofilin-S3A) demonstrated marked local invasiveness and spread across the corpus callosum to the contralateral hemisphere in all animals. Taken together, these data indicate that the cofilin activity pathway may represent a novel therapeutic target to diminish the invasion of these highly malignant tumors

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

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

Internalization of apo(a) by primary human hepatocytes.

<p>(A) Hepatocytes were plated on a collagen matrix and then incubated with 200 nM 17K apo(a) in the absence or presence of the indicated concentrations of PCSK9 or 200 mM ε-ACA 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. Representative blots are also shown. The results represent the means ± s.e.m. of at least 3 independent experiments. *: p < 0.05 for indicated pairwise comparisons by one-sample t-test. (B) Hepatocytes were transiently transfected with an expression vector encoding human LDLR (pIR-LDLR-v5) or the corresponding empty expression vector. Internalization of 17K apo(a) was determined as described for Panel A. Also shown are representative blots probed with an anti-apo(a) antibody for apo(a) internalization and an anti-v5 antibody for LDLR overexpression.</p

Effect of GOF and LOF variants of PCSK9 on Lp(a) internalization.

<p>HepG2 cells were treated with either no PCSK9, or 10 μg/mL purified PCSK9 variants. 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 blots are shown. The data represent the means ± s.e.m. of at least 4 independent experiments. *: <i>p</i> < 0.05 vs absence of PCSK9 by one-sample t-test; †: <i>p</i> < 0.05 versus wild-type (WT) PCSK9 by Student’s t-test.</p