Corey A. Scipione

Lipoprotein(a) Catabolism Is Regulated by Proprotein Convertase Subtilisin/Kexin Type 9 through the Low Density Lipoprotein Receptor

Background: Plasma lipoprotein(a) (Lp(a)) levels can be reduced through proprotein convertase subtilisin/kexin type 9 (PCSK9) through an unknown mechanism. Results: Lp(a) catabolism in hepatoma cells and primary fibroblasts is inhibited by PCSK9 via the low density lipoprotein receptor (LDLR). Conclusion: LDLR mediates the effects of PCSK9 on Lp(a) internalization. Significance: Our results provide a mechanistic explanation for the effects of PCSK9 inhibitors on plasma Lp(a) levels. Elevated levels of lipoprotein(a) (Lp(a)) have been identified as an independent risk factor for coronary heart disease. Plasma Lp(a) levels are reduced by monoclonal antibodies targeting proprotein convertase subtilisin/kexin type 9 (PCSK9). However, the mechanism of Lp(a) catabolism in vivo and the role of PCSK9 in this process are unknown. We report that Lp(a) internalization by hepatic HepG2 cells and primary human fibroblasts was effectively reduced by PCSK9. Overexpression of the low density lipoprotein (LDL) receptor (LDLR) in HepG2 cells dramatically increased the internalization of Lp(a). Internalization of Lp(a) was markedly reduced following treatment of HepG2 cells with a function-blocking monoclonal antibody against the LDLR or the use of primary human fibroblasts from an individual with familial hypercholesterolemia; in both cases, Lp(a) internalization was not affected by PCSK9. Optimal Lp(a) internalization in both hepatic and primary human fibroblasts was dependent on the LDL rather than the apolipoprotein(a) component of Lp(a). Lp(a) internalization was also dependent on clathrin-coated pits, and Lp(a) was targeted for lysosomal and not proteasomal degradation. Our data provide strong evidence that the LDLR plays a role in Lp(a) catabolism and that this process can be modulated by PCSK9. These results provide a direct mechanism underlying the therapeutic potential of PCSK9 in effectively lowering Lp(a) levels.

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 Asp55/57→Ala55/57 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 Asp57→Ala57 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.

Oxidized Phospholipids on Lipoprotein(a) Elicit Arterial Wall Inflammation and an Inflammatory Monocyte Response in Humans

Background: Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent cardiovascular risk factor, but the underlying mechanisms responsible for its pathogenicity are poorly defined. Because Lp(a) is the prominent carrier of proinflammatory oxidized phospholipids (OxPLs), part of its atherothrombosis might be mediated through this pathway. Methods: In vivo imaging techniques including magnetic resonance imaging, 18F-fluorodeoxyglucose uptake positron emission tomography/computed tomography and single-photon emission computed tomography/computed tomography were used to measure subsequently atherosclerotic burden, arterial wall inflammation, and monocyte trafficking to the arterial wall. Ex vivo analysis of monocytes was performed with fluorescence-activated cell sorter analysis, inflammatory stimulation assays, and transendothelial migration assays. In vitro studies of the pathophysiology of Lp(a) on monocytes were performed with an in vitro model for trained immunity. Results: We show that subjects with elevated Lp(a) (108 mg/dL [50–195 mg/dL]; n=30) have increased arterial inflammation and enhanced peripheral blood mononuclear cells trafficking to the arterial wall compared with subjects with normal Lp(a) (7 mg/dL [2–28 mg/dL]; n=30). In addition, monocytes isolated from subjects with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce proinflammatory cytokines on stimulation (n=15). In vitro studies show that Lp(a) contains OxPL and augments the proinflammatory response in monocytes derived from healthy control subjects (n=6). This effect was markedly attenuated by inactivating OxPL on Lp(a) or removing OxPL on apolipoprotein(a). Conclusions: These findings demonstrate that Lp(a) induces monocyte trafficking to the arterial wall and mediates proinflammatory responses through its OxPL content. These findings provide a novel mechanism by which Lp(a) mediates cardiovascular disease. Clinical Trial Registration: URL: http://www.trialregister.nl. Unique identifier: NTR5006 (VIPER Study).

Oxidized phospholipids are present on plasminogen, affect fibrinolysis, and increase following acute myocardial infarction.

OBJECTIVES This study sought to assess whether plasminogen, which is homologous to lipoprotein (a) [Lp(a)], contains proinflammatory oxidized phospholipids (OxPL) and whether this has clinical relevance. BACKGROUND OxPL measured on apolipoprotein B-100 (OxPL/apoB), primarily reflecting OxPL on Lp(a), independently predict cardiovascular disease (CVD) events. METHODS The authors examined plasminogen from commercially available preparations and plasma from chimpanzees; gorillas; bonobos; cynomolgus monkeys; wild-type, apoE(-/-), LDLR(-/-), and Lp(a)-transgenic mice; healthy humans; and patients with familial hypercholesterolemia, stable CVD, and acute myocardial infarction (AMI). Phosphocholine (PC)-containing OxPL (OxPC) present on plasminogen were detected directly with liquid chromatography-mass spectrometry (LC-MS/MS) and immunologically with monoclonal antibody E06. In vitro clot lysis assays were performed to assess the effect of the OxPL on plasminogen on fibrinolysis. RESULTS LC-MS/MS revealed that OxPC fragments were covalently bound to mouse plasminogen. Immunoblot, immunoprecipitation, density gradient ultracentrifugation, and enzyme-linked immunosorbent assay analyses demonstrated that all human and animal plasma samples tested contained OxPL covalently bound to plasminogen. In plasma samples subjected to density gradient fractionation, OxPL were present on plasminogen (OxPL/plasminogen) in non-lipoprotein fractions but on Lp(a) in lipoprotein fractions. Plasma levels of OxPL/apoB and OxPL/apo(a) varied significantly (>25×) among subjects and also strongly correlated with Lp(a) levels. In contrast, OxPL/plasminogen levels were distributed across a relatively narrow range and did not correlate with Lp(a). Enzymatic removal of OxPL from plasminogen resulted in a longer lysis time for fibrin clots (16.25 vs. 11.96 min, p = 0.007). In serial measurements over 7 months, OxPL/plasminogen levels did not vary in normal subjects or in patients with stable CVD, but increased acutely over the first month and then slowly decreased to baseline in patients following AMI. CONCLUSIONS These data demonstrate that plasminogen contains covalently bound OxPL that influence fibrinolysis. OxPL/plasminogen represent a second major plasma pool of OxPL, in addition to those present on Lp(a). OxPL present on plasminogen may have pathophysiological implications in AMI and atherothrombosis.

Mechanistic insights into Lp(a)-induced IL-8 expression: a role for oxidized phospholipid modification of apo(a)

Elevated lipoprotein (a) [Lp(a)] levels are a causal risk factor for coronary heart disease. Accumulating evidence suggests that Lp(a) can stimulate cellular inflammatory responses through the kringle-containing apolipoprotein (a) [apo(a)] component. Here, we report that recombinant apo(a) containing 17 kringle (17K) IV domains elicits a dose-dependent increase in interleukin (IL)-8 mRNA and protein expression in THP-1 and U937 macrophages. This effect was blunted by mutation of the lysine binding site in apo(a) kringle IV type 10, which resulted in the loss of oxidized phospholipid (oxPL) on apo(a). Trypsin-digested 17K had the same stimulatory effect on IL-8 expression as intact apo(a), while enzymatic removal of oxPL from apo(a) significantly blunted this effect. Using siRNA to assess candidate receptors, we found that CD36 and TLR2 may play roles in apo(a)-mediated IL-8 stimulation. Downstream of these receptors, inhibitors of MAPKs, Jun N-terminal kinase and ERK1/2, abolished the effect of apo(a) on IL-8 gene expression. To assess the roles of downstream transcription factors, luciferase reporter gene experiments were conducted using an IL-8 promoter fragment. The apo(a)-induced expression of this reporter construct was eliminated by mutation of IL-8 promoter binding sites for either NF-κB or AP-1. Our results provide a mechanistic link between oxPL modification of apo(a) and stimulation of proinflammatory intracellular signaling pathways.

Mechanistic insights into lipoprotein(a)-induced interleukin-8 expression: a role for oxidized phospholipid modification of apolipoprotein(a)

Elevated lipoprotein (a) [Lp(a)] levels are a causal risk factor for coronary heart disease. Accumulating evidence suggests that Lp(a) can stimulate cellular inflammatory responses through the kringle-containing apolipoprotein (a) [apo(a)] component. Here, we report that recombinant apo(a) containing 17 kringle (17K) IV domains elicits a dose-dependent increase in interleukin (IL)-8 mRNA and protein expression in THP-1 and U937 macrophages. This effect was blunted by mutation of the lysine binding site in apo(a) kringle IV type 10, which resulted in the loss of oxidized phospholipid (oxPL) on apo(a). Trypsin-digested 17K had the same stimulatory effect on IL-8 expression as intact apo(a), while enzymatic removal of oxPL from apo(a) significantly blunted this effect. Using siRNA to assess candidate receptors, we found that CD36 and TLR2 may play roles in apo(a)-mediated IL-8 stimulation. Downstream of these receptors, inhibitors of MAPKs, Jun N-terminal kinase and ERK1/2, abolished the effect of apo(a) on IL-8 gene expression. To assess the roles of downstream transcription factors, luciferase reporter gene experiments were conducted using an IL-8 promoter fragment. The apo(a)-induced expression of this reporter construct was eliminated by mutation of IL-8 promoter binding sites for either NF-κB or AP-1. Our results provide a mechanistic link between oxPL modification of apo(a) and stimulation of proinflammatory intracellular signaling pathways.

Identification of human thrombin-activatable fibrinolysis inhibitor in vascular and inflammatory cells

TAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.

Apolipoprotein(a) stimulates nuclear translocation of β-catenin: a novel pathogenic mechanism for lipoprotein(a)

Apolipoprotein(a) (apo(a)), the distinguishing component of lipoprotein(a), accelerates atherosclerosis by provoking endothelial cell dysfunction. This study unravels proatherosclerotic and proinflammatory apo(a)-mediated signaling pathways involving PTEN/PI3K/Akt/GSK3β that result in β-catenin nuclear translocation and up-regulation of COX-2.

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

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 bona fide 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.

Activation of liver X receptor attenuates lysophosphatidylcholine-induced IL-8 expression in endothelial cells via the NF-κB pathway and SUMOylation

The liver X receptor (LXR) is a cholesterol‐sensing nuclear receptor that has an established function in lipid metabolism; however, its role in inflammation is elusive. In this study, we showed that the LXR agonist GW3965 exhibited potent anti‐inflammatory activity by suppressing the firm adhesion of monocytes to endothelial cells. To further address the mechanisms underlying the inhibition of inflammatory cell infiltration, we evaluated the effects of LXR agonist on interleukin‐8 (IL‐8) secretion and nuclear factor‐kappa B (NF‐κB) activation in human umbilical vein endothelial cells (HUVECs). The LXR agonist significantly inhibited lysophosphatidylcholine (LPC)‐induced IL‐8 production in a dose‐dependent manner without appreciable cytotoxicity. Western blotting and the NF‐κB transcription activity assay showed that the LXR agonist inhibited p65 binding to the IL‐8 promoter in LPC‐stimulated HUVECs. Interestingly, knockdown of the indispensable small ubiquitin‐like modifier (SUMO) ligases Ubc9 and Histone deacetylase 4 (HDAC4) reversed the increase in IL‐8 induced by LPC. Furthermore, the LPC‐induced degradation of inhibitory κBα was delayed under the conditions of deficient SUMOylation or the treatment of LXR agonist. After enhancing SUMOylation by knockdown SUMO‐specific protease Sentrin‐specific protease 1 (SENP1), the inhibition of GW3965 was rescued on LPC‐mediated IL‐8 expression. These findings indicate that LXR‐mediated inflammatory gene repression correlates to the suppression of NF‐κB pathway and SUMOylation. Our results suggest that LXR agonist exerts the anti‐atherosclerotic role by attenuation of the NF‐κB pathway in endothelial cells.