Hagai Tavori

Serum Proprotein Convertase Subtilisin/Kexin Type 9 and Cell Surface Low-Density Lipoprotein Receptor Evidence for a Reciprocal Regulation

Background— Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. However, dysfunctional LDLR causes hypercholesterolemia without affecting PCSK9 clearance from the circulation. Methods and Results— To study the reciprocal effects of PCSK9 and LDLR and the resultant effects on serum cholesterol, we produced transgenic mice expressing human (h) PCSK9. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation was more evident in the liver. Adrenal LDLR levels were not affected, likely because of the impaired PCSK9 retention in this tissue. In addition, hPCSK9 expression increased hepatic secretion of apolipoprotein B–containing lipoproteins in an LDLR-independent fashion. Expression of hPCSK9 raised serum murine PCSK9 levels by 4.3-fold in wild-type mice and not at all in LDLR−/− mice, in which murine PCSK9 levels were already 10-fold higher than in wild-type mice. In addition, LDLR+/− mice had a 2.7-fold elevation in murine PCSK9 levels and no elevation in cholesterol levels. Conversely, acute expression of human LDLR in transgenic mice caused a 70% decrease in serum murine PCSK9 levels. Turnover studies using physiological levels of hPCSK9 showed rapid clearance in wild-type mice (half-life, 5.2 minutes), faster clearance in human LDLR transgenics (2.9 minutes), and much slower clearance in LDLR−/− recipients (50.5 minutes). Supportive results were obtained with an in vitro system. Finally, up to 30% of serum hPCSK9 was associated with LDL regardless of LDLR expression. Conclusions— Our results support a scenario in which LDLR represents the main route of elimination of PCSK9 and a reciprocal regulation between these 2 proteins controls serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels.

Proprotein Convertase Subtilisin Kexin Type 9 Promotes Intestinal Overproduction of Triglyceride-Rich Apolipoprotein B Lipoproteins Through Both Low-Density Lipoprotein Receptor–Dependent and –Independent Mechanisms

Background— Proprotein convertase subtilisin kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein (LDL) receptor (LDLR), and its deficiency in humans results in low plasma LDL cholesterol and protection against coronary heart disease. Recent evidence indicates that PCSK9 also modulates the metabolism of triglyceride-rich apolipoprotein B (apoB) lipoproteins, another important coronary heart disease risk factor. Here, we studied the effects of physiological levels of PCSK9 on intestinal triglyceride-rich apoB lipoprotein production and elucidated for the first time the cellular and molecular mechanisms involved. Methods and Results— Treatment of human enterocytes (CaCo-2 cells) with recombinant human PCSK9 (10 &mgr;g/mL for 24 hours) increased cellular and secreted apoB48 and apoB100 by 40% to 55% each (P<0.01 versus untreated cells), whereas short-term deletion of PCSK9 expression reversed this effect. PCSK9 stimulation of apoB was due to a 1.5-fold increase in apoB mRNA (P<0.01) and to enhanced apoB protein stability through both LDLR-dependent and LDLR-independent mechanisms. PCSK9 decreased LDLR protein (P<0.01) and increased cellular apoB stability via activation of microsomal triglyceride transfer protein. PCSK9 also increased levels of the lipid-generating enzymes FAS, SCD, and DGAT2 (P<0.05). In mice, human PCSK9 at physiological levels increased intestinal microsomal triglyceride transfer protein levels and activity regardless of LDLR expression. Conclusions— PCSK9 markedly increases intestinal triglyceride-rich apoB production through mechanisms mediated in part by transcriptional effects on apoB, microsomal triglyceride transfer protein, and lipogenic genes and in part by posttranscriptional effects on the LDLR and microsomal triglyceride transfer protein. These findings indicate that targeted PCSK9-based therapies may also be effective in the management of postprandial hypertriglyceridemia.

Loss of Plasma Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) After Lipoprotein Apheresis

Rationale: Lipoprotein apheresis (LA) reduces low-density lipoprotein (LDL) levels in patients with severe familial hypercholesterolemia (FH). We have recently reported that >30% of plasma proprotein convertase subtilisin/kexin 9 (PCSK9) is bound to LDL, thus we predicted that LA would also reduce plasma PCSK9 levels by removing LDL. Objective: Pre- and post-apheresis plasma from 6 patients with familial hypercholesterolemia on 3 consecutive treatment cycles was used to determine changes in PCSK9 levels. Methods and Results: LA drastically reduced plasma LDL (by 77±4%). Concomitantly, PCSK9 levels fell by 52±5%, strongly correlating with the LDL drop (P=0.0322; r2=0.26), but not with decreases in triglyceride (49±13%) or high-density lipoprotein levels (18±2%). Levels of albumin, creatinine, and CK-MB did not show significant changes after LA. Similar to LDL, PCSK9 levels returned to pretreatment values between cycles (2-week intervals). Fractionation of pre- and post-apheresis plasma showed that 81±11% of LDL-bound PCSK9 and 48±14% of apolipoprotein B–free PCSK9 were removed. Separation of whole plasma, purified LDL, or the apolipoprotein B–free fraction through a scaled-down, experimental dextran sulfate cellulose beads column produced similar results. Conclusions: Our results show, for the first time, that modulation of LDL levels by LA directly affects plasma PCSK9 levels, and suggest that PCSK9 reduction is an additional benefit of LA. Because the loss of PCSK9 could contribute to the LDL-lowering effect of LA, then (1) anti-PCSK9 therapies may reduce frequency of LA in patients currently approved for therapy, and (2) LA and anti-PCSK9 therapies may be used synergistically to reduce treatment burden.

Local effects of human PCSK9 on the atherosclerotic lesion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low‐density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE−/− and LDLR−/− mice, which were then placed on a high‐fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow‐derived PCSK9 progressively accumulated in lesions of apoE−/− recipient mice, while increasing the infiltration of Ly6Chi inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b‐ and Ly6Chi‐positive cell numbers in spleens of apoE−/− mice. In vitro, expression of hPCSK9 in LPS‐stimulated macrophages increased mRNA levels of the pro‐inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti‐inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR‐dependent, as PCSK9 protein was not detected in lesions of LDLR−/− recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR‐dependent but cholesterol‐independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction. Copyright

Human carotid atherosclerotic plaque increases oxidative state of macrophages and low-density lipoproteins, whereas paraoxonase 1 (PON1) decreases such atherogenic effects

Human atherosclerotic plaque contains a variety of oxidized lipids, which can facilitate further oxidation. Paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated esterase (lipolactonase), exhibiting antiatherogenic properties. The aims of the present study were to examine the oxidizing potency of the human carotid plaque lipid extract (LE), and the antiatherogenic role of PON1 on LE oxidation competence. Human carotid plaques were extracted by organic solvent, and the extract was incubated with lipoprotein particles, with macrophages, or with probes sensitive to oxidative stress, with or without preincubation with PON1, followed by oxidative-stress assessment. Our findings imply that the LE oxidized LDL, macrophages, and exogenous probes and decreases HDL-mediated cholesterol efflux from macrophages, in a dose-dependent manner. Incubation of PON1 with LE significantly affects LE composition, reduces LE atherogenic properties, and decreases the extracts total peroxide concentration by 44%, macrophage oxidation by 25%, and probe oxidation by up to 52%. We conclude that these results expand our understanding of how the plaque itself accelerates atherogenesis and provides an important mechanism for attenuation of atherosclerosis development by the antioxidant action of PON1 on the atherosclerotic plaque.

PCSK9 Association With Lipoprotein(a)

RATIONALE Lipoprotein(a) [Lp(a)] is a highly atherogenic low-density lipoprotein-like particle characterized by the presence of apoprotein(a) [apo(a)] bound to apolipoprotein B. Proprotein convertase subtilisin/kexin type 9 (PCSK9) selectively binds low-density lipoprotein; we hypothesized that it can also be associated with Lp(a) in plasma. OBJECTIVE Characterize the association of PCSK9 and Lp(a) in 39 subjects with high Lp(a) levels (range 39-320 mg/dL) and in transgenic mice expressing either human apo(a) only or human Lp(a) (via coexpression of human apo(a) and human apolipoprotein B). METHODS AND RESULTS We show that PCSK9 is physically associated with Lp(a) in vivo using 3 different approaches: (1) analysis of Lp(a) fractions isolated by ultracentrifugation; (2) immunoprecipitation of plasma using antibodies to PCSK9 and immunodetection of apo(a); (3) ELISA quantification of Lp(a)-associated PCSK9. Plasma PCSK9 levels correlated with Lp(a) levels, but not with the number of kringle IV-2 repeats. PCSK9 did not bind to apo(a) only, and the association of PCSK9 with Lp(a) was not affected by the loss of the apo(a) region responsible for binding oxidized phospholipids. Preferential association of PCSK9 with Lp(a) versus low-density lipoprotein (1.7-fold increase) was seen in subjects with high Lp(a) and normal low-density lipoprotein. Finally, Lp(a)-associated PCSK9 levels directly correlated with plasma Lp(a) levels but not with total plasma PCSK9 levels. CONCLUSIONS Our results show, for the first time, that plasma PCSK9 is found in association with Lp(a) particles in humans with high Lp(a) levels and in mice carrying human Lp(a). Lp(a)-bound PCSK9 may be pursued as a biomarker for cardiovascular risk.

On the Function and Homeostasis of PCSK9: Reciprocal Interaction with LDLR and Additional Lipid Effects

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a circulatory ligand that terminates the lifecycle of the low-density lipoprotein (LDL) receptor (LDLR) thus affecting plasma LDL-cholesterol (LDL-C) levels. Recent evidence shows that in addition to the straightforward mechanism of action, there are more complex interactions between PCSK9, LDLR and plasma lipoprotein levels, including: (a) the presence of both parallel and reciprocal regulation of surface LDLR and plasma PCSK9; (b) a correlation between PCSK9 and LDL-C levels dependent not only on the fact that PCSK9 removes hepatic LDLR, but also due to the fact that up to 40% of plasma PCSK9 is physically associated with LDL; and (c) an association between plasma PCSK9 production and the assembly and secretion of triglyceride-rich lipoproteins. The effect of PCSK9 on LDLR is being successfully utilized toward the development of anti-PCSK9 therapies to reduce plasma LDL-C levels. Current biochemical research has uncovered additional mechanisms of action and interacting partners for PCSK9, and this opens the way for a more thorough understanding of the regulation, metabolism, and effects of this interesting protein.

Characterization of the PON1 active site using modeling simulation, in relation to PON1 lactonase activity

Paraoxonase1 (PON1) is a HDL bound enzyme and many of the anti-atherogenic properties of HDL are attributed to PON1. The enzyme precise mechanism of protective action and its endogenous substrate remain elusive. PON1 hydrolyzes organophosphates, arylesters and lactones, whereas the lactones activity is assumed as the physio/pathological one. This study is aimed to predict the location of the PON1 active site within PON1 crystal structure, and the lactone structure suitability as PON1 ligand, by employing modeling techniques. Based on such calculations the ligands-PON1 interactions were characterized, and relating lactones rate of hydrolysis revealed an inverse correlation with the docking energy of the ligands-PON1 complex, and a direct correlation with the lactone side chain length. In conclusion, this study characterized the PON1 possible active site and proposes a tool which may make it possible to envisage the structure of potential endogenous and exogenous lactones such as the PON1 ligand.

Biology of proprotein convertase subtilisin kexin 9: beyond low-density lipoprotein cholesterol lowering

Proprotein convertase subtilisin kexin 9 (PCSK9) is a key regulator of low-density lipoprotein receptor levels and LDL-cholesterol levels. Loss-of-function mutations in PCSK9 gene are associated with hypocholesterolaemia and protection against cardiovascular disease, identifying PCSK9 inhibition as a valid therapeutic approach to manage hypercholesterolaemia and related diseases. Although PCSK9 is expressed mainly in the liver, it is present also in other tissues and organs with specific functions, raising the question of whether a pharmacological inhibition of PCSK9 to treat hypercholesterolaemia and associated cardiovascular diseases might be helpful or deleterious in non-hepatic tissues. For example, PCSK9 is expressed in the vascular wall, in the kidneys, and in the brain, where it was proposed to play a role in development, neurocognitive process, and neuronal apoptosis. A link between PCSK9 and immunity was also proposed as both sepsis and viral infections are differentially affected in the presence or absence of PCSK9. Despite the increasing number of observations, the debate on the exact roles of PCSK9 in extrahepatic tissues is still ongoing, and as very effective drugs that inhibit PCSK9 have become available to the clinician, a better understanding of the biological roles of PCSK9 is warranted.

Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms

AIMS Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of hepatic low-density lipoprotein (LDL) receptors (LDLR), thereby, decreasing hepatocyte LDL-cholesterol (LDL-C) uptake. However, it is unknown whether PCSK9 has effects on atherogenesis that are independent of lipid changes. The present study investigated the effect of human (h) PCSK9 on plasma lipids, hepatic lipogenesis, and atherosclerotic lesion size and composition in transgenic mice expressing hPCSK9 (hPCSK9tg) on wild-type (WT), LDLR⁻/⁻, or apoE⁻/⁻ background. METHODS AND RESULTS hPCSK9 expression significantly increased plasma cholesterol (+91%), triglycerides (+18%), and apoB (+57%) levels only in WT mice. The increase in plasma lipids was a consequence of both decreased hepatic LDLR and increased hepatic lipid production, mediated transcriptionally and post-transcriptionally by PCSK9 and dependent on both LDLR and apoE. Despite the lack of changes in plasma lipids in mice expressing hPCSK9 and lacking LDLR (the main target for PCSK9) or apoE (a canonical ligand for the LDLR), hPCSK9 expression increased aortic lesion size in the absence of apoE (268 655 ± 97 972 µm² in hPCSK9tg/apoE⁻/⁻ vs. 189 423 ± 65 700 µm(2) in apoE⁻/⁻) but not in the absence of LDLR. Additionally, hPCSK9 accumulated in the atheroma and increased lesion Ly6C(hi) monocytes (by 21%) in apoE⁻/⁻ mice, but not in LDLR⁻/⁻ mice. CONCLUSIONS PCSK9 increases hepatic lipid and lipoprotein production via apoE- and LDLR-dependent mechanisms. However, hPCSK9 also accumulate in the artery wall and directly affects atherosclerosis lesion size and composition independently of such plasma lipid and lipoprotein changes. These effects of hPCSK9 are dependent on LDLR but are independent of apoE.