Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptors (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque in 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 for 8 wk. 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 MPM from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. hPCSK9 was detected in 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.

Background Proprotein convertase subtilisin kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its deficiency in humans results in low plasma LDL-cholesterol and protection against coronary heart disease (CHD). Recent evidence indicates that PCSK9 also modulates the metabolism of triglyceride-rich apolipoprotein B (apoB) lipoproteins (TRL), another important CHD risk factor. Here we studied effects of physiological levels of PCSK9 on intestinal TRL 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 μg/mL, 24 hours) increased cellular and secreted apoB48 and apoB100 by 40–55% each (p<0.01 vs. untreated cells), whereas acute deletion of PCSK9 expression reversed this effect. PCSK9 stimulation of apoB was due to: (1) a 1.5-fold increase in apoB mRNA (p<0.01); and (2) 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 (MTP). PCSK9 also increased levels of the lipid-generating enzymes FAS, SCD and DGAT2 (p<0.05). In mice, human PCSK9 at physiologic levels increased intestinal MTP levels and activity regardless of LDLR expression. Conclusions PCSK9 markedly increases intestinal TRL apoB production through mechanisms mediated in part by transcriptional effects on apoB, MTP and lipogenic genes, and in part by post-transcriptional effects on the LDLR and MTP. These findings indicate that targeted PCSK9-based therapies may also be effective in the management of postprandial hypertriglyceridemia.

Rationale Lipoprotein(a) [Lp(a)] is a highly atherogenic low-density lipoprotein (LDL)-like particle characterized by the presence of apoprotein(a) [apo(a)] bound to apolipoprotein B (apoB). Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) selectively binds LDL, 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 co-expression of human apo(a) and human apoB). Methods and Results We show that PCSK9 is physically associated with Lp(a) in vivo using three different approaches: (i) Analysis of Lp(a) fractions isolated by ultracentrifugation; (ii) Immunoprecipitation of plasma using antibodies to PCSK9 and immunodetection of apo(a); (iii) 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) vs. LDL (1.7-fold increase) was seen in subjects with high Lp(a) and normal LDL. 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.

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.

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.

Absence of macrophage low-density lipoprotein receptor-related protein 1 unexpectedly accelerates atherosclerosis regression, enhances reverse cholesterol transport, and increases expression of the motility receptor CCR7, which drives macrophage egress from lesions.

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Objective Anti-atherosclerotic effects of TNFα blockade in patients with systemic inflammatory states are not conclusively demonstrated, which suggests that effects depend on the cause of inflammation. Macrophage LRP1 and apoE contribute to inflammation through different pathways. We studied the anti-atherosclerosis effects of TNFα blockade in hyperlipidemic mice lacking either LRP1 (MΦLRP1−/−) or apoE from macrophages. Approach and Results Lethally irradiated LDLR−/− mice were reconstituted with bone marrow from either wild type (WT), MΦLRP1−/−, apoE−/−, or apoE−/−/MΦLRP1−/−(DKO) mice, and then treated with the TNFα inhibitor adalimumab while fed a western-type diet. Adalimumab reduced plasma TNFα concentration, suppressed blood ly6Chi monocytes levels and their migration into the lesion, and reduced lesion cellularity and inflammation in both WT→LDLR−/− and apoE−/−→LDLR−/− mice. Overall adalimumab reduced lesion burden by 52%–57% in these mice. Adalimumab reduced TNFα and blood ly6Chi monocytes levels in MΦLRP1−/−→LDLR−/− and DKO→LDLR−/− mice, but it did not suppress ly6Chi monocyte migration into the lesion or atherosclerosis progression. Conclusions Our results show, for the first time, that TNFα blockade exerts anti-atherosclerotic effects that are dependent on the presence of macrophage LRP1.