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Objective: Antibody blockade of the “do not eat me” signal CD47 (cluster of differentiation 47) enhances efferocytosis and reduces lesion size and necrotic core formation in murine atherosclerosis. TNF (Tumor necrosis factor)-α expression directly enhances CD47 expression, and elevated TNF-α is observed in the absence of the proefferocytosis receptor LRP1 (low-density lipoprotein receptor-related protein 1), a regulator of atherogenesis and inflammation. Thus, we tested the hypothesis that CD47 blockade requires the presence of macrophage LRP1 to enhance efferocytosis, temper TNF-α-dependent inflammation, and limit atherosclerosis. Approach and Results: Mice lacking systemic apoE (apoE −/− ), alone or in combination with the loss of macrophage LRP1 (double knockout), were fed a Western-type diet for 12 weeks while receiving anti-CD47 antibody (anti-CD47) or IgG every other day. In apoE −/− mice, treatment with anti-CD47 reduced lesion size by 25.4%, decreased necrotic core area by 34.5%, and decreased the ratio of free:macrophage-associated apoptotic bodies by 47.6% compared with IgG controls ( P −/− phagocytes were incubated with anti-CD47 compared with IgG controls ( P −/− macrophages compared with wild type, but no differences were observed in inflammatory lipopolysaccharide-treated macrophages. Conclusions: The proefferocytosis receptor LRP1 in macrophages is necessary for anti-CD47 blockade to enhance efferocytosis, limit atherogenesis, and decrease necrotic core formation in the apoE −/− model of atherosclerosis.

Background: We previously showed that mice lacking MΦLRP1 -/- (low-density lipoprotein receptor–related protein 1 in macrophages) undergo accelerated atherosclerotic plaque formation due to changes in macrophages including increased apoptosis, decreased efferocytosis, and exaggerated transition to the inflammatory M1 phenotype. Here we sought to explore the role of macrophage low-density lipoprotein receptor–related protein 1 during regression of atherosclerosis since regressing plaques are characterized by transitioning of macrophages to M2 status as inflammation resolves. Methods: Apolipoprotein E -/- mice on a high-fat diet for 12 weeks were reconstituted with bone marrow from apolipoprotein E–producing wild-type or MΦLRP1 -/- mice, and then placed on a chow diet for 10 weeks (n=9 to 11 mice/group). A cohort of apolipoprotein E -/- mice reconstituted with apolipoprotein E -/- bone marrow served as baseline controls (n=9). Results: Plaques of both wild-type and MΦLRP1 -/- bone marrow recipients regressed compared with controls (11% and 22%, respectively; P -/- recipients were 13% smaller than those of wild-type recipients ( P -/- marrow had 36% fewer M1 macrophages ( P P + macrophages in mediastinal lymph nodes. Finally, in vivo studies of reverse cholesterol transport showed a 1.4-fold higher reverse cholesterol transport in MΦLRP1 -/- recipient mice ( P Conclusions: 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.

Unknown 15 years ago, PCSK9 (proprotein convertase subtilisin/kexin type 9) is now common parlance among scientists and clinicians interested in prevention and treatment of atherosclerotic cardiovascular disease. What makes this story so special is not its recent discovery nor the fact that it uncovered previously unknown biology but rather that these important scientific insights have been translated into an effective medical therapy in record time. Indeed, the translation of this discovery to novel therapeutic serves as one of the best examples of how genetic insights can be leveraged into intelligent target drug discovery. The PCSK9 saga is unfolding quickly but is far from complete. Here, we review major scientific understandings as they relate to the role of PCSK9 in lipoprotein metabolism and atherosclerotic cardiovascular disease and the impact that therapies designed to inhibit its action are having in the clinical setting.

Objective— Antiatherosclerotic effects of tumor necrosis factor-α (TNF-α) blockade in patients with systemic inflammatory states are not conclusively demonstrated, which suggests that effects depend on the cause of inflammation. Macrophage LRP1 (low-density lipoprotein receptor–related protein 1) and apoE contribute to inflammation through different pathways. We studied the antiatherosclerosis effects of TNF-α blockade in hyperlipidemic mice lacking either LRP1 (MΦLRP1 −/− ) or apoE from macrophages. Approach and Results— Lethally irradiated low-density lipoprotein receptor (LDLR) −/− mice were reconstituted with bone marrow from either wild-type, MΦLRP1 −/− , apoE −/− or apoE −/− /MΦLRP1 −/− (DKO) mice, and then treated with the TNF-α inhibitor adalimumab while fed a Western-type diet. Adalimumab reduced plasma TNF-α concentration, suppressed blood ly6C hi monocyte levels and their migration into the lesion, and reduced lesion cellularity and inflammation in both wild-type→LDLR −/− and apoE −/− →LDLR −/− mice. Overall, adalimumab reduced lesion burden by 52% to 57% in these mice. Adalimumab reduced TNF-α and blood ly6C hi monocyte levels in MΦLRP1 −/− →LDLR −/− and DKO→LDLR −/− mice, but it did not suppress ly6C hi monocyte migration into the lesion or atherosclerosis progression. Conclusions— Our results show that TNF-α blockade exerts antiatherosclerotic effects that are dependent on the presence of macrophage LRP1.

Aggressive lipid lowering halts atherosclerotic plaque progression but does not lead to bulk plaque regression in humans. We have previously reported that gene transfection of the HDL protein apoAI into macrophages (MΦ-apoAI) decrease the rate of plaque development in atherosclerosis-prone mice. In this study, we hypothesized that MΦ-apoAI can promote regression of atherosclerosis synergistically with lipid-lowering. Atherogenic mice (LDLR -/- and LDLR -/- /MΦ-apoAI) were fed a high-fat diet to promote stage II/III atherosclerotic lesions (as baseline comparator), and then switched to an extreme lipid-lowering intervention (chow with MTTP inhibitor). After 3 weeks on the lipid-lowering diet, both groups showed reduced systemic and lesion inflammation, and reduced macrophage content on histology when compared to baseline. Upon lipid-lowering, vascular ultrasound showed that LDLR -/- /MΦ-apoAI mice has 21% improvement in aortic pulse transit time, and a 37.1% improvement in instantaneous aortic compliance (a surrogate marker for arterial elastic modulus), compared to LDLR -/- mice. Histologic evidence showed that LDLR -/- /MΦ-apoAI mice on lipid-lowering diet also had reduced lesion size (-24%), reduced macrophage content (-27.5%), increased M2/M1 ratio (+51%) and reduced necrotic core area (-28.6%), compared to LDLR -/- mice. Using transplantation of bone marrow cells from MΦ-apoAI mice into recipient mice with established lesions, we show that newly recruited cells are not major contributors to the regression afforded by MΦ-apoAI. Thus, to study the kinetics of pre-existing cells in the lesion during regression, we developed a contrast ultrasound-mediated gene delivery system to tag and trace lesion cells in LDLR -/- mice, which revealed that lesion macrophages expressing apoAI specifically migrate to mediastinal LN in a CCR7-dependent manner during regression. Expression of apoAI in pre-existing lesion macrophages, promotes regression of atherosclerosis beyond lipid-lowering alone, and increases CCR7-dependent egress of macrophages to adjacent lymph nodes. Ultrasound-mediated gene delivery can be a useful device to study the kinetics of cell in the artery wall and also represent a novel approach with application to human therapy.

We report a case of acquired, progressive, and severe HDL cholesterol (HDL-C) deficiency that emerged in an individual with multiple myeloma. An 81 year-old white male presented with extremely low HDL-c (≤5 mg/dL). Between the ages of 69 - 73 years old, his annual lipid panels demonstrated HDL-c >70 mg/dL. At that point, his HDL-C levels progressively decreased until their virtual disappearance over a period of 12 years. During this time, he developed anemia, an IgG-kappa M-spike, and increased plasma cells on bone marrow biopsy, compatible with a diagnosis of multiple myeloma. Review of his medications, medical history, and metabolic status failed to identify a cause for his progressive decine of HDL-C. Parallel analyses of plasma samples using detergent to solubilize and release HDL-associated cholesterol (SYNCHRO LX, Beckman) and separation of non-HDL precipitated by dextran sulfate yielded HDL-C values of 5 mg/dL and 47 mg/dL, respectively. Furthermore, HDL-C levels of 52 mg/dL and 54 mg/dL were confirmed using ultracentrifugation and NMR-based methods, respectively. Serum apoAI concentration was 153 mg/dL, suggesting normal levels of HDL, in contrast with the ultra-low HDL-C reported in one method. The patient’s alpha migrating band on lipoprotein gel electrophoresis also appeared normal, suggesting the presence of roughly normal amounts of HDL. Methodological artifact alone, however, did not explain the significant “real” decrease in HDL-C (from the upper 70’s mg/dL to the 40’s mg/dL) recorded during 12 years of monitoring. Isolevuglandins (IsoLG) are highly reactive ketoaldehydes that interact with lysine residues of proteins and head groups of phosphatidylethanolamine. It was recently proposed that IsoLG adducts on HDL can result in deleterious structural and functional changes in the particle. Interestingly, the patient’s serum showed high titers of autoantibodies to IsoLG-apoAI, but low titers against native apoAI. We further studied the turnover of IsoLG-apoAI and native apoAI in mice. This case of artefactual and real HDL-C disappearance in a patient with multiple myeloma is likely due to paraprotein-related interference of HDL-C assays and increased plasma clearance of modified apoA1.

Background: We previously reported a marked (~7-fold) increase in plasma PCSK9 concentrations in patients treated with monoclonal antibodies blocking PCSK9 action. It is unclear whether this pronounced increase in plasma PCSK9 is due to increased hepatic production of PCSK9, slow plasma clearance of the antibody/PCSK9 complex, or both. Objectives: To determine whether hepatic PCSK9 gene expression increases after exposure to a therapeutic monoclonal antibody targeted to PCSK9. Methods: C57Bl/6 wild-type (WT) mice and human PCSK9 transgenic (PCSK9tg) mice were treated with a single injection (10 mg/kg) of a monoclonal antibody targeted to PCSK9. Plasma samples were collected at baseline and then at 1, 2, 4, 8, 24, 48, 72, 192 and 336 hours after injection. Livers were sampled from age-matched mice (n=4) at baseline and 24 hours post injection for gene expression analyses. Results: In WT mice (n=8), mean baseline plasma PCSK9 level was 69±28 ng/mL and mean peak plasma PCSK9 level was 719±239 ng/mL (a 10.4-fold increase). Peak PCSK9 levels were reached at 24 hours after injection of the monoclonal antibody. At 24 hours after injection a statistically significant 3-fold increase in hepatic PCSK9 gene expression was observed, compared with baseline, in WT mice. On the other hand, treatment with the antibody increased human PCSK9 levels only by 2-fold in our transgenic mice, whose human PCSK9 is expressed by extrahepatic tissues and in a stable and unregulated fashion. Conclusion: We observed a much larger increase in plasma PCSK9 levels in WT vs. PCSK9tg mice 24 hours after injection of a monoclonal antibody that blocks plasma PCSK9. This finding suggests that the striking increase in plasma PCSK9 in WT mice, which mirrors the effects seen in patients under treatment with PCSK9 blocking antibodies, is mostly due to increased hepatic production and only modestly due to reduced plasma clearance of the antibody/PCSK9 complex.

Background: Plasminogen, is a potent acceptor of cholesterol by the ABCA1 transporter in peripheral cells, and this function is inhibited by lipoprotein (a) [Lp(a)]. Patients with high Lp(a) have increased cardiovascular disease risk, but the mechanism for this effect is not known. The interplay of plasminogen and Lp(a) in contributing to the total sterol efflux capacity of whole serum (TSEC) could explain the influence of Lp(a) on vascular health. We investigated TSEC in patients with low and high plasma Lp(a) levels. Methods: TSEC (measured in cAMP-stimulated J774 murine macrophages), lipid profile, Lp(a), and plasminogen levels were measured in a cohort of patients (N=58) followed for standard-of-care in our lipid clinic. Weused a linear regression model with sex, age, Lp(a), plasminogen, LDL and HDL cholesterol levels, and use of lipid-lowering drugs to understand the predictors of TSEC. The interaction between plasminogen, ABCA1, and Lp(a) was further characterized by biochemical studies of Lp(a) isoforms, cell based assays, and label free protein-protein interaction assays. Results: TSEC was 30% lower (p=0.002) in patients with plasma Lp(a) levels >50mg/dl compared with those below 50mg/dl. Plasminogen, Lp(a), Lp(a)/plasminogen interaction, and LDL-C were significant predictors of TSEC (adjusted R 2 =0.48, p20mg/dl when compared to samples co-treated with plasma from patients with lower Lp(a) levels. Furthermore, two distinct Lp(a) isoforms isolated from different patients inhibited plasminogen mediated sterol efflux. Finally, label free protein-protein interaction assays showed that plasminogen binds to ABCA1 in a process inhibited by the presence of Lp(a). Conclusion: Our results support an interaction between plasminogen and Lp(a) that contribute to the total sterol efflux capacity of the serum, an atheroprotective process.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that plays a key role in the regulation of plasma low-density lipoprotein (LDL) cholesterol levels. PCSK9 binding to the LDL receptor (LDLR) leads to receptor-mediated endocytosis and lysosomal degradation of LDLR. Prior studies have shown that PCSK9 increases production of triglyceride-rich apoB-lipoproteins via up-regulation of lipogenic genes, and that PCSK9 inhibition reduces plasma triglyceride levels. However, the effect of PCSK9 inhibition on hepatic lipogenic genes expression remain unclear. Using a human hepatocellular carcinoma cell line (HepG2) we show that overexpression of PCSK9 upregulated the expression of several lipogenic genes, including a 1.5-fold increase in ATP citrate lyase (ACLY), a 1.2-fold increase in Fatty Acid Synthase (FAS), a 1.2-fold increase in HMG-CoA-reductase, and a 1.8-fold increase in microsomal triglyceride transfer protein (MTTP), when compared with untransfected HepG2 cells. Overexpression of a gain-of-function mutant of PCSK9 (PCSK9-D374Y) had stronger effects in the same direction: a 2.3-fold increase in ACLY, a 1.8-fold increase in FAS, a 1.6-fold increase in HMG-CoA-reductase, and a 2.1-fold increase in MTTP. In HepG2 cells overexpressing either normal PCSK9 or PCSK9-D374Y, inhibition of PCSK9 function using a monoclonal antibody blocked PCSK9-mediated degradation of LDLR, and reduced expression of SREBP-dependent genes (ACLY, FAS and HMG-CoA-reductase) to levels of untransfected HepG2 cells. Interestingly, while inhibition of PCSK9 function via monoclonal antibody did not affect MTTP gene expression in these cells, inhibition of PCSK9 production via RNA interference reduced MTTP gene expression by 66%. We conclude that inhibition of PCSK9 production decreases MTTP gene expression, whereas blockade of PCSK9 function (and cellular re-entry) only decreases SREBP-dependent genes expression, but does not affect MTTP gene expression. This study shows that while PCSK9 exerts an influence on genes related to both lipogenesis and lipoprotein assembly, any inhibition of PCSK9 controls SREPB-dependent genes but only inhibiting PCSK9 production controls MTTP gene expression.

Review novel insights into the biology of proprotein convertase subtilisin/kexin 9 (PCSK9) that may explain the extreme efficiency of PCSK9 inhibition and the unexpected metabolic effects resulting from PCSK9 monoclonal antibody therapy, and may identify additional patients as target of therapy.For over 20 years, the practical knowledge of cholesterol metabolism has centered around cellular mechanisms, and around the idea that statin therapy is the essential step to control metabolic abnormalities for cardiovascular risk management. This view has been embraced by the recent AHA/ACC guidelines, but is being challenged by recent studies including nonstatin medications and by the development of a new class of cholesterol-lowering agents that seems destined to early US Food and Drug Administration approval. The discovery of PCSK9 - a circulating protein that regulates hepatic low-density lipoprotein (LDL) receptor and serum LDL cholesterol levels - has led to a race for its therapeutic inhibition. Recent findings on PCSK9 regulation and pleiotropic effects will help identify additional patient groups likely to benefit from the inhibitory therapy and unravel the full potential of PCSK9 inhibition therapy.Injectable human monoclonal antibodies to block the interaction between PCSK9 and LDL receptor are demonstrating extraordinary efficacy (LDL reductions of up to 70%) and almost the absence of any side-effects. A more moderate effect is seen on other lipoprotein parameters, with the exception of lipoprotein(a) levels. We describe mechanisms that can explain the effect on lipoprotein(a), predict a potential effect on postprandial triglyderides, and suggest a new category of patients for anti-PCSK9 therapy.

We previously showed that mice lacking macrophage LDL receptor-related protein 1 (LRP1) undergo accelerated lesion formation due to increased apoptosis, decreased efferocytosis, and enhanced macrophage transformation into the inflammatory M1 phenotype. In vitro, LRP1-deficient macrophages (MΦLRP1 -/- ) show enhanced plasticity with exaggerated polarization towards either the inflammatory M1- or the anti-inflammatory M2-phenotype depending on the stimulant (LPS or IL-4, respectively). During atherosclerosis regression, the M2:M1 macrophage ratio increases as lesion M1 macrophages egress and inflammation resolves. Thus, we hypothesize that atherosclerosis regression is accelerated in MΦLRP1 -/- mice via enhanced macrophage M2 polarization and CCR7-dependent M1 macrophage egress. ApoE-/- mice on high fat diet for 12 weeks were reconstituted with bone marrow from wildtype (WT) or MΦLRP1 -/- mice and then placed on chow diet for 8 weeks. In this model, apoE is reintroduced into circulation to correct the hyperlipidemia and induce regression of atherosclerotic lesions. A cohort of apoE -/- mice reconstituted with apoE -/- bone marrow served as baseline controls. Lesions in both WT and MΦLRP1 -/- mice regressed relative to controls (11% and 22%, respectively; p-/- lesions were 13% smaller than those of WT mice (p-/- lesions contained 36% fewer M1 macrophages compared to WT (pIn vivo studies of reverse cholesterol transport (RCT) revealed that MΦLRP1 -/- have a 1.4-fold higher RCT compared to WT mice (p-/- lesions contained 2.5-fold more CCR7 + macrophages relative to WT lesions (pin vivo egress assay 4.6-fold more CCR7 + macrophages were found in mediastinal lymph nodes. In vitro , M1-differentiated MΦLRP1 -/- macrophages expressed 1.6-fold higher Ccr7 mRNA compared to WT controls (p

Gene therapy is a potential approach for inhibiting many of the processes that occur during atherogenesis, particularly when targeting key molecular regulators that have multiple adverse downstream signaling pathways. Local expression of human-apoAI in the artery wall has been shown to protect against atherosclerosis in mice, without affecting systemic HDL levels. We hypothesized that focal gene delivery in arterial plaque is possible using ultrasound (US)-mediated cavitation of acoustically-active cationic microbubble gene carriers. Lipid-shelled cationic microbubbles (MB) with a decafluorobutane gas core were prepared. Bicistronic plasmids (luciferase with either GFP or apoAI) were charge coupled to the MB and injected intravenously in 12 LDLR -/- mice fed high-fat diet for 8 weeks. The thoracic aortic root was exposed to US at 1.6MHz, a pulsing interval of 1Hz, and a mechanical index of 1.3 in order to produce repetitive cavitation of MB. In vivo optical imaging showed a focal luciferase activity only at the site of UMGD starting from a day after the procedure and lasting for 7-10 days. Activity was subsequently localized to the aortic root on ex vivo optical imaging. Immunohistochemistry of the aortic root confirmed luciferase transfection of both endothelial cells of the aortic root and base of the aortic cusps, and intramural cells in atherosclerotic lesions, likely macrophages. We further show that gene therapy with the luciferase-apoAI plasmid, results in migration of cells to the thymus 6-8 days after transfection. These results indicate that local US-mediated gene therapy approach for controlling atherosclerosis is feasible in this pre-clinical model.

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.

Recent data indicate that HDL function has effects independent of HDL cholesterol (HDL-C) levels that better predict cardiovascular risk. We aimed to characterize sterol efflux across a wide range of HDL-C, and to identify genetic determinants for both traits in a non-human primate model. To do this, we took advantage of rhesus macaque families that display spontaneous, extreme variation in HDL-C (range 13-106 mg/dL), but have total cholesterol, LDL-C, and triglyceride levels considered normal in humans. We hypothesized that genetic regulation of HDL metabolism is conserved between macaques and humans, and that sterol efflux is independent of HDL-C across the observed range of HDL-C values. We selected 16 macaque half-sib pairs matched for age-class and sex, based on maximum differences in HDL-C characterized in a larger sample (n=193), and conducted deep sequencing of exons and regulatory regions. We assessed predicted function for all variants in macaques found at 23 genes that regulate reverse cholesterol transport or are associated with HDL-C in humans, and that share ~93% identity with the corresponding human proteins. We assessed sterol efflux using J774 macrophages labeled with [ 3 H]cholesterol and stimulated with a cAMP analogue in 16 sequenced macaques, plus 6 macaques selected from the th />95 th percentiles of HDL-C (n=22). We found 27 predicted functional variants located at regulatory or coding regions in macaques that were within 10 bp of known variants in humans associated with HDL-C levels, coded protein deficiencies, or familial alpha-dyslipoproteinemias. Further, we show that 4 of these variants in CETP , LCAT , and ABCA1 are expected to inhibit normal protein function, as indicated by in silico prediction of changes in protein folding. Within these families, correlation between HDL-C and sterol efflux was 0.78 (P=1.65 X 10 -5 ), suggesting that a significant portion of the variation in efflux capacity cannot be accounted for by HDL-C levels. We conclude that functional genetic variation in pathways of HDL metabolism is conserved between humans and macaques, and is likely to influence both HDL-C and efflux capacity. However, a significant proportion of efflux capacity operates independently of HDL-C.

In our Circulation article,1 we stated that few studies have addressed the role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in the small intestine and in the metabolism of triglyceride-rich lipoproteins. In their letter, Drs Le May and Cariou point out the omission of 2 references. The first, published by Le May et al2 in 2009, reports that PCSK9−/− mice display reduced postprandial hypertriglyceridemia and apolipoprotein B levels after olive oil gavage and that adenoviral PCSK9 overexpression or targeted shRNA silencing of PCSK9 increased and reduced apolipoprotein B secretion, respectively. In that article, Le May et al2 acknowledge an earlier observation, published by 1 of us (S.R.) in 2005, that apolipoprotein B48 levels are reduced …

Monoclonal antibodies (mAb) against PCSK9 lower plasma LDL-cholesterol levels by increasing the lifespan of LDL receptors (LDLR). Significant effects on lipoprotein(a) [Lp(a)] have also been observed, but the mechanism for this is neither known nor intuitive since Lp(a) is not cleared by LDLR. Our and other laboratories have shown that PCSK9 associates with LDL in plasma. Here, we aimed to study whether PCSK9 also associates with Lp(a) particles and whether this association depends on Lp(a) levels and/or apo(a) size. Using sandwich ELISA we determined that plasma PCSK9 is indeed associated with Lp(a) particles in patient with high Lp(a) levels (>30mg/dl). We then isolated LDL and Lp(a) fractions from plasma of 8 patients with high Lp(a) levels, ranging from 36 to 224 mg/dl, and determined PCSK9 and apoB levels as well as apo(a) isoforms. Our results show a 17.8 fold higher PCSK9/apoB ratio for Lp(a) compared with LDL (Fig. 1A), which suggests a preferential distribution of PCSK9 with Lp(a), at least in high Lp(a) individuals. Surprisingly, the preferential association of PCSK9 with Lp(a) was inversely correlated to Lp(a) levels (Fig. 1B) and independent of LDL levels. Since Lp(a) levels are inversely correlated with the size of apo(a), we set out to determine whether PCSK9 association with Lp(a) is related to the molecular weight of apo(a). Using plasma of patients with two distinct apo(a) isoforms, we show that PCSK9 associates exclusively with the higher molecular weight of apo(a) (Fig. 1C). Our results suggest that Lp(a)-bound PCSK9 exists in plasma of patients with high Lp(a) and that this association depends on the size of apo(a). This provides a possible mechanism for the reduction in Lp(a) caused by PCSK9mAb, as immune complexes mAb-PCSK9-Lp(a) may be cleared via the mononuclear phagocyte system.

Duell et al1 report on the finding of a significant reduction in plasma proprotein convertase subtilisin/kexin 9 (PCSK9) levels in 4 subjects with heterozygous familial hypercholesterolemia after a single session of low-density lipoprotein (LDL) apheresis with the Liposorber system,1 thus providing complete support to the results we recently published in Circulation Research .2 We had worked with 6 patients undergoing multiple apheresis sessions with the same system, and obtained an aggregate PCSK9 reduction of 52% because of the almost complete loss of LDL-associated PCSK9 and a 40% loss of apolipoprotein B–free PCSK9. Duell et al1 also found a 52% PCSK9 reduction among the 3 patients who had a full apheresis session. As expected, in these subjects the …

Proprotein convertase subtilisin/kexin type 9 (PCSK9) modulates low-density lipoprotein (LDL) receptor (LDLR) degradation, thus influencing serum cholesterol levels. On the other hand, LDLR binds to and clears PCSK9 from the circulation, thus modulating its serum levels. To study the global and reciprocal effects of PCSK9 and LDLR on serum cholesterol, we developed transgenic mice expressing human (h) PCSK9 and characterized its activity, serum levels, and tissue distribution. Serum hPCSK9 concentration in transgenic mice was 2181±423 ng/ml, about 10 times higher than normal level in human serum. Although hPCSK9 was expressed mainly in the kidney, LDLR degradation activity was most evident in the liver and small intestine. In contrast, LDLR levels were not affected by hPCSK9 expression in the adrenals and large intestine. On a chow diet, hPCSK9 transgenic mice on either C57BL/6 (wild-type, WT) or LDLR -/- background had higher cholesterol levels than their non-transgenic counterparts. Human PCSK9 transgenic mice had over a 4-fold increase in murine (m) PCSK9 serum levels compared to WT controls. However, transgenic expression of hPCSK9 in LDLR -/- mice did not affect the already elevated levels of mPCSK9. On the other hand, induction of hLDLR expression in transgenic mice caused a dramatic decrease in mPCSK9 levels. In addition hPCSK9 levels were increased by 2 fold in transgenic mice under LDLR -/- compare to WT background. Turnover studies with native PCSK9 showed rapid serum clearance in WT mice (half-life 5.2 min), whereas clearance was much slower in LDLR -/- recipient mice (50.5 min), and faster in hLDLR transgenic mice (2.9 min). In WT mice the injected PCSK9 accumulated in the liver and kidney but not in the adrenal gland. Ultracentrifugation and FLPC analysis showed that approximately one quarter of circulating hPCSK9 is associated with LDL, and that the LDL-associated PCSK9 is mainly in monomeric form. Our results show a reciprocal regulation between LDLR and PCSK9, which determines serum PCSK9 levels, hepatic LDLR expression, and serum LDL levels. Understanding these interactions will increase our knowledge of serum cholesterol homeostasis and should provide the basis for an intelligent design of anti-PCSK9 therapies.

Renal disease is usually associated with lipoprotein abnormalities. Lipoprotein glomerulopathy (LPG) is a unique disease characterized by intraglomerular lipoprotein thrombi, often in the absence of dyslipidemia. Macrophages are not involved in lipid accumulation in LPG. ApoESendai is an apoE variant known to cause LPG in heterozygous carriers. It carries the same charge as the apoE2 common variant, but is due to a proline-for-arginine substitution at position 145 in the apoE3 sequence. The effects of macrophage expression of apoESendai on LPG and atherosclerosis have not been studied.We have characterized renal and arterial responses in apoE-/- or apoE-/-/LDLR-/- (DKO) mice expressing apoESendai exclusively in macrophages. Bone marrow from apoE-/- mice was transduced with lentiviral constructs expressing either human apoE2 or apoESendai, and then transplanted into apoE-/- or DKO recipient mice.Compared to BMT controls, both apoESendai and apoE2 macrophages significantly decreased plasma cholesterol levels in apoE-/- recipients (22.6% and 19.4%, respectively). No changes in serum cholesterol levels were found in DKO recipients compared to controls. Macrophage expression of apoESendai and apoE2 was associated with plasma human apoE levels of 6.9±1.9 and 5.4±1.7 μg/ml, respectively, in apoE-/- recipients, and 18.9±2.8 and 18.4±3.8 μg/ml, respectively, in DKO recipients. All mice displayed extensive kidney tubular lesions, which correlated with the severity of dyslipidemia. While none of the apoE-/- recipients showed any additional kidney damage, DKO mice expressing apoESendai showed clear features of LPG, with lesions in the glomerular capillaries characterized by mesangial cell expansion. Macrophage apoE2 expression by apoE-/- BMT recipients reduced aortic lesion size by 22.7% compare to BMT controls (p