PCSK9 inhibition fails to alter hepatic LDLR,circulating cholesterol,and atherosclerosis in the absence of ApoE

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15–30% lower circulating LDL-C and a disproportionately lower risk (47–88%) of experiencing a cardiovascular event. Here, we utilized pcsk9^−/− mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9^−/− mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (−45%) and TGs (−36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (−91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.     

Gypenoside LVI improves hepatic LDL uptake by decreasing PCSK9 and upregulating LDLR expression

BackgroundsAtherosclerotic Cardiovascular Disease (ASCVD) is defined as ischemic or endothelial dysfunction-various inflammatory diseases, which is mainly caused by excessive low-density lipoprotein cholesterol (LDL-C) in circulating blood. Gynostemma pentaphyllum is a traditional Chinese medicine, and total Gypenosides are used for the treatment of hyperlipidemia and to reduce circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) level. However, which gypenoside involved in the modulation of PCSK9 expression is still unknown.PurposeThis study aimed to discover effective PCSK9 inhibitors from Gypenosides in accordance with the 2019 ESC/EAS guidelines.MethodsHPLC was employed to determine major six components of Gypenosides. The inhibitory activity on secreted PCSK9 in HepG2 of six major compounds from Gypenosides were screened by ELISA. The level of low-density lipoprotein (LDL) receptor (LDLR) was determined by Flow cytometry and Immunofluorescence. The expression levels of PCSK9, LDLR and Sterol-regulatory element binding proteins-2 (SREBP-2) were analyzed by qPCR and Western blot. DiI-LDL was added to evaluated LDL uptake into HepG2.ResultsThe results suggested that the mRNA and protein levels of PCSK9 were down-regulated by Gypenoside LVI and the LDLR degradation in lysosomes system was inhibited, thereby leading to an increasing in LDL uptake into HepG2 cells. Furthermore, Gypenoside LVI decreased PCSK9 expression induced by stains. Altogether, Gypenoside LVI enhances LDL uptake into HepG2 cells by increased LDLR level on cell-surface and suppressed PCSK9 expression.ConclusionThis indicates that Gypenoside LVI can be used as a useful supplement for statins in the treatment of hypercholesterolemia. This is firstly reported that monomeric compound of G. pentaphyllum planted in Hunan province has the effect of increasing LDL-C uptake in hepatocytes via inhibiting PCSK9 expression.     

Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low density lipoprotein receptor (LDLR) levels. Naturally occurring mutations can lead to hyper- or hypocholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function and loss-of-function mutants as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. Although PCSK9 gain-of-function D374Y enhanced the degradation both receptors, D374H and D129N seemed to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-cysteine-histidine-rich domain segment primarily affected the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2 transmembrane anchor, the secretory N-terminal catalytic or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.     

A Novel,Orally Bioavailable,Small-Molecule Inhibitor of PCSK9 With Significant Cholesterol-Lowering Properties In Vivo

Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibits the clearance of low-density lipoprotein (LDL) cholesterol (LDL-C) from plasma by directly binding with the LDL receptor (LDLR) and sending the receptor for lysosomal degradation. As the interaction promotes elevated plasma LDL-C levels, and therefore a predisposition to cardiovascular disease, PCSK9 has attracted intense interest as a therapeutic target. Despite this interest, an orally bioavailable small-molecule inhibitor of PCSK9 with extensive lipid-lowering activity is yet to enter the clinic. We report herein the discovery of NYX-PCSK9i, an orally bioavailable small-molecule inhibitor of PCSK9 with significant cholesterol-lowering activity in hyperlipidemic APOE13-Leiden.CETP mice. NYX-PCSK9i emerged from a medicinal chemistry campaign demonstrating potent disruption of the PCSK9-LDLR interaction in vitro and functional protection of the LDLR of human lymphocytes from PCSK9-directed degradation ex vivo. APOE13-Leiden.CETP mice orally treated with NYX-PCSK9i demonstrated a dose-dependent decrease in plasma total cholesterol of up to 57%, while its combination with atorvastatin additively suppressed plasma total cholesterol levels. Importantly, the majority of cholesterol lowering by NYX-PCSK9i was in non-HDL fractions. A concomitant increase in total plasma PCSK9 levels and significant increase in hepatic LDLR protein expression strongly indicated on-target function by NYX-PCSK9i. Determinations of hepatic lipid and fecal cholesterol content demonstrated depletion of liver cholesteryl esters and promotion of fecal cholesterol elimination with NYX-PCSK9i treatment. All measured in vivo biomarkers of health indicate that NYX-PCSK9i has a good safety profile. NYX-PCSK9i is a potential new therapy for hypercholesterolemia with the capacity to further enhance the lipid-lowering activities of statins.     

Common Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Epitopes Mediate Multiple Routes for Internalization and Function

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a soluble protein that directs membrane-bound receptors to lysosomes for degradation. In the most studied example of this, PCSK9 binding leads to the degradation of low density lipoprotein receptor (LDLR), significantly affecting circulating LDL-C levels. The mechanism mediating this degradation, however, is not completely understood. We show here that LDLR facilitates PCSK9 interactions with amyloid precursor like protein 2 (APLP2) at neutral pH leading to PCSK9 internalization, although direct binding between PCSK9 and LDLR is not required. Moreover, binding to APLP2 or LDLR is independently sufficient for PCSK9 endocytosis in hepatocytes, while LDL can compete with APLP2 for PCSK9 binding to indirectly mediate PCSK9 endocytosis. Finally, we show that APLP2 and LDLR are also required for the degradation of another PCSK9 target, APOER2, necessitating a general role for LDLR and APLP2 in PCSK9 function. Together, these findings provide evidence that PCSK9 has at least two endocytic epitopes that are utilized by a variety of internalization mechanisms and clarifies how PCSK9 may direct proteins to lysosomes.     

Functional analysis of sites within PCSK9 responsible for hypercholesterolemia

Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. PCSK9 binds the LDL receptor (LDLR), and addition of PCSK9 to cells promotes degradation of LDLR. PCSK9 mutant proteins associated with hypercholesterolemia (S127R and D374Y) are more potent in decreasing LDL uptake than is wild-type PCSK9. To better understand the mechanism by which mutations at the Ser127 and Asp374 residues of PCSK9 influence PCSK9 function, a limited vertical scanning mutagenesis was performed at both sites. S127R and S127K proteins were more potent in decreasing LDL uptake than was wild-type PCSK9, and each D374 mutant tested was more potent in reducing LDL uptake when the proteins were added exogenously to cells. The potencies of D374 mutants in lowering LDL uptake correlated with their ability to interact with LDLR in vitro. Combining S127R and D374Y was also found to have an additive effect in enhancing PCSK9's ability to reduce LDL uptake. Modeling of PCSK9 S127 and D374 mutations indicates that mutations that enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at Ser127 and Asp374 residues modulate PCSK9's ability to regulate LDLR function through distinct mechanisms.     

Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels

The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.     

Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of hepatic low density lipoprotein receptors (LDLR), the major route of clearance of circulating cholesterol. Gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, whereas loss-of-function mutations result in hypocholesterolemia and protection from heart disease. Recombinant human PCSK9 binds the LDLR on the surface of cultured hepatocytes and promotes degradation of the receptor after internalization. Here we localized the site of binding of PCSK9 within the extracellular domain of the LDLR and determined the fate of the receptor after PCSK9 binding. Recombinant human PCSK9 interacted in a sequence-specific manner with the first epidermal growth factor-like repeat (EGF-A) in the EGF homology domain of the human LDLR. Similar binding specificity was observed between PCSK9 and purified EGF-A. Binding to EGF-A was calcium-dependent and increased dramatically with reduction in pH from 7 to 5.2. The addition of PCSK9, but not heat-inactivated PCSK9, to the medium of cultured hepatocytes resulted in redistribution of the receptor from the plasma membrane to lysosomes. These data are consistent with a model in which PCSK9 binding to EGF-A interferes with an acid-dependent conformational change required for receptor recycling. As a consequence, the LDLR is rerouted from the endosome to the lysosome where it is degraded.     

Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion

Proprotein convertase subtilisin/kexin type 9 (PCSK9) interferes with the recycling of low-density lipoprotein (LDL) receptor (LDLR). This leads to LDLR degradation and reduced cellular uptake of plasma LDL. Naturally occurring human PCSK9 loss-of-function mutations are associated with low levels of plasma LDL cholesterol and a reduced risk of coronary heart disease. PCSK9 gain-of-function mutations result in lower LDL clearance and increased risk of atherosclerosis. The exact mechanism by which PCSK9 disrupts the normal recycling of LDLR remains to be determined. In this study, we have assembled homologs of human PCSK9 from 20 vertebrates, a cephalochordate and mollusks in order to search for conserved regions of PCSK9 that may be important for the PCSK9-mediated degradation of LDLR. We found a large, conserved protrusion on the surface of the PCSK9 catalytic domain and have performed site-directed mutagenesis experiments for 13 residues on this protrusion. A cluster of residues that is important for the degradation of LDLR by PCSK9 was identified. Another cluster of residues, at the opposite end of the conserved protrusion, appears to be involved in the physical interaction with a putative inhibitor of PCSK9. This study identifies the residues, sequence segments and surface patches of PCSK9 that are under strong purifying selection and provides important information for future studies of PCSK9 mutants and for investigations on the function of this regulator of cholesterol homeostasis.     

Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein that binds to the epidermal growth factor-like-A domain of the low density lipoprotein receptor (LDLR) and mediates LDLR degradation in liver. Gain-of-function mutations in PCSK9 are associated with autosomal dominant hypercholesterolemia in humans. Size-exclusion chromatography of human plasma has shown PCSK9 to be partly associated with undefined high molecular weight complexes within the LDL size range. We used density gradient centrifugation to isolate LDL in plasma pooled from 5 normolipidemic subjects and report that >40% of total PCSK9 was associated with LDL. Binding of fluorophore-labeled recombinant PCSK9 to isolated LDL in vitro was saturable with a K_D ∼ 325 nm. This interaction was competed >95% by excess unlabeled PCSK9, and competition binding curves were consistent with a one-site binding model. An N-terminal region of the PCSK9 prodomain (amino acids 31–52) was required for binding to LDL in vitro. LDL dose-dependently inhibited binding and degradation of cell surface LDLRs by exogenous PCSK9 in HuH7 cells. LDL also inhibited PCSK9 binding to mutant LDLRs defective at binding LDL. These data suggest that association of PCSK9 with LDL particles in plasma lowers the ability of PCSK9 to bind to cell surface LDLRs, thereby blunting PCSK9-mediated LDLR degradation.     

Internalized PCSK9 dissociates from recycling LDL receptors in PCSK9-resistant SV-589 fibroblasts

Secreted PCSK9 binds to cell surface LDL receptor (LDLR) and directs the receptor for lysosomal degradation. PCSK9 is potent at inducing LDLR degradation in cultured liver-derived cells, but it is considerably less active in immortalized fibroblasts. We examined PCSK9 trafficking in SV-589 human skin fibroblasts incubated with purified recombinant wild-type PCSK9 or gain-of-function mutant PCSK9-D374Y with increased LDLR binding affinity. Despite LDLR-dependent PCSK9 uptake, cell surface LDLR levels in SV-589 fibroblasts were only modestly reduced by wild-type PCSK9, even at high nonphysiological concentrations (20 µg/ml). Internalized ¹²⁵I-labeled wild-type PCSK9 underwent lysosomal degradation at high levels, indicating its dissociation from recycling LDLRs. PCSK9-D374Y (2 µg/ml) reduced cell surface LDLRs by approximately 50%, but this effect was still blunted compared with HepG2 hepatoma cells. Radioiodinated PCSK9-D374Y was degraded less efficiently in SV-589 fibroblasts, and Alexa488-labeled PCSK9-D374Y trafficked to both lysosomes and endocytic recycling compartments. Endocytic recycling assays showed that more than 50% of internalized PCSK9-D374Y recycled to the cell surface compared with less than 10% for wild-type PCSK9. These data support that wild-type PCSK9 readily dissociates from the LDLR within early endosomes of SV-589 fibroblasts, contributing to PCSK9-resistance. Although a large proportion of gain-of-function PCSK9-D374Y remains bound to LDLR in these cells, degradative activity is still diminished.     

Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.     

An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.     

An Antibody against the C-Terminal Domain of PCSK9 Lowers LDL Cholesterol Levels In Vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.     

PCSK9: A novel inflammation modulator in atherosclerosis?

Proprotein convertase subtilisin/kexin 9 (PCSK9) is the ninth member of the secretory serine protease family. It binds to low-density lipoprotein receptor (LDLR) for endocytosis and lysosome degradation in the liver, resulting in an increasing in circulating LDL-cholesterol (LDL-c) level. Since a PCSK9 induced increase in plasma LDL-c contributes to atherosclerosis, PCSK9 inhibition has become a new strategy in preventing and treating atherosclerosis. However, in addition to the effect of PCSK9 on elevating blood LDL-c levels, accumulating evidence shows that PCSK9 plays an important role in inflammation, likely representing another major mechanism for PCSK9 to promote atherosclerosis. In this review, we discuss the association of PCSK9 and inflammation, and highlight the specific effects of PCSK9 on different vascular cellular components involved in the atherosclerotic inflammation. We also discuss the clinical evidence for the association between PCSK9 and inflammation in atherosclerotic cardiovascular disease. A better understanding of the direct association of PCSK9 with atherosclerotic inflammation might help establish a new role for PCSK9 in vascular biology and identify a novel molecular mechanism for PCSK9 therapy.     

PCSK9 is expressed in pancreatic δ-cells and does not alter insulin secretion

PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immunohistochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancreatic δ-cells, with no expression in α- and β-cells. PCSK9 seems not to be secreted by mouse isolated islets maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated islets, mainly in β-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin secretion in mouse islets. Finally, invivo glucose tolerance was similar in Pcsk9^+/+ and Pcsk9^−/− mice under basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that PCSK9 does not alter insulin secretion.     

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

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.     

Lipopolysaccharide Is Cleared from the Circulation by Hepatocytes via the Low Density Lipoprotein Receptor

Sepsis is the leading cause of death in critically ill patients. While decreased Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) function improves clinical outcomes in murine and human sepsis, the mechanisms involved have not been fully elucidated. We tested the hypothesis that lipopolysaccharide (LPS), the major Gram-negative bacteria endotoxin, is cleared from the circulation by hepatocyte Low Density Lipoprotein Receptors (LDLR)—receptors downregulated by PCSK9. We directly visualized LPS uptake and found that LPS is rapidly taken up by hepatocytes into the cell periphery. Over the course of 4 hours LPS is transported towards the cell center. We next found that clearance of injected LPS from the blood was reduced substantially in Ldlr knockout (Ldlr-/-) mice compared to wild type controls and, simultaneously, hepatic uptake of LPS was also reduced in Ldlr-/- mice. Specifically examining the role of hepatocytes, we further found that primary hepatocytes isolated from Ldlr-/- mice had greatly decreased LPS uptake. In the HepG2 immortalized human hepatocyte cell line, LDLR silencing similarly resulted in decreased LPS uptake. PCSK9 treatment reduces LDLR density on hepatocytes and, therefore, was another independent strategy to test our hypothesis. Incubation with PCSK9 reduced LPS uptake by hepatocytes. Taken together, these findings demonstrate that hepatocytes clear LPS from the circulation via the LDLR and PCSK9 regulates LPS clearance from the circulation during sepsis by downregulation of hepatic LDLR.