Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches.

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.

Macrophage LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Is Required for the Effect of CD47 Blockade on Efferocytosis and Atherogenesis-Brief Report.

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<0.05), confirming previous reports. Double knockout mice treated with anti-CD47 showed no differences in lesion size, necrotic core area, or the ratio of free:macrophage-associated apoptotic bodies compared with IgG controls. In vitro efferocytosis was 30% higher when apoE-/- phagocytes were incubated with anti-CD47 compared with IgG controls (P<0.05); however, anti-CD47 had no effect on efferocytosis in double knockout phagocytes. Analyses of mRNA and protein showed increased CD47 expression in anti-inflammatory IL (interleukin)-4 treated LRP1-/- 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.

Insights into the kinetics and dynamics of the furin-cleaved form of PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by inducing the degradation of hepatic low density lipoprotein receptors (LDLRs). Plasma PCSK9 has 2 main molecular forms: a 62 kDa mature form (PCSK9_62) and a 55 kDa, furin-cleaved form (PCSK9_55). PCSK9_55 is considered less active than PCSK9_62 in degrading LDLRs. We aimed to identify the site of PCSK9_55 formation (intracellular vs. extracellular) and to further characterize the LDLR-degradative function of PCSK9_55 relative to PCSK9_62. Coexpressing PCSK9_62 with furin in cell culture induced formation of PCSK9_55, most of which was found in the extracellular space. Under the same conditions, we found that i) adding a cell-permeable furin inhibitor preferentially decreased the formation of PCSK9_55 extracellularly; ii) using pulse-chase analysis, we observed the formation of PCSK9_55 exclusively extracellularly in a time-dependent manner. A recombinant form of PCSK9_55 was efficiently produced but displayed impaired secretion that resulted in its intracellular trapping. However, the nonsecreted PCSK9_55 was able to induce degradation of LDLR, though with 50% lower efficiency than PCSK9_62. Collectively, our data show that 1) PCSK9_55 is formed extracellularly; 2) PCSK9_55 has a shorter half-life; 3) there is a small intracellular pool of PCSK9_55 that is not secreted; and 4) PCSK9_55 retained within the cell maintains a reduced efficiency to cause LDLR degradation.

PMP22 Regulates Cholesterol Trafficking and ABCA1-Mediated Cholesterol Efflux.

The absence of functional peripheral myelin protein 22 (PMP22) is associated with shortened lifespan in rodents and severe peripheral nerve myelin abnormalities in several species including humans. Schwann cells and nerves from PMP22 knock-out (KO) mice show deranged cholesterol distribution and aberrant lipid raft morphology, supporting an unrecognized role for PMP22 in cellular lipid metabolism. To examine the mechanisms underlying these abnormalities, we studied Schwann cells and nerves from male and female PMP22 KO mice. Whole-cell current-clamp recordings in cultured Schwann cells revealed increased membrane capacitance and decreased membrane resistance in the absence of PMP22, which was consistent with a reduction in membrane cholesterol. Nerves from PMP22-deficient mice contained abnormal lipid droplets, with both mRNA and protein levels of apolipoprotein E (apoE) and ATP-binding cassette transporter A1 (ABCA1) being highly upregulated. Despite the upregulation of ABCA1 and apoE, the absence of PMP22 resulted in reduced localization of the transporter to the cell membrane and diminished secretion of apoE. The absence of PMP22 also impaired ABCA1-mediated cholesterol efflux capacity. In nerves from ABCA1 KO mice, the expression of PMP22 was significantly elevated and the subcellular processing of the overproduced protein was aberrant. In wild-type samples, double immunolabeling identified overlapping distribution of PMP22 and ABCA1 at the Schwann cell plasma membrane and the two proteins were coimmunoprecipitated from Schwann cell and nerve lysates. Together, these results reveal a novel role for PMP22 in regulating lipid metabolism and cholesterol trafficking through functional interaction with the cholesterol efflux regulatory protein ABCA1.SIGNIFICANCE STATEMENT Understanding the subcellular events that underlie abnormal myelin formation in hereditary neuropathies is critical for advancing therapy development. Peripheral myelin protein 22 (PMP22) is an essential peripheral myelin protein because its genetic abnormalities account for ∼80% of hereditary neuropathies. Here, we demonstrate that in the absence of PMP22, the cellular and electrophysiological properties of the Schwann cells' plasma membrane are altered and cholesterol trafficking and lipid homeostasis are perturbed. The molecular mechanisms for these abnormalities involve a functional interplay among PMP22, cholesterol, apolipoprotein E, and the major cholesterol-efflux transporter protein ATP-binding cassette transporter A1 (ABCA1). These findings establish a critical role for PMP22 in the maintenance of cholesterol homeostasis in Schwann cells.

Subcellular diversion of cholesterol by gain- and loss-of-function mutations in PMP22.

Abnormalities of the peripheral myelin protein 22 (PMP22) gene, including duplication, deletion and point mutations are a major culprit in Type 1 Charcot-Marie-Tooth (CMT) diseases. The complete absence of PMP22 alters cholesterol metabolism in Schwann cells, which likely contributes to myelination deficits. Here, we examined the subcellular trafficking of cholesterol in distinct models of PMP22-linked neuropathies. In Schwann cells from homozygous Trembler J (TrJ) mice carrying a Leu16Pro mutation, cholesterol was retained with TrJ-PMP22 in the Golgi, alongside a corresponding reduction in its plasma membrane level. PMP22 overexpression, which models CMT1A caused by gene duplication, triggered cholesterol sequestration to lysosomes, and reduced ATP-binding cassette transporter-dependent cholesterol efflux. Conversely, lysosomal targeting of cholesterol by U18666A treatment increased wild type (WT)-PMP22 levels in lysosomes. Mutagenesis of a cholesterol recognition motif, or CRAC domain, in human PMP22 lead to increased levels of PMP22 in the ER and Golgi compartments, along with higher cytosolic, and lower membrane-associated cholesterol. Importantly, cholesterol trafficking defects observed in PMP22-deficient Schwann cells were rescued by WT but not CRAC-mutant-PMP22. We also observed that myelination deficits in dorsal root ganglia explants from heterozygous PMP22-deficient mice were improved by cholesterol supplementation. Collectively, these findings indicate that PMP22 is critical in cholesterol metabolism, and this mechanism is likely a contributing factor in PMP22-linked hereditary neuropathies. Our results provide a basis for understanding how altered expression of PMP22 impacts cholesterol metabolism.

PCSK9: From Basic Science Discoveries to Clinical Trials.

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.

Deletion of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Accelerates Atherosclerosis Regression and Increases C-C Chemokine Receptor Type 7 (CCR7) Expression in Plaque Macrophages.

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<0.05), and plaques of MΦLRP1-/- recipients were 13% smaller than those of wild-type recipients ( P<0.05). Recipients of MΦLRP1-/- marrow had 36% fewer M1 macrophages ( P<0.01) and 2.5-fold more CCR7 (C-C chemokine receptor type 7)-positive macrophages in the plaque relative to wild-type mice ( P<0.01). Additionally, in vivo studies of cellular egress showed a 4.6-fold increase in 5-ethynyl-2´-deoxyuridine-labeled CCR7+ 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<0.01). 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.

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.

Loss of Macrophage Low-Density Lipoprotein Receptor-Related Protein 1 Confers Resistance to the Antiatherogenic Effects of Tumor Necrosis Factor-α Inhibition.

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.

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 Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -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.