Maxime Denis | University of Montreal (original) (raw)

Papers by Maxime Denis

The Journal of Lipid Research, 2007

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL bio... more It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced (125)I-apoA-I binding to the HCBS, whereas (125)I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Delta187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of (125)I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.

Journal of Clinical Lipidology, 2013

Impairment of acid sphingomyelinase (SMase) results in accumulation of sphingomyelin (SM) and cho... more Impairment of acid sphingomyelinase (SMase) results in accumulation of sphingomyelin (SM) and cholesterol in late endosomes, the hallmarks of a lysosomal storage disease. We describe cellular lipid metabolism in fibroblasts from two patients with novel compound heterozygote mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene manifesting as Niemann-Pick disease type B (NPB) and demonstrate mechanisms to overcome the storage defect. Using biochemical assays and confocal microscopy, we provide evidence that accumulated lysosomal SM and cholesterol can be released by different treatments. Defective SMase activity in these fibroblasts results in a 2.5-fold increased cellular mass of SM and cholesterol, increased de novo endogenous cholesterol synthesis, and decreased cholesterol esterification, demonstrating impaired intracellular cholesterol homeostasis. Depletion of exogenous addition of cholesterol for 24 hours or addition of the cholesterol acceptor apolipoprotein A-I are sufficient to restore normal homeostatic responses. In an effort to correct the lysosomal storage phenotype of NPB, we infected the fibroblasts with a lentivirus expressing the phosphotyrosine binding domain of the adapter protein GULP (PTB-GULP). We have previously shown that expression of PTB-GULP in Chinese hamster ovary cells promotes intracellular cholesterol trafficking and ABCA1-mediated cholesterol efflux. We find that expression of PTB-GULP in NPB fibroblasts results in increased ABCA1 expression, increased cellular cholesterol efflux and lysosomal cholesterol redistribution, independent of the impaired SMase and cholesterol presence. We provide extensive functional characterization of a novel compound heterozygote mutation and provide a novel functional mechanism to overcome lysosomal storage disease defects.

Angiology, 2015

Given the link between cholesterol and activation of inflammation via interleukin 1β (IL-1β), we ... more Given the link between cholesterol and activation of inflammation via interleukin 1β (IL-1β), we tested the effects of IL-1β inhibition on atherosclerotic calcification in mice. Patients with familial hypercholesterolemia develop extensive aortic calcification and calcific aortic stenosis. Although statins delay this process, low-density lipoprotein (LDL) cholesterol lowering alone is not enough to avert it. Data suggest that vascular inflammation initiated by hypercholesterolemia is followed by unchecked mineralization at sites of atherosclerotic plaques. The LDL-receptor (LDLR)-deficient (Ldlr(-/-)) and LDLR-attenuated Pcsk9(Tg) mice are available animal models for pharmacological testing. A mouse monoclonal antibody (mAb) against IL-1β or placebo was administered subcutaneously in Ldlr(-/-) and Pcsk9(Tg) models fed a Western diet. Drug level, anthropometric, lipid, and glucose profiles were determined. Expressions of proprotein convertase subtilisin/kexin type 9 (PCSK9), serum amyloid A1, and cytokine were measured by enzyme-linked immunosorbent assay. Aortic calcification was determined by microcomputerized tomography (micro-CT) and X-ray densitometry, and aortic flow velocity was assessed by ultrasound. Circulating levels of IL-1β in Ldlr(-/-) mice were significantly greater (2-fold) than observed in Pcsk9(Tg) mice. Placebo- and mAb-treated mice did not differ in their growth, lipid, glucose profiles, and other cytokines. Calcifications were significantly diminished in mAb-treatment Ldlr(-/-) mice (a reduction of ∼75% by X-ray and ∼90% by micro-CT) and reduced insignificantly in mAb-treatment Pcsk9(Tg) mice, whereas aortic flow velocity was unchanged in both models. Herein, we demonstrate that aortic calcifications can be inhibited by an IL-1β mAb in LDLR-deficient mice. These results have a translational component to prevent vascular calcification in human and represent new evidence to rationalize targeting inflammation in cardiovascular disease.

Journal of lipid research, 2005

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apo... more It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were inc...

The Journal of biological chemistry, 2004

The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nasce... more The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nascent apoA-I-containing particles (LpA-I) were investigated. Using n-dodecylmaltoside and perfluoro-octanoic acid combined with non-denaturing gel, the majority of ABCA1 was found as a tetramer in ABCA1-induced human fibroblasts. Furthermore, using chemical cross-linking and SDS-PAGE, ABCA1 dimers but not the tetramers were found covalently linked. Oligomeric ABCA1 was present in isolated plasma membranes as well as in intracellular compartments. Interestingly, apoA-I was found to be associated with both dimeric and tetrameric, but not monomeric, forms of ABCA1. Neither apoA-I nor lipid molecules did affect ABCA1 oligomerization. Immunoprecipitation analysis showed that oligomeric ABCA1 did not contain other associated proteins. We next investigated the relationship between the oligomeric ABCA1 complex and the structure of LpA-I. Lipid-free apoA-I incubated with normal cells generated LpA-I...

The Journal of biological chemistry, Jan 27, 2004

The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts in... more The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of (125)I-apoA-I to ABCA1 at 37 degrees C were determined: K(d) = 0.65 microg/ml, B(max) = 0.10 ng/microg cell protein. Lipid-free apoA-I inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than pre-beta(1)-LpA-I, reconstituted HDL particles r(LpA-I), or HDL(3) (IC(50) = 0.35 +/- 1.14, apoA-I; 1.69 +/- 1.07, pre-beta(1)-LpA-I; 17.91 +/- 1.39, r(LpA-I); and 48.15 +/- 1.72 microg/ml, HDL(3)). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither (125)I-apoA-I binding nor (125)I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of (125)I-apoA-I from normal cells at 37 degrees C was rapid (t((1/2)) ...

Molecular genetics and metabolism, 2003

Mutations in the ATP-binding cassette transporter A1 (ABCA1) gene cause familial high-density lip... more Mutations in the ATP-binding cassette transporter A1 (ABCA1) gene cause familial high-density lipoprotein deficiency and Tangier disease. ABCA1 plays a crucial role in active apolipoprotein A-I (apoA-I) lipidation, a key step in reverse cholesterol transport. We compared ABCA1 transcriptional regulation and cholesterol efflux in human skin fibroblasts, monocyte-derived macrophages and hepatocytes (HepG2). 8-Br-cAMP did not increase ABCA1 transcription in these tissues compared to mouse macrophages. We found that ABCA1 is differentially regulated among tissues. While transcription in HepG2 appears to be constitutive, sterols stimulate ABCA1 transcription in fibroblasts and monocyte-derived macrophages. ApoA-I promoted cholesterol efflux in fibroblasts, macrophages, and HepG2. Cholesterol homeostasis in fibroblasts is tightly regulated, and ABCA1 mRNA closely follows the cellular mass of free cholesterol (dose- and time-dependent manner). To further determine the mechanism used by fib...

Nature genetics, 1999

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) leve... more Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

PLoS ONE, 2012

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density ... more Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2 2/2 mice revealed: i) a ,1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ,2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2 2/2 tissues revealed that the LDLR was decreased by ,50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9;LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

The Lancet, 1999

A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients wit... more A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients with premature atherosclerosis. We have shown that Tangier disease, a rare and severe form of HDL deficiency characterised by a biochemical defect in cellular cholesterol efflux, is caused by mutations in the ATP-binding-cassette (ABC1) gene. This gene codes for the cholesterol-efflux regulatory protein (CERP). We investigated the presence of mutations in this gene in patients with familial HDL deficiency. Three French-Canadian families and one Dutch family with familial HDL deficiency were studied. Fibroblasts from the proband of each family were defective in cellular cholesterol efflux. Genomic DNA of each proband was used for mutation detection with primers flanking each exon of the ABC1 gene, and for sequencing of the entire coding region of the gene. PCR and restriction-fragment length polymorphism assays specific to each mutation were used to investigate segregation of the mutation in each family, and to test for absence of the mutation in DNA from normal controls. A different mutation was detected in ABC1 in each family studied. Each mutation either created a stop codon predicted to result in truncation of CERP, or altered a conserved aminoacid residue. Each mutation segregated with low concentrations of HDL-cholesterol in the family, and was not observed in more than 500 control chromosomes tested. These data show that mutations in ABC1 are the major cause of familial HDL deficiency associated with defective cholesterol efflux, and that CERP has an essential role in the formation of HDL. Our findings highlight the potential of modulation of ABC1 as a new route for increasing HDL concentrations.

The Journal of Lipid Research, 2005

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic propert... more It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only ␣ -nascent apolipoprotein A-I-containing particles ( ␣ -LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both ␣ -LpA-I and pre ␤ 1 -LpA-I. Furthermore, glyburide inhibits almost completely the formation of ␣ -LpA-I but not pre ␤ 1 -LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both pre ␤ 1 -LpA-I and ␣ -LpA-I; by contrast, CaCo-2 cells secreted only ␣ -LpA-I. To determine whether ␣ -LpA-I generated by fibroblasts is a good substrate for LCAT, isolated ␣ -LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V max (8.4 vs. 8.2 nmol cholesteryl ester/h/ g LCAT, respectively), the K m value was increased 2-fold for ␣ -LpA-I compared with r(HDL) (1.2 vs. 0.7 M apoA-I). These results demonstrate that 1 ) ABCA1 is required for the formation of ␣ -LpA-I but not pre ␤ 1 -LpA-I; and 2 ) ␣ -LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways. -Krimbou, L., H. Hajj Hassan, S. Blain, S. Rashid, M. Denis, M. Marcil, and J. Genest. Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines. J. Lipid Res. 2005. 46: 1668-1677.

The Journal of Lipid Research, 2005

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL.... more We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass (z50-100%). Analysis of LCAT kinetics parameters (V max and K m ) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [ 3 H]SM-labeled LpA-I and HDL 3 . Furthermore, expression of mutant SMase (DR608) in CHO cells revealed that DR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.-Lee, C. Y., A. Lesimple, M. Denis, J. Vincent, Å . Larsen, O. Mamer, L. Krimbou, J. Genest, and M. Marcil. Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B. J. Lipid Res. 2006. 47: 622-632.

The Journal of Lipid Research, 2008

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprot... more ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.

Journal of Biological Chemistry, 2004

It has been suggested that the signal transduction pathway initiated by apoA-I activates key prot... more It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (<or=45 min) increased ATP binding cassette A1 (ABCA1) phosphorylation in a concentration-dependent manner. Concomitantly, apoA-I increased the intracellular level of cAMP in a concentration- and time-dependent manner. The maximal cAMP level was reached within 10 min at 10 microg/ml apoA-I representing a 1-fold increase. The ability of apoA-I to mediate cAMP production was only observed in stimulated fibroblasts. Furthermore, overexpression of ABCA1 in Chinese hamster ovary cells resulted in a 1.5-fold increase in apoA-I-mediated cAMP accumulation as compared with untransfected cells. In contrast, forskolin increased cAMP production significantly in unstimulated fibroblasts as well as in untransfected Chinese hamster ovary cells. Pharmacological inhibition of protein kinase A (H89) completely blocked apoA-I-mediated ABCA1 phosphorylation. Naturally occurring mutations of ABCA1 associated with Tangier disease (C1477R, 2203X, and 2145X) severely reduced apoA-I-mediated cAMP production, ABCA1 phosphorylation, (125)I-apoA-I binding, and lipid efflux, without affecting forskolin-mediated cAMP elevation. In contrast, the protein kinase A catalytic subunit was able to phosphorylate ABCA1 similarly from mutant and normal cell lines in vitro. Together, our results indicate that apoA-I activates ABCA1 phosphorylation through the cAMP/protein kinase A-dependent pathway, apoA-I-mediated cAMP production required high level expression of functional ABCA1, and Tangier disease mutants have defective apoA-I-mediated cAMP signaling. These findings suggest that apoA-I may activate cAMP signaling through G protein-coupled ABCA1 transporter.

Circulation, 2012

Background-The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of it... more Background-The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in Ϸ7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results-We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E-deficient, and LDL receptor-deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor-deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions-Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective. (Circulation. 2012;125:894-901.) The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/

Canadian Journal of Cardiology, 2006

Atherosclerosis, 2011

Objective: Patients with familial hypercholesterolemia (FH) due mutations in the low-density lipo... more Objective: Patients with familial hypercholesterolemia (FH) due mutations in the low-density lipoprotein receptor (LDLR) suffer premature aortic calcification, an effect that is age-and gene dosage-dependent and cholesterol level independent later in life. To better understand this process, we examined a murine model. Methods: We compared chow fed Ldlr −/− mice to controls at 6, 12 and 18 months and on a Western diet (WD) at 6 months. Additionally, we compared controls to Ldlr −/− mice and transgenic mice Tg(Pcsk9) overexpressing PCSK9, which promotes LDLR degradation. Aortas were perfused-fixed, embedded in paraffin, and sections were stained with alizarin red. Micro-computerized tomography (micro-CT) was used to quantify vascular calcification. Results: Ldlr −/− mice develop calcification in the ascending, transverse aorta and neck vessels with a distribution similar to that of human. Calcification was most prominent in 18-month-old Ldlr −/− mice fed a chow diet and in 6-month-old Ldlr −/− mice fed a WD. Interestingly, Tg(Pcsk9) mice fed a WD develop aortic calcifications as well. Histology confirmed that the calcification were predominantly sub-intimal. Marked expression of LRP5 and WNT was observed in the Ldlr −/− and Tg(Pcsk9) models, but not in age-matched controls. Conclusions: The two mouse models develop aortic calcification in an age-and diet-dependent manner. Abnormal regulation of the LRP5/Wnt pathway may play a role in the calcification process. Further analysis of these aortic calcification models using this micro-CT imaging technique may provide a better understanding of the link between FH and arterial calcification.

The Journal of Lipid Research, 2007

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL bio... more It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced (125)I-apoA-I binding to the HCBS, whereas (125)I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Delta187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of (125)I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.

Journal of Clinical Lipidology, 2013

Impairment of acid sphingomyelinase (SMase) results in accumulation of sphingomyelin (SM) and cho... more Impairment of acid sphingomyelinase (SMase) results in accumulation of sphingomyelin (SM) and cholesterol in late endosomes, the hallmarks of a lysosomal storage disease. We describe cellular lipid metabolism in fibroblasts from two patients with novel compound heterozygote mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene manifesting as Niemann-Pick disease type B (NPB) and demonstrate mechanisms to overcome the storage defect. Using biochemical assays and confocal microscopy, we provide evidence that accumulated lysosomal SM and cholesterol can be released by different treatments. Defective SMase activity in these fibroblasts results in a 2.5-fold increased cellular mass of SM and cholesterol, increased de novo endogenous cholesterol synthesis, and decreased cholesterol esterification, demonstrating impaired intracellular cholesterol homeostasis. Depletion of exogenous addition of cholesterol for 24 hours or addition of the cholesterol acceptor apolipoprotein A-I are sufficient to restore normal homeostatic responses. In an effort to correct the lysosomal storage phenotype of NPB, we infected the fibroblasts with a lentivirus expressing the phosphotyrosine binding domain of the adapter protein GULP (PTB-GULP). We have previously shown that expression of PTB-GULP in Chinese hamster ovary cells promotes intracellular cholesterol trafficking and ABCA1-mediated cholesterol efflux. We find that expression of PTB-GULP in NPB fibroblasts results in increased ABCA1 expression, increased cellular cholesterol efflux and lysosomal cholesterol redistribution, independent of the impaired SMase and cholesterol presence. We provide extensive functional characterization of a novel compound heterozygote mutation and provide a novel functional mechanism to overcome lysosomal storage disease defects.

Angiology, 2015

Given the link between cholesterol and activation of inflammation via interleukin 1β (IL-1β), we ... more Given the link between cholesterol and activation of inflammation via interleukin 1β (IL-1β), we tested the effects of IL-1β inhibition on atherosclerotic calcification in mice. Patients with familial hypercholesterolemia develop extensive aortic calcification and calcific aortic stenosis. Although statins delay this process, low-density lipoprotein (LDL) cholesterol lowering alone is not enough to avert it. Data suggest that vascular inflammation initiated by hypercholesterolemia is followed by unchecked mineralization at sites of atherosclerotic plaques. The LDL-receptor (LDLR)-deficient (Ldlr(-/-)) and LDLR-attenuated Pcsk9(Tg) mice are available animal models for pharmacological testing. A mouse monoclonal antibody (mAb) against IL-1β or placebo was administered subcutaneously in Ldlr(-/-) and Pcsk9(Tg) models fed a Western diet. Drug level, anthropometric, lipid, and glucose profiles were determined. Expressions of proprotein convertase subtilisin/kexin type 9 (PCSK9), serum amyloid A1, and cytokine were measured by enzyme-linked immunosorbent assay. Aortic calcification was determined by microcomputerized tomography (micro-CT) and X-ray densitometry, and aortic flow velocity was assessed by ultrasound. Circulating levels of IL-1β in Ldlr(-/-) mice were significantly greater (2-fold) than observed in Pcsk9(Tg) mice. Placebo- and mAb-treated mice did not differ in their growth, lipid, glucose profiles, and other cytokines. Calcifications were significantly diminished in mAb-treatment Ldlr(-/-) mice (a reduction of ∼75% by X-ray and ∼90% by micro-CT) and reduced insignificantly in mAb-treatment Pcsk9(Tg) mice, whereas aortic flow velocity was unchanged in both models. Herein, we demonstrate that aortic calcifications can be inhibited by an IL-1β mAb in LDLR-deficient mice. These results have a translational component to prevent vascular calcification in human and represent new evidence to rationalize targeting inflammation in cardiovascular disease.

Journal of lipid research, 2005

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apo... more It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were inc...

The Journal of biological chemistry, 2004

The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nasce... more The oligomeric structure of ABCA1 transporter and its function related to the biogenesis of nascent apoA-I-containing particles (LpA-I) were investigated. Using n-dodecylmaltoside and perfluoro-octanoic acid combined with non-denaturing gel, the majority of ABCA1 was found as a tetramer in ABCA1-induced human fibroblasts. Furthermore, using chemical cross-linking and SDS-PAGE, ABCA1 dimers but not the tetramers were found covalently linked. Oligomeric ABCA1 was present in isolated plasma membranes as well as in intracellular compartments. Interestingly, apoA-I was found to be associated with both dimeric and tetrameric, but not monomeric, forms of ABCA1. Neither apoA-I nor lipid molecules did affect ABCA1 oligomerization. Immunoprecipitation analysis showed that oligomeric ABCA1 did not contain other associated proteins. We next investigated the relationship between the oligomeric ABCA1 complex and the structure of LpA-I. Lipid-free apoA-I incubated with normal cells generated LpA-I...

The Journal of biological chemistry, Jan 27, 2004

The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts in... more The dynamics of ABCA1-mediated apoA-I lipidation were investigated in intact human fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Specific binding parameters of (125)I-apoA-I to ABCA1 at 37 degrees C were determined: K(d) = 0.65 microg/ml, B(max) = 0.10 ng/microg cell protein. Lipid-free apoA-I inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than pre-beta(1)-LpA-I, reconstituted HDL particles r(LpA-I), or HDL(3) (IC(50) = 0.35 +/- 1.14, apoA-I; 1.69 +/- 1.07, pre-beta(1)-LpA-I; 17.91 +/- 1.39, r(LpA-I); and 48.15 +/- 1.72 microg/ml, HDL(3)). Treatment of intact cells with either phosphatidylcholine-specific phospholipase C or sphingomyelinase affected neither (125)I-apoA-I binding nor (125)I-apoA-I/ABCA1 cross-linking. We next investigated the dynamics of apoA-I lipidation by monitoring the kinetic of apoA-I dissociation from ABCA1. The dissociation of (125)I-apoA-I from normal cells at 37 degrees C was rapid (t((1/2)) ...

Molecular genetics and metabolism, 2003

Mutations in the ATP-binding cassette transporter A1 (ABCA1) gene cause familial high-density lip... more Mutations in the ATP-binding cassette transporter A1 (ABCA1) gene cause familial high-density lipoprotein deficiency and Tangier disease. ABCA1 plays a crucial role in active apolipoprotein A-I (apoA-I) lipidation, a key step in reverse cholesterol transport. We compared ABCA1 transcriptional regulation and cholesterol efflux in human skin fibroblasts, monocyte-derived macrophages and hepatocytes (HepG2). 8-Br-cAMP did not increase ABCA1 transcription in these tissues compared to mouse macrophages. We found that ABCA1 is differentially regulated among tissues. While transcription in HepG2 appears to be constitutive, sterols stimulate ABCA1 transcription in fibroblasts and monocyte-derived macrophages. ApoA-I promoted cholesterol efflux in fibroblasts, macrophages, and HepG2. Cholesterol homeostasis in fibroblasts is tightly regulated, and ABCA1 mRNA closely follows the cellular mass of free cholesterol (dose- and time-dependent manner). To further determine the mechanism used by fib...

Nature genetics, 1999

Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) leve... more Genes have a major role in the control of high-density lipoprotein (HDL) cholesterol (HDL-C) levels. Here we have identified two Tangier disease (TD) families, confirmed 9q31 linkage and refined the disease locus to a limited genomic region containing the gene encoding the ATP-binding cassette transporter (ABC1). Familial HDL deficiency (FHA) is a more frequent cause of low HDL levels. On the basis of independent linkage and meiotic recombinants, we localized the FHA locus to the same genomic region as the TD locus. Mutations in ABC1 were detected in both TD and FHA, indicating that TD and FHA are allelic. This indicates that the protein encoded by ABC1 is a key gatekeeper influencing intracellular cholesterol transport, hence we have named it cholesterol efflux regulatory protein (CERP).

PLoS ONE, 2012

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density ... more Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2 2/2 mice revealed: i) a ,1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ,2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2 2/2 tissues revealed that the LDLR was decreased by ,50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9;LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.

The Lancet, 1999

A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients wit... more A low concentration of HDL cholesterol is the most common lipoprotein abnormality in patients with premature atherosclerosis. We have shown that Tangier disease, a rare and severe form of HDL deficiency characterised by a biochemical defect in cellular cholesterol efflux, is caused by mutations in the ATP-binding-cassette (ABC1) gene. This gene codes for the cholesterol-efflux regulatory protein (CERP). We investigated the presence of mutations in this gene in patients with familial HDL deficiency. Three French-Canadian families and one Dutch family with familial HDL deficiency were studied. Fibroblasts from the proband of each family were defective in cellular cholesterol efflux. Genomic DNA of each proband was used for mutation detection with primers flanking each exon of the ABC1 gene, and for sequencing of the entire coding region of the gene. PCR and restriction-fragment length polymorphism assays specific to each mutation were used to investigate segregation of the mutation in each family, and to test for absence of the mutation in DNA from normal controls. A different mutation was detected in ABC1 in each family studied. Each mutation either created a stop codon predicted to result in truncation of CERP, or altered a conserved aminoacid residue. Each mutation segregated with low concentrations of HDL-cholesterol in the family, and was not observed in more than 500 control chromosomes tested. These data show that mutations in ABC1 are the major cause of familial HDL deficiency associated with defective cholesterol efflux, and that CERP has an essential role in the formation of HDL. Our findings highlight the potential of modulation of ABC1 as a new route for increasing HDL concentrations.

The Journal of Lipid Research, 2005

It is generally thought that the large heterogeneity of human HDL confers antiatherogenic propert... more It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only ␣ -nascent apolipoprotein A-I-containing particles ( ␣ -LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both ␣ -LpA-I and pre ␤ 1 -LpA-I. Furthermore, glyburide inhibits almost completely the formation of ␣ -LpA-I but not pre ␤ 1 -LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both pre ␤ 1 -LpA-I and ␣ -LpA-I; by contrast, CaCo-2 cells secreted only ␣ -LpA-I. To determine whether ␣ -LpA-I generated by fibroblasts is a good substrate for LCAT, isolated ␣ -LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Although both particles had similar V max (8.4 vs. 8.2 nmol cholesteryl ester/h/ g LCAT, respectively), the K m value was increased 2-fold for ␣ -LpA-I compared with r(HDL) (1.2 vs. 0.7 M apoA-I). These results demonstrate that 1 ) ABCA1 is required for the formation of ␣ -LpA-I but not pre ␤ 1 -LpA-I; and 2 ) ␣ -LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways. -Krimbou, L., H. Hajj Hassan, S. Blain, S. Rashid, M. Denis, M. Marcil, and J. Genest. Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines. J. Lipid Res. 2005. 46: 1668-1677.

The Journal of Lipid Research, 2005

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL.... more We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass (z50-100%). Analysis of LCAT kinetics parameters (V max and K m ) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [ 3 H]SM-labeled LpA-I and HDL 3 . Furthermore, expression of mutant SMase (DR608) in CHO cells revealed that DR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles.-Lee, C. Y., A. Lesimple, M. Denis, J. Vincent, Å . Larsen, O. Mamer, L. Krimbou, J. Genest, and M. Marcil. Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B. J. Lipid Res. 2006. 47: 622-632.

The Journal of Lipid Research, 2008

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprot... more ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.

Journal of Biological Chemistry, 2004

It has been suggested that the signal transduction pathway initiated by apoA-I activates key prot... more It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (<or=45 min) increased ATP binding cassette A1 (ABCA1) phosphorylation in a concentration-dependent manner. Concomitantly, apoA-I increased the intracellular level of cAMP in a concentration- and time-dependent manner. The maximal cAMP level was reached within 10 min at 10 microg/ml apoA-I representing a 1-fold increase. The ability of apoA-I to mediate cAMP production was only observed in stimulated fibroblasts. Furthermore, overexpression of ABCA1 in Chinese hamster ovary cells resulted in a 1.5-fold increase in apoA-I-mediated cAMP accumulation as compared with untransfected cells. In contrast, forskolin increased cAMP production significantly in unstimulated fibroblasts as well as in untransfected Chinese hamster ovary cells. Pharmacological inhibition of protein kinase A (H89) completely blocked apoA-I-mediated ABCA1 phosphorylation. Naturally occurring mutations of ABCA1 associated with Tangier disease (C1477R, 2203X, and 2145X) severely reduced apoA-I-mediated cAMP production, ABCA1 phosphorylation, (125)I-apoA-I binding, and lipid efflux, without affecting forskolin-mediated cAMP elevation. In contrast, the protein kinase A catalytic subunit was able to phosphorylate ABCA1 similarly from mutant and normal cell lines in vitro. Together, our results indicate that apoA-I activates ABCA1 phosphorylation through the cAMP/protein kinase A-dependent pathway, apoA-I-mediated cAMP production required high level expression of functional ABCA1, and Tangier disease mutants have defective apoA-I-mediated cAMP signaling. These findings suggest that apoA-I may activate cAMP signaling through G protein-coupled ABCA1 transporter.

Circulation, 2012

Background-The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of it... more Background-The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in Ϸ7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results-We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E-deficient, and LDL receptor-deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor-deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions-Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective. (Circulation. 2012;125:894-901.) The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/

Canadian Journal of Cardiology, 2006

Atherosclerosis, 2011

Objective: Patients with familial hypercholesterolemia (FH) due mutations in the low-density lipo... more Objective: Patients with familial hypercholesterolemia (FH) due mutations in the low-density lipoprotein receptor (LDLR) suffer premature aortic calcification, an effect that is age-and gene dosage-dependent and cholesterol level independent later in life. To better understand this process, we examined a murine model. Methods: We compared chow fed Ldlr −/− mice to controls at 6, 12 and 18 months and on a Western diet (WD) at 6 months. Additionally, we compared controls to Ldlr −/− mice and transgenic mice Tg(Pcsk9) overexpressing PCSK9, which promotes LDLR degradation. Aortas were perfused-fixed, embedded in paraffin, and sections were stained with alizarin red. Micro-computerized tomography (micro-CT) was used to quantify vascular calcification. Results: Ldlr −/− mice develop calcification in the ascending, transverse aorta and neck vessels with a distribution similar to that of human. Calcification was most prominent in 18-month-old Ldlr −/− mice fed a chow diet and in 6-month-old Ldlr −/− mice fed a WD. Interestingly, Tg(Pcsk9) mice fed a WD develop aortic calcifications as well. Histology confirmed that the calcification were predominantly sub-intimal. Marked expression of LRP5 and WNT was observed in the Ldlr −/− and Tg(Pcsk9) models, but not in age-matched controls. Conclusions: The two mouse models develop aortic calcification in an age-and diet-dependent manner. Abnormal regulation of the LRP5/Wnt pathway may play a role in the calcification process. Further analysis of these aortic calcification models using this micro-CT imaging technique may provide a better understanding of the link between FH and arterial calcification.