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Biomedical subjects

Jonathan C Cohen

Publications and source records attributed to Jonathan C Cohen.

At least 19 recordsLinked to original sources
PubMed2007

Molecular biology of PCSK9: its role in LDL metabolism.

Proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a newly discovered serine protease that destroys low density lipoprotein (LDL) receptors in liver and thereby controls the level of LDL in plasma. Mutations that increase PCSK9 activity cause hypercholesterolemia and coronary heart disease (CHD); mutations that inactivate PCSK9 have the opposite effect, lowering LDL levels and reducing CHD. Although the mechanism of PCSK9 action is not yet clear, the protease provides a new therapeutic target to lower plasma levels of LDL and prevent CHD.

Cholesterol, LDL↗
PubMed2007

Disruption of LDL but not VLDL clearance in autosomal recessive hypercholesterolemia.

Genetic defects in LDL clearance result in severe hypercholesterolemia and premature atherosclerosis. Mutations in the LDL receptor (LDLR) cause familial hypercholesterolemia (FH), the most severe form of genetic hypercholesterolemia. A phenocopy of FH, autosomal recessive hypercholesterolemia (ARH), is due to mutations in an adaptor protein involved in LDLR internalization. Despite comparable reductions in LDL clearance rates, plasma LDL levels are substantially lower in ARH than in FH. To determine the metabolic basis for this difference, we examined the synthesis and catabolism of VLDL in murine models of FH (Ldlr(-/-)) and ARH (Arh(-/-)). The hyperlipidemic response to a high-sucrose diet was greatly attenuated in Arh(-/-) mice compared with Ldlr(-/-) mice despite similar rates of VLDL secretion. The rate of VLDL clearance was significantly higher in Arh(-/-) mice than in Ldlr(-/-) mice, suggesting that LDLR-dependent uptake of VLDL is maintained in the absence of ARH. Consistent with these findings, hepatocytes from Arh(-/-) mice (but not Ldlr(-/-) mice) internalized beta-migrating VLDL (beta-VLDL). These results demonstrate that ARH is not required for LDLR-dependent uptake of VLDL by the liver. The preservation of VLDL remnant clearance attenuates the phenotype of ARH and likely contributes to greater responsiveness to statins in ARH compared with FH.

Animals↗
PubMed2006

Sterol transfer by ABCG5 and ABCG8: in vitro assay and reconstitution.

ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either half-transporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "inside-out" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5- and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP >> CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Sterol intermediates from cholesterol biosynthetic pathway as liver X receptor ligands.

The liver X receptors (LXRs) are ligand-activated transcription factors that regulate the expression of genes controlling lipid metabolism. Oxysterols bind LXRs with high affinity in vitro and are implicated as ligands for the receptor. We showed previously that accumulation of selected dietary sterols, in particular stigmasterol, is associated with activation of LXR in vivo. In the course of the defining of structural features of stigmasterol that confer LXR agonist activity, we determined that the presence of an unsaturated bond in the side chain of the sterol was necessary and sufficient for activity, with the C-24 unsaturated cholesterol precursor sterols desmosterol and zymosterol exerting the largest effects. Desmosterol failed to increase expression of the LXR target gene, ABCA1, in LXRalpha/beta-deficient mouse fibroblasts, but was fully active in cells lacking cholesterol 24-, 25-, and 27-hydroxylase; thus, the effect of desmosterol was LXR-dependent and did not require conversion to a side chain oxysterol. Desmosterol bound to purified LXRalpha and LXRbeta in vitro and supported the recruitment of steroid receptor coactivator 1. Desmosterol also inhibited processing of the sterol response element-binding protein-2 and reduced expression of hydroxymethylglutaryl-CoA reductase. These observations are consistent with specific intermediates in the cholesterol biosynthetic pathway regulating lipid homeostasis through both the LXR and sterol response element-binding protein pathways.

ATP Binding Cassette Transporter 1↗
PubMed2006

Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.

Elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) play a central role in the development of atherosclerosis. Mutations in proprotein convertase subtilisin/kexin type 9 (PCSK9) that are associated with lower plasma levels of LDL-C confer protection from coronary heart disease. Here, we show that four severe loss-of-function mutations prevent the secretion of PCSK9 by disrupting synthesis or trafficking of the protein. In contrast to recombinant wild-type PCSK9, which was secreted from cells into the medium within 2 hours, the severe loss-of-function mutations in PCSK9 largely abolished PCSK9 secretion. This finding predicted that circulating levels of PCSK9 would be lower in individuals with the loss-of-function mutations. Immunoprecipitation and immunoblotting of plasma for PCSK9 provided direct evidence that the serine protease is present in the circulation and identified the first known individual who has no immunodetectable circulating PCSK9. This healthy, fertile college graduate, who was a compound heterozygote for two inactivating mutations in PCSK9, had a strikingly low plasma level of LDL-C (14 mg/dL). The very low plasma level of LDL-C and apparent good health of this individual demonstrate that PCSK9 plays a major role in determining plasma levels of LDL-C and provides an attractive target for LDL-lowering therapy.

Adolescent↗
PubMed2006

Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.

BACKGROUND: A low plasma level of low-density lipoprotein (LDL) cholesterol is associated with reduced risk of coronary heart disease (CHD), but the effect of lifelong reductions in plasma LDL cholesterol is not known. We examined the effect of DNA-sequence variations that reduce plasma levels of LDL cholesterol on the incidence of coronary events in a large population. METHODS: We compared the incidence of CHD (myocardial infarction, fatal CHD, or coronary revascularization) over a 15-year interval in the Atherosclerosis Risk in Communities study according to the presence or absence of sequence variants in the proprotein convertase subtilisin/kexin type 9 serine protease gene (PCSK9) that are associated with reduced plasma levels of LDL cholesterol. RESULTS: Of the 3363 black subjects examined, 2.6 percent had nonsense mutations in PCSK9; these mutations were associated with a 28 percent reduction in mean LDL cholesterol and an 88 percent reduction in the risk of CHD (P=0.008 for the reduction; hazard ratio, 0.11; 95 percent confidence interval, 0.02 to 0.81; P=0.03). Of the 9524 white subjects examined, 3.2 percent had a sequence variation in PCSK9 that was associated with a 15 percent reduction in LDL cholesterol and a 47 percent reduction in the risk of CHD (hazard ratio, 0.50; 95 percent confidence interval, 0.32 to 0.79; P=0.003). CONCLUSIONS: These data indicate that moderate lifelong reduction in the plasma level of LDL cholesterol is associated with a substantial reduction in the incidence of coronary events, even in populations with a high prevalence of non-lipid-related cardiovascular risk factors.

Black People↗
PubMed2006

Multiple rare variants in NPC1L1 associated with reduced sterol absorption and plasma low-density lipoprotein levels.

An approach to understand quantitative traits was recently proposed based on the finding that nonsynonymous (NS) sequence variants in certain genes are preferentially enriched at one extreme of the population distribution. The NS variants, although individually rare, are cumulatively frequent and influence quantitative traits, such as plasma lipoprotein levels. Here, we use the NS variant technique to demonstrate that genetic variation in NPC1L1 contributes to variability in cholesterol absorption and plasma levels of low-density lipoproteins (LDLs). The ratio of plasma campesterol (a plant sterol) to lathosterol (a cholesterol precursor) was used to estimate relative cholesterol absorption in a population-based study. Nonsynonymous sequence variations in NPC1L1 were five times more common in low absorbers (n = 26 of 256) than in high absorbers (n = 5 of 256) (P < 0.001). The rare variants identified in low absorbers were found in 6% of 1,832 African-Americans and were associated with lower plasma levels of LDL cholesterol (LDL-C) (96 +/- 36 mg/dl vs. 105 +/- 36 mg/dl; P = 0.005). These data, together with prior findings, reveal a genetic architecture for LDL-C levels that does not conform to current models for quantitative traits and indicate that a significant fraction of genetic variance in LDL-C is due to multiple alleles with modest effects that are present at low frequencies in the population.

Absorption↗
PubMed2006

A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol.

Selected missense mutations in the proprotein convertase subtilisin/kexin type 9 serine protease gene (PCSK9) cause autosomal dominant hypercholesterolemia, whereas nonsense mutations in the same gene are associated with low plasma levels of low-density lipoprotein cholesterol (LDL-C). Here, DNA sequencing and chip-based oligonucleotide hybridization were used to determine whether other sequence variations in PCSK9 contribute to differences in LDL-C levels. The coding regions of PCSK9 were sequenced in the blacks and whites from the Dallas Heart Study (n=3,543) who had the lowest (<5th percentile) and highest (>95th percentile) plasma levels of LDL-C. Of the 17 missense variants identified, 3 (R46L, L253F, and A443T) were significantly and reproducibly associated with lower plasma levels of LDL-C (reductions ranging from 3.5% to 30%). None of the low-LDL-C variants were associated with increased hepatic triglyceride content, as measured by proton magnetic resonance spectroscopy. This finding is most consistent with the reduction in LDL-C being caused primarily by accelerating LDL clearance, rather than by reduced lipoprotein production. Association studies with 93 noncoding single-nucleotide polymorphisms (SNPs) at the PCSK9 locus identified 3 SNPs associated with modest differences in plasma LDL-C levels. Thus, a spectrum of sequence variations ranging in frequency (from 0.2% to 34%) and magnitude of effect (from a 3% increase to a 49% decrease) contribute to interindividual differences in LDL-C levels. These findings reveal that PCSK9 activity is a major determinant of plasma levels of LDL-C in humans and make it an attractive therapeutic target for LDL-C lowering.

Alleles↗
PubMed2006

WholePathwayScope: a comprehensive pathway-based analysis tool for high-throughput data.

BACKGROUND: Analysis of High Throughput (HTP) Data such as microarray and proteomics data has provided a powerful methodology to study patterns of gene regulation at genome scale. A major unresolved problem in the post-genomic era is to assemble the large amounts of data generated into a meaningful biological context. We have developed a comprehensive software tool, WholePathwayScope (WPS), for deriving biological insights from analysis of HTP data. RESULT: WPS extracts gene lists with shared biological themes through color cue templates. WPS statistically evaluates global functional category enrichment of gene lists and pathway-level pattern enrichment of data. WPS incorporates well-known biological pathways from KEGG (Kyoto Encyclopedia of Genes and Genomes) and Biocarta, GO (Gene Ontology) terms as well as user-defined pathways or relevant gene clusters or groups, and explores gene-term relationships within the derived gene-term association networks (GTANs). WPS simultaneously compares multiple datasets within biological contexts either as pathways or as association networks. WPS also integrates Genetic Association Database and Partial MedGene Database for disease-association information. We have used this program to analyze and compare microarray and proteomics datasets derived from a variety of biological systems. Application examples demonstrated the capacity of WPS to significantly facilitate the analysis of HTP data for integrative discovery. CONCLUSION: This tool represents a pathway-based platform for discovery integration to maximize analysis power. The tool is freely available at http://www.abcc.ncifcrf.gov/wps/wps_index.php.

Computer Graphics↗
PubMed2005

Functional asymmetry of nucleotide-binding domains in ABCG5 and ABCG8.

The ATP-binding cassette half-transporters ABCG5 (G5) and ABCG8 (G8) promote secretion of neutral sterols into bile, a major pathway for elimination of sterols. Mutations in either ABCG5 or ABCG8 cause sitosterolemia, a recessive disorder characterized by impaired biliary and intestinal sterol secretion, sterol accumulation, and premature atherosclerosis. The mechanism by which the G5G8 heterodimer couples ATP hydrolysis to sterol transport is not known. Here we examined the roles of the Walker A, Walker B, and signature motifs in the nucleotide-binding domains (NBD) of G5 and G8 using recombinant adenoviruses to reconstitute biliary sterol transport in G5G8-deficient mice. Mutant forms of each half-transporter were co-expressed with their wild-type partners. Mutations at crucial residues in the Walker A and Walker B domains of G5 prevented biliary sterol secretion, whereas mutations of the corresponding residues in G8 did not. The opposite result was obtained when mutations were introduced into the signature motif; mutations in the signature domain of G8 prevented sterol transport, but substitution of the corresponding residues in G5 did not. Taken together, these findings indicate that the NBDs of G5 and G8 are not functionally equivalent. The integrity of the canonical NBD formed by the Walker A and Walker B motifs of G5 and the signature motif of G8 is essential for G5G8-mediated sterol transport. In contrast, mutations in key residues of the NBD formed by the Walker A and B motifs of G8 and the signature sequence of G5 did not affect sterol secretion.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Dual roles for cholesterol in mammalian cells.

The structural features of sterols required to support mammalian cell growth have not been fully defined. Here, we use mutant CHO cells that synthesize only small amounts of cholesterol to test the capacity of various sterols to support growth. Sterols with minor modifications of the side chain (e.g., campesterol, beta-sitosterol, and desmosterol) supported long-term growth of mutant cells, but sterols with more complex modifications of the side chain, the sterol nucleus, or the 3-hydroxy group did not. After 60 days in culture, the exogenous sterol comprised >90% of cellular sterols. Inactivation of residual endogenous synthesis with the squalene epoxidase inhibitor NB-598 prevented growth in beta-sitosterol and greatly reduced growth in campesterol. Growth of cells cultured in beta-sitosterol and NB-598 was restored by adding small amounts of cholesterol to the medium. Surprisingly, enantiomeric cholesterol also supported cell growth, even in the presence of NB-598. Thus, sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective.

Animals↗
PubMed2005

The modular adaptor protein autosomal recessive hypercholesterolemia (ARH) promotes low density lipoprotein receptor clustering into clathrin-coated pits.

Autosomal recessive hypercholesterolemia is characterized by a cell type-specific defect in low density lipoprotein receptor (LDLR) endocytosis. LDLR-mediated uptake of LDL is impaired in the liver, but not in fibroblasts of subjects with this disorder. The disease is caused by mutations in ARH, which encodes a putative adaptor protein that interacts with the cytoplasmic tail of the LDLR, phospholipids, and two components of the clathrin endocytic machinery, clathrin and adaptor protein-2 (AP-2) in vitro. To determine the physiological relevance of these interactions, we examined the effect of mutations in the ARH on LDLR location and function in polarized hepatocytes (WIF-B). The integrity of the FDNPVY sequence in the LDLR cytoplasmic tail was required for ARH-associated LDLR clustering into clathrin-coated pits. The phosphotyrosine binding domain of ARH plus either the clathrin box or the AP-2 binding region were required for both clustering and internalization of the LDLR. Parallel studies performed in vivo with the same recombinant forms of ARH in livers of Arh(-/-) mice confirmed the relevance of the cell culture findings. These results demonstrate that ARH must bind the LDLR tail and either clathrin or AP-2 to promote receptor clustering and internalization of LDL.

Adaptor Protein Complex 2↗
PubMed2005

A phylogenetic survey of biliary lipids in vertebrates.

Biliary lipids (bile salts, phospholipids, cholesterol, plant sterols) were determined in 89 vertebrate species (cartilaginous and bony fish, reptiles, birds, and mammals), and individual phospholipid classes were measured in 35 species. All samples contained conjugated bile salts (C(27) bile alcohol sulfates and/or N-acyl amidates of C(27) and/or C(24) bile acids). Phospholipids were generally absent in the bile of cartilaginous fish and reptiles and were present in low amounts relative to bile salts in bony fish and most birds. In mammals, the phospholipid-bile salt ratio varied widely. The bile from species with low biliary phospholipid-bile salt ratios often contained a high proportion of sphingomyelin, confirmed by HPLC-MS. In species with a high phospholipid-bile salt ratio, the predominant biliary phospholipid was phosphatidylcholine (PC). The phospholipid-bile salt ratio correlated weakly with the calculated weighted hydrophobic index value. Cholesterol was present in the bile of virtually all species, with plant sterols uniformly being present in only trace amounts. The cholesterol-bile salt ratio tended to be higher in mammals than in non-mammals, but bile of all species was unsaturated. Thus, most nonmammalian vertebrates have relatively low levels of biliary phospholipid and cholesterol, suggesting that cholesterol is eliminated predominantly as bile salts. Mammals have a higher phospholipid and cholesterol to bile salt ratio, with the dominant phospholipid being PC.

Animals↗
PubMed2005

ABCG5 and ABCG8 require MDR2 for secretion of cholesterol into bile.

The major pathway for the removal of cholesterol from the body is via secretion into the bile. Three members of the ATP binding cassette (ABC) family, ABCG5 (G5), ABCG8 (G8), and ABCB4 (MDR2), are required for the efficient biliary export of sterols. Here, we examined the interdependence of these three ABC transporters for biliary sterol secretion. Biliary lipid levels in mice expressing no MDR2 (Mdr2-/- mice) were compared with those of Mdr2-/- mice expressing 14 copies of a human G5 (hG5) and hG8 transgene (Mdr2-/-;hG5G8Tg mice). Mdr2-/- mice had only trace amounts of biliary cholesterol and phospholipids. The Mdr2-/-;hG5G8Tg mice had biliary cholesterol levels as low as those of Mdr2-/- mice. Thus, MDR2 expression is required for G5G8-mediated biliary sterol secretion. To determine whether the reduction in fractional absorption of dietary sterols associated with G5G8 overexpression is secondary to the associated increase in biliary cholesterol, we compared the fractional absorption of sterols in Mdr2-/-;hG5G8Tg and hG5G8Tg animals. Inactivation of MDR2 markedly attenuated the reduction in fractional sterol absorption associated with G5G8 overexpression. These results are consistent with the notion that increased biliary cholesterol secretion contributes to the reduction in fractional sterol absorption associated with G5G8 overexpression.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2005

Ezetimibe normalizes metabolic defects in mice lacking ABCG5 and ABCG8.

The ATP binding cassette transporters ABCG5 (G5) and ABCG8 (G8) limit the accumulation of neutral sterols by restricting sterol uptake from the intestine and promoting sterol excretion into bile. Humans and mice lacking G5 and G8 (G5G8-/-) accumulate plant sterols in the blood and tissues. However, despite impaired biliary cholesterol secretion, plasma and liver cholesterol levels are lower in G5G8-/- mice than in wild-type littermates. To determine whether the observed changes in hepatic sterol metabolism were a direct result of decreased biliary sterol secretion or a metabolic consequence of the accumulation of dietary noncholesterol sterols, we treated G5G8-/- mice with ezetimibe, a drug that reduces the absorption of both plant- and animal-derived sterols. Ezetimibe feeding for 1 month sharply decreased sterol absorption and plasma levels of sitosterol and campesterol but increased cholesterol in both the plasma (from 60.4 to 75.2 mg/dl) and the liver (from 1.1 to 1.87 mg/g) of the ezetimibe-treated G5G8-/- mice. Paradoxically, the increase in hepatic cholesterol was associated with an increase in mRNA levels of HMG-CoA reductase and synthase. Together, these results indicate that pharmacological blockade of sterol absorption can ameliorate the deleterious metabolic effects of plant sterols even in the absence of G5 and G8.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2005

Lipoprotein(a) and apolipoprotein(a) isoforms: no association with coronary artery calcification in the Dallas Heart Study.

BACKGROUND: Elevated plasma levels of lipoprotein(a) [Lp(a)] are an independent risk factor for cardiovascular disease in whites. Blacks have 2- to 3-fold higher plasma levels of Lp(a) than whites and yet do not have a correspondingly higher rate of coronary events. It remains unclear whether elevated plasma levels of Lp(a) are an independent risk factor for coronary atherosclerosis in individuals of African descent. METHODS AND RESULTS: The relationship between plasma levels of Lp(a), apolipoprotein(a) isoform sizes, and the presence of coronary calcium was examined in 761 blacks and 527 whites (men aged >40 years, women aged >45 years) from a population-based sample. No relationship was found between plasma levels of Lp(a), apolipoprotein(a) isoform size, or a combination of these 2 variables and coronary artery calcium (CAC) in whites or blacks. No correlation was observed between plasma levels of Lp(a) and coronary calcium scores in any group, although all black men with very high plasma levels of Lp(a) (>300 micromol/L; n=7) were CAC-positive. Whites with high plasma levels of Lp(a) plus elevated plasma levels of LDL cholesterol (men) or reduced levels of HDL cholesterol (men and women) or who smoked (women) had a higher prevalence of CAC. In contrast, no joint effects between plasma levels of Lp(a) and other cardiovascular risk factors on coronary calcium were found in blacks. CONCLUSIONS: No consistent independent relationship between plasma levels of Lp(a) or apolipoprotein(a) isoform size and coronary calcium was found in whites or blacks.

Adult↗
PubMed2005

Lack of MEF2A mutations in coronary artery disease.

Mutations in MEF2A have been implicated in an autosomal dominant form of coronary artery disease (adCAD1). In this study we sought to determine whether severe mutations in MEF2A might also explain sporadic cases of coronary artery disease (CAD). To do this, we resequenced the coding sequence and splice sites of MEF2A in approximately 300 patients with premature CAD and failed to find causative mutations in the CAD cohort. However, we did identify the 21-bp MEF2A coding sequence deletion originally implicated in adCAD1 in 1 of 300 elderly control subjects without CAD. Further screening of approximately 1,500 additional individuals without CAD revealed 2 more subjects with the MEF2A 21-bp deletion. Genotyping of 19 family members of the 3 probands with the 21-bp deletion in MEF2A revealed that the mutation did not cosegregate with early CAD. These studies support that MEF2A mutations are not a common cause of CAD in white people and argue strongly against a role for the MEF2A 21-bp deletion in autosomal dominant CAD.

Adult↗