The Contribution of GWAS Loci in Familial Dyslipidemias.

Familial combined hyperlipidemia (FCH) is a complex and common familial dyslipidemia characterized by elevated total cholesterol and/or triglyceride levels with over five-fold risk of coronary heart disease. The genetic architecture and contribution of rare Mendelian and common variants to FCH susceptibility is unknown. In 53 Finnish FCH families, we genotyped and imputed nine million variants in 715 family members with DNA available. We studied the enrichment of variants previously implicated with monogenic dyslipidemias and/or lipid levels in the general population by comparing allele frequencies between the FCH families and population samples. We also constructed weighted polygenic scores using 212 lipid-associated SNPs and estimated the relative contributions of Mendelian variants and polygenic scores to the risk of FCH in the families. We identified, across the whole allele frequency spectrum, an enrichment of variants known to elevate, and a deficiency of variants known to lower LDL-C and/or TG levels among both probands and affected FCH individuals. The score based on TG associated SNPs was particularly high among affected individuals compared to non-affected family members. Out of 234 affected FCH individuals across the families, seven (3%) carried Mendelian variants and 83 (35%) showed high accumulation of either known LDL-C or TG elevating variants by having either polygenic score over the 90th percentile in the population. The positive predictive value of high score was much higher for affected FCH individuals than for similar sporadic cases in the population. FCH is highly polygenic, supporting the hypothesis that variants across the whole allele frequency spectrum contribute to this complex familial trait. Polygenic SNP panels improve identification of individuals affected with FCH, but their clinical utility remains to be defined.

Effects of angiopoietin-like protein 3 deficiency on postprandial lipid and lipoprotein metabolism.

The consequences of angiopoietin-like protein 3 (ANGPTL3) deficiency on postprandial lipid and lipoprotein metabolism has not been investigated in humans. We studied 7 homozygous (undetectable circulating ANGPTL3 levels) and 31 heterozygous (50% of circulating ANGPTL3 levels) subjects with familial combined hypolipidemia (FHBL2) due to inactivating ANGPTL3 mutations in comparison with 35 controls. All subjects were evaluated at fasting and during 6 h after a high fat meal. Postprandial lipid and lipoprotein changes were quantified by calculating the areas under the curve (AUCs) using the 6 h concentration data. Plasma changes of ß-hydroxybutyric acid (ß-HBA) were measured as marker of hepatic oxidation of fatty acids. Compared with controls, homozygotes showed lower incremental AUCs (iAUCs) of total TG (-69%, P < 0.001), TG-rich lipoproteins (-90%, P < 0.001), apoB-48 (-78%, P = 0.032), and larger absolute increase of FFA (128%, P < 00.1). Also, heterozygotes displayed attenuated postprandial lipemia, but the difference was significant only for the iAUC of apoB-48 (-28%; P < 0.05). During the postprandial period, homozygotes, but not heterozygotes, showed a lower increase of ß-HBA. Our findings demonstrate that complete ANGPTL3 deficiency associates with highly reduced postprandial lipemia probably due to faster catabolism of intestinally derived lipoproteins, larger expansion of the postprandial FFA pool, and decreased influx of dietary-derived fatty acids into the liver. These results add information on mechanisms underlying hypolipidemia in FHBL2.

HDL biogenesis, remodeling, and catabolism.

In this chapter, we review how HDL is generated, remodeled, and catabolized in plasma. We describe key features of the proteins that participate in these processes, emphasizing how mutations in apolipoprotein A-I (apoA-I) and the other proteins affect HDL metabolism. The biogenesis of HDL initially requires functional interaction of apoA-I with the ATP-binding cassette transporter A1 (ABCA1) and subsequently interactions of the lipidated apoA-I forms with lecithin/cholesterol acyltransferase (LCAT). Mutations in these proteins either prevent or impair the formation and possibly the functionality of HDL. Remodeling and catabolism of HDL is the result of interactions of HDL with cell receptors and other membrane and plasma proteins including hepatic lipase (HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor. Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown to form discrete HDL particles containing these apolipoproteins which may have important but still unexplored functions. Furthermore, several plasma proteins were found associated with HDL and may modulate its biological functions. The effect of these proteins on the functionality of HDL is the topic of ongoing research.

Angptl3 deficiency is associated with increased insulin sensitivity, lipoprotein lipase activity, and decreased serum free fatty acids.

OBJECTIVE: Angiopoietin-like 3 (Angptl3) is a regulator of lipoprotein metabolism at least by inhibiting lipoprotein lipase activity. Loss-of-function mutations in ANGPTL3 cause familial combined hypolipidemia through an unknown mechanism. APPROACH AND RESULTS: We compared lipolytic activities, lipoprotein composition, and other lipid-related enzyme/lipid transfer proteins in carriers of the S17X loss-of-function mutation in ANGPTL3 and in age- and sex-matched noncarrier controls. Gel filtration analysis revealed a severely disturbed lipoprotein profile and a reduction in size and triglyceride content of very low density lipoprotein in homozygotes as compared with heterozygotes and noncarriers. S17X homozygotes had significantly higher lipoprotein lipase activity and mass in postheparin plasma, whereas heterozygotes showed no difference in these parameters when compared with noncarriers. No changes in hepatic lipase, endothelial lipase, paraoxonase 1, phospholipid transfer protein, and cholesterol ester transfer protein activities were associated with the S17X mutation. Plasma free fatty acid, insulin, glucose, and homeostatic model assessment of insulin resistance were significantly lower in homozygous subjects compared with heterozygotes and noncarriers subjects. CONCLUSIONS: These results indicate that, although partial Angptl3 deficiency did not affect the activities of lipolytic enzymes, the complete absence of Angptl3 results in an increased lipoprotein lipase activity and mass and low circulating free fatty acid levels. This latter effect is probably because of decreased mobilization of free fatty acid from fat stores in human adipose tissue and may result in reduced hepatic very low density lipoprotein synthesis and secretion via attenuated hepatic free fatty acid supply. Altogether, Angptl3 may affect insulin sensitivity and play a role in modulating both lipid and glucose metabolism.

Genomic, transcriptomic, and lipidomic profiling highlights the role of inflammation in individuals with low high-density lipoprotein cholesterol.

OBJECTIVE: Low high-density lipoprotein cholesterol (HDL-C) is associated with cardiometabolic pathologies. In this study, we investigate the biological pathways and individual genes behind low HDL-C by integrating results from 3 high-throughput data sources: adipose tissue transcriptomics, HDL lipidomics, and dense marker genotypes from Finnish individuals with low or high HDL-C (n=450). APPROACH AND RESULTS: In the pathway analysis of genetic data, we demonstrate that genetic variants within inflammatory pathways were enriched among low HDL-C associated single-nucleotide polymorphisms, and the expression of these pathways upregulated in the adipose tissue of low HDL-C subjects. The lipidomic analysis highlighted the change in HDL particle quality toward putatively more inflammatory and less vasoprotective state in subjects with low HDL-C, as evidenced by their decreased antioxidative plasmalogen contents. We show that the focal point of these inflammatory pathways seems to be the HLA region with its low HDL-associated alleles also associating with more abundant local transcript levels in adipose tissue, increased plasma vascular cell adhesion molecule 1 (VCAM1) levels, and decreased HDL particle plasmalogen contents, markers of adipose tissue inflammation, vascular inflammation, and HDL antioxidative potential, respectively. In a population-based look-up of the inflammatory pathway single-nucleotide polymorphisms in a large Finnish cohorts (n=11 211), no association of the HLA region was detected for HDL-C as quantitative trait, but with extreme HDL-C phenotypes, implying the presence of low or high HDL genes in addition to the population-genomewide association studies-identified HDL genes. CONCLUSIONS: Our study highlights the role of inflammation with a genetic component in subjects with low HDL-C and identifies novel cis-expression quantitative trait loci (cis-eQTL) variants in HLA region to be associated with low HDL-C.

Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1).

Familial combined hyperlipidemia (FCHL), characterized by elevated levels of serum total cholesterol, triglycerides or both, is observed in about 20% of individuals with premature coronary heart disease. We previously identified a locus linked to FCHL on 1q21-q23 in Finnish families with the disease. This region has also been linked to FCHL in families from other populations as well as to type 2 diabetes mellitus. These clinical entities have several overlapping phenotypic features, raising the possibility that the same gene may underlie the obtained linkage results. Here, we show that the human gene encoding thioredoxin interacting protein (TXNIP) on 1q, which underlies combined hyperlipidemia in mice, is not associated with FCHL. We show that FCHL is linked and associated with the gene encoding upstream transcription factor 1 (USF1) in 60 extended families with FCHL, including 721 genotyped individuals (P = 0.00002), especially in males with high triglycerides (P = 0.0000009). Expression profiles in fat biopsy samples from individuals with FCHL seemed to differ depending on their carrier status for the associated USF1 haplotype. USF1 encodes a transcription factor known to regulate several genes of glucose and lipid metabolism.

Plasma homocysteine and the risk of venous thromboembolism: insights from the FIELD study.

BACKGROUND: The lipid-lowering effect of fenofibrate is accompanied by a rise in plasma homocysteine (HCY), a potential risk factor for venous thromboembolism (VTE).This study investigated the relationship between HCY and the risk of VTE in patients treated with fenofibrate. METHODS: The relationship between HCY and deep-vein thrombosis or pulmonary embolism was investigated in 9522 participants of the 5-year Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial. All subjects received fenofibrate during a 6-week active run-in phase before randomization.A Cox proportional-hazards model was used to assess the effect of HCY on risk of venous thromboembolic events. RESULTS: During active-drug run-in, HCY rose on average by 6.5 µ mol/L, accompanied by a substantial rise in plasma creatinine ( + 12 % ). Fenofibrate-induced changes in HCY and creatinine were fully reversible in the placebo group but persisted in the treatment group until reversing at the end of therapy. During follow-up, 1.8 % had at least one episode of deep-vein thrombosis or pulmonary embolism: 103 on fenofibrate and 68 on placebo (log-rank p = 0.006). In multivariate analysis, every 5 µ mol/L higher baseline HCY was associated with 19 % higher risk of VTE. Fenofibrate treatment was associated with 52 % higher risk, but the change in HCY with fenofibrate was not significantly associated with VTE after adjustment for baseline HCY. CONCLUSIONS: Hyperhomocysteinemia is prospectively associated with VTE. Fenofibrate may predispose individuals with high pretreatment HCY towards VTE. The fenofibrate induced increase in HCY did not, however, explain the risk associated with fenofibrate therapy.

Serum angiopoietin-like 4 protein levels and expression in adipose tissue are inversely correlated with obesity in monozygotic twins.

Animal studies have suggested that angiopoietin-like 4 (Angptl4) regulates adiposity through central and peripheral mechanisms. The aim of this study was to investigate whether serum concentration and adipose tissue expression of Angptl4 are associated with obesity-related parameters in humans. Altogether, 75 dizygotic (DZ) and 46 monozygotic (MZ) twin pairs were studied, from the FinnTwin12 and FinnTwin16 cohorts. Among the MZ pairs, 21 were discordant for body mass index (BMI) (intra-pair BMI-difference >2.5 kg/m², age 23-33 years). Serum Angptl4 (s-Angptl4) levels were measured by ELISA, and adipose tissue gene expression was analyzed by genome-wide transcript profiling. In MZ twin pairs discordant for BMI, s-Angptl4 and adipose tissue ANGPTL4 mRNA (at-ANGPTL4) levels were significantly decreased (P = 0.04 and P = 0.03, respectively) in obese twins as compared with their nonobese cotwins. In all twins, intra-pair differences in s-Angptl4 levels were inversely correlated with intra-pair differences in BMI (r = -0.27, P = 0.003). In individual MZ twins, at-ANGPTL4 expression was inversely correlated with BMI (r = -0.44, P = 0.001) and positively correlated with at-LIPE (r = 0.24, P = 0.01) and at-ABHD5 (r = 0.41, P = 0.005) expression. Our results demonstrated that variation in Angptl4 concentration was only modestly accounted for by genetic factors and suggest a role for Angptl4 in acquired obesity in humans.

Genetic association and interaction analysis of USF1 and APOA5 on lipid levels and atherosclerosis.

OBJECTIVE: USF1 is a ubiquitous transcription factor governing the expression of numerous genes of lipid and glucose metabolism. APOA5 is a well-established candidate gene regulating triglyceride (TG) levels and has been identified as a downstream target of upstream stimulatory factor. No detailed studies about the effect of APOA5 on atherosclerotic lesion formation have been conducted, nor has its potential interaction with USF1 been examined. METHODS AND RESULTS: We analyzed allelic variants of USF1 and APOA5 in families (n=516) ascertained for atherogenic dyslipidemia and in an autopsy series of middle-aged men (n=300) with precise quantitative measurements of atherosclerotic lesions. The impact of previously associated APOA5 variants on TGs was observed in the dyslipidemic families, and variant rs3135506 was associated with size of fibrotic aortic lesions in the autopsy series. The USF1 variant rs2516839, associated previously with atherosclerotic lesions, showed an effect on TGs in members of the dyslipidemic families with documented coronary artery disease. We provide preliminary evidence of gene-gene interaction between these variants in an autopsy series with a fibrotic lesion area in the abdominal aorta (P=0.0028), with TGs in dyslipidemic coronary artery disease subjects (P=0.03), and with high-density lipoprotein cholesterol (P=0.008) in a large population cohort of coronary artery disease patients (n=1065) in which the interaction for TGs was not replicated. CONCLUSIONS: Our findings in these unique samples reinforce the roles of APOA5 and USF1 variants on cardiovascular phenotypes and suggest that both genes contribute to lipid levels and aortic atherosclerosis individually and possibly through epistatic effects.

The relationship between plasma angiopoietin-like protein 4 levels, angiopoietin-like protein 4 genotype, and coronary heart disease risk.

OBJECTIVE: To investigate the relationship between angiopoietin-like protein 4 (Angptl4) levels, coronary heart disease (CHD) biomarkers, and ANGPTL4 variants. METHODS AND RESULTS: Plasma Angptl4 was quantified in 666 subjects of the Northwick Park Heart Study II using a validated ELISA. Seven ANGPTL4 single-nucleotide polymorphisms were genotyped, and CHD biomarkers were assessed in the whole cohort (N=2775). Weighted mean±SD plasma Angptl4 levels were 10.0±11.0 ng/mL. Plasma Angptl4 concentration correlated positively with age (r=0.15, P<0.001) and body fat mass (r=0.19, P=0.003) but negatively with plasma high-density lipoprotein cholesterol (r=-0.13, P=0.01). No correlation with triglycerides (TGs) was observed. T266M was independently associated with plasma Angptl4 levels (P<0.001) but was not associated with TGs or CHD risk in the meta-analysis of 5 studies (4061 cases/15 395 controls). E40K showed no independent association with plasma Angptl4 levels. In human embryonic kidney 293 and human hepatoma 7 cells compared with wild type, E40K and T266M showed significantly altered synthesis and secretion, respectively. CONCLUSIONS: Circulating Angptl4 levels may not influence TG levels or CHD risk for the following reasons: (1) Angptl4 levels were not correlated with TGs; (2) T266M, although associated with Angptl4 levels, showed no association with plasma TGs; and (3) TG-lowering E40K did not influence Angptl4 levels. These results provide new insights into the role of Angptl4 in TG metabolism.

Quantitation of serum angiopoietin-like proteins 3 and 4 in a Finnish population sample.

We have developed and validated quantitative ELISAs for human angiopoietin-like (ANGPTL)3 and 4 and correlated their serum levels with parameters of lipid and carbohydrate metabolism. For this study, we used a random subsample of the Health 2000 Health Examination Survey consisting of 125 men and 125 women, aged 30-94 years. The anthropometric and biochemical parameters of subjects were characterized in detail. ANGPTL 3 and 4 levels were determined using the developed ELISAs. The intra- and inter-assay coefficients of variation for the assays were less than 15%. The average serum concentration of ANGPTL3 was 368 +/- 168 ng/ml (mean +/- SD) and for ANGPTL4 it was 18 +/- 23 ng/ml (mean +/- SD). ANGPTL4 serum levels displayed high variability between individuals ranging from 2 to 158 ng/ml. In post-heparin plasma, both ANGPTL 3 and 4 were increased. Low levels of ANGPTL3 were associated with decreased HDL-cholesterol and increased triglyceride levels. ANGPTL4 levels were positively correlated with FFAs (P = 0.044) and waist-hip ratio (P = 0.016). The developed ELISAs will be important tools to clarify the role of ANGPTL 3 and 4 in human energy metabolism and partitioning of triglycerides between sites of storage (adipose tissue) and oxidation (skeletal and cardiac muscle).

Analysis of lipid transfer activity between model nascent HDL particles and plasma lipoproteins: implications for current concepts of nascent HDL maturation and genesis.

The specifics of nascent HDL remodeling within the plasma compartment remain poorly understood. We developed an in vitro assay to monitor the lipid transfer between model nascent HDL (LpA-I) and plasma lipoproteins. Incubation of alpha-(125)I-LpA-I with plasma resulted in association of LpA-I with existing plasma HDL, whereas incubation with TD plasma or LDL resulted in conversion of alpha-(125)I-LpA-I to prebeta-HDL. To further investigate the dynamics of lipid transfer, nascent LpA-I were labeled with cell-derived [(3 )H]cholesterol (UC) or [(3)H]phosphatidylcholine (PC) and incubated with plasma at 37 degrees C. The majority of UC and PC were rapidly transferred to apolipoprotein B (apoB). Subsequently, UC was redistributed to HDL for esterification before being returned to apoB. The presence of a phospholipid transfer protein (PLTP) stimulator or purified PLTP promoted PC transfer to apoB. Conversely, PC transfer was abolished in plasma from PLTP(-/-) mice. Injection of (125)I-LpA-I into rabbits resulted in a rapid size redistribution of (125)I-LpA-I. The majority of [(3)H]UC from labeled r(HDL) was esterified in vivo within HDL, whereas a minority was found in LDL. These data suggest that apoB plays a major role in nascent HDL remodeling by accepting their lipids and donating UC to the LCAT reaction. The finding that nascent particles were depleted of their lipids and remodeled in the presence of plasma lipoproteins raises questions about their stability and subsequent interaction with LCAT.

Relationships of HDL cholesterol, ApoA-I, and ApoA-II with homocysteine and creatinine in patients with type 2 diabetes treated with fenofibrate.

OBJECTIVE: The purpose of this study was to determine fenofibrate-induced changes in plasma high-density lipoprotein cholesterol (HDL-C), apolipoprotein (apo) A-I, and apolipoprotein (apo) A-II and how they relate to changes in plasma homocysteine and creatinine. METHODS AND RESULTS: FIELD was a double-blind placebo-controlled trial done in Australia, New Zealand, and Finland. All FIELD subjects were included except those who started statin therapy or permanently discontinued fenofibrate. Patients were randomized to receive daily micronized fenofibrate (200 mg) or matching placebo and were followed up for a median of 5 years. Plasma HDL-C, apoA-I, apoA-II, homocysteine, and creatinine were measured. There was an inverse relationship between baseline homocysteine levels and HDL-C in the placebo (P=0.07 for linear trend) and fenofibrate groups (P<0.0001) and apoA-I (P<0.001, both groups). The increases in homocysteine and creatinine in the fenofibrate group correlated positively (P<0.0001). The greater the increase in homocysteine induced by fenofibrate, the smaller the increases in HDL-c and apoA-I (P<0.0001 for linear trends). There was a highly significant and positive relationship between fenofibrate-induced changes in homocysteine and apoA-II levels. CONCLUSIONS: PPAR alpha agonists that have a more robust effect on HDL-C and apoA-I would be desirable.

Human apoA-I increases macrophage foam cell derived PLTP activity without affecting the PLTP mass.

BACKGROUND: phospholipid transfer protein (PLTP) plays important roles in lipoprotein metabolism and atherosclerosis and is expressed by macrophages and macrophage foam cells (MFCs). The aim of the present study was to determine whether the major protein from HDL, apoA-I, affects PLTP derived from MFCs. RESULTS: as cell model we used human THP-1 monocytes incubated with acetylated LDL, to generate MFC. The addition of apoA-I to the cell media increased apoE secretion from the cells, in a concentration dependent fashion, without affecting cellular apoE levels. In contrast, apoA-I had no effect on PLTP synthesis and secretion, but strongly induced the PLTP activity in the media. ApoA-I also increased phospholipid transfer activity of PLTP isolated from human plasma. This effect was dependent on apoA-I concentration but independent on apoA-I lipidation status. ApoE, ApoA-II and apoA-IV, but not immunoglobulins or bovine serum albumin, also increased PLTP activity. We also report that apoA-I protects PLTP from heat inactivation. CONCLUSION: apoA-I enhances the phospholipid transfer activity of PLTP secreted from macrophage foam cells without affecting the PLTP mass.

Important role for bone marrow-derived cholesteryl ester transfer protein in lipoprotein cholesterol redistribution and atherosclerotic lesion development in LDL receptor knockout mice.

Abundant amounts of cholesteryl ester transfer protein (CETP) are found in macrophage-derived foam cells in the arterial wall, but its function in atherogenesis is unknown. To investigate the role of macrophage CETP in atherosclerosis, LDL receptor knockout mice were transplanted with bone marrow from CETP transgenic mice, which express the human CETP transgene under control of its natural promoter and major regulatory elements. CETP production by bone marrow-derived cells induced a 1.8-fold (P<0.01) increase in atherosclerotic lesion development. The increase in lesion size coincided with an increase in VLDL/LDL cholesterol and a decrease in HDL cholesterol. The cholesterol redistribution in serum was a direct effect of the substantial serum CETP activity and mass (38+/-3 nmol/mL/h and 4.8+/-0.5 microg/mL, respectively) induced by CETP production by bone marrow-derived cells. Conversely, specific disruption of CETP production by bone marrow-derived cells in CETP transgenic mice resulted in a approximately 2-fold (P<0.0001) reduction in serum CETP activity and mass, demonstrating the quantitative relevance of bone marrow-derived CETP. Finally, we show that in liver Kupffer cells, hepatic macrophages, contribute approximately 50% to the total hepatic CETP expression. In conclusion, bone marrow-derived CETP induces a proatherogenic lipoprotein profile and promotes the development of atherosclerotic lesions in LDL receptor knockout mice. Most importantly, we show for the first time that bone marrow-derived CETP is an important contributor to total serum CETP activity and mass.

Oxysterol binding protein induces upregulation of SREBP-1c and enhances hepatic lipogenesis.

BACKGROUND: Oxysterol binding protein (OSBP) has previously been implicated as a sterol sensor that regulates sphingomyelin synthesis and the activity of extracellular signal-regulated kinases (ERK). METHODS AND RESULTS: We determined the effects of adenovirus-mediated hepatic overexpression of OSBP and its homologues ORP1L and ORP3 on mouse serum lipids. Whereas ORP1L and ORP3 had no effect on serum lipids, OSBP induced a marked increase of VLDL triglycerides (TG). Also, the liver tissue TG were elevated in the AdOSBP-injected mice, and their TG secretion rate was increased by 70%. The messenger RNAs for enzymes of fatty acid synthesis and their transcriptional regulator, SREBP-1c, as well as the Insig-1 mRNA, were upregulated two-fold in the OSBP-expressing livers. No change occurred in the messages of liver X receptor target genes ABCA1, ABCG5, and CYP7A1, and the Insig-2a mRNA was reduced. The phosphorylation of ERK was decreased in AdOSBP-infected liver and cultured hepatocytes. Importantly, silencing of OSBP in hepatocytes suppressed the induction of SREBP1-c by insulin and resulted in a reduction of TG synthesis. CONCLUSION: Our results demonstrate that OSBP regulates hepatic TG metabolism and suggest the involvement of OSBP in the insulin signaling pathways that control hepatic lipogenesis.

Expression of human OSBP-related protein 1L in macrophages enhances atherosclerotic lesion development in LDL receptor-deficient mice.

OBJECTIVE: The purpose of this study was to assess the role of macrophage OSBP-related protein 1L (ORP1L) in the development of atherosclerosis. METHODS AND RESULTS: C57BL/6 mice overexpressing human ORP1L in macrophages driven by scavenger receptor A promoter were generated. Bone marrow (BM) of the mice was transplanted into LDLr-/- animals, and aortic root lesion area in the recipients was determined after Western-type diet feeding. The recipients of ORP1L BM displayed 2.1-fold increase (P<0.001) in lesion size as compared with recipients of wild-type littermate BM. Macrophages of the ORP1L BM recipients showed a decrease in ABCG1 and APOE mRNAs and proteins, and an increase in PLTP message; also the plasma PLTP activity was elevated. The effect of ORP1L on cholesterol efflux was assessed using macrophages loaded with [3H]cholesterol oleate-acLDL or labeled with [3H]cholesterol. The ORP1L transgenic macrophages displayed 30% reduction (P<0.01) in cholesterol efflux to HDL2, but not to apoA-I. ORP1L was shown to bind 25- and 22(R)-hydroxycholesterol, identifying it as an oxysterol binding protein. Furthermore, ORP1L attenuated the response of ABCG1 mRNA to 22(R)-hydroxycholesterol, the effect on ABCA1 being less pronounced. CONCLUSIONS: The results demonstrate that macrophage ORP1L can act as a modulator of atherosclerotic lesion development and provide clues to the underlying mechanism.

Macrophage phospholipid transfer protein contributes significantly to total plasma phospholipid transfer activity and its deficiency leads to diminished atherosclerotic lesion development.

OBJECTIVE: Systemic phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, whereas overexpression of human PLTP in mice increases atherosclerotic lesion development. PLTP is also expressed by macrophage-derived foam cells in human atherosclerotic lesions, but the exact role of macrophage PLTP in atherosclerosis is unknown. METHODS AND RESULTS: To clarify the role of macrophage PLTP in atherogenesis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP(-/-)) mice into irradiated low-density lipoprotein receptor knockout mice. Selective deficiency of macrophage PLTP (PLTP(-M/-M)) resulted in a 29% (P<0.01 for difference in lesion area) reduction in aortic root lesion area as compared with mice possessing functional macrophage PLTP (384+/-36*10(3) microm2 in the PLTP(-M/-M) group (n=10), as compared with 539+/-35*10(3) microm2 in the PLTP(+M/+M) group (n=14)) after 9 weeks of Western-type diet feeding. The decreased lesion size in the PLTP(-M/-M) group coincided with significantly lower serum total cholesterol, free cholesterol, and triglyceride levels in these mice. Furthermore, plasma PLTP activity in the PLTP(-M/-M) group was 2-fold (P<0.001) lower than that in the PLTP(+M/+M) group. CONCLUSION: Macrophage PLTP is a significant contributor to plasma PLTP activity and deficiency of PLTP in macrophages leads to lowered atherosclerotic lesion development in low-density lipoprotein receptor knockout mice on Western-type diet.

Altered hepatic lipid status and apolipoprotein A-I metabolism in mice lacking phospholipid transfer protein.

The effect of PLTP deficiency on hepatic lipid status and apolipoprotein A-I (apoA-I) biosynthesis in PLTP knockout (PLTP-KO) mice was investigated. PLTP-KO mice exhibited a marked reduction in HDL levels, but also increased triglycerides (TG), phospholipids (PL), and cholesterol in very-low-density lipoproteins (VLDL). Both male and female PLTP-KO mice displayed increased hepatic PL and decreased TG, and in the females, increased hepatic cholesterol was also detected. Primary hepatocytes from PLTP-KO mice displayed a different PL molecular species composition to the wild type (WT) controls, with prominent changes being a reduction of long chain fatty acid-containing and an increase of medium chain mono- or di-unsaturated fatty acid containing PL species. Cultured PLTP-KO hepatocytes synthesized and secreted apoA-I in similar quantities as the WT cells. However, the apoA-I secreted by PLTP-KO hepatocytes contained less choline PL, differing also in phosphatidylcholine/sphingomyelin ratio and fatty acyl species composition when compared to apoA-I from WT hepatocytes. Furthermore, the PLTP-KO-derived PL-deficient apoA-I was less stable in the hepatocyte culture medium than that produced by WT cells. These results demonstrate a complex regulatory role of PLTP in serum and liver lipid homeostasis, as well as in the formation of nascent apoA-I-PL complexes from the liver.

Decreased high-density lipoprotein (HDL) particle size, prebeta-, and large HDL subspecies concentration in Finnish low-HDL families: relationship with intima-media thickness.

OBJECTIVE: High-density lipoprotein (HDL) cholesterol correlates inversely with the risk of coronary heart disease (CHD). The precise antiatherogenic mechanisms of HDL subspecies are not thoroughly elucidated. We studied the relationship between carotid intima-media thickness (IMT) and HDL subspecies distribution in Finnish families with low HDL cholesterol and premature CHD. METHODS AND RESULTS: Altogether, 148 members of Finnish low-HDL families and 133 healthy control subjects participated in our study. HDL particle size was significantly smaller in affected family members (HDL < or =10th Finnish age-sex specific percentile) compared with unaffected family members and control subjects (9.1+/-0.04 nm versus 9.5+/-0.05 nm, P<0.0001, versus 9.8+/-0.03 nm, P<0.0001 [mean+/-SE]). Large HDL2b particles as well as prebeta-HDL concentration were significantly decreased among the affected family members. Mean IMT was significantly higher in the affected family members than in the control subjects (0.85+/-0.01 mm versus 0.79+/-0.01 mm; P<0.0001). Age, HDL2b, systolic blood pressure, and prebeta-HDL were significant independent determinants of mean IMT. CONCLUSIONS: The decreased levels of HDL2b and prebeta-HDL reflect the potentially efflux-deficient HDL subspecies profile in the affected low-HDL family members. Decreased HDL particle size caused by the decrease of plasma concentration of HDL2b and decreased prebeta-HDL levels correlate with increased IMT.