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1.  Association Analysis of Allelic Variants of USF1 in Coronary Atherosclerosis 
Objective
USF1 regulates the transcription of more than 40 cardiovascular related genes and is well established as a gene associated with familial combined hyperlipidemia, a condition increasing the risk for coronary heart disease. No detailed data, however, exists on the impact of this gene to the critical outcome at the tissue level: different types of atherosclerotic lesions.
Methods and Results
We analyzed the USF1 in 2 autopsy series of altogether 700 middle-aged men (the Helsinki Sudden Death Study) with quantitative morphometric measurements of coronary atherosclerosis. SNP rs2516839, tagging common USF1 haplotypes, associated with the presence of several types of atherosclerotic lesions, particularly with the proportion of advanced atherosclerotic plaques (P=0.02) and area of calcified lesions (P<0.001) of the coronary arteries. Importantly, carriers of risk alleles of rs2516839 also showed a 2-fold risk for sudden cardiac death (genotype TT versus CC; OR 2.10, 95% CI 1.17 to 3.75, P=0.04). The risk effect of rs2516839 was present also in aorta samples of the men.
Conclusions
Our findings in this unique study sample suggest that USF1 contributes to atherosclerosis, the pathological arterial wall phenotype resulting in coronary heart disease and in its most dramatic consequence—sudden cardiac death.
doi:10.1161/ATVBAHA.107.156463
PMCID: PMC2687549  PMID: 18276913
atherosclerosis; coronary; genes; genetics; death; sudden
2.  Upstream Transcription Factor 1 (USF1) allelic variants regulate lipoprotein metabolism in women and USF1 expression in atherosclerotic plaque 
Scientific Reports  2014;4:4650.
Upstream transcription factor 1 (USF1) allelic variants significantly influence future risk of cardiovascular disease and overall mortality in females. We investigated sex-specific effects of USF1 gene allelic variants on serum indices of lipoprotein metabolism, early markers of asymptomatic atherosclerosis and their changes during six years of follow-up. In addition, we investigated the cis-regulatory role of these USF1 variants in artery wall tissues in Caucasians. In the Cardiovascular Risk in Young Finns Study, 1,608 participants (56% women, aged 31.9 ± 4.9) with lipids and cIMT data were included. For functional study, whole genome mRNA expression profiling was performed in 91 histologically classified atherosclerotic samples. In females, serum total, LDL cholesterol and apoB levels increased gradually according to USF1 rs2516839 genotypes TT < CT < CC and rs1556259 AA < AG < GG as well as according to USF1 H3 (GCCCGG) copy number 0 < 1 < 2. Furthermore, the carriers of minor alleles of rs2516839 (C) and rs1556259 (G) of USF1 gene had decreased USF1 expression in atherosclerotic plaques (P = 0.028 and 0.08, respectively) as compared to non-carriers. The genetic variation in USF1 influence USF1 transcript expression in advanced atherosclerosis and regulates levels and metabolism of circulating apoB and apoB-containing lipoprotein particles in sex-dependent manner, but is not a major determinant of early markers of atherosclerosis.
doi:10.1038/srep04650
PMCID: PMC3983598  PMID: 24722012
3.  Gene-Gene Interaction between APOA5 and USF1: Two Candidate Genes for the Metabolic Syndrome 
Obesity Facts  2009;2(4):235-242.
Summary
Objective
The metabolic syndrome, a major cluster of risk factors for cardiovascular diseases, shows increasing prevalence worldwide. Several studies have established associations of both apolipoprotein A5 (APOA5) gene variants and upstream stimulatory factor 1 (USF1) gene variants with blood lipid levels and metabolic syndrome. USF1 is a transcription factor for APOA5.
Methods
We investigated a possible interaction between these two genes on the risk for the metabolic syndrome, using data from the German population-based KORA survey 4 (1,622 men and women aged 55–74 years). Seven APOA5 single nucleotide polymorphisms (SNPs) were analyzed in combination with six USF1 SNPs, applying logistic regression in an additive model adjusting for age and sex and the definition for metabolic syndrome from the National Cholesterol Education Program's Adult Treatment Panel III (NCEP (AIII)) including medication.
Results
The overall prevalence for metabolic syndrome was 41%. Two SNP combinations showed a nominal gene-gene interaction (p values 0.024 and 0.047). The effect of one SNP was modified by the other SNP, with a lower risk for the metabolic syndrome with odds ratios (ORs) between 0.33 (95% CI = 0.13–0.83) and 0.40 (95% CI = 0.15–1.12) when the other SNP was homozygous for the minor allele. Nevertheless, none of the associations remained significant after correction for multiple testing.
Conclusion
Thus, there is an indication of an interaction between APOA5 and USF1 on the risk for metabolic syndrome.
doi:10.1159/000227288
PMCID: PMC2919429  PMID: 20054229
Metabolic syndrome; Cardiovascular risk; SNP; APOA5; USF1
4.  Risk Alleles of USF1 Gene Predict Cardiovascular Disease of Women in Two Prospective Studies 
PLoS Genetics  2006;2(5):e69.
Upstream transcription factor 1 (USF1) is a ubiquitously expressed transcription factor controlling several critical genes in lipid and glucose metabolism. Of some 40 genes regulated by USF1, several are involved in the molecular pathogenesis of cardiovascular disease (CVD). Although the USF1 gene has been shown to have a critical role in the etiology of familial combined hyperlipidemia, which predisposes to early CVD, the gene's potential role as a risk factor for CVD events at the population level has not been established. Here we report the results from a prospective genetic–epidemiological study of the association between the USF1 variants, CVD, and mortality in two large Finnish cohorts. Haplotype-tagging single nucleotide polymorphisms exposing all common allelic variants of USF1 were genotyped in a prospective case-cohort design with two distinct cohorts followed up during 1992–2001 and 1997–2003. The total number of follow-up years was 112,435 in 14,140 individuals, of which 2,225 were selected for genotyping based on the case-cohort study strategy. After adjustment for conventional risk factors, we observed an association of USF1 with CVD and mortality among females. In combined analysis of the two cohorts, female carriers of a USF1 risk haplotype had a 2-fold risk of a CVD event (hazard ratio [HR] 2.02; 95% confidence interval [CI] 1.16–3.53; p = 0.01) and an increased risk of all-cause mortality (HR 2.52; 95% CI 1.46–4.35; p = 0.0009). A putative protective haplotype of USF1 was also identified. Our study shows how a gene identified in exceptional families proves to be important also at the population level, implying that allelic variants of USF1 significantly influence the prospective risk of CVD and even all-cause mortality in females.
Synopsis
Better characterization of molecular events resulting in cardiovascular disease (CVD) requires elucidation of genetic background of CVD. After a CVD candidate gene is identified in family-based studies or case-control studies, population-based prospective studies are needed to demonstrate any potential impact of allelic variants on the CVD risk at the population level. This study addresses the role of different alleles of the upstream transcription factor 1 (USF1) gene, encoding a transcription factor and originally associated with familial combined hyperlipidemia in rare families with multiple affected individuals. The product of USF1 regulates numerous genes of lipid and glucose metabolism, and the authors show in large population cohorts that specific alleles of USF1 are associated with the risk of CVD and all-cause mortality among females. The study implies an interesting female-specific risk effect, and should stimulate additional studies of the sex-specific CVD risk genes in different populations.
doi:10.1371/journal.pgen.0020069
PMCID: PMC1458962  PMID: 16699592
5.  Candidate genetic analysis of plasma high-density lipoprotein-cholesterol and severity of coronary atherosclerosis 
BMC Medical Genetics  2009;10:111.
Background
Plasma level of high-density lipoprotein-cholesterol (HDL-C), a heritable trait, is an important determinant of susceptibility to atherosclerosis. Non-synonymous and regulatory single nucleotide polymorphisms (SNPs) in genes implicated in HDL-C synthesis and metabolism are likely to influence plasma HDL-C, apolipoprotein A-I (apo A-I) levels and severity of coronary atherosclerosis.
Methods
We genotyped 784 unrelated Caucasian individuals from two sets of populations (Lipoprotein and Coronary Atherosclerosis Study- LCAS, N = 333 and TexGen, N = 451) for 94 SNPs in 42 candidate genes by 5' nuclease assays. We tested the distribution of the phenotypes by the Shapiro-Wilk normality test. We used Box-Cox regression to analyze associations of the non-normally distributed phenotypes (plasma HDL-C and apo A-I levels) with the genotypes. We included sex, age, body mass index (BMI), diabetes mellitus (DM), and cigarette smoking as covariates. We calculated the q values as indicators of the false positive discovery rate (FDR).
Results
Plasma HDL-C levels were associated with sex (higher in females), BMI (inversely), smoking (lower in smokers), DM (lower in those with DM) and SNPs in APOA5, APOC2, CETP, LPL and LIPC (each q ≤0.01). Likewise, plasma apo A-I levels, available in the LCAS subset, were associated with SNPs in CETP, APOA5, and APOC2 as well as with BMI, sex and age (all q values ≤0.03). The APOA5 variant S19W was also associated with minimal lumen diameter (MLD) of coronary atherosclerotic lesions, a quantitative index of severity of coronary atherosclerosis (q = 0.018); mean number of coronary artery occlusions (p = 0.034) at the baseline and progression of coronary atherosclerosis, as indicated by the loss of MLD.
Conclusion
Putatively functional variants of APOA2, APOA5, APOC2, CETP, LPL, LIPC and SOAT2 are independent genetic determinants of plasma HDL-C levels. The non-synonymous S19W SNP in APOA5 is also an independent determinant of plasma apo A-I level, severity of coronary atherosclerosis and its progression.
doi:10.1186/1471-2350-10-111
PMCID: PMC2775733  PMID: 19878569
6.  The rs2516839 Polymorphism of the USF1 Gene May Modulate Serum Triglyceride Levels in Response to Cigarette Smoking 
Single nucleotide polymorphisms (SNPs) of the USF1 gene (upstream stimulatory factor 1) influence plasma lipid levels. This study aims to determine whether USF1 SNPs interact with traditional risk factors of atherosclerosis to increase coronary artery disease (CAD) risk. In the present study serum lipid levels and USF1 gene polymorphisms (rs2516839 and rs3737787) were determined in 470 subjects: 235 patients with premature CAD and 235 controls. A trend of increasing triglycerides (TG) levels in relation to the C allele dose of rs2516839 SNP was observed. The synergistic effect of cigarette smoking and C allele carrier state on CAD risk was also found (SIM = 2.69, p = 0.015). TG levels differentiated significantly particular genotypes in smokers (1.53 mmol/L for TT, 1.80 mmol/L for CT and 2.27 mmol/L for CC subjects). In contrast, these differences were not observed in the non-smokers subgroup (1.57 mmol/L for TT, 1.46 mmol/L for CT and 1.49 mmol/L for CC subjects). In conclusion, the rs2516839 polymorphism may modulate serum triglyceride levels in response to cigarette smoking. Carriers of the C allele seem to be particularly at risk of CAD, when exposed to cigarette smoking.
doi:10.3390/ijms160613203
PMCID: PMC4490492  PMID: 26068452
USF1; polymorphism; cigarette smoking; CAD; triglycerides; gene-traditional risk factors interactions
7.  Effects of native and myeloperoxidase-modified apolipoprotein A-I on reverse cholesterol transport and atherosclerosis in mice 
Objective
Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase (MPO)-mediated oxidation, leading in vitro to a loss of its ability to promote ABCA1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that MPO-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport (RCT) in vivo and the beneficial effects on atherosclerotic plaques.
Approach and Results
ApoA-I−/− or ApoE−/− mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (oxApoAI; MPO/hydrogen peroxide/chloride treated) or carrier. While early post injection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxApoA-I, native ApoA-I primarily resided within the HDL fraction, whereas the majority of oxApoA-I was highly cross-linked and not HDL particle associated, consistent with impaired ABCA1 interaction. In ApoA-I−/− mice, ApoA-I oxidation significantly impaired RCT in vivo. In advanced aortic root atherosclerotic plaques of ApoE−/− mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques.
Conclusions
MPO-mediated oxidation renders ApoA-I dysfunctional and unable to: (i) promote RCT; (ii) mediate beneficial changes in the composition of atherosclerotic plaques; and (iii) pacify the inflammatory status of plaque macrophages.
doi:10.1161/ATVBAHA.113.303044
PMCID: PMC3966977  PMID: 24407029
Atherosclerosis; ApoA-I; Myeloperoxidase; Dysfunctional HDL; Reverse Cholesterol Transport
8.  The Function and Distribution of Apolipoprotein A1 in the Artery Wall are Markedly Distinct from those in Plasma 
Circulation  2013;128(15):10.1161/CIRCULATIONAHA.113.002624.
Background
Prior studies show apolipoprotein A1 (apoA1) recovered from human atherosclerotic lesions is highly oxidized. Ex vivo oxidation of apoA1 or high density lipoprotein (HDL) cross-links apoA1 and impairs lipid binding, cholesterol efflux and lecithin cholesterol acyltransferase (LCAT) activities of the lipoprotein. Remarkably, no studies to date directly quantify either the function or HDL particle distribution of apoA1 recovered from the human artery wall.
Methods and Results
A monoclonal antibody (mAb 10G1.5) was developed that equally recognizes lipid-free and HDL-associated apoA1 in both native and oxidized forms. Examination of homogenates of atherosclerotic plaque-laden aorta showed >100-fold enrichment of apoA1 compared to normal aorta (P<0.001). Surprisingly, buoyant density fractionation revealed only a minority (<3% of total) of apoA1 recovered from either lesions or normal aorta resides within an HDL-like particle (1.063 ≤ d ≤ 1.21). In contrast, the majority (>90%) of apoA1 within aortic tissue (normal and lesions) was recovered within the lipoprotein-depleted fraction (d>1.21). Moreover, both lesion and normal artery wall apoA1 is highly cross-linked (50–70% of total), and functional characterization of apoA1 quantitatively recovered from aorta using mAb 10G1.5 showed ~80% lower cholesterol efflux activity and ~90% lower LCAT activity relative to circulating apoA1.
Conclusions
The function and distribution of apoA1 in human aorta are quite distinct from those found in plasma. The lipoprotein is markedly enriched within atherosclerotic-plaque, predominantly lipid-poor, not associated with HDL, extensively oxidatively cross-linked, and functionally impaired.
doi:10.1161/CIRCULATIONAHA.113.002624
PMCID: PMC3882895  PMID: 23969698
plaque; apolipoproteins; arteriosclerosis; cardiovascular diseases
9.  The WWOX Gene Modulates HDL and Lipid Metabolism 
Background
Low high-density lipoprotein-cholesterol (HDL-C) constitutes a major risk factor for atherosclerosis. Recent studies from our group reported a genetic association between the WW domain-containing oxidoreductase (WWOX) gene and HDL-C levels. Here, through next-generation resequencing, in vivo functional studies and gene microarray analyses, we investigated the role of WWOX in HDL and lipid metabolism.
Methods and Results
Using next-generation resequencing of the WWOX region, we first identified 8 variants significantly associated and perfectly segregating with the low-HDL trait in two multi-generational French Canadian dyslipidemic families. To understand in vivo functions of WWOX, we used liver-specific Wwoxhep−/− and total Wwox−/− mice models, where we found decreased ApoA-I and ABCA1 levels in hepatic tissues. Analyses of lipoprotein profiles in Wwox−/−, but not Wwox hep−/− littermates, also showed marked reductions in serum HDL-C concentrations, concordant with the low-HDL findings observed in families. We next obtained evidence of a gender-specific effect in female Wwoxhep−/− mice, where an increase in plasma triglycerides and altered lipid metabolic pathways by microarray analyses were observed. We further identified a significant reduction in ApoA-I and LPL, and upregulation in Fas, Angptl4 and Lipg, suggesting that the effects of Wwox involve multiple pathways, including cholesterol homeostasis, ApoA-I/ABCA1 pathway, and fatty acid biosynthesis/triglyceride metabolism.
Conclusions
Our data indicate that WWOX disruption alters HDL and lipoprotein metabolism through several mechanisms and may account for the low-HDL phenotype observed in families expressing the WWOX variants. These findings thus describe a novel gene involved in cellular lipid homeostasis, which effects may impact atherosclerotic disease development.
doi:10.1161/CIRCGENETICS.113.000248
PMCID: PMC4315188  PMID: 24871327
high-density lipoprotein cholesterol; gene; lipids and lipoprotein metabolism; gene expression/regulation; cardiovascular disease; WWOX; ApoA-I; ABCA1
10.  Insulin-Mediated Down-Regulation of Apolipoprotein A5 Gene Expression through the Phosphatidylinositol 3-Kinase Pathway: Role of Upstream Stimulatory Factor 
Molecular and Cellular Biology  2005;25(4):1537-1548.
The apolipoprotein A5 gene (APOA5) has been repeatedly implicated in lowering plasma triglyceride levels. Since several studies have demonstrated that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 is regulated by insulin. Here, we show that cell lines and mice treated with insulin down-regulate APOA5 expression in a dose-dependent manner. Furthermore, we found that insulin decreases human APOA5 promoter activity, and subsequent deletion and mutation analyses uncovered a functional E box in the promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that this APOA5 E box binds upstream stimulatory factors (USFs). Moreover, in transfection studies, USF1 stimulates APOA5 promoter activity, and the treatment with insulin reduced the binding of USF1/USF2 to the APOA5 promoter. The inhibition of the phosphatidylinositol 3-kinase (PI3K) pathway abolished insulin's effect on APOA5 gene expression, while the inhibition of the P70 S6 kinase pathway with rapamycin reversed its effect and increased APOA5 gene expression. Using an oligonucleotide precipitation assay for USF from nuclear extracts, we demonstrate that phosphorylated USF1 fails to bind to the APOA5 promoter. Taken together, these data indicate that insulin-mediated APOA5 gene transrepression could involve a phosphorylation of USFs through the PI3K and P70 S6 kinase pathways that modulate their binding to the APOA5 E box and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in men showed a decrease in the plasma ApoAV level. These results suggest a potential contribution of the APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.
doi:10.1128/MCB.25.4.1537-1548.2005
PMCID: PMC548024  PMID: 15684402
11.  Carboxyl-Terminal Cleavage of Apolipoprotein A-I by Human Mast Cell Chymase Impairs Its Anti-Inflammatory Properties 
Supplemental Digital Content is available in the text.
Objective—
Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I.
Approach and Results—
Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α–activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB–dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)– and M-CSF (macrophage colony-stimulating factor)–differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)–activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils.
Conclusions—
The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.
doi:10.1161/ATVBAHA.115.306827
PMCID: PMC4725095  PMID: 26681753
apolipoprotein A-I; carboxyl-terminal cleavage; chymase; endothelial cells; inflammatory; mast cell; proteases
12.  A Systems Genetics Approach Implicates USF1, FADS3, and Other Causal Candidate Genes for Familial Combined Hyperlipidemia 
PLoS Genetics  2009;5(9):e1000642.
We hypothesized that a common SNP in the 3' untranslated region of the upstream transcription factor 1 (USF1), rs3737787, may affect lipid traits by influencing gene expression levels, and we investigated this possibility utilizing the Mexican population, which has a high predisposition to dyslipidemia. We first associated rs3737787 genotypes in Mexican Familial Combined Hyperlipidemia (FCHL) case/control fat biopsies, with global expression patterns. To identify sets of co-expressed genes co-regulated by similar factors such as transcription factors, genetic variants, or environmental effects, we utilized weighted gene co-expression network analysis (WGCNA). Through WGCNA in the Mexican FCHL fat biopsies we identified two significant Triglyceride (TG)-associated co-expression modules. One of these modules was also associated with FCHL, the other FCHL component traits, and rs3737787 genotypes. This USF1-regulated FCHL-associated (URFA) module was enriched for genes involved in lipid metabolic processes. Using systems genetics procedures we identified 18 causal candidate genes in the URFA module. The FCHL causal candidate gene fatty acid desaturase 3 (FADS3) was associated with TGs in a recent Caucasian genome-wide significant association study and we replicated this association in Mexican FCHL families. Based on a USF1-regulated FCHL-associated co-expression module and SNP rs3737787, we identify a set of causal candidate genes for FCHL-related traits. We then provide evidence from two independent datasets supporting FADS3 as a causal gene for FCHL and elevated TGs in Mexicans.
Author Summary
By integrating a genetic polymorphism with genome-wide gene expression levels, we were able to attribute function to a genetic polymorphism in the USF1 gene. The USF1 gene has previously been associated with a common dyslipidemia, FCHL. FCHL is characterized by elevated levels of total cholesterol, triglycerides, or both. We demonstrate that this genetic polymorphism in USF1 contributes to FCHL disease risk by modulating the expression of a group of genes functionally related to lipid metabolism, and that this modulation is mediated by USF1. One of the genes whose expression is modulated by USF1 is FADS3, which was also implicated in a recent genome-wide association study for lipid traits. We demonstrated that a genetic polymorphism from the FADS3 region, which was associated with triglycerides in a GWAS study of Caucasians, was also associated with triglycerides in Mexican FCHL families. Our analysis provides novel insight into the gene expression profile contributing to FCHL disease risk, and identifies FADS3 as a new gene for FCHL in Mexicans.
doi:10.1371/journal.pgen.1000642
PMCID: PMC2730565  PMID: 19750004
13.  Upstream Stimulatory Factors 1 and 2 Mediate the Transcription of Angiotensin II Binding and Inhibitory Protein* 
The Journal of Biological Chemistry  2013;288(26):19238-19249.
Background: Regulation of angiotensin II type 1 receptor-interacting and inhibitory protein (ATRAP/Agtrap) is important in pathophysiology.
Results: Gene knockdown in cells and unilateral ureteral obstruction in mice indicate that Usf1 decreases and Usf2 increases Agtrap expression.
Conclusion: Interplay between E-box and Usf1/Usf2 is important for Agtrap regulation.
Significance: A strategy of modulating the E-box-Usf1/Usf2 interaction may have novel therapeutic potential.
The angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP/Agtrap) promotes constitutive internalization of the AT1R so as to specifically inhibit the pathological activation of its downstream signaling yet preserve the base-line physiological signaling activity of the AT1R. Thus, tissue-specific regulation of Agtrap expression is relevant to the pathophysiology of cardiovascular and renal disease. However, the regulatory mechanism of Agtrap gene expression has not yet been fully elucidated. In this study, we show that the proximal promoter region from −150 to +72 of the mouse Agtrap promoter, which contains the X-box, E-box, and GC-box consensus motifs, is able to elicit substantial transcription of the Agtrap gene. Among these binding motifs, we showed that the E-box specifically binds upstream stimulatory factor (Usf) 1 and Usf2, which are known E-box-binding transcription factors. It is indicated that the E-box-Usf1/Usf2 binding regulates Agtrap expression because of the following: 1) mutation of the E-box to prevent Usf1/Usf2 binding reduces Agtrap promoter activity; 2) knockdown of Usf1 or Usf2 affects both endogenous Agtrap mRNA and Agtrap protein expression, and 3) the decrease in Agtrap mRNA expression in the afflicted kidney by unilateral ureteral obstruction is accompanied by changes in Usf1 and Usf2 mRNA. Furthermore, the results of siRNA transfection in mouse distal convoluted tubule cells and those of unilateral ureteral obstruction in the afflicted mouse kidney suggest that Usf1 decreases but Usf2 increases the Agtrap gene expression by binding to the E-box. The results also demonstrate a functional E-box-USF1/USF2 interaction in the human AGTRAP promoter, thereby suggesting that a strategy of modulating the E-box-USF1/USF2 binding has novel therapeutic potential.
doi:10.1074/jbc.M113.451054
PMCID: PMC3696694  PMID: 23653383
Angiotensin; Gene Transcription; Receptors; Renin Angiotensin System; Transcription Regulation; Distal Tubule
14.  Low-Density Lipoprotein Apheresis 
Executive Summary
Objective
To assess the effectiveness and safety of low-density lipoprotein (LDL) apheresis performed with the heparin-induced extracorporeal LDL precipitation (HELP) system for the treatment of patients with refractory homozygous (HMZ) and heterozygous (HTZ) familial hypercholesterolemia (FH).
Background on Familial Hypercholesterolemia
Familial hypercholesterolemia is a genetic autosomal dominant disorder that is caused by several mutations in the LDL-receptor gene. The reduced number or absence of functional LDL receptors results in impaired hepatic clearance of circulating low-density lipoprotein cholesterol (LDL-C) particles, which results in extremely high levels of LDL-C in the bloodstream. Familial hypercholesterolemia is characterized by excess LDL-C deposits in tendons and arterial walls, early onset of atherosclerotic disease, and premature cardiac death.
Familial hypercholesterolemia occurs in both HTZ and HMZ forms.
Heterozygous FH is one of the most common monogenic metabolic disorders in the general population, occurring in approximately 1 in 500 individuals1. Nevertheless, HTZ FH is largely undiagnosed and an accurate diagnosis occurs in only about 15% of affected patients in Canada. Thus, it is estimated that there are approximately 3,800 diagnosed and 21,680 undiagnosed cases of HTZ FH in Ontario.
In HTZ FH patients, half of the LDL receptors do not work properly or are absent, resulting in plasma LDL-C levels 2- to 3-fold higher than normal (range 7-15mmol/L or 300-500mg/dL). Most HTZ FH patients are not diagnosed until middle age when either they or one of their siblings present with symptomatic coronary artery disease (CAD). Without lipid-lowering treatment, 50% of males die before the age of 50 and 25% of females die before the age of 60, from myocardial infarction or sudden death.
In contrast to the HTZ form, HMZ FH is rare (occurring in 1 case per million persons) and more severe, with a 6- to 8-fold elevation in plasma LDL-C levels (range 15-25mmol/L or 500-1000mg/dL). Homozygous FH patients are typically diagnosed in infancy, usually due to the presence of cholesterol deposits in the skin and tendons. The main complication of HMZ FH is supravalvular aortic stenosis, which is caused by cholesterol deposits on the aortic valve and in the ascending aorta. The average life expectancy of affected individuals is 23 to 25 years. In Ontario, it is estimated that there are 13 to 15 cases of HMZ FH. An Ontario clinical expert confirmed that 9 HMZ FH patients have been identified to date.
Diagnosis
There are 2 accepted clinical diagnostic criterion for the diagnosis of FH: the Simon Broome FH Register criteria from the United Kingdom and the Dutch Lipid Network criteria from the Netherlands. The criterion supplement cholesterol levels with clinical history, physical signs and family history. DNA-based-mutation-screening methods permit a definitive diagnosis of HTZ FH to be made. However, given that there are over 1000 identified mutations in the LDL receptor gene and that the detection rates of current techniques are low, genetic testing becomes problematic in countries with high genetic heterogeneity, such as Canada.
Treatment
The primary aim of treatment in both HTZ and HMZ FH is to reduce plasma LDL-C levels in order to reduce the risk of developing atherosclerosis and CAD.
The first line of treatment is dietary intervention, however it alone is rarely sufficient for the treatment of FH patients. Patients are frequently treated with lipid-lowering drugs such as resins, fibrates, niacin, statins and cholesterol absorption-inhibiting drugs (ezetimibe). Most HTZ FH patients require a combination of drugs to achieve or approach target cholesterol levels.
A small number of HTZ FH patients are refractory to treatment or intolerant to lipid-lowering medication. According to clinical experts, the prevalence of refractory HTZ FH in Ontario is between 1 to 5%. Using the mean of 3%, it is estimated that there are approximately 765 refractory HTZ FH patients in Ontario, of which 115 are diagnosed and 650 are undiagnosed.
Drug therapy is less effective in HMZ FH patients since the effects of the majority of cholesterol-lowering drugs are mediated by the upregulation of LDL receptors, which are often absent or function poorly in HMZ FH patients. Some HMZ FH patients may still benefit from drug therapy, however this rarely reduces LDL-C levels to targeted levels.
Existing Technology: Plasma Exchange
An option currently available in Ontario for FH patients who do not respond to standard diet and drug therapy is plasma exchange (PE). Patients are treated with this lifelong therapy on a weekly or biweekly basis with concomitant drug therapy.
Plasma exchange is nonspecific and eliminates virtually all plasma proteins such as albumin, immunoglobulins, coagulation factors, fibrinolytic factors and HDL-C, in addition to acutely lowering LDL-C by about 50%. Blood is removed from the patient, plasma is isolated, discarded and replaced with a substitution fluid. The substitution fluid and the remaining cellular components of the blood are then returned to the patient.
The major limitation of PE is its nonspecificity. The removal of HDL-C prevents successful vascular remodeling of the areas stenosed by atherosclerosis. In addition, there is an increased susceptibility to infections, and costs are incurred by the need for replacement fluid. Adverse events can be expected to occur in 12% of procedures.
Other Alternatives
Surgical alternatives for FH patients include portocaval shunt, ileal bypass and liver transplantation. However, these are risky procedures and are associated with a high morbidity rate. Results with gene therapy are not convincing to date.
The Technology Being Reviewed: LDL Apheresis
An alternative to PE is LDL apheresis. Unlike PE, LDL apheresis is a selective treatment that removes LDL-C and other atherogenic lipoproteins from the blood while minimally impacting other plasma components such as HDL-C, total serum protein, albumin and immunoglobulins. As with PE, FH patients require lifelong therapy with LDL apheresis on a weekly/biweekly basis with concomitant drug therapy.
Heparin-Induced Extracorporeal LDL Precipitation
Heparin-induced extracorporeal LDL precipitation (HELP) is one of the most widely used methods of LDL apheresis. It is a continuous closed-loop system that processes blood extracorporeally. It operates on the principle that at a low pH, LDL and lipoprotein (a) [Lp(a)] bind to heparin and fibrinogen to form a precipitate which is then removed by filtration. In general, the total duration of treatment is approximately 2 to 3 hours.
Results from early trials indicate that LDL-C concentration is reduced by 65% to 70% immediately following treatment in both HMZ and HTZ FH and then rapidly begins to rise. Typically patients with HTZ FH are treated every 2 weeks while patients with HMZ FH require weekly therapy. Heparin-induced extracorporeal LDL precipitation also produces small transient decreases in HDL-C, however levels generally return to baseline within 2 days. After several months of therapy, long-term reductions in LDL-C and increases in HDL-C have been reported.
In addition to having an impact on plasma cholesterol concentrations, HELP lowers plasma fibrinogen, a risk factor for atherosclerosis, and reduces concentrations of cellular adhesion molecules, which play a role in early atherogenesis.
In comparison with PE, HELP LDL apheresis does not have major effects on essential plasma proteins and does not require replacement fluid, thus decreasing susceptibility to infections. One study noted that adverse events were documented in 2.9% of LDL apheresis treatments using the HELP system compared with 12% using PE. As per the manufacturer, patients must weigh at least 30kgs to be eligible for treatment with HELP.
Regulatory Status
The H.E.L.P.® System (B.Braun Medizintechnologie GmbH, Germany) has been licensed by Health Canada since December 2000 as a Class 3 medical device (Licence # 26023) for performing LDL apheresis to acutely remove LDL from the plasma of 3 high-risk patient populations for whom diet has been ineffective and maximum drug therapy has either been ineffective or not tolerated. The 3 patient groups are as follows:
Functional hypercholesterolemic homozygotes with LDL-C >500 mg/dL (>13mmol/L);
Functional hypercholesterolemic heterozygotes with LDL-C >300 mg/dL (>7.8mmol/L);
Functional hypercholesterolemic heterozygotes with LDL-C >200 mg/dL (>5.2mmol/L) and documented CAD
No other LDL apheresis system is currently licensed in Canada.
Review Strategy
The Medical Advisory Secretariat systematically reviewed the literature to assess the effectiveness and safety of LDL apheresis performed with the HELP system for the treatment of patients with refractory HMZ and HTZ FH. A standard search methodology was used to retrieve international health technology assessments and English-language journal articles from selected databases.
The GRADE approach was used to systematically and explicitly make judgments about the quality of evidence and strength of recommendations.
Summary of Findings
The search identified 398 articles published from January 1, 1998 to May 30, 2007. Eight studies met the inclusion criteria. Five case series, 2 case series nested within comparative studies, and one retrospective review, were included in the analysis. A health technology assessment conducted by the Alberta Heritage Foundation for Medical Research, and a review by the United States Food and Drug Administration were also included.
Large heterogeneity among the studies was observed. Studies varied in inclusion criteria, baseline patient characteristics and methodology.
Overall, the mean acute1 relative decrease in LDL-C with HELP LDL apheresis ranged from 53 to 77%. The mean acute relative reductions ranged as follows: total cholesterol (TC) 47 to 64%, HDL-C +0.4 to -29%, triglycerides (TG) 33 to 62%, Lp(a) 55 to 68% and fibrinogen 56 to 65%.
The mean chronic2 relative decreases in LDL-C and TC with HELP LDL apheresis ranged from 9 to 46% and 5 to 34%, respectively. Familial hypercholesterolemia patients treated with HELP did not achieve the target LDL-C value set by international guidelines (LDL-C < 2.5mmol/L, 100mg/dL). The chronic mean relative increase in HDL-C ranged from 12 to 27%. The ratio of LDL:HDL and the ratio of TC:HDL are 2 measures that have been shown to be important risk factors for cardiac events. In high-risk patients, the recommended target LDL:HDL ratio is less than or equal to 2, and the target TC:HDL ratio is less than 4. In the studies that reported chronic lipid changes, the LDL:HDL and TC:HDL ratios exceeded targeted values.
Three studies investigated the effects of HELP on coronary outcomes and atherosclerotic changes. One noted that twice as many lesions displayed regression in comparison to those displaying progression. The second study found that there was a decrease in Agatston scores3 and in the volume of coronary calcium. The last study noted that 2 of 5 patients showed regression of coronary atherosclerosis, and 3 of the 5 patients showed no change as assessed by a global change score.
Adverse effects were typically mild and transient, and the majority of events were related to problems with vascular access. Of the 3 studies that provided quantitative information, the proportion of adverse events ranged from 2.9 to 5.1%.
GRADE Quality of Evidence
In general, studies were of low quality, i.e., case series studies (Tables 1-3). No controlled studies were identified and no studies directly compared the effectiveness of the HELP system with PE or with diet and drug therapy. Conducting trials with a sufficiently large control group would not have been feasible or acceptable given that HELP represents a last alternative in these patients who are resistant to conventional therapeutic strategies.
A major limitation is that there is limited evidence on the effectiveness and safety of HELP apheresis in HMZ FH patients. However, it is unlikely that better-quality evidence will become available, given that HMZ FH is rare and LDL apheresis is a last therapeutic option for these patients.
Lastly, there is limited data on the long-term effects of LDL apheresis in FH patients. No studies with HELP were identified that examined long-term outcomes such as survival and cardiovascular events. The absence of this data may be attributed to the rarity of the condition, and the large number of subjects and long duration of follow-up that would be needed to conduct such trials.
Homozygous Familial Hypercholesterolemia - Lipid Outcomes
Heterozygous Familial Hypercholesterolemia - Lipid Outcomes
Heterozygous Familial Hypercholesterolemia - Coronary Artery Disease Outcomes
Economic Analysis
A budget-impact analysis was conducted to forecast future costs for PE and HELP apheresis in FH patients. All costs are reported in Canadian dollars. Based on epidemiological data of 13 HMZ, 115 diagnosed HTZ and 765 cases of all HTZ patients (diagnosed + undiagnosed), the annual cost of weekly treatment was estimated to be $488,025, $4,332,227 and $24,758,556 respectively for PE. For HELP apheresis, the annual cost of weekly treatment was estimated to be $1,025,338, $9,156,209 and $60,982,579 respectively. Costs for PE and HELP apheresis were halved with a biweekly treatment schedule.
The cost per coronary artery disease death avoided over a 10-year period in HTZ FH-diagnosed patients was also calculated and estimated to be $37.5 million and $18.7 million for weekly and biweekly treatment respectively, when comparing HELP apheresis with PE and with no intervention. Although HELP apheresis costs twice as much as PE, it helped to avoid 12 deaths compared with PE and 22 deaths compared with no intervention, over a period of 10 years.
Ontario Health System Impact Analysis
Low-density lipoprotein apheresis using the HELP system is currently being funded by the provinces of Quebec and Alberta. The program in Quebec has been in operation since 2001 and is limited to the treatment of HMZ FH patients. The Alberta program is relatively new and is currently treating HMZ FH patients, but it is expanding to include refractory HTZ FH patients.
Low-density lipoprotein apheresis is a lifelong treatment and requires considerable commitment on the part of the patient, and the patient’s family and physician. In addition, the management of FH continues to evolve. With the advent of new more powerful cholesterol-lowering drugs, some HTZ patients may be able to sufficiently control their hypercholesterolemia. Nevertheless, according to clinical experts, HMZ patients will likely always require LDL apheresis.
Given the substantial costs associated with LDL apheresis, treatment has been limited to HMZ FH patients. However, LDL apheresis could be applied to a much larger population, which would include HTZ FH patients who are refractory to diet and drug therapy. HTZ FH patients are generally recruited in a more advanced state, demonstrate a longer natural survival than HMZ FH patients and are older.
Conclusions
For HMZ FH patients, the benefits of LDL apheresis clearly outweigh the risks and burdens. According to GRADE, the recommendation would be graded as strong, with low- to very low-quality evidence (Table 4).
In both HMZ and HTZ FH patients, there is evidence of overall clinical benefit of LDL apheresis from case series studies. Low-density lipoprotein apheresis has several advantages over the current treatment of PE, including decreased exposure to blood products, decreased risk of adverse events, conservation of nonatherogenic and athero-protective components, such as HDL-C and lowering of other atherogenic components, such as fibrinogen.
In contrast to HMZ FH patients, there remains a lot of uncertainty in the social/ethical acceptance of this technology for the treatment of refractory HTZ FH patients. In addition to the substantial costs, it is unknown whether the current health care system could cope with the additional demand. There is uncertainty in the estimates of benefits, risks and burdens. According to GRADE, the recommendation would be graded as weak with low- to very-low-quality evidence (Table 5).
GRADE Recommendation - Homozygous Patients
GRADE of recommendation: Strong recommendation, low-quality or very-low-quality evidence
Benefits clearly outweigh risk and burdens
Case series study designs
Strong, but may change when higher-quality evidence becomes available
GRADE Recommendation - Heterozygous Patients
GRADE of recommendation: Weak recommendation, low-quality or very-low-quality evidence
Uncertainty in the estimates of benefits, risks and burden, which these may be closely balanced
Case series study designs
Very weak; other alternatives may be equally reasonable
PMCID: PMC3377562  PMID: 23074505
15.  Comparison of High-Density Lipoprotein Cholesterol to Apolipoprotein A-I and A-II to Predict Coronary Calcium and the Effect of Insulin Resistance 
The American journal of cardiology  2011;107(3):393-398.
High-density lipoprotein (HDL) cholesterol and its apolipoproteins each capture unique lipid and cardiometabolic information important to risk quantification. It was hypothesized that metabolic factors, including insulin resistance and type 2 diabetes, would confound the association of HDL cholesterol with coronary artery calcification (CAC) and that apolipoprotein A-I (apoA-I) and/or apolipoprotein A-II (apoA-II) would add to HDL cholesterol in predicting CAC. Two community-based cross-sectional studies of white subjects were analyzed: the Penn Diabetes Heart Study (PDHS; n = 611 subjects with type 2 diabetes, 71.4% men) and the Study of Inherited Risk of Coronary Atherosclerosis (SIRCA; n = 803 subjects without diabetes, 52.8% men) using multivariable analysis of apoA-I, apoA-II, and HDL cholesterol stratified by diabetes status. HDL cholesterol was inversely associated with CAC after adjusting for age and gender in whites with type 2 diabetes (tobit ratio for a 1-SD increase in HDL cholesterol 0.58, 95% confidence interval [CI] 0.44 to 0.77, p <0.001) as well as those without diabetes (tobit ratio 0.72, 95% CI 0.59 to 0.88, p = 0.001). In contrast, apoA-I was a weaker predictor in subjects with (tobit ratio 0.64, 95% CI 0.45 to 0.90, p = 0.010) and without (tobit ratio 0.79, 95% CI 0.66 to 0.94, p = 0.010) diabetes, while apoA-II had no association with CAC. Control for metabolic variables, including triglycerides, waist circumference, and homeostasis model assessment of insulin resistance, attenuated these relations, particularly in subjects without diabetes. In likelihood ratio test analyses, HDL cholesterol added to apoA-I, apoA-II, and atherogenic apolipoprotein B lipoproteins but improved CAC prediction over metabolic factors only in subjects with diabetes. In conclusion, HDL cholesterol outperformed apoA-I and apoA-II in CAC prediction, but its association with CAC was attenuated by measures of insulin resistance.
doi:10.1016/j.amjcard.2010.09.033
PMCID: PMC3086062  PMID: 21257004
16.  Expression of sterol regulatory element-binding transcription factor (SREBF) 2 and SREBF cleavage-activating protein (SCAP) in human atheroma and the association of their allelic variants with sudden cardiac death 
Thrombosis Journal  2008;6:17.
Background
Disturbed cellular cholesterol homeostasis may lead to accumulation of cholesterol in human atheroma plaques. Cellular cholesterol homeostasis is controlled by the sterol regulatory element-binding transcription factor 2 (SREBF-2) and the SREBF cleavage-activating protein (SCAP). We investigated whole genome expression in a series of human atherosclerotic samples from different vascular territories and studied whether the non-synonymous coding variants in the interacting domains of two genes, SREBF-2 1784G>C (rs2228314) and SCAP 2386A>G, are related to the progression of coronary atherosclerosis and the risk of pre-hospital sudden cardiac death (SCD).
Methods
Whole genome expression profiling was completed in twenty vascular samples from carotid, aortic and femoral atherosclerotic plaques and six control samples from internal mammary arteries. Three hundred sudden pre-hospital deaths of middle-aged (33–69 years) Caucasian Finnish men were subjected to detailed autopsy in the Helsinki Sudden Death Study. Coronary narrowing and areas of coronary wall covered with fatty streaks or fibrotic, calcified or complicated lesions were measured and related to the SREBF-2 and SCAP genotypes.
Results
Whole genome expression profiling showed a significant (p = 0.02) down-regulation of SREBF-2 in atherosclerotic carotid plaques (types IV-V), but not in the aorta or femoral arteries (p = NS for both), as compared with the histologically confirmed non-atherosclerotic tissues. In logistic regression analysis, a significant interaction between the SREBF-2 1784G>C and the SCAP 2386A>G genotype was observed on the risk of SCD (p = 0.046). Men with the SREBF-2 C allele and the SCAP G allele had a significantly increased risk of SCD (OR 2.68, 95% CI 1.07–6.71), compared to SCAP AA homologous subjects carrying the SREBF-2 C allele. Furthermore, similar trends for having complicated lesions and for the occurrence of thrombosis were found, although the results were not statistically significant.
Conclusion
The results suggest that the allelic variants (SREBF-2 1784G>C and SCAP 2386A>G) in the cholesterol homeostasis regulating SREBF-SCAP pathway may contribute to SCD in early middle-aged men.
doi:10.1186/1477-9560-6-17
PMCID: PMC2645360  PMID: 19116028
17.  Amyloidogenic Mutations in Human Apolipoprotein A-I are not Necessarily Destabilizing: A Common Mechanism of ApoA-I Misfolding in Familial Amyloidosis and Atherosclerosis 
The FEBS journal  2014;281(11):2525-2542.
High-density lipoproteins (HDLs) and their major protein, apoA-I, remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, non-variant full-length apoA-I deposits as fibrils in atherosclerotic plaques; in familial amyloidosis, N-terminal fragments of variant apoA-I deposit in vital organs damaging them. Recently, we used the crystal structure of Δ(185-243)apoA-I to propose that amyloidogenic mutations destabilize apoA-I and increase solvent exposure of the extended strand 44-55 that initiates β-aggregation. Here we test this hypothesis by exploring naturally occurring human amyloidogenic mutations, W50R and G26R, within or close to this strand. The mutations caused small changes in the protein’s α-helical content, stability, proteolytic pattern, and protein-lipid interactions. These changes alone were unlikely to account for amyloidosis, suggesting the importance of other factors. Sequence analysis predicted several amyloid-prone segments that can initiate apoA-I misfolding. Aggregation studies using N-terminal fragments experimentally verified this prediction. Three predicted N-terminal amyloid-prone segments, mapped on the crystal structure, formed an α-helical cluster. Structural analysis indicates that amyloidogenic mutations or Met86 oxidation perturb native packing in this cluster. Together, the results suggest that structural perturbations in the amyloid-prone segments trigger α-helix-to-β-sheet conversion in the N-terminal ~75 residues forming the amyloid core. Polypeptide outside this core can be proteolysed to form 9-11 kDa N-terminal fragments found in familial amyloidosis. Our results imply that apoA-I misfolding in familial and acquired amyloidosis follows a similar mechanism that does not require significant structural destabilization or proteolysis. This novel mechanism suggests potential therapeutic interventions for apoA-I amyloidosis.
doi:10.1111/febs.12809
PMCID: PMC4047191  PMID: 24702826
High-density lipoprotein; α-helix to β-sheet conversion; amyloid self-recognition elements or “hot spots”; apoA-I oxidation and proteolysis; triglyceride reduction therapies
18.  Vasculoprotective Effects of Apolipoprotein Mimetic Peptides: An Evolving Paradigm In Hdl Therapy (Vascular Disease Prevention, In Press.) 
Vascular disease prevention  2009;6:122-130.
Anti-atherogenic effects of high density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) are principally thought to be due to their ability to mediate reverse cholesterol transport. These agents also possess anti-oxidant properties that prevent the oxidative modification of low density lipoprotein (LDL) and anti-inflammatory properties that include inhibition of endothelial cell adhesion molecule expression. Results of the Framingham study revealed that a reduction in HDL levels is an independent risk factor for coronary artery disease (CAD). Accordingly, there has been considerable interest in developing new therapies that specifically elevate HDL cholesterol. However, recent evidence suggests that increasing circulating HDL cholesterol levels alone is not sufficient as a mode of HDL therapy. Rather, therapeutic approaches that increase the functional properties of HDL may be superior to simply raising the levels of HDL per se. Our laboratory has pioneered the development of synthetic, apolipoprotein mimetic peptides which are structurally and functionally similar to apoA-I but possess unique structural homology to the lipid-associating domains of apoA-I. The apoA-I mimetic peptide 4F inhibits atherogenic lesion formation in murine models of atherosclerosis. This effect is related to the ability of 4F to induce the formation of pre-β HDL particles that are enriched in apoA-I and paraoxonase. 4F also possesses anti-inflammatory and anti-oxidant properties that are independent of its effect on HDL quality per se. Recent studies suggest that 4F stimulates the expression of the antioxidant enzymes heme oxygenase and superoxide dismutase and inhibits superoxide anion formation in blood vessels of diabetic, hypercholesterolemic and sickle cell disease mice. The goal of this review is to discuss HDL-dependent and -independent mechanisms by which apoA-I mimetic peptides reduce vascular injury in experimental animal models.
doi:10.2174/1567270000906010122
PMCID: PMC2806689  PMID: 20084185
keywords: ApoA-I; HDL; mimetic peptides; CAD risk; atherosclerosis; endothelial function
19.  A single infusion of MDCO-216 (ApoA-1 Milano/POPC) increases ABCA1-mediated cholesterol efflux and pre-beta 1 HDL in healthy volunteers and patients with stable coronary artery disease 
Aims
Apolipoprotein A-1 (ApoA-1), based on epidemiology, is inversely associated with cardiovascular (CV) events. Human carriers of the ApoA-1 Milano variant have a reduced incidence of CV disease. Regression of atherosclerotic plaque burden was previously observed on intravascular ultrasound (IVUS) with ETC-216, a predecessor of MDCO-216. MDCO-216, a complex of dimeric ApoA-1 Milano and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, is being developed to reduce atherosclerotic plaque burden and CV events. We investigated the efficacy and safety of a single infusion of MDCO-216 in healthy volunteers and in patients with coronary artery disease (CAD).
Methods and results
Twenty-four healthy volunteers and 24 patients with documented CAD received a 2-h infusion of MDCO-216 in a randomized, placebo controlled, single ascending dose study. Five cohorts of healthy volunteers and four cohorts of CAD patients received ApoA-1 Milano doses ranging from 5 to 40 mg/kg. Subjects were followed for 30 days. Dose-dependent increases in ApoA-1, phospholipid, and pre-beta 1 HDL and decreases in ApoE were observed. Prominent and sustained increases in triglyceride, and decreases in HDL-C, endogenous ApoA-1 and ApoA-II occurred at doses >20 mg/kg and profound increases in ABCA1-mediated cholesterol efflux were observed. Other lipid and lipoprotein parameters were generally unchanged. MDCO-216 was well tolerated.
Conclusions
MDCO-216-modulated lipid parameters profoundly increased ABCA1-mediated cholesterol efflux and was well tolerated. These single-dose data support further development of this agent for reducing atherosclerotic disease and subsequent CV events.
doi:10.1093/ehjcvp/pvv041
PMCID: PMC4900740  PMID: 27418968
Atherosclerosis; Coronary disease; Lipids; Lipoproteins; Cholesterol efflux
20.  Human Apolipoprotein A-I Natural Variants: Molecular Mechanisms Underlying Amyloidogenic Propensity 
PLoS ONE  2012;7(8):e43755.
Human apolipoprotein A-I (apoA-I)-derived amyloidosis can present with either wild-type (Wt) protein deposits in atherosclerotic plaques or as a hereditary form in which apoA-I variants deposit causing multiple organ failure. More than 15 single amino acid replacement amyloidogenic apoA-I variants have been described, but the molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here, we have investigated by fluorescence and biochemical approaches the stabilities and propensities to aggregate of two disease-associated apoA-I variants, apoA-IGly26Arg, associated with polyneuropathy and kidney dysfunction, and apoA-ILys107-0, implicated in amyloidosis in severe atherosclerosis. Results showed that both variants share common structural properties including decreased stability compared to Wt apoA-I and a more flexible structure that gives rise to formation of partially folded states. Interestingly, however, distinct features appear to determine their pathogenic mechanisms. ApoA-ILys107-0 has an increased propensity to aggregate at physiological pH and in a pro-inflammatory microenvironment than Wt apoA-I, whereas apoA-IGly26Arg elicited macrophage activation, thus stimulating local chronic inflammation. Our results strongly suggest that some natural mutations in apoA-I variants elicit protein tendency to aggregate, but in addition the specific interaction of different variants with macrophages may contribute to cellular stress and toxicity in hereditary amyloidosis.
doi:10.1371/journal.pone.0043755
PMCID: PMC3429494  PMID: 22952757
21.  Effect of apolipoprotein A1 genetic polymorphisms on lipid profiles and the risk of coronary artery disease 
Diagnostic Pathology  2015;10:102.
Background
The disorder of lipid metabolism and genetic predisposition are major risk factors for coronary artery disease (CAD). Variants in the apolipoprotein A1 (APOA1) gene play an important role in the regulation of lipids. The objective of the present study was to investigate the effect of two polymorphisms (-75 G/A and +83 C/T) of APOA1 on lipid profiles and the risk of CAD.
Methods
A total number of 300 subjects with CAD and 300 age and sex matched healthy controls were enrolled for the study. Genotyping of the APOA1 was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) combined with gel electrophoresis, and then confirmed by direct sequencing.
Results
The frequencies of APOA1 -75 AA genotype [odds ratio (OR) =0.50, 95 % confidence interval (CI) = 0.28, 0.88; P = 0.02] and APOA1 -75 A allele (OR =0.76, 95 % CI = 0.59, 0.98; P = 0.04) were significantly lower in CAD than in controls. The APOA1 -75 A allele was significantly associated with increasing serum concentrations of ApoA1 and high-density lipoprotein cholesterol (HDL-C) (P < 0.001).
Conclusions
The individuals with the APOA1 -75 A allele were likely to have a lower risk of CAD as a result of its effect on higher serum concentrations of ApoA1 and HDL-C.
doi:10.1186/s13000-015-0328-7
PMCID: PMC4502599  PMID: 26173491
Apolipoprotein A1; Gene polymorphism; Coronary artery disease; Lipid
22.  Polymorphisms in IL-10 and INF-γ genes are associated with early atherosclerosis in coronary but not in carotid arteries: A study of 122 autopsy cases of young adults 
BBA Clinical  2015;3:214-220.
Atherosclerosis is a complex disease, involving both genetic and environmental factors. However, the influence of genetic variations on its early development remains unclear. This study examined the association of 12 different polymorphisms with atherosclerosis severity in anterior descending coronary (DA, n = 103) and carotid arteries (CA, n = 66) of autopsied young adults (< 30 years old). Histological sections (H-E) were classified according to the American Heart Association. Polymorphisms in ACE, TNF-α (− 308G/A and − 238 G/A), IFN-γ (+ 874 A/T), MMP-9 (− 1562 C/T), IL-10 (− 1082 A/G and − 819 C/T), NOS3 (894 G/T), ApoA1 (rs964184), ApoE (E2E3E4 isoforms), and TGF-β (codons 25 and 10) genes were genotyped by gel electrophoresis or automatic DNA sequencing. Firearm projectile or car accident was the main cause of death, and no information about classical risk factors was available. Histological analysis showed high prevalence of type III atherosclerotic lesions in both DA (69%) and CA (39%) arteries, while severe type IV and V lesions were observed in 14% (DA) and 33% (CA). Allele frequencies and genotype distributions were determined. Among the polymorphisms studied, IFN-γ and IL-10 (− 1082 A/G) were related to atherosclerosis severity in DA artery. No association between genotypes and lesion severity was found in CA. In conclusion, we observed that the high prevalence of early atherosclerosis in young adults is associated with IFN-γ (p < 0.001) and IL-10 (p = 0.013) genotypes. This association is blood vessel dependent. Our findings suggest that the vascular system presents site specialization, and specific genetic variations may provide future biomarkers for early disease identification.
Highlights
•Twelve SNPs were associated with atherosclerosis severity in autopsied young adults.•We found high prevalence of type III lesions in coronary and carotid arteries.•Even severe lesions (types IV and V) were found in DA (14%) and CA (33%) arteries.•Lesion severity was associated with IL-10 and IFN-γ genotype.•The association was observed only in coronary, but not in carotid artery.
doi:10.1016/j.bbacli.2015.02.005
PMCID: PMC4661558  PMID: 26674973
Atherosclerosis; IL-10; INF-γ; Polymorphisms; Coronary artery disease
23.  Dysfunctional HDL Containing L159R ApoA-I Leads to Exacerbation of Atherosclerosis in Hyperlipidemic Mice 
Biochimica et biophysica acta  2011;1821(3):502-512.
The mutation L159R apoA-I or apoA-IL159R (FIN) is a single amino acid substitution within the sixth helical repeat of apoA-I. It is associated with a dominant negative phenotype, displaying hypoalphaproteinemia and an increased risk for atherosclerosis in humans. Mice lacking both mouse apoA-I and LDL receptor (LDL−/−, apoA-I−/−) (double knockout or DKO) were crossed > 9 generations with mice transgenic for human FIN to obtain L159R apoA-I, LDLr−/−, ApoA-I−/− (FIN-DKO) mice. A similar cross was also performed with human wild-type (WT) apoA-I (WT-DKO). In addition, FIN-DKO and WT-DKO were crossed to obtain WT/FIN-DKO mice. To determine the effects of the apoA-I mutations on atherosclerosis, groups of each genotype were fed either chow or an atherogenic diet for 12 weeks. Interestingly, the production of dysfunctional HDL-like particles occurred in DKO and FIN-DKO mice. These particles were distinct with respect to size, and their enrichment in apoE and cholesterol esters. Two-dimensional gel electrophoresis indicated that particles found in the plasma of FIN-DKO mice migrated as large α3-HDL. Atherosclerosis analysis showed that FIN-DKO mice developed the greatest extent of aortic cholesterol accumulation compared to all other genotypes, including DKO mice which lack any apoA-I. Taken together these data suggest that the presence of large apoE enriched HDL particles containing apoA-I L159R lack the normal cholesterol efflux promoting properties of HDL, rendering them dysfunctional and pro-atherogenic. In conclusion, large HDL-like particles containing apoE and apoA-IL159R contribute rather than protect against atherosclerosis, possibly through defective efflux properties and their potential for aggregation at their site of interaction in the aorta.
doi:10.1016/j.bbalip.2011.08.019
PMCID: PMC3690280  PMID: 21944998
Apolipoprotein A-1; Apolipoprotein A-1FIN; Atherosclerosis; Dysfunctional HDL; L159R ApoA-I
24.  Apolipoprotein A5 and lipoprotein lipase interact to modulate anthropometric measures in Hispanics of Caribbean origin 
Apolipoprotein A5 (APOA5) and lipoprotein lipase (LPL) proteins interact functionally to regulate lipid metabolism, and single nucleotide polymorphisms (SNPs) for each gene have also been associated independently with obesity risk. Evaluating gene combinations may be more effective than single SNP analyses in identifying genetic risk, but insufficient minor allele frequency (MAF) often limits evaluations of potential epistatic relationships. Populations with multiple ancestral admixtures may provide unique opportunities for evaluating genetic interactions. We examined relationships between LPL m107 (rs1800590) and APOA5 S19W (rs3135506) and lipid and anthropometric measures in Caribbean origin Hispanics (n=1019, aged 45–5 years) living in the Boston metropolitan area. Significant interaction terms between LPL m107 and APOA5 S19W were observed for BMI (P=0.003) and waist circumference (P=0.019). Higher BMI (P=0.001), waist (P=0.011) and hip (P=0.026) were observed in minor allele (G) carriers for LPL m107 who also carried the APOA5 S19W minor allele (G). Additionally, extreme obesity (BMI≥40 kg/m2) risk was higher (OR=4.02; 95% CI:1.81–.91; global P=0.008) for minor allele carriers for both SNPs (LPL TG+GG, APOA5 CG+GG) compared to major allele carriers for both SNPs. In summary, we identified significant interactions for APOA5 S19W and LPL m107 for obesity in Caribbean Hispanics. Population-specific MAFs increase the difficulties of replicating gene-gene interactions, but may support the hypothesis that combinations of frequencies in selected genes could heighten obesity susceptibility in a given population. Analyses of gene-gene interactions may improve understanding of genetically-based obesity risk, and underscore the need for further study of groups with multiple ancestral admixtures.
doi:10.1038/oby.2009.216
PMCID: PMC2813926  PMID: 19629056
25.  An abundant dysfunctional apolipoprotein A1 in human atheroma 
Nature medicine  2014;20(2):193-203.
Recent studies indicate high density lipoproteins (HDL) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma, are dysfunctional and extensively oxidized by myeloperoxidase (MPO), while in vitro oxidation of apoA1/HDL by MPO impairs its cholesterol acceptor function. We developed a high affinity monoclonal antibody (mAb) that specifically recognizes apoA1/HDL modified by the MPO/H2O2/Cl-system using phage display affinity maturation. An oxindolyl alanine (2-OH-Trp) moiety at tryptophan 72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirm a critical role for apoA1 Trp72 in MPO-mediated inhibition of ABCA1-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation, but accounts for 20% of the apoA1 in atherosclerotic plaque. OxTrp72-apoA1 recovered from human atheroma or plasma was lipid-poor, virtually devoid of cholesterol acceptor activity, and demonstrated both potent pro-inflammatory activities on endothelial cells and impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n=627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a pro-atherogenic process in the artery wall.
doi:10.1038/nm.3459
PMCID: PMC3923163  PMID: 24464187

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