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Author(s): Shadyab, Aladdin H; Kooperberg, Charles; Reiner, Alexander P; Jain, Sonia; Manson, JoAnn E; Hohensee, Chancellor; Macera, Caroline A; Shaffer, Richard A; Gallo, Linda C; LaCroix, Andrea Z | Abstract: BackgroundNo study has evaluated whether genetic factors are associated with longevity in African Americans or Hispanics, and it is unclear whether genetic factors are associated with healthy aging.MethodsIn this prospective study, we determined whether 14 genetic variants previously associated with longevity in genome-wide association studies were associated with survival to ages 85 and 90 in 11,053 postmenopausal white, African American, and Hispanic women from the Women's Health Initiative. The associations of these variants with healthy aging, defined as survival to age 85 without chronic diseases or disability, were also determined.ResultsAmong white women, three single nucleotide polymorphisms (SNPs) (rs2075650 [TOMM40], rs4420638 [APOC1], and rs429358 [APOE]) were significantly associated with survival to 90 years after correction for multiple testing (p l .001); rs4420638 and rs429358 were also significantly associated with healthy aging (p = .02). In African American women, no SNP was associated with longevity. In Hispanic women, 7 SNPs in linkage disequilibrium with a novel SNP, rs2149954, recently identified as being associated with increased longevity in a European population, were significantly associated with decreased survival to age 85 for carriers of the T versus C allele (p = .04). The association with decreased longevity was explained by higher risk of coronary heart disease in carriers of the T allele. There were no associations between FOXO3A SNPs and longevity in the analyses. In a meta-analysis, rs2075650 and rs429358 were significantly associated with longevity.ConclusionsFuture studies are needed to identify novel loci associated with longevity in African American and Hispanic women to determine biologic pathways regulating life span in these groups.

The aim of this study was to investigate the apolipoprotein E (APOE) chromosomal region in frontotemporal lobar degeneration (FTLD), and in particular in primary progressive aphasia (PPA) and the behavioral variant frontotemporal dementia (bvFTD). To this aim, we selected three single-nucleotide polymorphisms (SNPs) rs2075650 and rs157590 (TOMM40), and rs1064725 (APOC1), representative of the linkage disequilibrium (LD) blocks at the 19q13-q13.2 chromosomal region. The SNPs rs429358 and rs7412 forming the APOE polymorphism were also included in the study. The analysis was made in 282 patients with a clinical diagnosis of sporadic FTLD, namely 207 bvFTD and 75 PPA, and 296 cognitively healthy control subjects. LD (r2 = 0.35) between TOMM40 (rs2075650) and APOC1 (rs1064725) was observed in PPA, but not in controls and in bvFTD. Inside this region of 26.9 kb, LD (r2 ≥ 0.50) between TOMM40 (rs2075650) and APOE (rs429358) was observed in bvFTD and in controls, but not in PPA. Inside this region of 16.3 kb, LD (r2 = 0.14) between TOMM40 (rs157590) and APOE (rs429358) was observed in PPA, but not in bvFTD and in controls. Although the genetics of PPA and bvFTD needs further investigation, our results suggested the presence of a different genetic background underlying PPA and bvFTD at the 19q13-q13.2 chromosomal region.

Plasma cholesteryl ester transfer protein (CETP) promotes the cholesterol enrichment of apoB-containing lipoproteins (VLDL and LDL) at the expense of HDL. Recent studies demonstrated that apoC1 is a potent CETP inhibitor in plasma of healthy, normolipidemic subjects. Our goal was to establish whether the modulation of CETP activity by apoC1 is influenced by dyslipidemia in patients with documented coronary artery disease (CAD). In the total CAD population studied (n = 240), apoC1 levels correlated negatively with CETP activity, independently of apoE-epsilon, CETP-Taq1B, and apoC1-Hpa1 genotypes. In multivariate analysis, the negative relationship was observed only in normolipidemic patients, not in those with hypercholesterolemia, hypertriglyceridemia, or combined hyperlipidemia. In the normolipidemic subjects, apoC1 levels were positively associated with higher HDL- to LDL-cholesterol ratio (r = 0.359, P < 0.001). It is concluded that apoC1 as a CETP inhibitor no longer operates on cholesterol redistribution in high-risk patients with dyslipidemia, probably due to increasing amounts of VLDL-bound apoC1, which is inactive as a CETP inhibitor. Patients with dyslipidemia could experience major benefits from treatment with pharmacological CETP inhibitors, which might compensate for blunted endogenous inhibition.

We developed a quantitative strategy, named secretome-derived isotopic tag (SDIT), to concurrently identify and quantify the adipocyte-secreted plasma proteins from normal and high-fat-diet (HFD) induced obese mice, based on the application of isotope-labeled secreted proteins from cultured mouse adipocytes as internal standards. We detected 197 proteins with significant changes between normal and obese mice plasma. Importantly, a novel adipocyte-secreted plasma protein, apolipoprotein C-I (apoC-I), significantly increased in the obese mice plasma. The expression and secretion of adipocyte apoC-I was detected in differentiated 3T3-L1 and primary rat adipocytes. Our in vitro experiments proved that functional Golgi apparatus was required for apoC-I secretion. Additionally, obese mice had increased apoC-I production in adipose tissue. Population survey of 367 participants showed that the plasma level of apoC-I was significantly increased in obese individuals compared with healthy individuals. After multiple adjustments for age and sex, the odds ratios for risk factors of cardiovascular disease including high LDL cholesterol, hypercholesterolemia, and hypertriglyceridemia, respectively, were used to compare the highest with the lowest apoC-I quartile. Taken together, our studies provide a novel strategy to concurrently identify and quantify tissue-specific secreted proteins. This strategy can be used to identify the largest global characterization of adipocyte-derived plasma proteome and provides a potential disease-related biomarker for clinical diagnoses. By selectively analyzing adipocyte-secreted proteins in plasma from obese vs lean murine and/or human subjects, we discovered that apoC-I is an adipocyte-secreted plasma protein and a predictive marker for cardiovascular disease.

Apolipoprotein C-I (apoC-I) is an important constituent of high-density lipoprotein (HDL) and is involved in the accumulation of cholesterol ester in nascent HDL via inhibition of cholesterol ester transfer protein and potential activation of lecithin:cholesterol acyltransferase (LCAT). As the smallest exchangeable apolipoprotein (57 residues), apoC-I transfers between lipoproteins via a lipid-binding motif of two amphipathic α-helices (AαHs), spanning residues 7-29 and 38-52. To understand apoC-I's behavior at hydrophobic lipoprotein surfaces, oil drop tensiometry was used to compare the binding to triolein/water (TO/W) and palmitoyloleoylphosphatidylcholine/triolein/water (POPC/TO/W) interfaces. When apoC-I binds to either interface, the surface tension (γ) decreases by ~16-18 mN/m. ApoC-I can be exchanged at both interfaces, desorbing upon compression and readsorbing on expansion. The maximal surface pressures at which apoC-I begins to desorb (Π(max)) were 16.8 and 20.7 mN/m at TO/W and POPC/TO/W interfaces, respectively. This suggests that apoC-I interacts with POPC to increase its affinity for the interface. ApoC-I is more elastic on POPC/TO/W than TO/W interfaces, marked by higher values of the elasticity modulus (ε) on oscillations. At POPC/TO/W interfaces containing an increasing POPC:TO ratio, the pressure at which apoC-I begins to be ejected increases as the phospholipid surface concentration increases. The observed increase in apoC-I interface affinity due to higher degrees of apoC-I-POPC interactions may explain how apoC-I can displace larger apolipoproteins, such as apoE, from lipoproteins. These interactions allow apoC-I to remain bound to the interface at higher Π values, offering insight into apoC-I's rearrangement on triacylglycerol-rich lipoproteins as they undergo Π changes during lipoprotein maturation by plasma factors such as lipoprotein lipase.

The e4 allele of apolipoprotein E (APOE4), which is a well established genetic risk factor for development of Alzheimer's disease (AD), is in genetic disequilibrium with the H2 allele of apolipoprotein C1 (APOC1), giving rise to increased expression of apoC-I. This raises the possibility that the H2 allele of APOC1, either alone or in combination with APOE4, provides a major risk factor for AD. In line herewith, we previously showed that mice overexpressing human APOC1 display impaired learning and memory functions. Here, we tested the hypothesis that the absence of Apoc1 expression in mice may improve memory functions. In contrast with our expectations, Apoc1-/- mice showed impaired hippocampal-dependent memory functions, as judged from their performance in the object recognition task (p < 0.001) as compared to their wild-type littermates. No gross changes in brain morphology or in brain sterol concentrations were detected in knockout mice compared to wild-type littermates. Apoc1 deficiency reduced the expression of ApoE mRNA (-25%, p < 0.05), but not ApoE protein levels. In line with a role for apoC-I in inflammatory processes, we observed significantly increased mRNA concentrations of the proinflammatory marker tumor necrosis factor a and oxidative stress related heme oxygenase 1 (Hmox1) in the absence of glial activation. In conclusion, the absence of ApoC-I results in impaired memory functions, which is, together with previous data, suggestive of an important, bell-shaped gene-dose dependent role for ApoC-I in appropriate brain functioning. The relative contributions of the H2 allele of APOC1 and/or APOE4 in the risk assessment in AD remain to be determined. © 2011 - IOS Press and the authors. All rights reserved.

Timely sensing of lipopolysaccharide (LPS) is critical for the host to fight invading Gram-negative bacteria. We recently showed that apolipoprotein CI (apoCI) (apoCI(1-57)) avidly binds to LPS, involving an LPS-binding motif (apoCI(48-54)), and thereby enhances the LPS-induced inflammatory response. Our current aim was to further elucidate the structure and function relationship of apoCI with respect to its LPS-modulating characteristics and to unravel the mechanism by which apoCI enhances the biological activity of LPS. We designed and generated N- and C-terminal apoCI-derived peptides containing varying numbers of alternating cationic/hydrophobic motifs. ApoCI(1-38), apoCI(1-30), and apoCI(35-57) were able to bind LPS, whereas apoCI(1-23) and apoCI(46-57) did not bind LPS. In line with their LPS-binding characteristics, apoCI(1-38), apoCI(1-30), and apoCI(35-57) prolonged the serum residence of I-125-LPS by reducing its association with the liver. Accordingly, both apoCI(1-30) and apoCI(35-57) enhanced the LPS-induced TNF alpha response in vitro (RAW 264.7 macrophages) and in vivo (C57Bl/6 mice). Additional in vitro studies showed that the stimulating effect of apoCI on the LPS response resembles that of LPS-binding protein (LBP) and depends on CD14/ Toll-like receptor 4 signaling.(jlr) We conclude that apoCI contains structural elements in both its N-terminal and C-terminal helix to bind LPS and to enhance the proinflammatory response toward LPS via a mechanism similar to LBP.-Berbee, J. F. P., C. P. Coomans, M. Westerterp, J. A. Romijn, L. M. Havekes, and P. C. N. Rensen. Apolipoprotein CI enhances the biological response to LPS via the CD14/TLR4 pathway by LPS-binding elements in both its N- and C-terminal helix. J. Lipid Res. 2010. 51: 1943-1952

The role of common APOE variants on plasma lipids, particularly low density lipoprotein (LDL) levels, and coronary heart disease (CHD) risk is well known; the influence of variation in the other nearby apolipoprotein genes APOC1, APOC4 and APOC2 is unclear. This study examines the association between APOE/C1/C4/C2 gene cluster variation using tagging SNPs and plasma lipid concentration along with risk of CHD in a prospective cohort. Genotypes for 11 common APOE/C1/C4/C2 SNPs were determined in 2,767 middle-aged (49 to 64 years) men from the Second Northwick Park Heart Study, with 275 CHD events over a 15-year follow-up period. Seven SNPs showed significant associations with one or more lipid trait in univariate analysis. Multivariate and haplotype analysis showed that the APOE genotypes are most strongly associated with effects on LDL-C and apoB concentration (explaining 3.4% of the LDL-C variance) while the other SNPs in this gene cluster explained an additional 1.2%. Haplotypes in APOC2 and APOC4 were associated with modest effects on HDL-C and apoAI (explaining respectively 1.4% and 1.2%). Carriers of the APOE ɛ2 SNP had a significantly lower risk of CHD hazard ratio (HR) of 0.63 (95% confidence interval [CI]: 0.42–0.95), as did carriers of the APOC2 SNP rs5127 (HR = 0.72, 95% CI: 0.56–0.93), while carriers of APOC1 SNP rs4803770 had higher risk of CHD (HR = 1.36, 95% CI: 1.04–1.78) compared with noncarriers. While the common APOE polymorphism explains the majority of the locus genetic determinants of plasma lipid levels, additional SNPs in the APOC1/C2 region may contribute to CHD risk, but these effects require confirmation.

This perspective article provides an opportunity to explain a new genetic finding for late-onset Alzheimer's disease (LOAD). It is specifically written for physicians and scientists who are interested in LOAD, but it may be relevant to those interested in identifying susceptibility variants for other complex diseases. The significant finding discussed here is that a variable-length, deoxythymidine homopolymer (poly-T) within intron 6 of the TOMM40 gene is associated with the age of onset of LOAD [Roses AD, Lutz MW, Amrine-Madsen H, Saunders AM, Crenshaw DG, Sundseth SS, et al. A TOMM40 variable-length polymorphism predicts the age of late-onset Alzheimer's disease. Pharmacogenomics J 2009 December 22;[Epublication ahead of print]. This result was obtained with a phylogenetic study of the genetic polymorphisms that reside within the linkage disequilibrium (LD) block that contains the TOMM40, APOE, and APOC1 genes from patients with LOAD and age-matched subjects without disease. Although the data will have diagnostic, prognostic, and therapeutic strategy implications, this perspective is meant to place the inheritance pattern for this "complex" human disease into context, and to highlight the potential utility of applying phylogenetic tools to the study of the genetics of complex diseases.

Plasma cholesterol from low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors that either totally degrade lipoproteins as the LDL receptor or selectively take up their cholesteryl esters (CE) like the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-I on the uptake of LDL and HDL(3) by HepG2 cells. In experiments conducted with exogenously added purified apoC-I, no significant effect was observed on lipoprotein-protein association and degradation; however, LDL- and HDL(3)-CE selective uptake was significantly reduced in a dose-dependent manner. This study also shows that apoC-I has the ability to associate with HepG2 cells and with LDL and HDL(3). Moreover, pre-incubation of HepG2 cells with apoC-I reduces HDL(3)-CE selective uptake and pre-incubation of LDL and HDL(3) with apoC-I decreases their CE selective uptake by HepG2 cells. Thus, apoC-I can accomplish its inhibitory effect on SR-BI activity by either binding to SR-BI or lipoproteins. We conclude that by reducing hepatic lipoprotein-CE selective uptake, apoC-I has an atherogenic character.

Lipid peroxidation is considered a unifying mechanistic pathway through which known risk factors induce renal cell carcinoma (RCC). We hypothesized that genes selected a priori for their role in lipid peroxidation would modify cancer risk. We genotyped 635 single nucleotide polymorphisms (SNP) in 38candidate genes in 777 Caucasian RCC cases and 1,035 controls enrolled in a large European case-control study. Top candidate SNPs were confirmed among 718 Caucasian cases and 615 controls in a second study in the United States. Two of the three SNPs (rs8106822 and rs405509) that replicated in the U.S. study were within a regulatory region of the APOE promoter. The OR for rs8106822 A>G variant was 1.22AG and 1.41GG (Ptrend = 0.01) in the European study, 1.05AG and 1.51GG (Ptrend = 0.03) in the U.S. study, and 1.15AG and 1.44GG (Ptrend = 0.001) among 1,485 cases and 1,639 controls combined. The rs405509 G>T variant was associated with risk in the European (OR, 0.87TG; OR, 0.71TT; Ptrend = 0.02), the U.S. (OR, 0.68TG; OR, 0.71TT; Ptrend = 0.02), and both studies combined (ORTG, 0.79; ORTT, 0.71; Ptrend = 0.001), as was the G-G haplotype (r2 = 0.64; P= 4.7 × 10−4). This association is biologically plausible as SNP rs405509 was shown to modify protein binding and transcriptional activity of the APOE protein in vitro and is in linkage disequilibrium with key known variants defining the e2, e3, and e4 alleles that modify risk of atherosclerosis, Alzheimer's disease risk, and progression to AIDS. In two large case-control studies, our findings further define a functional region of interest at the APOE locus that increases RCC susceptibility. [Cancer Res 2009;69(20):8001–8]

Diagnosis of malignant pleural mesothelioma (MM) is limited. Novel proteomic technologies can be utilized to discover changes in expression of pleural proteins that might have diagnostic value. The objective of this study was to detect protein profiles that could be used to identify malignant pleural mesothelioma with surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Pleural effusions were collected from patients with confirmed mesothelioma (n = 41) and from patients with effusions due to other causes ([n = 48] cancerous and non-cancerous). Samples were fractionated using anion exchange chromatography and bound to different types of ProteinChip array surfaces. All samples were also subjected to other commercially available immunoassays (human epididymes protein 4 [HE4], osteopontin [OPN], soluble mesothelin-related proteins [SMRP], and the cytokeratin 19 fragment [CYFRA 21–1]). Peak intensity data obtained by SELDI-TOF were subjected to classification algorithms in order to identify potential classifier peaks. A protein peak at m/z 6614 was characterized as apolipoprotein (Apo) CI. In this setting, the sensitivity and specificity of this potential biomarker was 76 % and 69 %, respectively. The area under the receiver operating characteristic curve (AUC) for Apo CI was 0.755, thereby outperforming OPN, HE4, and CYFRA 21–1. SMRP performed best with an AUC of 0.860 with a sensitivity of 83% and specificity of 74%. Our study validates the use of SMRP as a diagnostic marker for pleural mesothelioma and furthermore suggests that Apo CI levels could be used in the future to discriminate pleural mesothelioma from other causes of exudates.

Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoprotein that binds to lipoprotein particles in vivo. In this study, we employed a LC-MS/MS assay to demonstrate that residues 38-51 of apoC-I are significantly protected from proteolysis in the presence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC). This suggests that the key lipid-binding determinants of apoC-I are located in the C-terminal region, which includes F42 and F46. To test this, we generated site-directed mutants substituting F42 and F46 for glycine or alanine. In contrast to wild-type apoC-I (WT), which binds DMPC vesicles with an apparent Kd [Kd(app)] of 0.89 microM, apoC-I(F42A) and apoC-I(F46A) possess 2-fold weaker affinities for DMPC with Kd(app) of 1.52 microM and 1.58 microM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A), apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weaker to DMPC with Kd(app) of 2.24 microM, 3.07 microM, 4.24 microM, and 10.1 microM, respectively. Sedimentation velocity studies subsequently show that the protein/DMPC complexes formed by these apoC-I mutants sediment at 6.5S, 6.7S, 6.5S, and 8.0S, respectively. This is compared with 5.0S for WT apoC-I, suggesting the shape of the particles was different. Transmission electron microscopy confirmed this assertion, demonstrating that WT forms discoidal complexes with a length-to-width ratio of 2.57, compared with 1.92, 2.01, 2.16, and 1.75 for apoC-I(F42G), apoC-I(F46G), apoC-I(F42A/F46A), and apoC-I(F42G/F46G), respectively. Our study demonstrates that the C-terminal amphipathic alpha-helix of human apoC-I contains the major lipid-binding determinants, including important aromatic residues F42 and F46, which we show play a critical role in stabilizing the structure of apoC-I, mediating phospholipid interactions, and promoting discoidal particle morphology.

Apolipoproteins (apo) E and C-I are components of triglyceride (TG)-rich lipoproteins and impact their metabolism. Functional polymorphisms have been established in apoE but not in apoC-I. We studied the relationship between apoE and apoC-I gene polymorphisms and plasma lipoproteins and coronary artery disease (CAD) in 211 African Americans and 306 Caucasians. In African Americans but not in Caucasians, apoC-I H2-carriers had significantly lower total and LDL cholesterol and apoB levels, and higher glucose, insulin, and HOMA-IR levels compared with H1 homozygotes. Differences across CAD phenotypes were seen for the apoC-I polymorphism. African-American H2-carriers without CAD had significantly lower total cholesterol (P < 0.001), LDL cholesterol (P < 0.001), and apoB (P < 0.001) levels compared with H1 homozygotes, whereas no differences were found across apoC-I genotypes for African Americans with CAD. Among African-American apoC-I H1 homozygotes, subjects with CAD had a profile similar to the metabolic syndrome (i.e., higher triglyceride, glucose, and insulin) compared with subjects without CAD. For African-American H2-carriers, subjects with CAD had a pro-atherogenic lipid pattern (i.e., higher LDL cholesterol and apoB levels), compared with subjects without CAD. ApoC-I genotypes showed an ethnically distinct phenotype relationship with regard to CAD and CAD risk factors.

The ɛ4 allele of APOE is a well-characterized genetic risk factor for late-onset Alzheimer disease (LOAD). Nevertheless, using high-density single nucleotide polymorphisms (SNPs), there have only been a few studies involving genetic association and linkage disequilibrium (LD) analyses of in and around the APOE. Here, we report fine mapping of a genomic region (about 200 kb) including the APOE in Japanese using 260 SNPs (mean intermaker distance, 0.77 kb). A case-control study demonstrated that 36 of these SNPs exhibited significance after adjustment for multiple testing. These SNPs are located in a genomic region including four genes, PVRL2, TOMM40, APOE and APOC1. Recombination rate estimation revealed that the associated region is firmly sandwiched between two recombination hotspots. Strong LD between these SNPs was observed (mean |D′| = 0.914). These data suggest that the three genes other than APOE, i.e. PVRL2, TOMM40 and APOC1, could also yield a predisposition to LOAD.

Twenty-four patients with Type IIa, IIb, III and IV hyperlipoproteinemia (HLP) were treated with 4 g of nicotinic acid daily with the purpose to study its effect on serum apolipoprotein B, C-I, C-II, C-III and E concentrations. Triglyceride and total cholesterol concentrations of whole serum and of different serum lipoprotein fractions were also determined. Analyses were performed prior to and after a drug treatment period of 6 weeks, during which all the patients were weight stable. Treatment caused a decrease in serum concentrations of apolipoproteins C-I, C-II, C-III and E. These highly significant reductions were all positively correlated to a reduction of very low density lipoprotein triglyceride levels of serum (r-values greater than 0.76, p less than 0.001). There were highly significant decreases in serum levels of apolipoprotein B and low density lipoprotein total cholesterol. These reductions were positively intercorrelated (r = 0.55; p less than 0.01). Similar effects were observed in the different HLP types and in both sexes. Treatment resulted in normolipidemia in 12 patients, who were hypercholesterolemic (7 type IIa, 3 type IIb, 2 type III hyperlipoproteinemia) prior to treatment. The serum apolipoprotein B concentrations of these 12 patients fell after therapy to values which, however, remained abnormally high. We suggest that serum lipid adjusting treatment should aim at a normalization not only of serum lipid concentrations but also of the serum apolipoprotein B concentration in order to achieve a maximal antiatherogenic effect.

Androgens play a central role in prostate cancer pathogenesis, and hence most of the patients respond to androgen deprivation therapies. However, patients tend to relapse with aggressive prostate cancer, which has been termed as hormone refractory. To identify the proteins that mediate progression to the hormone-refractory state, we used protein-chip technology for mass profiling of patients' sera. This study included 16 patients with metastatic hormone-refractory prostate cancer who were initially treated with androgen deprivation therapy. Serum samples were collected from each patient at five time points: point A, pre-treatment; point B, at the nadir of the prostate-specific antigen (PSA) level; point C, PSA failure; point D, the early hormone-refractory phase; and point E, the late hormone-refractory phase. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we performed protein mass profiling of the patients' sera and identified a 6 640-Da peak that increased with disease progression. Target proteins were partially purified, and by amino acid sequencing the peak was identified as a fragment of apolipoprotein C-I (ApoC-I). Serum ApoC-I protein levels increased with disease progression. On immunohistochemical analysis, the ApoC-I protein was found localized to the cytoplasm of the hormone-refractory cancer cells. In this study, we showed an increase in serum ApoC-I protein levels in prostate cancer patients during their progression to the hormone-refractory state, which suggests that ApoC-I protein is related to progression of prostate cancer. However, as the exact role of ApoC-I in prostate cancer pathogenesis is unclear, further research is required.

Objectives: The objective of this study was to investigate the possible association of clinical variables and apolipoprotein (APOE, APOCI and APOB) polymorphisms with the development of myocardial infraction (MI) and coronary heart disease (CHD) in Kuwaitis. Subjects and Methods: APOE, APOCI and APOB genotypes were determined by polymerase chain reaction followed by restriction fragment length polymorphism in 143 Kuwaiti CHD patients with (n = 88) and without (n = 55) MI and in 122 controls matched for gender and age. Statistical and genetic analyses of the genotype, allele and haplotype frequencies, as well as regression analyses of genetic and clinical variables were done. Results: There was a statistically significant association between CHD and medical history of diabetes mellitus (p < 0.001), hypertension (p < 0.01), high cholesterol (p < 0.05) and family history of CHD (p < 0.001). A highly significant association (p < 0.001) was found, with an adjusted odds ratio of 9.32, for family history and the development of MI. No significant differences were found for allele or genotype frequencies between CHD patients and controls. Conclusion: The strong effect of family history suggests a major genetic component for the development of CHD in Kuwaitis, but this association does not appear to be related to the APO genes studied here. The results in this study encourages future research into these and other polymorphisms and their potential association with MI and CHD in the Kuwaiti population.

Background: Wet-wrap treatment (WWT) with diluted topical steroids is widely used in atopic dermatitis (AD). Mice with transgenic overexpression of human apolipoprotein C1 (APOC1) in the liver and the skin are not only characterized by hyperlipidaemia and raised IgE levels, but also by pruritic dermatitis and a disturbed skin barrier function, providing a novel in vivo mouse model for AD. Objectives: We investigated an adapted WWT method in the AD model in APOC1 mice in order to establish its efficacy. Methods: The effect of topical 0.1% and 0.03% tacrolimus ointment, tacrolimus base ointment, different dilutions of 0.05% fluticasone propionate (FP) cream and emollient on the development of dermatitis in APOC1 mice was investigated. WWT was performed with 0.03% tacrolimus ointment or 0.017% FP cream. Results: AD in APOC1 mice responded to topical treatment with tacrolimus or FP. In contrast to tacrolimus treatment, FP treatment was associated with loss of body weight. WWT reinforced several therapeutic aspects, notably improvements in transepidermal water loss and in epidermal thickness. WWT using tacrolimus 0.03% ointment was more effective than WWT using FP 0.017% cream. Conclusions: AD in APOC1 mice responds to treatment with (diluted) tacrolimus or FP; treatment with FP cream, but not tacrolimus ointment, was associated with weight loss. In this study, the adapted WWT using tacrolimus or FP in mice had a limited improving effect as compared with open application of tacrolimus or FP. © 2008 The Authors.

Mice with transgenic expression of human apolipoprotein C1 (APOC1) in liver and skin have strongly increased serum levels of cholesterol, triglycerides, and free fatty acids, indicative of a disturbed lipid metabolism. Importantly, these mice display a disturbed skin barrier function, evident from increased transepidermal water loss, and spontaneously develop symptoms of dermatitis including scaling, lichenification, excoriations, and pruritus. Histological analysis shows increased epidermal thickening and spongiosis in conjunction with elevated numbers of inflammatory cells (eosinophils, neutrophils, mast cells, macrophages, and CD4+ T cells) in the dermis. In addition, affected mice have increased serum levels of IgE and show abundant IgE+ mast cells in the dermis. Partial inhibition of disease could be achieved by restoration of the skin barrier function with topical application of a lipophilic ointment. Furthermore, the development of atopic dermatitis in these mice was suppressed by corticosteroid treatment. These findings in APOC1(+/+) mice underscore the role of skin barrier integrity in the pathogenesis of atopic dermatitis. © 2007 The Society for Investigative Dermatology.

Endogenous yolk nutrients are crucial for embryo and larval development in fish, but developmental behavior of the genes that control yolk utilization remains unknown. Apolipoproteins have been shown to play important roles in lipid transport and uptake through the circulation system. In this study, EcApoC-I, the first cloned ApoC-I in teleosts, has been screened from pituitary cDNA library of female orange-spotted grouper (Epinephelus coioides), and the deduced amino acid sequence shows 43.5% identity to one zebrafish (Danio rerio) hypothetical protein similar to ApoC-I, and 21.2%, 21.7%, 22.5%, 20%, and 22.5% identities to Apo C-I of human (Homo sapiens), house mouse (Mus musculus), common tree shrew (Tupaia glis), dog (Canis lupus familiaris) and hamadryas baboon (Papio hamadryas), respectively. Although the sequence identity is low, amphipathic alpha-helices with the potential to bind to lipid were predicted to exist in the EcApoC-I. RT-PCR analysis revealed that it was first transcribed in gastrula embryos and maintained a relatively stable expression level during the following embryogenesis. During embryonic and early larval development, a very high level of EcApoC-I expression was in the yolk syncytial layer, indicating that it plays a significant role in yolk degradation and transfers nutrition to the embryo and early larva. By the day 7 after hatching, EcApoC-I transcripts were observed in brain. In adult, EcApoC-I mRNA was detected abundantly in brain and gonad. In transitional gonads, the EcApoC-I expression is restricted to the germ cells. The data suggested that EcApoC-I might play an important role in brain and gonad morphogenesis and growth.

In contrast to the notion established for many years that protein function depends on rigid 3D structures, nowadays there is important evidence suggesting that non-structured segments of proteins play important roles in protein function. Therefore, disorder-to-order dynamic conformational transitions have been proposed as an attractive mechanism involved in protein–protein recognition. Our laboratory using Langmuir monolayers of apolipoproteins has previously shown that upon lateral compression at the air/water and phospholipid/water interfaces, there is an important movement of the C-terminal segment of apolipoprotein CI towards the air, considered the hydrophobic region of the monolayer and the acyl-chain region of the interface when phospholipids are used. Here, in an attempt to define secondary structure changes that might occur within this C-terminal segment of apoCI while moving from the monolayer interface back and forth its hydrophobic region, employing three peptides derived from apoCI we studied by circular dichroism and dynamic light scattering their conformational properties when associated to a series of amphipathic lipids and lipid-like molecules. Our results show that a series of lysophospholipids present the ability to modulate the formation of an α helix at the C-terminal peptide of apoCI through a disorder-to-order transition while forming small lipid/peptide aggregates below 10 nm in diameter.

Background A number of the genes and proteins as the causes of carotid atherosclerotic disease have been recently reported, but the major factors for atherosclerosis have still not been identified. Methods The atherosclerotic atheromas were obtained during endarterectomy for each of 10 cases of diseased carotid and femoral arteries. As the nonatherosclerotic arteries, the iliac arteries were obtained during organ harvest from five cases of brain-dead donors, and the leg arteries were obtained during leg amputation from five cases of Buerger’s disease. The total RNAs and proteins were isolated from the atheromas and arteries. The annealing control primer method was used to screen the differentially expressed mRNAs. To identify if the mRNA expression of screened gene was associated with the protein expression, we performed an immunohistochemical analysis. Results We found that the apolipoprotein C1 (apo C1) gene was prominently expressed in the atheroma of the carotid and femoral arteries, as compared to the nonatherosclerotic arteries. Immunohistochemical analysis showed the high expression of apo C1 protein in the atheromas of the carotid and femoral arteries. Apo E protein was also highly expressed in atheromas compared with the nonatherosclerotic arteries, but there was no difference for apo C2 protein between those four groups of arteries. Discussion The expression of apo C1 and apo E are closely associated with the susceptibility to the pathogenesis of atherosclerosis. This study suggests that these factors might play important roles in the future to screen for preventing atherosclerosis and for diagnostic testing of patients.

SummaryHepatic steatosis, the accumulation of lipids in the liver, is widely believed to result in insulin resistance. To test the causal relationship between hepatic steatosis and insulin resistance, we generated mice that overexpress acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2), which catalyzes the final step of triacylglycerol (TG) biosynthesis, in the liver (Liv-DGAT2 mice). Liv-DGAT2 mice developed hepatic steatosis, with increased amounts of TG, diacylglycerol, ceramides, and unsaturated long-chain fatty acyl-CoAs in the liver. However, they had no abnormalities in plasma glucose and insulin levels, glucose and insulin tolerance, rates of glucose infusion and hepatic glucose production during hyperinsulinemic-euglycemic clamp studies, or activities of insulin-stimulated signaling proteins in the liver. DGAT1 overexpression in the liver also failed to induce glucose or insulin intolerance. Our results indicate that DGAT-mediated lipid accumulation in the liver is insufficient to cause insulin resistance and show that hepatic steatosis can occur independently of insulin resistance.

Objective Progression of atherosclerosis, the main reason of cardiovascular diseases, depends on multiple genetic and environmental factors. Polymorphic variants of genes involved in the lipids metabolism may genetically differentiate human populations and determine a susceptibility to the disease. The aim of the present study was to evaluate a possible interaction between R219K polymorphism of ABCA1 gene and G > C polymorphism in intron 7 of PPARA gene in determining the risk of the CAD. Methods We studied 358 subjects: 178 patients with angiographically confirmed CAD and 180 blood donors without history of CAD. Polymorphisms were genotyped using the PCR-RFLP method. Results In spite of a small or no correlation between single polymorphism and CAD we observed that the frequencies of AA + GC,CC genotype pattern (ABCA1 and PPARA gene) were significantly higher in CAD group than in controls. OR values were especially high in multiple logistic regression and were higher for male subgroups. The synergy index value equals 3.98 and indicates a quite strong synergistic effect between the analysed polymorphisms. We also observed that C allele carriers of PPARA gene had a significantly lower total and LDL-cholesterol level. Conclusion The present study shows that R219K polymorphism of ABCA1 gene and G > C polymorphism in intron 7 of PPARA gene act cumulatively and synergistically in determining the risk of premature CAD.

Binding of protein to a phospholipid surface is commonly mediated by amphipathic alpha-helices. To understand the role of alpha-helical structure in protein-lipid interactions, we used discoidal lipoproteins reconstituted from dimyristoylphosphatidylcholine (DMPC) and human apolipoprotein C-I (apoC-I, 6 kDa) or its mutants containing single Pro substitutions along the sequence and differing in their alpha-helical content in solution (0-48%) and on DMPC (40-75%). Thermal denaturation revealed that lipoprotein stability correlates weakly with the protein helix content: proteins with higher alpha-helical content on DMPC may form more stable complexes. Lipoprotein reconstitution upon cooling from the heat-denatured state and DMPC clearance studies revealed that protein secondary structure in solution and on DMPC correlates strongly with the maximal temperature of lipoprotein reconstitution: more helical proteins can reconstitute lipoproteins at higher temperatures. Interestingly, at Tc = 24 degrees C of the DMPC gel-to-liquid crystal transition, the clearance rate is independent of the protein helical content. Consequently, if the packing defects at the phospholipid surface are readily available (e.g., at the lipid phase boundary), insertion of protein into these defects is independent of the secondary structure in solution. However, if hydrophobic defects are limited, protein binding and insertion are aided by other surface-bound proteins and depend on their helical propensity: the larger the propensity, the faster the binding and the broader its temperature range. This positive cooperativity in binding of alpha-helices to phospholipid surface, which may result from direct and/or lipid-mediated protein-protein interactions, may be important for lipoprotein metabolism and for protein-membrane binding.

Purpose Apoptotic arterial wall vascular smooth muscle cell death is known to contribute to plaque vulnerability and rupture. Novel apoptotic markers like apolipoprotein C-I have been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway. In vivo relevance of these observations in an animal model of plaque rupture has not been shown. Methods and Results Using Watanabe rabbits, we investigated three different groups (group 1, three normal Watanabe rabbits; group 2, six Watanabe rabbits fed with high cholesterol diet for 3 months; group 3, five Watanabe rabbits with similar diet but additional endothelial denudation). We followed progression of atherosclerosis to pharmacologically induced plaque rupture non-invasively using novel 3D magnetic resonance Fast-Field-Echo angiography (TR=7.2, TE=3.6ms, matrix=512×512) and Fast-Spin-Echo vessel wall imaging methods (TR=3 heart beats, TE=10.5ms, matrix=304×304) on 1.5T MRI. MRI provided excellent image quality with good MRI versus histology vessel wall thickness correlation ( r =0.8). In six animals of group 2/3 MRI detected neo-intimal dissection in the abdominal aorta which was accompanied by immuno-histochemical demonstration of concomitant aforementioned novel apoptotic markers, previously implicated in the apoptotic smooth muscle cell death in vitro. Conclusions Our studies suggest a potential role for the signal transduction pathway involving apolipoprotein C-I for in vivo apoptosis and atherosclerotic plaque rupture visualized by MRI.

Interleukin (IL)-6 is a circulatory, pleiotropic cytokine with multiple roles in the immune system. Both IL-6 and the IL6 -174G>C promoter polymorphism have been linked to various diseases associated with inflammation. However, the mechanism by which the polymorphism influences disease risk is unclear. We postulated that serum proteome analysis of individuals with different IL6 -174G>C genotypes would provide insight on genotype-phenotype associations of this polymorphism and its role in disease susceptibility. Serum from a random sample of control participants in an ongoing population-based case-control study of non-Hodgkin lymphoma was pooled by IL6 genotype and used to screen for the optimal SELDI-TOF MS arrays for analysis. We report differences in serum protein expression of individuals with specific genotypes based on pooled and individual sample analysis. In particular, we report an association of the -174C allele with increased apolipoprotein C-I (ApoC-I). Additionally, we corroborate previous findings of an association of the -174C allele with lower autoantibodies to heat shock protein 60 and confirm the absence of any association between the IL6 -174G>C genotype and serum IL-6 levels. This study illustrates that proteome analysis can enhance our understanding of genotype-phenotype relationships. Additional studies are needed to clarify the interaction between the IL6 -174G>C polymorphism and ApoC-I.

Lipid-induced alpha-helix folding, which occurs in many lipid surface-binding proteins and peptides such as apolipoproteins and synucleins, has been proposed to provide an energy source for protein-lipid interactions. We propose that in a system comprised of a phospholipid surface and a small polypeptide that is unfolded in solution and binds reversibly to lipid surface, helical folding involves expenditure of free energy as compared to a similar polypeptide that is alpha-helical in solution. This is a consequence of the entropic cost of helix folding that is illustrated in a simple thermodynamic model and exemplifies the general "key-into-lock" paradigm of protein-ligand binding. Even though this simple model does not explicitly address the protein-induced lipid re-arrangement and may not directly apply to large proteins that undergo significant tertiary structural changes upon lipid binding, it suggests that the notion of helix folding as an energy source for lipid binding should be treated with caution.

The impact of apolipoprotein C-I (apoC-I) deficiency on hepatic lipid metabolism was addressed in mice in the presence or the absence of cholesteryl ester transfer protein (CETP). In addition to the expected moderate reduction in plasma cholesterol levels, apoCIKO mice showed significant increases in the hepatic content of cholesteryl esters (+58%) and triglycerides (+118%) and in biliary cholesterol concentration (+35%) as compared with wild-type mice. In the presence of CETP, hepatic alterations resulting from apoC-I deficiency were enforced, with up to 58% and 302% increases in hepatic levels of cholesteryl esters and triglycerides in CETPTg/apoCIKO mice versus CETPTg mice, respectively. Biliary levels of cholesterol, phospholipids, and bile acids were increased by 88, 77, and 20%, respectively, whereas total cholesterol, HDL cholesterol, and triglyceride concentrations in plasma were further reduced in CETPTg/apoCIKO mice versus CETPTg mice. Finally, apoC-I deficiency was not associated with altered VLDL production rate. In line with the previously recognized inhibition of lipoprotein clearance by apoC-I, apoCI deficiency led to decreased plasma lipid concentration, hepatic lipid accumulation, and increased biliary excretion of cholesterol. The effect was even greater when the alternate reverse cholesterol transport pathway via VLDL/LDL was boosted in the presence of CETP. Copyright ©2007 by the American Society for Biochemistry and Molecular Biology, Inc.

Reduced white adipose tissue (WAT) LPL activity delays plasma clearance of TG-rich lipoproteins (TRLs). We reported the secretion of apoC-I, an LPL inhibitor, from WAT ex vivo in women. Therefore we hypothesized that WAT-secreted apoC-I associates with reduced WAT LPL activity and TRL clearance. WAT apoC-I secretion averaged 86.9 ± 31.4 pmol/g/4 h and 74.1 ± 36.6 pmol/g/4 h in 28 women and 11 men with BMI ≥27 kg/m(2), respectively, with no sex differences. Following the ingestion of a (13)C-triolein-labeled high-fat meal, subjects with high WAT apoC-I secretion (above median) had delayed postprandial plasma clearance of dietary TRLs, assessed from plasma (13)C-triolein-labeled TGs and apoB48. They also had reduced hydrolysis and storage of synthetic (3)H-triolein-labeled ((3)H)-TRLs in WAT ex vivo (i.e., in situ LPL activity). Adjusting for WAT in situ LPL activity eliminated group differences in chylomicron clearance; while adjusting for plasma apoC-I, (3)H-NEFA uptake by WAT, or body composition did not. apoC-I inhibited in situ LPL activity in adipocytes in both a concentration- and time-dependent manner. There was no change in postprandial WAT apoC-I secretion. WAT apoC-I secretion may inhibit WAT LPL activity and promote delayed chylomicron clearance in overweight and obese subjects. We propose that reducing WAT apoC-I secretion ameliorates postprandial TRL clearance in humans.

Apolipoprotein E (APOE), translocase of outer mitochondrial membrane 40 homolog (TOMM40) and apolipoprotein C-I (APOC1) may extend lifespan by marked delay or escape from age-related diseases. This study aimed to elucidate the association of human longevity with genetic variations in TOMM40/APOE/APOC1 region in a Chinese population. Ten tag single-nucleotide polymorphisms (SNPs) in the TOMM40/APOE/APOC1 region were successfully genotyped in 616 unrelated long-lived individuals and 846 younger controls. Of the 10 SNPs, rs7254892 in 5' upstream of TOMM40 showed significant association with human longevity (G/A-A/A vs G/G: odds ratio (OR)=1.59, 95% confidence interval (CI)=1.20-2.09, P=0.0011, Bonferroni corrected P (Pc)=0.033). The haplotype analysis suggested that individuals carrying the haplotype A-A-A-A-T-A-T-G-C-A (rs7254892-rs157580-rs2075649-rs2075650-rs157582-rs8106922-rs1160985-rs405697-rs439401-rs445925) tended to have longer lifespan than those carrying the most common haplotype G-G-A-A-C-A-C-A-T-G (OR=1.59, 95% CI=1.19-2.12, P=0.0018, Pc=0.0216). These findings indicated that variants in TOMM40/APOE/APOC1 region might be associated with human longevity. Further studies are needed to identify the causal genetic variants influencing human longevity.

The serum proteomic profiles of mice exposed to terrified-sound-induced stress and after stress release were investigated. Serum samples from 32 mice were divided into four groups (n = 8 each) and analyzed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry techniques (MALDI-TOF MS) combined with magnetic bead-based weak cation-exchange chromatography. ClinProTools software identified several distinct markers that differed between the stressed and control groups and between the stress released and stressed released controls. Of 33 m/z peaks that differed among the four groups, 17 were significantly different (P 0.05). Five peaks (m/z: 2793.37, 2924.86, 1979.90, 3492.49, 3880.24) showed significant differences in expression after exposure to terrified-sound stress and returned to control levels after stress release. These were sequence identified as peptide regions of dimethylaniline monooxygenase, myosin-9, uncharacterized protein in Rattus norvegicus, apolipoprotein C-I, and plasma serine protease inhibitor (Serpina 5). Our study provides the first evidence of significant changes in serum proteomic profiles in mice exposed to terrified-sound stress, which suggests that protein expression profiles are affected by the stress. Normal expression levels were restored after stress release, suggesting the activation of self-adjustment mechanisms for the recovery of protein expression levels altered by this stress.

Protein profile analysis is increasingly used for identification of disease biomarkers. The approaches vary from surface-enhanced laser desorption/ionization to protein arrays. Newer platforms are constantly being developed. Almost all are based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and are often coupled with sophisticated software tools. Protein profiling has been applied to a variety of samples including plasma, urine, cerebrospinal fluid, saliva and solid tissue. This article focuses on those instances where it is possible to obtain sequential samples from the same individual. In the authors use of a profile method, many protein changes with highly significant correlations to disease have been found. The main challenge lies in the validation of the marker to demonstrate its adequacy for use in the clinical setting. The latter requires a methodology that is robust and amenable to high-throughput. One problem is that interindividual variability among the healthy population can mask major changes that occur on an intraindividual basis. Often, a large change for an individual may remain within the range of healthy individuals. Thus, one strategy to optimize biomarker discovery is to examine serial samples from a given individual, where a disease biomarker is established by comparison with the individual's own baseline sample. The focus of this review is to illustrate the principle and value of serial protein profiling using a rapid protein extraction method.

Previous studies have shown that overexpression of human apolipoprotein C-I (apoC-I) results in moderate hypercholesterolemia and severe hypertriglyceridemia in mice in the presence and absence of apoE. We assessed whether physiological endogenous apoC-I levels are sufficient to modulate plasma lipid levels independently of effects of apoE on lipid metabolism by comparing apolipoprotein E gene-deficient/apolipoprotein C-I gene-deficient (apoe -/-apoc1-/-), apoe-/-apoc +/-, and apoe-/-apoc1+/+ mice. The presence of the apoC-I gene-dose-dependently increased plasma cholesterol (+45%; P < 0.001) and triglycerides (TGs) (+137%; P < 0.001), both specific for VLDL. Whereas apoC-I did not affect intestinal [3H]TG absorption, it increased the production rate of hepatic VLDL-TG (+35%; P < 0.05) and VLDL-[ 35S]apoB (+39%; P < 0.01). In addition, apoC-I increased the postprandial TG response to an intragastric olive oil load (+120%; P < 0.05) and decreased the uptake of [3H]TG-derived FFAs from intravenously administered VLDL-like emulsion particles by gonadal and perirenal white adipose tissue (WAT) (-34% and -25%, respectively; P < 0.05). As LPL is the main enzyme involved in the clearance of TG-derived FFAs by WAT, and total postheparin plasma LPL levels were unaffected, these data demonstrate that endogenous apoC-I suffices to attenuate the lipolytic activity of LPL. Thus, we conclude that endogenous plasma apoC-I increases VLDL-total cholesterol and VLDL-TG dose-dependently in apoe-/- mice, resulting from increased VLDL particle production and LPL inhibition. Copyright © 2006 by the American Society for Biochemistry and Molecular Biology, Inc. Chemicals / CAS: cholesterol, 57-88-5; heparin, 37187-54-5, 8057-48-5, 8065-01-8, 9005-48-5; lipid, 66455-18-3; lipoprotein lipase, 83137-80-8, 9004-02-8; olive oil, 8001-25-0; Apolipoprotein C-I; Apolipoproteins C; Apolipoproteins E; Cholesterol, 57-88-5; Lipase, EC 3.1.1.3; Lipoprotein Lipase, EC 3.1.1.34; Lipoproteins, VLDL

Objective: To investigate associations of gene polymorphisms of the apoE-CI–CII gene cluster and the LDL-R gene on coronary artery disease (CAD) and their interactions with alcohol drinking and smoking in the Chinese Han population. Methods: A questionnaire survey of the behaviors of smoking and drinking, dietary patterns and anamnesis was conducted among 203 patients of CAD, aged 65.0±11.1 years, and 365 controls, aged 63.6±12.0 years. Peripheral blood samples were colleted and the total DNA was extracted. The apoE genotypes were identified by multiplex amplification refractory mutation system (multi-AMRS), the apoCI promoter polymorphisms and AvaII polymorphisms of the apoCII and LDL-R gene were detected by using PCR-RFLP. Pairwise linkage disequilibrium coefficients (D, D′) were estimated by the LINKAGE program. The interactions between genes with alcohol drinking and smoking were analyzed by using multivariate logistic regression models. Results: The differences of systolic/diastolic blood pressure, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) concentrations, smoking and drinking were significant between subjects with CAD and controls. The frequencies of apoE gene e 3/4 genotype (25.9%) and e 4 (13.9%) in CAD were significantly higher than those in controls (12.5% and 6.9%, respectively, p 0.0 5 ). A significant difference was also found for the apoCI locus, the frequencies of H2 allele were 20.5% in the CAD and 11.3% in the control. Linkage disequilibrium coefficient D′ was 0.672 ( p 0.0 1 ) between apoE and apoCI genes. Significant differences for a deficit of e 3-H1-T1 and excess of e 4-H2-T1 was found in CAD by estimation of the haplotype frequencies. After control for possible confounding factors, the multivariate logistic analysis showed that e 4, H2 allele, smoking and drinking were risk factors of CAD. A significant interaction among e 4, H2 and smoking was observed (OR 18.3, 95% CI: 2.35–150.81, p 0.0 5 ), it was a multiplicative model. An additive model was shown among e 4, H2 and drinking (OR12.7, 95% CI: 2.8–58.6, p 0.0 5 ). Conclusion: The results suggested that both apoE and apoCI on chromosome 19 were the susceptibility locus for CAD, their linkage disequilibrium should be responsible for the development of CAD. Drinking and smoking enhance the genetic predisposition to CAD.

Plasma cholesteryl ester transfer protein (CETP) has a profound effect on neutral lipid transfers between HDLs and apolipoprotein B (apoB)-containing lipoproteins when it is expressed in combination with human apoA-I in HuAI/CETP transgenic (Tg) rodents. In the present study, human apoA-I-mediated lipoprotein changes in HuAI/CETPTg rats are characterized by 3- to 5-fold increments in the apoB-containing lipoprotein-to-HDL cholesterol ratio, and in the cholesteryl ester-to-triglyceride ratio in apoB-containing lipoproteins. These changes occur despite no change in plasma CETP concentration in HuAI/CETPTg rats, as compared with CETPTg rats. A number of HDL apolipoproteins, including rat apoA-I and rat apoC-I are removed from the HDL surface as a result of human apoA-I overexpression. Rat apoC-I, which is known to constitute a potent inhibitor of CETP, accounts for approximately two-thirds of CETP inhibitory activity in HDL from wild-type rats, and the remainder is carried by other HDL-bound apolipoprotein inhibitors. It is concluded that human apoA-I overexpression modifies HDL particles in a way that suppresses their ability to inhibit CETP. An apoC-I decrease in HDL of HuAI/CETPTg rats contributes chiefly to the loss of the CETP-inhibitory potential that is normally associated with wild-type HDL.

We have recently shown that the predominant hypertriglyceridemia in human apolipoprotein C1 (APOC1) transgenic mice is mainly explained by apoCI-mediated inhibition of the lipoprotein lipase (LPL)-dependent triglyceride (TG)-hydrolysis pathway. Since the very-low-density lipoprotein receptor (VLDLr) and apoCIII are potent modifiers of LPL activity, our current aim was to study whether the lipolysis-inhibiting action of apoCI would be dependent on the presence of the VLDLr and apoCIII in vivo. Hereto, we employed liver-specific expression of human apoCI by using a novel recombinant adenovirus (AdAPOC1). In wild-type mice, moderate apoCI expression leading to plasma human apoCI levels of 12-33 mg/dl dose-dependently and specifically increased plasma TG (up to 6.6-fold, P < 0.001), yielding the same hypertriglyceridemic phenotype as observed in human APOC1 transgenic mice. AdAPOC1 still increased plasma TG in vldlr(-/-) mice (4.1-fold, P < 0.001) and in apoc3(-/-) mice (6.8-fold, P < 0.001) that were also deficient for the low-density lipoprotein receptor (LDLr) and LDLr-related protein (LRP) or apoE, respectively. Thus, irrespective of receptor-mediated remnant clearance by the liver, liver-specific expression of human apoCI causes hypertriglyceridemia in the absence of the VLDLr and apoCIII. We conclude that apoCI is a powerful and direct inhibitor of LPL activity independent of the VLDLr and apoCIII.

High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.

ApoC-I plays an important role in controlling plasma lipid metabolism, however little is known about factors regulating the hepatic synthesis and secretion of this apolipoprotein. In the present study, we have carried out experiments with human hepatoma (HepG2) cells, in order to determine the effect of different tissue culture conditions on cellular lipid levels and on the production of apoC-I (and apoE) at the protein and mRNA level. Cells incubated for 48 h with 10% human serum had significantly higher cellular triglyceride (22%, P0.05) and cholesterol levels (19%, P0.01), higher medium apoC-I and apoE levels (2.6- and 2.9-fold, respectively), but similar levels of apoC-I and apoE mRNA, compared to cells incubated with 10% human lipoprotein-deficient serum (LPDS). Serum containing only HDL, or containing HDL with LDL, also increased cellular lipids and increased secreted apoC-I and apoE levels without altering apoC-I and apoE mRNA levels. Incubation of cells with Intralipid triglyceride (625 microM), increased cellular triglyceride (2.8-fold, P0.001), decreased cellular cholesterol (32%, P0.01), decreased cellular and medium apoC-I (24 and 26%, P0.01) and had no effect on apoC-I mRNA levels. Additional experiments in which cells were loaded with cholesterol (incubation with 10 microg/ml cholesterol plus 1 microg/ml 25-hydroxycholesterol) or depleted of cholesterol (statin treatment) confirmed that secretion of apoC-I by HepG2 cells was dependent on cellular cholesterol levels and independent of changes in apoC-I mRNA levels. These results demonstrate that cellular cholesterol rather than triglyceride levels play a role in controlling apoC-I production by HepG2 cells and that this regulation occurs at a post-transcriptional level.

Early diagnosis and immediate therapeutic interventions are crucial factors to reduce the damage extent and the risk of death. Currently, the diagnosis of stroke relies on neurological assessment of the patient and neuro-imaging techniques including computed tomography and/or magnetic resonance imaging scan. An early diagnostic marker of stroke, ideally capable to discriminate ischemic from hemorrhagic stroke would considerably improve patient acute management. Using surface-enhanced laser desorption/ionization (SELDI) technology, we aimed at finding new early diagnostic plasmatic markers of stroke. Strong anionic exchange (SAX) SELDI profiles of plasma samples from 21 stroke patients were compared to 21 samples from healthy controls. Seven peaks appeared to be differentially expressed with significant p values (p < 0.05). Proteins were stripped from the SAX chips, separated on a one-dimensional electrophoresis (1-DE) gel and stained using mass spectrometry (MS)-compatible silver staining. Following in-gel tryptic digestion, the peptides were analyzed by MS. Four candidate proteins were identified as apolipoprotein CI (ApoC-I), apolipoprotein CIII (ApoC-III), serum amyloid A (SAA), and antithrombin-III fragment (AT-III fragment). Assessment of ApoC-I and ApoC-III levels in plasma samples using a sandwich enzyme-linked immunosorbent assay (ELISA) allowed to distinguish between hemorrhagic (n = 15) and ischemic (n = 16) stroke (p < 0.001). To the best of our knowledge, ApoC-I and ApoC-III are the first reported plasmatic biomarkers capable to accurately distinguish between ischemic and hemorrhagic stroke in a small number of patients. It requires further investigation in a large cohort of patients.

Objectives— Apolipoprotein C-I (apoC-I) influences lipoprotein metabolism, but little is known about its cellular effects in aortic smooth muscle cells (ASMC). Methods and Results— In cultured human ASMC, apoC-I and immunoaffinity purified apoC-I–enriched high-density lipoproteins (HDL) markedly induced apoptosis (5- to 25-fold), compared with control cells, apoC-I–poor HDL, and apolipoprotein C-III (apoC-III) as determined by 4′, 6-diamidino-2-phenylindole dihydrochloride staining and DNA ladder assay. Preincubation of cells with GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), blocked apoC-I–induced apoptosis, an effect that was bypassed by C-2 ceramide. The activity of N-SMase was increased 2- to 3-fold in ASMC by apoC-I, apoC-I–enriched HDL, and tumor necrosis factor α (TNF-α) (positive control) after 10 minutes and then decreased over 60 minutes, which is a kinetic pattern not seen with controls, apoC-III, and apoC-I–poor HDL. ApoC-I and apoC-I–enriched HDL stimulated the generation of ceramide, the release of cytochrome c from mitochondria, and activation of caspase-3 greater than that found in controls, apoC-III, and apoC-I–poor HDL. GW4869 inhibited apoC-I–induced production of ceramide and cytochrome c release. Conclusions— ApoC-I and apoC-I–enriched HDL activate the N-SMase-ceramide signaling pathway, leading to apoptosis in human ASMC, which is an effect that may promote plaque rupture in vivo.

Obese obob mice with strong overexpression of the human apolipoprotein C1 (APOC1) exhibit excessive free fatty acid (FFA) and triglyceride (TG) levels and severely reduced body weight (due to the absence of subcutaneous adipose tissue) and skin abnormalities. To evaluate the effects of APOC1 overexpression on hepatic and peripheral insulin sensitivity in a less-extreme model, we generated obob mice with mild overexpression of APOC1 (obob/APOC1+/-) and performed hyperinsulinemic clamp analysis. Compared with obob littermates, obob/APOC1+/- mice showed reduced body weight (-25%) and increased plasma levels of TG (+632%), total cholesterol (+134%), FFA (+65%), glucose (+73%, and insulin (+49%). Hyperinsulinemic clamp analysis revealed severe whole-body and hepatic insulin resistance in obob/APOC1+/- mice and, in addition, increased hepatic uptake of FFA and hepatic TG content. Treatment of obob/APOC1+/- mice with rosiglitazone strongly improved whole-body insulin sensitivity as well as hepatic insulin sensitivity, despite a further increase of hepatic fatty acid (FA) uptake and a panlobular increase of hepatic TG accumulation. We conclude that overexpression of APOC1 prevents rosiglitazone-induced peripheral FA uptake leading to severe hepatic steatosis. Interestingly, despite rosiglitazone-induced hepatic steatosis, hepatic insulin sensitivity improves dramatically. We hypothesize that the different hepatic fat accumulation and/or decrease in FA intermediates has a major effect on the insulin sensitivity of the liver. Chemicals / CAS: glucose, 50-99-7, 84778-64-3; insulin, 9004-10-8; rosiglitazone, 122320-73-4, 155141-29-0; Apolipoprotein C-I; Apolipoproteins C; Blood Glucose; Fatty Acids; Insulin, 11061-68-0

Apolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism.

CIII, which is associated with coronary heart disease (CHD). Because the incidence of CHD is increased in diabetic patients tive cohort study of patients with type 2 diabetes mellitus and is even higher when diabetic nephropathy is developed, has demonstrated that diabetic nephropathy is signifiwe measured apo CI levels in VLDL from type 2 diabetic cantly associated with subsequent mortality from CHD patients, with various degree of nephropathy, and compared even before end-stage renal disease (ESRD) occurs [4]. the results with those for healthy controls or nondiabetic paThe pathogenesis of CHD in diabetic nephropathy is tients with chronic renal failure (CRF). Methods. This study enrolled healthy control subjects, type multifactorial, but dyslipidemia is thought to be a strong 2 diabetic patients with normoalbuminuria, microalbuminuria, risk factor for coronary atherosclerosis [3‐8]. Several overt proteinuria, and CRF on hemodialysis and nondiabetic studies, including ours, suggested that an atherogenic lipohemodialyis patients. VLDL (density 1.006) was separated protein profile, such as an increase of very low-density by ultracentrifugation. Then the apo CI, CIII, and B concentrations in VLDL were measured by enzyme-linked immunosor- lipoprotein (VLDL) and intermediate-density lipoprobent assay (ELISA). tein (IDL) along with decrease of high-density lipoproResults. The apo CI, CIII, and B concentrations in VLDL tein (HDL)-cholesterol and LDL size, becomes more were respectively 3-, 2-, and 2-fold higher, respectively, in dia- prominent in type 2 diabetic patients who have diabetic betic patients with overt proteinuria than in controls. Hemodi- nephropathy [5‐10]. Although lipid metabolism has been alysis patients with diabetic nephropathy had levels of apo CI, CIII, and B in VLDL that were 2.6-, 2.7- and 2-fold higher, extensively investigated in diabetes, little information is respectively, than those in controls. Nondiabetic hemodialysis available concerning the influence of nephropathy on patients also had a 2.7-fold higher level of VLDL apo CIII, diabetic dyslipidemia. It remains unclear how lipoprotein whereas VLDL apo CI and VLDL apo B were not significantly abnormalities worsen with increasing severity of diabetic increased. VLDL apo CI was significantly correlated with VLDL nephropathy, and whether lipoprotein changes are atapo B independently of VLDL apo CIII level. Conclusion. An increase of VLDL apo CIII is a prominent tributable to diabetes-related metabolic abnormalities feature of dyslipidemia in CRF patients, regardless of whether or renal dysfunction. they are diabetic or nondiabetic, whereas an increase of VLDL We recently reported that the number of VLDL partiapo CI is more specific to diabetic nephropathy and is closely cles [as estimated from the apoprotein (apo) B concenassociated with an increase of VLDL particle numbers, a new risk factor for CHD. tration in VLDL] was markedly increased in Japanese men with CHD, and that this was a superior discriminator for CHD/non-CHD when compared with established coronary risk factors such as LDL-cholesterol level or LDL size [11]. That preliminary report also showed that the apo CI level in VLDL was markedly increased in

Alzheimer disease is a complex neurodegenerative disorder that is characterized by cognitive decline and distinct neuropathology. The gene for alpha-2 macroglobulin (A2M) on chromosome 12 has two polymorphisms that, in some cases, are associated with Alzheimer disease. We examined these two polymorphisms in our families in the DNA Bank (a collection of DNA samples from families with Alzheimer disease in Texas). Using both association studies and sib transmission/disequilibrium tests, we found no association of the two polymorphisms with the disease in our collection. In addition, we did not find an association of the disease with the two polymorphisms in A2M in a small subset of National Institute of Mental Health (NIMH) families. Thus, our studies do not support the A2M polymorphisms as a risk factor for Alzheimer disease.

The pathogenic relevance in Alzheimer's disease (AD) presents a decrease of cerebrospinal fluid amyloid-s42 (As42) burden and an increase in cerebrospinal fluid total tau (T-tau) levels. In this work, we performed genome-wide association study (GWAS) and genome-wide interaction study of T-tau/As42 ratio as an AD imaging quantitative trait on 843 subjects and 563,980 single-nucleotide polymorphisms (SNPs) in ADNI cohort. We aim to identify not only SNPs with significant main effects but also SNPs with interaction effects to help explain “missing heritability”. Linear regression method was used to detect SNP-SNP interactions among SNPs with uncorrected p-value ≤0.01 from the GWAS. Age, gender, and diagnosis were considered as covariates in both studies. The GWAS results replicated the previously reported AD-related genes APOE, APOC1, and TOMM40, as well as identified 14 novel genes, which showed genome-wide statistical significance. Genome-wide interaction study revealed 7 pairs of SNPs meeting the cell-size criteria and with bonferroni-corrected p-value ≤0.05. As we expect, these interaction pairs all had marginal main effects but explained a relatively high-level variance of T-tau/As42, demonstrating their potential association with AD pathology.

Metabolic syndrome (MetS) refers to the clustering of cardiometabolic risk factors, including dyslipidemia, central adiposity, hypertension, and hyperglycemia, in individuals. Identification of pleiotropic genetic factors associated with MetS traits may shed light on key pathways or mediators underlying MetS.Using the Metabochip array in 15 148 African Americans from the Population Architecture using Genomics and Epidemiology (PAGE) study, we identify susceptibility loci and investigate pleiotropy among genetic variants using a subset-based meta-analysis method, ASsociation-analysis-based-on-subSETs (ASSET). Unlike conventional models that lack power when associations for MetS components are null or have opposite effects, Association-analysis-based-on-subsets uses 1-sided tests to detect positive and negative associations for components separately and combines tests accounting for correlations among components. With Association-analysis-based-on-subsets, we identify 27 single nucleotide polymorphisms in 1 glucose and 4 lipids loci (TCF7L2, LPL, APOA5, CETP, and APOC1/APOE/TOMM40) significantly associated with MetS components overall, all P2.5e-7, the Bonferroni adjusted P value. Three loci replicate in a Hispanic population, n=5172. A novel African American-specific variant, rs12721054/APOC1, and rs10096633/LPL are associated with ≥3 MetS components. We find additional evidence of pleiotropy for APOE, TOMM40, TCF7L2, and CETP variants, many with opposing effects (eg, the same rs7901695/TCF7L2 allele is associated with increased odds of high glucose and decreased odds of central adiposity).We highlight a method to increase power in large-scale genomic association analyses and report a novel variant associated with all MetS components in African Americans. We also identify pleiotropic associations that may be clinically useful in patient risk profiling and for informing translational research of potential gene targets and medications.