apoA-I Gene Research Articles

Hepatic High-Density Lipoprotein Secretion Regulates the Mobilization of Cell-Surface Hepatic Lipase

HDL acts much like heparin to liberate hepatic lipase (HL) from cell surface proteoglycans and stimulate triglyceride clearance. Experiments were undertaken to evaluate the effects of factors that stimulate the secretion of HDL from the liver on the release of HL. Treatment of HepG2 cells with linoleic acid phospholipids (LAPL) (12 muM) promotes a similar increase in the accumulation of both HDL and HL in the cell media. LAPL also induce both apoA-I and HL release from primary human hepatocytes. Dilinoleoylphosphatidylcholine has a greater effect on both apoA-I secretion and HL release than palmitoyllinoleoylphosphatidylcholine. HL released from HepG2 cells is inactive and associated with a large HDL complex containing both apoA-I and apoA-II. Inclusion of the PPARalpha inhibitor, MK-886, or MAPK inhibitor, U0126, completely blocks the LAPL-induced apoA-I and HL accumulation in the media. LAPL-treated cell lysates, however, showed no change in HL protein expression nor HL mRNA. LAPL-induced HL release appears to be a consequence of the displacement ability of newly secreted HDL. Overexpression of pre-pro-apoA-I in HepG2 cells increased HL release, while siRNA inhibition of the apoA-I gene reduced HL in the media. The data show that factors that stimulate HDL secretion in hepatocytes act to also increase the release of HL. This may partly explain why HDL therapeutics often impact plasma triglyceride levels.

Abstract: P628 PRO-INFLAMMATORY EFFECTS OF APOLIPOPROTEIN CIII ON MONOCYTIC CELLS

This study was aimed to examine cholesteryl ester transfer protein (CETP), apolipoprotein AI and CIII gene polymorphisms, and to verify whether these genetic determinants are associated with the prevalence of myocardial infarction (MI) or type 2 diabetes. The TaqIB restriction fragment length polymorphism (RFLP) in intron I of the CETP gene, the MspI in the third intron of the APOAI gene, and also SstI in the 3′ untranslated region of the APOCIII gene were determined using standard methods. The prevalence of these polymorphisms was compared between diabetic (n = 119), and non-diabetic (n = 100) middle-aged individuals of both sexes. We found a higher prevalence of the B2B2 genotype of the CETP gene among diabetics than that observed in non-diabetics (P < 0.05), and a lower prevalence of this genotype among patients with previous MI (P < 0.02). The MspI polymorphisms of the APOAI gene showed that M1++ genotype was found mainly in diabetic patients (P < 0.04). Conversely, the SstI polymorphism of APOCIII gene was not significantly associated with either MI or diabetes. Therefore, among these genetic polymorphisms, TaqIB of CETP and MspI of apolipoprotein AI appeared to help significantly to identify diabetic individuals. In particular, the former may have an additional role in the primary prevention of coronary disease.

AAV gene therapy as a means to increase apolipoprotein (Apo) A‐I and high‐density lipoprotein‐cholesterol levels: correction of murine ApoA‐I deficiency

Inherited apolipoprotein (Apo) A-I deficiency is an orphan disorder characterized by high-density lipoprotein (HDL)-cholesterol deficiency and premature atherosclerosis. Constitutive over-expression of ApoA-I might provide a means to treat this disease. The present study provides a comprehensive evaluation of adeno-associated virus (AAV)-mediated ApoA-I gene delivery to express human (h)ApoA-I and correct the low HDL-cholesterol phenotype associated with ApoA-I deficiency. In an effort to maximize AAV-mediated gene expression, we performed head-to-head comparisons of recombinant AAVs with pseudotype capsids 1, 2, 6 and 8 administered by different routes with the use of five different liver-specific promoters in addition to cytomegalovirus as single-stranded or as self-complementary (sc) AAV vectors. Intravenous administration of 1 x 10(13) gc/kg scAAV8, in combination with the liver-specific promoter LP1, in female ApoA-I(-/-) mice resulted in hApoA-I expression levels of 634 +/- 69 mg/l, which persisted for the duration of the study (15 weeks). This treatment resulted in full recovery of HDL-cholesterol levels with correction of HDL particle size and apolipoprotein composition. In addition, we observed increased adrenal cholesterol content and a significant increase in bodyweight in treated mice. The present study demonstrates that systemic delivery of a scAAV8 vector provides a means for efficient liver expression of hApoA-I, thereby correcting the lipid abnormalities associated with murine ApoA-I deficiency. Importantly, the study demonstrates that AAV-based gene therapy can be used to express therapeutic proteins at a high level for a prolonged period of time and, as such, provides a basis for further development of this strategy to treat hApoA-I deficiency.

Marked high density lipoprotein deficiency due to apolipoprotein A-I Tomioka (codon 138 deletion)

We report a novel apolipoprotein A-I (apoA-I) mutation identified in a 64-year-old patient with marked plasma high density lipoprotein (HDL) cholesterol (4 mg/dl) and apoA-I (5 mg/dl) deficiency, prior myocardial infarction, and moderate corneal opacities. Coronary angiography revealed extensive atherosclerosis in all three major vessels. Genomic DNA sequencing of the proband revealed a homozygous novel deletion of two successive adenine residues in codon 138 in the apoA-I gene, resulting in a frameshift mutation at amino acid residues 138–178, which we have designated as apoA-I Tomioka. His elder brother was also homozygous for apoA-I Tomioka with marked HDL cholesterol and apoA-I deficiency, but had no clinical evidence of coronary heart disease. Other family members including three siblings and two sons were heterozygous for the mutation, and had approximately 50% of normal plasma HDL cholesterol, and apoA-I. Analysis of apoA-I-containing HDL particles by two-dimensional gel electrophoresis revealed undetectable apoA-I HDL particles in the homozygotes, while in heterozygotes, the mean concentrations of apoA-I in large α-1 and very small preβ-1 HDL subpopulations were significantly decreased at about 35% of normal. Thus, apoA-I Tomioka, a novel deletion mutation in codon 138 of the apoA-I gene, is the causative defect in this case of HDL deficiency.

Apolipoprotein A-I, a hyperosmotic adaptation-related protein in ayu (Plecoglossus altivelis)

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum and participates in the reverse transport of cholesterol from tissues to the liver for excretion. 2DE gel and protein identification by MALDI-TOF-MS revealed that an apoA-I-like molecule was significantly decreased in the liver of ayu transferred from freshwater (FW) to brackish water (BW). A full-length cDNA clone of this protein was subsequently isolated. It contains 1209 bp with an open reading frame of 825 bp, coding for 275 amino acids with MW 31.1 kDa and pI 5.25. Ayu apoA-I had highest similarity (88.6% amino acid identity) to that of rainbow smelt. Phylogenetic analysis showed that vertebrate apoA-Is formed two major groups, representing mammalian-bird-amphibian (M-A) and fish apoA-Is respectively. Ayu apoA-I was most closely related to rainbow smelt apoA-I. In FW ayu, apoA-I transcripts were present in all tested tissues including brain, spleen, liver, kidney, gill, muscle, heart and intestine, and highest in liver, brain and intestine. In BW ayu, the expression levels of apoA-I gene were significantly decreased and almost totally inhibited in spleen, kidney, gill, heart and muscle. Significant down-regulation of proteins and mRNA in ayu transferred from FW to BW suggests an involvement in hyperosmotic regulation in this species.

Induction of paraoxonase 1 and apolipoprotein A-I gene expression by aspirin

Low-dose aspirin therapy has become a standard in the treatment of cardiovascular diseases. Aspirin has been shown to inhibit atherosclerosis in mouse models. To determine the mechanisms by which aspirin might inhibit atherosclerosis, we incubated HEPG2 cells and rat primary hepatocytes with aspirin or salicylic acid and noted an increase in paraoxonase 1(PON1) activity in the medium, together with an induction of PON1 and apolipoprotein A-I (apoA-I) gene expression. Mice treated with aspirin also showed a 2-fold increase in plasma PON1 activity and a significant induction of both PON1 and apoA-I gene expression in the liver. The induction of the PON1 gene in cell culture was accompanied by an increase in arylhydrocarbon receptor (AhR) gene expression. Accordingly, aspirin treatment of AhR(-/-) animals failed to induce PON1 gene expression. We previously suggested that aspirin might be hydrolyzed by serum PON1, which could account for its short plasma half-life of 10 min. Taken together with the current studies, we suggest that the antiatherosclerotic effects of aspirin might be mediated by its hydrolytic product salicylate and that the induction of PON1 and apoA-I might be important in the cardioprotective effects of aspirin.

Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR(-/-)) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR(-/-) mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR(-/-) mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.

PPARδ increases expression of the human apolipoprotein A-II gene in human liver cells

The peroxisome proliferator-activated receptor delta (PPARdelta) is a transcription factor that regulates genes of importance in lipid and glucose metabolism. ApoA-II is one of the major proteins of the HDL-particle. The aim of this study was to investigate the regulation of apoA-II gene expression by PPARdelta. Treatment of HepG2 cells with the PPARdelta specific agonist GW501516 increased apoA-II mRNA expression. Likewise, reporter gene assays using a construct containing 2.7 kb of the proximal apoA-II promoter showed increased activity after treatment with GW501516, both in HepG2 and in HuH-7 cells. Mutation of two putative PPAR response elements (PPREs) in this region showed that the PPRE at position -737/-717 is the functional site. Binding of PPARdelta to this site was confirmed by chromatin immunoprecipitation and gel retardation analyses. In conclusion, PPARdelta increases the expression of the human apoA-II gene in liver cells via a PPRE in the proximal promoter.

Associations of the apolipoprotein A-I gene polymorphism and serum lipid levels in the Guangxi Hei Yi Zhuang and Han populations

Hei Yi Zhuang is an isolated subgroup of the Zhuang minority in China. Little is known about the effects of the genetic variants on serum lipid profiles in this population. The present study was undertaken to estimate the effects of the apolipoprotein (apo) A-I gene polymorphism adjacent to the initiate transcription site (-75 bp G/A) on the serum lipid levels in the Hei Yi Zhuang and Han populations. A total of 474 subjects of Hei Yi Zhuang and 564 subjects of Han Chinese were surveyed by a stratified randomized cluster sampling. Serum lipid levels were measured, and apoA-I gene polymorphism determined by polymerase chain reaction and restriction fragment length polymorphism. The frequencies of G and A alleles were 70.25 and 29.75% in Hei Yi Zhuang, and 65.96 and 34.04% in Han (P<0.05), respectively. The genotypic frequencies in Han were significantly different between males and females, subjects with normal TG (< or =1.70 mmol/l) and those with high TG (>1.70 mmol/l), or subjects with normal apoA-I (> or =1.20 g/l) and those with abnormal apoA-I (<1.20 g/l; P<0.05-0.01), respectively. The levels of LDL-C and apoA-I in Hei Yi Zhuang were higher in GG genotype than in AA or GA genotype (P<0.05 for each), but the levels of TG was lower in AA genotype than in GA genotype (P<0.05). There were also significant differences in serum TG levels among the three genotypes in Hei Yi Zhuang (P<0.05). The levels of HDL-C in Han were higher in GG genotype than in AA genotype (P<0.05), but the levels of TG in Han were lower in GG genotype than in GA genotype (P<0.05). The levels of apoA-I in Hei Yi Zhuang and the levels of HDL-C and apoB in Han were significantly correlated with genotype (P<0.05 for all). Hypertriglyceridemia was negatively associated with genotype in Hei Yi Zhuang (P<0.01). There were significant differences in the apoA-I -75 bp G/A between the Hei Yi Zhuang and Han populations. An association of the apoAI -75 bp G/A and serum TG, LDL-C and apoA-I levels in Hei Yi Zhuang and serum TG, HDL-C and apoB levels in Han was also observed in this study.

Role of apolipoproteins E and A-I: Epistatic villains of triglyceride mediation in coronary heart disease

The epistatic effects of ApoE (HhaI) and ApoA-I (PstI) genes as the genetic modulators of lipid levels were investigated in 165 angiographically verified CHD patients and 120 controls of Punjab, a northwest province of India. It has been revealed that of all the genotypic combinations of ApoE and ApoA-I, E4 allele carriers (E4+) with P1P2 genotype (ApoA-I/PstI) had higher risk of CHD (OR 2.99, CI 1.31–6.8, P < 0.01) which exacerbated (OR 3.44, CI 1.45–8.15, P < 0.01) after adjustment with the confounders. Individually, neither ApoA-I nor ApoE was found to be associated with TG levels however, pairwise epistasis (additive × additive model) explored their significant synergistic contributions with raised TG levels ( P < 0.01).

Ap1-like Cis elements in the 5′-regulatory region of the human apolipoprotein A-I gene

Several Ap1-like cis-acting elements were found within 5'-regulatory region (-2497...+173 versus transcription start point) of human apolipoprotein A-I gene (5'-apoA-I). Those elements are capable to interact with transcription factors belonging to Ap1 and CREB/ATF families. Those elements are localized outside of the hepatic enhancer (-220...-110) and the sequence responsible for apoA-I gene transcription in Caco2 cells (-595...-192). One of Ap1-like sites (5'-TGAGGTCT-3, Cre/jun2/apo) is present within 5'-apoA-I in two copies - distal (-1798 ...-1791) and proximal (+99...+106) ones. This and other Ap1-like sites - 5'-TGACTCT-3' (-1798...-1791, PF1) and 5'-TGACATCA-3' (-1171...-1163, Cre/jun1) were characterized by EMSA. It was shown by using the specific antibodies to c-Jun and ATF2 transcription factors in EMSA supershift experiments, that the DNA-protein complexes formed by Cre/jun2/apo, Cre/jun1 elements with nuclear proteins of human hepatoma HepG2 cells contain ATF2. The functional role of 5'-apoA-I regions containing Ap1-like sites was studied in cotransfection experiments of HepG2 cells (synthesize endogenous ApoA-I), human duodenum adenocarcinoma Hutu80 cells (do not synthesize endogenous ApoA-I), human neuroblastoma SK-N-SH cells (do not synthesize endogenous A-I) with expression vectors of c-jun and mekk1 genes. It was shown, that those Ap1-like sites appears to be responsible (the proximal Cre/jun2/apo is more efficient) for tissue-specific regulation of human apoA-I gene expression.

Glutathione S‐Transferase Null and Cholesteryl Ester Transfer Protein TaqI B Polymorphisms and Lipid Response to Atorvastatin in Greek Dyslipidaemic Patients

Impairment of high-density lipoprotein (HDL) cholesterolefflux capacity, coupled to attenuated antioxidative andanti-inflammatory properties of HDL particles, has beenlately recognized as intimately connected to atherogenicdyslipidaemia, a well-established cardiovascular risk factor[1]. Concomitantly, raising HDL-cholesterol (HDL-C) andapolipoprotein A-I (apoA-I) levels has been proposed as amajor anti-atherogenic therapeutic target [2]. While 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA)reductase inhibitors (statins) are very efficacious in reducingtotal cholesterol, triacylglycerol, low-density lipoproteincholesterol (LDL-C) and apolipoprotein B (apoB) levels indyslipidaemic patients, their effect on HDL-C and apoA-I ismoderate at best, and may be dependent on the type ofstatin used, dosage, duration of treatment, but also, geneticfactors [3,4]. In the past, pharmacogenetic studies of lipidresponse to statins have focused almost exclusively on poly-morphisms of genes related to lipoprotein turnover, with thegene coding for cholesteryl ester transfer protein (CETP)receiving most of the attention [5–7]. Other factors may beinvolved as well. Oxidative stress has been implicated in thepathophysiology of cardiovascular disease [8] and oxidativemodification has been recently reported to impair thelipid-binding capacity of apoA-I, resulting in reduced HDLcholesterol efflux [9]. In addition, the apoA-I gene promoteris known to contain an antioxidant response element andmay be responsive to oxidative stress [10]. Because statins arereportedly able to attenuate the production of reactiveoxygen species through mechanisms unrelated to theirlipid-lowering effect [11], the possibility that genes affectingthe antioxidant status of the organism can influence apoA-Iresponse to these drugs is an intriguing and as yet untestedhypothesis. On the other hand, such an effect could con-ceivably be modulated by polymorphisms affecting reversecholesterol transport – such as CETP TaqI B – especiallysince a growing body of evidence, from human and animalstudies, suggests that there is a functional interactionbetween apoA-I expression and CETP activity [12–14].Glutathione S-transferases (GST) are phase II biotrans-formation enzymes that play a major role in the antioxidantcapability of the organism, by catalysing the conjugation ofreduced GSH to a variety of endogenous and exogenoussubstrates [15]. Two common deletion polymorphisms ofthe GST T1 and M1 genes, resulting in complete absence ofactivity of the respected enzymes (null genotypes), haverecently become the focus of studies investigating their effecton the risk for myocardial infarction [16,17], vascularinflammation [18], hypertension [19] and smoking-relatedcardiovascular disease [20–23].In undertaking this study, we hypothesized that genotypingfor the GST T1 and M1 null, as well as the TaqI B poly-morphisms, may assist in the identification of strongerresponders to atorvastatin treatment, especially with respectto apoA-I and HDL-C levels that are only modestly affectedby this drug. Our study group consisted of 55 previouslyuntreated, Greek consecutive dyslipidaemic [total cholesterol(CHOL) > 240 mg/dl, LDL-C > 160 mg/dl and triacylglycerols(TG) < 250 mg/dl] patients. The latter were all examined in theoutpatient clinic of the First Propedeutic Clinic of AHEPAUniversity Hospital, Thessaloniki, Greece, from November2004 until May 2006. Exclusion criteria included: damage oftarget organs (coronary disease ruled out based on symptoms,electrocardiogram and history, renal failure, retinopathyand stroke), history of diabetes mellitus, hypothyroidismand impaired hepatic or renal function (fig. 1). During therun-in period (4–6 weeks), all patients underwent a completeclinical and laboratory examination to exclude secondary

Characterization of high density lipoprotein particles in familial apolipoprotein A-I deficiency

Our aim was to characterize HDL subspecies and fat-soluble vitamin levels in a kindred with familial apolipoprotein A-I (apoA-I) deficiency. Sequencing of the APOA1 gene revealed a nonsense mutation at codon -2, Q[-2]X, with two documented homozygotes, eight heterozygotes, and two normal subjects in the kindred. Homozygotes presented markedly decreased HDL cholesterol levels, undetectable plasma apoA-1, tuboeruptive and planar xanthomas, mild corneal arcus and opacification, and severe premature coronary artery disease. In both homozygotes, analysis of HDL particles by two-dimensional gel electrophoresis revealed undetectable apoA-I, decreased amounts of small alpha-3 migrating apoA-II particles, and only modestly decreased normal amounts of slow alpha migrating apoA-IV- and apoE-containing HDL, while in the eight heterozygotes, there was loss of large alpha-1 HDL particles. There were no significant decreases in plasma fat-soluble vitamin levels noted in either homozygotes or heterozygotes compared with normal control subjects. Our data indicate that isolated apoA-I deficiency results in marked HDL deficiency with very low apoA-II alpha-3 HDL particles, modest reductions in the separate and distinct plasma apoA-IV and apoE HDL particles, tuboeruptive xanthomas, premature coronary atherosclerosis, and no evidence of fat malabsorption.

The Effect of Retinoic Acid Receptor-Related Orphan Nuclear Receptor α on Apo AI and Malic Enzyme Gene Expression.

The Effect of Retinoic Acid Receptor-Related Orphan Nuclear Receptor α on Apo AI and Malic Enzyme Gene Expression.

Identifying Natural Derived Upregulators of Human ApoA-I Expression via a Cell-Based Drug Screening System

Apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein, is instrumental in promoting efflux of cholesterol from extrahepatic tissues to the liver. It has been proposed that upregulation of apoA-I gene expression could have anti-atherosclerosis effects. For the purpose of screening human apoA-I expression upregulators, we established a cell-based drug screening model in vitro to identifying the effects of natural derived upregulators of apoA-I. In the work, human apoA-I promoter gene was cloned into pGL3B-neo vector containing luciferase gene and neomycin resistance gene. The recombinant reporter gene vector pGL3B-neo/apo was transfected into HepG2 cells, and therefore the stable cell line SApoA-I was obtained. These cells were treated with extracts from traditional Chinese herbs. The luciferase activities were detected to identify which ones can activate apoA-I promoter. Baicalein extracted from dried roots of Scutellaria baicalensis Georgi (Labiatae, Scutellaria) showed significant dose-dependent upregulatory activity in cell model, which might become an effective basic compound for developing novel HDL-raising drugs because of its effects on proteins involved in reverse cholesterol transport.

Infertility and Hypergonadotropic Hypogonadism as First Evidence of Hereditary Apolipoprotein A-I Amyloidosis

We report that primary infertility and hypergonadotropic hypogonadism in young patients may be caused by testicular amyloidosis and it is associated with the presence of a mutation in the apoA-I gene, resulting in the replacement of proline for leucine at residue 75 of the protein. Ten patients presenting with infertility, gynecomastia, decreased libido, erectile dysfunction or a family history of amyloidosis underwent clinical evaluation, hormone assays, semen analysis, ultrasonographic investigation of the testicles, testicular biopsy and DNA sequencing of the apoA-I gene. All patients showed azoospermia and 9 had increased testicular volume. Massive amyloid deposition was observed in all testicular biopsies and the apoA-I mutation of replacement of proline for leucine at residue 75 of the protein was noted. Five patients showed hypergonadotropic hypogonadism and 5 had normal testosterone values with high gonadotropin levels. Nonobstructive azoospermia and macro-orchidism with or without hypogonadism may be caused by hereditary apoA-I amyloidosis in young patients. Testicular amyloidosis can be the first manifestation of this systemic disease. Specific staining for amyloid deposits and genetic analysis of apoA-I mutations are recommended in young, infertile patients with macro-orchidism. Finally, surveillance in asymptomatic mutation carriers is suggested to evaluate the opportunity to implement sperm retrieval and start androgen replacement therapy when necessary.

Electron Paramagnetic Resonance Spectroscopy of Site-directed Spin Labels Reveals the Structural Heterogeneity in the N-terminal Domain of ApoA-I in Solution

Apolipoprotein A-I (apoA-I) is the major protein constituent of high density lipoprotein (HDL) and plays a central role in phospholipid and cholesterol metabolism. This 243-residue long protein is remarkably flexible and assumes numerous lipid-dependent conformations. Consequently, definitive structural determination of lipid-free apoA-I in solution has been difficult. Using electron paramagnetic spectroscopy of site-directed spin labels in the N-terminal domain of apoA-I (residues 1-98) we have mapped a mixture of secondary structural elements, the composition of which is consistent with findings from other in-solution methods. Based on side chain mobility and their accessibility to polar and non-polar spin relaxers, the precise location of secondary elements for amino acids 14-98 was determined for both lipid-free and lipid-bound apoA-I. Based on intermolecular dipolar coupling at positions 26, 44, and 64, these secondary structural elements were arranged into a tertiary fold to generate a structural model for lipid-free apoA-I in solution.

Apolipoprotein A-IV Is an Independent Predictor of Disease Activity in Patients with Inflammatory Bowel Disease

ApoA-IV, an apolipoprotein (apo) with antioxidant, antiatherogenic, and antiinflammatory properties, was recently demonstrated to inhibit dextran sulfate sodium (DSS)-induced experimental colitis in mice. We therefore hypothesized that apoA-IV may be associated with disease activity in patients with inflammatory bowel disease (IBD). We addressed this question by testing for associations between apoA-IV genotypes, apoA-IV plasma levels, inflammatory parameters, and clinical disease activity in 206 patients with Crohn's disease (CD), 95 subjects with ulcerative colitis (UC), and 157 healthy controls. In CD patients, apoA-IV plasma levels were inversely associated with C-reactive protein (CRP) (P = 0.005) and disease activity (P = 0.01) in univariate analysis. In multiple logistic regression analysis, apoA-IV levels were identified as an independent predictor of elevated CRP (odds ratio [OR] 0.956, 95% confidence interval [CI]: 0.916-0.998, P = 0.04) and active disease (OR 0.957, 95% CI: 0.918-0.998, P = 0.04). In UC patients the apoA-IV gene variant 360 His (P = 0.03) but not apoA-IV levels (P = 0.15) were associated with increased disease activity in univariate analysis. This association, however, was lost in multiple logistic regression analysis (OR 3.435, 95% CI 0.995-11.853, P = 0.05). To our knowledge, this is the first study to demonstrate an association of apoA-IV with disease activity in patients with CD. Further studies are needed to define the relationship of apoA-IV to IBD.

Cyclosporin A inhibits apolipoprotein AI gene expression

Cyclosporin A (CsA), a calcineurin inhibitor, has been widely used as an immunosuppressant, and is known to induce hyperlipidemia and dyslipoproteinemia with low levels of high-density lipoprotein (HDL). Since apolipoprotein AI (apo AI) is a major protein component of HDL particles and reduction of apo AI results in low levels of HDL, we hypothesized that CsA inhibits apo AI gene expression contributing to its lipid effects. Therefore, we first measured the serum apo AI protein levels in rats with or without CsA treatment, and found that both serum apo AI protein and liver apo AI mRNA levels were significantly reduced in response to CsA treatment. In stably transfected Hep G2 cells harboring an apo AI-474-CAT reporter gene, we found that intracellular calcium mobilization by A23187 a calcium ionophore stimulated apo AI gene expression and the calcineurin inhibitors, CsA and FK605, selectively inhibited this stimulation. Therefore, we conclude that activation of the calcineurin pathway by intracellular calcium mobilization stimulates apo AI gene expression and calcineurin inhibition by CsA results in reduced apo AI gene expression.

Bone Marrow Transplantation Shows Superior Atheroprotective Effects of Gene Therapy With Apolipoprotein A-I Milano Compared With Wild-Type Apolipoprotein A-I in Hyperlipidemic Mice

We tested the hypothesis that gene therapy using apolipoprotein A-I Milano (apoA-IMilano) is more effective than that using wild-type apolipoprotein A-I (apoA-I) in reducing atherosclerosis. Apolipoprotein A-I Milano is a naturally occurring mutant with established antiatherogenic activity; however, its relative antiatherogenic efficacy compared with that of wild-type apoA-I remains unclear. We performed bone marrow transplantation in female double-knockout mice lacking both the apoE and apoA-I genes using male donor mice-derived bone marrow that had been transduced with a retroviral vector alone or retroviral vector expressing wild-type apoA-I or apoA-IMilano gene under the control of macrophage-specific scavenger receptor A promoter. Mice were fed a high-cholesterol diet and killed 24 weeks after transplantation, at which time the extent of aortic atherosclerosis was determined. Compared with vector control (n = 12), apoA-IMilano gene therapy (n = 15) reduced aortic atherosclerosis by 65% (p < 0.001) and plaque macrophage immunoreactivity by 58% (p < 0.0001), whereas wild-type apoA-I (n = 11) reduced atherosclerosis by 25% (p = 0.1) and plaque macrophage immunoreactivity by 23% (p < 0.05). The apoA-IMilano gene therapy was significantly more effective in reducing atherosclerosis (p < 0.05) and macrophage immunoreactivity (p < 0.001) compared with wild-type apoA-I. The circulating levels of cholesterol, lipoprotein profile, and apoA-IMilano or wild-type apoA-I were comparable among the groups. Apolipoprotein A-I Milano was more effective than wild-type apoA-I in promoting macrophage cholesterol efflux. Macrophage-specific expression of the apoA-IMilano gene is more effective than wild-type apoA-I in reducing atherosclerosis and plaque inflammation despite comparable circulating levels of the transgene and lipid profile.