Immune-regulation of the apolipoprotein A-I/C-III/A-IV gene cluster in experimental inflammation

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators' interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-alpha. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL.     

Association between coronary heart disease and the apolipoprotein A-I/C-III/A-IV complex in a Japanese population

Several studies have reported that a variant allele (S₂) of the apolipoprotein (apo) A-I/C-III/A-IV complex is associated with hyperlipoproteinemia in some populations and that the frequency of this allele is two- to fivefold higher in patients with premature coronary heart disease (CHD) than in healthy controls. In the present study in a Japanese population, we were unable to confirm the association of the S₂ allele with either coronary heart disease or elevated serum apo C-III levels, as has been previously reported in Caucasians. No genotype difference was observed among the severity of coronary heart disease, as determined by the number of involved vessels (one, two and three vessel disease), compared to controls. In addition, the frequency of the S₂ allele among Japanese, in both CHD (0.328) and controls (0.369), was quite different from that in many other populations.     

Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice reduces atherogenesis in response to a high fat-high cholesterol diet

Apoptotic proteases cleave and inactivate survival signaling molecules such as Akt/PKB, phospholipase C (PLC)-gamma1, and Bcl-2. We have found that treatment of A431 cells with tumor necrosis factor-alpha in the presence of cycloheximide resulted in the cleavage of epidermal growth factor receptor (EGFR) as well as the activation of caspase-3. Among various caspases, caspase-1, caspase-3 and caspase-7 were most potent in the cleavage of EGFR in vitro. Proteolytic cleavage of EGFR was inhibited by both YVAD-cmk and DEVD-fmk in vitro. We also investigated the effect of caspase-dependent cleavage of EGFR upon the mediation of signals to downstream signaling molecules such as PLC-gamma1. Cleavage of EGFR by caspase-3 significantly impaired the tyrosine phosphorylation of PLC-gamma1 in vitro. Given these results, we suggest that apoptotic protease specifically cleaves and inactivates EGFR, which plays crucial roles in anti-apoptotic signaling, to abrogate the activation of EGFR-dependent downstream survival signaling molecules.     

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.     

Production of a mouse strain with impaired glucose tolerance by systemic heterozygous knockout of the glucokinase gene and its feasibility as a prediabetes model

Exon II of glucokinase (Gk) was deleted to produce a systemic heterozygous Gk knockout (Gk^+/−) mouse. The relative expression levels of Gk in the heart, lung, liver, stomach, and pancreas in Gk^+/− mice ranged from 0.41–0.68 versus that in wild (Gk^+/+) mice. On the other hand, its expression levels in the brain, adipose tissue, and muscle ranged from 0.95–1.03, and its expression levels in the spleen and kidney were nearly zero. Gk knockout caused no remarkable off-target effect on the expression of 7 diabetes causing genes (Shp, Hnf1a, Hnf1b, Irs1, Irs2, Kir6.2, and Pdx1) in 10 organs. The glucose tolerance test was conducted to determine the blood glucose concentrations just after fasting for 24 h (FBG) and at 2 h after high-glucose application (GTT2h). The FBG-GTT2h plots obtained with the wild strain fed the control diet (CD), Gk^+/− strain fed the CD, and Gk^+/− strain fed the HFD were distributed in separate areas in the FBG-GTT2h diagram. The respective areas could be defined as the normal state, prediabetes state, and diabetes state, respectively. Based on the results, the criteria for prediabetes could be defined for the Gk^+/− strain developed in this study.     

Familial apolipoprotein A-I, C-III, and A-IV deficiency and premature atherosclerosis due to deletion of a gene complex on chromosome 11

The defect in a kindred with marked plasma high density lipoprotein (HDL) deficiency and premature atherosclerosis was examined. The homozygous proband died of coronary artery atherosclerosis at age 45 and had undetectable levels of plasma apolipoproteins A-I and C-III, proteins of HDL. In family studies 10 heterozygotes were identified whose mean apoA-I, apoC-III, apoA-IV, and HDL cholesterol levels were 67, 57, 65, and 62% of normal. These subjects were noted to have restriction fragment length polymorphisms following DNA digestion with a number of enzymes including BamHI, EcoRI, HindIII, XmnI, PstI, and PvuII, following hybridization with a probe spanning 1.1 kilobases approximately 2.5 kilobases 5' to the apoA-I gene. Cloning and sequence analysis of the abnormal allele indicated that the defect is due to the complete deletion of the apoA-I, -C-III, and -A-IV gene complex on chromosome 11, with both ends of the deletion being located in areas of highly repetitive DNA. The data support the concept of an independent role for HDL in the pathogenesis of atherosclerosis.     

Gene and gene-product variation in the apolipoprotein A-I/C-III/A-IV cluster in the Dogrib Indians of the Northwest Territories.

The Dogrib, an Amerindian tribe residing in the Northwest Territories of Canada, were typed for DNA and protein polymorphism at the apolipoprotein A-I/C-III/A-IV gene cluster. Variation was seen at three previously described RFLPs detected with the enzymes SstI, PstI, and XmnI, though frequencies of these polymorphisms differ significantly from those reported in other populations. They exhibit no variation at two previously reported PvuII sites. No variation was seen in the APO A-I or APO A-IV gene products, with the Dogrib showing the most common isoelectric-focusing/immunoblot patterns of other world populations. Haplotype frequencies computed from inferred haplotypes and by maximum likelihood estimation did not differ significantly. The extent of nonrandom association of these sites is highly significant (P less than .00001), though pairwise analysis shows significance between the SstI and XmnI sites only. Levels of fasting triglyceride and fasting total cholesterol were determined for each individual. Analysis of covariance shows that fasting triglyceride levels in women vary significantly with the XmnI genotype. These results suggest that genetic variation at the APO A-I/C-III/A-IV gene cluster may be a useful tool for the study of quantitative lipoprotein variation in the Dogrib.     

The nucleotide and derived amino acid sequence of human apolipoprotein A-IV mRNA and the close linkage of its gene to the genes of apolipoproteins A-I and C-III

Both cDNA and genomic clones encoding human apolipoprotein (apo-) A-IV have been isolated and characterized. Southern blot analyses of apo-A-IV gene-containing cosmids revealed that the apo-A-IV gene is linked to the apo-A-I and apo-C-III genes within a 20-kilobase span of chromosome 11 DNA. The apo-A-IV gene is located about 14 kilobases downstream from the apo-A-I gene in the same orientation, with the apo-C-III gene located between them in the opposite orientation. The nucleotide sequence of the corresponding human apo-A-IV mRNA was determined, and the derived amino acid sequence showed that mature plasma apo-A-IV contained 376 residues. Throughout most of its length, human apo-A-IV was found to contain multiple tandem 22-residue repeated segments having amphipathic, alpha-helical potential. Amino acid substitutions within these homologous segments were generally conservative in nature. A comparison of the sequences of human and rat apo-A-IV revealed a 79% identity of amino acid positions in the amino-terminal 60 residues and a 58% identity in the remainder of the sequences, with the human protein containing 5 extra residues near the carboxyl terminus. An examination of the distribution of apo-A-IV mRNA in different tissues of the rat, marmoset, and man showed that apo-A-IV mRNA was abundant in both the liver and small intestine of the rat, but abundant in both the liver and small intestine of the marmoset and man. It was expressed in only trace amounts in all other tissues that were examined. These findings on the structure and expression of apo-A-IV and the close linkage of its gene to those of apo-A-I and apo-C-III suggest a regulatory relationship between the three genes.     

Characterization of a new human apolipoprotein A-I Yame by direct sequencing of polymerase chain reaction-amplified DNA.

A new genetic variant apolipoprotein (apo), A-I Yame, was discovered during screening for apoA-I genetic variants with isoelectric focusing gel electrophoresis. To investigate the structural abnormality of apoA-I Yame, we amplified the apoA-I gene isolated from the proband's peripheral blood leukocytes with the polymerase chain reaction (PCR) method and directly sequenced these PCR fragments. ApoA-I Yame was found to have aspartic acid (GAC) substituted by tyrosine (TAC) at residue 13. We also identified this substitution by an automated DNA sequencer. This substitution was confirmed with amino acid sequencing of the isolated apoA-I Yame by Immobiline gel electrophoresis. This combined method, direct PCR from genomic DNA-derived individual peripheral blood leukocytes and subsequent direct sequencing, can be used to identify the entire sequence of apoA-I in a short period of time. Furthermore, with this method, it is possible to identify both alleles in heterozygous individuals.     

Identification of homozygosity for a human apolipoprotein A-I variant

An apolipoprotein (apo) A-I variant, previously described in two Norwegian families (Schamaun et al. 1983. Hum. Genet. 64: 380-383), represents a mutation in apoA-I in which a single amino acid substitution of lysine for glutamic acid has taken place at residue 136. An offspring resulting from intermarriage between the two families is genotypically homozygous for this variant. He is the first individual discovered to be homozygous for any of the apoA-I variants. Analysis of lipid data collected from these families indicates one or more lipid abnormalities. The low density lipoproteins (LDL) of subjects having this apoA-I variant demonstrate a compositional abnormality. The plasma cholesterol concentration in the homozygous subject is low because of the extremely reduced levels of LDL and apoB, a property shared by some of his first-degree relatives. However, because of the presence of apoE2 in this family, it is not possible to definitively link these lipid abnormalities to the presence of the A-I variant.     

Effect of apolipoprotein activators on the specificity of lecithin:cholesterol acyltransferase: determination of cholesteryl esters formed in A-I/C-III deficiency.

Although it is known that plasma lecithin:cholesterol acyltransferase (LCAT) is activated by several apolipoproteins (apo) including A-I, C-I, D, A-IV, and E, it is not clear what the physiological importance of having different apolipoprotein activators is. One possible explanation is that the activation by different apolipoproteins may result in the utilization of different species of phosphatidylcholine (PC), leading to the formation of different species of cholesteryl esters (CE). In order to determine this possibility, we analyzed the molecular species composition of PC and CE in two patients with familial deficiency of apoA-I and apoC-III. The LCAT activity, assayed by three different procedures, was found to be 36-63% of the control value. The lower LCAT activity, however, was due to deficiency of the enzyme rather than the absence of apoA-I. The patients' plasma was relatively enriched with sn-2 18:2 PC species reflecting the partial deficiency of LCAT activity. The fatty acid composition of plasma CE was not significantly different from that of controls. HPLC analysis of labeled CE formed after incubation of plasma with [C14]cholesterol showed no significant difference in the species of CE synthesized by the LCAT reaction. The transfer of pre-existing as well as newly formed CE from HDL to the apoB-containing lipoproteins was accelerated compared to control plasma. These results show that the absence of apoA-I does not significantly affect either the activity or the specificity of LCAT, and that the other apolipoprotein activators can substitute adequately for it.     

Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides

Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders.     

Serdin1/Lrrc10 is dispensable for mouse development

We have previously identified Serdin1/Lrrc10 as a cardiac-specific message that is expressed early in murine heart development and encodes a novel leucine-rich protein. A high degree of evolutionary conservation with respect to protein sequence, cardiac-specific expression, and cis-regulatory elements suggested that LRRC10 has an important and conserved function in cardiac development. Recently, the zebrafish lrrc10 knockdown models were described with a dramatic early defect in heart looping which supported the notion that Serdin1/Lrrc10 is likely to be essential for heart development in all vertebrates. To determine Lrrc10 function in mammalian cardiac development, we have disrupted the Lrrc10 gene in mice. We report here that, in striking contrast to the zebrafish lrrc10 knockdown, Lrrc10-null mice develop normally and exhibit no discernable phenotype.     

Administration of a PPARalpha agonist increases serum apolipoprotein A-V levels and the apolipoprotein A-V/apolipoprotein C-III ratio

Apolipoprotein A-V (apoA-V) first gained attention as a regulator of triglycerides through transgenic mouse studies. Furthermore, peroxisome proliferator-activated receptor alpha (PPARalpha) agonists such as fenofibrate increase apoA-V mRNA expression. Our group recently developed the first assay to quantitate serum apoA-V levels. Therefore, we sought to determine whether administration of a PPARalpha agonist would increase circulating apoA-V. Cynomolgus monkeys were dosed for 14 days with 0.3 mg/kg/day LY570977 L-lysine, a potent and selective PPARalpha agonist. Blood samples were drawn throughout the treatment period and after a 2 week washout. Administration of the PPARalpha agonist caused a 50% decrease in triglycerides that reversed at washout. Serum apoA-V concentrations increased 2-fold, correlated inversely with triglycerides, and were reversible at washout. The apoA-V/apoC-III ratio increased >2-fold, with this increase also reversible at washout. These data demonstrate for the first time that a PPARalpha agonist increases circulating apoA-V protein levels and the apoA-V/apoC-III ratio.     

Sandy: a new mouse model for platelet storage pool deficiency

Sandy (sdy) is a mouse mutant with diluted pigmentation which recently arose in the DBA/2J strain. Genetic tests indicate it is caused by an autosomal recessive mutation on mouse Chromosome 13 near the cr and Xt genetic loci. This mutation is different genetically and hematologically from previously described mouse pigment mutations with storage pool deficiency (SPD). The sandy mutant has diluted pigmentation in both eyes and fur, is fully viable and has prolonged bleeding times. Platelet serotonin levels are extremely low although ATP dependent acidification activity of platelet organelles appears normal. Also, platelet dense granules are extremely reduced in number when analysed by electron microscopy of unfixed platelets. Platelets have abnormal uptake and flashing of the fluorescent dye mepacrine. Secretion of lysosomal enzymes from kidney and from thrombin-stimulated platelets is depressed 2- and 3-fold, and ceroid pigment is present in kidney. Sandy platelets have a reduced rate of aggregation induced by collagen. The sandy mutant has an unusually severe dense granule defect and thus may be an appropriate model for cases of human Hermansky-Pudlak syndrome with similarly extreme types of SPD. It represents the tenth example of a mouse mutant with simultaneous defects in melanosomes, lysosomes and/or platelet dense granules.     

Adventitial fibroblasts are activated in the early stages of atherosclerosis in the apolipoprotein E knockout mouse

The role of the adventitia in vascular function and vascular lesion formation has been largely ignored. This study observed the activation of the adventitia and specifically the fibroblasts in the development of atherosclerosis in the apoE(-/-) mouse. The results showed a gradual increase in expression of collagen types I and III after 2, 4, and 8 weeks of hyperlipidic diet. The earliest expression of monocyte chemoattractant protein-1 (MCP-1) protein and mRNA was detected in the adventitial fibroblast before the formation of intimal lesions. Proliferation, too, was first found in the adventitial fibroblasts. We hypothesize that the adventitial fibroblast is activated in the early stage of atherosclerosis. Adventitial inflammation may be an early event in the development of atherosclerotic lesions.     

Neurological phenotype and reduced lifespan in heterozygous Tim23 knockout mice, the first mouse model of defective mitochondrial import

The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a Tim23 knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a neurological phenotype and a markedly reduced life span. Haploinsufficiency of the Tim23 mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function.     

雌性小鼠不适于构建HFD-STZ联合诱导的2型糖尿病模型

目的 探讨雌性小鼠在高脂饲料(HFD)-链脲佐菌素(STZ)联合诱导2型糖尿病模型中的可行性.方法 分别以高脂饲料、高脂饲料-果糖饮水(HFDF)和常规饲料(对照)喂养3周龄的封闭群ICR和近交系C57BL/6J (B6)小鼠6周后,HFD和HFDF组腹腔注射STZ,对照组注射相应体积柠檬酸钠溶液,然后分别以相应饲料继续喂养6周.每周测定小鼠体重,于注射前1周和注射后1～4周测定非空腹血糖浓度.结果 实验结束时各组小鼠体重较初始体重均显著增加.ICR小鼠HFD和HFDF组体重与对照组S无差异,HFD与HFDF组间也无显著变化.虽然B6小鼠HFD与HFDF组体重组间差异不显著,但两组体重均显著低于对照组.注射STZ后1～4周,两品系小鼠HFD与HFDF组血糖水平没有显著升高,组间也没有显著差异,且均没有达到2型糖尿病小鼠成模非空腹血糖标准(11 mmol/L).结论 果糖饮水不能促进高脂饲料诱导的育肥作用,而雌性小鼠也不是HFD-STZ联合诱导2型糖尿病模型的理性选择.     

Characterization of a novel mouse model with genetic deletion of CD177

Neutrophils play an essential role in the innate immune response to infection. Neutrophils migrate from the vasculature into the tissue in response to infection. Recently, a neutrophil cell surface receptor, CD177, was shown to help mediate neutrophil migration across the endothelium through interactions with PECAM1. We examined a publicly available gene array dataset of CD177 expression from human neutrophils following pulmonary endotoxin instillation. Among all 22,214 genes examined, CD177 mRNA was the most upregulated following endotoxin exposure. The high level of CD177 expression is also maintained in airspace neutrophils, suggesting a potential involvement of CD177 in neutrophil infiltration under infectious diseases. To determine the role of CD177 in neutrophils in vivo, we constructed a CD177-genetic knockout mouse model. The mice with homozygous deletion of CD177 have no discernible phenotype and no significant change in immune cells, other than decreased neutrophil counts in peripheral blood. We examined the role of CD177 in neutrophil accumulation using a skin infection model with Staphylococcus aureus. CD177 deletion reduced neutrophil counts in inflammatory skin caused by S. aureus. Mechanistically we found that CD177 deletion in mouse neutrophils has no significant impact in CXCL1/ KC- or fMLP-induced migration, but led to significant cell death. Herein we established a novel genetic mouse model to study the role of CD177 and found that CD177 plays an important role in neutrophils.