The pathogenic mutations of APOA5 in Chinese patients with hyperlipidemic acute pancreatitis.

BACKGROUND AND AIMS: To study the role of gene mutations in the development of severe hypertriglyceridemia (HTG) in patients with hyperlipidemic acute pancreatitis (HLAP), especially different apolipoprotein A5 (APOA5) mutations. METHODS: Whole-exome sequencing was performed on 163 patients with HLAP and 30 patients with biliary acute pancreatitis (BAP). The pathogenicity of mutations was then assessed by combining clinical information, predictions of bioinformatics programs, information from multiple gene databases, and residue location and conservation. The pathogenic mutations of APOA5 were visualized using the software. RESULTS: 1. Compared with BAP patients, pathogenic mutations of APOA5 were frequent in HLAP patients; among them, the heterozygous mutation of p.G185C was the most common. 2. All six pathogenic mutations of APOA5 identified in this study (p.S35N, p.D167V, p.G185C, p.K188I, p.R223C, and p.H182fs) were positively correlated with severe HTG; they were all in the important domains of apolipoprotein A-V (apoA-V). Residue 223 is strictly conserved in multiple mammals and is located in the lipoprotein lipase (LPL)-binding domain (Pro215-Phe261). When Arg 223 is mutated to Cys 223, the positive charge of this residue is reduced, which is potentially destructive to the binding function of apoA-V to LPL. 3. Four new APOA5 mutations were identified, namely c.563A > T, c.667C > T, c.788G > A, and c.544_545 insGGTGC. CONCLUSIONS: The pathogenic mutations of APOA5 were specific to the patients with HLAP and severe HTG in China, and identifying such mutations had clinical significance in elucidating the etiology and subsequent treatment.

Apolipoprotein A-IV-Deficient Mice in 129/SvJ Background Are Susceptible to Obesity and Glucose Intolerance.

Apolipoprotein A-IV (apoA-IV), synthesized by enterocytes, is potentially involved in regulating lipid absorption and metabolism, food intake, and glucose metabolism. In this study, we backcrossed apoA-IV knockout (apoA-IV-/-) mice onto the 129/SvJ background for eight generations. Compared to the wild-type (WT) mice, the 129/SvJ apoA-IV-/- mice gained more weight and exhibited delayed glucose clearance even on the chow diet. During a 16-week high-fat diet (20% by weight of fat) study, apoA-IV-/- mice were more obese than the WT mice, which was associated with their increased food intake as well as reduced energy expenditure and physical activity. In addition, apoA-IV-/- mice developed significant insulin resistance (indicated by HOMA-IR) with severe glucose intolerance even though their insulin levels were drastically higher than the WT mice. In conclusion, we have established a model of apoA-IV-/- mice onto the 129/SvJ background. Unlike in the C57BL/6J strain, apoA-IV-/- 129/SvJ mice become significantly more obese and insulin-resistant than WT mice. Our current investigations of apoA-IV in the 129/SvJ strain and our previous studies in the C57BL/6J strain underline the impact of genetic background on apoA-IV metabolic effects.

Association of Apolipoprotein A5 Gene Variants with Hyperlipidemic Acute Pancreatitis in Southeastern China.

Background: Apolipoprotein A5 (APOA5) is involved in serum triglyceride (TG) regulation. Several studies have reported that the rs651821 locus in the APOA5 gene is associated with serum TG levels in the Chinese population. However, no research has been performed regarding the association between the variants of rs651821 and the risk of hyperlipidemic acute pancreatitis (HLAP). Methods: A case-control study was conducted and is reported following the STROBE guidelines. We enrolled a total of 88 participants in this study (60 HLAP patients and 28 controls). APOA5 was genotyped using PCR and Sanger sequencing. Logistic regression models were conducted to calculate odds ratios and a 95% confidence interval. Results: The genotype distribution of the rs651821 alleles in both groups follow the Hardy-Weinberg distribution. The frequency of the "C" allele in rs651821 was increased in HLAP patients compared to controls. In the recessive model, subjects with the "CC" genotype had an 8.217-fold higher risk for HLAP (OR = 8.217, 95% CI: 1.023-66.01, p = 0.046) than subjects with the "TC+TT" genotypes. After adjusting for sex, the association remained significant (OR = 9.898, 95% CI: 1.176-83.344, p = 0.035). Additionally, the "CC" genotype was related to an increased TG/apolipoprotein B (APOB) ratio and fasting plasma glucose (FPG) levels. Conclusions: Our findings suggest that the C allele of rs651821 in APOA5 increases the risk of HLAP in persons from Southeastern China.

Association of S19W polymorphism in APOA5 gene and serum lipid levels in patients with type 2 diabetic nephropathy.

OBJECTIVES: Type 2 diabetic Mellitus (T2DM) is the most common systemic and endocrine disease in humans, and diabetic nephropathy is one of the most serious complications of this disorder. The polymorphisms in the apolipoprotein A5 (ApoA5) gene are strongly related to hypertriglyceridemia and are considered a predisposing factor for diabetic nephropathy. The current study proposed to examine the association of APOA5-S19W polymorphism with serum lipids levels in patients with type 2 diabetic nephropathy in Mazandaran province. METHODS: This case-control study was designed to determine the association of APOA5-S19W polymorphism with plasma lipid profile in 161 T2DM patients with nephropathy (DN+), without nephropathy (DN-), and in 58 healthy individuals. Lipid profile values were measured using Pars Azmoun commercial kits. S19W variant, one of the polymorphisms of the APOA5 gene, was determined by PCR-restriction fragment length polymorphism (PCR-RFLP) and Taq1 restriction enzyme. RESULTS: In comparison between the three groups, DN+ had a higher mean TG than DN- and the control group (p<0.001). The incidence of the G allele in DN+ was not significant compared to groups of DN-. Comparing the relationship between the mean of biochemical variables with CC and CG genotypes showed that the mean level of TG in people with CC genotype was increased compared to people with CG genotype in diabetic patients. However, this increase was not significant (p=0.19). CONCLUSIONS: There was no association between SNP APOA5 S19W and serum lipids in diabetic patients with and without nephropathy.

The kringle IV type 2 domain variant 4925G>A causes the elusive association signal of the LPA pentanucleotide repeat.

Lipoprotein(a) [Lp(a)] concentrations are regulated by the LPA gene mainly via the large kringle IV-type 2 (KIV-2) copy number variation and multiple causal variants. Early studies suggested an effect of long pentanucleotide repeat (PNR) alleles (10 and 11 repeats, PNR10 and PNR11) in the LPA promoter on gene transcription and found an association with lower Lp(a). Subsequent in vitro studies showed no effects on mRNA transcription, but the association with strongly decreased Lp(a) remained consistent. We investigated the isolated and combined effect of PNR10, PNR11, and the frequent splice site variant KIV-2 4925G>A on Lp(a) concentrations in the Cooperative Health Research in the Region of Augsburg F4 study by multiple quantile regression in single-SNP and joint models. Data on Lp(a), apolipoprotein(a) Western blot isoforms, and variant genotypes were available for 2,858 individuals. We found a considerable linkage disequilibrium between KIV-2 4925G>A and the alleles PNR10 and PNR11. In single-variant analysis adjusted for age, sex, and the shorter apo(a) isoform, we determined that both PNR alleles were associated with a highly significant Lp(a) decrease (PNR10: ß = -14.43 mg/dl, 95% CI: -15.84, -13.02, P = 3.33e-84; PNR11: ß = -17.21 mg/dl, 95% CI: -20.19, -14.23, P = 4.01e-29). However, a joint model, adjusting the PNR alleles additionally for 4925G>A, abolished the effect on Lp(a) (PNR10: ß = +0.44 mg/dl, 95% CI: -1.73, 2.60, P = 0.69; PNR11: ß = -1.52 mg/dl, 95% CI: -6.05, 3.00, P = 0.51). Collectively, we conclude that the previously reported Lp(a) decrease observed in pentanucleotide alleles PNR10 or PNR11 carriers results from a linkage disequilibrium with the frequent splicing mutation KIV-2 4925G>A.

Association of the APOA-5 Genetic Variant rs662799 with Metabolic Changes after an Intervention for 9 Months with a Low-Calorie Diet with a Mediterranean Profile.

In cross-sectional studies, the genetic variant rs662799 of the APOA5 gene is associated with high serum triglyceride concentrations, and in some studies, the effect of short-term dietary interventions has been evaluated. The aim of the present investigation was to evaluate the role of this genetic variant in metabolic changes after the consumption of a low-calorie diet with a Mediterranean pattern for 9 months. A population of 269 Caucasian obese patients was recruited. Adiposity and biochemical parameters were measured at the beginning (basal level) and after 3 and 9 months of the dietary intervention. The rs662799 genotype was assessed with a dominant analysis (TT vs. CT + CC). The APOA5 variant distribution was: 88.1% (n = 237) (TT), 11.5% (n = 31) (TC) and 0.4% (n = 1) (CC). There were significant differences only in triglyceride levels at all times of the study between the genotype groups. After 3 and 9 months of dietary intervention, the following parameters improved in both genotype groups: adiposity parameters, systolic pressure, total cholesterol, LDL cholesterol, leptin, adiponectin and the leptin/adiponectin ratio. The intervention significantly decreased insulin levels, HOMA-IR and triglyceride levels in non-C allele carriers (Delta 9 months TT vs. TC + CC). i.e., insulin levels (delta: -3.8 + 0.3 UI/L vs. -1.2 + 0.2 UI/L; p = 0.02), HOMA-IR levels (delta: -1.2 + 0.2 units vs. -0.3 + 0.1 units; p = 0.02), triglyceride levels (delta: -19.3 + 4.2 mg/dL vs. -4.2 + 3.0 mg/dL; p = 0.02). In conclusion, non-C allele carriers of rs662799 of the APOA5 gene showed a decrease of triglyceride, insulin and HOMA-IR levels after consuming a low-calorie diet with a Mediterranean pattern; we did not observe this effect in C allele carriers, despite a significant weight loss.

Lipoprotein(a) beyond the kringle IV repeat polymorphism: The complexity of genetic variation in the LPA gene.

High lipoprotein(a) [Lp(a)] concentrations are one of the most important genetically determined risk factors for cardiovascular disease. Lp(a) concentrations are an enigmatic trait largely controlled by one single gene (LPA) that contains a complex interplay of several genetic elements with many surprising effects discussed in this review. A hypervariable coding copy number variation (the kringle IV type-2 repeat, KIV-2) generates >40 apolipoprotein(a) protein isoforms and determines the median Lp(a) concentrations. Carriers of small isoforms with up to 22 kringle IV domains have median Lp(a) concentrations up to 5 times higher than those with large isoforms (>22 kringle IV domains). The effect of the apo(a) isoforms are, however, modified by many functional single nucleotide polymorphisms (SNPs) distributed over the complete range of allele frequencies (<0.1% to >20%) with very pronounced effects on Lp(a) concentrations. A complex interaction is present between the apo(a) isoforms and LPA SNPs, with isoforms partially masking the effect of functional SNPs and, vice versa, SNPs lowering the Lp(a) concentrations of affected isoforms. This picture is further complicated by SNP-SNP interactions, a poorly understood role of other polymorphisms such as short tandem repeats and linkage structures that are poorly captured by common R2 values. A further layer of complexity derives from recent findings that several functional SNPs are located in the KIV-2 repeat and are thus not accessible to conventional sequencing and genotyping technologies. A critical impact of the ancestry on correlation structures and baseline Lp(a) values becomes increasingly evident. This review provides a comprehensive overview on the complex genetic architecture of the Lp(a) concentrations in plasma, a field that has made tremendous progress with the introduction of new technologies. Understanding the genetics of Lp(a) might be a key to many mysteries of Lp(a) and booster new ideas on the metabolism of Lp(a) and possible interventional targets.

Apolipoprotein A-V is a potential target for treating coronary artery disease: evidence from genetic and metabolomic analyses.

Triglyceride (TG)-lowering LPL variants in combination with genetic LDL-C-lowering variants are associated with reduced risk of coronary artery disease (CAD). Genetic variation in the APOA5 gene encoding apolipoprotein A-V also strongly affects TG levels, but the potential clinical impact and underlying mechanisms are yet to be resolved. Here, we aimed to study the effects of APOA5 genetic variation on CAD risk and plasma lipoproteins through factorial genetic association analyses. Using data from 309,780 European-ancestry participants from the UK Biobank, we evaluated the effects of lower TG levels as a result of genetic variation in APOA5 and/or LPL on CAD risk with or without a background of reduced LDL-C. Next, we compared lower TG levels via APOA5 and LPL variation with over 100 lipoprotein measurements in a combined sample from the Netherlands Epidemiology of Obesity study (N = 4,838) and the Oxford Biobank (N = 6,999). We found that lower TG levels due to combined APOA5 and LPL variation and genetically-influenced lower LDL-C levels afforded the largest reduction in CAD risk (odds ratio: 0.78 (0.73-0.82)). Compared to patients with genetically-influenced lower TG via LPL, genetically-influenced lower TG via APOA5 had similar and independent, but notably larger, effects on the lipoprotein profile. Our results suggest that lower TG levels as a result of APOA5 variation have strong beneficial effects on CAD risk and the lipoprotein profile, which suggest apo A-V may be a potential novel therapeutic target for CAD prevention.

Apo(a) and ApoB Interact Noncovalently Within Hepatocytes: Implications for Regulation of Lp(a) Levels by Modulation of ApoB Secretion.

BACKGROUND: Elevated plasma Lp(a) (lipoprotein(a)) levels are associated with increased risk for atherosclerotic cardiovascular disease and aortic valve stenosis. However, the cell biology of Lp(a) biosynthesis remains poorly understood, with the locations of the noncovalent and covalent steps of Lp(a) assembly unclear and the nature of the apoB-containing particle destined for Lp(a) unknown. We, therefore, asked if apo(a) and apoB interact noncovalently within hepatocytes and if this impacts Lp(a) biosynthesis. METHODS: Using human hepatocellular carcinoma cells expressing 17K (17 kringle) apo(a), or a 17KΔLBS7,8 variant with a reduced ability to bind noncovalently to apoB, we performed coimmunoprecipitation, coimmunofluorescence, and proximity ligation assays to document intracellular apo(a):apoB interactions. We used a pulse-chase metabolic labeling approach to measure apo(a) and apoB secretion rates. RESULTS: Noncovalent complexes containing apo(a)/apoB are present in lysates from cells expressing 17K but not 17KΔLBS7,8, whereas covalent apo(a)/apoB complexes are absent from lysates. 17K and apoB colocalized intracellularly, overlapping with staining for markers of endoplasmic reticulum trans-Golgi, and early endosomes, and less so with lysosomes. The 17KΔLBS7,8 had lower colocalization with apoB. Proximity ligation assays directly documented intracellular 17K/apoB interactions, which were dramatically reduced for 17KΔLBS7,8. Treatment of cells with PCSK9 (proprotein convertase subtilisin/kexin type 9) enhanced, and lomitapide reduced, apo(a) secretion in a manner dependent on the noncovalent interaction between apo(a) and apoB. Apo(a) secretion was also reduced by siRNA-mediated knockdown of APOB. CONCLUSIONS: Our findings explain the coupling of apo(a) and Lp(a)-apoB production observed in human metabolic studies using stable isotopes as well as the ability of agents that inhibit apoB biosynthesis to lower Lp(a) levels.

Apolipoprotein A4 Defines the Villus-Crypt Border in Duodenal Specimens for Celiac Disease Morphometry.

Histological evaluation of the small intestinal mucosa is the cornerstone of celiac disease diagnostics and an important outcome in scientific studies. Gluten-dependent injury can be evaluated either with quantitative morphometry or grouped classifications. A drawback of mucosal readings is the subjective assessment of the border where the crypt epithelium changes to the differentiated villus epithelium. We studied potential immunohistochemical markers for the detection of the villus-crypt border: apolipoprotein A4 (APOA4), Ki-67, glucose transporter 2, keratin 20, cytochrome P450 3A4 and intestinal fatty-acid binding protein. Among these, villus-specific APOA4 was chosen as the best candidate for further studies. Hematoxylin-eosin (H&E)- and APOA4 stained duodenal biopsy specimens from 74 adult patients were evaluated by five observers to determine the villus-to-crypt ratio (VH : CrD). APOA4 delineated the villus to crypt epithelium transition clearly, and the correlation coefficient of VH : CrD values between APOA4 and H&E was excellent (r=0.962). The VH : CrD values were lower in APOA4 staining (p<0.001) and a conversion factor of 0.2 in VH : CrD measurements was observed to make the two methods comparable to each other. In the intraobserver analysis, the doubled standard deviations, representing the error ranges, were 0.528 for H&E and 0.388 for APOA4 staining, and the ICCs were 0.980 and 0.971, respectively. In the interobserver analysis, the average error ranges were 1.017 for H&E and 0.847 for APOA4 staining, and the ICCs were better for APOA4 than for H&E staining in all analyses. In conclusion, the reliability and reproducibility of morphometrical VH : CrD readings are improved with the use of APOA4 staining.

Augmenting and directing long-range CRISPR-mediated activation in human cells.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics.

Novel proteins associated with chronic intermittent hypoxia and obstructive sleep apnea: From rat model to clinical evidence.

OBJECTIVE: To screen for obstructive sleep apnea (OSA) biomarkers, isobaric tags for relative and absolute quantitation (iTRAQ)-labeled quantitative proteomics assay was used to identify differentially expressed proteins (DEPs) during chronic intermittent hypoxia (CIH). METHOD: The iTRAQ technique was applied to compare DEPs in the serum of a CIH rat model and control group. Biological analysis of DEPs was performed using Gene Ontology and Kyoto Encyclopedia to explore related biological functions and signaling pathways. Enzyme-linked immunosorbent assay (ELISA) was performed to validate their expression in sera from patients with OSA and CIH rats. RESULTS: Twenty-three DEPs (fold change ≥1.2 or ≤0.833, p<0.05) were identified, and two DEPs (unique peptides>3 and higher coverage) were further verified by ELISA in the CIH rat model and OSA subject: apolipoprotein A-IV (APOA4, p<0.05) and Tubulin alpha-1A chain (TUBA1A, p<0.05). Both groups showed significant differences in the expression levels of DEPs between the CIH and control groups and the severe OSA and non-OSA groups. APOA4 was found to be upregulated and TUBA1A downregulated in both the sera from OSA patients and CIH rats, on comparing proteomics results with clinical results. There were two pathways that involved three DEPs, the mitogen-activated protein kinase (MAPK) signaling pathway (p<0.05) and cytokine-cytokine receptor interaction (p<0.05). CONCLUSION: APOA4 and TUBA1A may be potential novel biomarkers for CIH and OSA, and may play an important role in the development of OSA complications.

Serum APOA4 Pharmacodynamically Represents Administered Recombinant Human Hepatocyte Growth Factor (E3112).

BACKGROUND: Hepatocyte growth factor (HGF) is an endogenously induced bioactive molecule that has strong anti-apoptotic and tissue repair activities. In this research, we identified APOA4 as a novel pharmacodynamic (PD) marker of the recombinant human HGF (rh-HGF), E3112. METHODS: rh-HGF was administered to mice, and their livers were investigated for the PD marker. Candidates were identified from soluble proteins and validated by using human hepatocytes in vitro and an animal disease model in vivo, in which its c-Met dependency was also ensured. RESULTS: Among the genes induced or highly enhanced after rh-HGF exposure in vivo, a soluble apolipoprotein, Apoa4, was found to be induced by rh-HGF in the murine liver. By using primary cultured human hepatocytes, the significant induction of human APOA4 was observed at the mRNA and protein levels, and it was inhibited in the presence of a c-Met inhibitor. Although mice constitutively expressed Apoa4 mRNA in the small intestine and the liver, the liver was the primary organ affected by administered rh-HGF to strongly induce APOA4 in a dose- and c-Met-dependent manner. Serum APOA4 levels were increased after rh-HGF administration, not only in normal mice but also in anti-Fas-induced murine acute liver failure (ALF), which confirmed the pharmacodynamic nature of APOA4. CONCLUSIONS: APOA4 was identified as a soluble PD marker of rh-HGF with c-Met dependency. It should be worthwhile to clinically validate its utility through clinical trials with healthy subjects and ALF patients.

A systemic apolipoprotein A-IV-associated amyloidosis confirmed by proteome analysis.

Amyloidosis is induced by extracellular deposition of certain proteins. Thirty-six proteins have so far been identified as amyloidogenic proteins in humans. Although it is very important to determine the specific amyloid protein type for the choice of therapy for amyloidosis patient, it might be difficult to identify specific proteins from amyloid-deposited tissue. Apolipoprotein A-IV is known as an amyloid-associated protein, but there have been few reports of apolipoprotein A-IV amyloidosis. Here we report a case of systemic apolipoprotein A-IV-associated amyloidosis that was confirmed by proteome analysis using formalin-fixed paraffin-embedded tissue and an immunohistochemical technique.

Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice.

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM's absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes.

Depression Augments Plasma APOA4 without Changes of Plasma Lipids and Glucose in Female Adolescents Carrying G Allele of APOA4 rs5104.

The association of apolipoprotein AIV (APOA4) with depression or plasma levels of lipids and glucose has been inconsistently reported. However, interplays between APOA4 and depression on the levels have not been explored yet. The present study aimed to investigate plasma levels of APOA4, lipids, and glucose in adolescents with different genotypes of APOA4 rs5104 and with or without depression. Depressive symptoms were assessed in 631 adolescents by Beck Depression Inventory (BDI). A total score of 14 was defined as the cutoff point for depression. Plasma levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), glucose, and insulin were measured by routine methods, and APOA4 by enzyme-linked immunosorbent assays. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism analyses and verified by DNA sequencing. Female adolescents had higher prevalence of depression than male subjects only in G allele carriers (p = 0.015), but not in AA homozygotes. Risk factors of depression and predictors of depression severity were different between G allele carriers and AA homozygotes. Lower levels of glucose (p = 0.003) were observed in male G allele carriers than those in male AA homozygotes and increased TG levels (p = 0.008) in female G allele carriers when compared with those in female AA homozygotes. When both APOA4 rs5104 and depression were taken into account, subjects with depression had higher levels of plasma APOA4 than adolescents without depression only in female G allele carriers (p = 0.043), but no significant changes of plasma lipids and glucose. Depression augments plasma APOA4 levels without changes of plasma lipids and glucose in female adolescents carrying G allele of APOA4 rs5104. These results may provide a novel explanation for the inconsistent relationship between depression, APOA4, and plasma levels of lipids and glucose in the literature.

Apolipoprotein A4 regulates the immune response in carbon tetrachloride-induced chronic liver injury in mice.

This article explores the role of ApoA4 in a CCl4-induced chronic liver injury (CLI) mouse model. C57BL/6J mice (WT) and ApoA4 knock-out (KO) mice were divided into CCl4 CLI (WT-CCl4 and KO-CCl4) and olive oil solvent control groups (WT-Veh and KO-Veh). Some of the KO-CCl4 mice were additionally treated with recombinant mouse ApoA4 and primary mouse T lymphocyte injections. After 6 weeks, histological analyses, biochemical and superoxide dismutase (SOD) and malondialdehyde (MDA) assays, flow cytometry of immune cells and qRT-PCR analyses were performed. KO mice after treatment with CCl4 showed reduced hepatic SOD and enhanced serum MDA activities leading to worsening liver injury and fibrosis compared with WT-CCl4, accompanied by enhanced hepatic alpha smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinases-1 (TIMP-1) and collagen type I alpha 1 chain (COL1A1) transcriptions, elevated macrophage M1 levels, enhanced tumor necrosis factor-alpha (TNF-α), Interleukin 6 (IL-6) and C-C Motif Chemokine Ligand 5 (CCL5), but reduced Interleukin 10 (IL-10), monocyte chemotactic protein 1 (MCP-1), C-C Motif Chemokine Receptor 2 (CCR2), C-X3-C Motif Chemokine Receptor 1 (CX3CR1) and C-X-C Motif Chemokine Ligand 9 (CXCL9) transcription, as well as reduced CD3+, CD4+ and CD8+ T cell percentages in hepatic tissue, blood cells and spleen. In addition, CD11b+CD115+, CD11b+/Ly6Chigh, CD11b+/LyC6- and CD11b+/Ly6Cint cells were enhanced, which partly reversed by ApoA4 protein and T cell injections. In conclusion, we propose that ApoA4 might be involved in liver protection via inhibiting fibrotic mediators and inflammatory cytokines, suppression of pro-inflammatory hepatic M1 cell invasion and regulation of CD8+ T and CD4+ T lymphocytes.

Hepatoprotective effect of apolipoprotein A4 against carbon tetrachloride induced acute liver injury through mediating hepatic antioxidant and inflammation response in mice.

Apolipoprotein A4 (ApoA4) regulates lipid and glucose metabolism and exerts anti-inflammatory effects in atherogenesis and colitis. The present study explored the presumed protective role of ApoA4 in carbon tetrachloride (CCl4)-induced acute liver injury (ALI) in mice. The ALI model in wild type (WT), ApoA4 knock-out (ApoA4-KO) and ApoA4 transgenic (ApoA4-TG) mice was induced by a single intraperitoneal administration of CCl4. Liver and blood were harvested from mice to assess liver functions, immunohistological changes, immune cell populations and cytokine profiles. ApoA4 deficiency aggravated, and ApoA4 overexpression alleviated CCl4-inflicted liver damage by controlling levels of anti-oxidant enzymes. ApoA4 deletion increased the recruitment of monocytes/macrophages into the injured liver and upregulated the plasma levels of IL-6, TNF-α and MCP-1, but lower IL-10 and IFN-γ. ApoA4 over-expression rescued this effect and resulted in lower percentages of monocytes/macrophages and dendritic cells, the ratio of blood pro-inflammatory to anti-inflammatory monocytes and reduced plasma concentrations of IL-6, but enhanced IL-10 and IFN-γ. We propose ApoA4 as a potential new therapeutic target for the management of liver damage.

Identification and characterization of a rare variant in apolipoprotein A-IV, p.(V336M), and evaluation of HDL functionality in a Greek cohort with extreme HDL cholesterol levels.

High-Density Lipoprotein cholesterol (HDL-C) levels do not correlate well with Coronary Artery Disease (CAD) risk, while HDL functionality affects atherogenesis and is a better prognostic marker for CAD. Often, the extreme HDL-C levels have a multigenic origin. Here, we searched for single-nucleotide polymorphisms (SNPs) in ten genes of HDL metabolism in a Greek cohort with very low (<10th percentile, n = 13) or very high (>90th percentile, n = 21) HDL-C. We also evaluated the association between HDL-C levels, HDL functionality (anti-oxidant capacity) and CAD in the subjects of this cohort. Individuals with low HDL-C levels had higher triglyceride levels, lower apoA-I levels, decreased HDL anti-oxidant capacity and higher incidence of CAD compared with individuals with control or high HDL-C levels. With next generation sequencing we identified 18 exonic SNPs in 6 genes of HDL metabolism and for selected amino acid changes we performed computer-aided structural analysis and modeling. A previously uncharacterized rare apolipoprotein A-IV variant, apoA-IV [V336M], present in a subject with low HDL-C (14 mg/dL) and CAD, was expressed in recombinant form and structurally and functionally characterized. ApoA-IV [V336M] had similar α-helical content to WT apoA-IV but displayed a small thermodynamic stabilization by chemical unfolding analysis. ApoA-IV [V336M] was able to associate with phospholipids but presented reduced kinetics compared to WT apoA-IV. Overall, we identified a rare apoA-IV variant in a subject with low HDL levels and CAD with altered biophysical and phospholipid binding properties and showed that subjects with very low HDL-C presented with HDL dysfunction and higher incidence of CAD in a Greek cohort.

Quantifying apolipoprotein(a) in the era of proteoforms and precision medicine.

Lipoprotein(a) (Lp(a)) is an independent risk factor in the development of atherosclerotic cardiovascular diseases (ASCVD) and calcific aortic valve disease (CAVD). Lp(a) is an LDL-like particle to which apolipoprotein (a) (apo(a)) is covalently bound. Apo(a) contains a variable number of kringle IV repeats, a kringle V and a protease domain. Serum/plasma Lp(a) concentrations are traditionally expressed as total particle mass in mg/L. Concern has arisen lately as flawed Lp(a) mass tests have masked its clinical utility. The determinants of variability in Lp(a) composition were investigated, including the apo(a) size polymorphism, post-translational modifications -N- and O-glycosylation- and the lipid:protein ratio. Depending on the number of kringle IV-2 repeats, the theoretical protein content of the Lp(a) particle varies between 30 and 46 (w/w) %, which inescapably confounds Lp(a) mass measurements. The authors advocate that reporting of Lp(a) particle concentrations in mass units is metrologically inappropriate and should be abandoned, as it results in systematically biased Lp(a) results. Enabling technology, such as mass spectrometry, allows unequivocal molecular characterization of the apo(a) measurand(s) and accurate quantitation of apo(a) in molar units, unaffected by apo(a) size polymorphism. To guarantee that Lp(a)/apo(a) tests are fit-for-clinical-purpose, basic metrology principles should be implemented upfront during test development.