Oxidant resistant human apolipoprotein A-I functions similarly to the unmodified human isoform in delaying atherosclerosis progression and promoting atherosclerosis regression in hyperlipidemic mice.

BACKGROUND: Transgenic overexpression of apolipoprotein A-I (apoA1) has been shown to delay atherosclerosis lesion progression and promote lesion regression in mouse models; however, apoA1 is subject to oxidation by myeloperoxidase (MPO) and loss of function. The activity of oxidant resistant human apoA1 was compared to unmodified human apoA1 in mouse models of atherosclerosis progression and regression. METHODS AND RESULTS: Human apoA1 and the MPO oxidant resistant 4WF isoform transgenic mice were bred to LDL receptor deficient (LDLr KO) mice and fed a western-type diet. High level expression of these human apoA1 isoforms did not lead to increased HDL-cholesterol levels on the LDLr KO background. In males and females, lesion progression was studied over time, and both apoA1 and 4WF transgenic mice vs. LDLr KO mice had significant and similar delayed lesion progression and reduced non-HDL cholesterol. Using time points with equivalent lesion areas, lesion regression was initiated by feeding the mice a low-fat control diet containing a microsomal triglyceride transfer protein inhibitor for 7 weeks. Lesions regressed more in the male apoA1 and 4WF transgenics vs. the LDLr KO, but the 4WF isoform was not superior to the unmodified isoform in promoting lesion regression. CONCLUSIONS: Both human apoA1 and the 4WF MPO oxidant resistant apoA1 isoform delayed lesion progression and promoted lesion regression in LDLr KO mice, with more pronounced effects in males than females; moreover, the 4WF isoform functioned similarly to the unmodified human apoA1 isoform.

Soat1 mediates the mouse strain effects on cholesterol loading-induced endoplasmic reticulum stress and CHOP expression in macrophages.

We previously demonstrated that AKR vs. DBA/2 mouse bone marrow derived macrophages have higher levels of free cholesterol and lower levels of esterified cholesterol after cholesterol loading, and that AKR, but not DBA/2, macrophages induced C/EBP homologous protein (CHOP) expression after cholesterol loading. We earlier determined that the free and esterified cholesterol level effect is due to a truncation in the sterol O-acyltransferase 1 (Soat1) gene, encoding acetyl-coenzyme A acetyltransferase 1 (ACAT1). Here we examined the mechanism for the differential induction of CHOP by cholesterol loading. CHOP was induced in both strains after incubation with tunicamycin, indicating both strains have competent endoplasmic reticulum stress pathways. CHOP was induced when DBA/2 macrophages were cholesterol loaded in the presence of an ACAT inhibitor, indicating that the difference in free cholesterol levels were responsible for this strain effect. This finding was confirmed in macrophages derived from DBA/2 embryonic stem cells. Cholesterol loading of Soat1 gene edited cells, mimicking the AKR allele, led to increased free cholesterol levels and restored CHOP induction. The upstream pathway of free cholesterol induced endoplasmic reticulum stress was investigated; and, RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1 α protein kinase (IRE1α) pathways were required for maximal CHOP expression.

Cytogenetic and molecular diagnostic testing associated with prenatal and postnatal birth defects.

Genetic testing is beneficial for patients and providers when in search of answers to medical problems related to the prenatal or early postnatal period. It can help to identify the cause or confirm a diagnosis associated with developmental delay, intellectual disability, dysmorphic features, heart defects, multiple malformations, short stature, stillbirth, neonatal death, or fertility problems. Genetic testing can be used to rule out single-gene or chromosome abnormalities. Different diagnostic cytogenetic and molecular genetic techniques are applied in clinical genetics laboratories, from conventional ones to the state of the art chromosomal microarrays and next-generation sequencing. Each of the genetic techniques or methods has its strengths and limitations, however different methods complement each-other in trying to identify the genetic variation(s) responsible for a medical condition, especially the ones related to birth defects.

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function.

HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo. Male mice had significantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol efflux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially (∼50%) protected from MPO-mediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice.

An abundant dysfunctional apolipoprotein A1 in human atheroma.

Recent studies have indicated that high-density lipoproteins (HDLs) and their major structural protein, apolipoprotein A1 (apoA1), recovered from human atheroma are dysfunctional and are extensively oxidized by myeloperoxidase (MPO). In vitro oxidation of either apoA1 or HDL particles by MPO impairs their cholesterol acceptor function. Here, using phage display affinity maturation, we developed a high-affinity monoclonal antibody that specifically recognizes both apoA1 and HDL that have been modified by the MPO-H2O2-Cl(-) system. An oxindolyl alanine (2-OH-Trp) moiety at Trp72 of apoA1 is the immunogenic epitope. Mutagenesis studies confirmed a critical role for apoA1 Trp72 in MPO-mediated inhibition of the ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. ApoA1 containing a 2-OH-Trp72 group (oxTrp72-apoA1) is in low abundance within the circulation but accounts for 20% of the apoA1 in atherosclerosis-laden arteries. OxTrp72-apoA1 recovered from human atheroma or plasma is lipid poor, virtually devoid of cholesterol acceptor activity and demonstrated both a potent proinflammatory activity on endothelial cells and an impaired HDL biogenesis activity in vivo. Elevated oxTrp72-apoA1 levels in subjects presenting to a cardiology clinic (n = 627) were associated with increased cardiovascular disease risk. Circulating oxTrp72-apoA1 levels may serve as a way to monitor a proatherogenic process in the artery wall.

Effects of native and myeloperoxidase-modified apolipoprotein a-I on reverse cholesterol transport and atherosclerosis in mice.

OBJECTIVE: Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques. APPROACH AND RESULTS: ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques. CONCLUSIONS: Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Transcriptome analysis of genes regulated by cholesterol loading in two strains of mouse macrophages associates lysosome pathway and ER stress response with atherosclerosis susceptibility.

Cholesterol loaded macrophages in the arterial intima are the earliest histological evidence of atherosclerosis. Studies of mouse models of atherosclerosis have shown that the strain background can have a significant effect on lesion development. We have previously shown that DBA/2 ApoE(-/-) mice have aortic root lesions 10-fold larger than AKR ApoE(-/-) mice. The current study analyzes the response to cholesterol loading of macrophages from these two strains. Macrophages from the atherosclerosis susceptible DBA/2 strain had significantly higher levels of total and esterified cholesterol compared to atherosclerosis resistant AKR macrophages, while free cholesterol levels were higher in AKR cells. Gene expression profiles were obtained and data were analyzed for strain, cholesterol loading, and strain-cholesterol loading interaction effects by a fitted linear model. Pathway and transcriptional motif enrichment were identified by gene set enrichment analysis. In addition to observed strain differences in basal gene expression, we identified many transcripts whose expression was significantly altered in response to cholesterol loading, including P2ry13 and P2ry14, Trib3, Hyal1, Vegfa, Ccr5, Ly6a, and Ifit3. Eight pathways were significantly enriched in transcripts regulated by cholesterol loading, among which the lysosome and cytokine-cytokine receptor interaction pathways had the highest number of significantly regulated transcripts. Of the differentially regulated transcripts with a strain-cholesterol loading interaction effect, we identified three genes known to participate in the endoplasmic reticulum (ER) stress response, Ddit3, Trib3 and Atf4. These three transcripts were highly up-regulated by cholesterol in AKR and either down-regulated or unchanged in loaded DBA/2 macrophages, thus associating a robust ER stress response with atherosclerosis resistance. We identified significant transcripts with strain, loading, or strain-loading interaction effect that reside within previously described quantitative trait loci as atherosclerosis modifier candidate genes. In conclusion, we characterized several strain and cholesterol induced differences that may lead to new insights into cellular cholesterol metabolism and atherosclerosis.

Red blood cells play a role in reverse cholesterol transport.

OBJECTIVE: Reverse cholesterol transport (RCT) involves the removal of cholesterol from peripheral tissue for excretion in the feces. Here, we determined whether red blood cells (RBCs) can contribute to RCT. METHODS AND RESULTS: We performed a series of studies in apolipoprotein AI-deficient mice where the high-density lipoprotein-mediated pathway of RCT is greatly diminished. RBCs carried a higher fraction of whole blood cholesterol than plasma in apolipoprotein AI-deficient mice, and as least as much of the labeled cholesterol derived from injected foam cells appeared in RBCs compared with plasma. To determine whether RBCs mediate RCT to the fecal compartment, we measured RCT in anemic and control apolipoprotein AI-deficient mice and found that anemia decreased RCT to the feces by over 35% after correcting for fecal mass. Transfusion of [(3)H]cholesterol-labeled RBCs led to robust delivery of the labeled cholesterol to the feces in apolipoprotein AI-deficient hosts. In wild-type mice, the majority of the blood cholesterol mass, as well as [(3)H]cholesterol derived from the injected foam cells, was found in plasma, and anemia did not significantly alter RCT to the feces after correction for fecal mass. CONCLUSIONS: The RBC cholesterol pool is dynamic and facilitates RCT of peripheral cholesterol to the feces, particularly in the low high-density lipoprotein state.

Changes in whole blood gene expression in obese subjects with type 2 diabetes following bariatric surgery: a pilot study.

BACKGROUND: A pilot study was performed in order to investigate the effects of bariatric surgery on whole blood gene expression profiles in obese subjects with type 2 diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Whole blood from eleven obese subjects with type 2 diabetes was collected in PAXgene tubes prior to and 6-12 months after bariatric surgery. Total RNA was isolated, amplified, labeled and hybridized to Illumina gene expression microarrays. Clinical and expression data were analyzed using a paired t-test, and correlations between changes in clinical trait and transcript levels were calculated. Pathways were identified using Ingenuity Pathway Analysis and DAVID gene ontology software. Overall, bariatric surgery resulted in significant reduction of body mass index, fasting plasma glucose, fasting plasma insulin, and normalization of glycosylated hemoglobin levels. The expression levels of 204 transcripts, representing 200 unique genes, were significantly altered after bariatric surgery. Among the significantly regulated genes were GGT1, CAMP, DEFA1, LCN2, TP53, PDSS1, OLR1, CNTNAP5, DHCR24, HHAT and SARDH, which have been previously implicated in lipid metabolism, obesity and/or type 2 diabetes. Selected findings were replicated by quantitative real-time-PCR. The changes in expression of seven transcripts, WDR35, FLF45244, DHCR24, TIGD7, TOPBP1, TSHZ1, and FAM8A1 were strongly correlated with the changes in body weight, fasting plasma glucose and glycosylated hemoglobin content. The top pathways associated with gene expression changes after bariatric surgery was lipid metabolism, small molecule biochemistry and gene expression. Two antimicrobial peptides were among the transcripts with the largest changes in gene expression after bariatric surgery. CONCLUSIONS/SIGNIFICANCE: Data from this pilot study suggest that whole blood expression levels of specific transcripts may be useful as biomarkers associated with susceptibility for type 2 diabetes and/or therapeutic response.

Zymosan-mediated inflammation impairs in vivo reverse cholesterol transport.

Inflammation has been proposed to impair HDL function and reverse cholesterol transport (RCT). We investigated the effects of inflammation mediated by zymosan, a yeast glucan, on multiple steps along the RCT pathway in vivo and ex vivo. Acute inflammation with 70 mg/kg zymosan impaired RCT to plasma, liver, and feces similarly by 17-22% (P < 0.05), with no additional block at the liver. Hepatic gene expression further demonstrated no change in ABCG5, ABCB4, and ABCB11 expression but a decline in ABCG8 mRNA (32% P < 0.05). Plasma from zymosan-treated mice had a 21% decrease in cholesterol acceptor ability (P < 0.01) and a 35% decrease in ABCA1-specific efflux capacity (P < 0.01) in vitro. Zymosan treatment also decreased HDL levels and led to HDL remodeling with increased incorporation of serum amyloid A. In addition, cholesterol efflux from cultured macrophages declined with zymosan treatment in a dose dependent manner. Taken together, our results suggest that zymosan impairs in vivo RCT primarily by decreasing macrophage-derived cholesterol entering the plasma, with minimal additional blocks downstream. Our study supports the notion that RCT impairment is one of the mechanisms for the increased atherosclerotic burden observed in inflammatory conditions.

Combining genome-wide data from humans and animal models of dyslipidemia and atherosclerosis.

PURPOSE OF REVIEW: Comparative genomics allows researchers to combine genome-wide association data from humans with studies in animal models in order to assist in the identification of the genes and the genetic variants that modify susceptibility to dyslipidemia and atherosclerosis. RECENT FINDINGS: Association and linkage studies in human and rodent species have been successful in identifying genetic loci associated with complex traits, but have been less robust in identifying and validating the responsible gene and/or genetic variants. Recent technological advancements have assisted in the development of comparative genomic approaches, which rely on the combination of human and rodent datasets and bioinformatics tools, followed by the narrowing of concordant loci and improved identification of candidate genes and genetic variants. Additionally, candidate genes and genetic variants identified by these methods have been further validated and functionally investigated in animal models, a process that is not feasible in humans. SUMMARY: Comparative genomic approaches have led to the identification and validation of several new genes, including a few not previously implicated, as modifiers of plasma lipid levels and atherosclerosis, yielding new insights into the biological mechanisms of these complex traits.