Stela Z. Berisha

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.

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.

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.

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.