HDL Improves Cholesterol and Glucose Homeostasis and Reduces Atherosclerosis in Diabetes-Associated Atherosclerosis.

BACKGROUND AND AIMS: Apolipoprotein A-I (ApoA-I), the main component of high-density lipoprotein (HDL), not only promotes reverse cholesterol transport (RCT) in atherosclerosis but also increases insulin secretion in pancreatic ß-cells, suggesting that interventions which raise HDL levels may be beneficial in diabetes-associated cardiovascular disease (CVD). Previously, we showed that TNF-related apoptosis-inducing ligand (TRAIL) deletion in Apolipoprotein Eknockout (Apoe-/- ) mice results in diabetes-accelerated atherosclerosis in response to a "Western" diet. Here, we sought to identify whether reconstituted HDL (rHDL) could improve features of diabetes-associated CVD in Trail-/-Apoe-/- mice. METHODS AND RESULTS: Trail-/-Apoe-/- and Apoe-/- mice on a "Western" diet for 12 weeks received 3 weekly infusions of either PBS (vehicle) or rHDL (containing ApoA-I (20 mg/kg) and 1-palmitoyl-2-linoleoyl phosphatidylcholine). Administration of rHDL reduced total plasma cholesterol, triglyceride, and glucose levels in Trail-/-Apoe-/- but not in Apoe-/- mice, with no change in weight gain observed. rHDL treatment also improved glucose clearance in response to insulin and glucose tolerance tests. Immunohistological analysis of pancreata revealed increased insulin expression/production and a reduction in macrophage infiltration in mice with TRAIL deletion. Furthermore, atherosclerotic plaque size in Trail-/-Apoe-/- mice was significantly reduced associating with increased expression of the M2 macrophage marker CD206, suggesting HDL's involvement in the polarization of macrophages. rHDL also increased vascular mRNA expression of RCT transporters, ABCA1 and ABCG1, in Trail-/-Apoe-/- but not in Apoe-/- mice. Conclusions. rHDL improves features of diabetes-associated atherosclerosis in mice. These findings support the therapeutic potential of rHDL in the treatment of atherosclerosis and associated diabetic complications. More studies are warranted to understand rHDL's mechanism of action.

Vascular transcriptome landscape of Trail-/- mice: Implications and therapeutic strategies for diabetic vascular disease.

Circulating plasma TRAIL levels are suppressed in patients with cardiovascular and diabetic diseases. To identify novel targets in vascular metabolic diseases, genome-wide transcriptome of aortic tissue from Trail-/- versus Trail+/+ mice were interrogated. We found 861 genes differentially expressed with TRAIL deletion. Gene enrichment analyses showed many of these genes were related to inflammation, cell-to-cell cytoskeletal interactions, and transcriptional modulation. We identified vascular protective and pathological gene clusters, with Ifi205 as the most significantly reduced vascular protective gene, whereas Glut1, the most significantly increased pathological gene with TRAIL deletion. We hypothesized that therapeutic targets could be devised from such integrated analysis and validated our findings from vascular tissues of diabetic mice. From the differentially expressed gene targets, enriched transcription factor (TF) and microRNA binding motifs were identified. The top two TFs were Elk1 and Sp1, with enrichment to eight gene targets common to both. miR-520d-3p and miR-377-3p were the top enriched microRNAs with TRAIL deletion; with four overlapping genes enriched for both microRNAs. Our findings offer an alternate in silico approach for therapeutic target identification and present a deeper understanding of gene signatures and pathways altered with TRAIL suppression in the vasculature.

TRAIL-Expressing Monocyte/Macrophages Are Critical for Reducing Inflammation and Atherosclerosis.

Circulating tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) levels are reduced in patients with cardiovascular disease, and TRAIL gene deletion in mice exacerbates atherosclerosis and inflammation. How TRAIL protects against atherosclerosis and why levels are reduced in disease is unknown. Here, multiple strategies were used to identify the protective source of TRAIL and its mechanism(s) of action. Samples from patients with coronary artery disease and bone-marrow transplantation experiments in mice lacking TRAIL revealed monocytes/macrophages as the main protective source. Accordingly, deletion of TRAIL caused a more inflammatory macrophage with reduced migration, displaying impaired reverse cholesterol efflux and efferocytosis. Furthermore, interleukin (IL)-18, commonly increased in plasma of patients with cardiovascular disease, negatively regulated TRAIL transcription and gene expression, revealing an IL-18-TRAIL axis. These findings demonstrate that TRAIL is protective of atherosclerosis by modulating monocyte/macrophage phenotype and function. Manipulating TRAIL levels in these cells highlights a different therapeutic avenue in the treatment of cardiovascular disease.

Non-alcoholic fatty liver disease, vascular inflammation and insulin resistance are exacerbated by TRAIL deletion in mice.

Non-alcoholic fatty liver disease (NAFLD) incorporates steatosis, non-alcoholic steato-hepatitis (NASH) and liver cirrhosis, associating with diabetes and cardiovascular disease (CVD). TNF-related apoptosis-inducing ligand (TRAIL) is protective of CVD. We aimed to determine whether TRAIL protects against insulin resistance, NAFLD and vascular injury. Twelve-week high fat diet (HFD)-fed Trail -/- mice had increased plasma cholesterol, insulin and glucose compared to wildtype. Insulin tolerance was impaired with TRAIL-deletion, with reduced p-Akt, GLUT4 expression and glucose uptake in skeletal muscle. Hepatic triglyceride content, inflammation and fibrosis were increased with TRAIL-deletion, with elevated expression of genes regulating lipogenesis and gluconeogenesis. Moreover, Trail -/- mice exhibited reduced aortic vasorelaxation, impaired insulin signaling, and >20-fold increased mRNA expression for IL-1ß, IL-6, and TNF-α. In vitro, palmitate treatment of hepatocytes increased lipid accumulation, inflammation and fibrosis, with TRAIL mRNA significantly reduced. TRAIL administration inhibited palmitate-induced hepatocyte lipid uptake. Finally, patients with NASH had significantly reduced plasma TRAIL compared to control, simple steatosis or obese individuals. These findings suggest that TRAIL protects against insulin resistance, NAFLD and vascular inflammation. Increasing TRAIL levels may be an attractive therapeutic strategy, to reduce features of diabetes, as well as liver and vascular injury, so commonly observed in individuals with NAFLD.

Comparative Evaluation of TRAIL, FGF-2 and VEGF-A-Induced Angiogenesis In Vitro and In Vivo.

Tumor necrosis-factor-related apoptosis-inducing ligand (TRAIL) has been implicated in angiogenesis; the growth of new blood vessels from an existing vessel bed. Our aim was to compare pro-angiogenic responses of TRAIL, vascular endothelial growth-factor-A (VEGF-A) and fibroblast growth-factor-2 (FGF-2) either separately (10 ng/mL) or in combination, followed by the assessment of proliferation, migration and tubule formation using human microvascular endothelial-1 (HMEC-1) cells in vitro. Angiogenesis was also measured in vivo using the Matrigel plug assay. TRAIL and FGF-2 significantly augmented HMEC-1 cell proliferation and migration, with combination treatment having an enhanced effect on cell migration only. In contrast, VEGF-A did not stimulate HMEC-1 migration at 10 ng/mL. Tubule formation was induced by all three factors, with TRAIL more effective compared to VEGF-A, but not FGF-2. TRAIL at 400 ng/mL, but not VEGF-A, promoted CD31-positive staining into the Matrigel plug. However, FGF-2 was superior, stimulating cell infiltration and angiogenesis better than TRAIL and VEGF-A in vivo. These findings demonstrate that each growth factor is more effective at different processes of angiogenesis in vitro and in vivo. Understanding how these molecules stimulate different processes relating to angiogenesis may help identify new strategies and treatments aimed at inhibiting or promoting dysregulated angiogenesis in people.

Insulin promotes vascular smooth muscle cell proliferation and apoptosis via differential regulation of tumor necrosis factor-related apoptosis-inducing ligand.

BACKGROUND: Insulin regulates glucose homeostasis but can also promote vascular smooth muscle (VSMC) proliferation, important in atherogenesis. Recently, we showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) stimulates intimal thickening via accelerated growth of VSMCs. The aim of the present study was to determine whether insulin-induced effects on VSMCs occur via TRAIL. METHODS: Expression of TRAIL and TRAIL receptor in response to insulin and glucose was determined by polymerase chain reaction. Transcriptional activity was assessed using wild-type and site-specific mutations of the TRAIL promoter. Chromatin immunoprecipitation studies were performed. VSMC proliferation and apoptosis was measured. RESULTS: Insulin and glucose exposure to VSMC for 24 h stimulated TRAIL mRNA expression. This was also evident at the transcriptional level. Both insulin- and glucose-inducible TRAIL transcriptional activity was blocked by dominant-negative specificity protein-1 (Sp1) overexpression. There are five functional Sp1-binding elements (Sp1-1, Sp1-2, Sp-5/6 and Sp1-7) on the TRAIL promoter. Insulin required the Sp1-1 and Sp1-2 sites, but glucose needed all Sp1-binding sites to induce transcription. Furthermore, insulin (but not glucose) was able to promote VSMC proliferation over time, associated with increased decoy receptor-2 (DcR2) expression. In contrast, chronic 5-day exposure of VSMC to 1 µg/mL insulin repressed TRAIL and DcR2 expression, and reduced Sp1 enrichment on the TRAIL promoter. This was associated with increased cell death. CONCLUSIONS: The findings of the present study provide a new mechanistic insight into how TRAIL is regulated by insulin. This may have significant implications at different stages of diabetes-associated cardiovascular disease. Thus, TRAIL may offer a novel therapeutic solution to combat insulin-induced vascular pathologies.