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Reversal of ischemia is mediated by neo-angiogenesis requiring endothelial cell (EC) and pericyte interactions to form stable microvascular networks. We describe an unrecognized role for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in potentiating neo-angiogenesis and vessel stabilization. We show that the endothelium is a major source of TRAIL in the healthy circulation compromised in peripheral artery disease (PAD). EC deletion of TRAIL in vivo or in vitro inhibited neo-angiogenesis, pericyte recruitment, and vessel stabilization, resulting in reduced lower-limb blood perfusion with ischemia. Activation of the TRAIL receptor (TRAIL-R) restored blood perfusion and stable blood vessel networks in mice. Proof-of-concept studies showed that Conatumumab, an agonistic TRAIL-R2 antibody, promoted vascular sprouts from explanted patient arteries. Single-cell RNA sequencing revealed heparin-binding EGF-like growth factor in mediating EC-pericyte communications dependent on TRAIL. These studies highlight unique TRAIL-dependent mechanisms mediating neo-angiogenesis and vessel stabilization and the potential of repurposing TRAIL-R2 agonists to stimulate stable and functional microvessel networks to treat ischemia in PAD.

The risk-factor-based prediction of atherosclerotic coronary artery disease (CAD) remains suboptimal, particularly in the absence of any of the standard modifiable cardiovascular risk factors (SMuRFs), making the discovery of biomarkers that correlate with atherosclerosis burden critically important. We hypothesized that cytokines and receptors associated with inflammation in CAD-tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interleukin-18 (IL-18), and osteoprotegerin (OPG)-would be independently associated with CAD. To determine this, we measured the serum biomarker levels of 993 participants from the BioHEART study who had CT coronary angiograms that were scored for severity of stenosis and plaque composition. We found that the quartiles of TRAIL, OPG, and IL-18 were significantly associated with disease scores, and that the IL-18/TRAIL and OPG/TRAIL ratios demonstrated significant differences between no CAD vs. STEMI whereas only the OPG/TRAIL ratio showed differences between no CAD and obstructive CAD (stenosis > 50%). However, these associations did not persist after adjustment for age, sex, SMuRFs, and a family history of CAD. In conclusion, TRAIL, IL-18, and OPG and the derived ratios of IL-18/TRAIL and OPG/TRAIL demonstrate significant associations with raw disease scores and risk factors, but these markers are not discriminatory biomarkers for the prediction of CAD when incorporated into multi-variable risk models.

Peripheral artery disease (PAD) is caused by blocked arteries due to atherosclerosis and/or thrombosis which reduce blood flow to the lower limbs. It results in major morbidity, including ischemic limb, claudication, and amputation, with patients also suffering a heightened risk of heart attack, stroke, and death. Recent studies suggest women have a higher prevalence of PAD than men, and with worse outcomes after intervention. In addition to a potential unconscious bias faced by women with PAD in the health system, with underdiagnosis, and lower rates of guideline-based therapy, fundamental biological differences between men and women may be important. In this review, we highlight sexual dimorphisms in endothelial cell functions and how they may impact PAD pathophysiology in women. Understanding sex-specific mechanisms in PAD is essential for the development of new therapies and personalized care for patients with PAD.

TNF-related apoptosis-inducing ligand (TRAIL) was originally discovered, almost 20 years ago, for its ability to kill cancer cells. More recent evidence has described pleiotropic functions, particularly in the cardiovascular system. There is potential for TRAIL concentrations in the circulation to act as prognostic and/or diagnostic factors for cardiovascular diseases (CVD). Pre-clinical studies also describe the therapeutic capacity for TRAIL signals, particularly in the context of atherosclerotic disease and diseases of the myocardium. Because diabetes mellitus significantly contributes to the progression and pathogenesis of CVDs, in this review we highlight recent evidence for the prognostic, diagnostic, and therapeutic potential of TRAIL signals in CVDs, and where relevant, the impact of diabetes mellitus. A greater understanding of how TRAIL signals regulate cardiovascular protection and pathology may offer new diagnostic and therapeutic avenues for patients suffering from CVDs.

Peripheral artery disease (PAD) is caused by occluded or narrowed arteries that reduce blood flow to the lower limbs. The treatment focuses on lifestyle changes, management of modifiable risk factors and vascular surgery. In this review we focus on how Endothelial Cell (EC) dysfunction contributes to PAD pathophysiology and describe the largely untapped potential of correcting endothelial dysfunction. Moreover, we describe current treatments and clinical trials which improve EC dysfunction and offer insights into where future research efforts could be made. Endothelial dysfunction could represent a target for PAD therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kavurma, Bursill, Stanley, Passam, Cartland, Patel, Loa, Figtree, Golledge, Aitken and Robinson.)

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 β -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 E knockout ( 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., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Belinda A. Di Bartolo et al.)

Aims/Hypothesis: Peripheral arterial disease (PAD) is a major burden, resulting in limb claudication, repeated surgical interventions and amputation. There is an unmet need for improved medical management of PAD that improves quality of life, maintains activities of daily life and reduces complications. Nitric oxide (NO)/redox balance is a key regulator of angiogenesis. We have previously shown beneficial effects of a β 3 adrenergic receptor ( β 3 AR) agonist on NO/redox balance. We hypothesized that β 3 AR stimulation would have therapeutic potential in PAD by promoting limb angiogenesis. Methods: The effect of the β 3 AR agonist CL 316,243 (1-1,000 nmol/L in vitro , 1 mg/kg/day s. c ) was tested in established angiogenesis assays with human endothelial cells and patient-derived endothelial colony forming cells. Post-ischemia reperfusion was determined in streptozotocin and/or high fat diet-induced diabetic and non-diabetic mice in vivo using the hind limb ischemia model. Results: CL 316,243 caused accelerated recovery from hind limb ischemia in non-diabetic and type 1 and 2 diabetic mice. Increased eNOS activity and decreased superoxide generation were detected in hind limb ischemia calf muscle from CL 316, 243 treated mice vs. controls. The protective effect of CL 316,243 in diabetic mice was associated with >50% decreases in eNOS glutathionylation and nitrotyrosine levels. The β 3 AR agonist directly promoted angiogenesis in endothelial cells in vitro . These pro-angiogenic effects were β 3 AR and NOS-dependent. Conclusion/Interpretation: β 3 AR stimulation increased angiogenesis in diabetic ischemic limbs, with demonstrable improvements in NO/redox balance and angiogenesis elicited by a selective agonist. The orally available β 3 AR agonist, Mirabegron, used for overactive bladder syndrome, makes translation to a clinical trial by repurposing of a β 3 AR agonist to target PAD immediately feasible., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bubb, Ravindran, Cartland, Finemore, Clayton, Tsang, Tang, Kavurma, Patel and Figtree.)

Systemic hypertension, characterized by elevated blood pressure ≥140/90 mm Hg, is a major modifiable risk factor for cardiovascular disease. Hypertension also associates with non-alcoholic fatty liver disease (NAFLD), which is becoming common due to a modern diet and lifestyle. The aim of the present study was to examine whether a high-fat "Western" diet had effects on hypertension and associated NAFLD. Normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were placed on a normal chow or high-fat diet for 8 weeks; blood pressure was measured fortnightly and body weight recorded weekly. As expected, SHR had elevated blood pressure compared to WKY. Diet did not influence blood pressure. Compared to SHR, WKY rats gained more weight, associating with increased white adipose tissue weight. Normotensive rats also had higher plasma cholesterol and triglycerides in response to a "Western" diet, with no changes in plasma glucose levels. Neither strain developed atherosclerosis. Interestingly, high-fat diet-fed SHR had increased liver weight, associating with a significant level of hepatic lipid accumulation not observed in WKY. Further, they exhibited hepatocellular ballooning and increased hepatic inflammation, indicative of steatohepatitis. These findings suggest that a high-fat "Western" diet promotes features of NAFLD in SHR, but not WKY rats. Importantly, the high-fat diet had no effect on blood pressure., (© 2020 Company of the International Journal of Experimental Pathology (CIJEP).)

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., (© 2020 Federation of American Societies for Experimental Biology.)

The extracellular matrix (ECM) is an essential part of the vasculature, not only providing structural support to the blood vessel wall, but also in its ability to interact with cells to regulate cell phenotype and function including proliferation, migration, differentiation and death - processes important in vascular remodelling. Increasing evidence implicates TNF-related apoptosis-inducing ligand (TRAIL) signalling in the modulation of vascular cell function and remodelling under normal and pathological conditions such as in atherosclerosis. TRAIL can also stimulate synthesis of multiple ECM components within blood vessels. This review explores the relationship between TRAIL signals, the ECM, and its implications in vessel remodelling in cardiovascular disease., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review., (© 2020 The authors.)

Atherosclerosis is a chronic inflammatory disease of the vasculature characterised by the infiltration of activated neutrophils and macrophages at sites of damage within the vessel wall, which contributes to lesion formation and plaque progression. Selenomethionine (SeMet) is an organic form of selenium (Se), an essential trace element that functions in the regulation of the immune response by both bolstering the endogenous thioredoxin and glutathione antioxidant defence systems and by directly scavenging damaging oxidant species. This study evaluated the effect of dietary SeMet supplementation within a high fat diet fed apolipoprotein E deficient (ApoE -/- ) mouse model of atherosclerosis. Dietary supplementation with SeMet (2 mg/kg) increased the tissue concentration of Se, and the expression and activity of glutathione peroxidase, compared to non-supplemented controls. Supplementation with SeMet significantly reduced atherosclerotic plaque formation in mouse aortae, resulted in a more stable lesion phenotype and improved vessel function. Concurrent with these results, SeMet supplementation decreased lesion accumulation of M1 inflammatory type macrophages, and decreased the extent of extracellular trap release from phorbol myristate acetate (PMA)-stimulated mouse bone marrow-derived cells. Importantly, these latter results were replicated within ex-vivo experiments on cultured neutrophils isolated from acute coronary syndrome patients, indicating the ability of SeMet to alter the acute inflammatory response within a clinically-relevant setting. Together, these data highlight the potential beneficial effect of SeMet supplementation as a therapeutic strategy for atherosclerosis., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

M3 is a broad-spectrum chemokine-binding protein that inactivates inflammatory chemokines, including CCL2, CCL5, and CX 3 CL1. The aim of this study was to compare whether M3 could inhibit angiogenesis driven by inflammation or ischemia. Here, apolipoprotein E -/- mice were injected with adenoviral M3 (AdM3) or control adenoviral green fluorescent protein (AdGFP) 3 d prior to stimulating angiogenesis using 2 established models that distinctly represent inflammatory or ischemia-driven angiogenesis, namely the periarterial femoral cuff and hind limb ischemia. AdM3 reduced intimal thickening, adventitial capillary density, and macrophage accumulation in femoral arteries 21 d after periarterial femoral cuff placement compared with AdGFP-treated mice ( P < 0.05). AdM3 also reduced mRNA expression of proangiogenic VEGF, inflammatory markers IL-6 and IL-1β, and vascular smooth muscle cell (VSMC)-activated synthetic markers Krüppel-like family of transcription factor 4 (KLF4) and platelet-derived growth factor receptor β (PDGFRβ) in the inflammatory cuff model. In contrast, capillary density, VSMC content, blood flow perfusion, and VEGF gene expression were unaltered between groups in skeletal muscle following hind limb ischemia. In vitro , AdM3 significantly reduced human microvascular endothelial cell 1 proliferation, migration, and tubule formation by ∼17, 71.3, and 8.7% ( P < 0.05) in macrophage-conditioned medium associating with reduced VEGF and hypoxia-inducible factor 1α mRNA but not in hypoxia (1% O 2 ). Compared with AdGFP, AdM3 also inhibited VSMC proliferation and migration and reduced mRNA expression of KLF4 and PDGFRβ under inflammatory conditions. In contrast, AdM3 had no effect on VSMC processes in response to hypoxia in vitro . Our findings show that broad-spectrum inhibition of inflammatory chemokines by M3 inhibits inflammatory-driven but not ischemia-driven angiogenesis, presenting a novel strategy for the treatment of diseases associated with inflammatory-driven angiogenesis.-Ravindran, D., Cartland, S. P., Bursill, C. A., Kavurma, M. M. Broad-spectrum chemokine inhibition blocks inflammation-induced angiogenesis, but preserves ischemia-driven angiogenesis.

We developed a universal method termed OnCELISA to detect cytokine secretion from individual cells by applying a capture technology on the cell membrane. OnCELISA uses fluorescent magnetic nanoparticles as assay reporters that enable detection on a single-cell level in microscopy and flow cytometry and fluorimetry in cell ensembles. This system is flexible and can be modified to detect different cytokines from a broad range of cytokine-secreting cells. Using OnCELISA we have been able to select and sort highly cytokine-secreting cells and identify cytokine-secreting expression profiles of different cell populations in vitro and ex vivo. We show that this system can be used for ultrasensitive monitoring of cytokines in the complex biological environment of atherosclerosis that contains multiple cell types. The ability to identify and select cell populations based on their cytokine expression characteristics is valuable in a host of applications that require the monitoring of disease progression., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Current treatment of ischaemic vascular diseases such as coronary and peripheral artery disease includes angioplasty and bypass grafting, as well as lipid lowering therapies and control of other cardiovascular risk factors. Numerous members of the tumour necrosis factor superfamily (TNFSF) have recently shown emerging roles in both the protection and progression of such diseases. Understanding the role TNFSF members play in ischaemic vascular disease may provide insight into the development of novel therapeutics to prevent or treat diseases relating to atherosclerosis and ischaemia. This review summarizes the most recent findings relating to TNFSF members and the mechanisms that precede ischaemic vascular disease progression, particularly endothelial dysfunction, chronic inflammation, and atherosclerotic plaque development. This review also explores recent translational research on the role of TNFSF therapies in cardiovascular disease., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The vascular endothelium is critical for maintenance of cardiovascular homeostasis. Endothelial dysfunction is a key event of atherosclerosis, with oxidative stress mediated by reactive oxygen species (ROS) playing a major role. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is increasingly recognised to play a protective role in atherosclerosis, however the molecular mechanisms by which it exerts its beneficial effects are unclear. Here we examined if TRAIL could attenuate vascular oxidative stress and improve endothelial cell (EC) function. In coronary artery disease patients, plasma TRAIL levels were significantly reduced compared to healthy individuals, and negatively correlated with the levels of circulating 8-iso Prostaglandin F 2α , a marker of in vivo oxidative stress. In vivo, high-fat fed, atherosclerotic Trail -/- Apoe -/- mice exhibited a significant impairment in endothelial-dependent vasorelaxation, which correlated with increased vascular ROS and 4-hydroxynonenal compared to Apoe -/- mice. Endothelial permeability measured by Evan's blue dye extravasation was increased in several organs of Trail -/- mice compared to wild-type mice, which correlated with a decrease in VE-cadherin expression. In vitro in ECs, angiotensin II (AngII)-induced ROS generation involving the mitochondria, NADPH oxidase-4 (NOX-4) and eNOS, was inhibited by pre-treatment with TRAIL. Furthermore, AngII-augmented VCAM-1 expression and monocyte adhesion to ECs was inhibited by TRAIL. Finally, AngII reduced VE-cadherin expression and redistributed this protein, all of which was brought back to baseline by TRAIL pre-treatment. These findings demonstrate for the first time that TRAIL protects against several forms of endothelial dysfunction involving its ability to control EC ROS generation. Understanding the role TRAIL plays in normal physiology and disease, may lead to potential new therapies to improve endothelial function and atherosclerosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Peripheral artery disease (PAD) is caused by narrowing of arteries in the limbs, normally occurring in the lower extremities, with severe cases resulting in amputation of the foot or leg. A potential approach for treatment is to stimulate the formation of new blood vessels to restore blood flow to limb tissues. This is a process called angiogenesis and involves the proliferation, migration, and differentiation of endothelial cells. Angiogenesis can be stimulated by reactive oxygen species (ROS), with NADPH oxidases (NOX) being a major source of ROS in endothelial cells. This review summarizes the recent evidence implicating NOX isoforms in their ability to regulate angiogenesis in vascular endothelial cells in vitro, and in PAD in vivo. Increasing our understanding of the involvement of the NOX isoforms in promoting therapeutic angiogenesis may lead to new treatment options to slow or reverse PAD., Competing Interests: The authors declare no conflict of interest.

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-1β, 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.

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., Competing Interests: The authors declare no conflict of interest.

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., (© 2015 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.)

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has the ability to inhibit angiogenesis by inducing endothelial cell death, as well as being able to promote pro-angiogenic activity in vitro. These seemingly opposite effects make its role in ischemic disease unclear. Using Trail(-/-) and wildtype mice, we sought to determine the role of TRAIL in angiogenesis and neovascularization following hindlimb ischemia., Methods and Results: Reduced vascularization assessed by real-time 3-dimensional Vevo ultrasound imaging and CD31 staining was evident in Trail(-/-) mice after ischemia, and associated with reduced capillary formation and increased apoptosis. Notably, adenoviral TRAIL administration significantly improved limb perfusion, capillary density, and vascular smooth-muscle cell content in both Trail(-/-) and wildtype mice. Fibroblast growth factor-2, a potent angiogenic factor, increased TRAIL expression in human microvascular endothelial cell-1, with fibroblast growth factor-2-mediated proliferation, migration, and tubule formation inhibited with TRAIL siRNA. Both fibroblast growth factor-2 and TRAIL significantly increased NADPH oxidase 4 (NOX4) expression. TRAIL-inducible angiogenic activity in vitro was inhibited with siRNAs targeting NOX4, and consistent with this, NOX4 mRNA was reduced in 3-day ischemic hindlimbs of Trail(-/-) mice. Furthermore, TRAIL-induced proliferation, migration, and tubule formation was blocked by scavenging H2O2, or by inhibiting nitric oxide synthase activity. Importantly, TRAIL-inducible endothelial nitric oxide synthase phosphorylation at Ser-1177 and intracellular human microvascular endothelial cell-1 cell nitric oxide levels were NOX4 dependent., Conclusions: This is the first report demonstrating that TRAIL can promote angiogenesis following hindlimb ischemia in vivo. The angiogenic effect of TRAIL on human microvascular endothelial cell-1 cells is downstream of fibroblast growth factor-2, involving NOX4 and nitric oxide signaling. These data have significant therapeutic implications, such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with cardiovascular disease and diabetes., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Rationale: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied., Objective: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process., Methods and Results: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions., Conclusions: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo., (© 2015 American Heart Association, Inc.)

Background: We recently demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) is protective of diet-induced diabetes in mice. While TRAIL has been implicated in chronic kidney disease, its role in vivo in diabetic nephropathy is not clear. The present study investigated the role of TRAIL in the pathogenesis of diabetic nephropathy using TRAIL(-/-)ApoE(-/-) mice., Methods: TRAIL(-/-)ApoE(-/-) and ApoE(-/-) mice were fed a high fat diet for 20 w. Plasma glucose and insulin levels were assessed over 0, 5, 8 and 20 w. At 20 w, markers of kidney function including creatinine, phosphate, calcium and cystatin C were measured. Changes in mRNA expression of MMPs, TIMP-1, IL-1β and IL-18 were assessed in the kidney. Functional and histological changes in kidneys were examined. Glucose and insulin tolerance tests were performed., Results: TRAIL(-/-)ApoE(-/-) mice had significantly increased urine protein, urine protein:creatinine ratio, plasma phosphorous, and plasma cystatin C, with accelerated nephropathy. Histologically, increased extracellular matrix, mesangial expansion and mesangial cell proliferation in the glomeruli were observed. Moreover, TRAIL(-/-)ApoE(-/-) kidneys displayed loss of the brush border and disorganisation of tubular epithelium, with increased fibrosis. TRAIL-deficient kidneys also had increased expression of MMPs, TIMP-1, PAI-1, IL-1β and IL-18, markers of renal injury and inflammation. Compared with ApoE(-/-) mice, TRAIL-/-ApoE-/- mice displayed insulin resistance and type-2 diabetic features with reduced renal insulin-receptor expression., Conclusions: Here, we show that TRAIL-deficiency in ApoE(-/-) mice exacerbates nephropathy and insulin resistance. Understanding TRAIL signalling in kidney disease and diabetes, may therefore lead to novel strategies for the treatment of diabetic nephropathy.

The osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) cytokine system, not only controls bone homeostasis, but has been implicated in regulating vascular calcification. TNF-related apoptosis-inducing ligand (TRAIL) is a second ligand for OPG, and although its effect in vascular calcification in vitro is controversial, its role in vivo is not yet established. This study aimed to investigate the role of TRAIL in vascular calcification in vitro using vascular smooth muscle cells (VSMCs) isolated from TRAIL(-/-) and wild-type mice, as well as in vivo, in advanced atherosclerotic lesions of TRAIL(-/-)ApoE(-/-) mice. The involvement of OPG and RANKL in this process was also examined. TRAIL dose-dependently inhibited calcium-induced calcification of human VSMCs, while TRAIL(-/-) VSMCs demonstrated accelerated calcification induced by multiple concentrations of calcium compared to wild-type cells. Consistent with this, RANKL mRNA was significantly elevated with 24 h calcium treatment, while OPG and TRAIL expression in human VSMCs was inhibited. Brachiocephalic arteries from TRAIL(-/-)ApoE(-/-) and ApoE(-/-) mice fed a high fat diet for 12 w demonstrated increased chondrocyte-like cells in atherosclerotic plaque, as well as increased aortic collagen II mRNA expression in TRAIL(-/-)ApoE(-/-) mice, with significant increases in calcification observed at 20 w. TRAIL(-/-)ApoE(-/-) aortas also had significantly elevated RANKL, BMP-2, IL-1β, and PPAR-γ expression at 12 w. Our data provides the first evidence that TRAIL deficiency results in accelerated cartilaginous metaplasia and calcification in atherosclerosis, and that TRAIL plays an important role in the regulation of RANKL and inflammatory markers mediating bone turn over in the vasculature.

It is recognized that the development of atherosclerosis involves many elements of an inflammatory process, involving components of both the innate and adaptive immune systems. The presence and roles of macrophages and T-cells in atherogenesis are well-established. More recently dendritic cells have been identified in the vasculature and in atherosclerotic lesions. This review summarises our current understanding of the roles of dendritic cells in the development and regression of atherosclerosis.