Aims/hypothesis TNF-related apoptosis-inducing ligand (TRAIL) is implicated in the regulation of diabetes and is reduced in patients with cardiovascular disease. Although TRAIL receptors are widespread, and TRAIL can promote cell proliferation and apoptosis, it is not known how TRAIL might protect against diabetes and atherosclerosis. Methods We examined the development of atherosclerosis and diabetes in Apoe −/−

Rationale: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is well reported as an inducer of apoptosis in tumor models; however, its role and function in vivo in atherosclerosis and vascular injury has not been established. Objective: We sought to study the function of TRAIL in cardiovascular pathology and its regulation in vivo. Methods and Results: Here, we show that TRAIL was upregulated in medial vascular smooth muscle cells (VSMCs) 24 hours following perivascular cuff placement around femoral arteries of mice. We also show that TRAIL mRNA and promoter activity was induced in VSMCs following in vitro mechanical injury. Intimal thickening 15 days after cuff placement was reduced 2-to 3-fold in TRAIL ؊/؊ compared to wild-type mice and was reversible by administration of recombinant TRAIL. Additionally, reduced VSMC proliferation was observed in injured arteries of TRAIL ؊/؊ mice. Fibroblast growth factor (FGF)-2, a potent growth factor released following vascular injury, was also reduced in arteries of TRAIL ؊/؊ mice, and VSMCs isolated from these animals did not respond to FGF-2 in vitro. Injury and FGF-2 regulated TRAIL transcriptional activity via 2 specificity protein (Sp)1 elements in the proximal TRAIL promoter, a binding site also shared by nuclear factor (NF)B. Mutational studies confirmed a role for Sp1 in injury-and FGF-2-inducible TRAIL transcription. Furthermore, increased NFB expression after injury transactivated the TRAIL promoter. Interestingly, following mechanical injury, Sp1 phosphorylation (Thr453) and an increase in the physical interaction of p-Sp1(Thr453) with NFB was observed. Conclusions: We conclude that TRAIL induction involves FGF-2, Sp1-phosphorylation and NFB and that TRAIL promotes VSMC proliferation and neointima formation after arterial injury. (Circ Res. 2010;106: 1061-1071.)Key Words: TRAIL Ⅲ VSMC proliferation Ⅲ injury Ⅲ transcriptional regulation T RAIL (tumor necrosis factor-related apoptosis inducing ligand) is expressed by a variety of cells, traditionally known to induce apoptosis via the death receptor pathway analogous to Fas ligand (FasL). Unlike FasL, however, TRAIL apoptotic signaling occurs following its engagement with 2 death domain-containing receptors, death receptors 4 and 5 (DR4 and DR5). Additional receptors are also present in humans, including decoy receptors 1 and 2 (DCR1 and DCR2) and osteoprotegerin, which may compete with DR4 and/or DR5 for TRAIL binding, thereby protecting cells from apoptosis. 1,2 TRAIL expression is regulated by insulin, interferons type I and II, and transcription factors Stat1, nuclear factor (NF)B, Egr2/3 (early growth response factor 2/3), and IRF-1/3/7 (interferon regulatory factor 1/3/7). 3 In the vessel wall, TRAIL is expressed in vascular smooth muscle cells (VSMCs), endothelial cells (ECs), macrophages, and T cells (reviewed 3 ); however, the role and regulation of TRAIL in cardiovascular disorders is currently controversial. Under certain conditions TRAIL can induce apoptosis of vascular cells which may...

Objective-The purpose of this study was to investigate the ability of high-density lipoproteins (HDLs) to upregulate genes with the potential to protect against inflammation in endothelial cells. Methods and Results-Human coronary artery endothelial cells (HCAECs) were exposed to reconstituted HDLs (rHDLs) for 16 hours before being activated with tumor necrosis factor-␣ (TNF-␣) for 5 hours. rHDLs decreased vascular cell adhesion molecule-1 (VCAM-1) promoter activity by 75% (PϽ0.05), via the nuclear factor-kappa B (NF-B) binding site. rHDLs suppressed the canonical NF-B pathway and decreased many NF-B target genes. Suppression of NF-B and VCAM-1 expression by rHDLs or native HDLs was dependent on an increase in 3␤-hydroxysteroid-⌬24 reductase (DHCR24) levels (PϽ0.05).

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BackgroundTumor 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 ResultsReduced 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.ConclusionsThis 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.

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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.

We recently reported that TNF-related apoptosis-inducing ligand (TRAIL) is important in atherogenesis, since it can induce vascular smooth muscle cell (VSMC) proliferation and arterial thickening following injury. Here we show the first demonstrate that TRAIL siRNA reduces platelet-derived growth factor-BB (PDGF-BB)-stimulated VSMC proliferation and migration. PDGF-BB-inducible VSMC proliferation was completely inhibited in VSMCs isolated from aortas of TRAIL(-/-) mice; whereas inducible migration was blocked compared to control VSMCs. TRAIL transcriptional control mediating this response is not established. TRAIL mRNA, protein and promoter activity was increased by PDGF-BB and subsequently inhibited by dominant-negative Sp1, suggesting that the transcription factor Sp1 plays a role. Sp1 bound multiple Sp1 sites on the TRAIL promoter, including two established (Sp1-1 and -2) and two novel Sp1-5/6 and -7 sites. PDGF-BB-inducible TRAIL promoter activity by Sp1 was mediated through these sites, since transverse mutations to each abolished inducible activity. PDGF-BB stimulation increased acetylation of histone-3 (ac-H3) and expression of the transcriptional co-activator p300, implicating chromatin remodelling. p300 overexpression increased TRAIL promoter activity, which was blocked by dominant-negative Sp1. Furthermore, PDGF-BB treatment increased the physical interaction of Sp1, p300 and ac-H3, while chromatin immunoprecipitation studies revealed Sp1, p300 and ac-H3 enrichment on the TRAIL promoter. Taken together, our studies demonstrate for the first time that PDGF-BB-induced TRAIL transcriptional activity requires the cooperation of Sp1, ac-H3 and p300, mediating increased expression of TRAIL which is important for VSMC proliferation and migration. Our findings have the promising potential for targeting TRAIL as a new therapeutic for vascular proliferative disorders.