Tumor Necrosis Factor–Related Apoptosis‐Inducing Ligand (TRAIL) Promotes Angiogenesis and Ischemia‐Induced Neovascularization Via NADPH Oxidase 4 (NOX4) and Nitric Oxide–Dependent Mechanisms

Abstract

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.

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

“…VEGF-A and FGF-2 are established pro-angiogenic growth factors, known to promote EC proliferation [6]. TRAIL can also induce EC proliferation in vitro and in vivo [4]. To date, a comprehensive analysis of TRAIL, VEGF-A and FGF-2 or the effect of combination treatment on EC proliferation has not been examined.…”

“…To date, a comprehensive analysis of TRAIL, VEGF-A and FGF-2 or the effect of combination treatment on EC proliferation has not been examined. We treated HMEC-1 cells with 10 ng/mL TRAIL, 50 ng/mL VEGF-A and 50 ng/mL FGF-2, doses known to stimulate cell proliferation [4]. TRAIL at 10 ng/mL promoted HMEC-1 proliferation just as effectively as VEGF-A at 50 ng/mL (Figure 1a).…”

“…Using an in vitro scratch assay, we assessed the effect of TRAIL, VEGF-A and FGF-2 on HMEC-1 migration into the wound area [4]. Exposure of HMEC-1 to TRAIL significantly augmented HMEC-1 migration into the denuded zone ~2.7-fold (Figure 2).…”

“…TRAIL can also promote microvessel growth in vitro by increasing EC migration, invasion and proliferation and is pro-angiogenic, with activity comparable to VEGF in Matrigel assays for tubule formation [3]. We recently demonstrated that Trail −/− mice have reduced vascularity and angiogenesis in response to ischemic injury compared to the wildtype, and adenoviral delivery of TRAIL not only stimulated angiogenesis, but also improved blood flow to the lower limbs after hindlimb ischemia [4]. Interestingly, TRAIL is expressed in highly vascularized malignant mesenchymal tumors [3] suggesting that an association between TRAIL and vascularity in cancer exists; however, there is no direct evidence of how TRAIL may regulate angiogenesis in these processes.…”

Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling

“…On the other hand, studies using systemic NOX4 KO mice cannot distinguish the effect of NOX4 in CMs from its effect in non-CMs. NOX4 enhances angiogenesis under ischemic conditions and is therefore protective against ischemic injury in lower limbs (35). Furthermore, Chen et al demonstrated that endothelial NOX4 mediates angiogenesis (36).…”