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International audience; Vascular endothelial growth factor-A (VEGF), a powerful factor involved in vasculogenesis and angiogenesis, is translationally regulated through 2 independent internal ribosome entry sites (IRESs A and B). IRESs enable an mRNA to be translated under conditions in which 5'-cap-dependent translation is inhibited, such as low oxygen stress. In the VEGF mRNA, IRES A influences translation at the canonical AUG codon, whereas the 5' IRES B element regulates initiation at an upstream, in frame CUG. In this study, we have developed transgenic mice expressing reporter genes under the control of these 2 IRESs. We reveal that although these IRESs display low activity in embryos and adult tissues, they permit efficient translation at early time points in ischemic muscle, a stress under which cap-dependent translation is inhibited. These results demonstrate the in vivo efficacy of the VEGF IRESs in response to a local environmental stress such as hypoxia.

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 CD 31 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 si RNA . Both fibroblast growth factor‐2 and TRAIL significantly increased NADPH oxidase 4 ( NOX 4) expression. TRAIL ‐inducible angiogenic activity in vitro was inhibited with si RNA s targeting NOX 4, and consistent with this, NOX 4 mRNA was reduced in 3‐day ischemic hindlimbs of Trail −/− mice. Furthermore, TRAIL ‐induced proliferation, migration, and tubule formation was blocked by scavenging H 2 O 2 , 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 NOX 4 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 NOX 4 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.

Background: Angiogenesis and neovascularization are essential processes that follow ischemia insults. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) not only induces endothelial cell (EC) death and inhibits angiogenesis, but also promotes EC migration, invasion and proliferation in vitro . These seemingly opposite effects make its role in angiogenesis in vivo unclear. Using TRAIL -/- and wild-type mice, we sought to determine the role of TRAIL in angiogenesis and neovascularisation. We also sought mechanisms in vitro . Methods and Results: Reduced vascularisation assessed by real-time in vivo 3D Vevo ultrasound imaging and CD31 staining was observed in TRAIL -/- mice 28 d after hindlimb ischemia. Moreover, reduced capillary formation and increased apoptosis was evident in TRAIL -/- muscles even at 3 d after ischemic surgery. We have previously shown that fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, regulates TRAIL gene expression in vascular smooth muscle cells. Indeed, FGF-2 also regulates TRAIL expression in ECs, and FGF-2-inducible proliferation, migration and tubule formation was inhibited with siRNA targeting TRAIL. Notably, both FGF-2 and TRAIL significantly increased NOX4 expression. TRAIL-inducible angiogenic activity in ECs was inhibited with siRNAs targeting NOX4, and consistent with these, NOX4 mRNA was reduced in 3 d ischemic hindlimbs of TRAIL -/- mice. TRAIL stimulated intracellular H 2 O 2 levels in ECs, and TRAIL-inducible proliferation, migration and tubule formation was inhibited with not only PEG-catalase, a H 2 O 2 scavenger, but also blocked with L-NAME, a nitric oxide synthase inhibitor. Conclusions: This is the first demonstration showing that TRAIL promotes angiogenesis in vivo . We show for the first time that the TRAIL stimulates NOX4 expression to mediate nitric oxide-dependent angiogenic effects. This has significant therapeutic implications such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with CVD and diabetes.