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Recombinant TRAIL induces miorelaxating activity in rat aortas, which is abrogated by the induction of diabetes mellitus

      TNF-related apoptosis inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of cytokines, which exist either as type II membrane or as a soluble protein [
      • Zauli G.
      • Secchiero P.
      The role of the TRAIL/TRAIL-receptors system in hematopoiesis and endothelial cell biology.
      ]. TRAIL interacts with four high-affinity trans-membrane receptors (TRAIL-R1, -R2, -R3 and -R4) as well as with the soluble decoy osteoprotegerin (OPG), all belonging to the apoptosis-inducing TNF-receptor family [
      • Zauli G.
      • Secchiero P.
      The role of the TRAIL/TRAIL-receptors system in hematopoiesis and endothelial cell biology.
      ]. Although the best-characterized activity of TRAIL is represented by its anti-cancer activity [
      • Almasan A.
      • Ashkenazi A.
      Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy.
      ], it has been recently shown that TRAIL serum levels are decreased in patients affected by atherosclerotic cardiovascular disease [
      • Michowitz Y.
      • Goldstein E.
      • Roth A.
      • et al.
      The involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in atherosclerosis.
      ,
      • Schoppet M.
      • Sattler A.M.
      • Schaefer J.R.
      • Hofbauer L.C.
      Osteoprotegerin (OPG) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) levels in atherosclerosis.
      ] raising the possibility that circulating soluble TRAIL might also affect vascular biology. In this respect, it has been previously shown that TRAIL induces the in vitro release of nitric oxide by vascular endothelial cells [
      • Zauli G.
      • Pandolfi A.
      • Gonelli A.
      • et al.
      TNF-related apoptosis-inducing ligand (TRAIL) sequentially up-regulates nitric oxide and prostanoid production in primary human endothelial cells.
      ]. In order to explore the in vivo relevance of these in vitro findings, we have assessed the vascular reactivity in response to recombinant TRAIL in a more intact paradigm. For this purpose, rings (4 mm long) from rat thoracic aorta were obtained as previously described [
      • Zanetti M.
      • Sato J.
      • Katusic Z.S.
      • O’Brien T.
      Gene transfer of superoxide dismutase isoforms reverses endothelial dysfunction in diabetic rabbit aorta.
      ] by both control (3-month-old male non-diabetic Wistar rats receiving vehicle infusion) and rats in which diabetes mellitus was induced by injecting 40 mg/kg streptozotocin (SZT, Sigma Chemicals, St. Louis, MO). All animal procedures were approved by the Animal Care Committee of the University of Trieste. After sacrifice of the animals, aortas were harvested, cleaned of connective tissue and vascular reactivity was analyzed in ex vivo immediately after harvest. During submaximal contraction with phenylephrine, incubation of aortic segments in the presence of increasing (up to 1000 ng/ml) concentrations of recombinant human TRAIL, produced as described [
      • Zanetti M.
      • Sato J.
      • Katusic Z.S.
      • O’Brien T.
      Gene transfer of superoxide dismutase isoforms reverses endothelial dysfunction in diabetic rabbit aorta.
      ], resulted in significant (p < 0.01) vaso-relaxation (Fig. 1A ). The effect was dose-dependent and was completely abolished by pre-incubation of the rat aortic rings with L-NAME, clearly indicating that the NOS pathway played a key role in mediating the myo-relaxating activity of TRAIL. In parallel, we have investigated whether the induction of diabetes mellitus might affect the relaxating activity of aortic rings in response to TRAIL. Diabetes mellitus was induced by destruction of pancreatic islet cells by treating rats with SZT (40 mg/kg). Non-fasting blood glucose concentrations of both SZT-diabetic rats (SZT, n = 10) and age-matched control non-diabetic rats treated with vehicle (n = 10) were measured at days 5 and 15, when animals were sacrificed with CO2. The loss of insulin secretion triggered stable hyperglycemia, as evaluated by blood glucose measurement using an AccuCheck glucose monitor (Roche, Indianapolis, IN): 260 ± 46 and 295 ± 55 in diabetic rats at days 5 and 15 after diabetic induction, respectively, versus 89 ± 8 in control rats. Relaxation to TRAIL was completely abrogated in aortic rings obtained from SZT-induced diabetic rats (Fig. 1B).
      Figure thumbnail gr1
      Fig. 1TRAIL-induced mio-relaxation in ex vivo experiments on isolated rat aortic rings. (A) During submaximal contraction with phenylephrine, aortic segments were incubated with increasing (up to 1000 ng/ml) concentrations of recombinant TRAIL, in the absence or in the presence of L-NAME. Data are expressed as means ± S.D. of eight separate experiments performed in duplicate. (B) During submaximal contraction with phenylephrine, aortic segments were incubated with increasing (up to 1000 ng/ml) concentrations of recombinant TRAIL. Data are expressed as means ± S.D. of three separate experiments performed in duplicate.

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