Background-TRAIL protein is expressed in the medial smooth cell layer of aorta and pulmonary artery, whereas endothelial cells express all TRAIL receptors (TRAIL-Rs). Methods and Results-The role of TRAIL/TRAIL-Rs in vascular biology was investigated in primary human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells, which showed comparable surface expression of death (TRAIL-R1 and -R2) and decoy (TRAIL-R3 and -R4) TRAIL-Rs. TRAIL activated the protein kinase Akt in HUVECs, as assessed by Western blot for phospho-Akt. Moreover, experiments performed with a pharmacological inhibitor of the phosphatidylinositol 3-kinase/Akt pathway (LY294002) or a dominant-negative Akt (K179M) demonstrated that TRAIL significantly protected HUVECs from apoptosis induced by trophic withdrawal via Akt and that inhibition of Akt sensitized HUVECs to TRAIL-induced caspase-dependent apoptosis. TRAIL also stimulated the ERK1/2 but not the p38 or the JNK pathways and induced a significant increase in endothelial cell proliferation in an ERK-dependent manner. Conversely, TRAIL did not activate NF-B or affect the surface expression of the inflammatory markers E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Conclusions-The

Human and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor-alpha + interleukin-1beta + interferon-gamma or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.

Adenosine displays contradictory effects on cell growth: it improves cell proliferation, but it may also induce apoptosis and impair cell survival. Following the pharmacologic characterization of adenosine receptor expression on the human melanoma cell line A375, we chose A375 as our cellular model to define how the extracellular adenosine signals are conveyed from each receptor. By using selective adenosine receptor agonists or antagonists, we found that A2A stimulation reduced cell viability and cell clone formation, whereas, at the same time, it improved cell proliferation. In support of this finding we demonstrated that the stimulation of A2A adenosine receptors stably expressed in Chinese hamster ovary cell clone reproduced deleterious effects observed in human melanoma cells. A3 stimulation counteracted A2A-induced cell death but also reduced cell proliferation. Furthermore, we found that A3 stimulation ensures cell survival. We demonstrated that adenosine triggers a survival signal via A3 receptor activation and it kills the cell through A2A receptor inducing a signaling pathway that involves protein kinase C and mitogen-activated protein kinases.

Treatment of the human HL-60 cell line with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) resulted in rapid (6-24 hours) cytotoxicity associated with progressive maturation of the surviving cells along the monocytic lineage. The occurrence of monocytic maturation was demonstrated by a significant increase of both CD14 and CD11b surface expression, the acquisition of morphologic features typical of mature monocytes, and phagocytic capacity in TRAIL-treated cultures. By using selective pharmacologic inhibitors, it was possible to demonstrate that activation of the caspase cascade played a crucial role in mediating TRAIL cytotoxicity and monocytic maturation of HL-60 cells. Moreover, experiments performed using agonistic polyclonal antibodies, which mimic the interactions between TRAIL and each TRAIL receptor, indicated that TRAIL-R1 was responsible for mediating the TRAILinduced maturation. Importantly, the maturational effects of TRAIL were observed also in primary normal CD34 ؉ cells, seeded in serum-free liquid cultures for 4 to 8 days in the presence of SCF ؉ GM؊CSF. After treatment with TRAIL for 3 additional days, a significant increase in CD14 and CD11b expression, coupled with an increased number of mature monocytes and macrophages, was noticed in the absence of cytotoxicity. These data disclose a novel role for TRAIL as a positive regulator of myeloid differentiation. Moreover, the dichotomous effect of TRAIL on malignant cells (early induction of apoptosis and monocytic maturation of the surviving cells) might have important therapeutic implications for the treatment of acute myeloid leukemia.