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

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Serum osteoprotegerin (OPG) is significantly increased in diabetic patients, prompting expanded investigation of the correlation between OPG production/release and glycemic levels. Serum levels of OPG, but not of its cognate ligand receptor activator of nuclear factor-B ligand (RANKL), were significantly increased in type 2 diabetes mellitus patients compared with healthy blood donors. Serum OPG was also significantly elevated in a subgroup of recently diagnosed diabetic patients (within 2 years). The relationship between serum OPG and diabetes mellitus onset was next investigated in apoE-null and littermate mice. Serum OPG increased early after diabetes induction in both mouse strains and showed a positive correlation with blood glucose levels and an inverse correlation with the levels of free (OPG-unbound) RANKL. The in vitro addition of tumor necrosis factor-␣ to human vascular endothelial cells, but not human peripheral blood mononuclear cells, markedly enhanced OPG release in culture. In contrast, high glucose concentrations did not modulate OPG release when used alone or in association with tumor necrosis factor-␣. Moreover, the ability of soluble RANKL to activate the extracellular signal-regulated kinase/mitogen-activated protein kinase and endothelial nitric-oxide synthase pathways in endothelial cells was neutralized by preincubation with recombinant OPG. Altogether, these findings suggest that increased OPG production represents an early Receptor activator of nuclear factor (NF)-B ligand (RANKL) is a member of the tumor necrosis factor (TNF) family of cytokines, which exists either as type II membrane or as soluble protein.1 RANKL was originally described as being expressed by activated T lymphocytes and osteoblasts, and it has been involved in the interaction between T lymphocytes and dendritic cells, osteoclast differentiation from monocytic precursor cells, and activation of mature osteoclasts.1-6 Two receptors for RANKL have been identified: transmembrane RANK and soluble osteoprotegerin (OPG).2,3,5 RANK mRNA is ubiquitously expressed in human tissues, but RANK protein expression has been characterized only in normal dendritic cells, CD4 and CD8 T lymphocytes, osteoclast monocytic precursors, and endothelial cells, suggesting that expression of this protein is posttranscriptionally regulated. 6 For the purpose of this study, it is noteworthy that, by interacting with RANK, RANKL induces a variety of biological effects on endothelial cells, such as promotion of cell survival and angiogenesis. [7][8][9] Although the affinity of RANKL for OPG is weaker than that for RANK,5 when present at high concentrations soluble OPG prevents RANKL interaction with transmembrane RANK, thus acting as a decoy receptor. 3,5 It has been shown that OPG is produced by a wide range of tissues, including the cardiovascular system, and that OPG levels are particularly high in aortic and renal arteries. 10 -12 Interestingly, different groups of investigators have reported that serum OPG is significantly increased in both t...

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Abstract-Endothelial cells express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors, but the function of TRAIL in endothelial cells is not completely understood. We explored the role of TRAIL in regulation of key intracellular signal pathways in endothelial cells. Key Words: tumor necrosis factor-related apoptosis-inducing ligand Ⅲ nitric oxide Ⅲ prostanoids T umor necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL)/Apo-2L is a member of the TNF family of cytokines, which are structurally related proteins playing important roles in regulating cell death, immune response, and inflammation. 1 TRAIL is a type II membrane protein, which can be proteolytically cleaved to a soluble form, 2 as previously shown also for TNF-␣ and CD95 (Apo-1/Fas)L. The unique feature of TRAIL, compared with other members of the TNF family, is its ability to induce apoptosis in a variety of malignant cells both in vitro and in vivo, displaying minimal toxicity on normal cells and tissues. 3,4 TRAIL interacts with 4 high affinity transmembrane receptors belonging to the apoptosis-inducing TNFreceptor (R) family. TRAIL-R1 (DR4) and TRAIL-R2 (DR5) transduce apoptotic signals on binding of TRAIL, whereas TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2) are homologous to DR4 and DR5 in their cysteine-rich extracellular domain, but they lack the intracellular death domain and apoptosis inducing capability. It has been proposed that TRAIL-R3 and TRAIL-R4 function as decoy receptors protecting normal cells, including endothelial cells, from apoptosis. 5,6 It has been shown that endothelial cells express TRAIL receptors, 6 and TRAIL protein is expressed in the medial smooth cell layer of the aorta and pulmonary arteries. 7 Whereas cleavage of Fas ligand from the cell surface requires the action of zinc-dependent metalloproteases, generation of soluble TRAIL involves the action of cysteine proteases. 2 Notably, the vessel wall is a rich source of cysteine proteases. 8 Using human umbilical vein endothelial cells (HUVECs) as a model system, the aim of this study was to investigate the ability of TRAIL to modulate intracellular pathways that play a key role in endothelial cell biology. In particular, we have analyzed whether TRAIL was able to modulate the production of nitric oxide (NO), which regulates vascular tone, promotes endothelial cell survival and migration, and inhibits platelet adhesion and aggregation, leukocyte adherence, and vascular smooth muscle cell proliferation, therefore providing antithrombotic and antiinflammatory activity. 9 -12 Moreover, we have investigated the expression and/or activity of cyclooxygenases (COX) in response to TRAIL, because also these enzymes, by catalyzing the rate-limiting step in the biosynthesis of prostanoids, 13 such as prostaglandin (PGE) 2 , prostacyclin (PGI) 2 , and thromboxane (TXA) 2 , have a profound influence on blood pressure, regional blood flow, vascular remodeling, and angiogenesis.

Limited knowledge exists about changes in follicle quality associated with age. The aim of this work was to investigate whether ageing may cause oxidative stress-mediated alterations in human granulosa cells (GCs) from periovulatory follicles. GCs employed in this study were obtained from follicular aspirates of 20 younger women (range 27-32 years) and 20 older women (range 38-41 years) undergoing an IVF treatment. Results obtained from comparative RT-PCR analysis revealed that the mean relative levels of mRNAs coding for superoxide dismutases, Cu, ZnSOD (SOD1), MnSOD (SOD2) and catalase were significantly decreased in women ³38 years (P < 0.05, Student's t-test). These changes were associated with a reduced expression of SOD1, SOD2 and catalase at the protein level. When examined at an ultrastructural level, most of the GCs from this group showed defective mitochondria and fewer lipid droplets than those observed in the younger group. These results indicate that GCs from older patients suffer from age-dependent oxidative stress injury and are taken as an evidence for reduced defence against reactive oxygen species (ROS) in GCs during reproductive ageing.

Abstract-Cyclooxygenase (COX)-2 is among the endothelial genes upregulated by uniform laminar shear stress (LSS), characteristically associated with atherosclerotic lesion-protected areas. We have addressed whether the induction of COX-2-dependent prostanoids in endothelial cells by LSS plays a role in restraining endothelial tumor necrosis factor (TNF)-␣ generation, a proatherogenic cytokine, through the induction of heme oxygenase-1 (HO)-1, an antioxidant enzyme. In human umbilical vein endothelial cells (HUVECs) exposed to steady LSS of 10 dyn/cm 2 for 6 hours, COX-2 protein was significantly induced, whereas COX-1 and the downstream synthases were not significantly modulated. This was associated with significant (PϽ0.05) increase of 6-keto-prostaglandin (PG)F 1␣ (the hydrolysis product of prostacyclin), PGE 2 , and PGD 2 . In contrast, TNF-␣ released in the medium in 6 hours (3633Ϯ882 pg) or detected in cells lysates (1091Ϯ270 pg) was significantly (PϽ0.05) reduced versus static condition (9100Ϯ2158 and 2208Ϯ300 pg, respectively). Coincident induction of HO-1 was detected. The finding that LSS-dependent reduction of TNF-␣ generation and HO-1 induction were abrogated by the selective inhibitor of COX-2 NS-398, the nonselective COX inhibitor aspirin, or the specific prostacyclin receptor (IP) antagonist RO3244794 illuminates the central role played by LSS-induced COX-2-dependent prostacyclin in restraining endothelial inflammation. Carbacyclin, an agonist of IP, induced HO-1. Similarly to inhibition of prostacyclin biosynthesis or activity, the novel imidazole-based HO-1 inhibitor QC15 reversed TNF-␣ reduction by LSS. These findings suggest that inhibition of COX-2-dependent prostacyclin might contribute to acceleration of atherogenesis in patients taking traditional nonsteroidal antiinflammatory drugs (NSAIDs) and NSAIDs selective for COX-2 through downregulation of HO-1, which halts TNF-␣ generation in human endothelial cells. Key Words: cyclooxygenase Ⅲ endothelial cells Ⅲ prostaglandins Ⅲ prostanoids Ⅲ shear stress Ⅲ tumor necrosis factor-␣ P rostanoids, including prostaglandin (PG)E 2 , PGF 2␣ , PGD 2 , prostacyclin (PGI 2 ), and thromboxane (TX)A 2 , are lipid autacoids, immediately released outside the cell after intracellular biosynthesis, that modulate a wide variety of physiological and pathological processes. 1 They are generated by 3 sequential enzymatic steps involving: (1) phospholipase A 2 enzymes, (2) cyclooxygenase (COX) enzymes (ie, COX-1 and COX-2), 2 and (3) [3][4][5][6][7][8][9] Two isoforms of COX (COX-1 and COX-2) have been cloned and characterized. 2 COX-1 is considered a "housekeeping gene" by virtue of constitutive low levels of expression in most cell types.Differently, the gene for COX-2 is a primary response gene with many regulatory sites; thus, COX-2 expression can be rapidly induced by bacterial endotoxin (lipopolysaccharide), cytokines, such as interleukin (IL)-1␤, and tumor necrosis factor (TNF)-␣, growth factors, and tumor promoter phorbol myristate acetate. 10 However, COX-2...

In end-stage renal disease (ESRD) endothelium may represent a key target for the action of circulating elements, such as modified erythrocytes (RBC) and/or plasmatic factors, that may facilitate inflammation and the vasculopathy associated with uremia. We have previously demonstrated that phosphatidylserine (PS) exposure on the surface of RBC from ESRD patients increases RBC-human umbilical vein endothelial cell (HUVEC) interactions and causes decreased nitric oxide (NO) production. We postulated that, besides the pro-inflammatory effects due to decreased NO bio-availability, enhanced ESRD-RBC-HUVEC interactions might directly stimulate proinflammatory pathways leading to increased vascular adhesion molecule expression. ESRD-RBC-endothelial cell interactions induced a time-dependent up-regulation of VCAM-1 and ICAM-1 (measured by Western blot (WB) and real-time PCR), associated with mitogenactivated protein kinase (MAPK) activation and impairment of the Akt/endothelial nitric oxide synthase (eNOS) signaling cascade, measured by WB. In reconstitution experiments, normal RBC incubated with uremic plasma showed increased PS exposure and significantly increased VCAM-1 and ICAM-1 mRNA levels when incubated on HUVEC. Interestingly, ESRD-RBC induced increased expression of adhesion molecules was prevented by Annexin-V (AnV, able to mask PS on RBC surface), anti-integrin-avb3, antithrombospondin-1 (TSP-1), and PD98059 (a selective inhibitor of MAPK phosphorylation). Moreover, AnV reversed the ESRD-RBC effects on MAPK and Akt/eNOS signaling pathways. Our data demonstrate that, possibly via a direct interaction with the endothelial thrombospondin-(avb3) integrin complex, ESRD-RBC-HUVEC adhesion induces a vascular inflammatory phenotype. Thus, intervention targeting ESRD-RBC increased adhesion to endothelium and/or MAPK and Akt/eNOS pathways may have the potential to prevent vascular lesions under uremic conditions.