Platelet-activating factor mediates CD40-dependent angiogenesis and endothelial-smooth muscle cell interaction.

The aim of the present study was to investigate whether stimulation of CD40 expressed by endothelial or smooth muscle cells triggers the synthesis of platelet-activating factor (PAF), an inflammatory mediator with angiogenic properties, and whether PAF contributes to CD40-induced neoangiogenesis. The results obtained indicate that the interaction of CD40 with soluble CD154 or with CD154 expressed on the membrane of leukocytes (CD154-transfected J558 cells) or of activated platelets, stimulated the synthesis of PAF by endothelial cells but not by smooth cells. The synthesis of PAF triggered by activated platelets was inhibited by a soluble CD40-murine Ig fusion protein that prevents the interaction between membrane CD40 and CD154. Studies with specific inhibitors and evaluation of protein phosphorylation indicated the involvement in PAF synthesis of two intracellular signaling pathways leading to cytosolic phospholipase A 2 activation: a phospholipase Cγ-protein kinase C-Raf-p42/p44-mitogen-activated protein kinase (MAPK) and a MAPK kinase-3/6-dependent activation of p38 MAPK. PAF synthesized by endothelial cells after CD40 stimulation was instrumental in the in vitro migration and vessel-like organization of endothelial cells, and in the interaction between endothelial cells and smooth muscle cells, as inferred by the inhibitory effect of two different PAF receptor antagonists, WEB2170 and CV3988. In vivo, blockade of PAF receptors prevented the angiogenic effect triggered by CD40 stimulation in a murine model of s.c. Matrigel implantation. In conclusion, these observations indicate that PAF synthesis induced by stimulation of endothelial CD40 contributes to the formation and organization of new vessels. This may be relevant in the vascular remodeling associated with tumor and inflammatory neoangiogenesis

HIV-1-Tat protein activates phosphatidylinositol 3-kinase/ AKT-dependent survival pathways in Kaposi's sarcoma cells.

In this study we found that Tat protected vincristine-treated Kaposi's sarcoma cells from apoptosis and from down-regulation of several anti-apoptotic genes such as AKT-1, AKT-2, BCL2, BCL-XL, and insulin-like growth factor I and induced the de novo expression of the interleukin-3 gene. Moreover, we found that Tat enhanced phosphorylation of AKT and BAD proteins. The inhibition of phosphatidylinositol 3-kinase with two unrelated pharmacological inhibitors, wortmannin and LY294002, abrogated both the anti-apoptotic effect and the phosphorylation of AKT induced by Tat. After treatment with Tat, the AKT enzymatic activity showed a biphasic increase: an early activation (15 min), independent from protein synthesis; and a delayed activation (24 h), which was significantly decreased upon blockage of protein synthesis. Experiments with a function blocking anti-vascular endothelial cell growth factor receptor-2 antibody suggested that both the early and delayed AKT activation and the protection from apoptosis were triggered by the interaction of Tat with vascular endothelial cell growth factor receptor-2. Moreover, experiments with function-blocking antibodies directed against insulin-like growth factor I/insulin-like growth factor I receptor or interleukin-3 indicated their involvement in the delayed activation of AKT and their contribution to the anti-apoptotic effect of Tat on vincristine-treated Kaposi's sarcoma cells

Platelet-Activating Factor Mediates CD40-Dependent Angiogenesis and Endothelial-Smooth Muscle Cell Interaction

The aim of the present study was to investigate whether stimulation of CD40 expressed by endothelial or smooth muscle cells triggers the synthesis of platelet-activating factor (PAF), an inflammatory mediator with angiogenic properties, and whether PAF contributes to CD40-induced neoangiogenesis. The results obtained indicate that the interaction of CD40 with soluble CD154 or with CD154 expressed on the membrane of leukocytes (CD154-transfected J558 cells) or of activated platelets, stimulated the synthesis of PAF by endothelial cells but not by smooth cells. The synthesis of PAF triggered by activated platelets was inhibited by a soluble CD40-murine Ig fusion protein that prevents the interaction between membrane CD40 and CD154. Studies with specific inhibitors and evaluation of protein phosphorylation indicated the involvement in PAF synthesis of two intracellular signaling pathways leading to cytosolic phospholipase A 2 activation: a phospholipase Cγ-protein kinase C-Raf-p42/p44-mitogen-activated protein kinase (MAPK) and a MAPK kinase-3/6-dependent activation of p38 MAPK. PAF synthesized by endothelial cells after CD40 stimulation was instrumental in the in vitro migration and vessel-like organization of endothelial cells, and in the interaction between endothelial cells and smooth muscle cells, as inferred by the inhibitory effect of two different PAF receptor antagonists, WEB2170 and CV3988. In vivo, blockade of PAF receptors prevented the angiogenic effect triggered by CD40 stimulation in a murine model of s.c. Matrigel implantation. In conclusion, these observations indicate that PAF synthesis induced by stimulation of endothelial CD40 contributes to the formation and organization of new vessels. This may be relevant in the vascular remodeling associated with tumor and inflammatory neoangiogenesis

Acute Tubular Injury is Associated With Severe Traumatic Brain Injury: in Vitro Study on Human Tubular Epithelial Cells

Abstract Acute kidney injury following traumatic brain injury is associated with poor outcome. We investigated in vitro the effects of plasma of brain injured patients with acute tubular kidney injury on kidney tubular epithelial cell function. we performed a prospective observational clinical study in ICU in a trauma centre of the University hospital in Italy including twenty-three ICU patients with traumatic brain injury consecutively enrolled. Demographic data were recorded on admission: age 39 ± 19, Glasgow Coma Score 5 (3–8). Neutrophil Gelatinase-Associated Lipocalin and inflammatory mediators were measured in plasma on admission and after 24, 48 and 72 hours; urine were collected for immunoelectrophoresis having healthy volunteers as controls. Human renal proximal tubular epithelial cells were stimulated with patients or controls plasma. Adhesion of freshly isolated human neutrophils and trans-epithelial electrical resistance were assessed; cell viability (XTT assay), apoptosis (TUNEL staining), Neutrophil Gelatinase-Associated Lipocalin and Megalin expression (quantitative real-time PCR) were measured. All patients with normal serum creatinine showed increased plasmatic Neutrophil Gelatinase-Associated Lipocalin and increased urinary Retinol Binding Protein and α1-microglobulin. Neutrophil Gelatinase-Associated Lipocalin was significantly correlated with both inflammatory mediators and markers of tubular damage. Patient’ plasma incubated with tubular cells significantly increased adhesion of neutrophils, reduced trans-epithelial electrical resistance, exerted a cytotoxic effect and triggered apoptosis and down-regulated the endocytic receptor Megalin compared to control. Plasma of brain injured patients with increased markers of subclinical acute kidney induced a pro-inflammatory phenotype, cellular dysfunction and apoptotic death in tubular epithelial cells

Prevention of acute rejection after rescue with Belatacept by association of low-dose Tacrolimus maintenance in medically complex kidney transplant recipients with early or late graft dysfunction.

BackgroundIncreased acute rejection risk in rescue protocols with Belatacept may limit its use particularly in medically complex patients where preexisting increased risk of rejection couples with CNI toxicity.MethodsRetrospective analysis was performed in 19 KTs shifted to a Belatacept-based immunosuppression with low-dose Tacrolimus (2-3 ng/mL) after evidence of allograft disfunction, including patients with primary non-function (PNF), chronic-active antibody-mediated rejection (cAMR), history of previous KTs and/or other concomitant transplants (liver, pancreas). Evaluation of CD28+ CD4+ effector memory T cell (TEM) before conversion was performed in 10/19.ResultsKidney function significantly improved (median eGFR 16.5 ml/min/1.73m2 before vs 25 ml/min after; p = 0.001) at a median time after conversion of 12.5 months (9.1-17.8). Overall graft and patient survival were 89.5% and 100% respectively. Definitive weaning from dialysis in 5/5 KTs with PNF was observed, whereas 7/8 patients lost their graft within first year in a control group. eGFR significantly ameliorated in re-trasplants (p = 0.001) and stabilized in KTs with other organ transplants or cAMR. No acute rejection episodes occurred, despite the significant risk suggested by high frequency of CD28+ CD4+ TEM in most patients. Opportunistic infections were limited and most common in early vs late-converted.ConclusionsRescue association of Belatacept with low-dose Tacrolimus in medically complex KTs is a feasible option that allows prevention of acute rejection and amelioration of graft function