Knowledge on the functional properties of tumor‐derived endothelial cells (TEC) can be relevant for the development of antiangiogenic therapeutic strategies. In the present study, we obtained and characterized endothelial cell lines from human renal carcinomas. TEC did not undergo senescence and showed constant expression of markers of endothelial activation and angiogenesis. In vitro, TEC, in contrast to normal endothelial cells, were resistant to apoptosis, proadhesive for renal carcinoma cells, and able to grow and organize in the absence of serum in persistent capillary‐like structures. In vivo, TEC were able to grow in immunodeficient mice and to form vascular structures connected with the circulation. At a molecular level, gene array analysis showed an increased expression of genes involved in survival and cell adhesion compared with expression in normal microvascular endothelial cells. Moreover, expression of angiopoietin‐1 and vascular endothelial growth factor (VEGF)‐D and the Akt survival pathway were up‐regulated. Inhibition of interaction of VEGFR‐2 or VEGFR‐3 with VEGF‐D but not of Tie‐2‐angiopoietin‐1 interaction with soluble receptors abrogated Akt activation and survival of TEC. These results indicate that at least some of the TEC within a tumor display abnormal characteristics in terms of survival and angiogenic properties and also indicate the presence of a functional autocrine pathway related to VEGF‐D.

In this study we found that Tat protected vincristinetreated 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 antivascular 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.

CD40 activation by CD154 may trigger diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death, in normal and malignant cells. However, the pathophysiologic role of CD154 expressed by tumor cells remains unclear. We have investigated the expression of the CD40-CD154 system in 24 primary cultures derived from renal cell carcinomas, its correlation with tumor stage and its potential functional significance. We found coexpression of CD40 and CD154 in most of the renal carcinoma cell lines. CD154, but not CD40 expression, significantly correlated with tumor stage. Moreover, renal carcinoma cell lines also released the soluble form of CD154 into the supernatant. CD40 engagement by CD154 did not affect apoptosis or survival. On the contrary, CD154 stimulated cell proliferation, motility and production of PAF, a phospholipid mediator of inflammation with angiogenic properties. Key words: CD154; platelet-activating factor; renal carcinoma; CD40; angiogenesis CD154, the ligand of CD40, is a type II integral membrane protein, related to TNF and FasL. 1 Its receptor, CD40, is a 50 kDa transmembrane glycoprotein of the TNF superfamily, which is expressed in a multitude of different cell types, including B cells, monocytes, dendritic cells, endothelial cells, fibroblasts and renal epithelial cells. 2,3 CD40 was originally identified in a bladder carcinoma cell line, 4 and it is also expressed in a number of carcinomas, such as ovarian, nasopharyngeal, bladder, lung, colon and breast carcinomas, whereas normal nonproliferating tissues are CD40-negative. 5 CD40 engagement by CD154 conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and apoptosis. 6,7 The CD40 cytoplasmic C terminus lacks intrinsic kinase activity and adapter proteins of the TRAF family appear to mediate the activation of CD40-signaling cascades. 8 Despite the large interest, the functional role of CD40 in cancer development remains undetermined. CD40 appears to possess an opposite effect on tumor cell survival and apoptosis. CD40-mediated signaling has been reported to induce apoptosis when expressed in certain transformed cells of mesenchymal origin 9 and in carcinomas of the breast and lung. 10,11 Conversely, Jakobson et al. 12 demonstrated that CD40 stimulation inhibits Fas-mediated apoptosis in human bladder carcinoma cells and we showed a similar protective effect of CD40 in Kaposi's sarcoma cells. 13 Moreover, immunohistochemic studies revealed that detection of CD40 in primary cutaneous malignant melanoma might have negative prognostic value, suggesting its role in tumor progression. 14 CD154 is coexpressed with CD40 in melanoma cells and some carcinomas. 10,11,14 However, the pathophysiologic role of CD40 -CD154 interaction within the tumor cell remains unclear.In the present study, we investigated expression of the CD40 -CD154 system in 24 primary cultures derived from renal clear cell carcinomas, its correlation with tumor st...

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 A2 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.

In autosomal dominant polycystic kidney disease, cysts arise focally and disrupt normal renal tissue leading to renal failure. In the present study, we show that cyst-lining cells express the stem cell marker CD133. CD133+ progenitor cells isolated from polycystic kidney, carrying mutations of PKD genes, showed a dedifferentiated phenotype similar to CD133+ progenitor cells from normal kidney. However, these cells were more proliferative and presented a defective epithelial differentiation phenotype with respect to normal renal CD133+ cells as they were not able to express all tubular epithelial cell markers when cultured in epithelial differentiation medium. Polycystic CD133+ cells, in contrast to normal renal CD133+ cells, formed cysts in vitro in a three-dimensional culture system and in vivo when injected subcutaneously within Matrigel in SCID mice. Rapamycin treatment reduced in vitro proliferation of polycystic CD133+ cells and decreased cystogenesis both in vitro and in vivo. The in vitro epithelial differentiation was only partially improved by rapamycin. These results indicate that polycystic CD133+ cells retain a dedifferentiated phenotype and the ability to generate cysts.

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.

Idiopathic membranous nephropathy (iMN) is considered an immune-mediated disease where circulating autoantibodies against podocyte targets (mainly the PLA2R) cause the deposition of in-situ subepithelial immune-complexes. The consequent podocyte damage may cause cell detachment in urine (Podocyturia-PdoU). PdoU has been assessed in different kidney diseases, but limited data are available in iMN. In this study all patients with a diagnosis of iMN between 15/12/1999–16/07/2014 were tested for PLA2R antibodies (Ab anti-PLA2R, ELISA kit) and PdoU by flow cytometry with anti-podocalyxin antibody. A semi-quantitative PdoU score was defined according to the percentage of podocalyxin positive cells normalized to the total volume of sample and set relative to the urine creatinine measured in the supernatant. PdoU was positive in 17/27 patients (63%; 1+ score in 6/27—22.2%, 2+ in 4/27—14.8%, 3+ in 2/27—7.4%, 4+ in 5/27—18.5%). Only 2/7 patients with complete remission showed a positive PdoU (1+) while all six patients without remission have significant PdoU. PdoU+ was statistically correlated with the absence of remission and Ab anti-PLA2R + (p < 0.05) but PdoU, analysed as a continuous variable, showed a non-linear correlation with proteinuria or PLA2R antibody levels also in the cohort of patients with two available PdoU tests. In conclusion, PdoU could be detected in iMN and seems to be associated with commonly considered markers of disease activity (proteinuria and Ab anti-PLA2R) with a non-linear correlation. Despite data should be confirmed in large and prospective cohorts, according to the podocyte depletion hypothesis PdoU may represent an early marker of immunological activation with potential prognostic utility.

Disruption of the CD40-CD154 interaction was found to be effective in the prevention and treatment of several immune-mediated diseases. The antibody-based strategy of inhibition was in humans limited by platelet activation leading to thrombotic effects. Other strategies different from antibody technology may be useful to create tools to interfere with CD40-CD154 pathway. In the present study, we selected and characterized from a phage display library, cyclic hepta-peptides specific for human CD154 through biopanning against plate-immobilized recombinant hCD154-muCD8. Nine phage clones were selected for the ability to bind CD154 expressed on the surface of J558L cells transfected with human CD154. From the nine selected phage clones, we obtained seven different amino acidic sequences, and the corresponding hepta-peptides rendered cyclic by two cysteines were synthesized. All the peptides specifically bound CD154 expressed on J558L. However, only the peptide 4.10 (CLPTRHMAC) was found to recognize the active binding site of CD154, as it competed with the blocking anti-CD154 antibody. When changes in the amino acid composition were introduced in the sequence of 4.10 peptide, the binding to CD154 was abrogated, suggesting that the amino acid sequence was critical for its specificity. This peptide was found to inhibit the CD40-CD154 interaction, preventing CD40-dependent activation of B lymphocytes in vitro as it was able, as the blocking anti-human CD154 mAb, to prevent the expression of CD80 and CD86 costimulatory molecules and switching of Ig isotype induced by CD154. Moreover, the peptide 4.10 inhibited the in vitro endothelial cell motility and organization into capillary-like structures, and the in vivo angiogenesis of human umbilical cord-derived endothelial cells implanted in Matrigel in severe combined immunodeficiency mice. In vitro studies on platelet activation demonstrated that the 4.10 peptide, at variance of the anti-CD154 mAb, was unable to prime human platelet activation and aggregation. In conclusion, we identify a cyclic hepta-peptide able to displace the binding of human CD154 to CD40 expressed on cell surface and to abrogate some biological effects related to the CD40 stimulation, such as B cell activation and endothelial triggered angiogenesis.