Marina Strasly

Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2.

Interaction between integrin αvβ3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin‐mediated outside‐in signals co‐operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between αvβ3 integrin and tyrosine kinase vascular endothelial growth factor receptor‐2 (VEGFR‐2) in human endothelial cells. We report that tyrosine‐phosphorylated VEGFR‐2 co‐immunoprecipitated with β3 integrin subunit, but not with β1 or β5, from cells stimulated with VEGF‐A165. VEGFR‐2 phosphorylation and mitogenicity induced by VEGF‐A165 were enhanced in cells plated on the αvβ3 ligand, vitronectin, compared with cells plated on the α5β1 ligand, fibronectin or the α2β1 ligand, collagen. BV4 anti‐β3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR‐2; (ii) the activation of downstream transductor phosphoinositide 3‐OH kinase; and (iii) biological effects triggered by VEGF‐A165. These results indicate a new role for αvβ3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, αvβ3 integrin participates in the full activation of VEGFR‐2 triggered by VEGF‐A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.

IL-12 Inhibition of Endothelial Cell Functions and Angiogenesis Depends on Lymphocyte-Endothelial Cell Cross-Talk

In vivo IL-12-dependent tumor inhibition rests on the ability of IL-12 to activate a CD8-mediated cytotoxicity, inhibit angiogenesis, and cause vascular injury. Although in vivo studies have shown that such inhibition stems from complex interactions of immune cells and the production of IFN-γ and other downstream angiostatic chemokines, the mechanisms involved are still poorly defined. Here we show that IL-12 activates an anti-angiogenic program in Con A-activated mouse spleen cells (activated spc) or human PBMC (activated PBMC). The soluble factors they release in its presence arrest the cycle of endothelial cells (EC), inhibit in vitro angiogenesis, negatively modulate the production of matrix metalloproteinase-9, and the ability of EC to adhere to vitronectin and up-regulate ICAM-1 and VCAM-1 expression. These effects do not require direct cell-cell contact, yet result from continuous interaction between activated lymphoid cells and EC. We used neutralizing Abs to show that the IFN-inducible protein-10 and monokine-induced by IFN-γ chemokines are pivotal in inducing these effects. Experiments with nu/nu mice, nonobese diabetic-SCID mice, or activated spc enriched in specific cell subpopulations demonstrated that CD4+, CD8+, and NK cells are all needed to mediate the full anti-angiogenetic effect of IL-12.

IL-12 Regulates an Endothelial Cell-Lymphocyte Network: Effect on Metalloproteinase-9 Production

IL-12 is key cytokine in innate immunity and participates in tumor rejection by stimulating an IFN-γ-mediated response characterized by CD8+ mediated-cytotoxicity, inhibition of angiogenesis, and vascular injury. We previously demonstrated that activated lymphocytes stimulated with IL-12 induced an angiostatic program in cocultured vascular endothelial cells. In this study, we have extended this observation showing that a reciprocal modulation of cellular responses occurs. Actually, the presence of endothelial cells enhanced the inhibitory effect of IL-12 on metalloproteinase-9 expression in activated PBMC as well as their ability to transmigrate across an extracellular matrix. IL-12 triggered intracellular signaling, as indicated by STAT-1 activation, appeared to mainly operative in activated CD4 + cells challenged with IL-12, but it was also initiated in CD8+ lymphocytes in the presence of endothelial cells. On the other hand, stimulated PBMC reduced the expression and the activity of metalloproteinase-9, up-regulated that of tissue inhibitor metalloproteinase-1, and stimulated the STAT-1 pathway in cocultured endothelial cells. We used neutralizing Abs to show that the IFN-inducible protein 10 (CXCL10) and monokine-induced by IFN-γ (CXCL9) chemokines produced by both PBMC and endothelial cells are pivotal in inducing these effects. Altogether these results suggest the existence of an IL-12-regulated circuit between endothelium and lymphocytes resulting in a shift of proteolytic homeostasis at site of tissue injury.

Removal of constitutive and inducible nitric oxide synthase-active compounds in a modified hemodiafiltration with on-line production of substitution fluid: The contribution of convection and diffusion

Chronic renal failure and the uremic state lead to accumulation of various endogenous inhibitors of nitric oxide synthase. Previous studies on end-stage uremic patients nitric oxide synthase activity in murine vascular endothelium and cytokine-induced macrophage cell lines was shown to be modulated during treatment (Nephrol Dial Transplant 1995; 10: 1386-96). Paired filtration dialysis, a modified hemodiafiltration technique, physically separates convection from diffusion. Plasmas, ultrafiltrates and dialysates from seven uremic patients undergoing paired filtration dialysis performed using ultrapure apyrogen substitution fluid in the absence (first 120 min) or presence (last 120 min) of extracellular fluid reduction were tested for their inhibitory/stimulatory effect on ecNOS, constitutively expressed on t. End 1 cell line, a murine vascular endothelium, or for their inducing effect on iNOS, inducible on J774 cells, a macrophage cell line. On ecNOS, Group 1 (stimulatory, 3/7 patients) markedly enhanced the ecNOS activity as compared to control plasma, whereas group 2 plasma (inhibitory, 4/7 patients) inhibited ecNOS plasma. Post-dialysis plasma samples from all Group 1 and 2 patients showed a marked decrease of the predialysis stimulatory and inhibitory activity, respectively. On iNOS: all patient plasmas stimulated iNOS activity. The UF and particularly the dialysate had a remarkable iNOS inducing effect (Group 1). The substitution fluid obtained at 120 min during treatment in Group 1 and 2 had no effect on NOS activity. No correlation was found between predialysis ecNOS or iNOS activity values with mean systolic or diastolic pressures. These studies suggest a complex balance of ecNOS inhibitors/stimulators and iNOS inducers in uremia. Dialysis may remove ecNOS inhibitors and stimulators by convection and, in the latter case, by diffusion. iNOS inducers are removed during dialysis, suggesting the biocompatibility of the dialysis system with the on-line production of ultrapure substitution fluid.

IL-12-dependent innate immunity arrests endothelial cells in G0-G1 phase by a p21 Cip1/Waf1 -mediated mechanism

Innate immunity may activate paracrine circuits able to entail vascular system in the onset and progression of several chronic degenerative diseases. In particular, interleukin (IL)-12 triggers a genetic program in lymphomononuclear cells characterized by the production of interferon-γ and specific chemokines resulting in an angiostatic activity. The aim of this study is to identify molecules involved in the regulation of cell cycle in endothelial cells co-cultured with IL-12-stimulated lymphomonuclear cells. By using a transwell mediated co-culture system we demonstrated that IL-12-stimulated lymphomonuclear cells induce an arrest of endothelial cells cycle in G1, which is mainly mediated by the up-regulation of p21Cip1/Waf1, an inhibitor of cyclin kinases. This effect requires the activation of STAT1, PKCδ and p38 MAPK, while p53 is ineffective. In accordance, siRNA-dependent silencing of these molecules in endothelial cells inhibited the increase of p21Cip1/Waf1 and the modification in cell cycle promoted by IL-12-stimulated lymphomonuclear cells. These results indicate that the angiostatic action of IL-12-stimulated lymphomononuclear cells may lie in the capability to arrest endothelial cells in G1 phase through a mechanisms mainly based on the specific up-regulation of p21Cip1/Waf1 induced by the combined activity of STAT1, PKCδ and p38 MAPK.