RESUMEN
Proangiogenic, proliferative effects of tumors have been extensively characterized in subconfluent endothelial cells (EC), but results in confluent, contact-inhibited EC are critically lacking. The present study examined the effect of tumor-conditioned medium (CM) of the malignant osteoblastic cell line MG63 on monolayer, quiescent bovine aorta EC. MG63-CM and MG63-CM + CoCl(2) significantly increased EC survival in serum-starved conditions, without inducing EC proliferation. Furthermore, MG63-CM and MG63-CM + CoCl(2), both containing high amounts of vascular endothelial growth factor (VEGF), induced relevant phenotypic changes in EC (all P < 0.01) involving increase of nucleoli/chromatin condensations, nucleus-to-cytosol ratio, capillary-like vacuolated structures, vessel-like acellular areas, migration through Matrigel, growth advantage in reseeding, and factor VIII content. All these actions were significantly inhibited by VEGF and VEGF receptor (VEGFR2) blockade. Of particular importance, a set of similar effects were detected in a human microvascular endothelial cell line (HMEC). With regard to gene expression, incubation with MG63-CM abolished endogenous VEGF mRNA and protein but induced a clear-cut increase in VEGFR2 mRNA expression in EC. In terms of mechanism, MG63-CM activates protein kinase B (PKB)/Akt, p44/p42-mitogen-activated protein kinase (MAPK)-mediated pathways, as suggested by both inhibition and phosphorylation experiments. In conclusion, tumor cells activate confluent, quiescent EC, promoting survival, phenotypic, and gene expression changes. Of importance, VEGF antagonism converts MG63-CM from protective to EC-damaging effects.
Asunto(s)
Medios de Cultivo Condicionados/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Aorta , Bovinos , Línea Celular Tumoral/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
BACKGROUND: Recent studies have shown that exogenous administration of vascular endothelial growth factor (VEGF) is protective against cyclosporine A (CsA) renal toxicity. No data are available, however, on the possible role of endogenous VEGF. Our objective was to examine whether endogenous VEGF has a significant role in the renal response against CsA toxicity. METHODS: In vivo, we used high-dose (50-150 mg/kg/day) CsA +/- specific goat anti-mouse VEGF blocking monoclonal antibody (alpha-VEGF) in mice. In vitro, we exposed mouse tubular cells (MCT) to CsA +/- alpha-VEGF. RESULTS: alpha-VEGF markedly enhanced CsA renal toxicity, inducing severe tubular damage and increased blood urea nitrogen. In animals treated with CsA + alpha-VEGF, damage progressed to generalized tubular injury (histology) and apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling) with associated anemia and reticulocytosis (18 days of treatment). CsA + alpha-VEGF treatments strikingly increased tubular VEGF and Bcl-xL proteins. In vitro, autocrine production of VEGF by MCT was identified by Western blot. Of specific interest, CsA toxicity in MCT increased significantly in the presence of alpha-VEGF. CONCLUSIONS: Endogenous VEGF has a relevant role in the renal tubular defense against CsA toxicity. Blockade of the VEGF effect by alpha-VEGF results in clear-cut intensification of the tubular injury and appearance of regenerative anemia in the CsA + alpha-VEGF-treated animals. The occurrence of both in vivo and in vitro effects of VEGF blockade provides evidence of a direct protective effect of VEGF on the tubular cell.
Asunto(s)
Ciclosporina/envenenamiento , Citoprotección/fisiología , Factores de Crecimiento Endotelial/fisiología , Inmunosupresores/envenenamiento , Péptidos y Proteínas de Señalización Intercelular/fisiología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/fisiología , Linfocinas/fisiología , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/farmacología , Western Blotting , Células Cultivadas , Sinergismo Farmacológico , Factores de Crecimiento Endotelial/inmunología , Femenino , Péptidos y Proteínas de Señalización Intercelular/inmunología , Túbulos Renales/patología , Linfocinas/inmunología , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Proteína bcl-XRESUMEN
The relative importance of cyclophilin (CyP) versus calcineurin (Cn)-mediated mechanisms in the effect of cyclosporin A (CsA) on endothelial cells (ECs) is largely unknown. In cultured ECs, CsA was cytotoxic/proapoptotic or cytoprotective/antiapoptotic at high or low concentrations, respectively. CsA analogs (MeVal-4-CsA and MeIle-4-CsA), which bind to CyP but do not inhibit Cn, closely reproduced the CsA effects. Based on our previous data, the role of vascular endothelial growth factor (VEGF) as a mediator of CsA-induced cytoprotection was further analyzed. The actions of CsA and CsA analogs were shifted from a protective to a cell-damaging pattern in the presence of a specific anti-VEGF monoclonal antibody (mAb). This positive interaction was further supported by a transient increase in cytosolic free calcium concentration ([Ca(2+)](i)) by VEGF after pretreatment with either CsA or MeVal-4-CsA and an increase in the expression and synthesis of VEGF receptor 2 (VEGFR2). Of functional importance, blockade of the interaction between VEGF and VEGFR2 by a VEGFR2 mAb abolished the cytoprotective effect of CsA. In addition, preconditioning with low concentrations of CsA or CsA analogs increased both cytoprotection and VEGFR2 mRNA expression when EC were exposed to higher concentrations of CsA. In summary, our results reveal that (1) the biphasic responses to CsA in EC are related to the interaction of CsA with CyP rather than with Cn and (2) VEGF is a critical factor in the cytoprotective effect of CsA, by a mechanism that involves VEGFR2.
Asunto(s)
Ciclofilinas/fisiología , Ciclosporina/farmacología , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/efectos de los fármacos , Linfocinas/fisiología , Proteínas Nucleares , Transducción de Señal , Animales , Calcineurina/fisiología , Calcio/metabolismo , Bovinos , Células Cultivadas , Ciclosporina/toxicidad , Citoprotección , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/metabolismo , Cinética , Linfocinas/genética , Linfocinas/farmacología , Factores de Transcripción NFATC , ARN Mensajero/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/fisiología , Receptores de Factores de Crecimiento Endotelial Vascular , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: Hydrogen peroxide (H2O2) formation is a critical factor in processes involving ischaemia/ reperfusion. However, the precise mechanism by which reactive oxygen species (ROS) induce vascular damage are insufficiently known. Specifically, activation of phospholipase C gamma (PLCgamma) is a probable candidate pathway involved in vascular smooth muscle cells (VSMC) activation by H2O2. METHODS: The activation of human venous VSMC was measured as cytosolic free calcium mobilization, shape change and protein phosphorylation, focusing on the role of tyrosine phosphorylation-activated PLCgamma. RESULTS: The exposure of VSMC to exogenous H2O2 caused a rapid increase in cytosolic free calcium concentration ([Ca2+]i), and induced a significant VSMC shape change. Both effects were dependent on a tyrosine kinase-mediated mechanism, as determined by the blockade of short-term treatment of VSMC with the protein tyrosine kinase inhibitor, genistein. Giving further support to the putative role of phospholipase C (PLC)-dependent pathways, the [Ca2+]i and VSMC shape change response were equally inhibited by the specific PLC blocker, 1-(6-((17-beta-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). In addition, U73122 had a protective effect against the deleterious action (24 h) of H2O2 on non-confluent VSMC. As a further clarification of the specific pathway involved, the exposure to H2O2 significantly stimulated the tyrosine phosphorylation of PLCgamma with a concentration- and time-profile similar to that of [Ca(2+)](i) mobilization. CONCLUSIONS: The present study reveals that H(2)O(2) activates PLCgamma on VSMC through tyrosine phosphorylation and that this activation has a major role in rapid [Ca(2+)](i) mobilization, shape-changing actions and damage by H(2)O(2) in this type of cells. These findings have direct implications for understanding the mechanisms of the vascular actions of H(2)O(2) and may help to design pharmacologically protective strategies.