RESUMEN
The WNT signaling enhancer R-spondin3 (RSPO3) is prominently expressed in the vasculature. Correspondingly, embryonic lethality of Rspo3-deficient mice is caused by vessel remodeling defects. Yet the mechanisms underlying vascular RSPO3 function remain elusive. Inducible endothelial Rspo3 deletion (Rspo3-iECKO) resulted in perturbed developmental and tumor vascular remodeling. Endothelial cell apoptosis and vascular pruning led to reduced microvessel density in Rspo3-iECKO mice. Rspo3-iECKO mice strikingly phenocopied the non-canonical WNT signaling-induced vascular defects of mice deleted for the WNT secretion factor Evi/Wls. An endothelial screen for RSPO3 and EVI/WLS co-regulated genes identified Rnf213, Usp18, and Trim30α. RNF213 targets filamin A and NFAT1 for proteasomal degradation attenuating non-canonical WNT/Ca(2+) signaling. Likewise, USP18 and TRIM5α inhibited NFAT1 activation. Consequently, NFAT protein levels were decreased in endothelial cells of Rspo3-iECKO mice and pharmacological NFAT inhibition phenocopied Rspo3-iECKO mice. The data identify endothelial RSPO3-driven non-canonical WNT/Ca(2+)/NFAT signaling as a critical maintenance pathway of the remodeling vasculature.
Asunto(s)
Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Transducción de Señal/genética , Trombospondinas/metabolismo , Factores de Transcripción/metabolismo , Animales , Calcio/metabolismo , Ratones Transgénicos , Factores de Transcripción NFATC/metabolismo , Trombospondinas/genética , Proteínas Wnt/metabolismoRESUMEN
Multiple cell types involved in the regulation of angiogenesis express Wnt ligands. Although ß-catenin dependent and independent Wnt signaling pathways have been shown to control angiogenesis, the contribution of individual cell types to activate these downstream pathways in endothelial cells (ECs) during blood vessel formation is still elusive. To investigate the role of ECs in contributing Wnt ligands for regulation of blood vessel formation, we conditionally deleted the Wnt secretion factor Evi in mouse ECs (Evi-ECKO). Evi-ECKO mice showed decreased microvessel density during physiological and pathological angiogenesis in the postnatal retina and in tumors, respectively. The reduced microvessel density resulted from increased vessel regression accompanied by decreased EC survival and proliferation. Concomitantly, survival-related genes were downregulated and cell cycle arrest- and apoptosis-inducing genes were upregulated. EVI silencing in cultured HUVECs showed similar target gene regulation, supporting a mechanism of EC-derived Wnt ligands in controlling EC function. ECs preferentially expressed non-canonical Wnt ligands and canonical target gene expression was unaffected in Evi-ECKO mice. Furthermore, the reduced vascularization of Matrigel plugs in Evi-ECKO mice could be rescued by introduction of non-canonical Wnt5a. Treatment of mouse pups with the non-canonical Wnt inhibitor TNP470 resulted in increased vessel regression accompanied by decreased EC proliferation, thus mimicking the proliferation-dependent Evi-ECKO remodeling phenotype. Taken together, this study identified EC-derived non-canonical Wnt ligands as regulators of EC survival, proliferation and subsequent vascular pruning during developmental and pathological angiogenesis.
Asunto(s)
Células Endoteliales/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica , Proteínas Wnt/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/genética , Recuento de Células , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclohexanos/farmacología , Proteínas de Unión al ADN/metabolismo , Células Endoteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ligandos , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Ratones Transgénicos , Modelos Biológicos , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , O-(Cloroacetilcarbamoil) Fumagilol , Fenotipo , Proto-Oncogenes , Retina/crecimiento & desarrollo , Retina/metabolismo , Sesquiterpenos/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
Telomerase reverse transcriptase (TERT) is fundamental in determining the life span by regulating telomere length of chromosomes. To address the question whether the enhancement of the proliferative potential hampers cell differentiation, we generated TERT-over-expressing endothelial cells (ECs) and analyzed in vitro their (1) barrier function; (2) low-density lipoprotein uptake; (3) expression pattern of six selected marker proteins; (4) angiogenic potential in four assays; and (5) inflammatory responses. In contrast to investigations with focus on other cell parameters, we demonstrate that immortalization of ECs by over-expression of TERT resulted in different angiogenic and inflammatory behavior in comparison to cells with low native telomerase levels.