Nuevas herramientas geneticas para el estudio de las vasculatura linfatica y de la linfagiogenesis in vivo

Resumen

The lymphatic system plays a critical role in many physiological and pathological processes such as tissue drainage, nutrient absorption, inflammation, immune response and metastatic spread. Despite of its relevance, little is known about lymphatic vessels development and function. The recent identification of genes that are specifically expressed in the lymphatic endothelium has made possible the generation of new tools that allow the study of this system. One of these genes is Flt4 (Fms-like-tyrosine kinase-4) that encodes for the vascular endothelial growth factor receptor 3 (VEGFR3), considered one of the best markers of lymphatic endothelial cells. We have generated a reporter mouse line in which an EGFP-luciferase fusion protein is expressed under the endogenous transcriptional control of the Flt4 gene without altering physiological VEGFR3 expression or regulation. EGFP expression in this model allows direct visualization of lymphatic vessels by fluorescence detection. In parallel, luciferase expression allows to trace and quantify in vivo lymphangiogenesis associated to different inflammatory processes as well as to trace the recruitment of VEGFR3-expressing cells to inflammation sites. Moreover, this model can be used to monitor tumourinduced lymphangiogenesis in vivo, both at the periphery of the tumour and at distant lymph nodes, and conceivably to predict/estimate the metastatic potential of tumour cells and grafts through the lymphatic system. As far as we know this is the first reporter mouse model for in vivo imaging of lymphangiogenesisinflammation and tumour metastasis in the mouse. In parallel, we have generated a mouse line that expresses the inducible Cre recombinase (CreERT2) under the endogenous transcriptional control of the Flt4 gene. This line provides an excellent genetic tool for the generation of lymphatic-endothelium specific- and conditional- mouse mutants and to study gene function in the lymphatic vasculature. This line has been extensively characterized by crosses with the Crereporter line Rosa26LSLlacZ using different protocols of tamoxifen administration and verified that the Cre recombinase activity is restricted to VEGFR3 positive cells. Moreover, we have used this line to verify, by lineage tracing, that embryonic progenitor cells are recruited to sites of tissue injury (inflammation) in the mother, and they get incorporated into newly generated lymphatic vessels at those sites, something that had not been previously demonstrated. Finally, we have used this genetic tool to study the relevance of hepatocyte growth factor /c-Met signalling in the lymphatic endothelium by inactivating c-Met specifically in lymphatic endothelial cells. The initial results of this study show that c-Met signalling is required for the proper development of the lymphatic system and for lymphatic system homeostasis and lymphagiogenesis in the adult.