Lymphangiogenomics in Xenopus tadpoles and in mice to elucidate the role of VEGF-D

Blood and lymphatic vessels form part of a circulatory system, which isessential to meet the high metabolic demands of the organisms, byallowing the transport of nutrients, cells and proteins in the body.Importantly, the lymphatic system is responsible for collecting andreturning the fluid that becomes trapped in the tissues of the body tothe blood vascular system. If the draining of this fluid (on average 2liters/day) would fail, our lives would be in danger. The lymphaticsystem is also the first line of defense against disease and infection,and lymph vessels of the gastrointestinal tract are crucial forabsorbing the dietary fat.A malfunction of the lymphatic system can lead to a severe conditioncalled lymphedema, a serious swelling, most often in the arms or legs,caused by the accumulation of fluid in the interstitial tissue. Thisdisfiguring disorder is currently untreatable and remains a lifelongdisability, which affects 170 million people worldwide. Lymphaticvessels are also associated with cancer, one of the most importantmedical concerns in our days and critically contribute to malignantprogression and death in cancer patients. Cancer cells can spread toother parts of the body using the lymphatics as a route, throughprocesses that are yet to be understood. Learning more about thelymphatic system and the players controling its formation will provideimportant clues to understand the mechanisms of cancer metastasis.Although the lymphatic system has been identified almost 400 years ago,the current knowledge about lymphatic biology is still very poor. Oneof the reasons has been the lack of an appropriate and affordableanimal model, which could be genetically manipulated in order torapidly unravel the function of genes involved in lymphatic vesselformation (lymphangiogenesis) and discover new molecular players inthis process, and thereby opening novel therapeutic avenues.Pioneer results from our group show that Xenopus (the African clawedfrog) possesses a well organized lymphatic system from early embryonicstages onwards. In the present study, we established Xenopus tadpolesas a novel genetic model to study the molecular basis oflymphangiogenesis by silencing (knocking-down) the expression of knownlymphangiogenic genes. Indeed, silencing the key lymphatic playersProx1 or VEGF-C impaired the formation of the lymphatic vessels andresulted in lymphedema. The usefulness and relevance of the tadpolemodel to study the role of lymphangiogenic candidates was evidenced bythe striking resemblance of these phenotypes in tadpoles to thecorresponding similar phenotypes in mutant mice lacking the same genes.VEGF-D, a close relative of VEGF-C, has been recently identified as apowerful lymphangiogenic factor. Both growth factors are ligands of theVEGF receptor-3, expressed on lymphatic endothelial cells. However, therole of endogenous VEGF-D in embryonic and pathological conditionsremained largely unknown. We therefore decided to unravel the role ofVEGF-D using our newly characterized Xenopus model. Knock-down ofVEGF-D resulted in a slight defect in the lymphatic cell migration,though not sufficiently severe to affect the functionality of thelymphatic system. In a next step, we took advantage of a uniqueopportunity offered by the tadpole model, i.e. the possibility ofstudying genetic interactions in an easy and rapid manner. Bysimultaneously silencing several lymphangiogenic genes, we were able todemonstrate that VEGF-D, VEGF-C and Prox1 cooperatively regulatelymphangiogenesis.While gene manipulation in mice, in contrast to the tadpole, is morelaborious, time-consuming and expensive, allowing only analysis on agene-per-gene basis, the mouse undeniably is an excellent model, moreclosely related to humans and valuable to study lymphangiogenenesis indisease. VEGF-D deficient mice have been generated and were availablein our laboratory. Consequently, we also studied the role of VEGF-D inphysiological and pathological conditions in these gene-targetedmice. However, no overt lymphangiogenesis defects were observedin the absence of VEGF-D in physiological conditions in embryonic orpostnatal stages, nor in the stress or pathological models performed inadult animals (skin wound healing, healing of myocardial infarcts,lymphangioma formation).Thus, this combined genetic study in mice and Xenopus tadpolesindicated that endogenous VEGF-D is only minimally involved inembryonic development, and in normal health after birth in mice. Inaddition, the mouse data revealed no overt lymphangiogenesis defects instress and pathological conditions in the absence of VEGF-D. A finding,not previously recognized in mouse mutants, is that, though their roleis not as important as that of Prox1, both VEGF-C and VEGF-Dco-determined the formation of lymphatic vessels.status: publishe