Ccbe1 function · Characterization of the role of CCBE1 during development and disease

Characterization of the role of CCBE1 during development and disease

The lymphatic vasculature is crucial for fluid homeostasis and immune function, and has a central role in many pathological conditions. During development, lymphangiogenesis is regulated by one major signaling pathway, Vegfc-Vegfr3, as well as by the secreted factor Ccbe1. Importantly, CCBE1 has been associated to Hennekam Syndrome where patients suffer from lymphedema, cardiovascular anomalies and mental retardation. During the project, we have investigated the biological role of Ccbe1 and Vegfc on a mechanistic level. We have found, that Ccbe1 is a crucial regulator of mature bioavailable Vegfc. We have established an in vivo assay expressing Vegfc and Ccbe1 in the floorplate of zebrafish to assess the biological function of these two molecules. Overexpression of Vegfc in the floorplate of zebrafish induces hypersprouting of veins and lymphatic vessels, while overexpression of Ccbe1 alone has no effect on vessels. Combined overexpression of both factors, Vegfc and Ccbe1, enhances Vegfc-driven sprouting and leads to bilateral sprouting of arteries and additional vessel formation. In absence of Ccbe1, Vegfc driven hypersprouting is suppressed. Furthermore, Ccbe1 and Vegfc double heterozygous embryos showed an increased frequency of lymphatic defects compared to single heterozygous embryos, consistent with genetic interaction in the same pathway. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo. Our findings that Ccbe1 is a critical modulator of the Vegfc/Vegfr3 pathway during embryo development, suggest that a deficiency of VEGFR3 signaling may be responsible for the lymphatic aspects of Hennekam Syndrome. In addition we have used the in vivo assay expressing VEGFC in the floorplate of zebrafish to assess the biological function of a VEGFC mutant in vivo. This mutant, a novel frameshift variant, c.571_572insTT in VEGFC was identified in patients with clinical signs resembling Milroy disease. Forced expression of wildtype human VEGFC in the floorplate of zebrafish embryos induces excessive lymphovenous hypersprouting. However, when overexpressing the human c.571_572insTT variant in the floorplate, no hypersprouting of neighboring vessels was observed. Furthermore, secretion of this mutant variant when expressed in vitro in 293T cells is strongly impaired as compared to wildtype VEGFC. Thus, the mutation has a marked effect on the activity of VEGFC indicating that the mutation in VEGFC is causative for the MD-like phenotype seen in this family. The topic of lymphangiogenesis is at the forefront of biomedical science and important in the context of various diseases such as tumor metastasis, inflammatory diseases or lymphedema. Thus, analysis of regulators of lymphangiogenesis is essential for the treatment and prevention of such diseases. Ccbe1 was recently discovered by the host lab as a key regulator of lymphangiogenesis and involved in the cause of a genetically inherited disease, Hennekam Syndrome. We have uncovered the molecular mechanisms of mode of action of CCBE1 as a critical regulator of VEGFC bioavailablility. This greatly increases understanding the role of CCBE1 in development and disease and is an important contribution to the field of lymphangiogenesis, thereby strengthening the European excellence in this research area. In addition we could identify we have identified a mutation in VEGFC to be causative for a Milroy Disease-like phenotype, the first time time a mutation in one of the ligands of VEGFR3 has been reported to cause primary lymphedema. These findings can lead to the development of new therapies for the treatment of various diseases related to lymphatic function.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз