Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD
Hung M. Bui, … , Kari Alitalo, Mark L. Kahn
Hung M. Bui, … , Kari Alitalo, Mark L. Kahn
Published May 9, 2016
Citation Information: J Clin Invest. 2016;126(6):2167-2180. https://doi.org/10.1172/JCI83967.
View: Text | PDF
Research Article Vascular biology Article has an altmetric score of 3

Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD

Abstract

Lymphangiogenesis is supported by 2 homologous VEGFR3 ligands, VEGFC and VEGFD. VEGFC is required for lymphatic development, while VEGFD is not. VEGFC and VEGFD are proteolytically cleaved after cell secretion in vitro, and recent studies have implicated the protease a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS3) and the secreted factor collagen and calcium binding EGF domains 1 (CCBE1) in this process. It is not well understood how ligand proteolysis is controlled at the molecular level or how this process regulates lymphangiogenesis, because these complex molecular interactions have been difficult to follow ex vivo and test in vivo. Here, we have developed and used biochemical and cellular tools to demonstrate that an ADAMTS3-CCBE1 complex can form independently of VEGFR3 and is required to convert VEGFC, but not VEGFD, into an active ligand. Consistent with these ex vivo findings, mouse genetic studies revealed that ADAMTS3 is required for lymphatic development in a manner that is identical to the requirement of VEGFC and CCBE1 for lymphatic development. Moreover, CCBE1 was required for in vivo lymphangiogenesis stimulated by VEGFC but not VEGFD. Together, these studies reveal that lymphangiogenesis is regulated by two distinct proteolytic mechanisms of ligand activation: one in which VEGFC activation by ADAMTS3 and CCBE1 spatially and temporally patterns developing lymphatics, and one in which VEGFD activation by a distinct proteolytic mechanism may be stimulated during inflammatory lymphatic growth.

Authors

Hung M. Bui, David Enis, Marius R. Robciuc, Harri J. Nurmi, Jennifer Cohen, Mei Chen, Yiqing Yang, Veerpal Dhillon, Kathy Johnson, Hong Zhang, Robert Kirkpatrick, Elizabeth Traxler, Andrey Anisimov, Kari Alitalo, Mark L. Kahn

×

Figure 8

Distinct mechanisms of proteolytic activation support distinct biological roles for VEGFC and VEGFD.

Options: View larger image (or click on image) Download as PowerPoint
Distinct mechanisms of proteolytic activation support distinct biologica...
(A) Molecular mechanisms of VEGFC and VEGFD activation. VEGFC is activated by the formation of a VEGFC-ADAMTS3-CCBE1 complex (left). CCBE1 binding to ADAMTS3 via the CCBE1 CT (left) is predicted to confer a conformational change that permits the enzyme to associate with and cleave VEGFC (middle). N-terminal cleavage of VEGFC releases the VHD that is able to activate VEGFR3 on the LEC. The CCBE1 NT may bind extracellular matrix (ECM) to localize the complex spatially during lymphatic growth. In contrast, VEGFD is activated independently of ADAMTS3 and CCBE1, most likely through a serine protease generated at sites of inflammation (right). (B) Proposed lymphangiogenic roles of VEGFC and VEGFD in vivo. VEGFC activation by ADAMTS3 and CCBE1 provides a mechanism for spatial patterning of the developing lymphatic vasculature (left). Schematic shows the mid-gestation cardinal vein with newly specified LECs that are in the process of sprouting to form the lymphatic network. The area encircled by the dotted line represents a zone of active VEGFC. VEGFD activation by inflammatory proteases such as those generated during wound healing in the skin provides a mechanism for lymphangiogenesis in mature animals with preexisting lymphatic networks (right).