title: Lymphatic endothelial heparan sulfate deficiency results in altered growth responses to vascular endothelial growth factor-C (VEGF-C).
abstract: Growth and remodeling of lymphatic vasculature occur during development and during various pathologic states. A major stimulus for this process is the unique lymphatic vascular endothelial growth factor-C (VEGF-C). Other endothelial growth factors, such as fibroblast growth factor-2 (FGF-2) or VEGF-A, may also contribute. Heparan sulfate is a linear sulfated polysaccharide that facilitates binding and action of some vascular growth factors such as FGF-2 and VEGF-A. However, a direct role for heparan sulfate in lymphatic endothelial growth and sprouting responses, including those mediated by VEGF-C, remains to be examined. We demonstrate that VEGF-C binds to heparan sulfate purified from primary lymphatic endothelia, and activation of lymphatic endothelial Erk1/2 in response to VEGF-C is reduced by interference with heparin or pretreatment of cells with heparinase, which destroys heparan sulfate. Such treatment also inhibited phosphorylation of the major VEGF-C receptor VEGFR-3 upon VEGF-C stimulation. Silencing lymphatic heparan sulfate chain biosynthesis inhibited VEGF-C-mediated Erk1/2 activation and abrogated VEGFR-3 receptor-dependent binding of VEGF-C to the lymphatic endothelial surface. These findings prompted targeting of lymphatic N-deacetylase/N-sulfotransferase-1 (Ndst1), a major sulfate-modifying heparan sulfate biosynthetic enzyme. VEGF-C-mediated Erk1/2 phosphorylation was inhibited in Ndst1-silenced lymphatic endothelia, and scratch-assay responses to VEGF-C and FGF-2 were reduced in Ndst1-deficient cells. In addition, lymphatic Ndst1 deficiency abrogated cell-based growth and proliferation responses to VEGF-C. In other studies, lymphatic endothelia cultured ex vivo from Ndst1 gene-targeted mice demonstrated reduced VEGF-C- and FGF-2-mediated sprouting in collagen matrix. Lymphatic heparan sulfate may represent a novel molecular target for therapeutic intervention.
authors.affiliation: Medicine and Research Services, Veterans Affairs San Diego Healthcare System, San Diego, California 92161, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Yin
authors.forename: Scott C
authors.initials: SC
authors.lastname: Johns
authors.forename: Roger
authors.initials: R
authors.lastname: Lawrence
authors.forename: Ding
authors.initials: D
authors.lastname: Xu
authors.forename: Krisanavane
authors.initials: K
authors.lastname: Reddi
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Bishop
authors.forename: Judith A
authors.initials: JA
authors.lastname: Varner
authors.forename: Mark M
authors.initials: MM
authors.lastname: Fuster
mesh_terms: D000818:Animals; D004729:Endothelium, Lymphatic; D006497:Heparan Sulfate / Q000172:deficiency; D042583:Lymphangiogenesis; D042601:Lymphatic Vessels; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D011485:Protein Binding; D015238:Sulfotransferases / Q000378:metabolism; D042582:Vascular Endothelial Growth Factor C / Q000502:physiology*; D040321:Vascular Endothelial Growth Factor Receptor-3
chemical_list: D006497:Heparan Sulfate; D048052:Mitogen-Activated Protein Kinase 3; D015238:Sulfotransferases; D042582:Vascular Endothelial Growth Factor C; D040321:Vascular Endothelial Growth Factor Receptor-3; C467483:vascular endothelial growth factor C, mouse; C021350:heparitin sulfotransferase