Deletion of tetraspanin CD9 diminishes lymphangiogenesis in vivo and in vitro.

Tetraspaninshave emerged as key players in malignancy and inflammatory diseases, yet little is known about their roles in angiogenesis, and nothing is known about their involvement in lymphangiogenesis. We found here that tetraspaninsare abundantly expressed in human lymphatic endothelial cells (LEC). After intrathoracic tumor implantation, metastasis to lymph nodes was diminished and accompanied by decreased angiogenesis and lymphangiogenesisin tetraspaninCD9-KO mice. Moreover, lymphangiomasinduced in CD9-KO mice were less pronounced with decreased lymphangiogenesiscompared with those in wild-type mice. Although mouse LEC isolated from CD9-KO mice showed normal adhesion, lymphangiogenesiswas markedly impaired in several assays (migration, proliferation, and cable formation) in vitro and in the lymphatic ring assay ex vivo. Consistent with these findings in mouse LEC, knocking down CD9 in human LEC also produced decreased migration, proliferation, and cable formation. Immunoprecipitation analysis demonstrated that deletion of CD9 in LEC diminished formation of functional complexes between VEGF receptor-3 and integrins (α5 and α9). Therefore, knocking down CD9 in LEC attenuated VEGF receptor-3 signaling, as well as downstream signaling such as Erk and p38 upon VEGF-C stimulation. Finally, double deletion of CD9/ CD81in mice caused abnormal development of lymphatic vasculature in the trachea and diaphragm, suggesting that CD9 and a closely related tetraspanin CD81coordinately play an essential role in physiological lymphangiogenesis. In conclusion, tetraspaninCD9 modulates molecular organization of integrins in LEC, thereby supporting several functions required for lymphangiogenesis.