Molecular mechanisms of cyclic phosphatidic acid-induced lymphangiogenic actions in vitro

The lymphatic system plays important roles in various physiological and pathological phenomena. As a bioactive phospholipid, lysophosphatidic acid (LPA) has been reported to function as a lymphangiogenic factor as well as some growth factors, yet the involvement of phospholipids including LPA and its derivatives in lymphangiogenesis is not fully understood. In the present study, we have developed an in-vitro lymphangiogenesis model (termed a collagen sandwich model) by utilizing type-I collagen, which exists around the lymphatic endothelial cells of lymphatic capillaries in vivo. The collagen sandwich model has revealed that cyclic phosphatidic acid (cPA), and not LPA, augmented the tube formation of human dermal lymphatic endothelial cells (HDLECs). Both cPA and LPA increased the migration of HDLECs cultured on the collagen. As the gene expression of LPA receptor 6 (LPA₆) was predominantly expressed in HDLECs, a siRNA experiment against LPA₆ attenuated the cPA-mediated tube formation. A synthetic LPA_1/3 inhibitor, Ki16425, suppressed the cPA-augmented tube formation and migration of the HDLECs, and the LPA-induced migration. The activity of Rho-associated protein kinase (ROCK) located at the downstream of the LPA receptors was augmented in both the cPA- and LPA-treated cells. A potent ROCK inhibitor, Y-27632, suppressed the cPA-dependent tube formation but not the migration of the HDLECs. Furthermore, cPA, but not LPA, augmented the gene expression of VE-cadherin and β-catenin in the HDLECs. These results provide novel evidence that cPA facilitates the capillary-like morphogenesis and the migration of HDLECs through LPA₆/ROCK and LPA_1/3 signaling pathways in concomitance with the augmentation of VE-cadherin and β-catenin expression. Thus, cPA is likely to be a potent lymphangiogenic factor for the initial lymphatics adjacent to type I collagen under physiological conditions.