Abstract 3943: Adrenomedullin Induces Lymphangiogenesis and Ameriolates Secondary Lymphedema
Background: Adrenomedullin (AM) stimulates angiogenesis by activation of extracellular signal-regulated kinase (ERK) and Akt in vascular endothelial cells (EC). Vascular endothelial growth factor C and D were major lymphangiogenesis factors, which enhance lymphatic EC activity via ERK and Akt pathways. Therefore, we investigated whether AM promotes lymphangiogenesis through activation of lymphatic EC and improves secondary lymphedema.
Methods and Results: RT-PCR analysis revealed that human lymphatic microvascular EC (HLMVEC) expressed mRNAs of all AM receptor complexes including calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP) 1–3. AM (10−9-10−7 M) significantly enhanced proliferation of HLMVEC (MTS assay; 194 ± 4% in 10−7 M, P < 0.05) with accumulation of intracellular cAMP in a dose dependent manner. Transwell migration assay revealed that AM (10−9-10−7 M) enhanced the chemotactic activity of HLMVEC (204 ± 7% in 10−7 M, P < 0.05). Proliferation of HLMVEC induced by AM (10−7 M) or a cAMP agonist, 8-Br-cAMP (10−4 M) was significantly inhibited by mitogen-activated protein kinase kinase (MEK) inhibitors, PD98059 (5 × 10−6 M) and U0126 (10−6 M). Western blot analysis revealed that AM (10−7 M) and 8-Br-cAMP (10−4 M) induced the phosphorylation of ERK in HLMVEC. A mouse model of tail lymphedema was developed in 8 –10 weeks old male BALB/c mice, and human AM (0.05 μg/kg/min) was delivered for 14 days using an osmotic minipump, which was subcutaneously implanted. AM significantly decreased the tail thicknesses at day 14 to 16 postoperation (Tail diameter at day 14; control vs. AM treatment, 4.19 ± 0.13 vs. 3.79 ± 0.08 mm, P = 0.023). Immunohistological analysis of injury sites using anti-LYVE-1 and anti-vWF antibodies revealed that AM treatment increased the number of lymphatic vessels (104 ± 15 vs. 188 ± 15 / mm2, P = 0.007) and vascular vessels (230 ± 11 vs. 315 ± 15 / mm2, P = 0.003).
Conclusions: AM enhanced proliferation and migration of lymphatic EC through activation of cAMP/MEK/ERK pathway. Infusion of AM induced lymphangiogenesis and improved secondary lymphedema in the mouse model of tail lymphedema. These results indicate that AM might represent a novel therapeutic strategy for treatment of lymphedema.