Angiogenic role of adrenomedullin through activation of Akt, mitogen‐activated protein kinase, and focal adhesion kinase in endothelial cells

Abstract

Adrenomedullin (AM) is a multifunctional peptide in human pheochromocytoma. To evaluate whether AM could be an angiogenic factor, we examined its effect on kinases and angiogenic processes. AM induced tyrosine phosphorylation of Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase1/2 (ERK1/2) by using distinct signaling pathways in human umbilical vein endothelial cells (HUVECs). AM also phosphorylated focal adhesion kinase, and phosphatidylinositol 3'-kinase inhibitor inhibited AM-induced focal adhesion kinase phosphorylation. Pretreatment with high concentrations of AM22-52, a putative AM receptor antagonist, partially suppressed AM-induced phosphorylation of Akt, ERK1/2, and focal adhesion kinase. AM and vascular endothelial growth factor produced increases in DNA synthesis and migration in HUVECs. AM induced tube formation in HUVECs, and its effect was inhibited by pretreatment with phosphatidylinositol 3'-kinase inhibitor or ERK1/2 inhibitor. AM induced sprouting in porcine pulmonary arterial endothelial cells and promoted neovessel formation in a mouse Matrigel plug assay. Inhibitors of phosphatidylinositol 3'-kinase and ERK1/2 inhibited AM-induced endothelial sprouting in vitro and angiogenesis in vivo. AM exerts angiogenic activity through activation of Akt, MAPK, and focal adhesion kinase in endothelial cells.

Adrenomedullin increases the expression of calcitonin-like receptor and receptor activity modifying protein 2 mRNA in human microvascular endothelial cells

“…In both reports, treatment of cells with AM leads to increased cAMP levels, decreases in ERK 1/2, and subsequently to inhibition of cell proliferation and protein synthesis. Other studies have shown that AM increases ERK 1/2 phosphorylation, thereby exerting angiogenic and proliferative effects in rat vascular smooth muscle cells and human umbilical vein endothelial cells (HUVECs) (Iwasaki et al 2001, Kim et al 2003.…”

“…Additionally, AM has also been implicated in physiological and pathological angiogenesis using different types of knockout mice, xenografted tumors, and in vitro models (Hague et al 2000, Nikitenko et al 2000, Oehler et al 2001, Martinez et al 2002, Kim et al 2003; AM has also been reported to be expressed as an angiogenic factor in tumors. For example, in some cancers, AM expression is associated with vascular density and endothelial cell proliferation (Hague et al 2000).…”

Adrenomedullin and CGRP interact with endogenous calcitonin-receptor-like receptor in endothelial cells and induce its desensitisation by different mechanisms

“…Desensitisation of endogenous AM and CGRP receptors Activation of Akt in endothelial cells plays an important role in AM-and vascular endothelial growth factor (VEGF)-induced angiogenesis (Ilan et al, 1998;Kim et al, 2003). We used an Akt phosphorylation assay to determine Akt activity and to investigate the dynamics of desensitisation of endogenous AM and CGRP receptors.…”

“…After agonist exposure, cells were washed twice in 0.5%FCS-EGM-2, left for 30 minutes and then re-exposed to 100 nM AM or CGRP in 0.5%FCS-EGM-2 for indicated time periods (up to 15 minutes). Desensitisation of endogenous AM and CGRP receptors was accessed using Akt phosphorylation, since activation of Akt in endothelial cells plays an important role in AM-and VEGF-induced angiogenesis (Ilan et al, 1998;Kim et al, 2003). Akt phosphorylation levels were analysed by immunoblotting using anti-Akt (Santa Cruz) and anti-phospho-specific Akt (Ser473; Cell Signalling Technology) antibodies.…”

“…On the basis of CL-RAMP cotransfectant models as reported above, it has been proposed that the known effects of AM and CGRP on vascular cells are mediated through pharmacologically distinct receptors that also mediate the actions of the endogenous CL (EndoCL) receptor in a similar manner (Kamitani et al, 1999;Kim et al, 2003). However, no interaction of either peptide with EndoCL in vascular cells has yet been demonstrated.…”