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Angiogenic role of adrenomedullin through activation of Akt, mitogen-activated protein kinase, and focal adhesion kinase in endothelial cells¹

Department of Internal Medicine, Research Institute of Clinical Medicine, Chonbuk National University Medical School, and
^* Department of Biotechnology, Woosuk University, Chonju, Republic of Korea

²Correspondence: Department of Internal Medicine, Chonbuk National University Medical School, 634-18, Keum-Am dong, Chonju, 560-180, Republic of Korea. E-mail: parksk{at}moak.chonbuk.ac.kr

SPECIFIC AIM

Although adrenomedullin (AM) is known to be a multifunctional peptide, its role in angiogenesis has not been defined. We examined the angiogenic effect of AM and its signaling pathway in endothelial cells.

PRINCIPAL FINDINGS

1. AM induces Akt, ERK1/2, and p125^FAK phosphorylation
Because Akt, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2), and focal adhesion kinase (p125^FAK) play an important role in angiogenesis in endothelial cells, we measured the effect of AM on the phosphorylation of these kinases in human umbilical vein endothelial cells (HUVECs). AM (10^-7 mol/L) increased Akt phosphorylation as early as 10 min and produced a maximal effect at 30 min (Fig. 1 A). AM increased ERK1/2 and p125^FAK phosphorylation as early as 5 min and produced a maximal effect at 10 min (Fig. 1B, C ). AM also induced Akt, ERK1/2, and p125^FAK phosphorylation in a dose-dependent manner (Fig. 1D-F) .

View larger version (44K): [in this window] [in a new window]   Figure 1. AM induces phosphorylation of Akt, ERK1/2, and p125FAK in HUVECs. HUVECs were incubated for 16 h in 1% serum-containing M199 medium, then incubated with AM (10-7 mol/L) for the indicated times (A–C) or incubated with varied concentrations of AM for 30 min (D) or 10 min (E, F). After treatment, cell lysates were harvested. Each lane contains 50 µg of total protein from the cell lysates. Blots were probed with anti-phospho-Akt (A, D), anti-phospho-ERK (B, E), or anti–phospho-p125FAK antibody (C, F) (upper panels). The membrane was stripped and reprobed with anti-Akt antibody, anti-actin antibody, or anti-p125FAK antibody to verify equal loading of protein in each lane (lower panels). Fold: Densitometric analyses are presented as the relative ratio of phospho-Akt to Akt or phospho-ERK2 to actin, or phospho-p125FAK to p125FAK. The relative ratio measured at time 0 is arbitrarily presented as 1. Numbers represent the mean ± SD from 3 experiments. *P <0.05 vs. time 0 or control buffer.

2. Phosphorylation effect of AM is partially inhibited by AM_22-52
Because AM_22-52 is a putative inhibitor of the AM receptor, we used AM_22-52 (10^-10, 10^-8 and 10^-6 mol/L) in HUVECs. Pretreatment with AM_22-52 at higher concentrations (10^-6 mol/L) partially suppressed AM (10^-7 mol/L) -induced phosphorylation of Akt, ERK1/2, and p125^FAK. These results support the idea that phosphorylation of Akt, ERK1/2, and p125^FAK by AM is partially mediated through an AM_22-52-sensitive receptor.

3. AM increased DNA synthesis, migration, and tube formation in endothelial cells
Because ERK1/2 and Akt activation might be involved in proliferation and migration in HUVECs, we examined the effect of AM on endothelial cell DNA synthesis and migration. AM (10^-7 mol/L) increased [³H] thymidine incorporation an average of 1.4-fold and increased migration 4-fold. These data demonstrated that AM increased DNA synthesis and endothelial migration.

Since our data indicated that AM is a relatively strong enhancer for migration, we examined the effect of AM on tube formation by HUVECs in type I collagen gel. AM (10^-7 mol/L) increased tube formation 3.1-fold. The MEK1/2 inhibitor, PD98059, and the phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitor, wortmannin, significantly suppressed AM-induced tube formation. These data suggested that AM-induced tube formation in endothelial cells is mediated through PI 3'-kinase- and MEK1/2-dependent pathways.

4. AM induces sprouting in PPAECs
Because sprouting is an essential step in the initial phase of angiogenesis and requires cell migration, cell proliferation, and tube formation, we evaluated the effect of AM on sprouting in porcine pulmonary artery endothelial cells (PPAECs). AM (10^-7 mol/L) increased the number of total endothelial sprouts 2.5-fold. MEK1/2 inhibitor or PI 3'-kinase inhibitor partially suppressed AM-induced sprouting (Fig. 2 A, B). However, the combination of PD98059 and wortmannin completely inhibited AM-induced endothelial sprouting formation in PPAECs. Thus, AM induces sprout formation, and this effect is mediated through PI 3'-kinase and MEK1/2 dependent pathways.

View larger version (73K): [in this window] [in a new window]   Figure 2. AM induces sprouting in PPAECs through PI 3'-kinase and the MEK1/2 pathway. Cells grown on microcarrier beads were placed in fibrin gels. Gels were incubated in the presence of the indicated combinations of: control buffer (CB), AM (10-7 mol/L), PD98059 (PD, 2 µmol/L), wortmannin (WT, 30 nmol/L), or VEGF (10 ng/mL). Growth factors and inhibitors were replenished every 24 h. A) Representative phase-contrast photographs of sprouting activity. x200. B) Quantification of sprouting activity. The total number of endothelial sprouts and number of endothelial sprouts with length exceeding the diameter of the microcarrier beads (175 µm) per 50 microcarrier beads were counted after 2 days. Data are mean ± SD from 5 experiments. *P < 0.05 vs. control, **P < 0.05 vs. AM.

5. AM promotes angiogenesis in vivo
To determine whether AM is capable of promoting angiogenesis in vivo, we used the mouse Matrigel plug assay. Matrigel containing AM (2x10^-7 mol/L) produced more neovessels than that containing control buffer. Addition of PD98059 (2 µmol/L) and wortmannin (30 nmol/L) suppressed the AM-induced angiogenic activity. Matrigel containing AM (2x10^-7 mol/L) had more hemoglobin content than Matrigel containing control buffer. Thus, AM has an angiogenic activity in vivo through activation of ERK and PI 3'-kinse/Akt dependent pathways.

CONCLUSIONS AND SIGNIFICANCE

In this report we demonstrate that AM induces angiogenesis through intracellular Akt, MAPK, and p125^FAK activation in endothelial cells. AM promoted endothelial DNA synthesis, migration, and tube formation, which are the essential steps for angiogenesis. Furthermore, AM promoted sprouting in vitro and neovessel formation in vivo in gel plugs (Fig. 3 ). Thus, we demonstrate a novel biological function of AM in angiogenesis.

View larger version (20K): [in this window] [in a new window]   Figure 3. Model for AM-induced angiogenesis in endothelial cells. On the plasma membrane of the endothelial cell, AM binds to the CRLR/RAMP 2 or 3 functional receptor shown. PI 3'-kinase/Akt, and mitogen-activated protein kinase (MAPK) pathways are activated. AM also activates focal adhesion kinase (p125FAK). The downstream pathways are associated with endothelial proliferation, migration, and tube formation. AM also induces sprouting in vitro. Taken together, these cellular processes promote angiogenesis in vivo.

The activations of Akt, MAPK, and p125^FAK in endothelial cells are important intracellular signaling steps for angiogenesis. Growth factors such as VEGF or angiopoietin-1 bind to their receptor tyrosine kinases in endothelial cells and induce angiogenesis through activation of these kinases. Western blot analyses indicated that AM induced phosphorylation of Akt, ERK, and p125^FAK in a time- and dose-dependent manner (Fig. 1) .

In the initial phase of angiogenesis and neovascularization, the sprouting of endothelial cells is an essential step. This process requires cell proliferation, cell migration, and tube formation. Our results showed that AM induced proliferation, migration, and tube formation in endothelial cells in vitro. We also confirmed that AM induced sprouting by endothelial cells (Fig. 2) . Our data demonstrated that the signal pathway in AM-induced sprouting was associated with the PI 3'-kinase/Akt and ERK 1/2 pathways. We demonstrated that the PI 3'-kinase inhibitors completely inhibit AM-stimulated tyrosine phosphorylation of p125^FAK in HUVECs. Thus, our results suggest that AM-induced tyrosine-phosphorylated p125^FAK occur through a PI 3'-kinase dependent pathway.

We found additional evidence on the angiogenic role of AM in HUVECs. AM has an antiapoptotic effect that is associated with Akt pathway activation. AM stimulated the hydrolytic activities of MMP-2 and MMP-9, measured by gelatin zymography, and these activities are inhibited by PI 3'-kinase inhibitor.

In tumor cells, inflammation and hypoxia increase AM expression and the elevated expression of AM is associated with tumor neovascularization in xenografted endometrial tumors and renal cell carcinoma. AM also acts as a tumor cell survival factor underlying human carcinogenesis. Thus, AM may have a significant role in tumor angiogenesis. As angiogenesis is an essential process in tumor–host interactions for tumor growth, maintenance, and metastasis, finding ways to regulate the action of AM may provide a new avenue for finding anti-cancer therapeutics.

Until recently, only fibroblast growth factor, platelet-derived growth factor, VEGF, and angiopoietin were known to have profound angiogenic effects in endothelial cells. In this study, we demonstrated that AM can be a new angiogenic factor and its signaling occurs through activation of Akt, MAPK, and focal adhesion kinase in endothelial cells. Further studies about the pathogenic role of AM in vascular diseases, therapeutic endothelial survival, and the counteractive or additive effect of AM with other angiogenic factors are needed.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1209fje; doi: 10.1096/fj.02-1209fje

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This Article Full Text (PDF) All Versions of this Article: 17/13/1937 02-1209fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KIM, W. Articles by PARK, S. K. Search for Related Content PubMed PubMed Citation Articles by KIM, W. Articles by PARK, S. K.