Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells

Abstract

Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.

Fig. 1. Adiponectin promotes endothelial cell migration and differentiation into tube-like structures

Tube formation assays were performed (A and B). HUVECs were seeded on Matrigel-coated culture dishes in the presence of adiponectin (30 µg/ml), VEGF (20 ng/ml), or BSA (30 µg/ml) (Control). A, representative cultures are shown. B, quantitative analysis of tube formation. C, a modified Boyden chamber assay was performed using HUVECs. HUVECs were treated with adiponectin (30 µg/ml), VEGF (20 ng/ml), or BSA (30 µg/ml) (Control). Results are shown as mean ± S.E. Results are expressed relative to control. *, p < 0.01 versus control.

Fig. 2. Adiponectin-stimulated signaling in endothelial cells

A, time-dependent changes in the phosphorylation (p-) of AMPK, Akt, eNOS, and extracellular signal-regulated kinase (ERK) after adiponectin treatment (30 µg/ml). B, role of AMPK in the regulation of adiponectin-induced protein phosphorylation. HUVECs were transduced with an adenoviral vector expressing dominant-negative AMPK tagged with c-Myc (dn-AMPK) or an adenoviral vector expressing green fluorescence protein (Control) 24 h before serum-starvation. After 16-h serum starvation, cells were treated with adiponectin (30 µg/ml) for the indicated lengths of time. C, role of Akt in the regulation of adiponectin-induced protein phosphorylation. HUVECs were transduced with an adenoviral vector expressing dominant-negative Akt (dn-Akt) or an adenoviral vector expressing green fluorescence protein (Control) 24 h before serum starvation. After 16 h of serum starvation, cells were treated with adiponectin (30 µg/ml) for the indicated lengths of time. Representative blots are shown.

Fig. 3. Contribution of AMPK and Akt to adiponectin-induced angiogenic cellular responses

HUVECs were transduced with an adenoviral vector expressing dn-AMPK (gray), dn-Akt (open) or green fluorescence protein (Control, solid) 24 h before the change to low-serum media. After 16 h of serum starvation, in vitro Matrigel (A and B) or modified Boyden chamber assays (C) were performed. Cells were treated with adiponectin (30 µg/ml) or BSA (30 µg/ml) (Vehicle). A, representative cultures displaying tube formation are shown. B, quantitative analysis of tube lengths. C, modified Boyden chamber assay was performed with adiponectin or VEGF as chemoattractant. Results are shown as the mean ± S.E. Results are expressed relative to control. *, p < 0.01 versus each control.

Fig. 4. PI3-kinase signaling is involved in adiponectin-induced angiogenic pathway

A, quantitative analysis of tube formation is shown. HUVECs were treated with adiponectin (30 µg/ml) or BSA (30 µg/ml) in the presence of LY294002 (10 µm) or vehicle at the time of seeding. B, a modified Boyden chamber assay was performed using adiponectin as the chemoattractant. HUVECs were pretreated with LY294002 (10 µm) or vehicle for 1 h and then incubated with adiponectin (30 µg/ml) or BSA (30 µg/ml) for 4 h. C, effects of LY294002 on adiponectin-stimulated protein phosphorylation (p-). Representative blots are shown. HUVECs were pretreated with LY294002 (10 µm) or vehicle for 1 h and then incubated with adiponectin (30 µg/ml) or BSA (30 µg/ml) for the indicated lengths of time. Results are presented as the mean ± S.E. For A and B, results are expressed relative to control. *, p < 0.01.

Fig. 5. Adiponectin promotes angiogenesis in vivo

An in vivo Matrigel plug assay was performed to evaluate the effect of adiponectin on angiogenesis (A and B). Matrigel plugs containing adiponectin (100 µg/ml, n = 3) or phosphate-buffered saline (Control, n = 3) were injected subcutaneously into mice. A, plugs were stained with the endothelial cell marker CD31. Bar, 100 µm. B, the frequency of CD31-positive cells in five low power fields was determined for each Matrigel plug. Data were presented as fold increase of CD31-positive cells relative to the control. A rabbit cornea assay was performed (C and D). Pellets containing adiponectin (1 and 10 µg, n = 8), VEGF (100 ng, n = 8), or phosphate-buffered saline (Control, n = 8) were implanted in the cornea. C, photographs of rabbit eyes are shown (Control, adiponectin 10 µg; VEGF, 100 ng). D, an angiogenic score was calculated (vessel density × distance from limbus). Results are shown as the mean ± S.E. *, p < 0.01 versus control.

Fig. 6. Proposed scheme for adiponectin-stimulated signaling in endothelial cells

Adiponectin activates AMPK, which in turn promotes Akt activation, eNOS phosphorylation, and angiogenesis. PI3-kinase is essential for adiponectin-mediated activation of Akt. Both AMPK and Akt can directly phosphorylate eNOS. However, inhibition of Akt or PI3-kinase was found to suppress adiponectin-stimulated eNOS phosphorylation without interfering with AMPK activation. Therefore, the data are most consistent with an AMPK-PI3-kinase-Akt-eNOS-signaling axis.