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Kimio Takeuchi, Yuki Morizane, Cynthia JACQUELINE Kamami-Levy, Fumiaki Kumase, Jun Suzuki, Maki Kayama, Wenyi Cai, Joan W Miller, Demetrios G Vavvas; The AMPK agonist AICAR suppresses VEGF stimulated tube formation, transcytosis, endocytosis, Caveolin-1 phosphorylation and Prdx1/c-abl dissociation.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4908.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effects of AMP-activated protein kinase (AMPK) activator AICAR on Vascular Endothelial Growth Factor (VEGF) dependent vascular tube formation and permeability.
Tube formation was evaluated at 3, 7, 10 days in co-cultures of normal human dermal fibroblasts and human umbilical vein endothelial cells (HUVECs) treated with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) in the presence or absence of VEGF. Endothelial cell permeability and endocytosis were evaluated in HUVECs cultured onto collagen-coated inserts in a 24-well tissue culture well plates using fluorescein-conjugated albumin. Dipyridamole (DPY) which blocks AICAR translocation into cells, 5-iodotubericidin (IODO), the inhibitor of the conversion of AICAR to ZMP, the direct activator of AMPK, and siRNAs were used to investigate the mechanisms of AICAR’s effects. Potential signaling pathways were evaluated with western blotting, co-immunoprecipitations and siRNA.
AICAR significantly inhibited VEGF induced tube formation, permeability, and albumin endocytosis in HUVECs as well as Caveolin-1, c-Abl and Akt phosphorylations. These effects were abolished by treatment with two inhibitors of AICAR stimulation of AMPK, dypiridamole and 5-iodotubericidin. Consistently, knock down of catalytic AMPKα subunit by siRNA abolished the inhibitory effect of AICAR on VEGF-induced phosphorylation of both caveolin-1 and c-Abl. Pretreatment with specific c-Abl inhibitor, imatinib, and knock down of c-Abl significantly decreased the caveolin-1 phosphorylation after VEGF exposure and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. AICAR had no effect on the association of VEGFR2 with caveolin-1, but suppressed the dissociation between Prdx1 and c-Abl in an AMPK dependent manner. Interestingly, knockdown of prdx-1, an antioxidant enzyme associated with c-Abl, increased phosphorylation of both caveolin-1 and c-Abl, and abolished the inhibitory effect of AICAR on the caveolin-1 phosphorylation. .
Our results suggest that up-regulation of AMPK by AICAR contributes to the suppression of VEGF induced tube formation, vascular endothelial cell permeability and caveolin-1 phosphorylation and these effects are mediated in part by stabilizing the interaction between c-Abl and prdx-1.
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