E in several endothelial cell types [8]. We next determined whetherDiscussion TheE in several endothelial

E in several endothelial cell types [8]. We next determined whetherDiscussion The
E in several endothelial cell types [8]. We next determined whetherDiscussion The principal findings of this study are that i) VEGF-B stimulates AMPK activity in HAECs and ii) AMPK is required for HAEC proliferation in response to either VEGF-A or VEGF-B. Incubation of HAECs with VEGFB robustly stimulated AMPK activity, yet the extent of activation was modest when compared with VEGF-A. There was no additive effect of VEGF-B on VEGF-A-stimulated AMPK activity. VEGF-B activates VEGF-R1, whereas VEGF-A activates both VEGF-R1 and VEGFR2. These findings suggest that VEGF-R1 stimulation leads to AMPK activation. Stimulation of AMPK by VEGF-B was sensitive to the CaMKK PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27385778 inhibitor, STO609, as we have previously demonstrated for VEGF-A [14], implying that VEGF-B, via VEGF-R1 stimulates an increase in intracellular Ca2+ required for activation of CaMKK. One previous study using BAECs has reported that siRNA-mediated downregulation of VEGF-R2 abrogated phosphorylation of AMPK and ACC in response to VEGF, although the particular VEGF used was not explicitly stated [16]. When these data are taken together with those in the current study, it suggests that stimulation of VEGF-R1 or VEGF-R2 activates AMPK via a CaMKK-mediated mechanism in HAECs. Angiogenesis involves endothelial cell proliferation as well as the migration of the cells toward an angiogenic stimulus. Although VEGF-A is a well-documented stimulus for proliferation and migration [2,4-6], there are few published studies of the effects of VEGF-B on endothelial cell proliferation, survival or migration. VEGF-B has been reported to stimulate proliferation in bovine carotid artery endothelial cells [21] and increase survival of mouse retinal or choroidal endothelial cells [7]. The stimulation of HAEC proliferation in response to VEGF-B observed in the current study represents, to the authors’ knowledge, the first demonstration that VEGF-B stimulates proliferation in isolated humanReihill et al. Vascular Cell 2011, 3:9 http://www.vascularcell.com/content/3/1/Page 6 ofFigure 3 AMPK is required for VEGF-stimulated HAEC proliferation. HAECs were incubated in the presence or absence PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28724915 of VEGF-A (10 ng/ ml), VEGF-B (100 ng/ml), Compound C (5 M), STO-609 (5 M), AICAR (2 mM), A769662 (10 M), L-NAME (1 mM) for 24 h and proliferation assessed. Ad.control, Ad.AMPK-DN and Ad.AMPK-CA adenoviruses were used to infect HAECs 24 h prior to the addition of VEGF. Results are expressed as the mean ?SEM basal proliferation for three independent experiments or six independent experiments in the case of Ad.CV205-502 hydrochloride cost AMPKDN. *p < 0.05 relative to absence of VEGF, **p < 0.05 relative to absence of AICAR or A769662, ***p < 0.05 relative to Ad.control.endothelial cells. HAEC proliferation in response to either VEGF-A or VEGF-B was abrogated by compound C, STO-609 or infection with Ad.AMPK-DN. These observations establish AMPK as a key regulator of cell proliferation in response to VEGF in HAECs. Such findings are consistent with previous observations demonstrating AMPK-mediated angiogenesis in response to both adiponectin [22] and hypoxia [15]. Despite the requirement for AMPK in VEGF-stimulated endothelial cell proliferation, activation of AMPK with AICAR, A769662 or Ad.AMPK-CA suppressed proliferation in the absence of VEGF. This effect ofAMPK activation alone is consistent with studies in non-endothelial cells, where AMPK activation has been reported to cause cell cycle arrest [23-25] and to facilitate apoptosis [26]. We propose t.