AMPK and hypoxia: crosstalk in the endothelium
File(s)
Author(s)
Kabir, Lida
Type
Thesis
Abstract
ECs are highly reliant on metabolic stimuli, integrating nutrient and oxygen (O2) availability to alter vascular function under physiological and pathological conditions. This remarkable metabolic plasticity is evident during angiogenesis, where metabolic changes promote the emergence of distinct EC subtypes. Hypoxia is a major stimulator of angiogenesis, a phenomenon frequently dysregulated in pathologies such as cancer and cardiovascular diseases. AMP-activated protein kinase (AMPK) is a master regulator of energy metabolism, restoring ATP levels during metabolic stress. Endothelial dysfunction and capillary rarefaction are hallmark features of cardiometabolic diseases; therefore, therapeutic targeting of AMPK holds promise in the treatment of these disorders. Despite extensive research having established the link between hypoxia signalling, and endothelial metabolism, the role of AMPK remains largely unexplored.
Examination of AMPK activity across different O2 tensions shows that short-term hypoxia does not appear to profoundly impact AMPK activity in HUVECs. There was no significant effect of 991 and/or BI-9774, a novel small molecule activator of AMPK, on HIF-1α stabilisation, although an increase in HIF-1α protein was noted. BI-9774 increased HRE-dependent transcription and enrichment in HIF-1α signalling based on luciferase reporter assays and transcriptomics. In vitro kinase assays and phospho-proteomics show that AMPK is capable of phosphorylating HIF-1α with putative phosphorylation sites identified by LC/MS2. Proximity ligation assays confirmed that AMPK and HIF-1α may directly interact in vitro. Despite suppressing endothelial proliferation, BI-9774 treatment still promoted Matrigel tube formation in vitro, with enhanced vascularisation observed in the global AMPK activating (γ1D316A-Tg) model. Endothelial-specific AMPK activation (EC-γ1D316A-Tg) promoted resistance against HFD-induced weight gain and hepatic steatosis. Further validation is required to determine (1) if HIF-1α is a target of AMPK in vivo; (2) whether the observed vascular and angiogenic phenotypes are dependent on HIF-1α activity; and (3) the possibility of AMPK rewiring the HIF-1α transcriptional response.
Examination of AMPK activity across different O2 tensions shows that short-term hypoxia does not appear to profoundly impact AMPK activity in HUVECs. There was no significant effect of 991 and/or BI-9774, a novel small molecule activator of AMPK, on HIF-1α stabilisation, although an increase in HIF-1α protein was noted. BI-9774 increased HRE-dependent transcription and enrichment in HIF-1α signalling based on luciferase reporter assays and transcriptomics. In vitro kinase assays and phospho-proteomics show that AMPK is capable of phosphorylating HIF-1α with putative phosphorylation sites identified by LC/MS2. Proximity ligation assays confirmed that AMPK and HIF-1α may directly interact in vitro. Despite suppressing endothelial proliferation, BI-9774 treatment still promoted Matrigel tube formation in vitro, with enhanced vascularisation observed in the global AMPK activating (γ1D316A-Tg) model. Endothelial-specific AMPK activation (EC-γ1D316A-Tg) promoted resistance against HFD-induced weight gain and hepatic steatosis. Further validation is required to determine (1) if HIF-1α is a target of AMPK in vivo; (2) whether the observed vascular and angiogenic phenotypes are dependent on HIF-1α activity; and (3) the possibility of AMPK rewiring the HIF-1α transcriptional response.
Version
Open Access
Date Issued
2024-01-15
Date Awarded
2024-04-01
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Carling, David
Birdsey, Graeme
Pericleous, Charis
Sponsor
British Heart Foundation
Publisher Department
Institute of Clinical Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)