Nina Zippel

The AMP-activated protein kinase (AMPK) is an energy sensing kinase that is activated by a drop in cellular ATP levels. Although several studies have addressed the role of the AMPKα1 subunit in monocytes and macrophages, little is known about the α2 subunit. The aim of this study was to assess the consequences of AMPKα2 deletion on protein expression in monocytes/macrophages, as well as on atherogenesis. A proteomics approach was applied to bone marrow derived monocytes from wild-type mice versus mice specifically lacking AMPKα2 in myeloid cells (AMPKα2^∆MC mice). This revealed differentially expressed proteins, including methyltransferases. Indeed, AMPKα2 deletion in macrophages increased the ratio of S-adenosyl methionine to S-adenosyl homocysteine and increased global DNA cytosine methylation. Also, methylation of the vascular endothelial growth factor and matrix metalloproteinase-9 (MMP9) genes was increased in macrophages from AMPKα2^∆MC mice, and correlated with their decreased expression. To link these findings with an in vivo phenotype, AMPKα2^∆MC mice were crossed onto the ApoE^-/- background and fed a western diet. ApoExAMPKα2^∆MC mice developed smaller atherosclerotic plaques than their ApoExα2^fl/fl littermates, that contained fewer macrophages and less MMP9 than plaques from ApoExα2^fl/fl littermates. These results indicate that the AMPKα2 subunit in myeloid cells influences DNA methylation and thus protein expression and contributes to the development of atherosclerotic plaques.

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα^−/−) or endothelial-specific deletion (AMPKα^ΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

Angiogenesis, an integral part of many physiological and pathological processes, is a tightly regulated multistep process. Angiogenesis assays are used to clarify the molecular mechanisms and screen for pharmacological inhibitors. However, most in vitro angiogenesis models measure only one aspect of this process, whereas in vivo assays are complex and difficult to interpret. The ex vivo aortic ring model allows the study of many key features of angiogenesis, such as endothelial activation, branching, and remodeling as well as later steps such as pericyte acquisition. This model can be modified to include genetic manipulation and can be used to assess the pro- or anti-angiogenic effects of compounds in a relatively controlled system.