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Background: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFKappaB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). Methodology/Principal Findings: Overexpression of a dominant negative AMPKalpha2 mutant in tumor necrosis factor-alpha-stimulated human endothelial cells resulted in increased NFKappaB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKalpha2-/- mice the interleukin (IL)-1beta induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFKappaB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKalpha2-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IKappaB and p65, indicating a link between AMPK and the IKappaB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKalpha2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKalpha2+/+ versus AMPKalpha2-/- mice. Conclusions: These results demonstrate that the IKK is a direct substrate of AMPKalpha2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFKappaB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.
Cytochrome P450 (CYP) epoxygenases generate bioactive lipid epoxides which can be further metabolized to supposedly less active diols by the soluble epoxide hydrolase (sEH). As the role of epoxides and diols in angiogenesis is unclear, we compared retinal vasculature development in wild-type and sEH−/− mice. Deletion of the sEH significantly delayed angiogenesis, tip cell, and filopodia formation, a phenomenon associated with activation of the Notch signaling pathway. In the retina, sEH was localized in Müller glia cells, and Müller cell–specific sEH deletion reproduced the sEH−/− retinal phenotype. Lipid profiling revealed that sEH deletion decreased retinal and Müller cell levels of 19,20–dihydroxydocosapentaenoic acid (DHDP), a diol of docosahexenoic acid (DHA). 19,20-DHDP suppressed endothelial Notch signaling in vitro via inhibition of the γ-secretase and the redistribution of presenilin 1 from lipid rafts. Moreover, 19,20-DHDP, but not the parent epoxide, was able to rescue the defective angiogenesis in sEH−/− mice as well as in animals lacking the Fbxw7 ubiquitin ligase, which demonstrate strong basal activity of the Notch signaling cascade. These studies demonstrate that retinal angiogenesis is regulated by a novel form of neuroretina–vascular interaction involving the sEH-dependent generation of a diol of DHA in Müller cells.
Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.
Rationale: The AMP-activated protein kinase (AMPK) is stimulated by hypoxia, and although the AMPKα1 catalytic subunit has been implicated in angiogenesis, little is known about the role played by the AMPKα2 subunit in vascular repair.
Objective: To determine the role of the AMPKα2 subunit in vascular repair.
Methods and Results: Recovery of blood flow after femoral artery ligation was impaired (>80%) in AMPKα2-/- versus wild-type mice, a phenotype reproduced in mice lacking AMPKα2 in myeloid cells (AMPKα2ΔMC). Three days after ligation, neutrophil infiltration into ischemic limbs of AMPKα2ΔMC mice was lower than that in wild-type mice despite being higher after 24 hours. Neutrophil survival in ischemic tissue is required to attract monocytes that contribute to the angiogenic response. Indeed, apoptosis was increased in hypoxic neutrophils from AMPKα2ΔMC mice, fewer monocytes were recruited, and gene array analysis revealed attenuated expression of proangiogenic proteins in ischemic AMPKα2ΔMC hindlimbs. Many angiogenic growth factors are regulated by hypoxia-inducible factor, and hypoxia-inducible factor-1α induction was attenuated in AMPKα2-deficient cells and accompanied by its enhanced hydroxylation. Also, fewer proteins were regulated by hypoxia in neutrophils from AMPKα2ΔMC mice. Mechanistically, isocitrate dehydrogenase expression and the production of α-ketoglutarate, which negatively regulate hypoxia-inducible factor-1α stability, were attenuated in neutrophils from wild-type mice but remained elevated in cells from AMPKα2ΔMC mice.
Conclusions: AMPKα2 regulates α-ketoglutarate generation, hypoxia-inducible factor-1α stability, and neutrophil survival, which in turn determine further myeloid cell recruitment and repair potential. The activation of AMPKα2 in neutrophils is a decisive event in the initiation of vascular repair after ischemia.
Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial-specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down-regulated in EVL-deficient P5-retinal endothelial cells. Consistently, EVL deletion impairs VEGF-induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor-2 internalization and signaling.
