Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover

Abstract

Vascular permeability regulated by the vascular endothelial growth factor (VEGF) through endothelial-barrier junctions is essential for inflammation. Mechanisms regulating vascular permeability remain elusive. Although ‘Akt’ and ‘Src’ have been implicated in the endothelial-barrier regulation, it is puzzling how both agents that protect and disrupt the endothelial-barrier activate these kinases to reciprocally regulate vascular permeability. To delineate the role of Akt1 in endothelial-barrier regulation, we created endothelial-specific, tamoxifen-inducible Akt1 knockout mice and stable ShRNA-mediated Akt1 knockdown in human microvascular endothelial cells. Akt1 loss leads to decreased basal and angiopoietin1-induced endothelial-barrier resistance, and enhanced VEGF-induced endothelial-barrier breakdown. Endothelial Akt1 deficiency resulted in enhanced VEGF-induced vascular leakage in mice ears, which was rescued upon re-expression with Adeno-myrAkt1. Furthermore, co-treatment with angiopoietin1 reversed VEGF-induced vascular leakage in an Akt1-dependent manner. Mechanistically, our study revealed that while VEGF-induced short-term vascular permeability is independent of Akt1, its recovery is reliant on Akt1 and FoxO-mediated claudin expression. Pharmacological inhibition of FoxO transcription factors rescued the defective endothelial-barrier due to Akt1 deficiency. Here we provide novel insights on the endothelial-barrier protective role of VEGF in the long-term and the importance of Akt1-FoxO signaling on tight-junction stabilization and prevention of vascular leakage through claudin expression.

Suppression of Akt1-β-catenin pathway in advanced prostate cancer promotes TGFβ1-mediated epithelial to mesenchymal transition and metastasis

“…Western blotting was performed as described previously[27] and protein concentration was determined by a Bradford protein assay kit (Bio-Rad, Hercules, USA). Samples were separated on 8–12 % SDS-PAGE gels, and then transferred onto PVDF membrane.…”

“…The TUNEL assay for the in situ detection of apoptosis in frozen sections was performed using the ApopTag® green In Situ apoptosis detection kit (Millipore, Billerica, MA) as described before[27]. …”

“…We have reported that Akt1 −/− mice exhibit increased vascular leakage and angiogenesis in response to vascular endothelial growth factor (VEGF) and melanoma xenografts[26]. Endothelial Akt1 −/− mice exhibit vascular leakage via suppression of tight-junction claudins[27]. Similarly, overexpression of MyrAkt1 (active) in the prostate[28] and Akt activation in PTEN −/− mice[29, 30] although developed PIN, it did not progress to metastasis.…”

RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G + -bacterial toxins

“…Dysfunction of the endothelial barrier arises from changes in cell behavior in response to signaling events that promote contraction, dissolution of adhesive complexes between cells and cell death. Many signaling pathways are involved including calcium, MLC (myosin light chain)-dependent mechanisms [4], cytoskeletal rearrangements [5], disassembly of junctional proteins between cells [6], activation of PKC (protein kinase C) [7], alteration of nitric oxide (NO) signaling [8] and numerous others [9,10]. Unresolved disruption of the endothelial barrier results in the inappropriate loss of fluid from the vasculature and extensive pulmonary edema which are the major complications of acute respiratory distress syndrome (ARDS) and CAP [11].…”

The E3 ubiquitin ligase MARCH3 controls the endothelial barrier

“…We thus unmasked MARCH3 as a novel regulator of the endothelial barrier, as its silencing allows endothelial cells to resist to cytokine‐triggered cortical actin remodeling and permeability increase. From a molecular standpoint, our results are reminiscent of the signaling pathway operating downstream of AJ and regulating claudin‐5 expression, where FoxO plays an instrumental role in steady state and/or recovery of the endothelial barrier integrity . Indeed, MARCH3 silencing causes an up‐regulation of both claudin‐5 and occludin expression, which in turn might strengthen TJ architecture.…”