AMP-activated protein kinase is a key regulator of acute neurovascular permeability

Abstract

12Pathological hyperpermeability accompanies many blinding retinopathies. Despite 13 important therapeutic breakthroughs benefitting many but not all retinopathy patients, 14 the intracellular signalling underlying retinal leakage is still poorly understood. We 15 have developed an ex-vivo model, which allowed us to measure and manipulate acute 16 vascular permeability in the intact rodent retina, and combined measurements with 17 immunohistochemical analyses of signal transduction. This ex-vivo retina platform 18 proved to be an easily accessible and reliable tool for systematic identification of 19 regulators of vascular permeability through small molecule antagonists/agonists and 20 siRNA. By using this model, we showed that AMPK was a key mediator of VEGF-and 21 bradykinin-induced permeability by acting downstream of Ca 2+ and CAMKK, and 22 upstream of eNOS/VE-cadherin as well as p38/HSP27. Accordingly, AMPK agonist 23 potently induced retinal vascular leakage, a finding of major importance for their use 24 as therapeutic agents in the treatment of e.g. cancer or metabolic syndrome. 25 26 489 490 1 Nagy, 500 5 Ford, J. A. et al. Current treatments in diabetic macular oedema: systematic review and meta-501 analysis. BMJ Open 3, 504 7 Canning, P. et al. Lipoprotein-associated phospholipase A2 (Lp-PLA2) as a therapeutic target 505 to prevent retinal vasopermeability during diabetes. of phospholipase C, protein 510 kinase C, and calcium in VEGF-induced venular hyperpermeability. Am J Physiol 276, H535-511 542,

CCL4 induces inflammatory signalling and barrier disruption in the neurovascular endothelium

“…To the best of our knowledge, this is the first report demonstrating that VE-cadherin is redistributed in response to a CCL chemokine. VE-cadherin redistribution was profound and its manifestations consistent with epitope masking (catenins bind within the C-terminal epitope recognition area) and more importantly internalisation, as also described previously ( Dragoni et al., 2021 ). VE-cadherin internalisation is generally triggered by its phosphorylation and is a pre-requisite of vascular leakage in the periphery ( Orsenigo et al., 2012 ) but also the retinal microvasculature ( Dragoni et al., 2021 ; Ninchoji et al., 2021 ) in response to VEGF.…”

“…VE-cadherin redistribution was profound and its manifestations consistent with epitope masking (catenins bind within the C-terminal epitope recognition area) and more importantly internalisation, as also described previously ( Dragoni et al., 2021 ). VE-cadherin internalisation is generally triggered by its phosphorylation and is a pre-requisite of vascular leakage in the periphery ( Orsenigo et al., 2012 ) but also the retinal microvasculature ( Dragoni et al., 2021 ; Ninchoji et al., 2021 ) in response to VEGF. Consistent with this, phosphorylation of VE-cadherin and loss of barrier integrity has also shown to facilitate increased leukocyte migration via the paracellular route ( Wessel et al., 2014 ; Martinelli et al., 2014 ; Turowski et al., 2008 ).…”

“…All of the above was compatible with signalling leading to endothelial barrier dysfunction, which is specifically linked to re-distribution of the adherens junction protein VE-cadherin ( Orsenigo et al., 2012 ; Dragoni et al., 2021 ; Wessel et al., 2014 ), including the interplay of VE-cadherin and actin filaments that can be seen in junctional remodelling ( Schnittler et al., 2014 ).…”

“…6 D and F). Indeed, this additional signal was observed as early as 5 ​min following CCL4 treatment and was localised in close proximity to the junctions spreading towards the inside of the cells, a localisation consistent with internalisation ( Dragoni et al., 2021 ).…”

Methamphetamine enhances caveolar transport of therapeutic agents across the rodent blood-brain barrier

“…Thus, this model allowed the study of an experimental condition and its control within the same brain. Preparation of rodent heads and the perfusion protocol was identical to that used to study a fully functional blood-retinal barrier in explants , 18,19 suggesting that this protocol left the BBB intact. When heads were perfused with Evans Blue-albumin (EB-Alb), this dye remained restricted to the vasculature of most areas of the brain, including the cortex, hippocampus, and thalamus, and did not leak for at least 1 h (Figure S1C), indicating that BBB properties were preserved.…”