Many neuronal and retinal disorders are associated with pathological hyperpermeability of the microvasculature. We have used explants of rodent retinae to study acute neurovascular permeability and signal transduction and the role of AMP-activated protein kinase (AMPK). Following stimulation with either vascular endothelial growth factor (VEGF-A) or bradykinin (BK), AMPK was rapidly and strongly phosphorylated and acted as a key mediator of permeability downstream of Ca2+. Accordingly, AMPK agonists potently induced acute retinal vascular leakage. AMPK activation led to phosphorylation of endothelial nitric oxide synthase (eNOS), which in turn increased VE-cadherin phosphorylation on Y685. In parallel, AMPK also mediated phosphorylation of p38 MAP kinase and HSP27, indicating that it regulated paracellular junctions and cellular contractility, both previously associated with endothelial permeability. Endothelial AMPK provided a missing link in neurovascular permeability, connecting Ca2+ transients to the activation of eNOS and p38, irrespective of the permeability-inducing factor used. Collectively, we find that, due to its compatibility with small molecule antagonists/agonists and siRNA, the ex-vivo retina model constitutes a reliable tool to identify and study regulators and mechanism of acute neurovascular permeability.
Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon’s capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon’s capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo .
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,
Diseases of the retina are major causes of visual impairment and blindness in developed countries and, due to an ageing population, their prevalence is continually rising. The lack of effective therapies and the limitations of those currently in use highlight the importance of continued research into the pathogenesis of these diseases. Vascular endothelial growth factor (VEGF) plays a major role in driving vascular dysfunction in retinal disease and has therefore become a key therapeutic target. Recent evidence also points to a potentially similarly important role of galectins, a family of β-galactoside-binding proteins. Indeed, they have been implicated in regulating fundamental processes, including vascular hyperpermeability, angiogenesis, neuroinflammation, and oxidative stress, all of which also play a prominent role in retinopathies. Here, we review direct evidence for pathological roles of galectins in retinal disease. In addition, we extrapolate potential roles of galectins in the retina from evidence in cancer, immune and neuro-biology. We conclude that there is value in increasing understanding of galectin function in retinal biology, in particular in the context of the retinal vasculature and microglia. With greater insight, recent clinical developments of galectin-targeting drugs could potentially also be of benefit to the clinical management of many blinding diseases.
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