AB018.Ocular hypertension promotes early mitochondrial fragmentation in retinal endothelial cells in a mouse model of glaucoma

Background:Retinal endothelial cells are very active and contribute to the integrity of the neurovascular unit.Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma.Here,we evaluated the hypothesis that ocular hypertension triggers mitochondrial alterations in endothelial cells impairing the integrity of the blood retinal barrier(BRB).Methods:Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice,a strain expressing green fluorescent protein selectively in the mitochondria of endothelial cells.Capillary density,mitochondrial volume,and the number of mitochondrial components were quantified in 3D-reconstructed images from whole-mounted retinas using Imaris software.Dynamin-related protein(DRP-1),mitofusin-2(MFN-2)and optic atrophy-1(OPA-1)expression were assessed by western blot analysis of enriched endothelial cells.Mitochondrial structure was evaluated by transmission electron microscopy(TEM)and oxygen consumption rate was monitored by Seahorse analysis.The integrity of the BRB was evaluated by quantifying Evans blue leakage.Results:Our data demonstrate that two and three weeks after ocular hypertension induction,the total mitochondria volume in endothelial cells decreased from 0.140±0.002µm3 from non-injured retinas to 0.108±0.005 and 0.093±0.007µm3,respectively in glaucomatous eyes(mean±S.E.M,ANOVA,P<0.001;N=6/group).Frequency distribution showed a substantial increase of smaller mitochondria complexes(<0.5µm3)in endothelial cells from glaucomatous retinas.Significant upregulation of DRP-1 was found in vessels isolated from glaucomatous retinas compared to the intact retinas,while MFN-2 and OPA-1 expression was not affected.Structural alteration in endothelial cell mitochondria was confirmed by TEM,which were accompanied by a 1.93-fold reduction in the oxygen consumption rate as well as 2.6-fold increase in vasculature leakage in glaucomatous retinas(n=3-6/group).In addition,this model did not trigger changes in the density of the vascular network,suggesting that mitochondrial fragmentation was not due to endothelial cell loss.Conclusions:This study shows that ocular hypertension leads to early alterations in the dynamic of endothelial cell mitochondria,contributing to vascular dysfunction in glaucoma.

AB039. BMP9 signaling maintains endothelial integrity and prevents hyperglycemia-induced retinal vascular permeability

Background:The maintenance of a quiescent retinal vascular endothelial barrier is paramount for tissue supply and homeostasis to ensure visual function.Chronic hyperglycemia in diabetes causes structural and functional alterations of the endothelium that are accelerated by the production of several mediators such as VEGF.The disturbance of interendothelial junction stability leading to retinal hyperpermeability is one of the changes leading to diabetic macular edema(DME)that can occur at any stage of diabetic retinopathy.Advances in our understanding of the pathophysiological mechanisms of DME have enabled effective new therapies such as anti-VEGF’s,which are however associated with non-negligible side effects.The discovery of endothelium-specific protective targets that could restore retinal endothelial quiescence could provide a therapeutic alternative.Signaling mediated by BMP9 circulating protein via its endothelium-specific receptor ALK1,is known for its role in the maintenance of vascular quiescence.However,its ability to protect the endothelium and prevent vascular permeability has not been tested in the context of diabetes.Methods:We investigated BMP9/ALK1 signalling pathway in the hyperglycemic endothelium and its effect on retinal permeability in a type 1 diabetes mouse model.Hyperglycemic endothelial cells and tissue were extracted to evaluate BMP9/ALK1 signaling.BMP9 overexpression was achieved using adenoviral vectors.Retinal permeability was measured using miles assay.Results:We found that BMP9/ALK1 signaling was inhibited in hyperglycemic endothelial cells and blood vessels of diabetic(DB)mice,and that this loss of function was directly associated with retinal hyperpermeability.Molecularly,inhibition of this pathway triggers the activation of the VEGFR2/SRC pathway reducing interendothelial adhesion junctions.Conversely,the activation of ALK1 by sustained BMP9 overexpression in DB mice enabled the restoration of physiological permeability by regulating the levels and localization of interendothelial junctions,in part by limiting the action of VEGF signalling.We also observed that BMP9 overexpression demonstrated a regulating effect of blood glucose levels in DB mice.Our results showed that BMP9 significantly ameliorates glucose control over a 4-week span in DB mice and that this regulation was mediated primarily via the ALK3 receptor inhibiting gluconeogenic gene expression and hepatic glucose production and hence hyperglycemia.Conclusions:Together,our data show that BMP9 acts on several levels to safeguard endothelial integrity preventing retinal hyperpermeability in DB mice.The effects are mediated by its endocrine effect by directly stabilizing the endothelial barrier through Alk1 and its hypoglycemic paracrine/autocrine action in the liver through Alk3.Thus,BMP9 could be used in the development of future therapeutic alternatives against several vascular diseases involving edematous complications.

AB025.Machine learning applied to the oxygen induced retinopathy model

Background:The oxygen induced retinopathy rodent model is widely used,notably for the assessment of developmental dystrophies in preclinical studies of vascular retinal diseases.Typically,the quantification of vessel tufts and avascular regions is computed manually from flat mounted retinas imaged using fluorescent probes that highlight the vascular network.However,such manual measurements are time-consuming and hampered by user variability and bias,thus a rapid and objective alternative is required.Methods:We employ a machine learning approach to segment and characterize vascular tufts.The proposed quantitative retinal vascular assessment(QuRVA)technique uses quadratic discrimination analysis and morphological techniques to provide reliable measurements of vascular density and pathological vascular tuft regions,devoid of user intervention within seconds.Our algorithms allow also delineating the whole vasculature network,and identifying and analyzing avascular regions.Results:Our first experiment shows the high degree of error and variability of manual segmentations.In consequence,we developed a set of algorithms to perform this task automatically.We benchmark and validate the results of our analysis pipeline using the consensus of several manually curated segmentations using commonly used computer tools.We describe the method,provide details for reproducing the algorithm,and validate all aspects of the analysis.Conclusions:Manual and semi-automated procedures for tuft detection present strong fluctuations among users,demonstrating the need for fast and unbiased tools in this highly active research field with tremendous implications for basic research and industry.

AB034. In vivo laser-mediated retinal ganglion cell optoporation using Kv1.1 conjugated gold nanoparticles

Background:There is a current void in efficient,cell-specific,retinal drug delivery systems,thus developing a safe,effective,selective drug delivery system would open novel therapeutic avenues.We previously demonstrated that femtosecond(fs)laser irradiation can transfect DNA plasmids into cultured cells in the presence of gold nanoparticles(AuNPs).These AuNPs locally amplify laser energy at a submicron range creating transient pores allowing exogenous genetic material or cell impermeable dyes to enter the cell.Here,we sought out to selectively optoporate retinal cells in vivo with functionalized AuNPs and a 800 nm femtosecond(fs)laser.Methods:The cell-surface Kv1.1 voltage-gated channel was chosen to selectively target retinal ganglion cells(RGC)in the rat retina.Citrate-capped spherical 100 nm AuNPs functionalized with orthopyridyl-disulfide-poly(ethylene glycol)(5 kDa)-N-hydroxysuccinimide(OPSS-PEG-NHS)conjugated to a Kv1.1 monoclonal antibody were injected intravitreally in Sprague Dawley rats 3 hours prior to irradiation,concomitantly to a FITC-dextran dye to detect optoporation.The eyes of anesthetized rats were placed in the beam path of a laser system consisting of an 800 nm,100 fs laser and a Heidelberg Spectralis HRA ophthalmoscope for fundus visualization.The rat retina was irradiated at powers ranging from 20-750 mW,the eyes fixed in 4%paraformaldehyde,dissected,rinsed,mounted and imaged by confocal microscopy.Results:Our novel laser system coupled to a Heidelberg ophthalmoscope allowed for a clear visualisation of the rat ocular fundus.A timecourse of AuNP intravitreal injections revealed that optimal nanoparticle dispersion on the retinal surface occurred at 3 hours post injection.Following Kv1.1-AuNP and FITC-dextran intravitreal injection and incubation,irradiation at 120-750 mW resulted in FITC uptake by retinal cells.Conclusions:Since living biological tissues absorb energy very weakly at 800 nm,this non-invasive tool may provide a safe,cost effective clinically relevant approach to selectively target retinal cells and limit complications associated with surgical interventions,and potential biological hazards associated with viral-based gene therapy.

AB008.Cellular senescence and retinal angiogenesis

Pathological retinal neovascularization is the hallmark of primary blinding diseases across all age groups,yet surprisingly little is known about the causative factors.These diseases include diabetic retinopathy and retinopathy of prematurity where progressive decay of retinal vasculature yields zones of neural ischemia.These avascular zones and the hypoxic neurons and glia that reside in them are the source of pro-angiogenic factors that mediate destructive pre-retinal angiogenesis.Central neurons such as retinal ganglion cells(RGCs),which are directly apposed to degenerating vasculature in ischemic retinopathies,require stable metabolic supply for proper function.However,we unexpectedly found that RGCs are resilient to hypoxia/ischemia and a generally compromised metabolic supply and instead of degenerating,trigger protective mechanisms of cellular senescence.Paradoxically,while potentially favoring neuronal survival,the senescent state of RGCs is incompatible with vascular repair as they adopt a senescence-associated secretory phenotype(SASP)that provokes release of a secretome of inflammatory cytokines that drives paracrine senescence and further exacerbates pathological angiogenesis.The mechanisms that lead to retinal cellular senescence and dormancy as well as the therapeutic potential of targeting these pathways will be discussed.