Retinal endothelial cell phenotypic modifications during experimental autoimmune uveitis: a transcriptomic approach

Abstract

Background: Blood-retinal barrier cells are known to exhibit a massive phenotypic change during experimental autoimmune uveitis (EAU) development. In an attempt to investigate the mechanisms of blood-retinal barrier (BRB) breakdown at a global level, we studied the gene regulation of total retinal cells and retinal endothelial cells during non-infectious uveitis. Methods: Retinal endothelial cells were isolated by flow cytometry either in Tie2-GFP mice (CD31 + CD45 − GFP + cells), or in wild type C57BL/6 mice (CD31 + CD45 − endoglin + cells). EAU was induced in C57BL/6 mice by adoptive transfer of IRBP1-20-specific T cells. Total retinal cells and retinal endothelial cells from naïve and EAU mice were sorted and their gene expression compared by RNA-Seq. Protein expression of selected genes was validated by immunofluorescence on retinal wholemounts and cryosections and by flow cytometry. Results: Retinal endothelial cell sorting in wild type C57BL/6 mice was validated by comparative transcriptome analysis with retinal endothelial cells sorted from Tie2-GFP mice, which express GFP under the control of the endothelial-specific receptor tyrosine kinase promoter Tie2. RNA-Seq analysis of total retinal cells mainly brought to light upregulation of genes involved in antigen presentation and T cell activation during EAU. Specific transcriptome analysis of retinal endothelial cells allowed us to identify 82 genes modulated in retinal endothelial cells during EAU development. Protein expression of 5 of those genes (serpina3n, lcn2, ackr1, lrg1 and lamc3) was validated at the level of inner BRB cells. Conclusion: Those data not only confirm the involvement of known pathogenic molecules but further provide a list of new candidate genes and pathways possibly implicated in inner BRB breakdown during non-infectious posterior uveitis.

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

“…Brain and retinal endothelial cells express AMPKα1 (also known as PRKAA1), but little to no AMPKα2 (PRKAA2) ( Vanlandewijck et al, 2018 ; Lipski et al, 2020 ). To assess neutralisation of AMPK more specifically, AMPKα1 was knocked down by intraocular injection of siRNA 72 h prior to preparing the retinae for further experimentation.…”

“…Retinal endothelial cells express AMPKβ1 (also known as PRKAB1) and AMPKβ2 (PRKAB2) ( Lipski et al, 2020 ), and are thus susceptible to AMPK activation by a wide variety of molecules binding to its allosteric drug and metabolite (ADaM) site ( Hardie, 2017 ). Stimulation of ex vivo retinae with the AMPKβ1 and AMPKβ2-specific activators PF739 and MK8722 ( Fig.…”

“…In order to specify the role of AMPK during VEGF-A-and BK-induced vascular permeability, AMPK activity was neutralised in the ex vivo retina. Preincubation of the ex-vivo retina with compound C, a widely used AMPK antagonist, significantly decreased VEGF-A-and BK-induced permeability by 80% and 93%, respectively Brain and retinal endothelial cells express AMPKα1, but little to no α2 (Vanlandewijck et al, 2018, Lipski et al, 2020. To assess neutralisation of AMPK more specifically, AMPKα1 was knocked down by intraocular injection of siRNA 72 h prior to preparing the retinae for further experimentation.…”

Immune-Mediated Retinal Vasculitis in Posterior Uveitis and Experimental Models: The Leukotriene (LT)B4-VEGF Axis

“…Modifications of retinal endothelial cell phenotypes have been described which correlated with inflammatory activity [ 86 , 87 ]. As it is well-established that blood–retinal barrier dysfunction occurs prior to any structural damage in EAU [ 88 ], it is likely that soluble factors such as LTs and VEGF play a role in vascular permeability and vascular dysfunction.…”

“…VEGF is the most specific mitogenic factor for vascular endothelial cells. Phenotypical modifications in retinal endothelial cells during EAU progression have been proposed to increase the level of angiogenic factors including VEGF [ 87 ]. Its role in the pathogenesis of uveitic complications such as choroidal-retinal neovascularisation has been described [ 91 ] and is further supported by the proven efficacy of intravitreal anti-VEGF therapies in uveitis [ 14 , 15 ].…”