The Vision Health Research Network Annual Research Day,and International Symposium on Retinal and Choroidal Angiogenesis(Montreal)

The Vision Health Research Network of Québec(the Network)was established in 1995 and funded by the Fonds de la recherche du Québec en santé(FRQS).In the past 23 years,it has been the major venue to foster collaborations within Quebec’s research community and beyond.The Network aims to increase research capacity,improve infrastructure and enhance competitiveness of Quebec’s vision research on the international stage.Focusing on key matters proposed by Canadian health policy,the Network strikes to improve visual outcomes among patients.

AB017.Investigation of the effect of lymphocyte-derived microparticles on retinal macrophages in the oxygen-induced retinopathy model

Background:Retinopathy of prematurity(ROP)is the major cause of blindness in children,mainly caused by the retinal neovascularization(NV).Mounting of evidences shown that macrophage plays a pivotal role in the regulation of angiogenesis in ROP.Numerous studies confirmed that the deletion of macrophage significantly reduce the neovascularized areas in the oxygen-induced retinopathy(OIR)model.We have been studied the effect of lymphocyte derived-microparticles(LMPs)over ten years.LMPs are extracellular vesicles derived from apoptotic human CEM T lymphocytes.Our previous studies demonstrated that LMPs possess strong anti-angiogenic effect.Recently we observed that LMPs are capable to switch the phenotype of macrophage,thus to suppress the choroidal neovascularization(CNV).However,the role of LMPs on macrophage in ROP has not been clarified.Thus,my project is to disclose the relationship between LMPs and macrophage in ROP using the OIR model.Hypothesis:LMPs may inhibit retinal NV in the OIR model through targeting at macrophage by affecting the migration of macrophage,thus to inhibit pathological angiogenesis in ROP.Methods:Cell culture[RAW 264.7 and bone marrow-derived macrophage(BMDM)]for cell migration and viability assay.Generate the OIR model for in vivo detection of macrophage recruitment.Quantification of retinal NV,immunohistostaining of the macrophage in vivo,ex vivo retinal explants for cell migration and qPCR.Results:LMPs do not affect RAW 264.7 and BMDM cell viability(P>0.05).LMPs significantly decrease the BMDM cell migration indirectly(P<0.05).I successfully generate the OIR model and confirm that more macrophages infiltrate during retinal angiogenesis with counting the F4/80 immunostaining in the retinal flat mount.LMPs exert inhibiting effect on retinal angiogenesis through decreasing the migration of macrophages in vivo.Conclusions:LMPs have the negative effect on retinal angiogenesis via reducing the infiltrated macrophages to the neovascularized areas in the OIR model.

AB041.The implication of miRNA let-7f in retinal pigment epithelium degeneration under oxidative stress

Background:Age-related macular degeneration(AMD)is the most suspected cause of vision loss in the elderly.Given the considerable evidence,oxidative stress is thought to be a primary contributing factor to AMD.Retinal pigment epithelium(RPE)could be detrimentally compromised by oxidative stress along which blebs called retinal microparticles(RMPs)start to shed.In continue these particles would be taken by retina,causing RPE senescence,dysfunction and ultimately cell death.Along with the intracellular damages,accumulative deposit of microparticles in subretinal region can cause most known histological hallmark of dry AMD namely drusen.Based on our preliminary study,of 20 present miRNAs,Let-7f is the most abundant microRNAs in RMPs.As the accused substrate of RMPs through which retina function is compromised has yet to be well understood,we aimed to investigate pathophysiological role of let-7f and specific signaling triggered in RPE dysfunction.In brief,the principal objective is to further understand how RMPs implicate in RPE dysfunction.Methods:By oxidative stress inducing,RMPs were isolated from cultured ARPE-19 cells.We considered the effect of RMPs on ARPE-19 cells viability using MTT assay.In addition,to see whether RMPs effect could be ascribed to let-7f,ARPE-19 cells were transfected by carrier containing miRNA Let-7f.These transfected cells were then subjected senescence(β-galactosidase)and cell cycle assay to explore the molecular events responsible for Let-7f induced RPE cell dysfunction.Results:Regarding result we found that RMPs adversely affected RPE cell growth and resulted in significant decrease(≥30%)in cell viability.Let-7f-treated cells also revealed considerable increases of the senescence-associatedβ-galactosidase activity.Alongside RMPs impact,let-7f treatment group also showed similar result in cell growth.Conclusions:To the best of our knowledge,RPE cells uptake microparticles derived from oxidative-injured retinal cells,deteriorating integrity of vision compartments.Not only these finding would suggest that RMP’s impression likely corresponds to the miRNA let-7f,but introduce Let-7f as a mediator exacerbating the oxidative damages to RPE cells.This undesirable interplay is followed probably by dry AMD.Taken together,it seems by finding involved downstream pathways under RMPs pathogenesis,we can inhibit AMD disease in the early stage as well.In this line,we plan to investigate consecutive effect of RMP-associated miRNA inhibition in oxidative damage of retinal pigment epithelium.

AB028.Mesenchymal stem cells repair retinal vascular damage in retinopathy of prematurity mouse model

Background:Retinopathy of prematurity is a leading cause of visual impairment and blindness in infants.Exposure of premature babies to the hyperoxic extrauterine environment leads to vaso-obliteration(VO),followed by ischemia,and subsequently pathological intravitreal neovascularization(NV)in the immature retina.Current treatments target only aberrant intravitreal vessel growth without repopulating the avascular regions of the retina.Thus,there is a dire need of new therapies that arrest pathological NV and promote normal retinal revascularization.Mesenchymal stem cells(MSCs)have shown the ability to migrate to the damaged tissue in different animal models and enhance vascularization.We,therefore,investigated whether MSCs can promote vascular repair in a mouse model of ROP.Methods:Oxygen-induced retinopathy(OIR)model was used herein.Postnatal day 7(P7)mice were subjected to 75%O2 until P12 to induce VO followed by 5 days of room air leading to NV.Compact bone-derived MSCs were isolated from adult C57BL/6 mice and cultured either in hypoxia(5%O2)or normoxia(21%O2).Conditioned media(CM)was collected 24 hours later and injected intravitreally in P12 OIR retinas to assess vascular repair.To determine possible factors involved in MSC-induced revascularization,gene expression analysis was performed on P17 OIR retinas.In vitro,we investigated the effect of MSC-CM on microglial polarization using quantitative PCR and flow cytometry.Results:Hypoxic MSC-CM significantly(P<0.01)decreased both VO and NV areas in comparison to the normoxic counterpart.Levels of IGF-1 and VEGF were significantly high in MSC-injected OIR retinas.Moreover,gene expression levels of pro-inflammatory cytokines(IL-1β,TNFα)dropped whereas levels of anti-inflammatory cytokines(IL-10,IL-4)increased.Treatment of pro-inflammatory M1 microglia with MSC-CM decreased the gene expression of IL-1βand TNFα,and iNOS(M1 marker)at the transcript and protein levels.Conclusions:In this study,we demonstrated that MSCs promote healthy vessel growth in OIR retinas via a paracrine fashion by regulating expression of angiogenic factors and modulating inflammation.

AB043.Long-standing choroidal thinning in oxygen-induced retinopathy

Background:Retinopathy of prematurity(ROP),the most common cause of blindness in premature infants,has long been associated with pathologic retinal vasculature.However,recent studies reveal choroidal involution in adolescent patients formerly afflicted with ROP.We have recently demonstrated that choroidal thinning occurs early in retinopathy and persists into adulthood.Unlike retinal vessels,the damaged choroidal vasculature in ROP is incapably to regenerate.Herein,we investigated the molecular mechanism implicated in the lack of choroidal repair in ischemic retinopathy.Methods:The oxygen-induced retinopathy(OIR)model was used.Newborn Sprague-Dawley(albino)or Long-Evans rats(pigmented)rats were placed under oxygen concentration which cycles at 50%±1%or 10%±1%every 24 hours(hr)from postnatal day(P)0 to P14.On P14,all rats were returned to room air.Western blotting and qPCR were used to quantify protein and RNA abundances,respectively.The Dual-Luciferase®Reporter Assay System was used to confirm microRNA(miRNA)-mRNA interaction.Results:We detected a substantial oxidative stress in retinal pigment epithelium(RPE)and choroidal tissue,accompanied by a drastic reduction in insulin-like growth factor 1 receptor(IGF1R),a critical player in post-injury revascularization.The mechanism of decreasing IGF1R involves the over-activation of the p53 tumor suppressor that regulates miRNA let-7b,which subsequently silences Igf1r mRNA in the RPE/choroid complex of OIR subjects.Luciferase reporter assay confirmed that let-7b directly targets Igf1r mRNA at its 3’untranslated region(UTR).Indeed,silencing p53 resulted in a decreased let-7b expression,and re-established IGF1R abundance that promoted choroidal regeneration.Conclusions:Together,this study sets forth new mechanistic notion by uncovering the novel p53/let-7b/IGF1R axis;timely intervention of this pathway facilitates healthy choroidal revascularization.Future investigations on anti-angiogenic miRNAs can better our understanding on degenerative choroidopathy,such as geographic atrophy.

AB033.Implication of beta-adrenergic receptor in choroidal neovascularization

Background:We investigated the role of beta-adrenergic receptor(B-AR)on choroidal neovascularization(CNV)in an animal model of age-related macular degeneration in mice.Methods:The angiogenic effect of the B-AR was evaluated in retinal pigment epithelium(RPE)-choroid explants from C57Bl6 mice stimulated with propranolol or isoproterenol(10μM)(respectively antagonist and agonist of the B-AR)during 24 h.Conversely,a classic choroidal neovascularization(CNV)model induced by laser burn in C57Bl6 mice(8 weeks)was used to assess the anti-angiogenic effect of propranolol.In this experiment,mice were treated with intraperitoneal propranolol(6 mg/kg/d)or vehicle(saline solution)daily for 10 days,starting on day 4 after laser burn and until sacrifice(day 14).Immunostaining analysis on retinal flatmounts and cryosections were performed to determine the surface of CNV,the distribution of B-AR and the number and morphology of microglia/macrophages associated with CNV.To explore if the antiangiogenic effect of propranolol involved the modulation of the inflammatory microenvironment associated with CNV,we used RPE primary cells,J774 macrophages cell line and polarized M1 and M2 bone marrow-derived macrophage(BMDM).Choroidal explants treated with conditioned media(CM)from J774 or polarized M1/M2 BMDM pre-treated with propranolol to confirm the anti-angiogenic effect of propranolol.Expression of angiogenic factors was evaluated by RT PCR and Elisa.Results:The expression and distribution of the B-1,B-2 and B-3 adrenergic receptors were localized in the choroid and RPE cells.The stimulation of RPE-choroid explants with isoproterenol increased CNV compared to vehicle,while propranolol decreased CNV.In vivo,propranolol inhibited significantly the levels of VEGF and CNV growth in laser burn model compared to the vehicle.Additionally,the treatment with propranolol decremented the number of activated(amoeboid shape)microglia/macrophages but surprisingly,the number of non-activated microglia/macrophages around the CNV was higher than with the vehicle treatment.In vitro,propranolol modulated the angiogenic balance in macrophages promoting anti-angiogenic factors expression,especially with M2 BMDM.CM from macrophages pre-treated with propranolol reduced CNV on choroidal explants.Conclusions:These ex vivo and in vivo studies highlight the importance of B-adrenergic receptor in the CNV.Propranolol can inhibit CNV by decreasing the levels of VEGF and modulating microglia/macrophages activation.Further work will investigate the role of B-adrenergic receptor on suppression of the inflammatory environment in order to understand the link between neovascularization and inflammation in CNV during age-related macular degeneration.

AB035. Lactate receptor GPR81 modulates epigenetic modification in the subretina

Background:Retinal pigment epithelium(RPE)is vital for the homeostasis of the subretina including photoreceptors and choroid.Interestingly,our previous results suggested that the recently discovered lactate receptor GPR81 is abundantly expressed in RPE.To date,only one previous study has shown that activating GPR81 could enhance DNA repair by activating HDAC1.Consequently,we investigated whether GPR81 exhibits epigenetic modification in the subretina by using GPR81−/−mice.Methods:GPR81−/−mice and wide type littermates were generated on a background of C57BL/6J mice.The thicknesses of their choroid were evaluated by immunohistochemistry.Meanwhile,Q-PCR,western blot and choroid sprout assay were performed.In vitro,primary retinal pigment epithelium(pRPE)cells were isolated from mice,and cultured for treatments.Results:The thickness of choroid was reduced in GPR81−/−mice compared to GPR81+/+mice,suggesting that GPR81 is important for the integrity of choroid.In the choroid sprout assay,lactate treated RPE/choroid complex showed a significant increase in angiogenesis compared to controls while lactate treated KO RPE/choroid complex showed no difference compared to their controls.For Q-PCR,most of the genes screened elevated their expression in GPR81−/−mice compared to WT mice,suggesting epigenetic modification may exist,which were confirmed by histone acetylation and HDACs activity assay.Conclusions:Taking together,the lactate receptor GPR81 in RPE is very important for maintaining homeostasis of the subretina.This novel discovery sheds new light on the relationship between metabolism and epigenetic modification.

A CK2-RNF4 interplay coordinates non-canonical SUMOylation and degradation of nuclear receptor FXR

Farnesoid X 受体(FXR ) 是在调整涉及胆汁酸动态平衡，和脂肪和葡萄糖新陈代谢的基因起一个中央作用的激活 ligand 的原子受体。这里，我们表明在在 hepatocytes 指导受体进一条激活降级小径的 FXR phosphorylation， SUMOylation，和 ubiquitination 之间的 translational 以后相互影响。我们识别一个不在经典中的 SUMOylation 主题在酷蛋白 kinase 的直接控制下面在 FXR 的 Lys-325 结合 SUMO2 的称为的 pSuM 2 (CK2 ) ，它提供要求的否定费用让 Ubc9 和 PIAS1 执行 SUMOylation，由 phosphorylating Ser-327。Lys-325 SUMOylation 对有效 ligand 激活的提升和 FXR 的 transcriptional coactivation 不可缺少。用 phospho 模仿 Ser-327 变异或催化的 CK2 表情的组成的 pSuM 激活强烈导致 SUMO2 变化形式，它指导 FXR ubiquitination 和 proteasome 依赖的降级。我们也决定 FXR 的如此的 SUMOylation 依赖的 ubiquitination 被 E3 ubiquitin ligase RNF4 调停，它被要求完成 FXR 的最大的正式就职并且在上面最佳 -- 或涉及胆汁酸动态平衡和肝新生的应答的基因的 downregulation。我们的调查结果识别服务协调 FXR transcriptional 胜任的一个高度调整的不正常的相扑变化形式主题，从而扩展到来的信号过去常管理新陈代谢的基因规定的 SUMOylation 过程的复杂动力学。

Pharmacogenetics of asparaginase in acute lymphoblastic leukemia

Asparaginase is a key component in leukemias and lymphomas treatment protocols and is suggested as a treatment for other malignancies in which an amino acid depletion strategy is indicated.Asparaginase intolerance is subject to inter-individual variability and can manifest as hypersensitivity reactions,pancreatitis,thrombosis,as well as metabolic abnormalities,and may affect treatment outcome.Pharmacogenetics aims at enhancing treatment efficacy and safety by better understanding the genetic basis of variability and its effect on the pharmacological responses.Many groups tried to tackle the pharmacogenetics of asparaginase but the potential implementation of such findings remains debatable.In this review,we highlight the most important findings reported in studies of the pharmacogenetics of asparaginase related complications and treatment outcome in acute lymphoblastic leukemia.