Background:Ischemic retinopathies(IRs)are ocular disorders associated to microvascular degeneration leading to visual impairments and blindness.microRNA(miRNAs)are a family of non-coding RNAs that regulate a wide rang...Background:Ischemic retinopathies(IRs)are ocular disorders associated to microvascular degeneration leading to visual impairments and blindness.microRNA(miRNAs)are a family of non-coding RNAs that regulate a wide range of gene expression involved in various biological process such blood vessel development and pathological NV.However,the post-transcriptional modulation of miRs and especially,their specific functions in the eyes during IRs remain to be evaluated.We aim to evaluate the potential role of miR-96 on microvascular degeneration in a rat model of oxygen-induced retinopathy(OIR).Methods:In vivo:next generation sequencing(NSG)was used to perform a complete miRNAs profiling in the retina and choroid from OIR and normoxia(CTL)rats.To evaluate the effects of miR-96 on microvasculature,OIR animals were treated with a miR-96 mimic(1 mg/kg)or a control-miR by intravitreal injection before hyperoxia-exposure(80%O2).Immunostaining analysis of retinal flatmounts and cryosections was used to explore the microvascular effects of miR-96.In vitro:Human Retinal Microvascular Endothelial Cells(HRMVEC)were subjected or not to hyperoxia(80%O2)and transfected with 50 nM of miR-96 mimic or antagomir-96.Angiogenic assay was performed(tube formation and migration)and molecular analysis evaluated by qRT-PCR and western blot.Results:NSG and qRT-PCR analyses identified miR-96 as one of most highly expressed miRNAs in retina and choroid during development.However,miR-96 showed a strong downregulation in OIR rats,and also in HRMVEC subjected to hyperoxia.In HRMVEC,we found that miR-96 regulates positively the expression of the key pro-angiogenic factors VEGF,FGF-2 and ANG-2.To better explore the role of miR-96 on HRMVEC angiogenic activity,we performed a gain/loss of function study.Similarly,to hyperoxia exposure,we observed a robust angiogenic impairment(tube formation and migration)on HMRVEC transfected with an antagomiR-96.Interestingly,overexpression of miR-96 completely recued the basal phenotype of HRMVEC and protected against hyperoxia-induced endothelial dysfunction.In vivo,intravitreal injection of miR-96 mimic(1 mg/kg)in OIR rats significantly restored retinal vascular density and choroidal tightness/sprouting hability.This was accompanied by the restoration in the physiological levels of VEGF,FGF-2 and ANG-2.Conclusions:This is the first study showing that reduced expression of miR-96 in OIR conditions lead to a reduction of VEGF/FGF/ANG-2 signaling,and inneficient post-ischemic revascularization in retinal/choroidal tissues.Intravitreal supplementation of miR-96 using a miR mimic could constitute a novel therapeutic strategy to improve vascular repair in IRs.展开更多
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 ado...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.展开更多
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 eva...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.展开更多
文摘Background:Ischemic retinopathies(IRs)are ocular disorders associated to microvascular degeneration leading to visual impairments and blindness.microRNA(miRNAs)are a family of non-coding RNAs that regulate a wide range of gene expression involved in various biological process such blood vessel development and pathological NV.However,the post-transcriptional modulation of miRs and especially,their specific functions in the eyes during IRs remain to be evaluated.We aim to evaluate the potential role of miR-96 on microvascular degeneration in a rat model of oxygen-induced retinopathy(OIR).Methods:In vivo:next generation sequencing(NSG)was used to perform a complete miRNAs profiling in the retina and choroid from OIR and normoxia(CTL)rats.To evaluate the effects of miR-96 on microvasculature,OIR animals were treated with a miR-96 mimic(1 mg/kg)or a control-miR by intravitreal injection before hyperoxia-exposure(80%O2).Immunostaining analysis of retinal flatmounts and cryosections was used to explore the microvascular effects of miR-96.In vitro:Human Retinal Microvascular Endothelial Cells(HRMVEC)were subjected or not to hyperoxia(80%O2)and transfected with 50 nM of miR-96 mimic or antagomir-96.Angiogenic assay was performed(tube formation and migration)and molecular analysis evaluated by qRT-PCR and western blot.Results:NSG and qRT-PCR analyses identified miR-96 as one of most highly expressed miRNAs in retina and choroid during development.However,miR-96 showed a strong downregulation in OIR rats,and also in HRMVEC subjected to hyperoxia.In HRMVEC,we found that miR-96 regulates positively the expression of the key pro-angiogenic factors VEGF,FGF-2 and ANG-2.To better explore the role of miR-96 on HRMVEC angiogenic activity,we performed a gain/loss of function study.Similarly,to hyperoxia exposure,we observed a robust angiogenic impairment(tube formation and migration)on HMRVEC transfected with an antagomiR-96.Interestingly,overexpression of miR-96 completely recued the basal phenotype of HRMVEC and protected against hyperoxia-induced endothelial dysfunction.In vivo,intravitreal injection of miR-96 mimic(1 mg/kg)in OIR rats significantly restored retinal vascular density and choroidal tightness/sprouting hability.This was accompanied by the restoration in the physiological levels of VEGF,FGF-2 and ANG-2.Conclusions:This is the first study showing that reduced expression of miR-96 in OIR conditions lead to a reduction of VEGF/FGF/ANG-2 signaling,and inneficient post-ischemic revascularization in retinal/choroidal tissues.Intravitreal supplementation of miR-96 using a miR mimic could constitute a novel therapeutic strategy to improve vascular repair in IRs.
文摘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.
文摘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.
