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Absence of ephrin-A2/A3 increases retinal regenerative potential for Muller cells in Rhodopsin knockout mice.

ABSTRACT: Müller cells (MC) are considered dormant retinal progenitor cells in mammals. Previous studies demonstrated ephrin-As act as negative regulators of neural progenitor cells in the retina and brain. It remains unclear whether the lack of ephrin-A2/A3 is sufficient to promote the neurogenic potential of MC. Here we investigated whether the MC is the primary retinal cell type expressing ephrin-A2/A3 and their role on the neurogenic potential of Müller cells. In this study, we showed that ephrin-A2/A3 and their receptor EphA4 were expressed in retina and especially enriched in MC. The level of ephrinAs/EphA4 expression increased as the retina matured that is correlated with the reduced proliferative and progenitor cell potential of MC. Next, we investigated the proliferation in primary MC cultures isolated from wild-type and A2^-/- A3^-/- mice by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. We detected a significant increase of EdU⁺ cells in MC derived from A2^-/- A3^-/- mice. Next, we investigated the role of ephrin-A2/A3 in mice undergoing photoreceptor degeneration such as Rhodopsin knockout (Rho^-/-) mice. To further evaluate the role of ephrin-A2/A3 in MC proliferation in vivo, EdU was injected intraperitoneally to adult wild-type, A2^-/- A3^-/- , Rho^-/- and Rho^-/- A2^-/- A3^-/- mice and the numbers of EdU⁺ cells distributed among different layers of the retina. EphrinAs/EphA4 expression was upregulated in the retina of Rho^-/- mice compared to the wild-type mice. In addition, cultured MC derived from ephrin-A2^-/- A3^-/- mice also expressed higher levels of progenitor cell markers and exhibited higher proliferation potential than those from wild-type mice. Interestingly, we detected a significant increase of EdU⁺ cells in the retinas of adult ephrin-A2^-/- A3^-/- mice mainly in the inner nuclear layer; and these EdU⁺ cells were co-localized with MC marker, cellular retinaldehyde-binding protein, suggesting some proliferating cells are from MC. In Rhodopsin knockout mice (Rho^-/- A2^-/- A3^-/- mice), a significantly greater amount of EdU⁺ cells were located in the ciliary body, retina and RPE than that of Rho^-/- mice. Comparing between 6 and 12 weeks old Rho^-/- A2^-/- A3^-/- mice, we recorded more EdU⁺ cells in the outer nuclear layer in the 12-week-old mice undergoing severe retinal degeneration. Taken together, Ephrin-A2/A3 are negative regulators of the proliferative and neurogenic potentials of MC. Absence of ephrin-A2/A3 promotes the migration of proliferating cells into the outer nuclear layer and may lead to retinal cell regeneration. All experimental procedures were approved by the Animal Care and Use Committee at Schepens Eye Research Institute, USA (approval No. S-353-0715) on October 24, 2012.

SUBMITTER: Zhu RL

PROVIDER: S-EPMC8284269 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:To determine whether ?-adrenergic receptors require insulin receptor substrate (IRS)-1 activity to regulate apoptosis in retinal Müller cells.Müller cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium grown in normal (5 mm) or high glucose (25 mM) conditions. The medium was supplemented with 10% fetal bovine serum and antibiotics. Cells were allowed to reach 80%-90% confluence. After becoming appropriately confluent, cells were placed in medium with reduced serum (2%) for 18-24 h to eliminate any effects of fetal bovine serum. Cells were then transfected with 10 ug of IRS-1 small hairpin RNA (shRNA). Forty-eight hours following transfection, cells were lysed and harvested for protein analysis using western blotting. In additional experiments, some cells were treated with 10 uM salmeterol for 24 h following transfection with IRS-1 shRNA. To determine whether IRS-1 directly regulates apoptotic events in the insulin-signaling pathway in retinal Müller cells, a cell death assay kit was used. In tumor necrosis factor (TNF)? inhibitory studies, cells were treated with 5 ng/ml of TNF? alone for 30 min or 30 min pretreatment with TNF? followed by salmeterol for 4 h.Müller cells treated with 5 ng/ml TNF? in 25 mM glucose significantly increased phosphorylation of IRS-1(Ser307). Treatment with the selective beta-2-adrenergic receptor agonist, salmeterol, significantly decreased phosphorylation of IRS-1(Ser307). Following IRS-1 shRNA transfection+salmeterol treatment, Bcl-2-associated X protein (Bax) and cytochrome c levels were significantly decreased. Salmeterol+IRS-1 shRNA also decreased cell death and increased protein levels of B-cell lymphoma-extra large (Bcl-xL), an anti-apoptotic factor.In these studies, we show for the first time that salmeterol, a beta-2-adrenergic receptor agonist, can reduce retinal Müller cell death through IRS-1 actions. These findings also suggest the importance of IRS-1 in beta-adrenergic receptor signaling in the prevention of cell death in retinal Müller cells.

Project description:AIMS/HYPOTHESIS:Retinal Müller cells are known to produce inflammatory and angiogenic cytokines, which play important roles in diabetic retinopathy. Hypoxia-inducible factor (HIF)-1 has been shown to play a crucial role in retinal inflammation and neovascularisation. We sought to determine the role of Müller cell-derived HIF-1 in oxygen-induced retinopathy (OIR) and diabetic retinopathy using conditional Hif-1? (also known as Hif1a) knockout (KO) mice. METHODS:Conditional Hif-1? KO mice were generated by crossing mice expressing cyclisation recombinase (cre, also known as P1_gp003) in Müller cells with floxed Hif-1? mice and used for OIR and streptozotocin-induced diabetes to induce retinal neovascularisation and inflammation, respectively. Abundance of HIF-1? and pro-angiogenic and pro-inflammatory factors was measured by immunoblotting and immunohistochemistry. Retinal neovascularisation was visualised by angiography and quantified by counting pre-retinal nuclei. Retinal inflammation was evaluated by leucostasis and vascular leakage. RESULTS:While the Hif-1? KO mice showed significantly decreased HIF-1? levels in the retina, they exhibited no apparent histological or visual functional abnormalities under normal conditions. Compared with wild-type counterparts, Hif-1? KO mice with OIR demonstrated attenuated overproduction of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1, reduced vascular leakage and alleviated neovascularisation in the retina. Under diabetes conditions, disruption of Hif-1? in Müller cells attenuated the increases of retinal vascular leakage and adherent leucocytes, as well as the overproduction of VEGF and ICAM-1. CONCLUSIONS/INTERPRETATION:Müller cell-derived HIF-1? is a key mediator of retinal neovascularisation, vascular leakage and inflammation, the major pathological changes in diabetic retinopathy. Müller cell-derived HIF-1? is therefore a promising therapeutic target for diabetic retinopathy.

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Absence of ephrin-A2/A3 increases retinal regenerative potential for Muller cells in Rhodopsin knockout mice.

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