In retinal proliferative diseases, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloprotease 2 (MMP‐2) and MMP‐9 also present increased levels of the protease inhibitor α ₂ ‐macroglobulin (α ₂ M) and its receptor, the low‐density lipoprotein receptor‐related protein 1 (LRP1). In the present work, we investigated whether the protease activated form of α ₂ M, α ₂ M∗, and LRP1 are involved with the MGC migratory process. By performing wound‐scratch migration and zymography assays, we demonstrated that α ₂ M∗ induced cell migration and proMMP‐2 activation in the human Müller glial cell line, MIO‐M1. This induction was blocked when LRP1 and MT1‐MMP were knocked down with siRNA techniques. Using fluorescence microscopy and biochemical procedures, we found that α ₂ M∗ induced an increase in LRP1 and MT1‐MMP accumulation in early endosomes, followed by endocytic recycling and intracellular distribution of MT1‐MMP toward cellular protrusions. Moreover, Rab11‐dominant negative mutant abrogated MT1‐MMP recycling pathway, cell migration, and proMMP‐2 activation induced by α ₂ M∗. In conclusion, α ₂ M∗, through its receptor LRP1, induces cellular migration of Müller glial cells by a mechanism that involves MT1‐MMP intracellular traffic to the plasma membrane by a Rab11‐dependent recycling pathway.—Barcelona, P. F., Jaldín‐Fincati, J. R., Sánchez, M. C.Chiabrando, G. A., Activated α2‐macroglobulin induces Müller glial cell migration by regulating MT1‐MMP activity through LRP1. FASEBJ. 27, 3181‐3197 (2013). www.fasebj.org

Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As retinal impairment involves abnormal neovascularization and neuronal degeneration, we evaluated here the involvement of galectin-1 in vascular and non-vascular alterations associated with retinopathies, using the oxygen-induced retinopathy (OIR) model. Postnatal day 17 OIR mouse retinas showed the highest neovascular profile and exhibited neuro-glial injury as well as retinal functional loss, which persisted until P26 OIR. Concomitant to VEGF up-regulation, galectin-1 was highly expressed in P17 OIR retinas and it was mainly localized in neovascular tufts. In addition, OIR induced remodelling of cell surface glycophenotype leading to exposure of galectin-1-specific glycan epitopes. Whereas VEGF returned to baseline levels at P26, increased galectin-1 expression persisted until this time period. Remarkably, although anti-VEGF treatment in P17 OIR improved retinal vascularization, neither galectin-1 expression nor non-vascular and functional alterations were attenuated. However, this functional defect was partially prevented in galectin-1-deficient (Lgals1 -/-) OIR mice, suggesting the importance of targeting both VEGF and galectin-1 as non-redundant independent pathways. Supporting the clinical relevance of these findings, we found increased levels of galectin-1 in aqueous humor from patients with proliferative diabetic retinopathy and neovascular glaucoma. Thus, using an OIR model and human samples, we identified a role for galectin-1 accompanying vascular and non-vascular retinal alterations in neovascular retinopathies.

Purpose: In ischemic proliferative retinopathies, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloproteinases (MMPs) also involve insulin-like growth factor (IGF)-1 participation. Therefore, the main interest of this work was to investigate the IGF-1 effect on the extracellular proteolytic activity in MGCs. Methods: Cell culture supernatants and cell lysates of the human MGC line MIO-M1 stimulated with IGF-1 were analyzed for MMP-2 by zymographic and Western blot analysis. The MGCs' motility was evaluated by scratch wound assay. The MMP-2, β1-integrin, and focal adhesions were detected by confocal microscopy. The localization of active MMPs and actin cytoskeleton were evaluated by in situ zymography. Results: The IGF-1 induced the activation of canonical signaling pathways through the IGF-1R phosphorylation. Culture supernatants showed a relative decrease in the active form of MMP-2, correlating with an increased accumulation of MMP-2 protein in the MGCs' lysate. The IGF-1 effect on MMP-2 was abolished by an IGF-1R blocking antibody, αIR3, as well as by the PI3-kinase inhibitor, LY294002. The IGF-1 increased the migratory capacity of MGCs, which was blocked by the GM6001 MMP inhibitor, LY294002 and αIR3. Finally, IGF-1 induced the intracellular distribution of MMP-2 toward cellular protrusions and the partial colocalization with β1-integrin and phospo-focal adhesion kinase signals. Gelatinase activity was concentrated along F-actin filaments. Conclusions: Taken together, these data indicate that IGF-1, through its receptor activation, regulates MGCs' motility by a mechanism that involves the MMP-2 and PI3K signaling pathway.

Hypoxia and hypoxia-reoxygenation are frequently developed through the course of many retinal diseases of different etiologies. Müller glial cells (MGCs), together with microglia and astrocytes, participate firstly in response to the injury and later in the repair of tissue damage. New pharmacological strategies tend to modulate MGCs ability to induce angiogenesis and gliosis in order to accelerate the recovery stage. In this article, we investigated the variation in autophagy flux under hypoxia during 4 h, employing both gas culture chamber (1% O₂) and chemical (CoCl₂) hypoxia, and also in hypoxia-reoxygenation. Then, we delineated a strategy to induce autophagy with Rapamycin and Resveratrol and analysed the gliotic and pro-angiogenic response of MGCs under hypoxic conditions. Our results showed an increase in LC3B II and p62 protein levels after both hypoxic exposure respect to normoxia. Moreover, 1 h of reoxygenation after gas hypoxia upregulated LC3B II levels respect to hypoxia although a decreased cell survival was observed. Exposure to low oxygen levels increased the protein expression of the glial fibrillary acid protein (GFAP) in MGCs, whereas Vimentin levels remained constant. In our experimental conditions, Rapamycin but not Resveratrol decreased GFAP protein levels in hypoxia. Finally, supernatants of MGCs incubated in hypoxic conditions and in presence of the autophagy inductors inhibited endothelial cells (ECs) tubulogenesis. In agreement with these results, reduced expression of vascular endothelial growth factor (VEGF) mRNA was observed in MGCs with Rapamycin, whereas pigment epithelium-derived factor (PEDF) mRNA levels significantly increased in MGCs incubated with Resveratrol. In conclusion, this research provides evidence about the variation of autophagy flux under hypoxia and hypoxia-reoxygenation as a protective mechanism activated in response to the injury. In addition, beneficial effects were observed with Rapamycin treatment as it decreased the gliotic response and prevented the development of newly formed blood vessels.