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Gopalan Gnanaguru, Germán A. Pinzón-Duarte, Johnny Chew, Galina Bachay, William J. Brunken; Astrocyte Migration And Vascular Development In The Retina are Regulated By Laminin-Mediated Signaling Mechanisms. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2021.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal vascular development is dependent on astrocyte function. Our aim is to study the mechanism by which basement membrane (BM) molecules regulate astrocyte migration and subsequent retinal vascular growth.
Expression of laminin β2 and γ3 chains, integrins and integrin-linked kinase (ILK) were studied by immunohistrochemistry (IHC). Astrocyte migration and inhibition assays were studied using ex vivo culture methods. Vascular growth was analyzed by IHC.
Laminins containing β2 and γ3 chains are important for inner limiting membrane formation and are differentially expressed in vascular BM. Analysis of Lamc3 nulls showed slower astrocyte migration, reduced vascular growth progression and excessive vascular branching. Deletion of Lamb2 and Lamb2:c3 genes severely affected astrocyte migration and distribution, as well as subsequent vascular growth. Furthermore, in vitro study revealed that laminins act as haptotactic factors in an isoform specific manner and exogenous addition of laminins rescued astrocyte migration and spatial patterning in Lamb2:c3 null retina. Integrin β1 has shown to be critical for cell migration. We found that in the Lamb2 and Lamb2:c3 nulls integrin β1 expression was specifically affected in astrocytes. To test if laminin-integrin β1 interactions regulate astrocyte migration, we analyzed the effect of integrin β1 antibody on ex-vivo WT retinal explants cultured on EHS-Laminin coated coverslips. Anti-integrin β1 treatment affected both astrocyte migration and cytoskeletal organization. This result demonstrates that laminins regulate astrocyte migration via β1 integrins. Because integrin β1-BM interactions activate integrin-linked kinase (ILK), which further activates cdc42 and Rac-1 promoting cytoskeletal reorganization and migration. We are currently investigating if these BM mediated intracellular signaling events are affected in laminin nulls. Our preliminary results demonstrate that WT astrocytes express ILK. In contrast, ILK protein was absent from Lamb2 null astrocytes demonstrating a defect in coupling of BM-integrin β1 mediated signaling during migration.
These data demonstrate that laminins containing β2 and γ3 chains regulate astrocyte migration through an integrin β1 receptor-mediated mechanism. Deletion of these laminin chains affects localization of integrin β1 receptors and recruitment of ILK during astrocyte migration, which is critical for subsequent angiogenesis.
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