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William Brunken, Galina Bachay, Saptarshi Biswas, Gopalan Gnanaguru; Laminin Regulation of Retinal Vascularization: Role in Astrocyte Migration and Differentiation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4541.
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© ARVO (1962-2015); The Authors (2016-present)
A thorough understanding of the regulation of retinal vascular development is required to adequately design therapies to treat retinal neovascularization. Astrocytes are key cells regulating retinal vascularization. Here we address the role of laminin signaling, particularly those containing the β2 and γ3 chains.
Immunohistochemistry was employed to determine the temporal and spatial distribution of laminins, their receptors and scaffolding components. Deletions of laminin genes, Lamb2 and Lamc3, were used to study their function, and siRNA was used to manipulate the effector mechanisms of laminin signaling cascades.
Deletion of the Lamc3 gene slowed astrocyte migration from P0 to P3; by P5 migration had recovered. In contrast, deletion of Lamb2 alone, or with Lamc3, permanently hinders astrocyte migration, reducing retinal coverage to less than 50% of control. Astrocytes in the Lamb2 null retinas fasciculate instead of tiling over the retina. Using ex vivo systems, we show that laminin regulation of astrocyte migration is isoform specific. Exogenous treatment with laminin 111 and 521 rescues the migratory behavior, and cellular organization of Lamb2:c3-/- astrocytes in situ. Our data suggest that integrin-β1 and its downstream kinases, ILK and FAK, serve as the laminin-response elements in astrocyte migration. Lamb2-/- and Lamb2:c3-/- astrocytes down-regulate integrin β1 expression and their cytoskeletal structure is disrupted as compared to wild-type or Lamc3-/-. Anti-integrin β1 antibodies reduce astrocyte migration and expression of these kinases. siRNA knockdown of FAK inhibits astrocyte migration, filopodial extension and α-actinin organization. Thus, laminin promotes astrocyte migration via integrin-β1-FAK signaling. Endothelial growth in the laminin mutants was disrupted. In Lamc3-/- retina, the vascular tree development was delayed and had an increased branch density. In Lamb2-/- and Lamb2:c3-/- retinas, endothelial growth was disrupted severely resulting in a persistent hyaloid vasculature. Fluorescein angiography demonstrated that vessels in the Lamb2 mutants were tortuous and leaky.
In summary, our study clearly shows that laminin-mediated signaling is critical for astrocyte migration, spatial organization and subsequent vascular growth.
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