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Gopalan Gnanaguru, Johnny Chew, Germán Pinzón-Duarte, Galina Bachay, William J. Brunken; Laminins Regulate Astrocyte Migration and Retinal Vascular Growth. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4834.
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The laminin chains β2 and γ3 are key components of the ILM and the vascular basement membrane. Our aim is to study the laminin regulation of retinal angiogenesis and to begin to identify the mechanisms of laminin action.
Expression of laminin β2 and γ3 was analyzed in wild type whole mount retinas using immunohistochemistry (IHC). Migration of astrocytes was studied from optic nerve head (ONH) explants. Expression of integrins was studied by IHC and western blotting. VEGF protein levels were quantified using western blot from bulk tissue and following laminin immunoprecipitation (IP) assays.
Previously we showed that astrocyte distribution is disrupted in laminin mutants. Deletion of either Lamb2 alone, or in combination with Lamc3, disrupts astrocyte migration and vascular growth and patterning. Deletion of Lamc3 alone reduces astrocyte migration rate, slows vascular growth, and causes excessive branching. Next, we analyzed the expression of laminin binding integrins in WT and laminin null retinas. At P5, both integrin α6 and β1 were expressed by WT astrocytes and on vascular endothelium. Lamc3 deletion did not affect integrin expression in astrocytes; however, at the leading edges of the growing vessels an increase in integrin β1 was observed. In contrast, deletion of Lamb2 alone or with Lamc3 abolished integrin expression in astrocytes; however, integrin expression persisted in endothelial cells. Since we showed that VEGF levels, in an isoform specific manner, are dysregulated in laminin mutants, we assayed whether VEGF binds directly to laminins. All isoforms of VEGF were found in β2 laminin IPs. We are currently using a similar approach to determine whether VEGF isoforms bind to γ3 laminin.
In Lamc3 mutants, an increased integrin mediated adhesion is a likely explanation for the delay in vascular development, whereas in Lamb2 mutants, integrin down-regulation appears to account for the failure in astrocyte migration. The direct binding of VEGF isoforms to the vascular BM is of obvious importance in the pathobiology of retinal neovascularization.
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