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G. Gnanaguru, G. Bachay, W. J. Brunken; Laminins of the Inner Limiting Membrane Regulate Retinal Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):31.
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© ARVO (1962-2015); The Authors (2016-present)
Cell migration and spatial patterning are regulated by basement membrane (BM) derived cues. Here, we test the hypothesis that laminins guide astrocyte migration and vascular patterning.
Expression of laminin β2 and γ3 was analyzed using standard immunostaining methods. Astrocyte migration was studied using optic nerve (ON) explants. Whole mount preparations of wild type, β2-/-, γ3-/-, and β2-/-γ3-/- retinae were used to study vascular development.
We have previously shown that the β2 and γ3 laminin chains are deposited in the ILM and differentially in vascular BM, and that the deletion of laminin β2 and γ3 genes disrupts astrocyte migration and angiogenesis, suggesting a haptotactic role for laminins. We assayed whether astrocyte migration is laminin-dependent in vitro. ON explants were treated with exogenous laminins. EHS laminin promoted astrocyte migration while laminin α3β3γ2 did not. Astrocyte migration/patterning in β2-/- and β2-/-γ3-/- retinae were dysmorphic while the γ3-/- retina was normal; in contrast, endothelial migration was slowed in all the mutants. This latter result was surprising given the lack of an effect on astrocyte patterning in γ3-/- mice. Developmental studies demonstrate that retinal angiogenesis in γ3-/- mice was delayed up to 2 days and the capillary network branched excessively. As the γ3 chain is spatially restricted to the branch points of sprouting vessels, we posit that γ3 regulates branching. Thus, we measured vessel density and showed it was significantly increased in the γ3-/- retina. We also tested if laminin deletion affected VEGF expression: VEGF isoforms 188/164 regulate branching while 120 regulates tip cell guidance. VEGF188/164 expression was increased and VEGF120 was decreased in γ3-/-, whereas all the isoform levels were reduced in β2-/- and β2-/-γ3-/- mice.
These data indicate that laminin β2 and γ3 play independent roles in regulating retinal angiogenesis; moreover, they suggest that laminins are upstream of VEGF regulation and that the β2 chain is important for astrocyte patterning and migration, whereas γ3 regulates endothelial migration rate and branching behavior.
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