Role of laminins containing beta2 and gamma3 chains in regulating retinal angiogenesis

Abstract

Retinal vasculature related pathologies account for much of the global blinding disease. Proliferative diabetic retinopathy (PDR) is the main cause of acquired blindness in working adults and retinopathy of prematurity (ROP) is the leader cause of acquired blindness in children. A thorough understanding of the cell intrinsic and extrinsic regulation of vascular development in the retina is required to address such vasculature related pathologies. Unlike other organs, specialized glial cells called astrocytes guide vascular development in the retina. The astrocytes invade the retina around birth and form a template in the retina for endothelial cells to migrate on. It is well known that an astrocyte-derived template is crucial for vascular development in the retina; however, the factors that guide astrocytes into the retina remain unknown. It is important to know the mechanism by which astrocytes are guided, because models for ROP and diabetic retinopathy (DR) show degeneration of astrocytes. Maintaining astrocytes under such conditions reduces neovascularization events. In this study, we show that astrocyte migration into the retina is dependent upon laminins deposited at the inner limiting membrane (ILM). Deletion of Lamc3 gene reduced astrocyte migration rate at early stages and recovers at later stages. Deletion of Lamb2 and Lamb2:c3 genes severely affect astrocyte migration and spatial patterning. Astrocytes in these null retinas clump together and appear like a honeycomb. Interestingly, these Lamb2 and Lamb2:c3- also show a loss of integrin beta1 expression specifically in astrocytes. Using both in vitro and ex vivo techniques we show that laminins induce astrocyte migration in an isoform specific manner. Exogenous treatment of Lamb2:c3- null retinas with EHS-laminin rescues both migration and spatial patterning of astrocytes. Moreover, we show that exogenous laminin restores integrin-beta1 in Lamb2:c3- null astrocytes in culture. Functional blocking of integrin-beta1 receptor affects astrocyte migration in the presence of laminin, suggesting that laminin- integrin-beta1 interaction is critical for astrocyte migration. In addition, functional blocking of integrin-beta1 affects the recruitment of both ILK and FAK, which are downstream kinases that are important for cell migration. siRNA knockdown of FAK affects astrocyte filopodial extension and alpha-actinin organization during migration. This suggests that laminin likely orchestrates astrocyte migration through integrin-beta1- FAK mediated signaling mechanism. As mentioned above, an astrocyte template is critical for vascular development in the retina. As astrocyte migratory pattern were affected in laminin nulls, we analyzed vascular growth in these nulls. Lamc3- null retinas show a delay in vascular growth progression at early stages with increased vascular branches at the leading edges. At later stages vascular growth was complete and appeared as similar to WT. Deletion of Lamb2 and Lamb2:c3- genes however, severely affected vascular growth with persistent hyaloid vessels (commonly observed in ROP). These nulls also show abnormal astrocyte-endothelial interactions, which resulted in leaky vasculature, analyzed by fluorescein angiography. In summary, our study clearly suggests that laminin mediated signaling mechanism is critical for astrocyte migration and spatial organization and subsequent vascular growth.