Abstract
Purpose:
During development of the mammalian eye, nourishment of the immature lens, inner retina and vitreous is provided by the hyaloid vascular system. Failure of all or part of these vessels to regress, a condition called Persistent Fetal Vasculature (PFV), leads to serious congenital pathologies. Our goal is to elucidate regulatory mechanisms responsible for fetal vascular regression and the underlying cause of failure of regression.
Methods:
Astrocytes were isolated from wild type (WT; mouse and rat), Nuc1 rat (a mutation in Cryba1 gene encoding βA3/A1-crystallin) and Cryba1 floxed mice for primary culture. Migration of astrocytes was analyzed by transwell assay. Notch signaling pathway genes/proteins were studied using quantitative PCR, Notch expression plasmids, western blots, immunofluorescence and reporter-tagged markers. Lysosomal function was assessed by measuring acidification and activities of V-ATPase and γ-secretase. Sections through the optic nerve head from Nuc1 eyes were compared to WT and used for localization of GFAP and crystallins by immunofluorescence. Expression of VEGF was assessed by western blotting.
Results:
Western analysis showed expression of βA3/A1-crystallin in astrocytes of the neural retina. Loss of βA3/A1-crystallin in astrocytes decreased V-ATPase activity, thereby inducing alkalization of endolysosomal compartments, leading to reduced γ-secretase-mediated processing and release of the Notch intracellular domain. GFAP reporter activity was also reduced. Astrocytes from Nuc1 retinas displayed a 3-fold increase in migration rate compared to WT astrocytes. The astrocytes in Nuc1 rat abnormally ensheath the persisting hyaloid artery. VEGF was increased in the Nuc1 lens relative to WT.
Conclusions:
It is important to note that human PFV disease is a very complex and heterogeneous condition, and multiple factors may be involved in its etiology. We postulate that βA3/A1-crystallin expressed in the astrocytes plays a crucial role in remodeling during hyaloid regression and the astrocytes are involved in the pathogenesis of PFV. We hypothesize that the severity of PFV depends on lens pathology. βA3/A1-crystallin might be a relevant therapeutic target in PFV, a potentially blinding disease in an otherwise normal child, for which there are limited treatment options at the present time.
Keywords: 488 crystallins •
429 astrocyte •
636 pathobiology