Abstract
Purpose:
We have previously shown that in both genetically engineered animal models lacking βA3/A1-crystallin, and in eyes from patients with Persistent Fetal Vasculature (PFV), astrocytes abnormally ensheath the hyaloid artery. We have postulated that this contributes to the persistence of the hyaloid vasculature in PFV. The focus of this study was to identify molecular targets modulated by βA3/A1-crystallin that could affect astrocyte migration and contribute to development of PFV, a potentially blinding disease in an otherwise normal child, for which there are limited treatment options at the present time.
Methods:
To study changes in the expression of genes involved in cell motility in wild type (wt) and Nuc1 (a spontaneous mutation in the Cryba1 gene that encodes for βA3/A1-crystallin) astrocytes, we performed RT-PCR analysis using Rat Cell Motility PCR array (Qiagen). RNA from wt and Nuc1 astrocytes was extracted using RNeasy kit (Qiagen). 5 μg of RNA was reverse transcribed using the RT2 First Strand Kit (Qiagen) according to the manufacturer’s instructions. The Rat Cell Motility RT² Profiler™ PCR Array was used to study the expression of 84 key genes involved in cell motility following the manufacturer’s instructions. Quantitative real time PCR (qRT-PCR) using TaqmanTM gene expression probes (Applied Biosystems) was performed to confirm the array results.
Results:
Cell motility PCR array showed decreased expression of Rho family proteins (RhoA, RhoB and RhoC) and Rho-associated protein kinase 1 (ROCK1) in Nuc1 astrocytes compared to wt astrocytes. qRT-PCR analysis confirmed a significant decrease in expression of RhoA and RhoB, as well as of ROCK1, in the Nuc1 astrocytes.
Conclusions:
Human PFV disease is a very complex and heterogeneous condition, and multiple factors may be involved in its etiology. Our previous data showed that loss of βA3/A1-crystallin in astrocytes leads to rapid migration of astrocytes beyond the retinal nerve fiber layer into the vitreous chamber, where they ensheath the hyaloid artery, thereby affecting the normal regression of the hyaloid vascular system. In this study, we found that loss of βA3/A1-crystallin was associated with markedly decreased expression of Rho GTPases, which have been shown to stimulate cell migration.