A novel bA3/A1-crystallin gene mutation results in persistent fetal vasculature

描述(申请人提供)：在哺乳动物眼睛的发育过程中，未成熟的晶状体、视网膜和玻璃体的营养由玻璃体血管系统提供。持续性胎儿血管系统(PFV)是人类眼睛的一种先天性发育障碍，是这种血管退化完全或部分失败的结果。对玻璃体血管退变失败的细胞和分子机制的了解非常有限。我们的研究提供了新的证据，表明视网膜发育过程中星形胶质细胞的异常可以抑制玻璃体动脉的退化。在人类PFV病和许多小鼠模型中，星形胶质细胞包裹着玻璃体动脉。我们现在已经开发了模拟人类PFV疾病临床症状的新模型。因此，在这次竞争性更新中，我们建议进行研究，以使我们能够调查：具体目标1：评估A3/A1-晶体蛋白对视网膜星形胶质细胞Notch信号的影响。星形胶质细胞从视神经迁移到视网膜内部，形成一个模板，视网膜血管将在该模板上发育。正常模板形成的缺陷可能导致星形胶质细胞形态和行为的显著差异，并可能扰乱发育过程中玻璃体血管的正常程序性回归。调节星形胶质细胞模板形成的分子信号仍然难以捉摸。我们的初步数据表明，Notch信号是视网膜星形胶质细胞模板形成所必需的，A3/A1-晶体蛋白可能是星形胶质细胞Notch信号的调节因子。在焦点1中，我们将比较野生型星形胶质细胞和缺乏A3/A1-晶体蛋白的星形胶质细胞中-分泌酶抑制的效果，而焦点2将确定A3/A1-晶体蛋白是否参与活性Noch的蛋白水解性释放，从而激活Noth靶基因。重点3将是研究A3/A1-晶体蛋白在Notch降解中的可能作用。具体目标2：确定A3/A1-晶体蛋白的缺失如何增加视网膜星形胶质细胞中的水通道蛋白-4。我们建议寻找A3/A1-晶体蛋白调节Aqp4表达和星形胶质细胞在发育中的视网膜迁移的直接证据(焦点1)。也有可能A3/A1-晶体蛋白不直接调节Aqp4，但通过与Aqp4的联合作用影响Aqp4水平 一个结合的伙伴；我们将在焦点2中讨论这种可能性。具体目标3：研究星形胶质细胞在玻璃体动脉异常滞留中的作用。我们的方法将是(1)确定A3/A1-晶体蛋白是否调节星形胶质细胞分泌血管内皮生长因子，从而有助于血管稳定；(2)研究周细胞在发育过程中玻璃体动脉异常滞留中的可能作用；(3)分析玻璃体血管的结构完整性和细胞组织。我们提出的研究将帮助我们阐明星形胶质细胞在血管重塑中扮演关键角色的机制。了解玻璃体退行性变过程中重塑的分子基础可能会导致开发新的治疗方法来治疗PFV，PFV是一种潜在的致盲疾病，在其他方面正常的儿童中，目前对这种疾病的治疗选择有限。

DESCRIPTION (provided by applicant): During development of the mammalian eye, nourishment of the immature lens, inner retina and vitreous is provided by the hyaloid vascular system. One of the congenital, developmental disorders of the human eye, persistent fetal vasculature (PFV), results from the complete or partial failure of this vascular regression. Knowledge of the cellular and molecular mechanisms by which hyaloid vascular regression fails is very limited. Our studies have provided novel evidence that abnormalities in astrocytes during retinal development can inhibit regression of the hyaloid artery. Astrocytes ensheath the hyaloid artery in human PFV disease and in many mouse models. We have now developed novel models that mimic the clinical signs of human PFV disease. In this competitive renewal, we therefore propose, studies that will allow us to investigate: Specific Aim 1: To evaluate the impact of ¿A3/A1-crystallin on Notch signaling in retinal astrocytes. Astrocytes migrate from the optic nerve into the inner retina to form a template on which the retinal vessels will develop. A defect in the normal template formation may lead to dramatic differences in astrocyte morphology and behavior and may disrupt the normal programmed regression of the hyaloid vessels during development. The molecular signals that mediate astrocyte template formation remain elusive. Our preliminary data indicate that Notch signaling is necessary for astrocyte template formation in the retina and that A3/A1-crystallin may be a regulator of notch signaling in astrocytes. In Focus 1, we will compare the effects of -secretase inhibition in wild type astrocytes and astrocytes lacking A3/A1-crystallin, while Focus 2 will determine if A3/A1-crystallin is involved in the proteolytic release of active notch and thereby the activation of Noth target genes. Focus 3 will be to investigate a possible role for A3/A1-crystallin in Notch degradation. Specific Aim 2: To determine how loss of A3/A1-crystallin increases Aquaporin-4 in retinal astrocytes. We propose to seek direct evidence that A3/A1-crystallin regulates Aqp4 expression and astrocyte migration in the developing retina (Focus 1). It is also possible that A3/A1-crystallin does not directly regulate Aqp4, but affects Aqp4 levels through joint action with a binding partner; we will address this possibility in Focus 2. Specific Aim 3: To investigate the role of astrocytes in the abnormal retention of the hyaloid artery. Our approach will be (1) to determine whether A3/A1-crystallin regulates VEGF secretion by astrocytes and thereby contributes to vessel stabilization; (2) to investigate the possible role of pericytes in this abnormal retention of the hyaloid artery during development; and (3) to analyze the structural integrity and cellular organization of the hyaloid vessels. Our proposed investigations will help us to elucidate mechanisms whereby astrocytes play a crucial role in vascular remodeling. Understanding the molecular basis of remodeling during hyaloid regression may lead to development of novel therapeutic approaches for PFV, a potentially blinding disease in an otherwise normal child, for which there are limited treatment options at the present time.