Extracellular Matrix and Outflow Resistance

项目摘要。 青光眼是一种常见的致盲性疾病，影响全世界超过6600万人，并且经常在 与眼内压（IOP）升高相关。然而，一种调节升高的 眼压必须存在，因为少于10%的人发展为青光眼。眼压升高是由于 对通过小梁网（TM）流出的房水的阻力。当TM细胞受到 持续升高的IOP，它们通过释放特异性的细胞外基质（ECM）来启动细胞外基质（ECM）的重塑。 蛋白酶，包括基质金属蛋白酶（MMP）。ECM周转产生了一个新的，减少阻力， 允许更多的房水流出TM并降低IOP。分子检查 参与重塑，它们的蛋白水解靶点和作用方式和位点是至关重要的， 了解IOP是如何调节的。我们的长期目标是确定 升高的IOP在正常眼睛中被稳态调节。目前这项提案的目的是进一步 阐明两种ECM成分ADAMTS 4和透明质酸的功能及其在IOP中的作用 体内平衡 ADAMTS 4（A Disintegrin and Metalloproteinase with Thrombospondin motifs）是一种蛋白水解酶 使TM中的ECM成分降解。ADAMTS 4增加眼前节灌注的流出功能 提示ADAMTS 4在对升高的IOP的正常稳态反应中的作用。在这里，我们提出一个 工作模型，其中ADAMTS 4在专门的细胞结构中被激活，然后切割其目标 分子，并在核内体中内化到细胞中。这些核内体可以再循环到细胞中 表面以将活性ADAMTS 4释放回ECM。我们将研究ADAMTS 4是如何被蛋白水解的， 在TM细胞中活化后的细胞命运。TM中的Hybryonan浓度在 衰老和POAG，这可能是由于合成减少，降解增加或细胞增殖增加， 透明质酸摄取。最近，三个基因参与透明质酸的合成和六个基因负责 透明质酸降解已经被鉴定，我们将研究它们的mRNA水平， 压力我们还将确定透明质酸浓度的降低如何影响前牙区的流出功能。 节段灌流培养。最后，研究了透明质酸浓度对ADAMTS 4/MMP表达的影响， 将确定TM细胞中的定位和/或活化。 研究ADAMTS 4和透明质酸在肿瘤细胞中调节的分子机制。 TM将提供有关导致IOP稳态调节的复杂系列事件的新信息。 研究正常眼压稳态的分子细节有助于开发新的治疗方法 旨在降低青光眼患者的IOP。

Project Summary. Glaucoma is a common blinding disease that affects over 66 million people worldwide and is frequently associated with elevated intraocular pressure (IOP). However, a homeostatic mechanism to adjust elevated IOP must exist since less than 10% of people develop glaucoma. Elevated IOP is caused by increased resistance to aqueous humor outflow through the trabecular meshwork (TM). When TM cells are subjected to sustained elevated IOP, they initiate remodeling of the extracellular matrix (ECM) by releasing specific proteinases including matrix metalloproteinases (MMPs). ECM turnover produces a new, reduced resistance to allow greater aqueous humor outflow through the TM and decrease IOP. Examination of the molecules involved in remodeling, their proteolytic targets and modes and sites of action is critical in order for us to understand how IOP is adjusted. Our long-term goal is to determine the molecular mechanisms by which elevated IOP is homeostatically adjusted in normal eyes. The goal of this current proposal is to further elucidate the function of two ECM components, ADAMTS4 and hyaluronan, and their roles in IOP homeostasis. ADAMTS4 (A Disintegrin and Metalloproteinase with Thrombospondin motifs) is a proteolytic enzyme that degrades ECM components in the TM. ADAMTS4 increases outflow facility in anterior segment perfusion culture suggesting a role for ADAMTS4 in normal homeostatic responses to elevated IOP. Here, we propose a working model whereby ADAMTS4 is activated at specialized cellular structures, then cleaves its target molecules and is internalized into the cell in endosomes. These endosomes may be recycled to the cell surface to release active ADAMTS4 back into the ECM. We will investigate how ADAMTS4 is proteolytically activated and its cellular fate following activation in TM cells. Hyaluronan concentrations in TM decrease during aging and in POAG, which may be due to decreased synthesis, increased degradation or increased cellular hyaluronan uptake. Recently, three genes involved in hyaluronan synthesis and six genes responsible for hyaluronan degradation have been identified and we will investigate their mRNA levels in response to pressure. We will also determine how decreased hyaluronan concentration affects outflow facility in anterior segment perfusion culture. Finally, the effects of hyaluronan concentration on ADAMTS4/MMP expression, localization and/or activation in TM cells will be determined. Investigation into the molecular mechanisms by which ADAMTS4 and hyaluronan are regulated in the TM will provide new information on the complex series of events that leads to homeostatic adjustment of IOP. Studying the molecular details of normal IOP homeostasis should facilitate development of novel therapies targeted at reducing IOP in glaucoma patients.