Extracellular Matrix and Outflow Resistance

项目摘要。 青光眼是一种常见的致盲疾病，在全球范围内影响着6600万人，而且经常 与眼压升高有关。然而，一种调节体内平衡的机制升高 眼压肯定存在，因为只有不到10%的人会患上青光眼。高眼压是由眼压升高引起的 抗房水流出通过小梁网(TM)。当TM细胞受到 持续的高眼压，它们通过释放特定的细胞外基质(ECM)来启动细胞外基质的重塑 蛋白水解酶包括基质金属蛋白酶(MMPs)。ECM周转产生了新的、减少的阻力 允许更多的房水流出TM，降低眼压。对分子的检测 它们的蛋白分解靶点、作用方式和作用部位对我们来说至关重要。 了解眼压是如何调整的。我们的长期目标是确定 在正常眼睛中，高眼压是稳态调节的。目前这项提议的目标是进一步 阐明两种细胞外基质成分ADAMTS4和透明质酸的功能及其在眼压中的作用 动态平衡。 ADAMTS4(一种带有凝血酶反应蛋白基序的去整合素和金属蛋白酶)是一种蛋白水解酶 这会降低TM中的ECM组件。ADAMTS4增加眼前段灌注的流出功能 培养提示ADAMTS4在对高眼压的正常内环境平衡反应中起作用。在这里，我们提出一种 ADAMTS4在特定的细胞结构上被激活，然后切割其靶标的工作模型 分子，并在内吞体内进入细胞内。这些内小体可能被回收到细胞中。 表面将活性ADAMTS4释放回ECM。我们将研究ADAMTS4是如何蛋白分解的 在TM细胞中被激活后其细胞命运。TM中的透明质酸浓度在 衰老和POAG，可能是由于合成减少、降解增加或细胞数量增加所致 透明质酸摄取。目前，有3个基因参与透明质酸合成，6个基因参与透明质酸合成 已经确定了透明质酸的降解，我们将研究它们的mRNA水平，以响应 压力。我们还将确定降低的透明质酸浓度如何影响前路的流出功能。 节段灌流培养。最后，研究了透明质酸浓度对ADAMTS4/基质金属蛋白酶表达的影响。 TM细胞中的定位和/或激活将被确定。 ADAMTS4和透明质酸调控的分子机制研究 TM将提供有关导致眼压稳态调整的一系列复杂事件的新信息。 研究正常眼压稳态的分子细节应有助于开发新的治疗方法 旨在降低青光眼患者的眼压。

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.