Extracellular Matrix and Outflow Resistance

描述（由申请人提供）：青光眼是一种常见的致盲疾病，影响全球超过6600万人，通常与眼压升高（IOP）相关。然而，必须存在调节IOP升高的稳态机制，因为只有不到10%的人会患上青光眼。IOP升高是由于房水通过小梁网（TM）流出的阻力增加引起的。当TM细胞遭受持续升高的IOP时，它们通过释放包括基质金属蛋白酶（MMPs）在内的特定蛋白酶来启动细胞外基质（ECM）的重塑。ECM翻转产生新的、降低的阻力，允许更多房水通过TM流出并降低IOP。检查参与重塑的分子，它们的蛋白水解目标、模式和作用位点对于我们了解眼压是如何调节的至关重要。我们的长期目标是确定正常眼睛内眼压升高的分子机制。本研究的目的是进一步阐明两个ECM成分ADAMTS4和透明质酸的功能及其在IOP稳态中的作用。ADAMTS4（一种带有血栓反应蛋白基元的崩解素和金属蛋白酶）是一种降解TM中ECM成分的蛋白水解酶。ADAMTS4增加前段灌注培养的流出设施，表明ADAMTS4在IOP升高的正常稳态反应中起作用。在这里，我们提出了一个工作模型，其中ADAMTS4在特定的细胞结构中被激活，然后切割其靶分子并内化到细胞内体中。这些核内体可以循环到细胞表面，将活性ADAMTS4释放回ECM。我们将研究ADAMTS4是如何被蛋白水解激活的，以及它在TM细胞中激活后的细胞命运。TM和POAG中的透明质酸浓度随着衰老而降低，这可能是由于合成减少、降解增加或细胞透明质酸摄取增加。最近，三个参与透明质酸合成的基因和六个负责透明质酸降解的基因已经被确定，我们将研究它们的mRNA水平对压力的反应。我们还将确定透明质酸浓度降低如何影响前段灌注培养的流出设施。最后，我们将确定透明质酸浓度对TM细胞中ADAMTS4/MMP表达、定位和/或激活的影响。通过对ADAMTS4和透明质酸在TM中调控的分子机制的研究，将为了解导致IOP内稳态调节的一系列复杂事件提供新的信息。研究正常IOP动态平衡的分子细节有助于开发针对青光眼患者降低IOP的新疗法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Glaucoma is a leading cause of vision loss, affecting approximately 67 million people worldwide, but the underlying cause(s) of this disease is poorly understood. Current treatments for glaucoma are directed toward treating the symptoms but not the cause of the disease. This study focuses on understanding how glaucoma develops at the molecular level and thus may facilitate development of new therapies targeted at reducing elevated intraocular pressure.