Mechanotransduction in Aqueous Outflow Regulation and Open Angle Glaucoma

房水流出调节和开角型青光眼中的机械传导

• 批准号: 10091442
• 负责人: TED S ACOTT
• 金额: $ 37.22万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091442
• 关键词: Agreement; Anterior; Aqueous Humor; Area; Atomic Force Microscopy; Binding; Biochemical; Biological Assay; Biomechanics; Biophysics; Blindness; Cell Surface Receptors; Cells; Characteristics; Complex; Cultured Cells; Data; Deposition; Development; Disease; Enzymes; Etiology; Extracellular Matrix; Extracellular Matrix Proteins; Eye; Foundations; Gene Proteins; Glaucoma; Homeostasis; Human; Hydrogels; ITGA7 gene; In Vitro; Individual; Integrin Binding; Integrins; LOX gene; Laminin; Link; Measurement; Measures; Mechanics; Mediating; Modeling; Molecular; Nature; Open-Angle Glaucoma; Organ Culture Techniques; Pathway interactions; Perfusion; Phenotype; Physiologic Intraocular Pressure; Property; Proteins; Proteomics; Regulation; Resistance; Risk Factors; Role; Signal Transduction; Small Interfering RNA; Stimulus; Structure of sinus venosus of sclera; System; Therapeutic; Time; Tissues; Trabecular meshwork structure; Western Blotting; aqueous; base; biophysical analysis; cell behavior; crosslink; enzyme activity; inhibitor/antagonist; interdisciplinary approach; mRNA Expression; mechanotransduction; novel; novel therapeutic intervention; novel therapeutics; pressure; protein expression; response

PROJECT SUMMARY Glaucoma is a leading cause of irreversible blindness worldwide. While the etiology of the disease is complex, it is typically associated with elevated intraocular pressure (IOP) due to increased resistance to aqueous humor outflow through the trabecular meshwork (TM). The human TM is approximately 20 fold stiffer in glaucoma, suggesting a prominent role of TM mechanobiology. Although current outflow pathway models estimate that the juxtacanalicular region of the TM contributes to 90-95% of the outflow resistance, it is widely accepted that outflow itself is not uniform around the circumference of the TM, but is highly segmental with regions of relatively high flow (HF), and low flow (LF). Although this has been recognized previously, nearly all studies over the past few decades have essentially ignored this fact. Whether there are inherent differences in TM cells of HF and LF regions and between non-glaucomatous and glaucomatous individuals remains unclear. Preliminary data in support of this proposal shows that, with glaucoma tissues, there are more LF regions, they are stiffer, and are associated with elevated matrix crosslinking enzyme activity. Conversely, HF regions are softer, fewer, and have lower levels of crosslinking activity. Based on these and other observations, we have hypothesized that there are innate differences in cells between the segmental flow regions, and these directly regulate extracellular matrix (ECM) turnover, crosslinking, and outflow. The precise mechanism that underlies the relative shift to increased LF regions is unclear. In order to mechanistically understand the regulatory link between matrix biomechanics, composition, and segmental outflow, we will use two general experimental approaches, (A) using perfused human anterior segment organ culture, we will compare biomechanical and biochemical properties of HF and LF regions, measure crosslinking, and, isolate cells from these; and (B) use cell derived matrices to determine cell-matrix interactions. Specifically, in Aim 1, we will isolate TM cells from different flow regions of glaucomatous and non-glaucomatous TM, characterize cell surface receptor distribution, and investigate their mechanotransduction response to biophysical stimuli. We will also obtain and characterize cell derived ECM, and determine the effect that these ECM have on cellular behavior. In Aim 2, we will ascertain and quantify the nature of ECM crosslinks, document differences in crosslinking enzyme activity, and determine if inhibiting crosslinks changes the biomechanics and composition of segmental regions in both normal and glaucomatous eyes. We will also determine if substratum biomechanics modulates crosslinking in segmental flow cells. Finally, in Aim 3, we will use a targeted approach to identify regulators of the homeostatic response and manipulate outflow regions. Particularly we will target the specific role that ECM binding integrin α7β1 has in mediating outflow, ECM remodeling, and shifts in segmental flow. Accomplishment of these aims will reveal a mechanism for TM cell-ECM interactions and identify novel targets to reduce elevated IOP by increasing areas of active outflow, and treat glaucoma.

项目摘要 青光眼是全球不可逆失明的主要原因。虽然该疾病的病因很复杂，但 由于对水性的抗性增加，它通常与眼内压（IOP）相关 通过小梁网（TM）的幽默出口。人类TM大约20倍 青光眼，表明TM机械生物学的重要作用。尽管当前的出口通路模型 据估计，TM的近去核区域贡献了90-95％的出口电阻，它是广泛的 接受插座本身并不统一在TM的循环周围，而是高度分段的 流动相对较高（HF）和低流量（LF）的区域。尽管以前已经认可了这一点，但几乎全部 在过去的几十年中，研究基本上忽略了这一事实。是否存在继承差异 HF和LF区域的TM细胞以及非葡萄糖和青光眼个体之间的TM细胞尚不清楚。 支持该提案的初步数据表明，有了青光眼组织，有更多的LF区域，它们 被僵硬，与升高的基质交联酶活性有关。相反，HF地区是 较软，较少，交联活动的水平较低。基于这些和其他观察，我们有 假设，节段流区域之间的细胞存在差异， 直接调节细胞外基质（ECM）周转，交联和出口。精确机制 这是尚不清楚与LF区域增加的相对转变的基础。为了机械理解 基质生物力学，组成和节段插座之间的调节链接，我们将使用两个一般 实验方法，（a）使用灌注的人体前部器官培养，我们将比较 HF和LF区域的生物力学和生化特性，测量交联和分离细胞中的细胞 这些; （b）使用细胞衍生的矩阵确定细胞 - 矩阵相互作用。具体来说，在AIM 1中，我们将 来自青光眼和非葡萄糖良好TM的不同流动区域的分离TM细胞，表征细胞 表面受体分布，并研究其对生物物理刺激的机械传导反应。我们 还将获得并表征派生的ECM细胞，并确定这些ECM对细胞的影响 行为。在AIM 2中，我们将确定和量化ECM交联的性质，文件差异 交联酶活性，并确定抑制交联是否改变了生物力学和组成 在正常和青光眼中的节段区域。我们还将确定底层是否 生物力学调节节段流中的交联。最后，在AIM 3中，我们将使用目标 识别体内稳态响应和操纵输出流区域的调节器的方法。特别是我们 将针对ECM结合α7β1在介导出口，ECM重塑和 节段流的变化。这些目标的完成将揭示TM细胞ECM相互作用的机制 并确定新的靶标，通过增加活性出口区域并治疗青光眼来降低IOP的升高。