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.

项目概要 青光眼是全世界不可逆失明的主要原因，虽然该疾病的病因很复杂， 由于对房水的抵抗力增加，它通常与眼内压（IOP）升高有关 体液通过小梁网 (TM) 流出，人类 TM 的硬度约为 20 倍。 青光眼，表明 TM 机械生物学的重要作用，尽管目前的流出途径模型。 据估计，TM 的近小管区域贡献了 90-95% 的流出阻力，因此广泛认为 接受流出本身在 TM 圆周上并不均匀，而是高度分段的 相对高流量 (HF) 和低流量 (LF) 的区域虽然先前已经认识到，但几乎所有区域。 过去几十年的研究基本上忽略了这一事实：是否存在内在差异。 HF 和 LF 区域以及非青光眼个体和青光眼个体之间的 TM 细胞仍不清楚。 支持这一提议的初步数据表明，青光眼组织中存在更多的 LF 区域，它们 HF 区域更硬，并且与离线基质交联酶活性升高相关。 根据这些和其他观察，我们得到了更柔软、更少且交联活性水平更低的材料。 证实分段流动区域之间的细胞存在先天差异，并且这些 直接调节细胞外基质（ECM）周转、交联和流出的精确机制。 为了机械地理解 LF 区域相对转移的原因尚不清楚。 基质生物力学、组成和节段流出之间的调节联系，我们将使用两个通用的 实验方法，（A）使用灌注的人眼前段器官培养，我们将比较 HF 和 LF 区域的生物力学和生化特性，测量交联，并从其中分离细胞 这些；以及 (B) 使用细胞衍生矩阵来确定细胞-基质相互作用。具体来说，在目标 1 中，我们将。 从青光眼和非青光眼 TM 的不同流动区域分离 TM 细胞，表征细胞 表面受体分布，并研究它们对生物物理刺激的机械转导反应。 还将获得并表征细胞衍生的 ECM，并确定这些 ECM 对细胞的影响 在目标 2 中，我们将确定并量化 ECM 交联的性质，记录差异。 交联酶活性，并确定抑制交联是否会改变生物力学和成分 我们还将确定正常眼和青光眼眼的节段区域是否为基质。 最后，在目标 3 中，我们将使用有针对性的生物力学调节分段流动池中的交联。 方法来识别稳态反应的调节因子并操纵流出区域。 将针对 ECM 结合整合素 α7β1 在介导流出、ECM 重塑和 这些目标的实现将揭示TM细胞-ECM相互作用的机制。 并确定新的目标，通过增加主动流出面积来降低眼压升高，并治疗青光眼。