Mechanotransduction in Aqueous Outflow Regulation and Open Angle Glaucoma

项目摘要 青光眼是世界范围内不可逆性失明的主要原因。虽然这种疾病的病因很复杂， 它通常与眼内压（IOP）升高有关，这是由于对房水的抵抗力增加， 体液通过小梁网（TM）流出。人类TM的硬度大约是20倍， 青光眼，表明TM机械生物学的突出作用。虽然目前的外流途径模型 据估计，TM的近端小管区域贡献了90-95%的流出阻力，它被广泛应用于 接受流出本身在TM圆周周围不均匀，但高度分段， 相对高流量（HF）和低流量（LF）的区域。虽然这一点以前已经认识到，但几乎所有 过去几十年的研究基本上忽略了这一事实。是否存在内在的差异， HF和LF区域的TM细胞以及非昏迷和昏迷个体之间的TM细胞尚不清楚。 支持这一提议的初步数据显示，青光眼组织中有更多的LF区域， 更硬，并与基质交联酶活性升高有关。相反，HF区域是 更软、更少，并且具有更低水平的交联活性。根据这些和其他观察，我们有 假设在节段性流动区域之间的细胞存在先天差异，这些 直接调节细胞外基质（ECM）的周转、交联和流出。的精确机制 导致LF区域相对增加的原因尚不清楚。为了机械地理解 基质生物力学，成分和节段性流出之间的调节联系，我们将使用两个一般 实验方法，（A）使用灌注的人眼前节器官培养，我们将比较 HF和LF区域的生物力学和生物化学性质，测量交联，并从 这些;和（B）使用细胞衍生的基质来确定细胞-基质相互作用。具体而言，在目标1中，我们将 从昏迷和非昏迷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。我们 还将获得和表征细胞衍生的ECM，并确定这些ECM对细胞的影响。 行为在目标2中，我们将确定和量化ECM交联的性质，记录 交联酶活性，并确定抑制交联是否会改变生物力学和组成 正常眼和青光眼眼的节段性区域。我们还将确定底层是否 生物力学调节节段流动池中的交联。最后，在目标3中，我们将使用目标 方法来确定调节器的稳态反应和操纵流出区域。特别是我们 将靶向ECM结合整合素α7β1在介导流出、ECM重塑和 节段性血流的变化这些目标的实现将揭示TM细胞与ECM相互作用的机制 并确定新的靶点，以通过增加主动流出面积来降低升高的IOP，并治疗青光眼。

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.