Substrate Stiffness, Topography, and TRPV4 in AF Mechanotransduction

摘要 在过去的十年中(S)，研究强调了衬底硬度和建筑/地形可以 由细胞识别，并作为最终驱动细胞行为的机械和地形线索 机械感受器。底物的变化也会影响细胞的机械刺激，从而影响它们的 对装载的响应。这些细胞反应很大程度上是通过机械敏感的离子通道来控制的，如 作为瞬时受体电位(Trp)通道。TRPV4由于其激活和表达而备受关注 会受到基质硬度和地形的影响。此外，它的激活控制着细胞外基质 不同细胞类型的细胞外基质(ECM)合成、基质降解酶表达和ECM重塑。 纤维环(AF)是椎间盘(IVD)的外部区域，是一种机械敏感组织，其中 在退化过程中，地形和机械信号会发生变化，因此可能会影响细胞命运、细胞活动、 和疾病的进展。房协在……的发展中起着至关重要的作用。 腰背部疼痛是其结构性故障 可能导致IVD 疝气。 令人惊讶的是，到目前为止，只有很少的研究对细胞底物进行了研究 房颤细胞内的相互作用，目前还没有关于底物硬度/地形对TRPV4激活的相关性的数据 在房颤细胞中。目前也不清楚TRPV4是否调节房颤中ECM的合成/重塑，这将在 反过来，影响它的激活，从而创造一个关键的反馈循环。 我们的长期目标是揭示细胞-底物过程在IVD健康和疾病中的相关性，并使用 这种知识在再生方法的发展中发挥着重要作用。具体地说，本项目旨在：(1)确定 底物硬度与房颤细胞激活TRPV4的相关性 激动剂和(B)循环拉伸。(2)确定底物形貌与TRPV4激活的相关性 房颤细胞对(A)药物TRPV4激动剂和(B)循环拉伸的反应。(3)确定 房颤细胞TRPV4激活在调节细胞外基质合成和重塑中的作用 拟议的项目将使用一种创新的拉伸室设计，允许调查 底物提示(硬度、地形)和机械刺激在调节细胞功能中的综合作用 和命运。TRPV4将被特定的激动剂激活或在这些小室和细胞中播种时被拉伸 对根据RNA-SEQ数据选择的靶标，将通过定量聚合酶链式反应(QPCR)、酶联免疫吸附试验(ELISA)和Western Blot确定反应。 此外，还将评估TRPV4激活后ECM的合成和重塑。 这将是第一次在房颤细胞的底物硬度和地形的背景下研究TRPV4。 由于开发的工具也将适用于其他研究领域，我可以帮助推进基本的理解 机械转导过程在健康和疾病中的作用。所获得的知识将应用于组织 设计和支持识别与失调的机械转导相关的新药靶点。

SUMMARY Over the past decade(s), research has highlighted that substrate stiffness and architecture/topography can be recognized by cells and serve as mechanical and topographical cues that ultimately drive cell behavior through mechanoreceptors. Substrate changes can also affect the mechanical stimulation of cells and thus their response to loading. These cell responses are largely governed through mechanosensitive ion channels, such as the transient receptor potential (TRP) channels. TRPV4 is of specific interest as its activation and expression can be affected by matrix stiffness and topography. Furthermore, its activation controls extracellular matrix (ECM) synthesis, matrix-degrading enzyme expression, and ECM remodeling in various cell types. The annulus fibrosus (AF), the outer area of the intervertebral disc (IVD), is a mechanosensitive tissue in which topographical and mechanical cues change during degeneration, thus likely affecting cell fate, cellular activity, and disease progression. The AF plays a crucial role in the development of low back pain as its structural failure can lead to IVD herniation. Surprisingly, only very few studies have thus far investigated cell-substrate interactions in AF cells and no data exists on the relevance of substrate stiffness/topography on TRPV4 activation in AF cells. It is also unknown whether TRPV4 regulates ECM synthesis/remodeling in the AF, which would, in turn, affect its activation and hence create a crucial feedback loop. Our long-term goal is to reveal the relevance of cell-substrate processes in IVD health and disease and to use this knowledge in the development of regenerative approaches. Specifically, this project aims to: (1) Determine the relevance of substrate stiffness on TRPV4 activation in AF cells in response to (a) a pharmacological TRPV4 agonist and (b) cyclic stretching. (2) Determine the relevance of substrate topography on TRPV4 activation in AF cells in response to (a) a pharmacological TRPV4 agonist and (b) cyclic stretching. (3) Determine the importance of TRPV4 activation in AF cells in regulating ECM synthesis and remodeling The proposed project will use an innovative design of stretching chambers that allows investigating the integrative role of substrate cues (stiffness, topography) and mechanical stimulation in modulating cell function and fate. TRPV4 will be activated by specific agonists or stretching upon seeding in these chambers and cell responses will be determined by qPCR, ELISA, and Western Blot for targets selected based on RNA-seq data. Furthermore, ECM synthesis and remodeling following TRPV4 activation will be evaluated. This will be the first study to investigate TRPV4 in the context of substrate stiffness and topography in AF cells. As the developed tools will also apply to other research areas, I can help advance the fundamental understanding of mechanotransduction processes in health and disease. The gained knowledge will be applicable in tissue engineering and support the identification of new drug targets related to dysregulated mechanotransduction.