Substrate Stiffness, Topography, and TRPV4 in AF Mechanotransduction

AF 机械传导中的基底刚度、形貌和 TRPV4

• 批准号: 10797825
• 负责人: Karin Wuertz-Kozak
• 金额: $ 9.8万
• 依托单位: ROCHESTER INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797825
• 关键词: Affect; Agonist; Architecture; Area; Cell physiology; Cells; Cues; Data; Development; Disease; Disease Progression; Enzyme-Linked Immunosorbent Assay; Enzymes; Extracellular Matrix; Failure; Feedback; Goals; Health; Intervertebral disc structure; Ion Channel; Knowledge; Low Back Pain; Mechanical Stimulation; Mechanics; Mechanoreceptors; Periodicity; Play; Process; Research; Role; Slipped Disk; Spinal; Stretching; Substrate Interaction; TRP channel; Tissue Engineering; Tissues; Vertebral column; Western Blotting; cell behavior; cell type; design; innovation; interest; mechanical signal; mechanotransduction; new therapeutic target; pharmacologic; regenerative approach; response; tool; transcriptome sequencing

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.

概括 在过去的十年中，研究强调基材的刚度和结构/形貌可以 被细胞识别并作为机械和地形线索，最终驱动细胞行为 机械感受器。基质的变化也会影响细胞的机械刺激，从而影响它们的活性。 对加载的响应。这些细胞反应很大程度上是通过机械敏感离子通道控制的，例如 作为瞬时受体电位（TRP）通道。 TRPV4 由于其激活和表达而受到特别关注 可能受到基体刚度和地形的影响。此外，它的激活控制细胞外基质 (ECM) 合成、基质降解酶表达以及各种细胞类型中的 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 变性过程中形貌和机械线索发生变化，从而可能影响细胞命运、细胞活动， 和疾病进展。 AF在发展中起着至关重要的作用 腰痛是其结构性故障 可能导致 IVD 疝气。 令人惊讶的是，迄今为止只有极少数研究调查了细胞基质 AF 细胞中的相互作用，并且没有关于基质硬度/形貌与 TRPV4 激活的相关性的数据 在 AF 细胞中。目前还不清楚 TRPV4 是否调节 AF 中的 ECM 合成/重塑，这会在 转动，影响其激活，从而创建一个关键的反馈循环。 我们的长期目标是揭示细胞基质过程在 IVD 健康和疾病中的相关性，并利用 这些知识用于再生方法的开发。具体而言，该项目旨在： (1) 确定 基质刚度与 AF 细胞中 TRPV4 激活的相关性，以响应 (a) 药理学 TRPV4 激动剂和（b）循环拉伸。 (2) 确定底物形貌对 TRPV4 激活的相关性 AF 细胞对 (a) 药理学 TRPV4 激动剂和 (b) 循环拉伸的反应。 (3) 确定 AF 细胞中 TRPV4 激活在调节 ECM 合成和重塑中的重要性 拟议的项目将使用拉伸室的创新设计，可以研究 基质线索（硬度、形貌）和机械刺激在调节细胞功能中的综合作用 和命运。 TRPV4 将被特定激动剂激活或在这些室和细胞中接种后拉伸 将通过 qPCR、ELISA 和 Western Blot 确定基于 RNA-seq 数据选择的靶标的反应。 此外，还将评估 TRPV4 激活后的 ECM 合成和重塑。 这将是第一项在 AF 细胞的基底刚度和形貌背景下研究 TRPV4 的研究。 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理解 健康和疾病中的机械传导过程。所获得的知识将适用于组织 设计并支持识别与机械转导失调相关的新药物靶点。