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: 10689826
• 负责人: Karin Wuertz-Kozak
• 金额: $ 18.5万
• 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/10689826
• 关键词: Affect; Aging; Agonist; Apoptosis; Architecture; Area; Atomic Force Microscopy; Cattle; Cell physiology; Cells; Collagen; Communities; Cues; Data; Detection; Development; Disease; Disease Progression; Economic Burden; Enzyme-Linked Immunosorbent Assay; Enzymes; Extracellular Matrix; Failure; Feedback; Fibroblasts; Focal Adhesions; Genes; Goals; Government; Health; Individual; Intervertebral disc structure; Ion Channel; Knowledge; Low Back Pain; Measures; Mechanical Stimulation; Mechanics; Mechanoreceptors; Microfilaments; Pain management; Pathway interactions; Periodicity; Play; Polarization Microscopy; Prevalence; Process; Proteins; Research; Role; Slipped Disk; Spinal; Stretching; Structure; Substrate Interaction; TRP channel; Tissue Engineering; Tissues; Vertebral column; Western Blotting; Work; cell behavior; cell type; cost; culture plates; design; disability; in silico; innovation; interest; mechanical load; mechanical properties; mechanical signal; mechanotransduction; multi-scale modeling; new therapeutic target; pharmacologic; polydimethylsiloxane; regenerative approach; release of sequestered calcium ion into cytoplasm; 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.

总结 在过去的十年中，研究已经强调了衬底刚度和架构/形貌可以是可调节的。 被细胞识别，并作为机械和地形线索，最终驱动细胞行为， 机械感受器底物的变化也会影响细胞的机械刺激，从而影响细胞的功能。 对负载的响应。这些细胞反应在很大程度上是通过机械敏感离子通道，如 瞬时受体电位（TRP）通道。TRPV 4是特别感兴趣的，因为它的激活和表达 可受基质硬度和形貌的影响。此外，它的激活控制细胞外基质 (ECM)合成、基质降解酶表达和各种细胞类型中的ECM重塑。 纤维环（AF），椎间盘（IVD）的外部区域，是机械敏感组织，其中 在变性过程中，地形和机械线索发生变化，从而可能影响细胞命运，细胞活性， 和疾病进展。AF在发展中起着至关重要的作用， 下背痛是由于其结构性故障 可能导致IVD 脑疝 令人惊讶的是，到目前为止，只有很少的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/地形对TRPV 4活化的相关性的数据 在AF细胞中。TRPV 4是否调节AF中的ECM合成/重塑也是未知的， 转，影响其激活，从而创建一个至关重要的反馈回路。 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， 这方面的知识在再生方法的发展。具体而言，本项目旨在：（1）确定 基底硬度与AF细胞中TRPV 4激活的相关性，以响应（a）药理学TRPV 4 激动剂和（B）环状伸展。(2)确定基质形貌对TRPV 4活化的相关性， AF细胞响应于（a）药理学TRPV 4激动剂和（B）周期性拉伸。(3)确定 房颤细胞中TRPV 4活化在调节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， 基质线索（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TRPV 4将在接种到这些室和细胞中时被特异性激动剂或拉伸激活。 对于基于RNA-seq数据选择的靶标，将通过qPCR、ELISA和蛋白质印迹确定应答。 此外，将评价TRPV 4激活后的ECM合成和重塑。 这将是第一项研究TRPV 4在AF细胞中的基底硬度和形貌的背景下。 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 在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 工程和支持与失调的机械转导相关的新药靶点的鉴定。