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; 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.

概括 在过去的十年中，研究表明，底物僵硬和建筑/地形可以是 被细胞识别并用作机械和地形线索，最终通过 机械感受器。底物变化也会影响细胞的机械刺激，从而影响它们 对加载的响应。这些细胞反应在很大程度上通过机械敏感的离子通道来控制 作为瞬态受体电位（TRP）通道。 TRPV4引起了特定的关注，因为其激活和表达 可能会受到基质刚度和地形的影响。此外，其激活控制细胞外基质 （ECM）在各种细胞类型中的合成，降解酶表达和ECM重塑。 环纤维（AF），椎间盘（IVD）的外部区域，是一种机械敏感的组织 地形和机械提示在变性过程中发生变化，因此可能影响细胞命运，细胞活性， 和疾病进展。 AF在发展中起着至关重要的作用 腰痛作为结构性故障 可以导致IVD 疝气。 令人惊讶的是，到目前为止，只有很少的研究研究了细胞基底 AF细胞中的相互作用且没有关于底物刚度/地形在TRPV4激活的相关性的数据 在AF细胞中。 TRPV4是否调节AF中的ECM合成/重塑，这也未知，这将在 转弯，影响其激活，因此产生了关键的反馈回路。 我们的长期目标是揭示IVD健康和疾病中细胞基底过程的相关性，并使用 在开发再生方法中的这种知识。具体而言，该项目的目的是：（1）确定 底物刚度对AF细胞中TRPV4激活的相关性，响应（a）药理TRPV4 激动剂和（b）环状拉伸。 （2）确定底物地形在TRPV4激活中的相关性 AF细胞响应（a）药理TRPV4激动剂和（b）环状拉伸。 （3）确定 在调节ECM合成和重塑时，AF细胞中TRPV4激活的重要性 拟议的项目将使用伸展室的创新设计，允许研究 底物线索（刚度，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腔室和细胞中播种时伸展 响应将由QPCR，ELISA和Western印迹确定基于RNA-Seq数据选择的靶标。 此外，将评估TRPV4激活后的ECM合成和重塑。 这将是第一项在AF细胞中底物刚度和地形的背景下研究TRPV4的研究。 由于开发的工具也将适用于其他研究领域，因此我可以帮助提高基本理解 健康和疾病中的机械转导过程。获得的知识将适用于组织 工程和支持与机械转导失调相关的新药物靶标识别。