Temporary plasma membrane disruptions in corneal epithelium and keratocytes.

角膜上皮和角膜细胞的暂时质膜破坏。

• 批准号: 10630403
• 负责人: MITCHELL A WATSKY
• 金额: $ 38.5万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10630403
• 关键词: Actins; Affect; Apoptosis; Basic Science; Biomechanics; Calcium; Calcium Oscillations; Cell membrane; Cells; Contact Lenses; Cornea; Corneal Diseases; Corneal Stroma; Data; Diabetes Mellitus; Diabetic mouse; Event; Eye; Future; Gap Junctions; Genetic; Health; Homeostasis; Human; Individual; Injury; Keratopathy; Light Cell; Location; Maintenance; Mechanics; Mediating; Membrane; Messenger RNA; Microscope; Microscopy; Minor; Mus; Osteocytes; Pathology; Pathway interactions; Physiological; Predisposition; Proliferating; Protein Biosynthesis; Proteins; Publishing; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Small Interfering RNA; Stress; System; TRPV1 gene; Testing; Time; Tissues; Trauma; Work; bone; corneal epithelial wound healing; corneal epithelium; design; diabetic; environmental stressor; improved; knock-down; migration; multi-photon; novel; pharmacologic; receptor; response; stressor

Transient plasma membrane disruptions (TPMD) are minor, quickly repairable plasma membrane tears that result from normal stresses on cells within tissues. We hypothesize that keratocyte and cornea epithelial (CE) TPMDs represent a novel cornea mechanosensation pathway, with TPMDs routinely occurring in keratocytes and CE following mechanical stressors such as eye rubbing and application of contact lenses. We further hypothesize that the resulting TPMD-induced Ca++ waves (TPMD-Ca++ Wvs) initiate signaling cascades that coordinate specific activities of keratocytes and CE, including corneal matrix maintenance and cell actin dynamics, respectively, which become disrupted in pathologies such as diabetes. The first Aim of this proposal is to identify the specific Ca++ channels responsible for keratocyte and CE TPMD-Ca++ Wvs. The only Ca++ transporters identified to date in keratocytes are TRPV1 and TRPM8. We determined that neither are significantly involved in TPMD-Ca++ Wvs, and our preliminary data indicate that L- type Ca++ channels are likely involved. Identifying the specific Ca++ channels involved will allow us to determine the signaling pathways triggering these Ca++ waves. Channel identification will be carried out by creating TPMDs in individual mouse and human keratocytes and CE using a multiphoton microscope, either in culture or within ex-vivo corneas, and pharmacological blockers and genetic knockdown of the identified channels will be utilized to identify channel subtypes contributing to the TPMD-Ca++ Wvs. The second Aim is to identify the cell signaling pathways responsible for initiating and propagating TPMD- Ca++ Wvs in keratocytes and CE in normal and diabetic corneas. We previously examined the general contribution of several signaling pathways leading to keratocyte TPMD-Ca++Wvs, including ATP, release of intracellular Ca++ stores, and gap junctions. This aim is designed to identify the specific signaling molecules responsible for keratocyte and CE TPMD-Ca++Wvs. Pharmacological blockers and genetic knockdown of the signaling molecules we have identified to date and their different receptor subtypes will be employed to determine which specific receptors and signaling proteins are responsible for initiating and propagating keratocyte and CE TPMD-Ca++ Wvs. Aim 3 will test the hypothesis that TPMD-Ca++ Wvs initiate Ca++-activated activities associated with cornea matrix remodeling and CE actin dynamics, and that these are altered in diabetic corneas. Our preliminary data indicate that keratocyte and CE TPMD-Ca++ Wvs influence cornea matrix and actin remodeling, respectively, along with other physiological activities. We also find that diabetes alters TPMD responses. TPMDs will be created by gentle manipulation of healthy and diabetic cells in culture and in diabetic mice. TPMD-Ca++ Wvs will be blocked pharmacologically or with genetic knockdown, and matrix protein synthesis and CE actin remodeling will be examined, along with additional activities including ATP release, apoptosis, migration, and proliferation.

瞬时质膜破裂（TPMD）是一种轻微的、可快速修复的质膜撕裂， 是由组织内细胞的正常应力引起的。我们假设角膜细胞和角膜上皮（CE） TPMD代表了一种新的角膜机械感觉通路，TPMD通常发生在角膜细胞中 以及在机械压力刺激（例如眼睛摩擦和隐形眼镜的应用）之后的CE。我们进一步 假设由此产生的TPMD诱导的Ca++波（TPMD-Ca++ Wvs）启动信号级联， 协调角膜细胞和CE的特定活动，包括角膜基质维持和细胞肌动蛋白 动力学，其在诸如糖尿病的病理中被破坏。 本研究的第一个目的是鉴定负责角膜细胞和CE的特异性Ca++通道。 TPMD-Ca++ Wvs.迄今为止，在角膜细胞中发现的唯一Ca++转运蛋白是TRPV 1和TRPM 8。我们 确定两者都不显著参与TPMD-Ca++ Wvs，我们的初步数据表明，L- 型Ca++通道可能参与。识别所涉及的特定Ca++通道将使我们能够确定 触发这些Ca++波的信号通路。将通过创建TPMD进行通道识别 在单个小鼠和人角膜细胞中，使用多光子显微镜进行CE，无论是在培养物中还是在 将使用离体角膜和药理学阻断剂以及所鉴定通道的遗传敲除 以鉴定对TPMD-Ca++ Wvs有贡献的通道亚型。 第二个目的是确定负责启动和传播TPMD的细胞信号传导途径。 正常和糖尿病角膜中角膜细胞的Ca++ Wvs和CE。我们之前检查了将军 导致角膜细胞TPMD-Ca++Wvs的几种信号通路的贡献，包括ATP， 细胞内Ca++储存和间隙连接。这一目的是为了确定特定的信号分子 负责角膜细胞和CE TPMD-Ca++Wvs。药理学阻断剂和基因敲除 我们迄今为止已经鉴定的信号分子及其不同的受体亚型将被用于 确定哪些特定的受体和信号蛋白负责启动和传播 角膜基质细胞和CE TPMD-Ca++ Wvs. 目的3将验证TPMD-Ca++ Wvs启动与角膜相关的Ca++激活活动的假设 基质重塑和CE肌动蛋白动力学，并且这些在糖尿病角膜中改变。我们的初步数据 表明角膜基质细胞和CE TPMD-Ca++ Wvs分别影响角膜基质和肌动蛋白重塑， 沿着其他生理活动。我们还发现糖尿病改变了TPMD反应。TPMD将是 通过温和地操作培养中的健康细胞和糖尿病细胞以及糖尿病小鼠。TPMD-Ca++ Wvs将 通过基因敲除、基质蛋白合成和CE肌动蛋白重塑来阻断细胞凋亡 沿着检查其他活性，包括ATP释放、凋亡、迁移和增殖。