Validation of Fibroblast-Derived PI16 as a Novel Target for pain Treatment

验证成纤维细胞衍生的 PI16 作为疼痛治疗新靶点

• 批准号: 9974863
• 负责人: Andrew John Shepherd
• 金额: $ 290.84万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9974863
• 关键词: Affect; American; Blood-Nerve Barrier; Brain; Conditioned Culture Media; Cyclic AMP; Data; Databases; Family; Female; Fibroblasts; Freund' s Adjuvant; Gene Expression; Genetic; Health; Human; Immunofluorescence Immunologic; In Vitro; Infiltration; Laboratories; Lead; Leukocyte Trafficking; Leukocytes; Mass Spectrum Analysis; Mediating; Meningeal; Meninges; Messenger RNA; Modeling; Molecular; Mus; Myofibroblast; Nerve; Neuraxis; Neuroglia; Neurons; Neuropathy; Opioid; Paclitaxel; Pain; Pain management; Pathway interactions; Patients; Perineuriums; Peripheral Nerves; Permeability; Precipitation; Production; Protease Inhibitor; Proteins; Public Health; Regulation; Research; Resistance; Resolution; Role; Sampling; Spinal Cord; Spinal Ganglia; Testing; Tissues; Transcript; Transforming Growth Factor beta; Validation; Vascular Permeabilities; addiction; base; chronic pain; human tissue; in vitro Model; inflammatory pain; innovation; male; mechanical allodynia; member; migration; monocyte; mouse model; nerve injury; new therapeutic target; novel; opioid use; overexpression; pain model; painful neuropathy; prevent; response; sciatic nerve; spared nerve; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Chronic pain and the addictive effects of opioids used to control pain are major health problems affecting millions of Americans. Validation of novel targets for the safe treatment of chronic pain is urgently needed. We identified peptidase inhibitor 16 (PI16) as a novel regulator of chronic pain in an unbiased RNA seq screen. PI16 is a putative peptidase inhibitor that has not been studied in the context of pain. We showed that male and female Pi16-/- mice are protected against mechanical allodynia in the spared nerve injury (SNI) and paclitaxel models of neuropathic pain. Along the neuraxis, PI16 is only detectable in fibroblasts around peripheral nerves (perineurium), and in the meninges of dorsal root ganglia (DRG), spinal cord, and brain, but not in neurons, glia or leukocytes. . PI16 levels in perineurial and DRG meningeal fibroblasts increase during neuropathic pain. The overall objective of this project is to validate PI16 as a novel target for the treatment of chronic pain using mouse models and human tissues of neuropathy patients and controls and to identify the underlying mechanisms. Our central hypothesis is that increased PI16 secretion by DRG meningeal and perineurial fibroblasts promotes chronic pain by increasing blood nerve barrier (BNB) permeability and leukocyte trafficking into nerve and DRG. The significance is in the validation of PI16 as a novel, potentially druggable, regulator of chronic pain and the discovery of fibroblasts as key regulators of chronic pain. We propose the following three specific aims: 1. Validate the key role of PI16 in chronic pain. In two independent laboratories, we will test the hypothesis that genetic deletion of Pi16 in male and female mice protects against chronic pain in well-established models of chronic pain. 2. Investigate expression and regulation of PI16 in fibroblasts around mouse DRG and mouse and human peripheral nerves. The hypothesis is that PI16 production by fibroblasts in DRG meninges and perineurium is increased via a TGFβ− and Epac1-mediated pathway to promote chronic pain. We will use tissues from the neuraxis in mouse pain model and nerves from humans with neuropathic pain. 3. Determine the contribution of PI16 to blood nerve barrier integrity in mouse pain models and in a human in vitro model. Using a model of the human BNB, we will test the hypothesis that PI16 increases BNB permeability and leukocyte transmigration. Using co-immuno-precipitation and mass spectrometry we will identify novel PI16 targets. This proposal is conceptually innovative because successful completion will validate PI16 as a novel key regulator of neuropathic and inflammatory pain and establish a novel role of perineurial fibroblasts in regulating BNB function with major consequences for chronic pain. The expected findings are significant because they will advance our fundamental understanding of cellular and molecular mechanisms that contribute to chronic pain and will identify a novel target for treatment of pain that is not expressed in the brain, and therefore is unlikely to lead to addiction.

项目概要/摘要 慢性疼痛和用于控制疼痛的阿片类药物的成瘾作用是影响健康的主要问题 我们迫切需要验证安全治疗慢性疼痛的新靶点。 在无偏 RNA seq 筛选中确定肽酶抑制剂 16 (PI16) 是慢性疼痛的新型调节剂。 PI16 是一种推定的肽酶抑制剂，尚未在疼痛背景下进行研究。 雌性 Pi16-/- 小鼠在免神经损伤 (SNI) 和紫杉​​醇中可免受机械性异常性疼痛 沿着神经轴，PI16 只能在周围神经周围的成纤维细胞中检测到。 （神经束膜）、背根神经节（DRG）、脊髓和大脑的脑膜中，但不在神经元中， 神经性疼痛期间神经周围和背根神经节脑膜成纤维细胞中的 PI16 水平升高。 该项目的总体目标是验证 PI16 作为治疗慢性疼痛的新靶点 使用小鼠模型和神经病患者和对照的人体组织来识别潜在的 我们的中心假设是 DRG 脑膜和神经周围的 PI16 分泌增加。 成纤维细胞通过增加血神经屏障（BNB）通透性和白细胞来促进慢性疼痛 贩运到神经和 DRG 中的意义在于验证 PI16 作为一种潜在的新型药物。 可药物化，慢性疼痛的调节剂以及成纤维细胞作为慢性疼痛的关键调节剂的发现。 我们提出以下三个具体目标： 1. 验证 PI16 在慢性疼痛中的关键作用。 独立实验室，我们将测试雄性和雌性小鼠中 Pi16 基因缺失的假设 在成熟的慢性疼痛模型中预防慢性疼痛 2. 研究表达和。 PI16 对小鼠 DRG 周围成纤维细胞以及小鼠和人类周围神经的调节。 假设 DRG 脑膜和神经束膜中成纤维细胞产生的 PI16 通过以下途径增加： TGFβ− 和 Epac1 介导的途径可促进慢性疼痛 我们将使用小鼠神经轴的组织。 3. 确定 PI16 对神经性疼痛的影响。 使用模型在小鼠疼痛模型和人体体外模型中实现血神经屏障完整性。 对于人类 BNB，我们将检验 PI16 增加 BNB 通透性和白细胞的假设 使用免疫共沉淀和质谱分析，我们将新颖地鉴定 PI16 靶点。 该提案在概念上具有创新性，因为成功完成将验证 PI16 作为新颖的密钥 神经病理性疼痛和炎性疼痛的调节剂，并建立神经周围成纤维细胞的新作用 调节 BNB 功能对慢性疼痛产生重大影响预期的结果是重要的。 因为它们将增进我们对细胞和分子机制的基本理解 有助于慢性疼痛，并将确定治疗疼痛的新靶点，而该靶点未在 脑，因此不太可能导致成瘾。

项目成果

Response to comment on: Fibroblast-derived PI16 sustains inflammatory pain via regulation of CD206+ myeloid cells.

对评论的回应：成纤维细胞衍生的 PI16 通过调节 CD206 骨髓细胞维持炎性疼痛。

• DOI: 10.1016/j.bbi.2024.01.224
• 发表时间: 2024
• 期刊: Brain, behavior, and immunity
• 作者: Shepherd,AndrewJ