Local modulation of S1P receptor signaling with nanofibrous hyaluronic acid scaffolds as a regenerative immunotherapy following critical volumetric muscle loss injury

使用纳米纤维透明质酸支架局部调节 S1P 受体信号作为关键体积肌肉损失损伤后的再生免疫疗法

• 批准号: 10591401
• 负责人: Lauren Alexandra Hymel
• 金额: $ 4.77万
• 依托单位: GEORGIA 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 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10591401
• 关键词: Affect; Allogenic; Anti-Inflammatory Agents; Attention; Attenuated; Autologous; Award; Bone Marrow; Cells; Characteristics; Chemotactic Factors; Chimera organism; Chronic; Clinical; Clinical Treatment; Coculture Techniques; Complex; Cues; Cytometry; Data; Defect; Ensure; Environment; Excision; Fellowship; Fibrosis; Frequencies; Future; G-Protein-Coupled Receptors; Histologic; Hyaluronic Acid; Immune; Immune response; Immunotherapy; Impairment; In Vitro; Infiltration; Inflammation; Inflammatory; Injury; Integrins; Invaded; Isometric Exercise; Knockout Mice; Life; Macrophage; Mediating; Medical; Military Personnel; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Muscle function; Muscle satellite cell; Muscular Atrophy; Necrosis; Outcome Measure; Pain; Pathologic; Pathology; Phenotype; Play; Production; Profibrotic signal; Proliferating; Quality of life; Receptor Signaling; Recovery; Recovery of Function; Regenerative engineering; Role; SPHK1 enzyme; Scientist; Signal Transduction; Site; Sorting; Sphingolipids; Sphingosine; Stimulus; Structure; TNF gene; Techniques; Testing; Therapeutic; Thick; Time; Tissues; Torque; Training; Transforming Growth Factor beta; Trauma; Traumatic injury; Work; analytical method; antagonist; bone; cell injury; design; disability; draining lymph node; experience; experimental study; functional disability; functional outcomes; functional restoration; histological stains; injured; innovation; inorganic phosphate; limb injury; lipid metabolism; lipidomics; mesenchymal stromal cell; monocyte; muscle form; muscle regeneration; myogenesis; nanofiber; novel; pharmacologic; pre-clinical; progenitor; quadriceps muscle; receptor; recruit; regenerative; regenerative therapy; repaired; response; scaffold; severe injury; sphingosine kinase; standard care; standard of care; success; tissue reconstruction; trafficking; volumetric muscle loss

PROJECT SUMMARY Extremity trauma is an increasingly significant clinical challenge among both civilian and military populations, particularly in cases that result in volumetric muscle loss (VML). Current standards of treatment for VML fail to successfully restore muscle function after injury and result in fibrosis rather than newly formed muscle fibers. Many approaches aimed to treat VML fail to pay attention to the local endogenous immune response of the host which underlies the aberrant chronic inflammation and fibrotic signaling characteristic of VML pathology. VML injury rapidly leads to degeneration and necrosis of damaged myofibers and the invasion and activation of a broad range of immune cells, including monocytes and macrophages. This creates an environment rich in both pro- and anti-inflammatory cues that most often leads to pathological fibrosis. Designing anti-inflammatory strategies to reduce overall macrophage burden and promote their removal from sites of injury is critical to restore function. The study's hypothesis is that sphingosine-1-phophate (S1P), a bioactive signaling sphingolipid that is produced in tissue upon inflammation, plays a crucial role in the pro-longed immune cell retention following VML, as S1P is a potent chemoattractant towards injury. S1P signals through 5 known G protein-coupled receptors (S1PR1-5) and therefore S1P-dependent immune cell responses are dependent on their S1PR profile. S1P has been implicated in propagating tissue fibrosis via the S1P/S1PR3 signaling axis and our previous studies reveal a crucial role for S1PR3 in promoting immune cell niche occupancy or egress. In Aim 1, the role of S1P on aberrant immune cell retention and macrophage-mediated fibrosis will be evaluated in a murine quadriceps VML model via lipidomic analysis of injured muscle and single-cell time-of-flight mass cytometry (CyTOF) from injured muscle tissue and its draining lymph node. Sphingosine kinase 1 knockout (SPHK1-/-) mice will be utilized to directly assess the role of S1P in impairing efficient immune cell egress and mediating pro-fibrotic macrophage signaling on fibroadipogenic progenitors (FAPs) which drives pathological fibrosis. In Aim 2, the effect of S1PR3 antagonism on promoting immune cell egress and abrogating macrophage-induced fibrosis to enhance overall muscle recovery after VML injury will be assessed. This will be accomplished by creating bone marrow chimeras between C57/BL6 mice and S1PR3-/- mice to determine the contribution of S1PR3 signaling on immune cell recruitment vs egress in a microenvironment of chronic inflammatory stimuli. Moreover, local, pharmacological antagonism of S1PR3 by delivery of VPC01091 (S1PR3 antagonist) from novel, nanofibrous hyaluronic acid scaffolds to the injury milieu of critically sized VML defects will be evaluated. Lipidomic and single-cell CyTOF analysis will be performed to analyze how S1PR3 antagonism affects local lipid metabolism and inflammation following injury. In addition, structural muscle assessments via histological staining for regenerative muscle markers will be assessed. Isometric torque production will be quantified as a functional outcome measure to determine if our therapeutic strategy enhances functional muscle recovery. This study will demonstrate how S1P receptor modulators can be re-purposed to locally target endogenous repair cells in the host as a novel form of regenerative immunotherapy.

项目概要 肢体创伤对于平民和军人来说都是一个日益重要的临床挑战，特别是 在导致体积肌肉损失（VML）的情况下。目前 VML 的治疗标准未能成功恢复 损伤后肌肉功能受损并导致纤维化而不是新形成的肌纤维。许多方法旨在治疗 VML未能关注宿主的局部内源性免疫反应，这是异常慢性疾病的基础 VML 病理学的炎症和纤维化信号传导特征。 VML损伤迅速导致变性和坏死 受损的肌纤维以及广泛的免疫细胞（包括单核细胞和免疫细胞）的入侵和激活 巨噬细胞。这创造了一个富含促炎和抗炎信号的环境，最常导致病理性的 纤维化。设计抗炎策略以减少巨噬细胞的总体负担并促进其从部位清除 损伤对于恢复功能至关重要。该研究的假设是，1-磷酸鞘氨醇 (S1P) 是一种生物活性信号传导 炎症时组织中产生的鞘脂在延长免疫细胞保留中起着至关重要的作用 VML 之后，因为 S1P 是一种针对损伤的有效化学引诱剂。 S1P信号通过5个已知的G蛋白偶联 受体 (S1PR1-5)，因此 S1P 依赖性免疫细胞反应取决于其 S1PR 特征。 S1P有 与通过 S1P/S1PR3 信号轴传播组织纤维化有关，我们之前的研究揭示了一个关键的 S1PR3 在促进免疫细胞生态位占据或流出中的作用。在目标 1 中，S1P 对异常免疫细胞的作用 将通过脂质组学分析在鼠股四头肌 VML 模型中评估滞留和巨噬细胞介导的纤维化 受伤的肌肉和来自受伤肌肉组织及其引流淋巴液的单细胞飞行时间质谱流式细胞仪 (CyTOF) 节点。鞘氨醇激酶 1 敲除 (SPHK1-/-) 小鼠将用于直接评估 S1P 在损害效率方面的作用 免疫细胞出口并介导纤维脂肪形成祖细胞（FAP）上的促纤维化巨噬细胞信号传导，从而驱动 病理性纤维化。在目标2中，S1PR3拮抗作用对促进免疫细胞流出和消除的作用 将评估巨噬细胞诱导的纤维化以增强 VML 损伤后的整体肌肉恢复。这将是 通过在 C57/BL6 小鼠和 S1PR3-/- 小鼠之间创建骨髓嵌合体来确定贡献来完成 在慢性炎症刺激的微环境中，S1PR3 信号传导对免疫细胞招募与排出的影响。 此外，通过从新型、 将评估纳米纤维透明质酸支架对临界尺寸 VML 缺陷损伤环境的影响。脂质组学和 将进行单细胞CyTOF分析来分析S1PR3拮抗作用如何影响局部脂质代谢和 受伤后出现炎症。此外，通过再生肌肉的组织学染色进行结构肌肉评估 将评估标记。等距扭矩产生将被量化为功能结果测量，以确定是否 我们的治疗策略可增强功能性肌肉的恢复。这项研究将展示 S1P 受体调节剂如何 可以重新定位宿主体内的内源性修复细胞，作为一种新型的再生免疫疗法。