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: 10390016
• 负责人: Lauren Alexandra Hymel
• 金额: $ 4.6万
• 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/10390016
• 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; Gold; Histologic; Hyaluronic Acid; Immune; Immune response; Immunotherapy; Impairment; In Vitro; Infiltration; Inflammation; Inflammatory; Injury; Integrins; Isometric Exercise; Knock-out; Life; Mediating; Medical; Military Personnel; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Muscle function; Muscle satellite cell; Muscular Atrophy; Necrosis; Outcome Measure; Pain; Pathologic; Pathology; Pharmacology; Phenotype; Play; Production; Profibrotic signal; Quality of life; Receptor Signaling; Recovery; Recovery of Function; Regenerative engineering; Role; SPHK1 enzyme; Scientist; Signal Transduction; Site; Sphingolipids; Sphingosine; Stimulus; Structure; Surgical Flaps; 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; macrophage; mesenchymal stromal cell; monocyte; muscle regeneration; myogenesis; nanofiber; novel; 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.

项目摘要 肢体创伤是平民和军事人口中越来越重大的临床挑战，尤其是 在导致体积肌肉损失（VML）的情况下。 VML的当前治疗标准无法成功恢复 受伤后的肌肉功能并导致纤维化，而不是新形成的肌肉纤维。许多旨在治疗的方法 VML未能注意宿主的局部内源性免疫反应，该反应是慢性异常的基础 VML病理的炎症和纤维化信号传导特征。 VML损伤迅速导致退化和坏死 受损的肌纤维以及侵袭和激活广泛的免疫细胞，包括单核细胞和 巨噬细胞。这创造了一个富含促炎和抗炎的环境，最经常导致病理 纤维化。设计抗炎策略，以减轻整体巨噬细胞负担并促进其从现场移走 伤害对于恢复功能至关重要。该研究的假设是生物活性信号传导鞘氨醇-1-磷酸盐（S1P） 炎症后在组织中产生的鞘脂，在亲长的免疫细胞保留中起着至关重要的作用 在VML之后，由于S1P是针对损伤的有效趋化剂。通过5个已知G蛋白耦合的S1P信号 因此，受体（S1PR1-5），因此S1P依赖性免疫细胞反应取决于其S1PR谱。 S1P具有 与通过S1P/S1PR3信号轴传播组织纤维化有关，我们先前的研究表明至关重要 S1PR3在促进免疫细胞生态位占用或出口中的作用。在AIM 1中，S1P在异常免疫细胞中的作用 保留和巨噬细胞介导的纤维化将在鼠股四头肌VML模型中评估 受伤的肌肉组织及其排水淋巴受伤的肌肉和单细胞飞行时间质量细胞术（Cytof） 节点。鞘氨醇激酶1敲除（SPHK1 - / - ）小鼠将被用来直接评估S1P在有效降低效率下的作用 免疫细胞出口和促进纤维化巨噬细胞信号传导在纤维生成祖细胞（FAP）上，该信号驱动 病理纤维化。在AIM 2中，S1PR3拮抗作用对促进免疫细胞出口和废除的影响 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 在慢性炎症刺激的微环境中，免疫细胞募集的S1PR3信号传导与出口相对于出口。 此外，通过从小说 将评估纳米纤维透明质酸支架，以评估至关重要的VML缺陷的损伤环境。脂质组和 将进行单细胞细胞分析，以分析S1PR3拮抗作用如何影响局部脂质代谢和 受伤后的炎症。另外，通过组织学染色的结构肌肉评估再生肌肉 标记将进行评估。等距扭矩的产生将被量化为功能结果指标，以确定是否是否 我们的治疗策略增强了功能性肌肉恢复。这项研究将证明S1P受体调节剂如何 可以将重新置于宿主中的局部靶向内源性修复细胞，作为一种新型的再生免疫疗法形式。