Using axolotls to define innate mechanisms for combatting fibrosis

使用蝾螈来定义对抗纤维化的先天机制

• 批准号: 10260449
• 负责人: Fallon Durant
• 金额: $ 0.85万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2021-09-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10260449
• 关键词: Affect; Ambystoma; Animals; Area; Biological; Bleomycin; Blood Vessels; CRISPR/Cas technology; Cells; Characteristics; Chemicals; Cicatrix; Clinical; Collaborations; Complex; Connective Tissue; Cutaneous; Deposition; Dermal; Development; Drug usage; Equilibrium; Exhibits; Explosion; Extracellular Matrix; Face; Fibroblasts; Fibrosis; Future; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Models; Histologic; Human; Hydrogels; Implant; Infection; Injury; Israel; Laboratories; Lead; Limb structure; Liquid substance; Mammals; Medical; Medical center; Medicine; Methods; Modeling; Molecular; Molecular Genetics; Molecular Target; Natural regeneration; Outcome; Pathway interactions; Patients; Pattern; Peptides; Phenocopy; Phenotype; Preventive; Regenerative response; Research; Salamander; Silicones; Silk; Speed; Techniques; Testing; Therapeutic; Thick; Thrombospondins; Time; Tissue Engineering; Tissue Harvesting; Tissue-Specific Gene Expression; Tissues; Transplantation; Work; base; bioelectricity; blastema; cohort; combat; density; design; experience; experimental study; genome editing; healing; human tissue; improved; in vivo; inhibitor/antagonist; knock-down; limb amputation; loss of function; medical implant; mutant; novel; novel strategies; prevent; regenerative; response; soft tissue; therapeutic development; therapeutic target; tissue regeneration; tool; transcriptome sequencing; wound closure; wound healing

Soft tissue fibrosis and cutaneous scarring represent huge clinical burdens to 100 million patients per year, and therapeutic options are currently quite limited. Novel approaches to combat fibrosis and scarring are necessary. Efficient wound closure is a crucial part of wound healing. Without it, injuries would be far more susceptible to fluid loss and infection. It has been hypothesized that scarring evolved as a solution to maximize healing speed. This hypothesis, however, does not explain why regenerative animals, with arguably the most remarkable healing abilities, are capable of scar-free healing. Here, I propose that understanding how axolotl salamanders heal wounds scarlessly and antagonize fibrosis during regeneration will provide critical therapeutic approaches. To overcome the impressive ability of these animals to combat fibrosis, I will create both genetic- and chemical-based fibrotic models in axolotls. I have already successfully managed to develop a chemical-based model using the drug bleomycin, which limits the animals' regenerative ability, and a genetic- based model using tsp-1 loss-of-function mutants, which reduces regenerative rate and blastema size. In Aim 1, I will perform hypothesis-driven, as well as discovery-based, studies to interrogate the balance between fibrosis and regeneration. I will use genome editing to create tsp-1/tsp-2 and tsp-1/tsp-4 double-mutant axolotls, which are predicted to have exacerbated fibrotic phenotypes. In parallel, I will use established Thrombospondin-inhibiting peptides (shown to promote fibrotic phenotypes in cultured human dermal fibroblasts). For both models, I will identify candidate molecular targets that antagonize fibrosis in axolotls using RNAseq and differential gene expression analysis. In collaboration with tissue engineers, I will then use these findings to develop micro-patterned, silk-based hydrogels loaded with biologicals designed to inhibit fibrosis that could be later developed toward human treatments. In Aim 2, I will identify mechanisms whereby axolotls might maintain scar-free tissue in the presence of silicone implants that routinely lead to fibrosis in human patients, necessitating their replacement. These mechanisms represent novel approaches for future therapies that might be preventively employed in human patients necessitating medical implants. They might also be active in remediating existing fibrosis around implants. In parallel, I will test the axolotl extracellular matrix's capability to remodel transplanted human fibrotic tissues. Together, these approaches are extremely novel. They leverage both the explosion of molecular genetic tools now available in these remarkable animals, and they capitalize on natural solutions to fibrotic insults that have yet to be applied to humans. These strategies could provide powerful new approaches to improving fibrosis outcomes in human patients.

软组织纤维化和皮肤瘢痕形成每年给1亿患者带来巨大的临床负担， 目前治疗选择相当有限。对抗纤维化和瘢痕形成的新方法是 必要有效的伤口闭合是伤口愈合的关键部分。如果没有它， 容易脱水和感染据推测，瘢痕形成是一种解决方案， 治愈速度然而，这一假设并不能解释为什么再生动物，可以说是最多的， 卓越的愈合能力，能够无疤痕愈合。在这里，我建议理解美西蝾螈 蝾螈愈合伤口无疤痕，并在再生过程中对抗纤维化，将提供关键的 治疗方法。为了克服这些动物对抗纤维化的惊人能力， 遗传和化学纤维化模型在蝾螈。我已经成功地开发了 一种基于化学的模型，使用药物博来霉素，这限制了动物的再生能力， 使用tsp-1功能丧失突变体的基于模型，其降低再生率和芽基大小。在Aim中 1，我将进行假设驱动，以及基于发现的研究，以询问 纤维化和再生。我将使用基因组编辑来创建tsp-1/tsp-2和tsp-1/tsp-4双突变体 蝾螈，其被预测具有恶化的纤维化表型。同时，我将使用已建立的 血小板反应蛋白抑制肽（显示促进培养的人真皮纤维化表型） 成纤维细胞）。对于这两种模型，我将确定拮抗蝾螈纤维化的候选分子靶点 使用RNAseq和差异基因表达分析。与组织工程师合作，我将使用 这些发现开发了微图案，丝基水凝胶装载生物制剂，旨在抑制 纤维化，以后可以发展为人类治疗。在目标2中，我将确定 美西蝾螈可能在硅胶植入物存在下保持无疤痕组织，而硅胶植入物通常会导致纤维化， 人类患者，需要更换。这些机制代表了未来的新方法 这些疗法可以预防性地用于需要医疗植入物的人类患者。他们可能 也可以积极治疗植入物周围现有的纤维化。与此同时，我会在细胞外检测美西螈 基质重塑移植的人类纤维化组织的能力。总之，这些方法非常 小说他们利用了现在在这些非凡的动物中可用的分子遗传工具的爆炸， 他们利用天然的方法来解决纤维化的问题，而这些方法还没有应用到人类身上。这些 这些策略可以为改善人类患者的纤维化结局提供强大的新方法。