Characterization of Keloid Specific Exosomes and Determination of Exosomal Critical Signaling Pathways in the Keloid Microenvironment

项目摘要/摘要：世界上有1100多万人患有瘢痕疙瘩或更多 在美国，每年有超过425,000次相关的诊所就诊。瘢痕疙瘩是良性的纤维增生性病变 引起疼痛、瘙痒、精神痛苦和功能丧失的肿瘤。目前的治疗方法是 不满意，复发率高得令人无法接受，主要是因为对 瘢痕疙瘩的发病机制。成纤维细胞在瘢痕疙瘩的发病机制中起关键作用，但其驱动因素尚不清楚。 瘢痕疙瘩受到异常信号通路的影响。然而，没有明确的信号通路 已被确认身份。胞外体介导细胞间的通讯，行使主要的生理和 病理生理功能。外体物质，如microRNAs(MiRNAs)，调节细胞功能。 我们的小组发现RAB27，对外体分泌很重要，在 瘢痕疙瘩与正常皮肤相比。我们的团队已经分离出了瘢痕疙瘩特异的外切体。到目前为止，还没有 已发表的关于瘢痕疙瘩外切体或RAB27甲基化对外切体功能的贡献的研究。 我们建议检验中心假说，即外体在细胞内传递关键信号事件 RAB27基因甲基化介导的瘢痕疙瘩微环境目标1：确定 RAB27甲基化对瘢痕疙瘩外切体生产和miRNA货物轮廓的影响。假设：(1)瘢痕疙瘩 胞外体的产生与RAB27基因甲基化有关。(2)瘢痕疙瘩外体货物miRNA 表达谱与RAB27基因甲基化相关。(3)瘢痕疙瘩外切体miRNA的可能靶点 基因存在于伤口愈合和/或纤维化所必需的途径中。目标2：确定影响 对瘢痕疙瘩微环境的影响。假设：比较了瘢痕疙瘩成纤维细胞外切体 对于正常的成纤维细胞，外切体会引起正常成纤维细胞的促纤维化表型改变。目标3： 目的：探讨瘢痕疙瘩外切体对体内瘢痕形成的影响。假设：外体 在AIM 1中产生并在AIM 2中测试将增加兔耳疤痕模型中的瘢痕形成。 意义：这个项目将有助于加深对瘢痕疙瘩发病机制和 基于外显子的治疗的可能性。创新：(I)从初步数据进行理性演进 支持外切体在瘢痕疙瘩发病机制中的新作用；(Ii)研究RAB27的影响 瘢痕疙瘩外切体的功能和生产上的甲基化可能是 新的表观遗传生物标志物；(Iii)使用独特的资源，包括来自原代的成纤维细胞系 未治疗的瘢痕疙瘩(25例)和匹配的正常皮肤(25例)，来自多民族患者和活体 动物模型允许结果的实用翻译应用；(Iv)瘢痕疙瘩的全新领域 调查。总而言之，这个项目、指导和职业发展计划将定位于Lamont R 琼斯，医学博士，MBA，成为一名独立的临床科学家和瘢痕疙瘩发病机制的领导者。

Project Summary/Abstract: There are more than 11 million people in the world with keloids and more than 425,000 associated clinic visits, yearly, in the United States. Keloids are benign fibroproliferative tumors which cause pain, pruritus, emotional distress and loss of function. Current therapies are unsatisfactory with unacceptably high recurrence rates, mainly because of an incomplete understanding of keloid pathogenesis. Fibroblasts are a key player in keloid pathogenesis, but the drivers are unknown. Keloid disease is influenced by aberrant signaling pathways. However, no clear signaling pathway has been identified. Exosomes mediate cell-cell communication, exercising primary physiological and pathophysiological function. Exosomal cargo, such as microRNAs (miRNAs), regulate cellular function. Our group identified RAB27, important for exosome secretion, as being differentially hypomethylated in keloid compared to normal skin. Our group has isolated keloid-specific exosomes. To date, there are no published studies on keloid exosomes or the contribution of RAB27 methylation on exosome function. We propose to test the central hypothesis that exosomes communicate critical signaling events in the keloid microenvironment mediated by RAB27 gene methylation. Aim 1: To determine the effect of RAB27 methylation on keloid exosome production and miRNA cargo profiles. Hypotheses: (1) Keloid exosomal production correlates with RAB27 gene methylation. (2) Keloid exosomal cargo miRNA expression profiles correlates with RAB27 gene methylation. (3) Keloid exosome miRNA’s putative target genes lie within pathways essential for wound healing and/or fibrosis. Aim 2: To determine the effects of keloid exosomes on the keloid microenvironment. Hypothesis: Keloid fibroblast exosomes compared to normal fibroblast exosomes will cause pro-fibrotic phenotype changes in normal fibroblasts. Aim 3: To determine the effect of keloid exosomes on scar formation in vivo. Hypothesis: Exosomes generated in aim 1 and tested in aim 2 will increase scar formation in a rabbit ear scar model. Significance: This project will lead to an enhanced understanding of keloid pathogenesis and the potential for exosome-based therapy. Innovation: (i) rational progression from preliminary data supporting the novel role of exosomes in keloid pathogenesis; (ii) investigating the influence of RAB27 methylation on the function and production of keloid exosomes would suggest a mechanistic basis for novel epigenetic biomarkers;(iii) using unique resources which includes fibroblast cell lines from primary untreated keloid (25) and matched normal skin (25) from a multi-ethnic group of patients and an in vivo animal model allow for the pragmatic translational application of results; (iv) entirely new field of keloid investigation. In summary, this project, mentoring and career development plan will position, Lamont R Jones, MD, MBA, to become an independent clinician scientist and leader in keloid pathogenesis.