Advancement of a lead small molecule gp130 modulator for improving outcomes in joint fibrosis

项目摘要/摘要 纤维化是一种病理性的伤口愈合，其特征是紊乱的胶原过度沉积(Col)。 分化的成纤维细胞(称为肌成纤维细胞)形成的纤维，这种细胞通常来自纤维成脂前体细胞 (FAP)。关节纤维化是一种专门发生在关节的纤维性疾病，通常被归因于外科手术。 疤痕组织反应过度导致关节活动受限和疼痛的手术或损伤。 关节纤维化(AF)显著降低了患者的生活质量，目前治疗选择有限 手术和普通消炎药。在关节纤维化过程中，各种信号级联信号被激活 包括IL-6/gp130炎症信号。IL-6参与慢性纤维化的发生发展，IL-6 在组织损伤后，小鼠的缺乏导致较少的纤维化改变。此外，IL-6还负责 转化生长因子-β-1是FAP纤维化和纤维化转化的主要驱动力。另一种IL-6 家族成员，抑癌素M(OSM)也参与了纤维化过程的不同阶段，包括 成纤维细胞的炎症和激活。在慢性炎症期间，滑膜和滑膜中的FAP 膝下脂肪垫可分化为纤维化细胞，产生疤痕组织，限制正常关节功能。我们的 该小组先前已经证明，用小分子调节IL-6/gp130信号可以刺激缺陷修复 具有良好的透明组织，并防止在伤口愈合过程中纤维软骨基质的沉积。 增厚大鼠骨软骨缺损模型。然而，gp130调节剂的作用从未被研究过。 在关节纤维化方面。我们现在已经合成了一个具有不同功能特性的新型类似物库 原型原始分子。基于先前分析的人类间充质细胞的信号特性 在肺纤维化的小鼠模型中，我们选择了CX-159来 前进的步伐。因此，我们假设在早期用CX-159调制gp130信号。 损伤诱导的关节纤维化可以阻止和/或阻止导致关节纤维化的过程。 在这里，我们将定义在存在IL-6细胞因子的情况下阻止纤维化的CX-159的最佳浓度 和/或转化生长因子-β-1。我们将通过定量聚合酶链式反应和酶联免疫吸附试验评估纤维化生物标志物(COLI、COLIII)，以及 人和大鼠FAP向肌成纤维细胞的分化将通过免疫细胞化学染色进行评估 α-SmA/Coli和天狼星红染色检测ColI/ColIII。然后，我们将在已建立的大鼠身上评估CX-159 房颤模型。关节的活动范围和痉挛程度将首先使用经过验证的仪器进行评估 结合滑膜的行为结果和免疫组织学分析来评估房颤。 这项拟议研究的总体目标是开发一种有效的药物治疗方法 减少创伤后关节纤维化的进展。

Project summary/abstract Fibrosis is a pathological wound healing characterized by excessive deposition of disorganized collagen (COL) fibers by differentiated fibroblasts known as myofibroblasts that often originate from fibro-adipogenic progenitors (FAPs). Arthrofibrosis is a fibrotic disorder that occurs specifically in joints and is often attributed to surgical procedures or injury in which an excessive scar tissue response leads to restriction of joint motion and pain. Arthrofibrosis (AF) significantly decreases quality of life for patients and treatment options are currently limited to surgery and general anti-inflammatories. Various signaling cascades are activated during arthrofibrosis including IL-6/gp130 inflammatory signaling. IL-6 is involved in the development of chronic fibrosis, and IL-6 deficiency in mice results in fewer fibrotic changes after tissue injury. In addition, IL-6 is responsible for upregulation of TGF-β1, which is the primary driver of fibrosis and fibrogenic transition of FAPs. Another IL-6 family member, oncostatin M (OSM), has also been implicated in various stages of the fibrotic process including inflammation and activation of fibroblasts. During chronic inflammation, FAPs in the synovial capsule and infrapatellar fat pad differentiate into fibrogenic cells that generate scar tissue, limiting normal joint function. Our group has previously shown that modulation of IL-6/gp130 signaling with small molecules stimulated defect repair with superior hyaline tissue and prevented deposition of fibrocartilaginous matrix during wound healing in a full- thickness rat osteochondral defect model. However, the effects of gp130 modulators have never been studied in arthrofibrosis. We have now synthesized a library of novel analogs with different functional properties from the prototypic original molecule. Based on previously analyzed signaling properties in human mesenchymal progenitors and compelling in vivo data in a mouse model of pulmonary fibrosis, we have chosen CX-159 to progress forward. Thus, we hypothesize that modulating gp130 signaling with CX-159 early in the process of injury-induced arthrofibrosis may prevent and/or halt the processes that lead to arthrofibrosis. Here we will define the optimal concentration of CX-159 that blocks fibrosis in the presence of IL-6 cytokines and/or TGF-β1. We will evaluate the fibrotic biomarker profile (COLI, COLIII) via qPCR and ELISA, and differentiation of human and rat FAPs to myofibroblasts will be assessed via immunocytochemistry staining for α-SMA/COLI and Picrosirius Red staining for COL I/COLIII. We will then evaluate CX-159 in an established rat model of AF. Joint range of motion and contracture will be evaluated using a validated apparatus we first published, coupled with behavioral outcomes and immunohistological analysis of the synovium to assess AF. The overall goal of the proposed study is to develop an effective pharmacological therapeutic approach for reducing the progression of post-traumatic arthrofibrosis.