Small Molecules Promote Tendon Regeneration by Targeting Endogenous Stem Cells

摘要 肌腱和韧带损伤是美国严重的医疗负担，造成300亿美元的损失。 每年一次。肌腱损伤经常导致物理性质较差的疤痕样组织--然而 到目前为止，再生疗法已经存在。最近，我们已经确定并表征了血管周围(CD146+) 肌腱干/祖细胞(TSCs)通过FAK和ERK1/2在肌腱愈合中发挥重要作用 发信号。 在我们的初步研究中，我们从FAK和ERK1/2激动剂的库中筛选出小分子，并 鉴定了能刺激TSCs向再生方向发展的氧化曲莫林M(Oxo-M)和顺丁烯二酸PPBP(4-PPBP 肌腱愈合。Oxo-M和4-PPBP最初是为治疗神经元疾病而开发的，但从未 在肌肉骨骼系统中进行了测试。在体外，Oxo-M和4-PPBP联合使用可显著诱导 参与肌腱修复的肌腱相关基因表达增加。Oxo-M和4-PPBP显示 工作剂量高达10倍时，无细胞毒性。Western印迹和siRNA敲除(KD)证实FAK和 ERK1/2信号调节Oxo-M和4-PPBP诱导的TSCs向腱细胞分化。活体内，直接 OXO-M和4-PPBP在大鼠全横断髌腱(PT)上的局部给药显著改善 肌腱愈合，如组织学观察到的密集重组的胶原纤维，以及功能上的 显著提高了抗拉强度。这一过程是由快速但短暂的增长所引导的 肌腱分化过程中的内源性TSCs数量。此外，Oxo-M和4-PPBP还特别 乙酰胆碱受体(AChRs)和σ-1受体(σ-1R)靶向CD146+TSC 通路，对其他类型的肌腱细胞影响最小。这些发现证明了一种新颖的和 特异性靶向Oxo-M和4-PPBP在肌腱愈合中的应用前景 内源性TSCs。 这项资助的总体目标是开发一种可靠和有效的基于小分子的 一过性激活内源性神经再生通路治疗肌腱损伤 TSCS并修复肌腱撕裂。STTR第一阶段赠款的首要目标是优化 OXO-M和4-PPBP的结合，这2个化合物并测定它们的组合药效性。这两个 第一阶段STTR的目标是获得可靠的概念验证和初步安全数据，以建立 创新技术的技术优点、可行性和商业潜力。

Abstract Tendon and ligament injuries represent an acute healthcare burden in the United States, costing >$30 billion annually. Tendon injuries frequently result in scar-like tissue with inferior physical properties - however no regenerative therapy exists to date. Recently, we have identified and characterized perivascular (CD146+) tendon stem/progenitor cells (TSCs) that play an essential role in tendon healing via FAK and ERK1/2 signaling. In our preliminary study, we screened small molecules from a library of FAK and ERK1/2 agonists and identified Oxotremorine M (Oxo-M) and PPBP maleate (4-PPBP) that stimulated TSCs toward regenerative tendon healing. Oxo-M and 4-PPBP were originally developed for treating neuronal diseases but have never been tested in the musculoskeletal system. In vitro, a combination of Oxo-M and 4-PPBP induced significant increases in the expression of tendon-related genes involved in tendon repair. Oxo-M and 4-PPBP showed no cytotoxicity up to 10X working doses. Western blot and siRNA knockdown (KD) confirmed that FAK and ERK1/2 signaling regulate Oxo -M & 4-PPBP-induced tenogenic differentiation of TSCs. In vivo, direct topical delivery of Oxo-M and 4-PPBP onto full-transected rat patellar tendons (PT) significantly improved tendon healing, as observed histologically as densely reorganized collagen fibrils, and functionally as significantly enhanced tensile strength. This process was guided by a rapid but transient increase in the number endogenous TSCs undergoing tenogenic differentiation. In addition, Oxo-M and 4-PPBP specifically targeted CD146+ TSCs through muscarinic acetylcholine receptors (AChRs) and σ1 receptor (σ1R) pathways, with minimal effect on other types of tendon cells. These findings demonstrate a novel and promising activity of the combination of Oxo-M and 4-PPBP in tendon healing by specifically targeting endogenous TSCs. The overall objectives of this STTR grant are to develop a reliable and effective small molecule-based regenerative therapy for tendon injuries by transiently activating regenerative pathways of endogenous TSCs and repair tendon tears. The overarching goal of the STTR phase I grant is to optimize the combination of Oxo-M and 4-PPBP, the 2 compounds and determine their drugability in combination. The 2 aims of the phase I STTR are to obtain robust proof-of-concept and preliminary safety data to establish technical merit, feasibility, and commercial potential of the innovative technology.