Novel roles for articular cartilage vesicles in osteoarthritis

描述(由申请人提供)： 关节软骨基质囊泡(ACV)是在正常软骨和软骨细胞培养中发现的一种小的膜结合的细胞外细胞器。到目前为止，它们几乎都是根据它们在关节软骨病理性基质矿化中的作用来描述的。然而，它们在正常软骨中的存在以及除了矿物质形成之外对ACV的作用仍未被探索。我们最近证实，ACV含有功能性RNA，并能够将标记的RNA和蛋白质转移到NAVE软骨细胞。在正常软骨细胞单层中加入1-105克/毫升的正常ACV，可增加骨关节炎(OA)软骨中肥大软骨细胞的标志物。这些令人兴奋的发现表明，ACV直接与软骨细胞相互作用，并可能参与骨性关节炎的细胞变化。初步数据表明，蛋白多糖在ACV与细胞外基质结合中起着重要作用。我们推测，在早期骨性关节炎中蛋白多糖降解酶的活性增加促进了ACV的“流动性”，并促进了ACV与软骨细胞的相互作用。然而，ACV与基质成分结合的机制以及调节基质结合的因素的同一性尚未被研究。同样，人们对介导ACV细胞效应的因素知之甚少。正常关节软骨细胞暴露于外源性谷氨酰胺转氨酶(Tgase)酶以整合素依赖的方式促进软骨细胞肥大。由于在ACV中存在高水平的TGAS，我们假设ACV中或ACV上的TGAS是导致暴露于ACV的软骨细胞肥大表型的原因，并且这种作用是整合素依赖的。本研究以猪ACV和软骨细胞为研究对象，通过对人软骨细胞ACV的实验研究，提出了以下假设：1.研究ACV与软骨基质中的蛋白多糖结合以及ADAMTS4和5从软骨基质中释放ACV的假说。具体目的2：研究ACV中的TGAS是ACV暴露后软骨细胞出现肥大表型的诱因，并且这是整合素依赖的假说。这项工作的最终目的是了解ACV在软骨中的作用，希望控制这些细胞器的含量或可获得性可能有助于治疗OA。 公共卫生相关性： 简介：骨性关节炎是成人中最常见的关节炎形式，影响着4000万美国人。它目前无法治愈，往往会导致严重丧失独立性，并增加受影响人群的医疗费用。骨性关节炎的风险因素，包括高龄、既往创伤和肥胖，在美国退伍军人中尤其普遍，骨性关节炎是退伍军人残疾的主要来源。这项建议涉及到小的细胞外小泡，称为关节软骨小泡(ACV)在骨关节炎中的作用。ACV携带的RNA和蛋白质可以转移到软骨细胞。ACV内容物向软骨细胞的转移模拟了骨性关节炎的变化。了解ACV在软骨中的作用，并最终控制ACV的含量可能会导致新的治疗OA的方法。

DESCRIPTION (provided by applicant): Articular cartilage matrix vesicles (ACVs) are small membrane-bound extracellular organelles found in normal cartilage and chondrocyte cultures. To date, they have almost exclusively been described in reference to their role in pathologic matrix mineralization in articular cartilage. Yet, their presence in normal cartilage and roles for ACVs other than mineral formation remain unexplored. We recently demonstrated that ACVs contain functional RNA and are able to transfer labeled RNA and protein to naove chondrocytes. The addition of 1-10 5g/ml normal ACVs to normal chondrocyte monolayers increased markers characteristic of the hypertrophic chondrocytes seen in osteoarthritic (OA) cartilage. These exciting findings suggest that ACVs directly interact with chondrocytes and may participate in the cellular changes seen in OA. Preliminary data suggest an important role for proteoglycans in binding ACVs to pericellular matrix. We hypothesize that increased activity of proteoglycan-degrading enzymes in early OA promotes ACV "mobility" and fosters ACV-chondrocyte interactions in cartilage. However, the mechanisms through which ACVs bind to matrix components and the identity of the factors modulating matrix binding are unstudied. Similarly, little is known about the factors that mediate the cellular effects of ACVs. Exposure of normal articular chondrocytes to exogenous transglutaminase (Tgase) enzymes promotes chondrocyte hypertrophy in an integrin-dependent manner. As Tgases are present in high levels in ACVs, we hypothesize that Tgases in or on ACVs are responsible for inducing the hypertrophic phenotype in chondrocytes exposed to ACVs and that this effect is integrin-dependent. Using porcine ACV and chondrocytes, and replicating key experiments with ACVs from purchased human chondrocytes, we will investigate the following hypotheses: Specific aim 1: To investigate the hypothesis that ACVs bind to proteoglycans in matrix and ADAMTS4 and 5 release ACVs from cartilage matrix. Specific aim 2: To investigate the hypothesis that Tgases in ACVs are responsible for induction of the hypertrophic phenotype seen in chondrocytes after ACV exposure and that this is integrin-dependent. The ultimate goal of this work is to understand the role of ACVs in cartilage in the hope that manipulating the contents or availability of these organelles might contribute to the treatment of OA. PUBLIC HEALTH RELEVANCE: Narrative: Osteoarthritis is the most common form of arthritis in adults and affects 40 million Americans. It is currently untreatable, and often results in significant loss of independence and increased medical costs for affected people. Risk factors for OA, including advanced age, prior trauma, and obesity are particularly prevalent in the US veteran population and OA is a leading source of disability in veterans. This proposal deals with the role of small extracellular vesicles, called articular cartilage vesicles (ACVs) in OA. ACVs carry RNA and protein that can be transferred to chondrocytes. Transfer of ACV contents to chondrocytes mimics changes seen in OA. Understanding the role of ACVs in cartilage and ultimately manipulating the contents of ACVs could lead to novel therapies for OA.