Probing biochemical/biophysical influences on endothelial-mesenchymal transition

描述(申请人提供)：最近的几份报告显示内皮细胞可以分化为间充质细胞类型[1-8]，这一过程被称为内皮-间充质转化(EndoMT或EndMT)。反过来，内皮细胞来源的间充质细胞能够分化为多种细胞类型，并已被证明分别形成脂肪、软骨和骨[8]。最近的研究表明，EndMT有助于发育后组织的疾病进展，深入的研究表明，EndMT在纤维化[1，3-5]、癌症[2]以及与进行性纤维发育不良骨化症相关的异位骨化(FOP)[8]中起作用。因此，影响EndMT的因素在理解和控制疾病进展方面可能具有特殊的相关性。细胞外基质(ECM)提供广泛的生化和生物物理信号来指导细胞功能，在组织发育过程中，几种基质成分参与了EndMT，包括蛋白多糖[9]、玻璃体连接蛋白、胶原、纤维连接蛋白和层粘连蛋白[10-11]。此外，转化生长因子-b超家族生长因子可以诱导EndMT[3，8]，而可溶性转化生长因子-b对内皮细胞行为的影响依赖于培养物中存在的细胞外基质成分[12]。此外，生物物理因素也是内皮细胞行为、间充质细胞分化、纤维化和骨病的重要决定因素[5，14-24]。综上所述，这些先前的研究暗示，ECM来源的信号可能是EndMT的调节因子，进而导致疾病进展。然而，ECM在引导EndMT中的作用在很大程度上是一个未被探索的研究领域，可能是因为相对较新的发现，这一过程发生在成人组织中。在这里，我们提出了一种方法来识别影响EndMT的ECM模拟信号。这种实验方法是基于我们创造材料以有效控制细胞与材料相互作用的经验。该应用的指导性假设是，模拟细胞外基质属性的生化和生物物理因素都将显著影响EndMT的过程。拟议的研究具有重要意义，因为它们将为细胞外基质如何促进EndMT以及它所涉及的疾病提供关键的新见解。已知的影响成人组织EndMT的几个因素(如BMP-4、转化生长因子-β1、转化生长因子-β2)以及由此导致的内皮细胞表型的变化(如肌成纤维细胞激活、异位成骨)通常与一系列具有实质性和广泛相关性的疾病有关。因此，更好地了解EndMT的作用可能对人类健康有很大的好处。拟议的研究是创新的，因为他们使用新的、化学定义的细胞培养阵列来有效地探索ECM模拟信号的影响。这种探测ECM模拟信号的方法在EndMT过程中是理想的，因为ECM在EndMT中发挥着鲜为人知但可能是关键的作用。此外，了解合成生物材料如何影响EndMT最终可能与医疗植入物设计相关，特别是在限制纤维化或异位骨化很重要的情况下。

DESCRIPTION (provided by applicant): Several recent reports have demonstrated that endothelial cells can differentiate into mesenchymal cell types [1-8], a process called endothelial-mesenchymal transition (EndoMT or EndMT). In turn, endothelial cell-derived mesenchymal cells are capable of differentiating into multiple cell types, and have been shown to form fat, cartilage, and bone, respectively[8]. Recent studies indicate that EndMT contributes to disease progression in post-developmental tissue, and thorough investigations have shown roles in fibrosis[1, 3-5], cancer[2], and heterotopic ossification associated with fibrodysplasia ossificans progressiva (FOP) [8]. Therefore, the factors that influence EndMT may be of particular relevance in understanding and manipulating disease progression. The extracellular matrix (ECM) provides a wide variety of biochemical and biophysical cues that guide cell function, and several matrix components have been implicated in EndMT during tissue development, including proteoglycans [9], vitronectin, collagen, fibronectin and laminin [10-11]. In addition, TGF-b superfamily growth factors can induce EndMT[3, 8] and the influence of soluble TGF-b on endothelial cell behavior is dependent on ECM components present in culture [12]. Further, biophysical factors are also known to be important determinants of endothelial cell behavior, mesenchymal cell differentiation [13], fibrosis, and bone disease [5, 14-24]. Taken together, these previous studies implicate ECM-derived signals as likely regulators of EndMT and, in turn, disease progression. However, the role for the ECM in guiding EndMT is a largely unexplored area of research, perhaps due to the relatively recent discovery that this process occurs in adult tissue. Here, we propose an approach to identify ECM-mimetic signals that influence EndMT. The experimental approach is based on our experience creating materials to efficiently control cell-material interactions. The guiding hypothesis of the application is that biochemical and biophysical factors that mimic extracellular matrix properties will each significantly influence the process of EndMT. The proposed studies are Significant, as they will provide critical new insights into how the extracellular matrix contributes to EndMT, and the diseases in which it is implicated. The few factors that are known to influence EndMT in adult tissue (e.g., BMP-4, TGF-b1, TGF-b2) and the changes in endothelial cell phenotype that result (e.g., myofibroblast activation, heterotopic osteogenesis) are generally implicated in a range of diseases that are of substantial and broad relevance. Therefore, a better understanding of the role of EndMT could have a substantial benefit for human health. The proposed studies are Innovative, as they use novel, chemically-defined cell culture arrays to efficiently probe the effects of ECM-mimetic signals. This approach for probing ECM-mimetic signals is ideal in the case of the EndMT process, as the ECM plays a poorly understood but likely critical role in EndMT. Further, understanding how synthetic biomaterials influence EndMT could ultimately have relevance to medical implant design, particularly in scenarios in which it is important to limit fibrosis or heterotopic ossification.