Assessment of Corneal Fibroblast Biomechanical Behavior

描述(申请人提供)：细胞和细胞外基质(ECM)之间的机械相互作用驱动基本过程，如发育形态发生、伤口愈合和生物工程组织的组织。本项目致力于通过发展新的三维培养模型以及应用定量三维和四维成像技术来研究这些相互作用如何调节角膜基质细胞的机械行为及其在角膜透明度中的作用。在之前的资助期间进行的研究为调节角膜基质中角膜基质细胞的扩散和迁移提供了重要的见解。我们证明，角膜基质细胞在伤口愈合过程中表达的特定生长因子的反应下，分化为不同的机械表型(树突和收缩)，这一过程在一定程度上受到Rho和Rac激活之间的平衡以及ECM的机械硬度的调节。我们还首次证明，尽管角膜成纤维细胞通常在胶原基质中独立运动，但纤维蛋白诱导细胞切换到相互连接的集体扩散和迁移模式，这与纤维连接蛋白的局部分泌、钙粘附素的表达和细胞内应激纤维的发展有关。使用我们定制的体内HRT-RCM共聚焦显微镜，我们还生成了第一个试点数据，表明在兔机械擦伤后，树突可以在体内发生迁移，在这种情况下，愈合过程中不会有明显的透明度损失。相反，在经角膜冷冻损伤(这导致细胞光散射和纤维化增加)后观察到集体的成纤维细胞形态。在切开手术或准分子激光屈光性角膜切削术(PRK)后，角膜肌成纤维细胞也被证明组织成一个相互连接的网状结构。在目前的应用中，我们将在体外(3D培养)进一步表征调节角膜基质细胞机械行为的机制，并将这些机制与体内观察到的伤口愈合表型相关联。具体目标1将确定生长因子如何调节细胞收缩、正常和纤维化ECM成分的基质重组产生以及不同硬度的胶原和纤维蛋白基质中ALDH1A1的表达水平，并确定这些表达谱是否通过抑制Rho激酶来调节。这些研究将首次确定基质组成和硬度、生长因子和Rho激酶激活如何在3D培养中调节正常和纤维化角质细胞标志物和角膜晶体蛋白的表达；这些因素与体内伤口愈合期间角膜透明度的维持或丧失直接相关。具体目标2将使用静止的角膜基质细胞和激活的真皮和角膜成纤维细胞，确定集体细胞迁移对生长因子的依赖性、纤维连接蛋白、5整合素和钙粘附素的表达和定位、细胞外基质的降解和图案化，以及Rho激酶介导的细胞收缩。角膜成纤维细胞和肌成纤维细胞在切开手术、板层角膜切除术和PRK后的愈合过程中形成相互连接的网络。我们研究3D培养中集体细胞迁移的独特模型应该会为这一基本过程的机械和生物化学调控提供重要的新见解。特异靶3将在体内共聚焦成像和免疫细胞化学将细胞形态、连通性和后向散射与兔机械刮除、经角膜冷冻和板层角膜切除术后成纤维细胞和肌成纤维细胞标志物的表达相关联，并确定是否可以通过抑制Rho激酶(使用Y-27632)来调节这些反应。静止的角膜基质细胞的成纤维细胞和肌成纤维细胞转化导致损伤或手术后角膜混浊的形成。我们的研究表明，Y-27632是在体内抑制这种转化的天然候选药物(可能是除了翻译一种新的疗法外)。在资助过程中，总共将为两名生物医学工程研究生(博士)、两名博士后以及4名暑期医学生和3名本科生提供培训。

DESCRIPTION (provided by applicant): The mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as developmental morphogenesis, wound healing, and the organization of bioengineered tissues. This project is focused on investigating how these interactions regulate corneal keratocyte mechanical behavior and its role in corneal transparency, through the development of novel 3-D culture models, and the application of quantitative 3-D and 4-D imaging techniques. Research conducted in the prior grant period has provided important insights into the regulation of corneal keratocyte spreading and migration within 3-D matrices. We demonstrated that corneal keratocytes differentiate into distinct mechanical phenotypes (dendritic vs. contractile) in response to specific growth factors expressed during wound healing, and that this process is regulated, in part, by the balance between Rho and Rac activation and the mechanical stiffness of the ECM. We also demonstrated for the first time that whereas corneal fibroblasts generally move independently within collagen matrices, fibrin induces a switch to an interconnected, collective mode of cell spreading and migration, which is associated with localized fibronectin secretion, cadherin expression and development of intracellular stress fibers. Using an in vivo HRT- RCM confocal microscope that we custom-modified to allow quantitative full-thickness corneal imaging, we also generated pilot data suggesting for the first time that dendritic migration can occur in vivo following mechanical scrape injury in the rabbit, in which healing occurs without significant loss of transparency. In contrast, a collective, fibroblastic morphology was observed following transcorneal freeze injury (which leads to increased cellular light scattering and fibrosis). Corneal myofibroblasts have also been shown to organize into an interconnected mesh following incisional surgery or photorefractive keratectomy (PRK). In the current application, we will further characterize the mechanisms regulating corneal keratocyte mechanical behavior in vitro (in 3-D culture), and correlate these with wound healing phenotypes observed in vivo. Specific Aim 1 will establish how growth factors modulate cell contractility, matrix reorganization production of normal and fibrotic ECM components and ALDH1A1 expression levels in collagen and fibrin matrices of varying stiffness, and determine whether these expression profiles are modulated by inhibiting Rho kinase. These studies will be the first to determine how matrix composition and stiffness, growth factors and Rho kinase activation regulate expression of normal and fibrotic keratocyte markers and corneal crystallins in 3-D culture; factors that are directly associated with maintenance or loss of corneal transparency during in vivo wound healing. Specific Aim 2 will determine the dependency of collective cell migration on growth factors, expression and localization of fibronectin, ¿5¿1 integrin and cadherin, ECM degradation and patterning, and Rho kinase-mediated cell contractility, using both quiescent corneal keratocytes and activated dermal and corneal fibroblasts. Corneal fibroblasts and myofibroblasts have previously been shown to form an interconnected network during healing after incisional surgery, lamellar keratectomy and PRK. Our unique model for studying collective cell migration in 3-D culture should provide important new insights into the mechanical and biochemical regulation of this fundamental process. Specific Aim 3 will in vivo confocal imaging and immunocytochemistry to correlate cell morphology, connectivity and backscattering with expression of fibroblast and myofibroblast markers after mechanical scrape, transcorneal freeze and lamellar keratectomy in the rabbit, and determine whether these responses can be modulated by inhibiting Rho kinase (using Y-27632). Fibroblast and myofibroblast transformation of quiescent corneal keratocytes lead to corneal haze development following injury or surgery. Our research suggests that Y-27632 is a natural candidate for inhibiting this transformation in vivo (possible bench to beside translation of a new therapy). During the course of the grant, training will be provided to a total of two biomedical engineering graduate (PhD) students, two post-docs, as well as 4 summer medical students and 3 undergraduate research fellows.