Assessment of Corneal Fibroblast Biomechanical Behavior

描述（由申请人提供）：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化。该项目的重点是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，通过开发新的3-D培养模型，以及应用定量3-D和4-D成像技术。在前一个资助期进行的研究为3-D基质内角膜基质细胞扩散和迁移的调节提供了重要的见解。我们证明，角膜基质细胞分化成不同的机械表型（树突状与收缩）在伤口愈合过程中表达的特定生长因子，这一过程是受调节的，部分，通过Rho和Rac激活和ECM的机械刚度之间的平衡。我们还首次证明，虽然角膜成纤维细胞通常在胶原基质内独立移动，但纤维蛋白诱导细胞扩散和迁移的相互关联的集体模式的转变，这与局部纤连蛋白分泌、钙粘蛋白表达和细胞内应力纤维的发育有关。使用我们定制修改以允许定量全层角膜成像的体内HRT-RCM共焦显微镜，我们还生成了首次表明在兔中机械刮伤后可以在体内发生树突迁移的试验数据，其中愈合发生而没有显著的透明度损失。相反，在经角膜冷冻损伤后观察到集体成纤维细胞形态（这导致细胞光散射和纤维化增加）。角膜肌成纤维细胞也已被证明在切口手术或屈光性角膜切除术（photorefractive keratectomy，PRK）后组织成相互连接的网状结构。在本申请中，我们将进一步表征体外（3-D培养）调节角膜基质细胞机械行为的机制，并将其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性、正常和纤维化ECM组分的基质重组产生以及不同硬度的胶原蛋白和纤维蛋白基质中的ALDH1A1表达水平，并确定这些表达谱是否通过抑制Rho激酶来调节。这些研究将是第一个确定基质成分和刚度，生长因子和Rho激酶激活如何调节正常和纤维化角膜基质细胞标志物和角膜晶体蛋白在3-D培养中的表达;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将确定集体细胞迁移对生长因子的依赖性，纤连蛋白、5 <$1整合素和钙粘蛋白的表达和定位，ECM降解和图案化，以及Rho激酶介导的细胞收缩性，使用静止的角膜角膜细胞和活化的真皮和角膜成纤维细胞。角膜成纤维细胞和肌成纤维细胞先前已被证明在切口手术、板层角膜切除术和角膜移植后的愈合期间形成互连网络。我们独特的模型，研究集体细胞迁移的3-D文化应该提供重要的新的见解，这一基本过程的机械和生化调节。具体目标3将在兔中进行体内共聚焦成像和免疫细胞化学，以将细胞形态、连接性和反向散射与机械刮擦、经角膜冷冻和板层角膜切除术后的成纤维细胞和肌成纤维细胞标志物的表达相关联，并确定这些反应是否可以通过抑制Rho激酶（使用Y-27632）来调节。静止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发展。我们的研究表明，Y-27632是抑制这种体内转化的天然候选物（可能是一种新疗法的旁边翻译）。在赠款期间，将向总共两名生物医学工程研究生（博士）、两名博士后以及4名暑期医科学生和3名本科研究员提供培训。

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.