Fibroblast Differentiation During Eye Development

描述（由申请人提供）：拟议研究的长期目标是了解胚胎发育期间角膜细胞外基质（ECM）的生物物理化学，并能够在成人中修复该ECM。这在医学上具有直接的重要性，原因有两个：1)仅在美国，就有数百万患者接受了LASIK手术。角膜的LASIK操作创造了一个透明的，但永久不附着的基质瓣。LASIK还显著减少角膜神经支配，并可大大增加感觉性干眼症。在一般人群中，干眼症影响11%的30岁以上人群和15%的65岁以上人群，并且与角膜神经病变有关。LASIK疤痕界面蛋白多糖与邻近基质相比异常。现有的非附着性LASIK皮瓣在面部创伤中存在物理不稳定性问题。在此，我们开发了通过化学交联正常和异常基质ECM成分来减少这种不稳定性的方法，并评估了相邻角膜基质成纤维细胞（角质细胞）、雪旺细胞和神经中的基因表达。2)另一种临床上重要的角膜ECM物理操作，核黄素和紫外线（UVA）联合给药，已经在美国开始试验，用于治疗圆锥角膜。这种治疗改变角膜ECM的机制假设，但尚未证实，涉及胶原交联，蛋白多糖参与。由此产生的周围存活的角膜神经和角化细胞的再生和伤口愈合反应涉及基质内的细胞迁移以及角化细胞和神经分化状态的变化，目前尚不清楚。在此，我们开始对角膜ECM的生物物理化学进行研究，描述正常合成的ECM大分子的分子生物学特征以及对临床治疗的反应。蛋白质组学和糖基微阵列将用于确定ECM分子的物理结合特异性。激光捕获显微解剖，MALDI-TOF/TOF和其他质谱技术将用于详细分析，可以检测LASIK和核黄素+UVA治疗后非常有限的角膜ECM区域和细胞群的变化，以及其他临床重要的角膜手术和条件，例如LASEK， PRK，角膜移植和干眼症。具体目的：1)确定核黄素+UVA治疗交联角膜基质并影响角膜细胞的化学机制，从而设计共价固定LASIK皮瓣的化学机制。2)使用糖糖学和蛋白质组学微阵列和表面等离子体共振分析来确定角膜蛋白聚糖对神经营养因子、生长因子、神经递质、激素和角膜结构大分子的结合偏好、关联和解离动力学。3)表征三叉神经嵴源性神经及其非髓鞘雪旺细胞生长锥与硫酸角蛋白聚糖（KS）和KS蛋白聚糖核心蛋白接触后的基因表达。公共卫生相关性：角膜是人体内唯一既透明又受感觉神经末梢高度支配的活组织，这意味着对角膜的任何医疗治疗，无论是选择性的还是强制性的，都有可能对视力造成永久性损害，并产生巨大的疼痛。拟议研究的长期目标是了解和学习修复眼睛角膜的结缔组织，基于研究胚胎发育和成人期间角膜基质的结缔组织成纤维细胞（“角质细胞”）分化的经验教训。这对医疗公共健康具有直接的重要性，首先，因为目前最流行的选择性眼科手术，LASIK，产生的角膜虽然在大多数患者中是透明的，但从未真正愈合，并且在2-3%的患者中成为问题，其次，因为一种令人兴奋的新治疗方法，涉及核黄素（维生素B2）和长波紫外线（UVA），其主要机制和潜在的副作用尚不清楚。但其显著的主要作用是使角膜“变韧”，大大减缓了一种非常严重的进展性角膜圆锥综合征（使角膜变薄、锥形和穿孔）的发展速度，并为LASIK角膜的“愈合”提供了一种机制。

DESCRIPTION (provided by applicant): The long-range goal of the proposed research is to understand the biological physical chemistry of the corneal extracellular matrix (ECM) during embryonic development and be able to repair that ECM in adults. This is of immediate medical importance for two reasons: 1) In the U.S. alone, several million patients have undergone LASIK. LASIK manipulations of the cornea create a transparent, but permanently non-attached stromal flap. LASIK also significantly reduces corneal innervation, and can greatly increase perceived Dry Eye. In the general population, Dry Eye affects 11% of people over 30 and 15% of people over 65, and has been linked to corneal neuropathy. Proteoglycans of the LASIK scar interface are abnormal compared with adjacent stroma. Existing non-attached LASIK flaps are of concern for their physical instability during facial trauma. Herein, we develop methods to reduce that instability by chemically crosslinking normal and abnormal stromal ECM components, and assess gene expression in adjacent corneal stroma fibroblasts (keratocytes), Schwann cells, and nerves. 2) Another clinically important physical manipulation of corneal ECM, combined riboflavin and ultraviolet light (UVA) administration, has begun trials in the U.S. as a treatment for keratoconus. This treatment alters corneal ECM by mechanisms assumed, but not yet proven, to involve collagen crosslinking, with proteoglycan involvement. Resulting regeneration and wound healing reactions of surrounding surviving corneal nerves and keratocytes involve cellular migrations within the stroma and changes in keratocyte and nerve differentiated states yet to be characterized. Here, we begin a study of the biological physical chemistry of the corneal ECM, characterizing the molecular biology of the ECM macromolecules synthesized normally and also in response to clinical treatments. Proteomic and glycomic microarrays will be used to determine physical binding specificities of ECM molecules. Laser Capture Microdissection, MALDI-TOF/TOF and other mass spectrometry techniques will be used for detailed analyses that can detect changes in very restricted corneal ECM regions and cell populations following LASIK and riboflavin+UVA treatments, as well as after other clinically important corneal procedures and conditions, e.g., LASEK, PRK, corneal transplantation, and Dry Eye. Specific Aims: 1) Determine chemical mechanisms by which riboflavin+UVA treatment cross-links the corneal stroma and affects corneal cells, and thereby devise chemical mechanisms for covalently immobilizing LASIK flaps. 2) Use glycomic and proteomic microarrays and surface plasmon resonance assays to determine binding preferences and association and dissociation kinetics of corneal proteoglycans for neurotrophins, growth factors, neurotransmitters, hormones, and structural macromolecules of the cornea. 3) Characterize gene expression in trigeminal neural crest-derived nerves and in their non-myelinating Schwann cells following neuronal growth cone contact with keratan sulfate (KS) and with KS-proteoglycan core proteins. PUBLIC HEALTH RELEVANCE: The cornea is the only living tissue in the body that is both transparent and highly innervated with sensory nerve endings, meaning that any medical treatment of the cornea, elective or required, has the potential for permanently damaging sight and producing great pain. The long-range goal of the proposed research is to understand and to learn to repair the connective tissue of the cornea of the eye, based upon lessons learned from studying the differentiation of the connective tissue fibroblasts of the corneal stroma (the "keratocytes") during embryonic development and in adults. This is of immediate medical public health importance, first, because the most currently popular elective eye surgery, LASIK, produces corneas that, while transparent in most patients, never truly heal and become problematic in 2-3% of patients, and, second, because an exciting new treatment involving riboflavin (vitamin B2) and long wavelength-ultraviolet light (UVA) has been devised whose primary mechanism and potential side effects remain unknown, but whose remarkable primary effect of "toughening" the cornea greatly slows the rate at which a very serious, progressive syndrome, keratoconus, makes corneas thin, cone-shaped, and perforated, and could provide a mechanism for "healing" LASIK corneas.