Sphingolipids and their Impact in Corneal Wound Healing

全世界多达四分之一的失明病例可归因于由以下因素产生的角膜混浊： 瘢痕或纤维化，代表了巨大的经济和社会负担。没有有效的角膜治疗方法 纤维化已经发展，最可靠的治疗选择是角膜移植， 许多局限性/和并发症，包括术后角膜纤维化。角膜纤维化是 其特征在于由于无序的细胞外基质的过度积累而形成角膜瘢痕 (ECM)由成纤维细胞和肌成纤维细胞在它们被损伤或感染激活后产生。角膜 伤口愈合以及纤维化过程是由多个复杂的途径驱动的， 细胞因子、生长因子和趋化因子，其尚未完全理解。缺乏知识 是开发新的治疗策略以最大限度地减少疤痕并保留 或恢复角膜透明度。我们最近的进展，在NIH/NEI R21（EY 025256）的帮助下， 发现一种新的潜在机制，结合鞘脂（SPL）信号转导与经典的转化， 生长因子-β（TGF-β）途径介导角膜纤维化，通过活化/分化角膜细胞， 肌成纤维细胞我们还证明SPL代谢在“受损”角膜基质细胞中改变， 以及用1-磷酸鞘氨醇（S1 P），一种生物活性SPL， 诱导TGF-β1表达和纤维化，通过S1 P受体3（S1 P3）进行信号传导。此外，我们发现， TGF-β亚型可通过增加鞘氨醇激酶1（SPHK 1）的表达来增加S1 P3信号传导， 合成S1 P和S1 P3的酶。基于这些发现，我们假设S1 P是一个关键， 通过激活TGF-β介导角膜纤维化，TGF-β反过来诱导S1 P信号传导的表达 蛋白质，从而形成正反馈环，其驱动角膜成纤维细胞的不可逆活化， 向肌成纤维细胞分化。拟议的研究旨在明确界定这些领域的主要参与者， 通路，并使用我们的体外2D和3D模型描述它们在角膜纤维化背景下如何相互作用 人和小鼠角膜基质细胞（Aim 1）;以及野生型角膜伤口愈合的体内模型， Sphk 1和S1 P3敲除小鼠，沿着测试靶向S1 P和TGF-β的治疗潜力 在这些模型中使用选择性抑制剂进行信号传导（Aim 2）。S1 P在角膜纤维化中的作用尚未得到证实。 我们目前是唯一一个寻求以苏人解为基础的进程作为潜在的 机制如果成功，拟议研究的结果可能具有深远的科学和临床意义。 重要意义，作为理解角膜纤维化机制和S1 P作为重要介质的作用 不仅对角膜纤维化的临床管理/治疗很重要， 纤维化是许多疾病的主要病理结果，例如肝、肺和心脏纤维化。

Up to one fourth of all cases of blindness worldwide are attributable to corneal opacities generated by scarring, or fibrosis, representing a huge economic and societal burden. No effective therapies for corneal fibrosis have been developed and the most reliable treatment option is corneal transplantation, which has numerous limitations/ and complications, including post-surgical corneal fibrosis. Corneal fibrosis is characterized by the formation of corneal scars from over-accumulation of disorganized extracellular matrix (ECM) produced by fibroblasts and myofibroblasts after they are activated by injury or infection. The corneal wound-healing, as well as the process of fibrosis, is driven by multiple complex pathways involving many cytokines, growth factors, and chemokines, which are not completely understood. The lack of knowledge regarding this process is a critical barrier to developing new treatment strategies to minimize scarring and retain or restore corneal transparency. Our recent advances, with the aid of an NIH/NEI R21 (EY025256), have led us to discover a novel potential mechanism that combines sphingolipid (SPL) signaling with classical transforming growth factor-β (TGF-β) pathways to mediate corneal fibrosis via activation/differentiation of keratocytes into myofibroblasts. We have also demonstrated that SPL metabolism is altered in “injured” corneal stromal cells, and that stimulation of healthy corneal stromal cells with Sphingosine 1-Phosphate (S1P), a bioactive SPL, induces TGF-β1 expression and fibrosis, signaling through the S1P receptor 3 (S1P3). Furthermore, we found that TGF-β isoforms can increase S1P3 signaling by increasing expression of Sphingosine kinase 1 (SPHK1; an enzyme that synthesizes S1P) and S1P3. Based on these discoveries, we hypothesize that S1P is a key mediator of corneal fibrosis via activation of TGF-β, and TGF-β in turn induces expression of S1P signaling proteins and thus forms a positive feedback loop which drives irreversible activation of corneal fibroblasts and differentiation to myofibroblasts. The proposed studies are designed to clearly define the key players in these pathways and delineate how they interact in the context of corneal fibrosis using our in vitro 2D and 3D models of human and mouse corneal stromal cells (Aim 1); and in vivo models of corneal wound healing in wild type, Sphk1, and S1P3 knockout mice, along with testing the therapeutic potential of targeting S1P and TGF-β signaling using selective inhibitors in these models (Aim 2). The role of S1P in corneal fibrosis has not received substantial attention and we are currently the only group pursuing SPL-based processes as part of the potential mechanism. If successful, the results from the proposed studies could have far-reaching scientific and clinical significance, as the understanding of corneal fibrotic mechanisms and the role of S1P as an important mediator would not only be important for clinical management/treatment of corneal fibrosis, but could also be applicable to many diseases in which fibrosis is a major pathological outcome, such as liver, lung, and cardiac fibrosis.