Sphingolipids and their Impact in Corneal Wound Healing

全世界高达四分之一的失明病例可归因于由 疤痕形成或纤维化，代表着巨大的经济和社会负担。目前尚无有效的角膜治疗方法 纤维化已经发展起来，最可靠的治疗选择是角膜移植，它已经 许多限制和并发症，包括手术后的角膜纤维化。角膜纤维化是 以散乱的细胞外基质过度堆积而形成角膜疤痕为特征的 (ECM)由成纤维细胞和肌成纤维细胞在损伤或感染后激活而产生。角膜 伤口愈合以及纤维化过程是由多条复杂的途径驱动的，涉及许多 细胞因子、生长因子和趋化因子，目前还不完全清楚。知识的匮乏 这一过程是开发新的治疗策略的关键障碍，以尽量减少疤痕和保留 或者恢复角膜的透明度。我们最近的进展，在NIH/NEI R21(EY025256)的帮助下，使我们 发现一种将鞘磷脂(SPL)信号与经典转换相结合的新的潜在机制 生长因子-β(转化生长因子-β)途径通过激活/分化角膜基质细胞介导角膜纤维化 肌成纤维细胞。我们还证明了在“受损”的角膜基质细胞中SPL的代谢会发生改变。 1-磷酸鞘氨醇(S1P)，一种生物活性的SPL，刺激健康的角膜基质细胞， 诱导转化生长因子-β1的表达和纤维化，通过S1P受体3(S1P3)传递信号。此外，我们发现， 转化生长因子-β亚型可通过增加鞘氨醇激酶1的表达而增加S1P3信号传导。 合成S1P)和S1P3的酶。基于这些发现，我们假设S1P是一个关键 转化生长因子-β激活介导角膜纤维化，转化生长因子-β诱导S1P信号的表达 蛋白质，从而形成一个正反馈环，驱动角膜成纤维细胞不可逆转的激活和 分化为肌成纤维细胞。拟议的研究旨在明确定义这些研究中的关键参与者 使用我们的体外2D和3D模型，描述在角膜纤维化的背景下它们是如何相互作用的 人和小鼠角膜基质细胞(目标1)；以及野生型角膜伤口愈合的活体模型， Sphk1和S1P3基因敲除小鼠，以及测试靶向S1P和转化生长因子-β的治疗潜力 在这些模型中使用选择性抑制剂的信号传递(目标2)。S1P在角膜纤维化中的作用尚未得到研究 我们受到了极大的关注，我们目前是唯一一个将基于SPL的进程作为潜在的 机制。如果成功，这些拟议研究的结果可能会对科学和临床产生深远的影响 意义，因为了解角膜纤维化的机制和S1P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，而且还可能适用 到许多以纤维化为主要病理结果的疾病，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.