Modulation of Hsp90 signaling to limit corneal fibrosis and improve ocular drug penetration

摘要 包括准分子激光屈光性角膜切削术(PRK)、准分子激光治疗性角膜切削术(PTK)、 和准分子激光原位角膜磨镶术(LASIK)是矫正屈光不正的常用手术 前房基质角膜疾病的误诊和治疗。这些手术的成功需要良好的协调 限制术后角膜纤维化的角膜伤口愈合反应。热休克蛋白90(Hsp90)是一种 负责许多细胞蛋白质正确折叠的关键分子伴侣。此外，Hsp90还具有 已被证明调节两个对伤口愈合至关重要的信号通路，转化生长因子β (1)稳定激活的转化生长因子-β受体复合体 (2)通过蛋白酶体对YAP和TAZ进行靶向降解。我们的实验室和其他实验室已经证明了 细胞活性因子和生物物理信号在决定角膜基质反应中的重要性 细胞。例如，角膜成纤维细胞在转化生长因子-β刺激下生长在坚硬的基质上，模拟 角膜创面床，将上调αSMA的表达，并向肌成纤维细胞转化。在临床上， 过多的肌成纤维细胞或持续存在的肌成纤维细胞可能与角膜的发育有关 纤维化和雾霾。YAP和TAZ，两个重要的机械换能器，“感应”周围的基质硬度 牢房。当生长在更坚硬的底物上时，YAP和TAZ会使细胞核局部化，导致表达 多个下游分子，包括转化生长因子-β。我们建议抑制热休克蛋白90来调节转化生长因子-β 和TAZ信号通路，以限制αSMA表达和角膜纤维化/雾化。基因敲除实验 小鼠和带有化学抑制剂的这些通路被设计来帮助剖析 转化生长因子-β和他氮卓酮在角膜伤口愈合中的作用。此外，我们还研究了Hsp90的作用 对角膜上皮细胞的抑制作用。我们证明了分层的角膜上皮细胞的治疗 Hsp90抑制剂可导致细胞旁紧密连接的破坏，其特征是反式连接减少。 上皮电阻和ZO-1在上皮细胞边界的定位丢失。我们建议定义 Hsp90抑制剂对角膜上皮细胞的毒性、时程及对通透性的影响 体外和体内。这些实验的结果可能会导致一种新的方法的开发 增加药物渗透性，帮助克服局部给药的最大障碍之一 角膜上皮紧密连接。这项提议集中于两个独立的结果，(1)限制角膜 伤口愈合期间的纤维化/雾化，以及(2)增加角膜渗透性以促进局部药物渗透， 它们通过抑制Hsp90来协调。总体而言，这项提案的结果可能被证明是 具有临床意义，并导致新的治疗方法的发展，使患者受益。

ABSTRACT Keratoablative surgeries, including photorefractive keratectomy (PRK), phototherapeutic keratectomy (PTK), and laser assisted in situ keratomileusis (LASIK), are commonly performed procedures to correct refractive error and treat anterior stromal corneal diseases. The success of these surgeries requires a well-coordinated corneal wound healing response to limit post-operative corneal fibrosis. Heat shock protein 90 (Hsp90) is a key molecular chaperone responsible for the correct folding of many cellular proteins. In addition, Hsp90 has been shown to regulate two signaling pathways important to wound healing, transforming growth factor β (TGF-β) and the Hippo (mainly YAP and TAZ) pathways, by (1) stabilizing the activated TGF-β receptor complex and (2) targeting YAP and TAZ for degradation by the proteasome. Our lab and others have demonstrated the importance of both cytoactive factors and biophysical cues on determining the responses of corneal stromal cells. For example, corneal fibroblasts stimulated with TGF-β and grown on stiff substrates, mimicking a corneal wound bed, will upregulate αSMA expression and transdifferentiate to myofibroblasts. Clinically, excessive numbers or sustained persistence of myofibroblasts can be associated with development of corneal fibrosis and haze. YAP and TAZ, two important mechanotransducers, “sense” the matrix stiffness surrounding the cell. When grown on stiffer substrates, YAP and TAZ will localize the nucleus, resulting in the expression of multiple downstream molecules, including TGF-β. We propose to inhibit Hsp90 to modulate both the TGF-β and TAZ signaling pathways to limit αSMA expression and corneal fibrosis/haze. Experiments with knockout mice and with chemical inhibitors of these pathways are designed to help dissect the interaction between TGF-β and TAZ in the context of corneal wound healing. In addition, we have investigated the role Hsp90 inhibition in corneal epithelial cells. We demonstrate that treatment of stratified corneal epithelial cells with an Hsp90 inhibitor can result in the disruption of paracellular tight junctions, characterized by reduced trans- epithelial electrical resistance and loss of ZO-1 localization at the epithelial cell borders. We propose to define the toxicity, time course and effect on permeability of an Hsp90 inhibitor on corneal epithelial cells, both in vitro and in vivo. Results from these experiments could lead to the development of a novel method for increasing drug permeability, helping to overcome one the of the largest barrier to topical drug delivery, the corneal epithelial tight junction. This proposal is focused on two independent outcomes, (1) limiting corneal fibrosis/haze during wound healing and (2) increase corneal permeability to promote topical drug penetration, that are harmonized through the inhibition of Hsp90. Overall, findings from this proposal could prove to be clinically significant and lead to the development of novel therapeutic approaches to the benefit of patients.