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

摘要 角膜切削手术，包括屈光性角膜切除术（PRK）、光治疗性角膜切除术（PTK）， 和激光辅助原位角膜磨镶术（LASIK）是通常进行的手术以矫正屈光不正 错误和治疗前基质角膜疾病。这些手术的成功需要一个协调良好的 角膜伤口愈合反应，以限制术后角膜纤维化。热休克蛋白90（Hsp 90）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，HSP 90还具有 已被证明可调节两条对伤口愈合至关重要的信号通路，即转化生长因子β (TGF-β）和Hippo（主要是雅普和TAZ）途径，通过（1）稳定活化的TGF-β受体复合物 和（2）靶向雅普和TAZ以被蛋白酶体降解。我们的实验室和其他人已经证明了 细胞活性因子和生物物理因素对角膜基质反应的重要性 细胞例如，用TGF-β刺激角膜成纤维细胞，并在坚硬的基质上生长，模拟角膜成纤维细胞。 角膜伤口床，将上调αSMA表达并转分化为肌成纤维细胞。临床上， 肌成纤维细胞数量过多或持续存在可能与角膜上皮细胞的发育有关。 纤维化和混浊。雅普和TAZ是两种重要的力学传感器，它们“感知”周围的基体刚度， 牢房当在较硬的基质上生长时，雅普和TAZ将定位于细胞核，导致表达 包括TGF-β在内的多个下游分子。我们建议通过抑制Hsp 90来调节TGF-β 和TAZ信号通路，以限制αSMA表达和角膜纤维化/混浊。敲除实验 小鼠和这些途径的化学抑制剂的设计，以帮助解剖之间的相互作用， TGF-β和TAZ在角膜伤口愈合中的作用此外，我们还研究了热休克蛋白90（Hsp 90）在细胞凋亡中的作用。 抑制角膜上皮细胞。我们证明，用角膜上皮细胞治疗复层角膜上皮细胞， Hsp 90抑制剂可导致细胞旁紧密连接的破坏，其特征在于减少反式- 上皮电阻和上皮细胞边缘ZO-1定位的丧失。我们建议定义 Hsp 90抑制剂对角膜上皮细胞的毒性、时程和对通透性的影响， 体外和体内。这些实验的结果可能会导致开发一种新的方法， 增加药物渗透性，有助于克服局部给药的最大障碍之一， 角膜上皮紧密连接该建议集中在两个独立的结果，（1）限制角膜 伤口愈合期间的纤维化/混浊和（2）增加角膜渗透性以促进局部药物渗透， 通过抑制热休克蛋白90来协调。总的来说，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.