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

调节 Hsp90 信号传导以限制角膜纤维化并改善眼部药物渗透

• 批准号: 9370775
• 负责人: Brian C. Leonard
• 金额: $ 21.5万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9370775
• 关键词: Affect; Aftercare; Anterior; Beds; Biophysics; Cell Nucleus; Cell Survival; Cells; Chemicals; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complement Factor B; Complex; Cornea; Corneal Diseases; Corneal Injury; Cues; Data; Development; Disease; Drug Delivery Systems; Electrical Resistance; Epithelial; Epithelial Cells; Equilibrium; Extracellular Matrix; Fibroblasts; Fibrosis; Gene Expression; Goals; HSP 90 inhibition; Heat-Shock Proteins 90; In Situ; In Vitro; Intercellular Junctions; Knockout Mice; Lasers; Lateral; Lead; Mediating; Methods; Modeling; Molecular Chaperones; Myofibroblast; Operative Surgical Procedures; Oryctolagus cuniculus; Outcome; Pathway interactions; Patients; Penetration; Permeability; Pharmaceutical Preparations; Photorefractive Keratectomy; Play; Postoperative Period; Procedures; Process; Proteins; Recovery; Refractive Errors; Resistance; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Smooth Muscle Actin Staining Method; Stromal Cells; Surface; System; Therapeutic; Tight Junctions; Time; Topical application; Toxic effect; Transcriptional Coactivator with PDZ-Binding Motif; Transforming Growth Factor beta Receptors; Transforming Growth Factors; Visual; Work; Wound Healing; absorption; clinical application; clinically significant; corneal epithelium; corneal scar; design; experimental study; improved; in vivo; inhibitor/antagonist; keratomileusis; multicatalytic endopeptidase complex; novel; novel therapeutic intervention; ophthalmic drug; response; single molecule; success; transdifferentiation; translational impact; wound

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.

抽象的 角膜切削术，包括屈光性角膜切除术（PRK）、光疗性角膜切除术（PTK）、 和激光辅助原位角膜磨镶术 (LASIK) 是矫正屈光的常用手术 这些手术的成功需要良好的协调。 热休克蛋白 90 (Hsp90) 是一种限制术后角膜纤维化的角膜愈合伤口反应。 此外，Hsp90 还具有负责许多细胞蛋白质正确折叠的关键分子伴侣。 已被证明可以调节对伤口愈合很重要的两条信号通路：转化生长因子 β (TGF-β) 和 Hippo（主要是 YAP 和 TAZ）途径，通过 (1) 稳定激活的 TGF-β 受体复合物 (2) 通过蛋白酶体降解 YAP 和 TAZ 我们的实验室和其他人已经证明了这一点。 细胞活性因子和生物物理线索对确定角膜基质反应的重要性 例如，用 TGF-β 刺激角膜成纤维细胞并在坚硬的基质上生长，模仿细胞。 临床上，角膜伤口床会上调αSMA表达并转分化为肌成纤维细胞。 肌成纤维细胞数量过多或持续存在可能与角膜的发育有关 纤维化和雾霾，两种重要的机械传感器，“感知”周围的基质刚度。 当细胞在较硬的基质上生长时，YAP 和 TAZ 将定位细胞核，导致表达。 我们建议抑制 Hsp90 来调节 TGF-β。 和 TAZ 信号通路限制 αSMA 表达和角膜纤维化/混浊实验。 小鼠和这些途径的化学抑制剂旨在帮助剖析之间的相互作用 TGF-β 和 TAZ 在角膜伤口愈合中的作用 此外，我们还研究了 Hsp90 的作用。 我们证明了对分层角膜上皮细胞的抑制作用。 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 我们建议定义上皮电阻和 ZO-1 在上皮细胞边界定位的丧失。 Hsp90 抑制剂对角膜上皮细胞的毒性、时程和通透性影响 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，有助于克服局部药物输送的最大障碍之一， 该提案侧重于两个独立的结果，（1）限制角膜。 纤维化/雾霾伤口愈合和（2）增加角膜通透性以促进局部药物渗透， 总体而言，该提案的结果可能被证明是通过抑制 Hsp90 来协调的。 临床意义重大，并导致新的治疗方法的开发，造福患者。