PDF(Pubmed)
Abstract：
Interactions between trabecular meshwork (TM) cells and their extracellular matrix (ECM) are critical for normal outflow function in the healthy eye. Multifactorial dysregulation of the TM is the principal cause of elevated intraocular pressure that is strongly associated with glaucomatous vision loss. Key characteristics of the diseased TM are pathologic contraction and actin stress fiber assembly, contributing to overall tissue stiffening. Among first-line glaucoma medications, the Rho-associated kinase inhibitor (ROCKi) netarsudil is known to directly target the stiffened TM to improve outflow function via tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no in vitro studies have explored the effect of netarsudil on human TM (HTM) cell contractility and actin remodeling in a 3D ECM environment. Here, we use our bioengineered HTM cell-encapsulated ECM hydrogel to investigate the efficacy of different netarsudil-family ROCKi compounds on reversing pathologic contraction and actin stress fibers. Netarsudil and all related experimental ROCKi compounds exhibited significant ROCK1/2 inhibitory and focal adhesion disruption activities. Furthermore, all ROCKi compounds displayed potent contraction-reversing effects on HTM hydrogels upon glaucomatous induction in a dose-dependent manner, relatively consistent with their biochemical/cellular inhibitory activities. At their tailored EC50 levels, netarsudil-family ROCKi compounds exhibited distinct effect signatures of reversing pathologic HTM hydrogel contraction and actin stress fibers, independent of the cell strain used. Netarsudil outperformed the experimental ROCKi compounds in support of its clinical status. In contrast, at uniform EC50-levels using netarsudil as reference, all ROCKi compounds performed similarly. Collectively, our data suggest that netarsudil exhibits high potency to rescue HTM cell pathobiology in a tissue-mimetic 3D ECM microenvironment, solidifying the utility of our bioengineered hydrogel model as a viable screening platform to further our understanding of TM pathophysiology in glaucoma.
摘要：
小梁网（TM）细胞和它们的细胞外基质（ECM）之间的相互作用对于健康眼睛的正常流出功能至关重要。TM的多因素失调是与青光眼性视力丧失密切相关的眼内压升高的主要原因。病变TM的关键特征是病理性收缩和肌动蛋白应力纤维组装，有助于整体组织变硬。在一线青光眼药物中，已知Rho相关激酶抑制剂（ROCKi）netarsudil直接靶向硬化的TM，通过涉及局灶性粘连和肌动蛋白应力纤维分解的组织松弛来改善流出功能。然而，没有体外研究探索netarsudil对3DECM环境中的人TM（HTM）细胞收缩性和肌动蛋白重塑的影响。这里,我们使用我们的生物工程HTM细胞包裹的ECM水凝胶来研究不同netarsudil家族ROCKi化合物逆转病理收缩和肌动蛋白应力纤维的功效。Netarsudil和所有相关的实验性ROCKi化合物均表现出显着的ROCK1/2抑制和粘着斑破坏活性。此外，所有ROCKi化合物均以剂量依赖性方式在青光眼诱导时对HTM水凝胶显示出有效的收缩逆转作用，与其生化/细胞抑制活性相对一致。在他们量身定制的EC50水平，netarsudil家族ROCKi化合物表现出逆转病理性HTM水凝胶收缩和肌动蛋白应力纤维的明显效应特征，与所用的细胞株无关。Netarsudil在支持其临床状态方面优于实验性ROCKi化合物。相比之下,在使用netarsudil作为参考的统一EC50水平下，所有ROCKI化合物的性能相似。总的来说，我们的数据表明，netarsudil在组织模拟的3DECM微环境中表现出挽救HTM细胞病理生物学的高效力，巩固了我们的生物工程水凝胶模型作为可行的筛选平台的实用性，以进一步了解青光眼中的TM病理生理学。
