Bagué Tyler, Singh Ayushi, Ghosh Rajanya, Yoo Hannah, Kelly Curtis, deLong Mitchell A, Kopczynski Casey C, Herberg Samuel
Department of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY, United States.
Department of Cell and Developmental Biology, SUNY Upstate Medical University, Syracuse, NY, United States.
Front Ophthalmol (Lausanne). 2022 Jul 15;2:948397. doi: 10.3389/fopht.2022.948397. eCollection 2022.
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 tissue relaxation involving focal adhesion and actin stress fiber disassembly. Yet, no 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 EC 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 EC-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.
小梁网（TM）细胞与其细胞外基质（ECM）之间的相互作用对于健康眼睛的正常房水流出功能至关重要。TM的多因素失调是眼压升高的主要原因，而眼压升高与青光眼性视力丧失密切相关。患病TM的关键特征是病理性收缩和肌动蛋白应力纤维组装，导致整体组织硬化。在一线青光眼药物中，Rho相关激酶抑制剂（ROCKi）奈他地尔已知可直接作用于硬化的TM，以改善房水流出功能，促进涉及粘着斑和肌动蛋白应力纤维解体的组织松弛。然而，尚无研究探讨奈他地尔在三维ECM环境中对人TM（HTM）细胞收缩性和肌动蛋白重塑的影响。在此，我们使用生物工程化的HTM细胞包封ECM水凝胶，研究不同奈他地尔家族ROCKi化合物在逆转病理性收缩和肌动蛋白应力纤维方面的功效。奈他地尔和所有相关实验性ROCKi化合物均表现出显著的ROCK1/2抑制和粘着斑破坏活性。此外，所有ROCKi化合物在青光眼诱导后对HTM水凝胶均表现出剂量依赖性的强效收缩逆转作用，这与其生化/细胞抑制活性相对一致。在其特定的有效浓度水平下，奈他地尔家族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独特效应特征，与所用细胞株无关。奈他地尔在支持其临床地位方面优于实验性ROCKi化合物。相比之下，以奈他地尔为参照，在统一的有效浓度水平下，所有ROCKi化合物的表现相似。总体而言，我们的数据表明，奈他地尔在模拟组织的三维ECM微环境中具有高效拯救HTM细胞病理生物学的能力，巩固了我们生物工程水凝胶模型作为一个可行的筛选平台的实用性，有助于我们进一步了解青光眼的TM病理生理学。
