Department of Basic Sciences, University of Houston College of Optometry, Houston, Texas, United States.
Department of Biomedical Engineering, Cullen College of Engineering, University of Houston, Houston, Texas, United States.
Invest Ophthalmol Vis Sci. 2020 Nov 2;61(13):16. doi: 10.1167/iovs.61.13.16.
To determine the temporal effects of dexamethasone (DEX) and glucocorticoid-induced matrix (GIM) on integrins/integrin adhesomes, caveolins, cytoskeletal-related proteins, and stiffness in human trabecular meshwork (hTM) cells.
Primary hTM cells were plated on plastic dishes (TCP), treated with vehicle (Veh) or 100 nM DEX in 1% serum media for 1, 3, 5, and 7 day(s). Concurrently, hTM cells were also plated on vehicle control matrices (VehMs) and GIMs for similar time points; VehMs and GIMs had been generated from chronic cultures of Veh-/DEX-stimulated hTM cells and characterized biochemically. Subsets of cells prior to plating on TCP or VehMs / GIMs served as baseline. Protein expression of mechanoreceptors, cytoskeletal-related proteins, and elastic moduli of hTM cells were determined.
Compared with Veh, DEX temporally overexpressed αV, β3, and β5 integrins from day 3 to day 7, and integrin linked kinase at day 7, in hTM cells. However, DEX decreased β1 integrin at day 1 and day 7, while increasing Cavin1 at day 7, in a time-independent manner. Further, DEX temporally upregulated α-smooth muscle actin(α-SMA) and RhoA at day 7 and day 5, respectively; while temporally downregulating Cdc42 at day 3 and day 7 in hTM cells. Conversely, GIM showed increased immunostaining of fibronectin extra-domain A and B isoforms. Compared with VehM, GIM temporally increased αV integrin, Cavin1, and RhoA from day 3 to day 7, at day 3 and day 7, and at day 5, respectively, in hTM cells. Further, GIM overexpressed α-SMA at day 3 and day 7, and stiffened hTM cells from day 1 to day 7, in a time-independent fashion.
Our data highlight crucial mechanoreceptors, integrin adhesomes, and actin-related proteins that may temporally sustain fibrotic phenotypes precipitated by DEX and/or GIM in hTM cells.
研究地塞米松（DEX）和糖皮质激素诱导基质（GIM）对人眼小梁网（hTM）细胞整合素/整合素黏附斑、窖蛋白、细胞骨架相关蛋白和硬度的时间效应。
将原代 hTM 细胞种植于塑料培养皿（TCP）上，用含 1%血清的培养液中的 vehicle（Veh）或 100 nM DEX 处理 1、3、5 和 7 天。同时，hTM 细胞也种植于 Veh 对照基质（VehMs）和 GIMs 上，培养时间相同；VehMs 和 GIMs 是由 Veh-/DEX 刺激的 hTM 细胞的慢性培养中生成的，并进行了生化特征分析。在种植于 TCP 或 VehMs / GIMs 之前的细胞亚群作为基线。测定 hTM 细胞的机械感受器蛋白、细胞骨架相关蛋白的表达和弹性模量。
与 Veh 相比，DEX 在 hTM 细胞中于第 3 天至第 7 天时间依赖性地上调了 αV、β3 和 β5 整合素，并于第 7 天上调了整合素连接激酶。然而，DEX 在第 1 天和第 7 天下调了 β1 整合素，而在时间上独立地上调了 Cavin1。此外，DEX 于第 7 天和第 5 天时间依赖性地上调了 α-平滑肌肌动蛋白（α-SMA）和 RhoA，而于第 3 天和第 7 天时间依赖性地下调了 Cdc42。相反，GIM 显示出纤维连接蛋白外显子 A 和 B 异构体的免疫染色增加。与 VehM 相比，GIM 在 hTM 细胞中于第 3 天至第 7 天、第 3 天和第 7 天、第 5 天时间依赖性地上调了 αV 整合素、Cavin1 和 RhoA。此外，GIM 在第 3 天和第 7 天上调了 α-SMA，并在第 1 天至第 7 天使 hTM 细胞硬度增加，均呈时间非依赖性。
我们的数据突出了重要的机械感受器、整合素黏附斑和肌动蛋白相关蛋白，它们可能在 hTM 细胞中时间依赖性地维持 DEX 和/或 GIM 诱导的纤维化表型。
