Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.
Glaucoma Center of Excellence, The Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.
Exp Eye Res. 2020 May;194:107999. doi: 10.1016/j.exer.2020.107999. Epub 2020 Mar 13.
Scleral fibroblast activation occurs in glaucomatous and myopic eyes. Here we perform an unbiased screen to identify kinase inhibitors that reduce fibroblast activation to diverse stimuli in vitro and to in vivo intraocular pressure (IOP) elevation. Primary cultures of peripapillary scleral (PPS) fibroblasts from two human donors were screened using a library of 80 kinase inhibitors to identify compounds that inhibit TGFβ-induced extracellular matrix (ECM) synthesis. Inhibition of myofibroblast differentiation was verified by alpha smooth muscle actin (αSMA) immunoblot and collagen contraction assay. Inhibition of IOP-induced scleral fibroblast proliferation was assessed by ELISA assay for proliferating cell nuclear antigen (PCNA). The initial screen identified 7 inhibitors as showing>80% reduction in ECM binding. Three kinase inhibitors were verified to reduce TGFβ-induced αSMA expression and cellular contractility (rottlerin, PP2, tyrphostin 9). The effect of three Src inhibitors, bosutinib, dasatinib, and SU-6656, on myofibroblast differentiation was evaluated, with only dasatinib significantly inhibiting TGFβ-induced ECM synthesis, αSMA expression, and cellular contractility at nanomolar dosages. Subconjunctival injection of dasatinib reduced IOP-induced scleral fibroblast proliferation compared to control (4.9 ± 11.1 ng/sclera with 0.1 μM versus 88.7 ± 38.6 ng/sclera in control, P < 0.0001). Dasatinib inhibits scleral myofibroblast differentiation and there is pharmacologic evidence that this inhibition is not solely due to Src-kinase inhibition.
巩膜成纤维细胞的激活发生在青光眼和近视眼中。在这里，我们进行了一项无偏筛选，以确定能够减少成纤维细胞对不同刺激物（包括转化生长因子β (TGFβ)）体外激活以及体内眼内压 (IOP) 升高的激酶抑制剂。我们使用 80 种激酶抑制剂文库筛选了来自两位人类供体的视周巩膜 (PPS) 成纤维细胞的原代培养物，以鉴定抑制 TGFβ 诱导的细胞外基质 (ECM) 合成的化合物。通过α平滑肌肌动蛋白 (αSMA) 免疫印迹和胶原蛋白收缩试验验证肌成纤维细胞分化的抑制。通过 ELISA 检测增殖细胞核抗原 (PCNA) 评估抑制 IOP 诱导的巩膜成纤维细胞增殖的效果。初始筛选确定了 7 种抑制剂，其 ECM 结合减少超过 80%。有 3 种激酶抑制剂（罗特林、PP2 和 tyrphostin 9）被验证可降低 TGFβ 诱导的 αSMA 表达和细胞收缩性。评估了三种Src 抑制剂（博舒替尼、达沙替尼和 SU-6656）对肌成纤维细胞分化的影响，只有达沙替尼在纳摩尔剂量下显著抑制 TGFβ 诱导的 ECM 合成、αSMA 表达和细胞收缩性。与对照相比，结膜下注射达沙替尼可降低 IOP 诱导的巩膜成纤维细胞增殖（0.1 μM 时为 4.9 ± 11.1 ng/巩膜，而对照中为 88.7 ± 38.6 ng/巩膜，P < 0.0001）。达沙替尼抑制巩膜肌成纤维细胞分化，并且有药理学证据表明这种抑制不仅仅是由于 Src-激酶抑制。
