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.

中文翻译：

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达沙替尼抑制乳头状巩膜肌成纤维细胞分化。

巩膜成纤维细胞活化发生在青光眼和近视眼中。在这里，我们进行无偏筛选，以鉴定可降低成纤维细胞活化至体外各种刺激和体内眼内压（IOP）升高的成纤维细胞活化的激酶抑制剂。使用80种激酶抑制剂库筛选了来自两个人类供体的乳头状巩膜（PPS）成纤维细胞的原代培养，以鉴定抑制TGFβ诱导的细胞外基质（ECM）合成的化合物。肌成纤维细胞分化的抑制作用通过α平滑肌肌动蛋白（αSMA）免疫印迹和胶原蛋白收缩试验得以证实。通过ELISA分析评估IOP诱导的巩膜成纤维细胞增殖的抑制作用，以检测增殖细胞核抗原（PCNA）。初步筛选确定了7种抑制剂，这些抑制剂显示ECM结合降低> 80％。证实了三种激酶抑制剂可降低TGFβ诱导的αSMA表达和细胞收缩力（rottlerin，PP2，tyrphostin 9）。评估了三种Src抑制剂波舒替尼，达沙替尼和SU-6656对成肌纤维细胞分化的影响，只有达沙替尼以纳摩尔剂量显着抑制TGFβ诱导的ECM合成，αSMA表达和细胞收缩。与对照组相比，结膜下注射达沙替尼降​​低了IOP诱导的巩膜成纤维细胞增殖（0.1μM时为4.9±11.1 ng / sclera，对照组为88.7±38.6 ng / scle，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作用不仅是由于Src激酶抑制作用引起的。评估了三种Src抑制剂波舒替尼，达沙替尼和SU-6656对成肌纤维细胞分化的影响，只有达沙替尼以纳摩尔剂量显着抑制TGFβ诱导的ECM合成，αSMA表达和细胞收缩。与对照组相比，结膜下注射达沙替尼降​​低了IOP诱导的巩膜成纤维细胞增殖（0.1μM时为4.9±11.1 ng / sclera，对照组为88.7±38.6 ng / scle，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作用不仅是由于Src激酶抑制作用引起的。评估了三种Src抑制剂（波舒替尼，达沙替尼和SU-6656）对成纤维细胞分化的影响，只有达沙替尼以纳摩尔剂量显着抑制了TGFβ诱导的ECM合成，αSMA表达和细胞收缩力。与对照组相比，结膜下注射达沙替尼降​​低了IOP诱导的巩膜成纤维细胞增殖（0.1μM时为4.9±11.1 ng / sclera，对照组为88.7±38.6 ng / scle，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作用不仅是由于Src激酶抑制作用引起的。和纳摩尔剂量的细胞收缩力。与对照组相比，结膜下注射达沙替尼降​​低了IOP诱导的巩膜成纤维细胞增殖（0.1μM时为4.9±11.1 ng / sclera，对照组为88.7±38.6 ng / scle，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作用不仅是由于Src激酶抑制作用引起的。和纳摩尔剂量的细胞收缩力。与对照组相比，结膜下注射达沙替尼降​​低了IOP诱导的巩膜成纤维细胞增殖（0.1μM时为4.9±11.1 ng / sclera，对照组为88.7±38.6 ng / scle，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作用不仅是由于Src激酶抑制作用引起的。