McClellan Sharon A, Ekanayaka Sandamali A, Li Cui, Jiang Xiaoyu, Barrett Ronald P, Hazlett Linda D
Invest Ophthalmol Vis Sci. 2015 Dec;56(13):8091-100. doi: 10.1167/iovs.15-18393.
Thrombomodulin (TM) is a multidomain, transmembrane protein with anti-inflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity.
Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only).
Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1β, CXCL2, NF-κB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1β, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls.
These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic.
血栓调节蛋白（TM）是一种具有抗炎特性的多结构域跨膜蛋白。血栓调节蛋白结构域（D）1 具有凝集素样结构，可与脂多糖上的 Lewis Y 抗原以及高迁移率族蛋白 B1（HMGB1）相互作用，而跨膜结构域 23（TMD23）则与血管生成和抗炎功能相关。因此，我们测试了 TM 是否对铜绿假单胞菌性角膜炎具有保护作用，以及它是否会增强角膜血管化。
在感染铜绿假单胞菌之前，对 C57BL/6（B6）小鼠的眼睛进行结膜下注射重组 TM（rTM）、rTMD1 或 PBS，并在感染后进行腹腔注射。采用临床评分、裂隙灯摄影、逆转录-聚合酶链反应（RT-PCR）、酶联免疫吸附测定（ELISA）、髓过氧化物酶（MPO）测定、活菌平板计数以及印度墨汁灌注来评估疾病反应和角膜血管化（仅针对 rTM）。
与 PBS 治疗相比，重组 TM 治疗降低了临床评分和角膜混浊程度。HMGB1 的角膜 mRNA 水平未发生变化，但促炎分子白细胞介素-1β（IL-1β）、趋化因子配体 2（CXCL2）、核因子κB（NF-κB）、Toll 样受体 4（TLR4）和晚期糖基化终末产物受体（RAGE）水平降低；抗炎分子单免疫球蛋白白细胞介素-1 相关受体（SIGIRR）和白细胞介素-33 受体（ST2）水平升高。ELISA 证实了 HMGB1、IL-1β 和 CXCL2 蛋白的 mRNA 数据。rTM 治疗后中性粒细胞浸润和活菌平板计数也均降低。在感染后第 5 天测量血管生成分子血管内皮生长因子（VEGF）、血管内皮生长因子受体-1（VEGFR-1）和血管内皮生长因子受体-2（VEGFR-2）的蛋白水平，rTM 治疗后这些水平未出现差异或显著降低。此外，印度墨汁灌注显示两组之间的血管向内生长情况相似。对 rTMD1 进行了类似研究，但疾病严重程度、mRNA、蛋白质、MPO 和平板计数与对照组相比未发生变化。
这些数据证明 rTM 治疗对细菌性角膜炎具有保护作用，不会降低 HMGB1，且不具有血管生成作用。
