Methylene Blue Modulates Transendothelial Migration of Peripheral Blood Cells

Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose/time-dependent manner.血管麻痹（Vasoplegia）是心脏手术后的严重并发症。近年来，一氧化氮合酶抑制剂亚甲蓝（methylene blue，MB）的给药已成为一种新型治疗策略。本研究旨在探讨亚甲蓝对循环血细胞跨内皮迁移的作用、环磷酸鸟苷（cyclic cGMP）、内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）及诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）在该过程中的潜在作用，以及亚甲蓝对内皮细胞凋亡的影响。本研究采用不同浓度的亚甲蓝处理人血管内皮细胞（HuMEC-1），处理时长分别为30分钟或2小时。在亚甲蓝处理前后，采用脂多糖（LPS）刺激以模拟炎症环境。随后检测外周血单个核细胞（PBMCs）及T淋巴细胞经处理后的内皮细胞的跨膜迁移情况。采用流式细胞术（flow-cytometry）分析跨膜迁移后外周血单个核细胞各亚群（粒细胞、T淋巴细胞、B淋巴细胞及单核细胞）对亚甲蓝的响应情况。采用膜联蛋白V（Annexin-V）与碘化丙啶（Propidium Iodide）染色法评估亚甲蓝对细胞凋亡的影响。通过逆转录聚合酶链反应（RT-PCR）及蛋白质印迹（Western Blot）检测环磷酸鸟苷、内皮型一氧化氮合酶及诱导型一氧化氮合酶的表达水平。结果采用非配对Student t检验进行统计分析。亚甲蓝诱导的内皮细胞凋亡分析显示，凋亡细胞数量呈剂量依赖性升高。本研究观察到亚甲蓝对循环血细胞的跨内皮迁移具有时间及剂量依赖性影响。预防性给予亚甲蓝可增加外周血单个核细胞的跨内皮迁移能力，但对Jurkat细胞无此调控效果。此外，亚甲蓝给药后，人血管内皮细胞分泌的环磷酸鸟苷水平与诱导型一氧化氮合酶的表达呈正相关，而与内皮型一氧化氮合酶的表达无显著关联。亚甲蓝给药后，上述分子在蛋白水平的表达均有所下调。本研究明确证实，内皮细胞对亚甲蓝的响应呈剂量依赖性，尤其呈时间依赖性。亚甲蓝对循环血细胞各亚群具有不同的调控作用，并可改变内皮型一氧化氮合酶、诱导型一氧化氮合酶及环磷酸鸟苷的释放模式。经亚甲蓝处理后，细胞跨内皮迁移过程受到调控。此外，亚甲蓝可通过剂量及时间依赖性方式诱导内皮细胞凋亡。