AIMS Sphingosine kinase 1 (Sphk1) and the signaling molecule sphingosine-1-phosphate (S1P) are known to be key regulators of a variety of important biological processes, such as neovascularization. Nitric oxide (NO) is also known to play a role in vasoactive properties, whether Sphk1/S1P signaling is able to alter angiogenesis in the context of cerebral ischemia-reperfusion injury (IRI), and whether such activity is linked with NO production, however, remains uncertain. METHODS We used immunofluorescence to detect the expression of Sphk1 and NOS in cerebral epithelial cells (EC) after IR or oxygen-glucose deprivation (OGDR). Western blotting was used to detect the Sphk1 and NOS protein levels in brain tissues or HBMECs. Adenovirus transfection was used to inhibit Sphk1 and NOS. An NO kit was used to detect NO contents in brain tissues and epithelial cells. Tube formation assays were conducted to measure angiogenesis. RESULTS We determined that EC used in a model of cerebral IRI expressed Sphk1, and that inhibiting this expression led to decreased expression of two isoforms of NO synthase (eNOS and iNOS), as well as to decrease neovascularization density and NO production following injury. In HBMECs, knocking down Sphk1 markedly reduced NO production owing to reduced eNOS activity, and inhibiting eNOS directly similarly decreased NO production in a manner which could be reversed via exogenously treating cells with S1P. We further found that knocking down Sphk1 reduced HBMEC eNOS expression, in addition to decreasing the adhesion, migration, and tube formation abilities of these cells under OGDR conditions. CONCLUSIONS Based on these results, we therefore postulate that Sphk1/S1P signaling is able to mediate angiogenesis following cerebral IRI via the regulation of eNOS activity and NO production. As such, targeting these pathways may potentially represent a novel means of improving patient prognosis in those suffering from cerebral IRI.

中文翻译：

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Sphk1/SlP 通路通过脑缺血再灌注后的 NOS/NO 合成来调节血管生成。

AIMS 鞘氨醇激酶 1 (Sphk1) 和信号分子 1-磷酸鞘氨醇 (S1P) 是各种重要生物过程（如新血管形成）的关键调节剂。一氧化氮 (NO) 也已知在血管活性中起作用，Sphk1/S1P 信号是否能够在脑缺血再灌注损伤 (IRI) 的情况下改变血管生成，以及这种活性是否与 NO 的产生有关，然而，仍然不确定。方法我们使用免疫荧光检测IR或氧糖剥夺（OGDR）后脑上皮细胞（EC）中Sphk1和NOS的表达。使用蛋白质印迹法检测脑组织或 HBMECs 中 Sphk1 和 NOS 蛋白水平。腺病毒转染用于抑制 Sphk1 和 NOS。NO试剂盒用于检测脑组织和上皮细胞中的NO含量。进行管形成测定以测量血管生成。结果我们确定在脑 IRI 模型中使用的 EC 表达 Sphk1，并且抑制该表达导致 NO 合酶的两种同工型（eNOS 和 iNOS）的表达降低，并降低损伤后的新生血管形成密度和 NO 产生。在 HBMECs 中，由于 eNOS 活性降低，敲低 Sphk1 显着减少了 NO 的产生，直接抑制 eNOS 类似地减少了 NO 的产生，这种方式可以通过用 S1P 外源性处理细胞来逆转。我们进一步发现，除了降低这些细胞在 OGDR 条件下的粘附、迁移和管形成能力之外，敲低 Sphk1 还降低了 HBMEC eNOS 表达。结论 基于这些结果，我们因此假设 Sphk1/S1P 信号能够通过调节 eNOS 活性和 NO 产生来介导脑 IRI 后的血管生成。因此，靶向这些通路可能代表一种改善脑 IRI 患者预后的新方法。