Pyruvate minimizes rtPA toxicity from in vitro oxygen-glucose deprivation and reoxygenation
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- 作者：Myoung-Gwi Ryou ; Gourav Roy Choudhury ; Ali Winters ; Luokun Xie ; Robert T. Mallet ; Shao-Hua Yang
- 关键词：BBB ; blood&ndash ; brain barrier ; MCT ; monocarboxylate transporter ; MMP ; matrix metalloproteinase ; OGD ; oxygen-glucose deprivation ; ROS ; reactive oxygen species ; rtPA ; recombinant tissue plasminogen activator ; TIMP ; tissue inhibitor of metalloproteinase
- 刊名：Brain Research
- 出版年：2013
- 出版时间：12 September, 2013
- 年：2013
- 卷：1530
- 期：Complete
- 页码：66-75
- 全文大小：3450 K
文摘
Clinical application of recombinant tissue plasminogen activator (rtPA) for stroke is limited by hemorrhagic transformation, which narrows rtPA¡¯s therapeutic window. In addition, mounting evidence indicates that rtPA is potentially neurotoxic if it traverses a compromised blood brain barrier. Here, we demonstrated that pyruvate protects cultured HT22 neuronal and primary microvascular endothelial cells co-cultured with primary astrocytes from oxygen glucose deprivation (OGD)/reoxygenation stress and rtPA cytotoxicity. After 3 or 6 h OGD, cells were reoxygenated with 11 mmol/L glucose¡Àpyruvate (8 mmol/L) and/or rtPA (10 ¦Ìg/ml). Measured variables included cellular viability (calcein AM and annexin-V/propidium iodide), reactive oxygen species (ROS; mitosox red and 2¡ä,7¡ä-dichlorofluorescein diacetate), NADPH, NADP+ and ATP contents (spectrophotometry), matrix metalloproteinase-2 (MMP2) activities (gelatin zymography), and cellular contents of MMP2, tissue inhibitor of metalloproteinase-2 (TIMP2), and phosphor-activation of anti-apoptotic p70s6 kinase, Akt and Erk (immunoblot). Pyruvate prevented the loss of HT22 cells after 3 h OGD¡ÀrtPA. After 6 h OGD, rtPA sharply lowered cell viability; pyruvate dampened this effect. Three hours OGD and 4 h reoxygenation with rtPA increased ROS formation by about 50 % . Pyruvate prevented this ROS formation and doubled cellular NADPH/NADP+ ratio and ATP content. In endothelial cell monolayers, 3 h OGD and 24 h reoxygenation increased FITC-dextran leakage, indicating disruption of intercellular junctions. Although rtPA exacerbated this effect, pyruvate prevented it while sharply lowering MMP2/TIMP2 ratio and increasing phosphorylation of p70s6 kinase, Akt and Erk. Pyruvate protects neuronal cells and microvascular endothelium from hypoxia-reoxygenation and cytotoxic action of rtPA while reducing ROS and activating anti-apoptotic signaling. These results support the proposed use of pyruvate as an adjuvant to dampen the side effects of rtPA treatment, thereby extending rtPA¡¯s therapeutic window.