Pyruvate minimizes rtPA toxicity from in vitro oxygen-glucose deprivation and reoxygenation

Abstract

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 6h OGD, cells were reoxygenated with 11mmol/L glucose±pyruvate (8mmol/L) and/or rtPA (10µg/ml). Measured variables included cellular viability (calcein AM and annexin-V/propidium iodide), reactive oxygen species (ROS; mitosox red and 2′,7′-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 3h OGD±rtPA. After 6h OGD, rtPA sharply lowered cell viability; pyruvate dampened this effect. Three hours OGD and 4h 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, 3h OGD and 24h 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.