title: 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.
authors.affiliation: Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX, 76107-2699 USA. myoung-gwi.ryou@unthsc.edu
authors.forename: Myoung-Gwi
authors.initials: MG
authors.lastname: Ryou
authors.forename: Gourav Roy
authors.initials: GR
authors.lastname: Choudhury
authors.forename: Ali
authors.initials: A
authors.lastname: Winters
authors.forename: Luokun
authors.initials: L
authors.lastname: Xie
authors.forename: Robert T
authors.initials: RT
authors.lastname: Mallet
authors.forename: Shao-Hua
authors.initials: SH
authors.lastname: Yang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001812:Blood-Brain Barrier / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D000860:Hypoxia / Q000378:metabolism; D051379:Mice; D018696:Neuroprotective Agents / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D019289:Pyruvic Acid / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D018696:Neuroprotective Agents; D017382:Reactive Oxygen Species; D019289:Pyruvic Acid; D010959:Tissue Plasminogen Activator; D005947:Glucose; D010100:Oxygen
keywords: Antioxidant; BBB; Blood brain barrier; Cell death; MCT; MMP; Matrix metalloproteinases; Monocarboxylate transporter; OGD; ROS; Reactive oxygen species; TIMP; blood–brain barrier; matrix metalloproteinase; monocarboxylate transporter; oxygen-glucose deprivation; reactive oxygen species; recombinant tissue plasminogen activator; rtPA; tissue inhibitor of metalloproteinase
references.citation: Stroke. 2011 Jan;42(1):196-203
references.pmid: 21106952
references.citation: Br J Pharmacol. 2008 Mar;153 Suppl 1:S325-38
references.pmid: 18059324
references.citation: Am J Physiol Heart Circ Physiol. 2001 Dec;281(6):H2568-74
references.pmid: 11709424
references.citation: Biochim Biophys Acta. 2008 Jan;1784(1):116-32
references.pmid: 17913600
references.citation: Int J Stroke. 2012 Jul;7(5):407-18
references.pmid: 22394615
references.citation: Am J Physiol Heart Circ Physiol. 2009 Nov;297(5):H1914-22
references.pmid: 19767525
references.citation: Brain Res. 2003 Nov 14;990(1-2):1-7
references.pmid: 14568323
references.citation: J Neurosci Res. 2003 Jul 15;73(2):260-9
references.pmid: 12836169
references.citation: Mol Cell Biochem. 2003 Nov;253(1-2):103-11
references.pmid: 14619960
references.citation: IUBMB Life. 2012 Feb;64(2):109-19
references.pmid: 22162139
references.citation: Stroke. 2012 Apr;43(4):1101-7
references.pmid: 22282883
references.citation: N Engl J Med. 2008 Sep 25;359(13):1317-29
references.pmid: 18815396
references.citation: Ann N Y Acad Sci. 2010 Oct;1207:123-33
references.pmid: 20955435
references.citation: J Chem Neuroanat. 2003 Oct;26(2):109-18
references.pmid: 14599660
references.citation: Brain Res. 2007 Feb 9;1132(1):1-9
references.pmid: 17174285
references.citation: Stroke. 2009 Dec;40(12):e647-56
references.pmid: 19834012
references.citation: Stroke. 2002 Jun;33(6):1717-21
references.pmid: 12053017
references.citation: Neuroscience. 2012 Sep 27;221:47-55
references.pmid: 22766232
references.citation: Cerebrovasc Dis. 2012;34(4):249-62
references.pmid: 23052179
references.citation: Stroke. 2010 Sep;41(9):2071-6
references.pmid: 20671252
references.citation: Ann Neurol. 2006 Mar;59(3):467-77
references.pmid: 16453316
references.citation: Proc Soc Exp Biol Med. 2000 Feb;223(2):136-48
references.pmid: 10654616
references.citation: Cell Commun Signal. 2012 Nov 23;10(1):35
references.pmid: 23176293
references.citation: J Neurotrauma. 2009 Jul;26(7):1095-110
references.pmid: 19594384
references.citation: Eur J Pharmacol. 2012 Sep 15;691(1-3):190-7
references.pmid: 22796671
references.citation: Exp Mol Pathol. 2013 Feb;94(1):1-9
references.pmid: 23073243
references.citation: Rev Neurosci. 2012 Apr 18;23(3):279-87
references.pmid: 22752785
references.citation: Neurobiol Dis. 2010 Jun;38(3):376-85
references.pmid: 20302940
references.citation: Neurobiol Dis. 2007 Apr;26(1):94-104
references.pmid: 17261368
references.citation: Brain Res. 2012 Oct 30;1482:81-90
references.pmid: 22982730
references.citation: Pediatr Res. 2012 Nov;72(5):479-89
references.pmid: 22885415