Skip to main content

Pyrrolidine Dithiocarbamate Activates p38 MAPK and Protects Brain Endothelial Cells From Apoptosis: A Mechanism for the Protective Effect in Stroke?

Neurochemical Research volume 35, pages 1391–1401 (2010)Cite this article

Abstract

The antioxidant and inhibitor of nuclear factor κB pyrrolidine dithiocarbamate (PDTC) potently reduces infarct size in various experimental stroke models. In addition, it has been shown to have a favourable safety profile in humans. In this study, we further investigated the mechanistic actions of PDTC on cerebral microvascular endothelial cells as main components of the blood–brain barrier. We propose activation of p38 MAPK by PDTC as an additional protective mechanism. C57/BL6 mice were subjected to transient MCAO for 2 h and treated with PDTC (100 mg/kg) or vehicle i.p. before reperfusion. Infarct size was determined after 24 h. Apoptosis was induced in a cerebral microvascular endothelial cell line and the effect of pretreatment with PDTC and its dependency on p38 MAPK activity was assayed. PDTC administered 2 h after MCAO reduced infarct size by 61% (P < 0.05) and reduces the apoptotic death rate in vivo. In vitro, PDTC reduces the apoptotic death rate of bEnd.3 cells. p38 MAPK was activated by PDTC and its inhibition abrogated the protective effect of PDTC. PDTC reduces infarct size after stroke with a reasonable time window and decreases apoptotic cell death in vivo and in vitro. The attenuation of apoptotic cell death in brain microvascular endothelial cells is dependent on p38 MAPK activity.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

Bad:

Bcl-2 Antagonist of cell death

Bcl-2:

B-Cell lymphoma 2

CHX:

Cycloheximide

CO:

Carbon monoxide

DETA-NO:

Diethylenetriamine-NO

GSK3:

Glycogen synthase kinase 3

HO-1:

Heme oxygenase-1

IAP:

Inhibitor of apoptosis protein

IκBα:

Inhibitory protein of NF-κB

IKK:

IκB Kinase

JNK:

c-Jun N-terminal kinase

MCAO:

Middle cerebral artery occlusion

MIP-2:

Macrophage inflammatory protein-2

NF-κB:

Nuclear factor κB

p38 MAPK:

p38 Mitogen-activated protein kinase/stress-activated protein kinase

PBS:

Phosphate-buffered solution

PDTC:

Pyrrolidine dithiocarbamate

PI3K:

Phosphatidylinositol 3-kinase

PKB:

Protein kinase B

rt-PA:

Recombinant tissue plasminogen activator

SEM:

Standard error of the mean

STAT:

Signal transducer and activator of transcription

TNFα:

Tumour necrosis factor α

TUNEL:

TdT-Mediated dUTP-biotin nick end labelling

References

  1. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group (1995) Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 333:1581–1587

    Article  Google Scholar 

  2. Hacke W, Kaste M, Bluhmki E et al (2008) Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med 359:1317–1329

    Article  CAS  PubMed  Google Scholar 

  3. Schenkel J, Weimar C, Knoll T et al (2003) R1—Systemic thrombolysis in German stroke units. The experience from the German stroke data bank. J Neurol 250:320–324

    Article  CAS  PubMed  Google Scholar 

  4. O’Collins VE, Macleod MR, Donnan GA et al (2006) 1, 026 experimental treatments in acute stroke. Ann Neurol 59:467–477

    Article  PubMed  Google Scholar 

  5. Nurmi A, Vartiainen N, Pihlaja R et al (2004) Pyrrolidine dithiocarbamate inhibits translocation of nuclear factor kappa-B in neurons and protects against brain ischemia with a wide therapeutic time window. J Neurochem 91:755–765

    Article  CAS  PubMed  Google Scholar 

  6. Nurmi A, Goldsteins G, Närvainen J et al (2006) Antioxidant pyrrolidine dithiocarbamate activates Akt-GSK signaling and is neuroprotective in neonatal hypoxia-ischemia. Free Radic Biol Med 40:1776–1784

    Article  CAS  PubMed  Google Scholar 

  7. Schneider A, Martin-Villalba A, Weih F et al (1999) NF-kappaB is activated and promotes cell death in focal cerebral ischemia. Nat Med 5:554–559

    Article  CAS  PubMed  Google Scholar 

  8. Hermann O, Baumann B, de Lorenzi R et al (2005) IKK mediates ischemia-induced neuronal death. Nat Med 11:1322–1329

    Article  Google Scholar 

  9. Zhang W, Potrovita I, Tarabin V et al (2005) Neuronal activation of NF-kappaB contributes to cell death in cerebral ischemia. J Cereb Blood Flow Metab 25:30–40

    Article  PubMed  Google Scholar 

  10. Reisinger EC, Kern P, Ernst M et al (1990) Inhibition of HIV progression by dithiocarbamates. German DTC Study Group. Lancet 335:679–682

    Article  CAS  PubMed  Google Scholar 

  11. Graham SH, Chen J (2001) Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 21:99–109

    Article  CAS  PubMed  Google Scholar 

  12. Sarainen T, Karjalainen-Lindsberg ML, Paetau A et al (2006) Apoptosis dominant in the periinfarct area of human ischaemic stroke—a possible target of antiapoptotic treatments. Brain 129:189–199

    Article  Google Scholar 

  13. Sharp FR, Lu A, Tang Y et al (2000) Multiple molecular penumbras after focal cerebral ischemia. J Cereb Blood Flow Metab 20:1011–1032

    Article  CAS  PubMed  Google Scholar 

  14. Irving EA, Barone FC, Reith AD et al (2000) Differential activation of MAPK/ERK and p38/SAPK in neurones and glia following focal cerebral ischaemia in the rat. Brain Res Mol Brain Res 77:65–75

    Article  CAS  PubMed  Google Scholar 

  15. Barone FC, Irving EA, Ray AM et al (2001) SB 239063, a second generation p38 mitogen-activated protein kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia. JPET 296:312–321

    CAS  Google Scholar 

  16. Piao CS, Kim JB, Han PL et al (2003) Administration of the p38 MAPK inhibitor SB203580 affords brain protection with a wide therapeutic window against focal ischemic insult. J Neurosci Res 73:537–544

    Article  CAS  PubMed  Google Scholar 

  17. Nito C, Kamada H, Endo H et al (2008) Role of the p38 mitogen-activated protein kinase/cytosolic phospholipase A2 signaling pathway in blood-brain barrier disruption after focal cerebral ischemia and reperfusion. J Cereb Blood Flow Metab 28:1686–1696

    Article  CAS  PubMed  Google Scholar 

  18. Lennmyr F, Ericsson A, Gerwins P et al (2003) Increased brain injury and vascular leakage after pretreatment with p38-inhibitor SB203580 in transient ischemia. Acta Neurol Scand 108:339–345

    Article  CAS  PubMed  Google Scholar 

  19. Czech B, Pfeilschifter W, Mazaheri-Omrani N et al (2009) The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia. Biochem Biophys Res Comm 389:251–256

    Article  CAS  PubMed  Google Scholar 

  20. Hirata I, Yasumoto S, Toshina K (2007) Evaluation of the effect of pyrrolidine dithiocarbamate in suppressing inflammation in mice with dextran sodium sulfate-induced colitis. World J Gastroenterol 13:1666–1671

    CAS  PubMed  Google Scholar 

  21. Zehendner CM, Luhmann HJ, Kuhlmann CR (2009) Studying the neurovascular unit: an improved blood–brain barrier model. J Cereb Blood Flow Metab 29:1879–1884

    Article  PubMed  Google Scholar 

  22. Hossmann KA (1994) Viability thresholds and the penumbra of cerebral ischemia. Ann Neurol 36:557–565

    Article  CAS  PubMed  Google Scholar 

  23. Zaremba J, Skrobanski P, Losy J (2001) Tumor necrosis factor alpha is increased in cerebrospinal fluid and serum of ischemic stroke patients and correlates to volume of evolving brain infarct. Biomed Pharmacother 55:258–263

    Article  CAS  PubMed  Google Scholar 

  24. Szotowski B, Antoniak S, Goldin-Lang P et al (2007) Antioxidative treatment inhibits the release of thrombogenic tissue factor from irradiation- and cytokine-induced endothelial cells. Cardiovasc Res 73:806–812

    Article  CAS  PubMed  Google Scholar 

  25. Trickler WJ, Mayhan WG, Miller DW (2005) Brain microvessel endothelial cell responses to tumor necrosis factor-alpha involve a nuclear factor kappa B (NF-kappaB) signal transduction pathway. Brain Res 1048:24–31

    Article  CAS  PubMed  Google Scholar 

  26. Ho FM, Lin WW, Chen BC et al (2006) High glucose-induced apoptosis in human vascular endothelial cells is mediated through NF-κB and c-Jun NH2-terminal kinase pathway and prevented by PI3K/Akt/eNOS pathway. Cell Signal 18:391–399

    Article  CAS  PubMed  Google Scholar 

  27. Nooteboom A, Bleichrodt RP, Hendriks T (2006) Modulation of endothelial monolayer permeability induced by plasma obtained from lipopolysaccharide-stimulated whole blood. Clin Exp Immunol 144:362–369

    Article  CAS  PubMed  Google Scholar 

  28. Harper SJ, LoGrasso P (2001) Signalling for survival and death in neurones: the role of stress-activated kinases, JNK and p38. Cell Signal 13:299–310

    Article  CAS  PubMed  Google Scholar 

  29. Ono K, Han J (2000) The p38 signal transduction pathway: activation and function. Cell Signal 12:1–13

    Article  CAS  PubMed  Google Scholar 

  30. Zhang X, Shan P, Otterbein LE et al (2003) Carbon monoxide inhibition of apoptosis during ischemia-reperfusion lung injury is dependent on the p38 mitogen-activated protein kinase pathway and involves caspase 3. J Biol Chem 278:1248–1258

    Article  CAS  PubMed  Google Scholar 

  31. Zhang X, Shan P, Alam J et al (2003) Carbon monoxide modulates Fas/Fas ligand, Caspases, and Bcl-2 family proteins via the p38α mitogen-activated protein kinase pathway during ischemia-reperfusion lung injury. J Biol Chem 278:22061–22070

    Article  CAS  PubMed  Google Scholar 

  32. Davies SP, Reddy H, Caivano M et al (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105

    Article  CAS  PubMed  Google Scholar 

  33. Zhang X, Shan P, Alam J et al (2005) Carbon monoxide differentially modulates STAT1 and STAT3 and inhibits apoptosis via a phosphatidylinositol 3-kinase/Akt and p38 kinase-dependent STAT3 pathway during anoxia-reoxygenation injury. J Biol Chem 280:8714–8721

    Article  CAS  PubMed  Google Scholar 

  34. Otterbein LE, Zuckerbraun BS, Haga M et al (2003) Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat Med 9:183–190

    Article  CAS  PubMed  Google Scholar 

  35. Hamanoue M, Sato K, Takamatsu K (2007) Inhibition of p38 mitogen-activated protein kinase-induced apoptosis in cultured mature oligodendrocytes using SB 202190 and SB 203580. Neurochem Int 51:16–24

    Article  CAS  PubMed  Google Scholar 

  36. Furusu A, Nakayama K, Xu Q et al (2007) MAP Kinase dependent, NF-κB-independent regulation of inhibitor of apoptosis protein genes by TNFα. J Cell Physiol 210:703–710

    Article  CAS  PubMed  Google Scholar 

  37. Barone FC, Irving EA, Ray AM et al (2001) Inhibition of p38 mitogen-activated protein kinase provides neuroprotection in cerebral focal ischemia. Med Res Rev 21:129–145

    Article  CAS  PubMed  Google Scholar 

  38. Su EJ, Fredriksson L, Geyer M et al (2008) Activation of PDGF-CC by tissue plasminogen activator impairs blood-brain barrier integrity during ischemic stroke. Nat Med 14:731–737

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The technical advice of Dr. Peter Lüdike on the MCAO model is gratefully acknowledged. We thank Dr. Alf Theisen and Christa Tandi for providing the infrastructure for the animal studies. W.P. was supported by a grant from the Medical faculty of Johann Wolfgang Goethe University (Patenschaftsstipendium), the Novartis Stiftung für Therapeutische Forschung and by the trust of endowments of Marie Christine Held and Erika Hecker.

Author information

Affiliations

  1. Klinik für Neurologie, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany

    Waltraud Pfeilschifter, Timo Kahles, Helmuth Steinmetz & Tobias Neumann-Haefelin

  2. Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany

    Waltraud Pfeilschifter, Bożena Czech, Britta P. Hoffmann, Marian Sujak & Josef Pfeilschifter

Authors
  1. Waltraud Pfeilschifter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bożena Czech
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Britta P. Hoffmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marian Sujak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Timo Kahles
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Helmuth Steinmetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tobias Neumann-Haefelin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Josef Pfeilschifter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Waltraud Pfeilschifter.

Additional information

Waltraud Pfeilschifter and Bozena Czech contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pfeilschifter, W., Czech, B., Hoffmann, B.P. et al. Pyrrolidine Dithiocarbamate Activates p38 MAPK and Protects Brain Endothelial Cells From Apoptosis: A Mechanism for the Protective Effect in Stroke?. Neurochem Res 35, 1391–1401 (2010). https://doi.org/10.1007/s11064-010-0197-0

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-010-0197-0

Keywords

  • Stroke
  • Inflammation
  • MCAO
  • p38 MAPK
  • Blood–brain barrier