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Digestive Diseases and Sciences

, Volume 46, Issue 1, pp 54–61 | Cite as

Effects of Genotypically Different Strains of Helicobacter pylori on Human Microvascular Endothelial Cells In Vitro

  • N. Kalia
  • C. Jones
  • K.D. Bardhan
  • M.W.R. Reed
  • J.C. Atherton
  • N.J. Brown
Article

Abstract

Helicobacter pylori induces a number of disturbances in rodent gastric microcirculation in vivo. These events may result from direct necrotic or apoptotic damage to endothelial cells. This study therefore aimed to investigate the effects of genotypically different H. pylori strains on microvascular endothelial cell (MVEC) viability in vitro. Four H. pylori extracts were prepared from strains with different cagA or vacA status. MVECs were plated into 96-well plates and coincubated with 50 μl of extract or vehicle for 24, 48, 72, or 96 hr. An MTT assay quantified overall MVEC viability. The dual labeling of MVECs with propidium iodide and Hoechst 33342 distinguished between necrotic and apoptotic cell death, respectively, and allowed total number of viable cells to be determined. All strains of H. pylori decreased cell viability after 72 and 96 hr. Neither necrosis or apoptosis was observed. Counting total number of viable cells revealed decreased cell proliferation with all strains when compared to controls, again reaching significance at 72 and 96 hr. In conclusion, both the MTT assay and the direct cell counting technique demonstrated that all H. pylori strains induced cytostatic but not cytotoxic effects on MVECs. This suggests that microcirculatory disturbances observed in vivo may not be the result of direct endothelial cell damage. However, inhibition of angiogenesis may explain why ulcer healing is delayed in H. pylori-infected patients.

Helicobacter pylori microvascular endothelial cells apoptosis cytostasis cagA vacA 

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REFERENCES

  1. 1.
    Neilson H, Anderson LP: Chemotactic activity of Helicobacter pylori sonicate for human polymorphonuclear leukocytes and monocytes. Gut 33:738–742, 1992Google Scholar
  2. 2.
    Craig PM, Territo MC, Karnes WE, Walsh JH: Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils. Gut 33:1020–1023, 1992Google Scholar
  3. 3.
    Kurose I, Granger DN, Evans GJ, Evans DG, Graham DY, Miyasaka M, Anderson DC, Wolf RE, Cepinskas G, Kvietys PR: Helicobacter pylori induced microvascular protein leakage in rats: Role of neutrophils, mast cells and platelets. Gastroenterology 104:7–79, 1994Google Scholar
  4. 4.
    Kalia N, Jacob S, Brown NJ, Reed MWR, Morton D, Bardhan KD: Studies on the gastric mucosal microcirculation 2. Helicobacter pylori water soluble extracts induce platelet aggregation in the gastric mucosal microcirculation in vivo. Gut 41:748–752, 1997Google Scholar
  5. 5.
    Braquet P, Touqui L, Shen TY, Vargaftig BB: Perspective in platelet-activation factor research. Pharmacol Rev 39:97–145, 1987Google Scholar
  6. 6.
    Elizalde JI, Gomez J, Panes J, Lozano M, Casadevall M, Ramirez J, Pizcueta P, Marco F, Rojas FD, Granger DN, Pique JM: Platelet activation in mice and human Helicobacter pylori infection. J Clin Invest.100:996–1005, 1997Google Scholar
  7. 7.
    Hatz RA, Reider G, Stolte M, Bayerdörtter E, Meimarakis G, Wilhelm-Schildberg F, Ender G: Pattern of adhesion molecule expression on vascular endothelium in Helicobacter pylori associated antral gastritis. Gastroenterology 112:1905–1919, 1997Google Scholar
  8. 8.
    Bombeli T, Karsen A, Tait JF, Harlan JM: Apoptotic vascular endothelial cell become procoagulant. Blood 89:2429–2442, 1997Google Scholar
  9. 9.
    Hansson GK, Chaos S, Schwartz SM, Reidy MA: Aortic endothelial cell death and replication in normal and LPS-treated rats. Am J Pathol 121:123–127, 1985Google Scholar
  10. 10.
    Buchman TG, Abello PA, Smith EH, Bulkley GB: Induction of heat shock protein response leads to apoptosis in endothelial cells previously exposed to endotoxin. Am J Physiol 93:H165–H170, 1993Google Scholar
  11. 11.
    Robaye B, Mosselmans R, Fiers W, Dumont JE, Galand P: TNF induces apoptosis (programmed cell death) in normal endothelial cells in vitro. Am J Pathol 138:447–453, 1991Google Scholar
  12. 12.
    Wagner S, Beil W, Westermann J, Logan RP, Bock CT, Trautwein C, Bleck JS, Manns MP: Regulation of gastric epithelial cell growth by Helicobacter pylori: Evidence for a major role of apoptosis. Gastroenterology 113:1836–1847, 1997Google Scholar
  13. 13.
    Chen G, Sordillo EM, Ramey WG, Reidy J, Holt PR, Krajewski S, Reed JC, Blaser MJ, Moss SF: Apoptosis in gastric epithelial cells is induced by Helicobacter pylori and accompanied by increased expression of Bak. Biochem Biophys Res Commun 39:626–632, 1997Google Scholar
  14. 14.
    Shirin H and Moss SF: Helicobacter pylori induced apoptosis. Gut 43:592–594, 1998Google Scholar
  15. 15.
    Moss SF, Calam J, Agarwal B, Wangs S, Holt PR: Induction of gastric epithelial apoptosis by Helicobacter pylori. Gut 38:498–501, 1996Google Scholar
  16. 16.
    Piotrowski J, Skrudzka D, Slomiany A, Slomiany BL: Helicobacter pylori LPS induces epithelial cell apoptosis. Biochem Mol Biol Int 40:597–602, 1996Google Scholar
  17. 17.
    Wyatt J: Histopathology of gastroduodenal inflammation: The impact of Helicobacter pylori. Histopathology 26:1–15, 1995Google Scholar
  18. 18.
    Cover TL: The vacuolating cytotoxin of Helicobacter pylori. Mol Microbiol 20:241–246, 1996Google Scholar
  19. 19.
    Atherton JC: The clinical relevance of strain types in Helicobacter pylori. Gut 40:701–703, 1997Google Scholar
  20. 20.
    Atherton JC: H. pylori virulence factors. Br Med Bull 554:105–120, 1998Google Scholar
  21. 21.
    Craig LE, Spelman JP, Strandberg JD, Zirik MC: Endothelial cells from diverse tissues exhibit differences in growth and morphology. Microvasc Res 55:65–76, 1998Google Scholar
  22. 22.
    Folkman J: Angiogenesis-retrospect and outlook. In Angiogenesis: Key Principles, Sciences, Technology and Medicine. R Steiner, PB Weisz, R Langer, (eds). Boston, Birkhäuser Verlag, 1992, pp 4–13Google Scholar
  23. 23.
    Tarnawski A, Hollander D, Stachura J, Gergely H, Krause WJ, Sarfeh IJ: Role of angiogenesis in healing of experimental gastric ulcer. In: Mechanisms of Peptic Ulcer Healing, Edited by F. Halter, A. Garner and GNJ. Tytgat. Kluwer Academic Publishers pp 1650–171Google Scholar
  24. 24.
    Arnold F, West DC: Angiogenesis in wound healing. Pharmacol Ther 52:407–422, 1991Google Scholar
  25. 25.
    Auerbach W, Auerbach R: Angiogenesis inhibition: A review. Pharmacol Ther 63:265–311, 1994Google Scholar
  26. 26.
    Hudson N, Balsitis M, Everitt S, Hawkey CJ: Angiogenesis in gastric ulcers: Impaired in patients taking non-steroidal anti inflammatory drugs. Gut 37:191–194, 1995Google Scholar
  27. 27.
    Tarnawski A, Hollander D, Stachura J, Sarfeh IJ, Gergely H, Krause WJ: Angiogenic response of gastric mucosa to ethanol injury is abolished by indomethacin. Gastroenterology 96:A505, 1989Google Scholar
  28. 28.
    Konturek SJ, Brzozowski T, Mjka J, Szlachcic A, Bielanski W, Stachura J, Otto W: Fibroblast growth factor in gastroprotection and ulcer healing: interaction with sucralfate. Gut 34:881–887, 1993Google Scholar
  29. 29.
    Szabo S, Folkman J, Vattay P, Morales RE, Pinkus GS, Kato K: Accelerated healing of duodenal ulcers by oral administration of a mutein of basic fibroblast growth factor in rats. Gastroenterology 106:1106–1111, 1994Google Scholar
  30. 30.
    Wagner S, Beil W, Mai UE, Gebel M, Nietert M, Manns M: Cytotoxicity of Helicobacter pylori on human gastric epithelial cells. Gut 37(suppl 1):325A, 1995Google Scholar
  31. 31.
    Knipp U, Kaup W, Birkholz S, Opferkuch W: Characterisation of a cell proliferation inhibiting factor produced by Helicobacter pylori. Gut 37(suppl 1):313A, 1995Google Scholar
  32. 32.
    Lamarque D, Kiss J, Tankovic J, Delchier JC, Whittle BJR: Induction of nitric oxide by an extract of Helicobacter pylori in rat duodenum. Gut 37(suppl 1):120A, 1995Google Scholar
  33. 33.
    Melichar B, Karlicek R, Bures J, Komarkova O, Rejchrt S, Fixa B: Gastric juice nitrate and Helicobacter pylori infection. Gut 37(suppl 1):131A, 1995Google Scholar
  34. 34.
    Mannick EE, Bravo LE, Zarama G, Realpe JL, Zhang XJ, Ruiz B, Fontham ET, Mera R. Miller MJ, Correa P: Inducible nitric oxide synthase, nitrotyrosine and aopotosis in Helicobacter pylori gastritis: Effects of antibiotics and antioxidants. Cancer Res 56:3238–3243, 1996Google Scholar
  35. 35.
    Sakkaoula E, Synetos P, Maragoudakis ME: Involvement of nitric oxide in the inhibition of angiogenesis by IL-2. Br J Pharmacol 122:793–795, 1997Google Scholar
  36. 36.
    Gerol M, Curry L, McCarroll L, Doctrow S: Growth regulation of cultured endothelial cells by inflammatory cytokines: Mitogenic, antiproliferative and cytotoxic effects. Comp Biochem Physiol 120:397–404, 1998Google Scholar
  37. 37.
    Cozzolino F, Torcia M, Aldinucci D, Ziche M, Almerigogna F, Bani D, Stern DM: Interleukin-1 is an autocrine inhibitor of human endothelial cell growth. Proc Natl Acad Sci USA 87:6487–6491, 1990Google Scholar
  38. 38.
    Shimayama T, Crabtree JE: Mucosal chemokines in Helicobacter pylori infection. J Physiol Pharmacol 48:315–323, 1997Google Scholar
  39. 39.
    Buzas G, Illyes G, Jozan J, Hamar J: Gastric mucosal apoptosis in chronic gastritis and Helicobacter pylori infection. Gut 41(suppl 3):13.09, 1997Google Scholar
  40. 40.
    Caselli M, Ruina M, Trevisani L, Sartori S, Chiamenti CM, Dentale A, Gallerani L, Alvisi V: Patterns of necrosis in gastric epithelial cells associated with both Helicobacter pylori and NSAIDS. Gut 41(suppl 3):13.10, 1997Google Scholar
  41. 41.
    Brzozowski T, Konturek PC, Konturek SJ, Kwiecieu S, Pajdo R, Karczewska E, Stachura J, Hahn EG: Water extracts of Helicobacter pylori delay healing of chronic gastric ulcers in rats: role of cytokines and gastrin-somatostatin link. Digestion 60:22–33, 1999Google Scholar
  42. 42.
    Makino M, Koga T, Ito K, Kawada H, Tobata K: Delayed healing of chronic ulcers after Helicobacter pylori infection in mice. J Pharm Pharmacol 50:943–948, 1998Google Scholar
  43. 43.
    Ross JS, Bui HX, del Rosario A, Sonbati H, George M, Lee CY: Helicobacter pylori. Its role in the pathogenesis of peptic ulcer disease in a new animal model. Am J Pathol 141:721–727, 1992Google Scholar

Copyright information

© Plenum Publishing Corporation 2001

Authors and Affiliations

  • N. Kalia
    • 1
  • C. Jones
    • 1
  • K.D. Bardhan
    • 1
  • M.W.R. Reed
    • 1
  • J.C. Atherton
    • 1
  • N.J. Brown
    • 1
  1. 1.Department of Surgical and Anaesthetic Sciences, Royal Hallamshire HospitalUniversity of SheffieldSheffieldUK

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