Skip to main content

Advertisement

SpringerLink
Log in

In vitro effects of preserved and unpreserved antiglaucoma drugs on apoptotic marker expression by human trabecular cells

  • Laboratory Investigation
  • Published:
Graefe's Archive for Clinical and Experimental Ophthalmology Aims and scope Submit manuscript

Abstract

Background

The mechanisms of trabecular cell loss in glaucoma patients are poorly understood. In order to determine whether drug-induced apoptosis could be one of the mechanisms by which trabecular cells die in glaucoma, we evaluated the effect of benzalkonium-preserved (BAC+) or preservative-free (BAC−) antiglaucoma medications on apoptotic marker expression by cultured human trabecular meshwork (HTM) cells.

Methods

Normal and glaucomatous trabecular cell lines were treated for 15 min with antiglaucoma drugs (1/100 and 1/10 dilutions): timolol BAC+ or BAC−, betaxolol BAC+ or BAC−, latanoprost BAC+ or pure BAC. Apo2.7 expression, annexin V binding and DNA content were evaluated by flow cytometry and confocal microscopy.

Results

Results obtained in the two cell lines were similar for all tested drugs and criteria. In a 1/100 dilution, unpreserved beta-blockers had no apoptotic effect, preserved beta-blockers and latanoprost significantly increased Apo2.7 expression only, while BAC significantly increased all three apoptotic markers. When tested in a 1/10 dilution, all drugs except unpreserved timolol triggered a 2- to 3.5-fold increase in apoptotic features, whereas up to 95% of the cells underwent apoptosis upon treatment with BAC (representing a 9-fold increase over the background level).

Conclusion

At concentrations higher than those supposed to be found in the aqueous humor after instillation (1/100 dilution), unpreserved beta-blocker exhibited no proapoptotic activity on HTM cells in vitro. Benzalkonium-containing beta-blockers and prostaglandin analogue triggered mild expression of one out of three apoptotic markers, while the pro-apoptotic effect observed with BAC appeared to be largely hindered by active compounds in the preserved eyedrops.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3a–d

Similar content being viewed by others

References

  1. Alvarado J, Murphy C, Polansky J, Juster R (1981) Age-related changes in trabecular meshwork cellularity. Invest Ophthalmol Vis Sci 21:714–727

    CAS  PubMed  Google Scholar 

  2. Alvarado J, Murphy C, Juster R (1984) Trabecular meshwork cellularity in primary open-angle glaucoma and non-glaucomatous normals. Ophthalmology 91:564–579

    CAS  PubMed  Google Scholar 

  3. Baudouin C, Pisella PJ, Fillacier K, Goldschild M, Becquet F, De Saint Jean M, Béchetoille A (1999) Ocular surface inflammatory changes induced by topical antiglaucoma drugs: human and animal studies. Ophthalmology 106:556–563

    Google Scholar 

  4. Brignole F, De Saint Jean M, Goldschild M, Becquet F, Baudouin C (1998) Expression of Fas-ligand antigens and apoptotic marker APO2.7 by the human conjunctival epithelium: positive correlation with class II HLA DR expression in inflammatory ocular surface disorders. Exp Eye Res 67:687–697

    Article  CAS  PubMed  Google Scholar 

  5. Champeau EJ, Edelhauser HF (1986) Effect of ophthalmic preservatives on the ocular surface: conjunctival and corneal uptake of benzalkonium chloride and chlorhexidine disconjugate. In: Holly FJ (ed). The preocular tear film in health, disease and contact lens wearers. The Dry Eye Institute, Lubbock, Texas, pp 292–302

  6. De Saint Jean M, Debbasch C, Brignole F, Rat P, Warnet JM, Baudouin C (2000) Toxicity of preserved and unpreserved antiglaucoma topical drugs in an in vitro model of conjunctival cells. Curr Eye Res 20:85–94

    Article  PubMed  Google Scholar 

  7. Debbasch C, Brignole F, Pisella PJ, Warnet JM, Rat P, Baudouin C (2001a) Quaternary ammoniums and other preservatives’ contribution in oxidative stress and apoptosis on Chang conjunctival cells. Invest Ophthalmol Vis Sci 42:642–652

    CAS  PubMed  Google Scholar 

  8. Debbasch C, Pisella PJ, De Saint Jean M, Rat P, Warnet JM, Baudouin C (2001b) Mitochondrial activity and glutathione injury in apoptosis induced by unpreserved and preserved β-blockers on Chang conjunctival cells. Invest Ophthalmol Vis Sci 42:2525–2533

    CAS  PubMed  Google Scholar 

  9. Ellis PP, Wu PY, Riegel M (1991) Aqueous humor pilocarpine and timolol levels after instillation of the single drug or in combination. Invest Ophthalmol Vis Sci 32:520–522

    PubMed  Google Scholar 

  10. Grierson I (1987) What is open angle glaucoma? Eye 1:15–28

    PubMed  Google Scholar 

  11. Grierson I, Howes RC (1987) Age-related depletion of the cell population in the human trabecular meshwork. Eye 1:204–210

    PubMed  Google Scholar 

  12. Hamard P, Sourdille P, Valtot F, Baudouin C (2002) Confocal microscopic examination of trabecular meshwork removed during ab externo trabeculectomy. Br J Ophthalmol 86:1046–1052

    Article  CAS  PubMed  Google Scholar 

  13. Hogg P, Calthorpe M, Batterbury M, Grierson Y (2000) Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro. Invest Ophthalmol Vis Sci 41:1091–1098

    CAS  PubMed  Google Scholar 

  14. Johnson DH, Richardson TM, Epstein DL (1989) Trabecular meshwork recovery after phagocytic challenge. Curr Eye Res 8:1121–1130

    CAS  PubMed  Google Scholar 

  15. Johnson MC, Kamm RD (1983) The role of Schlemm’s canal in aqueous outflow from the human eye. Invest. Ophthalmol Vis Sci 24:320–325

    CAS  Google Scholar 

  16. Kawa JE, Higginbotham EJ, Chang IL, Yue BY (1993) Effects of antiglaucoma medications on bovine trabecular meshwork cells in vitro. Exp Eye Res 57:557–565

    Article  CAS  PubMed  Google Scholar 

  17. Mäepea O, Bill A (1989) The pressures in the episcleral veins, Schlemm’s canal, and the trabecular meshwork in monkeys: effects of changes in intraocular pressure. Exp Eye Res 49:645–663

    PubMed  Google Scholar 

  18. Mäepea O, Bill A (1992) Pressure in the juxtacanalicular tissue and Schlemm’s canal in monkeys. Exp Eye Res 54:879–883

    PubMed  Google Scholar 

  19. Miyake K, Ota I, Ibaraki N, Ibaraki N, Akura J, Ichihashi S, Shibuya Y, Maekubo K, Miyake S (2002) Enhanced disruption of the blood-aqueous barrier and the incidence of angiographic cystoid macular edema by topical timolol and its preservative in early postoperative pseudophakia. Arch Ophthalmol 119:397–394

    Google Scholar 

  20. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279

    CAS  PubMed  Google Scholar 

  21. Ormerod MG (1998) The study of apoptotic cells by flow cytometry. Leukemia 12:1013–1025

    CAS  PubMed  Google Scholar 

  22. Pang IH, Shade DI, Clark AL, Steely HT, DeSantis L (1994) Preliminary characterization of a transformed cell strain derived from trabecular meshwork. Curr Eye Res 13:51–63

    CAS  PubMed  Google Scholar 

  23. Phan TM, Nguyen KP, Giacomini JC, Lee DA (1991) Ophthalmic beta-blockers: determination of plasma and aqueous humor levels by radioreceptor assay following multiple doses. J Ocul Pharmacol 7:243–252

    CAS  PubMed  Google Scholar 

  24. Rohen JW, Lutjen-Drecoll E, Flugel C, Meyer M, Grierson I (1993) Ultrastructure of the trabecular meshwork in untreated cases of primary open-angle glaucoma. Exp Eye Res 56:683–692

    CAS  PubMed  Google Scholar 

  25. Samples JR, Binder PS, Nayak S (1989) The effect of epinephrine and benzalkonium chloride on cultured corneal endothelial and trabecular meshwork cells. Exp Eye Res 49:1-12

    CAS  PubMed  Google Scholar 

  26. Sibayan SA, Latina MA, Sherwood ME, Flotte TJ, White K (1998) Apoptosis and morphologic changes in drug-treated trabecular meshwork cells in vitro. Exp Eye Res 66:521–529

    CAS  PubMed  Google Scholar 

  27. Sjoquist B, Sternschantz J (2002) Ocular and systemic pharmacokinetics of latanoprost in human. Surv Ophthalmol 47 [Suppl] 1:S6–12

    Google Scholar 

  28. Tripathi BJ, Tripathi R, Millard CB (1989) Epinephrine-induced toxicity of human trabecular cells in vitro. Lens Eye Toxic Res 6:141–156

    CAS  PubMed  Google Scholar 

  29. Verhoven AJ, Schlegel RA, Williamson P (1995) Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182:1597–1601

    CAS  PubMed  Google Scholar 

  30. Wood JP, Desantis L, Chao H, Osborne NN (2001) Topically applied betaxolol attenuates ischaemia-induced effects to the rat retina and stimulates BDNF mRNA. Exp Eye Res 72:79–86

    Article  CAS  PubMed  Google Scholar 

  31. Zhang C, Ao Z, Seth A, Schlossmann SF (1996) A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells. J Immunol 157:3980–3987

    CAS  PubMed  Google Scholar 

  32. Zhou L, Fukuchi T, Kawa JE, Higginbotham EJ, Yue BY(1995) Loss of cell-matrix cohesiveness after phagocytosis by trabecular meshwork cells. Invest Ophthalmol Vis Sci 36:787–795

    CAS  PubMed  Google Scholar 

  33. Zhou L, Li Y, Yue BY (1999) Alteration of cytoskeletal structure, integrin distribution, and migratory activity by phagocytic challenge in cells from an ocular tissue—the trabecular meshwork. In Vitro Cell Dev Biol Anim 35:144–149

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank Alcon Laboratories, Fort Worth, Texas, USA for kindly providing the cell lines HTM-5 and HTM-3.

Author information

Authors and Affiliations

  1. Department of Ophthalmology, Quinze-Vingts Hospital, 28 rue de Charenton, 75012 , Paris, France

    Pascale Hamard & Christophe Baudouin

  2. Department of Ophthalmology, Ambroise Paré Hospital, AP-HP, Paris, France

    Pascale Hamard & Christophe Baudouin

  3. Immuno-ophthalmology Unit, Quinze-Vingts National Hospital and University of Paris VI, Paris, France

    Pascale Hamard, Catherine Blondin, Christophe Baudouin & Françoise Brignole

  4. Laboratory of Toxicology, Faculty of Pharmacological and Biological Sciences, University of Paris V, Paris, France

    Pascale Hamard, Caroline Debbasch, Jean-Michel Warnet & Françoise Brignole

Authors
  1. Pascale Hamard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Catherine Blondin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Caroline Debbasch
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jean-Michel Warnet
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christophe Baudouin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Françoise Brignole
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pascale Hamard.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hamard, P., Blondin, C., Debbasch, C. et al. In vitro effects of preserved and unpreserved antiglaucoma drugs on apoptotic marker expression by human trabecular cells. Graefe's Arch Clin Exp Ophthalmol 241, 1037–1043 (2003). https://doi.org/10.1007/s00417-003-0777-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00417-003-0777-7

Keywords

Search

Navigation