Korean J Ophthalmol.  2004 Jun;18(1):23-28. 10.3341/kjo.2004.18.1.23.

Effects of Hyaluronic Acid on the Polymorphonuclear Leukocyte (PMN) Release of Active Oxygen and Protection of Bovine Corneal Endothelial Cells from Activated PMNs

• Lym HS
• Suh Y
• Park CK

• KMID: 1115779
• DOI: http://doi.org/10.3341/kjo.2004.18.1.23

Abstract

The goal of this study was to evaluate the function of hyaluronic acid (HA) on the active oxygen release from polymorphonuclear leukocytes (PMNs) and the protective effect of bovine corneal endothelial cells (BCEC) from activated PMNs. We used HA with three different molecular weights (MW 700, 000, 2, 000, 000, and 4, 000, 000) and five different concentrations (0, 0.1, 1, 2, and 3 mg/ml). We evaluated the amount of released superoxide from activated PMNs by using dismutase-inhibitable ferricytochrome C reduction. To compare the property and protective effect of HA with those of other viscoelastic substances, we used the same concentration of methylcellulose. HA suppressed superoxide release from PMNs and protected BCEC from activated PMNs in a dose-dependent, rather than a molecular weight-dependent, manner. The effect of HA reached almost a plateau at concentration above 2 mg/ml. However, methylcellulose, another viscoelastic substance, showed a similar effect. Therefore, it seems that the suppression of superoxide released from PMNs is not a property that is unique to HA, but is a general property of viscoelastic substances. Our results indicate that the action mechanism of HA proceeds not only through cell surface HA-receptor. We think that HA also acts as a physical barrier and/or a scavenger of superoxide.

Keyword

corneal endothelial cell; hyaluronic acid; polymorphonuclear leukocyte; superoxide

MeSH Terms

Animals
Cattle
Cell Survival
Cells, Cultured
Comparative Study
Cytochromes c/metabolism
Cytoprotection
Dose-Response Relationship, Drug
Endothelium, Corneal/cytology/*drug effects
Humans
Hyaluronic Acid/*pharmacology
Methylcellulose
Molecular Weight
*Neutrophil Activation
Neutrophils/*drug effects/metabolism
Superoxides/*metabolism

Figure

• 

  Fig. 1 Amount of superoxide release (percentage score of reduced cytochrome C) depending on the concentration of viscoelastic substances. The inhibitory effects of superoxide release were influenced by both the molecular weight and concentration of hyaluronic acid. Specifically, hyaluronic acid inhibited superoxide release in proportion to its concentration over 0.1 mg/ml. HA70: Hyaluronic Acid, molecular weight 700,000 HA200: Hyaluronic Acid, molecular weight 2,000,000 HA400: Hyaluronic Acid, molecular weight 4,000,000 *P < 0.05 compare to control concentration (0 mg/ml) by Duncan's multiple pairwise comparison test.

• 

  Fig. 2 Viability of bovine corneal endothelial cells (percentage score of MTT assay) depending on the concentration of viscoelastic substances. There was no significant difference in BCEC viability except for methylcellulose wells in which more BCEC survived than in other wells. HA70: Hyaluronic Acid, molecular weight 700,000 HA200: Hyaluronic Acid, molecular weight 2,000,000 HA400: Hyaluronic Acid, molecular weight 4,000,000 *P < 0.05 compare to control concentration (0 mg/ml) by Duncan's multiple pairwise comparison test.

Reference

1. Liesegang TJ. Viscoelastic substances in ophthalmology. Surv Ophthalmol. 1990. 34:268–293.

2. Ayako A, Michael EY. Posttrabeculectomy anterior subcapsular cataract formation induced by anterior chamber air. Ophthalmic Surg. 1993. 24:314–319.

3. Suzuki Y, Yamaguchi T. Effect of hyaluronic acid on macrophage phagocytosis and active oxygen release. Agents Actions. 1993. 38:32–37.

4. Koichi T, Masahito T, Sachigy S, Hiromi N, Yoki M. Effects of high-molecular-weight hyaluronates on the functions of guinea pig polymorphonuclear leukocytes. Seminars in Arthritis and Rheumatism. 1993. 2:4–8.

5. Crawford KM, Ernst SA, Meyer RF, MacCallumDK . Na/K-ATPase pump sites in cultured bovine corneal endothelium of varying cell density at confluence. Invest Ophthalmol Vis sci. 1995. 36:1317–1326.

6. Wagner M, Benson MT, Rennie IG, MacNeil S. Intracellular regulation of retinal pigment epithelial cell attachment to extracellular matrix proteins. Curr Eye Res. 1995. 14:374–384.

7. Ueno N. Changes in vitreous structure caused by oxygen free radicals. Nippon Ganka Gakkai Zasshi. 1995. 99:1342–1360.

8. Saari H, Konttinen YT, Friman C, Sorsa T. Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate. Inflammation. 1993. 17:403–415.

9. Schenck P, Schneider S, Miehlke R, Prehm P. Synthesis and degradation of hyaluronate by synovia from patients with rheumatoid arthritis. J Rheumatol. 1995. 22:400–405.

10. Fragonas E, Degrassi A, Kvam C, Matulova M, Pollesello P, Zanetti F, Vittur F. Oxygen-derived free radical (ODFR) action on hyaluronic acid (HA), on two HA ester derivatives, and on the metabolism of articular chondrocytes. Exp Cell Res. 1995. 218:79–86.

11. Lacy BE, Underhill CB. The hyaluronate receptor is associated with actin filaments. J Cell Biol. 1987. 105:1395–1404.

12. Miyake K, Underhill CB, Lesley J. Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition. J Exp Med. 1990. 172:69–75.

13. Culty M, Miyake K, Kincade PW. The hyaluronate receptor is a member of the CD44 (H-CAM) family of cell surface glycoproteins. J Cell Biol. 1990. 111:2765–2774.

14. Alberto A, Jorge LA, Juan LB, Jose MR. Protective properties of viscoelastic substances (sodium hyaluronate and 2% hydroxymethylcellulose) against experimental free radical damage to the corneal endothelium. Cornea. 1993. 12:109–114.