Korean J Ophthalmol.  2010 Feb;24(1):29-34. 10.3341/kjo.2010.24.1.29.

A Novel Therapeutic Target in Inflammatory Uveitis: Transglutaminase 2 Inhibitor

Affiliations
  • 1Department of Ophthalmology, Hangil Eye Hospital, Incheon, Korea.
  • 2Department of Ophthalmology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. dropkim@dreamwiz.com
  • 3Molecular Oncology Branch, Division of Basic & Applied Sciences, National Cancer Center, Seoul, Korea.

Abstract

PURPOSE: Our goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU)
METHODS
EIU was induced in female Lewis rats by single footpad injections of 200 microgram of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients.
RESULTS
Clinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region.
CONCLUSIONS
TGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.

Keyword

Endotoxin-induced uveitis; Lipopolysaccharides; Transglutaminases

MeSH Terms

Animals
Disease Progression
Enzyme Inhibitors/*therapeutic use
Female
GTP-Binding Proteins/*antagonists & inhibitors/*metabolism
Lipopolysaccharides
Rats
Rats, Inbred Lew
Transglutaminases/*antagonists & inhibitors/*metabolism
Uveitis/chemically induced/*enzymology/pathology

Figure

  • Fig. 1 Disease activity and increasing transglutaminase 2 (TGase 2) activity over time in endotoxin-induced uveitis (EIU). EIU as induced in female Lewis rats by a single foot pad injection of 200 µg lipopolysaccharide (LPS), and disease activity and TGase 2 activity in aqueous humor (AqH) were determined every 4 hour after LPS administration. (A) Clinical findings were assessed on a scale of 0 to 4 according to the extent of inflammation and tissue damage. Peak inflammation was seen 24 hr after LPS stimulation (mean±SD of 10 eyes). (B) Cellular infiltration over time. Infiltration peaked 24 to 28 hr after LPS stimulation (mean±SD of 10 eyes). (C) TGase 2 activity over time. TGase activity increased 130-fold 24 hr after LPS stimulation (mean± SD, n=3). (D) Western blotting using a TGase cross-linked antibody, revealing that a 23-kDa protein increased concomitantly with increases in TGase 2 activity.

  • Fig. 2 Effect of transglutaminase (TGase) inhibitors on disease activity and TGase 2 activity in Endotoxin-induced uveitis Rats were injected intraperitoneally with R2 peptide (KVLDGQDP,10 mg/kg, 500 µm), cystamine (CTM, 6.0 mg/kg, 500 µm) or dexamethasone (2 mg/kg) 30 minutes before and at the time of lipopolysaccharide administration. Disease activity and TGase 2 activity in aqueous humor were determined 24 hr later. (A) Grading of inflammation, showing a two-fold reduction in the R2-treated rats (mean±SD of 6-8 eyes). (B) Number of infiltrating cells on histologic sections, showing a three-fold reduction in the R2-treated group (mean±SD of 6-8 eyes). (C) TGase 2 activity, showing a ten-fold reduction in the R2 treated rats (mean±SD of 6-8 eyes). *Significantly different from control. p<0.001 by unpaired Student t-test.

  • Fig. 3 MS/MS identification of transglutaminase 2 (TGase 2) substrate in Aqueous humor (AqH) from uveitis patients. (A) AqH samples from the eyes of human patients with uveitis were obtained using a 30-gauge needle. Protein samples (10 g) were resolved in 4-12% gels and stained with Coomassie blue. The indicated bands were excised for in-gel tryptic digestion. (B) MS/MS spectra of 1797.9 peptide. (C) MS/MS spectra of 1946.1 peptide. (D) MS/MS spectra of 1876.0 peptide. (E) Amino acid sequence of Ig chain C region. The identified peptides are indicated in bold.


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