Koung Hoon Kook

Antifibrotic effect of Pirfenidone on orbital fibroblasts of patients with thyroid-associated ophthalmopathy by decreasing TIMP-1 and collagen levels.

PURPOSE The aim of this study was to determine the antifibrotic effects of pirfenidone in orbital fibroblasts of patients with thyroid-associated ophthalmopathy (TAO). METHODS The effects of interleukin (IL)-1beta and of fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF)-beta on the induction of tissue inhibitors of metalloproteinases (TIMP)-1 were assessed in orbital fibroblasts of TAO patients. TIMP-1 protein levels were measured by ELISA and Western blot analyses, and TIMP-1 activity was assessed by reverse zymography. The effect of pirfenidone on TIMP-1 induction in orbital fibroblasts was evaluated with the same methods using dexamethasone as a reference agent. A hydroxyproline assay was used to determine the effect of pirfenidone and dexamethasone on collagen production in orbital fibroblasts, and the tetrazolium-based MTT assay was used to assess pirfenidone cytotoxicity. RESULTS IL-1beta strongly and dose dependently increased the level of active TIMP-1 protein, whereas FGF, PDGF, and TGF-beta did not significantly induce TIMP-1 protein. Pirfenidone was more effective than dexamethasone in inhibiting IL-1beta-induced increases in TIMP-1, reducing TIMP-1 levels to less than those in untreated controls at a minimal concentration (5 mM). Moreover, pirfenidone effectively decreased hydroxyproline levels in orbital fibroblasts, whereas dexamethasone had no effect on hydroxyproline levels. Pirfenidone exhibited no toxicity in orbital fibroblasts at the concentrations used. CONCLUSIONS These results indicate that nontoxic concentrations of pirfenidone have significant antifibrotic effects on orbital fibroblasts from patients with TAO.

Pirfenidone attenuates the IL-1β-induced hyaluronic acid increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy.

PURPOSE This study aimed to investigate the effect of pirfenidone on the IL-1β-induced hyaluronic acid (HA) increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). METHODS Primary cultured orbital fibroblasts were obtained from patients with TAO, and the excreted levels of HA from IL-1β-treated cells with or without pirfenidone were measured. The effect of pirfenidone on IL-1β-induced hyaluronic acid synthase (HAS) expression was evaluated. The relevance of the mitogen-activated protein kinase (MAPK)-mediated signaling pathway in IL-1β-induced HAS expression was assessed using specific inhibitors to p38, extracellular signal-regulated kinase (ERK), or c-Jun N-terminal kinase (JNK). The phosphorylation level of each MAPK in IL-1β-treated cells with or without pirfenidone and the level of AP-1 DNA binding were measured. The inhibitory potency of pirfenidone on HA production was evaluated using dexamethasone as a reference agent. RESULTS Pirfenidone strongly attenuated the IL-1β-induced HA release in a dose-dependent manner. The IL-1β-induced HAS expression was decreased significantly following cotreatment with pirfenidone at the mRNA and protein levels. The production of mRNAs was halted by cotreatment with inhibitors of ERK and p38, but not by inhibitors of JNK. The IL-1β-induced ERK and p38 phosphorylation, and AP-1 DNA binding were attenuated in the presence of pirfenidone. Pirfenidone showed greater potency than dexamethasone in inhibiting increases in IL-1β-induced HA. CONCLUSIONS Pirfenidone attenuates the IL-1β-induced HA production in orbital fibroblasts from patients with TAO, at least in part, through suppression of the MAPK-mediated HAS expression. These results support the potential use of pirfenidone for treatment of patients with TAO.

Pirfenidone attenuates IL-1β-induced COX-2 and PGE2 production in orbital fibroblasts through suppression of NF-κB activity.

The aim of this study was to determine the effect of pirfenidone on interleukin (IL)-1β-induced cyclooxygenase (COX)-2 and prostaglandin (PG)E2 expression in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). Primary cultures of orbital fibroblasts from patients with TAO (n = 4) and non-TAO subjects (n = 4) were prepared. The level of PGE2 in orbital fibroblasts treated with IL-1β in the presence or absence of pirfenidone was measured using an enzyme-linked immunosorbent assay. The effect of pirfenidone on IL-1β-induced COX-2 expression in orbital fibroblasts from patients with TAO was evaluated by reverse transcription-polymerase chain reaction (PCR) and quantitative real-time PCR analyses, and verified by Western blot. Activation of nuclear factor-κB (NF-κB) was evaluated by immunoblotting for inhibitor of κB (IκB)α and phosphorylated IκBα, and DNA-binding activity of p50/p65 NF-κB was analyzed by electrophoretic mobility shift assay. In addition, IL-1 receptor type 1 (IL-1R1) expression was assessed by RT-PCR in IL-1β-treated cells with or without pirfenidone. Pirfenidone significantly attenuated IL-1β-induced PGE2 release in both TAO and non-TAO cells. IL-1β-induced COX-2 mRNA and protein expression decreased significantly following co-treatment with pirfenidone. IL-1β-induced IκBα phosphorylation and degradation decreased in the presence of pirfenidone and led to decreased nuclear translocation and DNA binding of the active NF-κB complex. In our system, neither IL-1β nor pirfenidone co-treatment influenced IL-1R1 expression. Our results suggest that pirfenidone attenuates the IL-1β-induced PGE2/COX-2 production in TAO orbital fibroblasts, which is related with suppression of the NF-κB activation.

Protective Effect of Grape Seed Extract against Oxidative Stress-Induced Cell Death in a Staurosporine-Differentiated Retinal Ganglion Cell Line

Objective: Grape seed extract (GSE) is a potent antioxidant. We examined the effect of GSE on oxidative stress-induced cell death in a transformed retinal ganglion cell line, RGC-5. Methods: Staurosporine-differentiated RGC-5 (ssdRGC-5) cells obtained by treating RGC-5 cells with 1 µM staurosporine were incubated with GSE for 2 h and then exposed to buthionine sulfoximine plus glutamate (B/G) for 24 h. Cell death was detected using the LIVE/DEAD viability assay and the type of cell death was evaluated using fluorescein isothiocyanate-conjugated Annexin–V/propidium iodide staining. To investigate the mechanism underlying cell death, we determined the caspase-3 activity and level of reactive oxygen species (ROS) formation. Results: Treatment of ssdRGC-5 cells with B/G increased intracellular ROS and induced apoptosis (not necrosis) with increasing caspase-3 activity. GSE rescued the ssdRGC-5 cells from oxidative stress-induced cell death by inhibiting both intracellular ROS production and caspase-3 activation. Conclusion: GSE had a neuroprotective effect against oxidative stress-induced apoptotic death in ssdRGC-5 cells.

Electronic enhancement of tear secretion.

OBJECTIVE To study electrical stimulation of the lacrimal gland and afferent nerves for enhanced tear secretion, as a potential treatment for dry eye disease. We investigate the response pathways and electrical parameters to safely maximize tear secretion. APPROACH We evaluated the tear response to electrical stimulation of the lacrimal gland and afferent nerves in isofluorane-anesthetized rabbits. In acute studies, electrical stimulation was performed using bipolar platinum foil electrodes, implanted beneath the inferior lacrimal gland, and a monopolar electrode placed near the afferent ethmoid nerve. Wireless microstimulators with bipolar electrodes were implanted beneath the lacrimal gland for chronic studies. To identify the response pathways, we applied various pharmacological inhibitors. To optimize the stimulus, we measured tear secretion rate (Schirmer test) as a function of pulse amplitude (1.5-12 mA), duration (0.1-1 ms) and repetition rate (10-100 Hz). MAIN RESULTS Stimulation of the lacrimal gland increased tear secretion by engaging efferent parasympathetic nerves. Tearing increased with stimulation amplitude, pulse duration and repetition rate, up to 70 Hz. Stimulation with 3 mA, 500 μs pulses at 70 Hz provided a 4.5 mm (125%) increase in Schirmer score. Modulating duty cycle further increased tearing up to 57%, compared to continuous stimulation in chronically implanted animals (36%). Ethmoid (afferent) nerve stimulation increased tearing similar to gland stimulation (3.6 mm) via a reflex pathway. In animals with chronically implanted stimulators, a nearly 6 mm increase (57%) was achieved with 12-fold less charge density per pulse (0.06-0.3 μC mm(-2) with 170-680 μs pulses) than the damage threshold (3.5 μC mm(-2) with 1 ms pulses). SIGNIFICANCE Electrical stimulation of the lacrimal gland or afferent nerves may be used as a treatment for dry eye disease. Clinical trials should validate this approach in patients with aqueous tear deficiency, and further optimize electrical parameters for maximum clinical efficacy.

Hyaluronic acid induces COX-2 expression via CD44 in orbital fibroblasts from patients with thyroid-associated ophthalmopathy.

PURPOSE The aim of this study was to determine the effect of hyaluronic acid (HA) on cyclooxygenase (COX)-2 expression in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). METHODS Primary cultured orbital fibroblasts were obtained from patients with TAO and non-TAO subjects. Dermal and conjunctival fibroblasts were cultured from the eyelid skin of subjects undergoing cosmetic lid surgery or cataract surgery, respectively. The cells were treated with HA and the transcriptional and translational levels of COX-2 were measured. The expression of CD44 on each type of cells was determined, and the involvement of CD44 in the HA-induced COX-2 increase in orbital fibroblasts from patients with TAO was evaluated by using CD44 knockdown cells and by pretreatment with neutralizing antibody. The relevance of the mitogen-activated protein kinase (MAPK) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated signaling pathway was assessed by immunoblotting for the phosphorylated form of each MAPK or IκB and by using specific inhibitors to these pathways. RESULTS Hyaluronic acid increased COX-2 expression in orbital fibroblasts from patients with TAO, which was not observed in the cells from non-TAO subjects and conjunctival or dermal fibroblasts. Orbital fibroblasts from patients with TAO expressed significantly higher level of CD44 than non-TAO cells, and the increased COX-2 expression by HA in these cells was attenuated by knockdown or neutralizing of CD44. Hyaluronic acid induced MAPK and IκB phosphorylation; and cotreatment with specific MAPK or NF-κB inhibitors halted HA-induced transcription of COX-2, suggesting the involvement of these signaling pathways. CONCLUSIONS Hyaluronic acid induced COX-2 expression in orbital fibroblasts from patients with TAO via CD44 through the MAPK and NF-κB-mediated signaling pathways. These results suggest that HA may have a proinflammatory role in the pathogenesis of TAO by inducing COX-2.

Altered ganglioside expression modulates the pathogenic mechanism of thyroid-associated ophthalmopathy by increase in hyaluronic acid.

PURPOSE The aim of this study was to determine the role of gangliosides in pathogenic mechanisms of thyroid-associated ophthalmopathy (TAO). METHODS The gangliosides profile and mRNA level of sialyltransferases of the orbital tissues from TAO patients (n = 5) and non-TAO subjects (n = 4) were investigated. In addition, the effect of exogenous gangliosides on the expression of hyaluronic acid was examined in orbital fibroblasts. For in vitro experiments, we used four different strains of cells obtained from non-TAO subjects with at least three replicates for each strain. RESULTS Trisialoganglioside 1b (GT1b) was significantly overexpressed in the orbital tissue of TAO patients compared with control tissue, whereas no significant difference was observed for either monosialoganglioside 1 (GM1) or disialoganglioside 1a (GD1a) by digital analyses of immunohistochemical images. Moreover, mRNA levels of sialyltransferase (SAT)-I and SAT II were increased in TAO patients compared with control. Exogenous GT1b strongly induced the morphologic changes related to an accumulation of sparse flocculent precipitates in lysosomes and increased the extracellular hyaluronic acid level in orbital fibroblasts with the induction of hyaluronic acid synthase, which were less by GD1a but not by GM1. The GT1b-induced morphologic changes of cells were due, at least in part, to an increase of intracellular hyaluronic acid. Co-treatment of hyaluronidase nicely attenuated the morphologic changes in orbital fibroblasts. Thy-1(+) orbital fibroblasts were more capable of producing hyaluronic acid by exogenous GT1b. CONCLUSIONS The results suggest that gangliosides, particularly GT1b, may play a role in the pathologic mechanisms of TAO by stimulating an increase in hyaluronic acid.

Use of the Bispectral Index to Predict Eye Position of Children during General Anesthesia

Purpose To assess the relationship between eye position and anesthesia depth using the bispectral index (BIS) value, a parameter derived from electroencephalography data. Methods We investigated the relationship between BIS value and eye position in 32 children who underwent surgery for epiblepharon under general anesthesia. BIS values were recorded continuously throughout the procedure (from induction to awakening). Eye positions were video-recorded and analyzed after surgery. The vertical position of each eye was scored according to its height in relation to the medial canthus. An eye position in which the upper eyelid covered one-third of the cornea was defined as a significant ocular elevation. Results The BIS value correlated inversely with the end-tidal concentration of each anesthetic agent, whereas it correlated positively with the eye elevation score (eye position = 0.014 × BIS + 0.699, p = 0.011). The mean eye position score was significantly greater in patients whose BIS values were over 65. Eleven patients (34.4%) had significant ocular elevation; their mean concurrent BIS value was 61.6. Two of these patients had elevation during surgery and 9 had elevation during emergence from anesthesia. Conclusions We found that high BIS values were correlated with low levels of anesthetic concentration and high eye position, suggesting that BIS monitoring may be useful for predicting eye position during anesthesia. Particular attention must be given to eye position during ophthalmic surgery. Anesthesia depth can be maintained by assuring that the BIS value remains below 65.

Identification and Functional Characterization of ST3GAL5 and ST8SIA1 Variants in Patients with Thyroid-Associated Ophthalmopathy

Purpose This study was conducted to identify and to functionally characterize genetic variants in ST3GAL5 and ST8SIA1 in Korean patients with thyroid-associated ophthalmopathy (TAO). Materials and Methods Genetic analyses were conducted using DNA samples from TAO patients (n=50) and healthy subjects (n=48) to identify TAO-specific genetic variants of ST3GAL5 or ST8SIA1. The effect of each genetic variant on the transcription or expression of these genes was examined. Additionally, correlations between functional haplotypes of ST3GAL5 or ST8SIA1 and clinical characteristics of the patients were investigated. Results Six promoter variants and one nonsynonymous variant of ST3GAL5 were identified, and four major promoter haplotypes were assembled. Additionally, three promoter variants and two major haplotypes of ST8SIA1 were identified. All ST3GAL5 and ST8SIA1 variants identified in TAO patients were also found in healthy controls. Promoter activity was significantly decreased in three promoter haplotypes of ST3GAL5 and increased in one promoter haplotype of ST8SIA1. Transcription factors activating protein-1, NKX3.1, and specificity protein 1 were revealed as having roles in transcriptional regulation of these haplotypes. The nonsynonymous variant of ST3GAL5, H104R, did not alter the expression of ST3GAL5. While no differences in clinical characteristics were detected in patients possessing the functional promoter haplotypes of ST3GAL5, exophthalmic values were significantly lower in patients with the ST8SIA1 haplotype, which showed a significant increase in promoter activity. Conclusion These results from genotype-phenotype analysis might suggest a possible link between the ST8SIA1 functional promoter haplotype and the clinical severity of TAO. However, further studies with larger sample sizes are warranted.