J Korean Ophthalmol Soc JKOS Journal of the Korean Ophthalmological Society 0378-6471 2092-9374 The Korean Ophthalmological Society 10.3341/jkos.2019.60.10.975 Original Article Role of Hydrogen Sulfide in the Survival of Fibroblasts and Fibroblast-mediated Contraction of Collagen Gel Park Hyeon Jin MD Kim Jae Woo MD PhD Department of Ophthalmology, Daegu Catholic University School of Medicine, Daegu, Korea. Address reprint requests to Jae Woo Kim, MD, PhD. Department of Ophthalmology, Daegu Catholic University Medical Center, #33 Duryugongwon-ro 17-gil, Nam-gu, Daegu 42472, Korea. Tel: 82-53-650-4728, Fax: 82-53-627-0133, jwkim@cu.ac.kr 10 2019 21 10 2019 60 10 975 981 24 01 2019 26 02 2019 19 09 2019 ©2019 The Korean Ophthalmological Society 2019 The Korean Ophthalmological Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Purpose

To investigate the role of hydrogen sulfide in the survival and collagen gel contraction of cultured human Tenon's capsule fibroblasts (HTCFs).

 Methods

Primarily cultured HTCFs were exposed to 0, 100, 200, or 300 µM hydrogen sulfide (sodium hydrogen sulfide, NaHS) for 2 days. Cellular survival was assessed by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. Degree of apoptosis was assessed with flow cytometry using annexin-V/propidium iodide double staining. To evaluate the effect of NaHS on cellular transdifferentiation, HTCFs were stimulated with 5 ng/mL TGF-β1 and the level of expression of α-smooth muscle actin (SMA) mRNA was assessed using reverse-transcription polymerase chain reaction. The cells were embedded in collagen gel, and the amount of gel contraction was measured.

 Results

NaHS at 300 µM reduced HTCF survival (p = 0.013); NaHS at both 200 and 300 µM increased apoptosis in a dose-dependent manner (p = 0.013 and p = 0.016). TGF-β1 increased the expression of α-SMA mRNA (p = 0.041); co-treatment with 100 µM NaHS decreased TGF-β1-induced α-SMA mRNA expression (p = 0.039) and inhibited collagen gel contraction.

 Conclusions

NaHS at high concentration reduced cellular survival and increased HTCF apoptosis. NaHS decreased TGF-β 1-induced increases in α-SMA mRNA expression and collagen gel contraction. Thus, hydrogen sulfide may suppress scar formation by inhibiting HTCF transdifferentiation and contraction of collagen gels.

 Apoptosis Hydrogen sulfide Survival Transdifferentiation Tenon capsule fibroblasts

The authors have no conflicts to disclose.

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Exposure to 300 µM NaHS decreased cellular survival significantly compared to non-exposed control (<sup>*</sup><italic>p</italic> < 0.05). NaHS = sodium hydrogen sulfide. Effect of NaHS on the apoptosis of human Tenon's capsule fibroblasts. Exposure to 200 or 300 µM NaHS increased cellular apoptosis significantly compared to non-exposed control (<sup>**</sup><italic>p</italic> < 0.05). NaHS = sodium hydrogen sulfide. Effect of 100 µM NaHS and 5 ng/mL TGF-β1 on the survival of human Tenon's capsule fibroblasts. Exposure to TGF-β1 increased cellular survival significantly (<sup>*</sup><italic>p</italic> < 0.05). Co-exposing 100 µM NaHS and TGF-β1 suppressed cellular survival significantly compared to exposing TGF-β1 alone (<sup>**</sup><italic>p</italic> < 0.05). NaHS = sodium hydrogen sulfide; TGF = transforming growth factor. Effect of 100 µM NaHS and 5 ng/mL TGF-β1 on the expression of α-SMA mRNA in human Tenon's capsule fibroblasts. Exposure to TGF-β1 increased the level of α-SMA mRNA expression significantly (<sup>*</sup><italic>p</italic> < 0.05). Co-exposing 100 µM NaHS and TGF-β1 suppressed the TGF-β1 induced expression α-SMA mRNA significantly compared to exposing TGF-β1 alone (<sup>**</sup><italic>p</italic> < 0.05). NaHS = sodium hydrogen sulfide; TGF = transforming growth factor; SMA = smooth muscle actin. Effect of NaHS and 5 ng/mL TGF-β1 (T) on the contraction of collagen gels. At day 1 and day 2, exposure to 100 or 300 µM NaHS inhibited collagen gel contraction significantly compared to exposure to TGF-β1 alone (<sup>*</sup><italic>p</italic> < 0.05). NaHS = sodium hydrogen sulfide; TGF = transforming growth factor.