J Korean Ophthalmol Soc.  2019 Oct;60(10):975-981. 10.3341/jkos.2019.60.10.975.

Role of Hydrogen Sulfide in the Survival of Fibroblasts and Fibroblast-mediated Contraction of Collagen Gel

• Park HJ
• Kim JW

• KMID: 2460516
• DOI: http://doi.org/10.3341/jkos.2019.60.10.975

Abstract

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.

Keyword

Apoptosis; Hydrogen sulfide; Survival; Transdifferentiation; Tenon capsule fibroblasts

MeSH Terms

Actins
Apoptosis
Cicatrix
Collagen*
Fibroblasts*
Flow Cytometry
Gels
Humans
Hydrogen Sulfide*
Hydrogen*
Polymerase Chain Reaction
RNA, Messenger
Tenon Capsule
Actins
Collagen
Gels
Hydrogen
Hydrogen Sulfide
RNA, Messenger

Figure

• 

  Figure 1 Effect of NaHS on the survival of human Tenon's capsule fibroblasts. Exposure to 300 µM NaHS decreased cellular survival significantly compared to non-exposed control (*p < 0.05). NaHS = sodium hydrogen sulfide.

• 

  Figure 2 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 (**p < 0.05). NaHS = sodium hydrogen sulfide.

• 

  Figure 3 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 (*p < 0.05). Co-exposing 100 µM NaHS and TGF-β1 suppressed cellular survival significantly compared to exposing TGF-β1 alone (**p < 0.05). NaHS = sodium hydrogen sulfide; TGF = transforming growth factor.

• 

  Figure 4 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 (*p < 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 (**p < 0.05). NaHS = sodium hydrogen sulfide; TGF = transforming growth factor; SMA = smooth muscle actin.

• 

  Figure 5 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 (*p < 0.05). NaHS = sodium hydrogen sulfide; TGF = transforming growth factor.

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