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Nitric Oxide Attenuated Transforming Growth Factor-Β Induced www.nature.com/scientificreports OPEN Nitric oxide attenuated transforming growth factor‑β induced myofbroblast diferentiation of human keratocytes Joo‑Hee Park1,3, Martha Kim2,3, Bora Yim2 & Choul Yong Park2* Nitric oxide (NO) has the potential to modulate myofbroblast diferentiation. In this study, we investigated the efect of exogenous NO on the myofbroblast diferentiation of human keratocytes using sodium nitrite as a NO donor. Myofbroblasts were induced by exposing resting keratocytes to transforming growth factor (TGF)‑β1. N‑cadherin and α‑smooth muscle actin (αSMA) were used as myofbroblast markers. Both resting keratocytes and ‑stimulated keratocytes were exposed to various concentrations of sodium nitrite (1 μM to 1000 mM) for 24 to 72 h. Exposure to sodium nitrite did not alter keratocytes’ viability up to a 10 mM concentration for 72 h. However, signifcant cytotoxicity was observed in higher concentrations of sodium nitrite (over 100 mM). The expression of αSMA and N‑cadherin was signifcantly increased in keratocytes by TGF‑β1 stimulation after 72 h incubation. The addition of sodium nitrite (1 mM) to TGF‑β1‑stimulated keratocytes signifcantly decreased αSMA and N cadherin expression. Smad3 phosphorylation decreased after sodium nitrite (1 mM) exposure in TGF‑β1‑stimulated keratocytes. The efect of NO was reversed when NO scavenger, 2‑4‑carboxyphenyl‑4,4,5,5‑tetramethylimidazoline‑1‑oxyl‑3‑oxide (cPTIO) was added in the culture medium. Application of sodium nitrite resulted in signifcant decrease of corneal opacity when measured at 2 weeks after the chemical burn in the mouse. These results verifed the potential therapeutic efect of NO to decrease myofbroblast diferentiation of human keratocytes and corneal opacity after injury. Abbreviations NO Nitric oxide cPTIO 2-4-Carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide cGMP Cyclic guanosine monophosphate αSMA Alpha smooth muscle actin TGFβ1 Transforming growth factor beta 1 mTOR Mammalian target of rapamycin Nitric oxide (NO) is a small signaling molecule with various biological functions. NO is endogenously produced by the activation of NO synthases (NOSs) under various conditions 1. In pathologic states, NO acts as a free radi- cal messenger and mediates infammation and vasodilatation1,2. NO is also an important physiological regula- tor of cellular proliferation3–5. In a skin wound model, the exogenous NO supply facilitated the wound healing response1,2. In addition, NO defciency leads to impaired wound healing, as shown in nitric oxide synthase (NOS) knock-out mice6,7. Furthermore, a potent anti-fbrotic action of exogenous NO was also reported 8. NO activates soluble guanylate cyclase and increases the cyclic guanosine monophosphate (cGMP) level in cells 9. Te activa- tion of the NO-guanylate cyclase-cGMP pathway was verifed to attenuate fbrotic responses in organs, such as the liver, kidney, prostate, heart, skin, and lung 10–12. 1Department of Biochemistry, College of Medicine, Dongguk University, Gyeongju, South Korea. 2Department of Ophthalmology, Ilsan Hospital, Dongguk University, 814, Siksadong, Ilsan-dong-gu, Goyang, Gyunggido 410-773, South Korea. 3These authors contributed equally: Joo-Hee Park and Martha Kim. *email: [email protected] Scientifc Reports | (2021) 11:8183 | https://doi.org/10.1038/s41598-021-87791-x 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Keratocytes’ viability was measured afer diferent concentrations (1 μM to 1000 mM) of sodium nitrite exposure for 24 (A), 48 (B) and 72 h (C). Sodium nitrite did not harm cultured keratocyte viability up to 10 mM concentration for 72 h. However, high concentrations (100 mM or 1000 mM) of sodium nitrite decreased keratocyte viability. *p < 0.05, ***p < 0.001. Te results of previous studies suggest the potential therapeutic efect of NO in the healing process of cor- neal wounds. Recently, the application of exogenous NO in the ophthalmic feld was actively investigated. Te permissive role of NO in corneal epithelial wound healing was reported previously 13–17. Te topical application of NO successfully promoted the corneal epithelial wound healing process13,17. In addition, NO’s antibacterial efect is another beneft to prevent further corneal damage from bacterial infection afer injury18,19. Incidentally, a recent development of NO as a promising anti-glaucoma medication further increased the clinical interest of NO in the ophthalmic feld9,20,21. Although NO ameliorates corneal epithelial wound healing, corneal injury usually results in both corneal epithelial and stromal damage simutaneously. Of course, keratocytes are the major cell component of corneal stroma. In corneal scars, keratocytes diferentiate into myofbroblasts and lay down abnormal collagen fbers that can deteriorate corneal transparency. Terefore, the modulation of myofbroblast diferentiation in an injured cornea is a critical therapeutic target to minimize corneal opacity and preserve clear vision. From this perspec- tive, the evaluation of the efect of NO on keratocytes is a necessary step for the development of NO as a corneal wound healing modulator. Although the anti-fbrotic action of exogenous NO was reported in various human tissues, its role in corneal fbrosis, especially myofbroblasts’ diferentiation from keratocytes, has not been fully elucidated. If NO is found to beneft both corneal epithelial cells and stromal cells, the further development of new drugs using NO can be more efective. In this study, we investigated the efect of exogenous NO on primarily cultured human keratocytes. Diferent concentrations of NO donors (sodium nitrite) were applied in the culture media, and the cellular viability of keratocytes was measured. We induced keratocytes’ myofbroblast diferentiation by adding transforming growth factor β1 (TGF-β1) to the culture media and investigated the efect of NO on myofbroblast markers’ expressions, N cadherin and α-smooth muscle actin (αSMA) from TGF-β1-stimulated keratocytes. Finally, the efect of NO on corneal opacity development was evaluated using murine chemical corneal burn model. Results Keratocytes’ viability with diferent concentrations of NO donors. We investigated any toxic efect of NO on keratocytes. Keratocyte viability was not deteriorated at low concentrations (up to 10 mM) of sodium nitrite. Rather mild increase of cell viability was observed with the addition of 10 mM of sodium nitrite. However, sodium nitrite decreased keratocytes’ viability at high concentrations (equal to or more than 100 mM). Tis toxicity increased with a longer incubation period. Te decrease of viability in high concentrations (over 100 mM) of sodium nitrite is attributed to the hyper-osmolar stress induced by excess sodium in the culture media (Fig. 1). Intracellular NO concentration afer exposure to diferent concentrations of sodium nitrite was measured (Fig. 2). A mild increase of intracellular NO concentration was observed afer 24, 48 and 72 h incu- bation. Addition of 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 10 μM) in the culture medium scavenged NO and decreased intracellular NO concentration. NO activates guanylate cyclase and increase the production of cGMP. Signifcant increase of intracellular cGMP was observed afer exposure to sodium nitrite. No signifcant change of oncogene activation related proteins, p53 and p21 was observed with the NO donor treatment keratocytes. (Supplement Fig. S1). mTOR pathway activation and cellular autophagy. Two critical cell survival pathways (mTOR and autophagy) were not afected by sodium nitrite up to a 1000 μM concentration afer 24-h exposure (Fig. 3). Keratocyte stimulation with TGFβ1. Myofbroblast diferentiation from resting keratocyte afer TGFβ1 stimulation for 24 h was evaluated by examining the expression level of pSmad3/Smad3, αSMA and N-cadherin. Te phosphorylation of Smad 3 is an important pivotal step of the TGFβ1 pathway. Exposure of keratocytes to TGFβ1 signifcantly increased mlSmad3 phosphorylation (pSmad3) (Fig. 3A). Stimulation with TGFβ1 (2, 5, and 10 ng/) induced a dose-dependent increase of the αSMA expression in keratocytes (Fig. 4A,B). N-cadherin expression was also activated afer TGFβ1 stimulation (Fig. 4A). Scientifc Reports | (2021) 11:8183 | https://doi.org/10.1038/s41598-021-87791-x 2 Vol:.(1234567890) www.nature.com/scientificreports/ Figure 2. Te efect of sodium nitrite on the intracellular NO and cyclic guanine monophosphate (cGMP) concentrations. (A–C) Sodium nitrite in the culture medium resulted in mild increase of intracellular NO concentration afer 24 (A), 48 (B) and 72 h (C) incubation. Addition of 2-4-carboxyphenyl-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 10 μM) in the culture medium scavenged NO and decreased intracellular NO concentration. (D) cGMP increased with the addition of sodium nitrite and addition of cPTIO blunted cGMP generation. *p < 0.05, **p < 0.01, ###p < 0.001. ROS production in keratocyte stimulated with TGFβ1. Slight increase of total ROS was observed in TGFβ1 (10 ng/mL) stimulated keratocytes compared to control keratocytes. Treatment with sodium nitrite (10, 100 or 1000 μM) signifcantly alleviated total ROS production in TGFβ1 stimulated keratocytes (Fig. 5). Nitrite reductase activity in keratocyte. Nitrite reductase activity in keratocyte was measured in kerat- ocytes using mitochondrial amidoxime reducing component 1 (mARC1), xanthine dehydrogenase and xanthine oxidase. Te mRNA expression level of xanthine dehydrogenase was signifcantly increased
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