Nox4 Facilitates TGFβ1-Induced Fibrotic Response in Human Tenon’s Fibroblasts and Promotes Wound Collagen Accumulation in Murine Model of Glaucoma Filtration Surgery
Abstract
:1. Introduction
2. Materials and Methods
2.1. Human Tenon’s Fibroblast Culture
2.2. Pharmacological Drug Treatment
2.3. Adenovirus Infection
2.4. Determination of Extracellular H2O2
2.5. Gene Expression Analysis
2.6. Western Blot Analysis
2.7. Picro-Sirius Red Spectrophotometric Assay
2.8. Cell Proliferation Assay
2.9. Mouse Model of Glaucoma Filtration Surgery
2.10. Statistical Analysis
3. Results
3.1. TGFβ1 Cause Increase in Nox4 mRNA, H2O2 Generation, and Collagen Synthesis in HTFs
3.2. TGFβ1-Induced Nox4 Expression Requires Smad3-Activation in HTFs
3.3. Suppression of Nox4 and H2O2 Generation Decreased TGFβ1-Induced Responses in HTFs
3.4. Nox4 and H2O2 Generation Is Involved in TGFβ1-Induced Proliferation of HTFs
3.5. Collagen Deposition at the Wound Is Reduced in Nox4-Deficient Mice with Glaucoma Filtration Surgery
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
Adv-Ctrl siRNA | adenovirus expressing scrambled siRNA |
Adv-Nox4i | adenovirus carrying siRNA targeting Nox |
GFS | glaucoma filtration surgery |
H2O2 | hydrogen peroxide |
HTF | human Tenon’s fibroblasts |
MOI | multiplicity of infection |
mRNA | messenger ribonucleic acid |
NADPH oxidase 4 | Nox4 |
PCR | polymerase chain reaction |
RNA | ribonucleic acid |
ROS | reactive oxygen species |
SIS3 | specific inhibitor of Smad3 |
TGFβ | transforming growth factor β |
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Gene Name | Gene ID | TaqMan Gene Expression Assays |
---|---|---|
Nox1 | 27035 | Hs002455589_m1 |
Nox2 (CYBB) | 1536 | Hs00166163_m1 |
Nox4 | 50507 | Hs01558199_m1 |
Nox5 | 79400 | Hs00225846_m1 |
GAPDH | 2597 | 4326317E |
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Shah, M.H.; Chan, E.C.; Van Bergen, N.J.; Pandav, S.S.; Ng, S.; Crowston, J.G.; Peshavariya, H.M. Nox4 Facilitates TGFβ1-Induced Fibrotic Response in Human Tenon’s Fibroblasts and Promotes Wound Collagen Accumulation in Murine Model of Glaucoma Filtration Surgery. Antioxidants 2020, 9, 1126. https://doi.org/10.3390/antiox9111126
Shah MH, Chan EC, Van Bergen NJ, Pandav SS, Ng S, Crowston JG, Peshavariya HM. Nox4 Facilitates TGFβ1-Induced Fibrotic Response in Human Tenon’s Fibroblasts and Promotes Wound Collagen Accumulation in Murine Model of Glaucoma Filtration Surgery. Antioxidants. 2020; 9(11):1126. https://doi.org/10.3390/antiox9111126
Chicago/Turabian StyleShah, Manisha H., Elsa C. Chan, Nicole J. Van Bergen, Surinder S. Pandav, Sze Ng, Jonathan G. Crowston, and Hitesh M. Peshavariya. 2020. "Nox4 Facilitates TGFβ1-Induced Fibrotic Response in Human Tenon’s Fibroblasts and Promotes Wound Collagen Accumulation in Murine Model of Glaucoma Filtration Surgery" Antioxidants 9, no. 11: 1126. https://doi.org/10.3390/antiox9111126
APA StyleShah, M. H., Chan, E. C., Van Bergen, N. J., Pandav, S. S., Ng, S., Crowston, J. G., & Peshavariya, H. M. (2020). Nox4 Facilitates TGFβ1-Induced Fibrotic Response in Human Tenon’s Fibroblasts and Promotes Wound Collagen Accumulation in Murine Model of Glaucoma Filtration Surgery. Antioxidants, 9(11), 1126. https://doi.org/10.3390/antiox9111126