Effect of vitamin D3 on shear stress‐driven cell proliferation in human tenon’s fibroblasts
Authors: Jia Shuo 
Published: December 2019

Purpose

The project seeks to evaluate the effect of vitamin D3 on cell proliferation in shear stress treated human Tenon’s fibroblasts (HTF). Vitamin D3 has been shown in previous studies to have a regulatory effect on the activity of cutaneous fibroblasts and play a protective role in ocular health. A similar effect on ocular fibroblasts would be greatly beneficial after surgeries where excessive postoperative subconjunctival fibrosis is the main cause of surgical failure.

Methods

The effect of vitamin D3 on HTF cytotoxicity and cell viability was determined by lactate dehydrogenase (LDH) cytotoxic assay and PrestoBlue cell viability assay. The effect of vitamin D3 on cell proliferation in shear stress treated HTF was determined via CRYSTAL VIOLET and CYQUANT proliferation assay. Cell cycle progression was evaluated using flow cytometry. RNA sequencing, quantitative real‐time PCR and western blot analysis were carried out to determine the effect of vitamin D3 on the expression of vitamin D3 and PDGF receptors, and the respective downstream signaling pathways regulated by these receptors. The effect of vitamin D3 on shear stress driven cell proliferation in HTF was confirmed by in vitro scratch wound healing assay.

Results

A variety of HTF from squint, pterygium and glaucoma surgeries were successfully primary‐cultured and passaged. Doses of vitamin D3 from 0.001 to 10 µM were found to be non‐cytotoxic to HTF. From our experiments, 10 000 cells and 96 h were determined to be the optimal cell concentration and exposure time, respectively. Shear stress (shaking at 25 rpm) condition significantly induces HTF proliferation. Shear stress stimulated cell cycle of HTF was delayed by specific dose of vitamin D3. The inhibitory effect of vitamin D3 (specific dose) on cell proliferation was confirmed via in vitro scratch wound healing assay. The effect of vitamin D3 on shear stress driven HTF may be mediated via the down‐regulation of vitamin D3 and PDGF receptor‐regulated signaling pathway.

Conclusions

Vitamin D3 (specific dose) could inhibit cell proliferation and delay cell cycle progression in shear stress treated HTF in vitro, without noticeable cell toxicity. Our findings suggest that specific dose of vitamin D3 has potential therapeutic applications in glaucoma filtering surgery.

References

1. Aydinlik S, Dere E, Ulukaya E. Induction of autophagy enhances apoptotic cell death via epidermal growth factor receptor inhibition by canertinib in cervical cancer cells. Biochimica et biophysica acta General subjects. 2019.

2. Amoozgar B, Lin SC, Han Y. A role for antimetabolites in glaucoma tube surgery: current evidence and future directions. Curr Opin Ophthalmol. 2016 Mar;27(2):164‐9.

3. Ameeramja J, Panneerselvam L, Govindarajan V, Jeyachandran S, Baskaralingam V,

4. Chen S, Zhu J, Zuo S. 1,25(OH)2D3 attenuates TGF‐β1/β2‐induced increased migration and invasion via inhibiting epithelial–mesenchymal transition in colon cancer cells. Biochemical and Biophysical Research Communications, 2015 Dec; 468 (1–2), 130‐135.

5. Chen YH, Chen CL, Lu DW, Liang CM, Tai MC, Chen JT. Vitamin D3 Inhibits Platelet‐Derived Growth Factor‐Driven Cell Proliferation via Downregulation of N‐Glycosylation in Human Tenon’s Fibroblasts in a Proteasome‐Dependent Manner. PloS one. 2016;11(12):e0168765.

6. Dang Sentao, LU Xiaohe, ZHOU Jin, BAI Lang, Effects of 1a, 25dehydroxyvitamin D3 on the acute immune rejection and corneal neovascularization in high‐risk penetrating keratoplasty in rats, J First Mil Med Univ, 2004;24(8):892‐903

7. De Los Reyes MM, Oyong GG, VA SN, Shen CC, Ragasa CY. Cytotoxic Compounds from Wrightia pubescens (R.Br.). Pharmacognosy research. 2018;10(1):9‐15.

8. Fei Leng, Ping Liu, Hulun Li & Jiaying Zhang. Long‐Term Topical Antiglaucoma Medications Cause Enhanced Tenon’s Capsule Fibroblast Proliferation and Abnormal TGF‐β and MMP Expressions: Potential Effects on Glaucoma Filtering Surgery. Current Eye Research. 2011 Feb:36(4): 301‐309

9. Galina D.KutuzovaaB’Ann T.GabeltbJulie A.KilandbElizabeth A.Hennes‐BeannbPaul L.KaufmanbHector F.DeLucaa.1α,25‐Dihydroxyvitamin D3 and its analog, 2‐methylene‐19‐nor‐(20S)‐1α,25‐dihydroxyvitamin D3 (2MD), suppress intraocular pressure in non‐human primates. J Archives of Biochemistry and Biophysics. 1 February 2012;518(1):53‐60.

10. Kanazawa S, Fujiwara T, Matsuzaki S, Shingaki K, Taniguchi M, Miyata S, et al. bFGF Regulates PI3‐Kinase‐Rac1‐JNK Pathway and Promotes Fibroblast Migration in Wound Healing. PloS one. 2010;5(8):e12228.

11. Lee DE et al. High‐mobility group box protein‐1, matrix metalloproteinases, and vitamin D in keloids and hypertrophic scars. PRS Global Open, 2015 Apr.

12. Marcella Nebbioso, Giuseppe Buomprisco, Antonia Pascarella & Nicola Pescosolido. Modulatory effects of 1,25‐dihydroxyvitamin D3 on eye disorders: A critical review. J Critical Reviews in Food Science and Nutrition. 09 Jun 2015; 57(3) : 559‐565

13. Pandey S, Walpole C, Shaw PN, Cabot PJ, Hewavitharana AK, Batra J. Bio‐Guided Fractionation of Papaya Leaf Juice for Delineating the Components Responsible for the Selective Anti‐proliferative Effects on Prostate Cancer Cells. Frontiers in pharmacology. 2018;9:1319.

14. Perumal E. Tamarind seed coat ameliorates fluoride induced cytotoxicity, oxidative stress, mitochondrial dysfunction and apoptosis in A549 cells. Journal of hazardous materials. 2016;301:554‐65.

15. Przekora A, Zarnowski T, Ginalska G. A simple and effective protocol for fast isolation of human Tenon’s fibroblasts from a single trabeculectomy biopsy ‐ a comparison of cell behaviour in different culture media. Cell Mol Biol Lett. 2017;22:5. Published 2017 Mar 9. doi:10.1186/s11658‐017‐0034‐4

16. Reins, Rose Y., and Alison M. McDermott. Vitamin D: Implications for Ocular Disease and Therapeutic Potential. Experimental eye research 134 Sept. 2018: 101–110.

17. Stahnke, Thomas et al. Suppression of TGF‐Β Pathway by Pirfenidone Decreases Extracellular Matrix Deposition in Ocular Fibroblasts in Vitro. Ed. Carol Feghali‐Bostwick. PLoS ONE Sept. 2018.

18. Silva‐Carvalho R, Silva JP, Ferreirinha P, Leitao AF, Andrade FK, Gil da Costa RM, et al. Inhalation of Bacterial Cellulose Nanofibrils Triggers an Inflammatory Response and Changes Lung Tissue Morphology of Mice. Toxicological research. 2019;35(1):45‐63.

19. Song H, Zhang L, Luo Y, Zhang S, Li B. Effects of collagen peptides intake on skin ageing and platelet release in chronologically aged mice revealed by cytokine array analysis. Journal of cellular and molecular medicine. 2018;22(1):277‐88.

20. Vitamin D Deficiency And Glaucoma.Website: newglaucoma treatments, Dec 1, 2014

21. Wang Y, Wang J, Wei LJ. Biological function and mechanism of lncRNA‐MEG3 in Tenon’s capsule fibroblasts proliferation: By MEG3‐Nrf2 protein interaction. Biomedicine & Pharmacotherapy. 2017 Mar; 87, 548‐554.

22. Wang Q, Li J, Guo H. Transcriptome analysis and discovery of genes involved in immune pathways in Solen strictus (Gould, 1861) under Vibrio anguillarum. Fish & shellfish immunology. 2019.

Clinical trial registration: IRB Reference Number: UW 19‐366

Grant Support: Seed Fund for Basic Research, University Research Committee, The University of Hong Kong