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

AbstractPurposeThe 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.MethodsThe 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.ResultsA 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.ConclusionsVitamin 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.References1. 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. 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