Tetramethylpyrazine Attenuates Transdifferentiation of TGF-β2–Treated Human Tenon's Fibroblasts

Abstract

Citation: Cai X, Yang Y, Chen P, et al. Tetramethylpyrazine attenuates transdifferentiation of TGF-b2-treated human Tenon's fibroblasts. Invest Ophthalmol Vis Sci. 2016;57:4740-4748. DOI:10.1167/iovs.16-19529 PURPOSE. To investigate the pharmacologic effect of tetramethylpyrazine (TMP) on human Tenon's fibroblasts (HTFs), the cells implicated in scarring after filtration surgery. METHODS.Transforming growth factor-b2 (TGF-b2) was used to stimulate a fibrotic phenotype in primary HTFs, and the influence of TMP on the fibrotic phenotype was assessed. Cell proliferation and cell cycle regulation were profiled. Immunofluorescence staining tracked proliferating cell nuclear antigen (PCNA) expression. Transwell assays monitored cell migration. Flow cytometry measured TMP toxicity. In addition, in TGF-b2-treated HTFs, Western blot and immunofluorescence were employed to assess the expression of a-smooth muscle actin (a-SMA). The TMP-mediated activity on cytoskeletal arrangements and extracellular matrix (ECM) accumulation in HTFs was evaluated using actin polymerization and Western blot assays. Moreover, TGF-b-dependent activation of Smad3 and p38 was examined by Western blot analysis. RESULTS.In TGF-b2-treated HTFs, TMP reduced proliferation and migration but did not induce apoptosis. Moreover, TMP attenuated expression of a-SMA and suppressed stress fiber formation stimulated by profibrotic cytokine; it also counteracted TGF-b2-induced cytoskeletal rearrangements, morphologic changes, and ECM accumulation. Smad3 and p38 mitogenactivated protein kinase (MAPK) signaling were downstream of the TMP-sensitive effect.CONCLUSIONS. Tetramethylpyrazine counteracts TGF-b2-mediated myofibroblast transdifferentiation and attenuates ECM component deposition and cell proliferation in HTFs, implicating TMP as a potential antifibrosis agent in glaucoma filtration surgery.

Essential role of connective tissue growth factor (CTGF) in transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation from Graves’ orbital fibroblasts

“…TGF-β1 has been shown to stimulate myofibroblast differentiation, extracellular matrix synthesis, or fibrosis in corneal-fibroblast 25 conjunctival fibroblasts 26 , 27 , human retinal pigment epithelial cell 28 , and orbital fibroblasts 29 . TGF-β2 has also been reported to induce expression of extracellular matrix and/or fibrosis in cultured astrocytes of the human optic nerve head 30 , human Tenon’s fibroblasts 31 , and trabecular meshwork 32 . Current study revealed TGF-β1, but not TGF-β2, could induce CTGF, fibronectin, and α-SMA in Graves’ orbital fibroblasts.…”

M2 macrophages promote subconjunctival fibrosis through YAP/TAZ signalling

“…Primary cultures of HTFs were established as previously described [ 28 , 29 ]. Briefly, Tenon’s capsule samples were cut into small pieces and cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12, Solarbio) containing 10% FBS (Gibco, Gaithersburg, MD) and 1% penicillin/streptomycin.…”

Secreted protein, acidic and rich in cysteine is crucial for the vascular endothelial growth factor-stimulated fibrotic process in human Tenon’s fibroblasts

“…Small biopsied Tenon's capsule specimens were isolated from five patients when they underwent a strabismus surgery. After being washed, the tissues were cultured for preparation of HTFs, as described previously 11 . The HTFs were cultivated in Dulbecco's Modified Eagle's medium (DMEM) containing 10% of fetal bovine serum (FBS) and the cells were passaged every 3-5 days.…”