PhD Scientific Days 2023

Budapest, 22-23 June 2023

Molecular Sciences II.

Fluvoxamine reduces TGF-β2-induced fibrotic changes in trabecular meshwork cells

Timea Medveczki* 1,2, Minh N. Tran* 1,2, Balazs Besztercei3, Gyorgy Torok4, Illes Kovacs5,6, Attila J. Szabo2, Andrea Fekete 1,2, Judit Hodrea 1,2

1 MTA-SE Lendület “Momentum” Diabetes Research Group, Semmelweis University, Budapest;
2 Pediatric Center, MTA Center of Excellence, Semmelweis University; Budapest;
3 Institute of Clinical Experimental Research, Semmelweis University, Budapest;
4 Department of Biophysics and Radiation Biology, Semmelweis University, Budapest;
5 Department of Ophthalmology, Semmelweis University, Budapest;
6 Department of Ophthalmology, Weill Cornell Medical College, New York, NY
* these authors contributed equally to this work

Text of the abstract

Introduction: Trabecular meshwork (TM) is the main pathway of aqueous humor drainage. Fibrotic-like remodeling of the actin cytoskeleton in TM cells results in extracellular matrix protein accumulation, increased stiffness and impaired outflow. These are the primary causes of increased intraocular pressure, which is the main risk factor of glaucoma, however, the exact pathomechanism is not known. The level of transforming growth factor-β2 (TGF-β2), a main driver of eye fibrosis, is increased in the aqueous humor of glaucoma patients. Sigma-1 receptor (S1R) was shown to be protective in the retina, however, its role in the TM region is unknown. Recently, we proved that the specific S1R agonist fluvoxamine (FLU) is antifibrotic in the kidney.

Aims: Here we investigated the effects of FLU on the TGF-β2-induced fibrotic response in cultured human trabecular meshwork (HTM) cells and mice anterior segment (AS).

Methods: Fibrosis of the AS was induced in C57BL/6J mice by intracameral TGF-β2 injection. AS were collected and F-actin was visualized with Phalloidin and detected with confocal microscopy. The effect of FLU on TGF-β2-induced cell proliferation, cytoskeletal rearrangement, and fibrosis-related protein levels was investigated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, Phalloidin staining and Western blotting respectively.

Results: F-actin enhancement was observed upon TGF-β2 injection in vivo, indicating fibrotic-like changes. In vitro, TGF-β2 induced cytoskeletal rearrangement with increased F-actin- bundles and clumps formation (p<0.001) that were reduced by FLU (p<0.001). Furthermore, FLU also inhibited cell proliferation (p<0.05) in TGF-β2-treated cells. The profibrotic protein levels of connective tissue growth factor, fibronectin, collagen type IV, and alpha-smooth muscle actin were all elevated by TGF-β2 treatment (p<0.001), while the S1R agonist prevented the elevation of these fibrosis elements (p<0.01; p<0.001). TGF-β2 also reduced the expression of Matrix Metalloproteinas-2 (p<0.05), which was inhibited when TGF-β2 was combined with FLU (p<0.05).

Conclusions: FLU may reduce the fibrotic response of trabecular meshwork and could be a potential candidate for the treatment of fibrosis-induced ocular hypertension.

Grants: OTKA- K135398, LP2021-3/2021, TKP2021-EGA-24, Stipendium Hungaricum