Overexpression of ALK5 Induces Human Tenon’s Capsule Fibroblasts Transdifferentiation and Fibrosis In Vitro

ABSTRACT

Purpose: To investigate the involvement of activin receptor-like kinase 5 (ALK5) in human Tenon’s capsule fibroblasts (HTFs) transdifferentiation and fibrosis.

Methods: (1) Cultured HTFs were treated with transforming growth factor beta 1 (TGF-β1) at different concentrations for different durations, mRNA expression of ALK5 and plasminogen activator inhibitor-1 (PAI-1) was measured by quantitative polymerase chain reaction (PCR) while protein expression of ALK5, α-smooth muscle actin (α-SMA), and extracellular matrix deposition including fibronectin (FN) and collagen I (Col1) was assessed by western blot. HTFs with or without TGF-β1 were also treated with an ALK5 activity inhibitor, SB-431542, and fibrosis-related genes were assessed.

(2) HTFs were transduced with ALK5 lentivirus (ALK5-OE group) or empty lentivirus (NC-OE) with or without the treatment of SB-431542. Protein expression of ALK5, α-SMA, FN, and Col1 was evaluated.

(3) HTFs in the ALK5-OE group and NC-OE group were subjected to a scratch-wound assay and their migratory activities assessed.

Results: (1) TGF-β1, in a concentration-dependent manner, upregulated ALK5 and PAI-1 expressions in the HTFs, which peaked between 24 and 36 h. These changes were associated with increases in protein levels of FN, Col1, and α-SMA. These TGF-β1 effects were blocked by the ALK5 inhibitor SB-431542.

(2) Similarly, overexpression of ALK5 by lentiviral vector significantly increased protein expression of α-SMA, FN, and Col1. Addition of TGF-β1 to the ALK5-OE cells did not produce additional expression of any of the marker proteins. The upregulation of extracellular matrix and α-SMA can be reduced by SB-431542.

(3) In ALK5-OE group, HTFs migration was significantly increased compared with normal control and TGF-β1 could still promote ALK5-OE cells migration.

Conclusions: Our findings suggest that ALK5 is an important mediator of HTFs fibrosis. ALK5 is a potential therapeutic target to suppress scar formation after filtration surgery.

Keywords:

• ALK5
• extracellular matrix
• human Tenon’s capsule fibroblasts
• subconjunctival fibrosis
• TGF-β
• transdifferentiation

Acknowledgments

We would like to thank Professor Ji-ming Zhang, Department of Infectious Disease, Huashan Hospital, Fudan University, for expert advice on the experimental procedure.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Funding

This work has been supported by the National Natural Science Foundation of China (Grant No. 30901651).

Additional information

Funding

This work has been supported by the National Natural Science Foundation of China (Grant No. 30901651).