Effects of hyperglycemia on the TGF-β pathway in trabecular meshwork cells

Hyperglycemia, which can lead to apoptosis, hypertrophy, fibrosis, and induces hyperinflammation in diabetic vascular complications due to oxidative stress. In order to elucidate the potential dual roles and regulatory signal transduction of TGF-β1 and TGF-β2 in human trabecular meshwork cells (HTMCs), we established an oxidative cell model in HTMCs using 5.5, 25, 50, and 100 mM d-glucose-supplemented media and characterized the TGF-β-related oxidative stress pathway.

Further analysis was conducted to investigate oxidative damage and protein alterations in the HTMC caused by the signal transduction. This was done through a series of qualitative cell function studies, such as cell viability/apoptosis analysis, intracellular reactive oxygen species (ROS) detection, analysis of calcium release concentration, immunoblot analysis to detect the related protein expression alteration, and analysis of cell fibrosis to study the effect of different severities of hyperglycemia. Also, we illustrated the role of TGF-β1/2 in oxidative stress-induced injury by shRNA-mediated knockdown or stimulation with recombinant human TGF-β1 protein (rhTGF-β1).

Results from the protein expression analysis showed that p-JNK, p-p38, p-AKT, and related SMAD family members were upregulated in HTMCs under hyperglycemia. In the cell functional assays, HTMCs treated with rhTGFβ-1 (1 ng/mL) under hyperglycemic conditions showed higher proliferation rates and lower ROS and calcium levels.

To summarize, mechanistic analyses in HTMCs showed that hyperglycemia-induced oxidative stress activated TGF-β1 along with its associated pathway.

While at low concentrations, TGF-β1 protects cells from antioxidation, whereas at high concentrations, it accumulates in the extracellular matrix, causing further HTMC dysfunction.