Hepatocyte Growth Factor Confers Trabecular Meshwork Stem Cell Resilience and Paracrine Protection of Trabecular Meshwork Cells in Glaucoma

Purpose: We have reported that trabecular meshwork stem cells (TMSCs) are effective for trabecular meshwork (TM) regeneration. This study aims to investigate different responses of TMSCs and TM cells to TGF-β2 and protective factors in TMSCs. Methods: Human TMSCs and TM cells were exposed to TGF-β2 with or without antioxidant N-acetylcysteine, TMSC-derived secretome (TMSC-Scr), hepatocyte growth factor (HGF), or HGF receptor inhibitor (HGFI). Cell viability was assessed by the MTT assay. Intracellular reactive oxygen species (ROS) and lipid peroxidation were measured using CellROX and C11-BODIPY, respectively. Mitochondrial membrane potential was assessed using tetramethylrhodamine methyl ester (TMRM). Fibrotic markers (fibronectin, α-SMA, CTGF) were evaluated by immunofluorescent staining and Western blotting. RNA sequencing was performed to compare transcriptional profiles between TMSCs and TM cells. A cytokine array was used to compare TMSC-Scr and TM-Scr. A t-test or ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis, with significance defined as P < 0.05. Results: Compared with TM cells, TMSCs exhibited greater survival, better-preserved mitochondrial function, and lower levels of ROS, lipid peroxidation, and fibrotic marker expression after TGF-β2 treatment. N-acetylcysteine partially prevented TGF-β2 effects on TM cells. RNA sequencing and cytokine analyses identified significant enrichment of HGF in TMSCs and TMSC-Scr. TMSC-Scr and HGF markedly reduced oxidative and fibrotic responses to TGF-β2 in TM cells, whereas an HGFI abolished this protective effect. Conclusions: Our findings confirm that TMSC-Scr and HGF are promising candidates for cell-free therapies in glaucoma. HGF confers intrinsic resilience in TMSCs and functions as a key, but partial, mediator of their protection of TM cells.