Assistant Professor University of California, Riverside, United States
Introduction:: Introduction: Primary open-angle glaucoma (POAG) is the second leading cause of irreversible blindness worldwide. While the etiology of glaucoma is complex, dysfunction of the human trabecular meshwork (HTM) is a known contributor. As humans age, the HTM and its cells become more fibrotic, senescent, and apoptotic; additionally, there is increased oxidative stress. These changes are exacerbated in POAG. In both POAG and normal aging TM cells also suffer from several forms of mitochondrial damage, including damage to mitochondrial proteins, in particular Complex I. Mitochondrial DNA is known to become damaged as well, manifesting as oxidized bases (e.g. 8-Oxo-2'-deoxyguanosine), the mitochondrial common deletion, and a decrease of mitochondrial DNA per cell. In the current work, we sought to quantify the metabolic impacts of dexamethasone (Dex) and transforming growth factor β2 (TGF-β2), commonly used to induce glaucomatous phenotypes in HTM. Measurements include the rate of ATP production and the proportion of ATP from oxidative phosphorylation (mitochondria) versus glycolysis. We additionally assessed basal respiration, ATP-linked respiration, mitochondrial spare capacity, proton leak, and non-mitochondrial oxygen consumption.
Materials and Methods:: Materials and Methods: All work involving human tissue was performed in a manner consistent with the Declaration of Helsinki. Primary cultures of HTM cells were isolated from donor human corneoscleral rims deemed unsuitable for transplant (Saving Sight, St. Louis, MO) as previously described. Briefly, the iris and ciliary body were removed from the corneoscleral rim, allowing dissection of the meshwork. The meshwork was then carefully dissected out nd placed with 0.2% Cytodex beads (Sigma-Aldrich, St. Louis, MO) in supplemented media. Cells that migrated out of the tissue were maintained in the aforementioned media and cultured normally. All cultures used were confirmed as HTM by examining myocilin mRNA (MYOC) upregulation in response to a 3-day 100 nM Dex treatment. Equivolume treatments of ethanol were used as vehicle controls. Cells were seeded in six wells of a Seahorse XFp miniplate (Agilent Technologies, Santa Clara, CA) at 30,000 to 50,000 cells per well and allowed to attach for 24 h. Three wells were treated with 100 nM of Dex for 72 hrs, and three wells were treated with equivolume ethanol as vehicle control. Similarly, in plates treated with TGF-β2 (Peprotech, Cranbury, NJ), three wells were treated with 1 ng/ml for 72 hours was used and equivolume of water vehicle control. After 72 hours the cell metabolic activity was assayed on a Seahorse XFp (Agilent Technologies, Santa Clara, CA) using either the Mito Stress Test Kit or the Real-Time ATP Rate Assay according to manufacturer instructions.
Results, Conclusions, and Discussions:: Results and Discussion: HTM cells treated with 100 nM Dex for 72 h were assayed using the ATP Rate Assay. In absolute terms, there was a decrease in ATP production from glycolysis (92.2 ± 9.5 pmol/min/104 cells) compared to the vehicle control (150.0 ± 36.5 pmol/min/104 cells). This was also the case for ATP produced by mitochondria, which after Dex treatment was lower (49.1 ± 6.6 pmol/min/104 cells) than vehicle control (98.0 ± 18.9 pmol/min/104 cell). These values are consistent with data from similar cell types. Further, the ratio of glycolysis to oxidative phosphorylation is consistent with prior results in calf and human TM showing approximately one quarter to one third of ATP generation is from mitochondrial respiration. In relative terms, there was statistically significant decrease in overall ATP production, in Dex treated cells with levels 60% ± 7.6% of control (p = 0.0349). This decrease came both glycolysis and mitochondria. ATP from glycolysis was 66.5% ± 10% of control (p = 0.083) while a greater proportion of the decrease in ATP came from mitochondria, with levels 51% ± 5.4% of control (p = 0.0123). In contrast, we found no statistically significant or consistent effect from treating HTM cells with 1 ng/mL TGF-β2, although both Dex and TGF-β2 have been linked to similar glaucomatous phenotypes. Additionally, we assayed Dex treated HTM cells using the Mito Stress Test, which further allows quantification of maximal respiration (achieved by uncoupling ATP synthesis from electron transport using the protonophore FCCP). Maximal respiration for control cells was 120.13 ± 26.8 pmol/min/104, which decreased after Dex treatment to 74.9 ± 17.2 pmol/min/104 cells (p = 0.048). The observed decrease in maximal respiration is consistent with mitochondrial damage previously observed in POAG. These studies further highlight mitochondria as a potential druggable target in POAG.
Acknowledgements (Optional): : Acknowledgements: The authors thank the University of Riverside Stem Cell Core facility, especially Dr. Rachel Behar, for technical support and equipment access. This work was partially supported by the National Science Foundation under Grant No. CAREER 2046093. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. This work was partially supported by a Grant-In-Aid from The Glaucoma Foundation.
