A Novel Therapeutic Agent for Modulating Oxidative Stress-Induced Ocular Fibroblast Activity

Authors: Victoria Leung¹, Anastasiya Vinokurtseva¹, James J. Armstrong¹, Hong Liu¹, Eric K. Chin², David R. P. Almeida³, Cindy M. L. Hutnik¹. ¹Department of Ophthalmology, Schulich School of Medicine and Dentistry, ²Loma Linda University Eye Institute, ³Erie Retinal Surgery.

Author Disclosures: V. Leung: None. A. Vinokurtseva: None. J.J. Armstrong: None. H. Liu: None. E.K. Chin: Any direct financial payments including receipt of honoraria; Name of for-profit or not-for-profit organization(s); Alimera Sciences, Allergan. Funded grants or clinical trials; Name of for-profit or not-for-profit organization(s); Bayer, Chengdu Kanghong Biotechnology, Genentech, Hexal AG and Sandoz Inc, Iveric, Kodiak Sciences Inc, Novartis, Oculus, Opthea, Valo Health, Inc. Patents on a drug, product or device; Name of for-profit or not-for-profit organization(s); Citrus Therapeutics. D.R.P. Almeida: Any direct financial payments including receipt of honoraria; Name of for-profit or not-for-profit organization(s); ACYLERIN, ALCON, ALIMERA SCIENCES, ALLERGAN/ABBVIE, BAUSCH + LOMB, BAYER, BOEHRINGER INGELHEIM, CITRUS THERAPEUTICS, CLINICAL TRIALS NETWORK, EYEPOINT PHARMACEUTICALS, GENENTECH, GYROSCOPE THERAPEUTICS, NOVARTIS, OCUGEN, OPTHEA, REGENERON, REGENXBIO, ROCHE, SAMSARA VISION, SPHERIX CONSULTING GROUP. C.M.L. Hutnik: None.


Abstract Body: 

Purpose: Oxidative stress is a significant factor in the two most common causes of irreversible blindness, namely age-related macular degeneration (ARMD), and glaucoma. In both diseases, this stress triggers fibrosis which contributes to the ultimate visual demise. CTZ1 is a novel carbazole-nicotine analog combination molecule designed in silico with antioxidant properties and the potential to reduce fibrosis. The purpose of the current study was to assess whether CTZ1 will mitigate the effects of experimental oxidative stress in fibroblasts by salvaging ROS-impaired cellular metabolic activity, reducing cellular cytotoxicity and inhibiting TGFb1-induced fibrosis. 

Study Design: In vitro experimental. 

Methods: Primary cultures of human Tenon’s Capsule fibroblasts (HTCF) were prepared. A dose-response curve for the oxidative stressor tert -butyl hydroperoxide (t-BOOH) was determined via an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Next, the non-cytotoxic dose range of CTZ1 was determined using a lactate dehydrogenase (LDH) assay. Afterward, the efficacy of CTZ1 at mitigating the aberrant metabolic activity and cytotoxicity induced by t-BOOH was assessed using MTT and LDH assays respectively. Expression of contractile protein alpha-smooth muscle actin (α-SMA) was assessed in fibroblasts treated with pro-fibrotic cytokine TGFb1 (2ng/ml), t-BOOH and CTZ1 via Western Immunoblotting. 

Results: t-BOOH was found to induce a 40% reduction in metabolic activity at 1 mM for 1 hour, and CTZ1 was determined to be safe and effective over a dose range between 10-100 μM for 1-hour exposure. Both co-treatment and post-treatment with CTZ1 were able to reverse the effects of t-BOOH. Western blot analysis revealed that treatment with t-BOOH or with TGFb1 in HTCF increased α-SMA expression, while the addition of CTZ1 at 100uM to t-BOOH or TGFb1 reduced expression of α-SMA. 

Conclusions: Data demonstrate that CTZ1 may reduce the effects of experimental oxidative stress in human Tenon’s Capsule fibroblasts by salvaging ROS-impaired metabolic activity and decreasing cell proliferation, as well as counteracting TGFb1-induced pro-fibrotic myofibroblast activity. These data support the strong potential for CTZ1 to mitigate the pathophysiology of oxidative stress.

X

X