Counteracting subconjunctival scarring from glaucoma surgery by ALK5 inhibition

Authors: Jack E. Teplitsky , Anastasiya Vinokurtseva, James J. Armstrong, James Denstedt, Hong Liu, Cindy M. L. Hutnik. University of Western Ontario.

Author Disclosures: J.E. Teplitsky: None. A. Vinokurtseva: None. J.J. Armstrong: None. J. Denstedt: None. H. Liu: None. C.M.L. Hutnik: None.


Abstract Body: 

Purpose: Subconjunctival glaucoma surgeries, such as microinvasive bleb-forming surgeries and trabeculectomies, are often reserved for patients whose intraocular pressure (IOP) is not adequately controlled with maximally tolerated medical therapy. Unfortunately, these surgeries have a suboptimal long-term success rate with failure secondary to aberrant postsurgical ocular scarring resulting in IOP elevation. The chemotherapeutic agent Mitomycin C (MMC) was introduced in the 1980s to reduce surgical failure rates. Unfortunately, its off-target cytotoxic effects on surrounding ocular tissue are known to be associated with a number of complications. To find a safer alternative to MMC we investigated the novel usage of the TGFβ1 ALK5 receptor inhibitor, SB-431542 (SB) in a primary culture of human Tenon’s capsule fibroblasts (HTCFs). This molecule is known to prevent the downstream promotion of fibrosis-associated gene expression. We hypothesize that SB will have comparable anti-fibrotic efficacy to MMC while being significantly less cytotoxic. 

Study Design: In vitro experimental. 

Methods: HTCFs embedded in a 3D collagen lattice were pre-treated with 20 μM SB or 0.2 mg/mL MMC for varying durations and then treated with TGFβ1 for 72 hours to measure collagen contraction and assess cellular viability by LIVE/DEAD staining. HTCFs were also cultured in 2D and similarly pre-treated with SB or MMC, followed by TGFβ1 exposure for 48 hours to immunoblot for EDA splice variant fibronectin (EDA-FN), matrix metallopeptidase-9 (MMP 9) and alpha smooth muscle actin (α-SMA) protein expression. The LDH and MTT assays in 2D culture were used to assess HTCF cell death and cellular metabolism, respectively. 

Results: Treatment with TGFβ1 alone significantly increased collagen contraction and protein expression of EDA-FN, MMP-9 and α-SMA. Pre-treatment of TGFβ1-induced HTCFs with SB or MMC resulted in a significant lowering of collagen contraction and EDA-FN, MMP-9 and α-SMA protein expression. SB had no significant effect on cellular viability, cell death and cellular metabolism in HTCFs. In contrast, MMC significantly increased cell death and significantly lowered cellular viability and cellular metabolism in HTCFs. 

Conclusions: Our in vitro study demonstrates that SB has comparable anti-fibrotic effects on HTCFs, while being a significantly safer alternative to MMC.

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