TGFβ1-SMAD 2 and 3 Pathway Inhibition Leads to Inflammatory Attenuation in Preclinical Model of surgical Aqueous Outflow Tract

Authors: Matthew Fung1, James Armstrong1, Hong Liu2, Cindy Hutnik1. 1Schulich School of Medicine, 2Lawson Health Research Institute.

Author Disclosure Block: M. Fung: None. J. Armstrong: None. H. Liu: None. C. Hutnik: None.

Abstract Title: TGFβ1-SMAD 2 and 3 Pathway Inhibition Leads to Inflammatory Attenuation in Preclinical Model of surgical Aqueous Outflow Tract

Abstract Body:

Purpose: Surgically created outflow tracts lead to subconjunctival exposure to surgical inflammation and endogenous proinflammatory cytokines in the aqueous. These factors lead to post-operative fibrosis resulting in impaired outflow and eventual surgical failure. Although surgical modalities have improved over recent decades, interventions to control post-surgical inflammation-driven fibrosis have not followed suit. Current practice utilizes non-specific cytotoxic agents with limited therapeutic windows, such as mitomycin C. Therefore, there is a need for new surgical adjuvants that can more safely and consistently address post-operative inflammation-driven fibrosis. The Transforming Growth Factor Beta (TGFβ1)/SMAD2/3 pathway has been implicated in fibroblast transformation into its inflammatory myofibroblast phenotype, which is believed to contribute to surgical failure. Previously, we reported on a perfused in vitro 3D cell culture model of the subconjunctival outflow tract. Resistance to perfusion through the 3D cell culture could be approximated via pressure readings (mmHg) of the perfusate afferent to the 3D cell culture chamber. The aim of the present report is to compare the effects within this model of TGFβ1, an aqueous humor growth factor thought to negatively impact surgical outcome and a small molecule inhibitor of the TGFβ1 pathway. Study Design: Subconjunctival Human Tenon’s Capsule Fibroblasts (HTCFs) were treated with exogenous TGFβ1 to represent the pro-inflammatory subconjunctival milieu following surgery. Verteporfin, an inhibitor of the TGFβ1-SMAD2/3 pathway was used to attenuate the pro-inflammatory effects of TGFβ1. TGFβ1 was initially compared with vehicle control with subsequent experiments comparing the effects of TGFβ1 to TGFβ1 with verteporfin. Real-time monitoring of afferent perfusion pressure served as an analogue for intra-ocular pressure. Methods: HTCFs were cultured inside a 3D collagen matrix within a microfluidics chamber. Experimental groups were determined by perfusate additives: 1) culture media, 2) culture media with TGFβ1 ( 2ng/mL), or 3) culture media with TGFβ1 (2ng/mL) and verteporfin (20μM/L). Culture media was perfused through the HTCF-containing collagen matrix at 2.6 μL/min. A pressure transducer was placed afferent to the HTCF-containing matrix and recorded in real-time over 78 hours. Slides were then stained with DAPI and F-actin molecular probes. Afferent perfusion pressure curves were compared between groups using repeated measures ANOVA and morphology was compared semi-quantitatively on microscopy.

Results: TGFβ1 lead to significantly increased mean afferent perfusion pressure and induced significant cytoskeletal expansion versus control. Verteporfin attenuated the effects of TGFβ1 on afferent perfusion pressure and cellular cytoskeleton. Conclusions: TGFβ1 is found in the aqueous humor and associated with glaucoma surgery failure. This model demonstrates the role of TGFβ1 on the pressure generated by a perfused 3D collagen culture of HTCFs and how its impact can be mitigated by verteporfin. These findings can be leveraged to support further investigation of verteporfin as a post-operative glaucoma surgery adjuvant as well as to test other inhibitors for pre-clinical development.