Fig 2: Remodelling of ECM components in SCEC and HTM cell monolayers. Immunocytochemistry shows various remodelling artefacts on core ECM components in SCEC and HTM cells in response to MMP-3 treatment. (A,B) Collagen IV appears to have reduced intensity in both cell types after treatment. Collagen IV is concentrated around cells in controls but shows reduced spread after treatment, fibrils barely protruding past the cell nuclei. (C) Alpha smooth muscle fibres extend the width of the cell towards a neighbouring cell. Treated samples show that these fibre bundles have constricted, leading to multiple thin connections between cells. (D) HTM F-actin staining depicts a slight thinning of filament bundles and a reduction of filament branching post MMP-3 treatment. (E,F) Laminin expression exhibits a modest reduction in staining intensity in both cell types, and a reduction in network complexity in TM cells. (G,H) Fibronectin was visualised after decellularisation, depicting linear and organised strands in PBS controls, as denoted by an asterisk. Treatment groups lacked a linear network, and instead showed a disjointed, porous network. Scale bars represent 50 μm. Left column pairs = SCEC, right column pairs = HTM.

Fig 3: Transmission electron microscopy (TEM) analysis of ECM remodelling in outflow tissues. Semi-thin sections of the iridocorneal angle in mouse eyes treated with either (A) AAV-Null or (B) AAV-MMP-3. AAV-MMP-3 treated eyes show greater inter-trabecular spaces in outer trabecular meshwork (TM) than controls. Scale bar denotes 50 μm. (C,D) Transmission electron micrograph of the inner wall of Schlemm’s Canal (SC) and the outer TM. (C) Control eye illustrating normal attachment between foot-like extensions of the inner wall endothelium and subendothelial cells (arrowheads), as well as with the discontinuous basement-membrane material underlying the inner wall endothelium (arrows). (D) Representative TEM image of an MMP-3 treated eye showing a disconnection of the inner wall endothelium from the subendothelial cells and the ECM (arrowheads). The widened subendothelial region lacks basement-membrane material and other ECM components. (E,F) Higher magnification of the inner wall of a treated eye. (E) Foot-like extensions of the inner wall endothelium (E) have disconnected from the subendothelial cells and the ECM (arrowheads), and the lack of ECM in this region is shown. (F) In other regions of treated eyes, clumps of presumably degraded ECM-material are localised underneath the inner wall of SC (asterisk). Such clumps of ECM are not present in controls. Scale bars are denoted on each image. CB = ciliary body, I = iris, R = retina. (G). Morphometric measurements of the optically empty space immediately underlying SC from four regions of contralateral eyes treated with AAV-MMP-3 (red data points) or AAV-Null (blue data points). Bars indicate average values for each eye. Contralateral eyes are presented immediately next to one another.

Fig 4: Effect of recombinant human MMP-3 on paracellular permeability in HTM and SCEC cell monolayers. SCEC and HTM cells were treated with 10 ng/ml recombinant MMP-3 for 24 h, using PBS and inactivated MMP-3 (incubation with TIMP-1, MMP(−)) as vehicle and negative controls respectively. (A) SCEC and (B) HTM both show reductions in TEER values after treatment of 4.6 [2.9, 6.2] and 5 [2.2, 7.8] Ohms.cm2 respectively. Permeability to a 70 kDa dextran was increased in treated cells (MMP (+)) in both (C) SCEC and (D) HTM. (E) An average viability of 85% was expected for SCEC with MMP-3 concentrations up to 36 ng/ml. (F) 85% viability is retained on average in HTM cells at concentrations up to 151 ng/ml MMP-3. A, C and E in blue represent SCEC data, whereas B, D and F in red represent HTM data.

Fig 5: Effect of ECM remodelling on outflow facility and IOP. (A) ‘Cello’ plot depicting individual outflow facility values for eyes at 8 mmHg (Cr) and statistical distribution of both control (AAV-Null) and experimental (AAV-MMP-3) groups. Each point represents a single eye with 95% CI on Cr. Log normal distribution is shown, with the central white band showing the geometric mean and the thinner white bands showing two geometric standard deviations from the mean. The shaded region represents the 95% CI on the mean. (B) Paired outflow facility plot. Each inner point represents an eye pair, with log-transformed facilities of the control eye plotted on the x axis, and treated eye on the y axis. Outer blue and green ellipses show uncertainties generated from fitting the data to a model, intra-individual and cannulation variability respectively. Average increase is denoted by the red line, enclosed by a grey 95% CI, indicating significantly increased facility (does not overlap the blue unity line). (C) Box plots showing the change in IOP in treated and control eyes. Boxes show interquartile range and error bars represent the 5th and 95th percentiles. A significant reduction in IOP is observed in AAV-MMP-3 treated eyes (Wilcoxon signed-rank test). (D-E) Cello and paired facility plots for inducible AAV data sets.