Rho-kinase inhibition reduces subretinal fibrosis

Abstract

Subretinal fibrosis, a consequence of choroidal neovascularization (CNV) in age-related macular degeneration (AMD), leads to irreversible vision loss due to excessive accumulation of extracellular matrix (ECM) proteins and fibrotic scarring. Anti-VEGF therapy can reverse neovascularization, but its effect on fibrosis is relatively limited. To reduce the visual impact of the fibrosis that remains after CNV. Our study investigated the use of ROCK inhibitors, fasudil and belumosudil, to treat subretinal fibrosis after CNV. The results confirmed that levels of key fibrotic markers (TGF-β1, fibronectin, vimentin, α-SMA and pMYPT1) were lower after treatment. IMC provided detailed spatial mapping of protein expression, revealing significant changes in structure and cellular composition before and after the treatment. We found that fasudil and belumosudil are effective in attenuating subretinal fibrosis by modulating the ROCK-signaling pathway, reducing ECM remodeling and attenuating the expression of markers associated with fibrosis. We hope to provide a basis for maximizing clinical benefit, focusing on optimizing dose and timing of treatment, exploring combination therapies for future anti-subretinal fibrosis research.

Fig. 1

ROCK1/2 pathway diagram.

Fig. 2. Area with ROCK1 (red) staining, corrected total cell fluorescence (CTCF) changes between before and after treatment in HDF cells.

A Panels ai-cii depict the expression of ROCK1 in HDF prior to treatment and after ROCK inhibitors treatment. ROCK1 is identified by red staining, actin filaments are marked in green by phalloidin, and cell nuclei are stained blue with DAPI. A yellow dotted outline highlights an enlarged section of the relevant area for detailed examination. Scale bar = 50 µm. B1–3 The change in percentage of cells occupying the ROCK1-stained region before and after ROCK inhibitor treatment. Red indicates ROCK1-stained regions, green denotes phalloidin staining, blue corresponds to DAPI, and gray represents background and other non-stained areas. Each pie chart is calibrated to a total of 100% to represent the entire cellular area. C White represents the vehicle, orange represents the fasudil group, and mint represents the belumosudil group. While the CTCF values for DAPI and phalloidin exhibited minimal, non-significant changes, ROCK1 expression decreased significantly following treatment. D The CTCF value of ROCK1 is significantly reduced after fasudil and belumosudil treatment. E, F1–3, G, H The expression and CTCF value of ROCK2 in HDF before and after treatment. I Changes in the number and morphology of MC3T3 cells before and after ROCK treatment. Actin filaments stained with phalloidin are green and nuclei stained with DAPI are blue. Their corresponding enlarged versions are shown in bi- biii. Scale bar = 50 µm. J Schematic illustration of morphology changes in MC3T3 cells. K MC3T3 cell number/region of interest changes after treatment.

Fig. 3. Temporal changes in laser-induced retinal lesions in mice, assessed using various imaging modalities.

A An overview of the fibrosis model in C57BL/6 mice and the subsequent evaluation timeline. B Weekly serial images collected using AF, FA, and OCT from day 7 to day 49 for the vehicle group and after fasudil and belumosudil treatment. Flatmounts were stained immunohistochemically to visualize CNV and fibrosis at the same time points. Green staining for isolectin B identified CNV regions and red staining for type1 collagen highlighted areas of fibrosis. The lesions had their peripheries marked with yellow dotted lines. C1–2 The volume changes in the lesion area pre- and post-treatment. D1–2 CNV volume changes before and after treatment. E1–2 The proportion of lesions grading classification. F1–2 Fibrosis volume comparison pre- and post-treatment.

Fig. 4. Histology (H&E) and immunofluorescent detection of type I collagen and α-SMA in mouse choroid/retina, pre- vs post-treatment.

A Display of the temporal alterations of fibrotic makers in H&E and fluorescent staining of mouse choroid and retina paraffin sections without- and with-ROCK inhibitor treatment. Red signifies type 1 collagen, green denotes α-SMA, and blue represents DAPI staining. Scale bar = 50 µm. GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, IS = inner segment of photoreceptors, OS = outer segment of the photoreceptors, RPE = retinal pigment epithelium. B The difference in the percentage of stained regions per ROI expressing type1 collagen and α-SMA between the Vehicle and Fasudil groups. C The difference in the percentage of stained regions per ROI expressing type1 collagen and α-SMA between the Vehicle and Belumosudil groups.

Fig. 5. Time-course fluorescence staining of mouse choroid and retina with quantitative results.

A Fluorescence staining images of mouse retina post-laser injury. Red indicates type1 collagen, green represents α-SMA, and blue denotes DAPI. Scale bar = 50 μm. B, C The percentage of stained regions per ROI by α-SMA. D, E The percentage of stained regions per ROI by type1 collagen. Red indicates type1 collagen, green is α-SMA, blue represents DAPI staining, and the gray- bordered blank areas denote background and other unstained regions.

Fig. 6. Western blot images and statistical analysis of the mouse choroid–RPE complex and retina.

A Representative Western blot images of the mouse choroid-RPE complex. Relative expression of antibodies in the fasudil and belumosudil groups in comparison with the vehicle group at different time points. B Representative Western blot images of the mouse retina, relative expression of antibodies in the fasudil and belumosudil groups compared to the vehicle group at different time points.

Fig. 7. IMC-based retinal cell profiling and antibody detection dynamics pre- and post-ROCK inhibition.

A IMC flow chart. B UMAP visualization of cell types in the mouse retina. C Comparative analysis of antibody detection ratios across various cell types pre- and post-Rock inhibitor treatment. Different colors correspond to the annotations in B. D Panels di-div depict the expression distribution images of different antibodies in the retina of normal healthy mouse by IMC, they represent in order: C, LC, Fas, Bel groups. Blue-DNA, yellow-CD44, green-fibronectin, red-vimentin. Panels dv-diii are the corresponding images of di-div. E Variations in cell type distribution in the retina pre- and post-Rock inhibitor treatment. C-Control, LC-Laser control, Fas-Fasudil, Bel-Belumosudil. Different colors correspond to the annotations in B, C. Scale bar = 60 µm.