Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Structures of netarsudil-family ROCKi test compounds. Color code used throughout all figures.

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques:

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Experimental design. HTM hydrogels were treated with vehicle control (=baseline) or TGFβ2 for 5 d to induce a glaucoma-like cell phenotype (=induction) before adding netarsudil-family ROCKi test compounds for the next 5 d in absence of TGFβ2 (=induction + rescue), with fresh media supplied every 2-3 days.

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Control

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Dose response effects of netarsudil-family ROCKi treatment following TGFβ2-induction on HTM hydrogel contraction. Representative brightfield micrographs of HTM hydrogels encapsulated with HTM07 subjected to (A) netarsudil, (C) compound A, (E) compound B, (G) compound C, or (I) compound D over a broad dose range for 10 d, with Y27632 serving as refence control (dashed lines outline original construct size at 0 d). Scale bars, 1 mm. Construct size quantification of HTM hydrogels subjected to (B) netarsudil, (D) compound A, (F) compound B, (H) compound C, or (J) compound D (N = 3-8 replicates per group). (K) ROCKi dose response curves with calculated EC 50 values. Data shown as Mean ± SD with individual data points. Significance was determined by one-way ANOVA using multiple comparisons tests (*p<0.05, ***p<0.001, ****p<0.0001; ns, not significant).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Control, Construct

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Effects of netarsudil-family ROCKi treatment at EC 50 following TGFβ2-induction on HTM hydrogel contraction. Representative brightfield micrographs of HTM hydrogels encapsulated with (A) HTM05, (C) HTM07, or (E) HTM36 subjected to the different treatments for 10 d, with max level netarsudil serving as refence control (dashed lines outline original construct size at 0 d). Scale bars, 1 mm. Construct size quantification of HTM hydrogels encapsulated with (B) HTM05, (D) HTM07, or (F) HTM36 (N = 3-8 replicates per group). (G) Construct size quantification of HTM hydrogels encapsulated with HTM05 (purple), HTM07 (maroon), or HTM36 (gray). Data shown as Mean ± SD with individual data points. Significance was determined by one- or two-way ANOVA using multiple comparisons tests (*p<0.05, ***p<0.001, ****p<0.0001; ns, not significant; # p<0.0001 vs. all other groups).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Control, Construct

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Effects of netarsudil-family ROCKi treatment at EC 50 following TGFβ2-induction on HTM cell F-actin stress fibers within ECM hydrogels. Representative confocal fluorescence micrographs of F-actin in HTM hydrogels encapsulated with (A) HTM05, (C) HTM07, or (E) HTM36 subjected to the different treatments for 10 d (F-actin = green). Scale bar, 200 μm. Quantification of relative F-actin signal intensity in HTM hydrogels encapsulated with (B) HTM05, (D) HTM07, or (F) HTM36 (N = 4 replicates per group). (G) Pooled quantification of relative F-actin signal intensity in HTM hydrogels encapsulated with HTM05 (purple), HTM07 (maroon), or HTM36 (gray) (N = 4 replicates per group and HTM cell strain). Data shown as Mean ± SD with individual data points. Significance was determined by one- or two-way ANOVA using multiple comparisons tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant; # p<0.01 vs. Ctrl, TGFβ2, netarsudil, and compounds A-C).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Fluorescence

Journal: Frontiers in Ophthalmology

Article Title: Effects of Netarsudil-Family Rho Kinase Inhibitors on Human Trabecular Meshwork Cell Contractility and Actin Remodeling Using a Bioengineered ECM Hydrogel

doi: 10.3389/fopht.2022.948397

Figure Lengend Snippet: Effects of netarsudil-family ROCKi treatment at uniform netarsudil-EC 50 following TGFβ2-induction on HTM hydrogel contraction. Representative brightfield micrographs of HTM hydrogels encapsulated with (A) HTM07 or (C) HTM36 subjected to the different treatments for 10 d (dashed lines outline original construct size at 0 d). Scale bars, 1 mm. Construct size quantification of HTM hydrogels encapsulated with (B) HTM07 or (D) HTM36 (N = 3-4 replicates per group). (E) Construct size quantification of HTM hydrogels encapsulated with HTM07 (maroon) or HTM36 (gray). Data shown as Mean ± SD with individual data points. Significance was determined by one- or two-way ANOVA using multiple comparisons tests (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant).

Article Snippet: In addition to the clinically-used netarsudil, two sets of experimental ROCKi compounds related to netarsudil were selected and provided by Aerie Pharmaceuticals Inc. One set included two compounds that performed similarly or better than netarsudil in Aerie’s in vitro screening but did not perform as well in intraocular pressure-lowering studies using normotensive Dutch Belted rabbits (unpublished data): compounds A and C. The other set included two compounds that did not perform as well as netarsudil both in vitro and in vivo : compound B and D. First, we investigated the specific inhibitory activity of the different netarsudil-family ROCKi compounds against ROCK1 and ROCK2 together with their ability to disrupt HTM cell focal adhesions.

Techniques: Construct