Anti-fibrotic activity of a rho-kinase inhibitor restores outflow function and intraocular pressure homeostasis
- Department of Ophthalmology, Duke University, United States
- Department of Biomedical Engineering, Georgia Institute of Technology/Emory University, United States
- Department of Mechanical Engineering, Georgia Institute of Technology, United States
- Aerie Pharmaceuticals, Inc, United States
- Department of Biomedical Engineering, Duke University, United States
- Washington State University Floyd Elson School of Medicine, United States
Figures
Figure 1 with 1 supplement
Netarsudil (NT) efficaciously lowered intraocular pressure (IOP) in patients with steroid-induced elevated IOP poorly controlled with standard glaucoma medications.
IOPs were measured by Goldmann applanation tonometry in patients who demonstrated ocular hypertension (OHT) after steroid treatment for a variety of ocular conditions (Table 1). These individuals …
Figure 1—figure supplement 1
Time course of netarsudil (NT) effects on intraocular pressure (IOP) in patients with steroid-induced elevated IOP poorly controlled with standard glaucoma medications.
IOPs were measured by Goldmann applanation tonometry in patients who demonstrated ocular hypertension (OHT) after steroid treatment for a variety of ocular conditions (Table 1). These individuals …
Figure 2 with 1 supplement
Netarsudil (NT) prevented and reversed steroid-induced ocular hypertension by improving outflow function.
(A) In the prevention study, intraocular pressure (IOP) was measured in two groups of age- and gender-matched wild-type C57BL/6 mice receiving NT or placebo (PL) unilaterally by subconjunctival …
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Figure 2—source data 1
Source data for IOPs and outflow facilities in Figure 2.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Netarsudil (NT) reversed dexamethasone-loaded nanoparticle (DEX-NP)-induced intraocular pressure (IOP) elevation even after 3 months.
Two groups of age- and gender-matched wild-type C57BL/6 mice were treated unilaterally with DEX-NPs twice per month for 3 months. During the last week, eyes were treated with NT or placebo (PL) once …
Figure 3 with 3 supplements
Netarsudil (NT) reduced steroid-induced trabecular meshwork (TM) stiffening visualized in living mice by spectral domain-optical coherence tomography (SD-OCT) and estimated by inverse finite element modeling (iFEM) and measured directly by atomic force microscopy (AFM).
Wild-type C57BL/6 mice received dexamethasone-loaded nanoparticles (DEX-NPs) bilaterally for 4 weeks. During the last week, mice received either NT or placebo (PL) unilaterally for 4 consecutive …
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Figure 3—source data 1
Source data for segmentation of area of SC lumen in Figure 3.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig3-data1-v1.xlsx
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Figure 3—source data 2
Source data for FEM in Figure 3.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig3-data2-v1.xlsx
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Figure 3—source data 3
Source data for AFM measurements in Figure 3.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Netarsudil (NT) enhanced intraocular pressure (IOP)-induced collapse of Schlemm’s canal (SC) lumen in ocular hypertensive eyes as visualized by spectral domain-optical coherence tomography (SD-OCT).
Mice were treated with dexamethasone-loaded nanoparticles (DEX-NPs) in both eyes for 4 weeks and unilaterally with NT or placebo (PL) for 4 consecutive days. On day 5, living mouse eyes were …
Figure 3—figure supplement 2
Quantitative comparison of normalized Schlemm’s canal (SC) lumen areas in netarsudil (NT) and placebo (PL) treatment groups at five clamped intraocular pressures (IOPs) (10, 12, 15, 17, and 20 mmHg).
This figure is similar to Figure 3G, but also includes results from eyes with dexamethasone-loaded nanoparticle (DEX-NP) treatment without administration of either NT or PL (DEX-NP, red) and from …
Figure 3—figure supplement 3
A representative cryosection showing the limbal region during AFM measurements of TM stiffness, acquired on a tissue sample immersed in PBS.
AFM, atomic force microscopy; PBS, phosphate buffered saline; SC, Schlemm’s canal; TM, trabecular meshwork. Section thickness 10 µm.
Figure 4 with 1 supplement
Netarsudil (NT) decreased fibrotic markers in ocular hypertensive mouse eyes.
Two groups of age- and gender-matched wild-type C57BL/6 mice had dexamethasone-loaded nanoparticles (DEX-NPs) delivered bilaterally 1–2 times per week for 4 weeks to create ocular hypertension. …
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Figure 4—source data 1
Source data for quantification of FN in Figure 4.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig4-data1-v1.xlsx
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Figure 4—source data 2
Source dat for quantification of SMA in Figure 4.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Quantitative analysis of fibronectin (FN) and alpha-smooth muscle actin (αSMA) expression in trabecular meshwork (TM).
Wild-type C57BL/6 mice were injected subconjunctivally/periorbitally with dexamethasone-loaded nanoparticles (DEX-NPs), delivered bilaterally 1–2 times per week for 3 weeks to induce ocular …
Figure 5 with 1 supplement
Netarsudil (NT) partially normalized ultrastructure of conventional outflow tissues in ocular hypertensive eyes.
Two groups of age- and gender-matched wild-type C57BL/6 mice were bilaterally treated with dexamethasone-loaded nanoparticles (DEX-NPs) 1–2 times per week for 4 weeks. During the last week of …
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Figure 5—source data 1
Source data for semi-quantitative scoring of JCT in Figure 5.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig5-data1-v1.xlsx
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Figure 5—source data 2
Source data for quantitative measurements of BMM in Figure 5.
- https://cdn.elifesciences.org/articles/60831/elife-60831-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Quantification of basement membrane material (BMM) underlying the inner wall of Schlemm’s canal.
Shown is a representative example of the quantification approach that was performed on 97 images. The lengths of the continuous BMM (in red) and the total length of the inner wall (in blue) were …
Figure 6
Schematic representation of a feed-forward model of fibrotic disease in the conventional outflow pathway responsible for ocular hypertension, incorporating a number of pathophysiological aspects of ocular hypertension (Stamer and Acott, 2012, Schmidl et al., 2015).
The broader literature suggests that this feed-forward loop can be triggered by multiple factors, including aging, oxidative stress, or genetic predisposition. In this work we triggered this loop by …
Tables
Table 1
Netarsudil (NT) effectively lowered steroid-induced ocular hypertension in patients whose IOP was not well controlled with standard glaucoma medications.
| Patient/ eye | Age (years) | Gender | Race | Ocular condition | Steroid type | Glaucoma medications | IOP before NT (mmHg) | IOP < 1 month after NT (mmHg) |
|---|---|---|---|---|---|---|---|---|
| Cohort 1 | ||||||||
| 1/OD | 50 | M | AA | POAG | Durezol | Brim, Cos | 23 | 16 |
| 2/OD | 74 | F | AA | CACG | PF | Apra, Meth, Lat | 33 | 22 |
| 3/OD | 27 | M | HIS | TG | PF | Bet, Brim, Dor; Lum | 36 | 28 |
| 4/OD | 66 | F | CAU | Uveitis | Retisert | Cos, Brim | 17 | 9 |
| 5/OS | 76 | F | CAU | POAG | PF | Lum, Cos | 17 | 11 |
| 6/OS | 53 | M | CAU | SR | Ozurdex | Com, Dor | 31 | 12 |
| 7/OS | 74 | F | CAU | SR | PF | Cos, Brim | 25 | 16 |
| 8/OS | 61 | F | AA | Uveitis | Durezol | Cos, Brim, Lat | 34 | 26 |
| 9/OD | 81 | M | UNK | POAG | PF | Cos, Brim, Lat | 25 | 19 |
| 9/OS | 20 | 16 | ||||||
| 10/OS | 51 | F | CAU | Uveitis; SR | PF; Fluti | Cos, Brim | 26 | 15 |
| 11/OS | 78 | F | CAU | POAG; SR | Oral Pred | Lat, Tim, Dor, Dmx | 22 | 12 |
| 12/OD | 76 | F | CAU | SIG | Lotemax | Brim, Cos, Vyzulta | 28 | 20 |
| 12/OS | 27 | 19 | ||||||
| 13/OD | 62 | F | CAU | SIG | PF | Tim, Dor, Lat | 21 | 15 |
| 14/OD | 78 | M | AA | POAG; SR | PF | Com, Dor, Lat | 30 | 13 |
| 15/OS | 72 | F | CAU | LTG; SIG | PF | Brim, Lat | 15 | 13 |
| 16/OS | 83 | F | AA | Glaucoma | PF; Retisert | Com, Lat | 21 | 18 |
| 17/OD | 76 | M | CAU | POAG; SIG | Durezol | Cos, Brim | 12 | 10 |
| 18/OS | 83 | F | CAU | POAG; SIG | PF | Cos, Brim | 29 | 18 |
| 19/OD | 49 | F | CAU | POAG; SIG | Lotemax | Cos, Brim | 18 | 17 |
| 66.8 | 6M/13F | 2.7 ± 0.7 | 24.3 ± 6.6 | 16.4 ± 4.9 | ||||
| Cohort 2 | ||||||||
| 20/OS | 60 | M | CAU | Uveitis | Ivt. Dex | Com, Lat | 60 | 44 |
| 21/OS | 92 | F | CAU | SR | Ozurdex | Lat, Alph, Dor, Tim | 17 | 12 |
| 22/OD | 77 | F | CAU | SR | Oral Pred | Lat | 24 | 19 |
| 22/OS | 24 | 18 | ||||||
| 23/OD | 65 | F | CAU | SR | Ozurdex | Lum | 30 | 26 |
| 23/OS | 33 | 22 | ||||||
| 24/OD | 16 | F | CAU | PCG | Topical Pred | Tim, Azo, Lat | 21 | 16 |
| 25/OD | 67 | M | CAU | POAG | Topical Pred | Lat, Azo, Bet | 28 | 18 |
| 26/OS | 3d | M | CAU | POAG | Oral Pred | Tim, Lat, Alph | 44 | 32 |
| 27/OD | 90 | M | CAU | POAG | Topical Pred | Azo, Lat | 22 | 16 |
| 27/OS | 22 | 16 | ||||||
| 58.4 | 4M/4F | 2.4 ± 1.1 | 26.5 ± 7.7 | 19.5 ± 5.8 | ||||
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IOP, intraocular pressure; OD, right eye; OS, left eye; M, male; F, female; AA, African American; HIS, Hispanic; CAU, Caucasian; UNK, unknown; POAG, primary open-angle glaucoma; CACG, chronic angle-closure glaucoma; TG, traumatic glaucoma; OHT, ocular hypertension; PCG primary congenital glaucoma; PF=Pred Forte; Dex=Dexamethasone; Pred=Prednisone; Ivt=intravitreal; Brim=Brimonidine; Com=Combigan; Cos=Cosopt; Apra=Apraclonidine; Meth=Methazolamide; Lat=Latanoprost, Bet=Betaxolol; Dor=Dorzolamide; Lum=Lumigan; Com=Combigan; Alph=Alphagan; Tim=Timolol; Azo=Azopt. IOPs of patient 10/OS were statistically determined to be outliers and the numbers are removed from the analysis.
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Software, algorithm | Schlemm3_2 | PMID:30651311 | Version 3.0 | Schlemm’s canal segmentation |
| Software, algorithm | FEBio | https://febio.org/ | Version 3.0 | Finite element modeling |
| Antibody | Anti-αSMA (rabbit polyclonal) | Abcam, Cambridge, MA | ab5694 | IF (1:100) |
| Antibody | Anti-FN (mouse monoclonal) | Santa Cruz, Dallas, TX | sc8422 | IF (1:50) |
Additional files
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Supplementary file 1
Estimated pressures within Schlemm’s canal (SC) lumen as a function of clamped intraocular pressure (IOP) levels using two-series resistor model of conventional outflow pathway described previously.
- https://cdn.elifesciences.org/articles/60831/elife-60831-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60831/elife-60831-transrepform-v1.pdf
