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. Author manuscript; available in PMC: 2025 May 1.
Published in final edited form as: Vet Ophthalmol. 2023 Sep 1;27(3):238–247. doi: 10.1111/vop.13143

Relative ability of aqueous humor from dogs with and without primary angle-closure glaucoma and ADAMTS10-open angle glaucoma to catalyze or inhibit collagenolysis

Stephanie A Pumphrey 1, Christine D Harman 2, Amanda L Anderson 2, Benjamin Sweigart 3, András M Komáromy 2
PMCID: PMC10904665  NIHMSID: NIHMS1936890  PMID: 37658474

Abstract

Objective:

To compare the ability of aqueous humor (AH) from dogs with primary angle closure glaucoma (CPACG), companion dogs without overt evidence of CPACG, and Beagles with and without ADAMTS10-open angle glaucoma (ADAMTS10-OAG) to catalyze or inhibit collagenolysis.

Animals studied:

17 normal pet dogs, 27 dogs with CPACG, 19 Beagles with ADAMTS10-OAG, and 4 unaffected Beagles.

Procedures:

A fluorescein-based substrate degradation assay was used to assess AH proteolytic capacity. Samples were then assayed using the same substrate degradation assay, with recombinant activated matrix metalloproteinase-2 (MMP-2) added to measure protease inhibition effects.

Results:

For the protease activity assay, relative fluorescence (RF) for AH from normal pet dogs was 13.28 +/− 2.25% of control collagenase while RF for AH from dogs with CPACG was 17.47 +/− 4.67%; RF was 8.57 +/− 1.72% for ADAMTS10-OAG Beagles and 7.99 +/− 1.15% for unaffected Beagles. For the MMP-2 inhibition assay, RF for AH from normal dogs was 34.96 +/− 15.04% compared to MMP-2 controls, while RF from dogs with CPACG was 16.69 +/− 7.95%; RF was 85.85 +/− 13.23% for Beagles with ADAMTS10-OAG and 94.51 +/− 8.36% for unaffected Beagles. Significant differences were found between dogs with CPACG and both normal pet dogs and dogs with ADAMTS10-OAG, and between normal pet dogs and both groups of Beagles.

Conclusions:

AH from dogs with CPACG is significantly more able to catalyze proteolysis and inhibit MMP-2 than AH from normal dogs or dogs with ADAMTS10-OAG. Results suggest that pathogenesis may differ between CPACG and ADAMTS10-OAG.

Keywords: canine, glaucoma, extracellular matrix, matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP)

Glaucoma is a painful, blinding disease that affects many dogs. Treatment outcomes are often disappointing, and enucleation is frequently performed for palliative reasons.1,2 Primary angle closure glaucoma (PACG) is the most common type of primary glaucoma seen in companion dogs.1 Open-angle glaucoma (OAG), the most common primary glaucoma in people, is less common in pet dogs but has been well documented in association with mutations in genes coding for members of the ADAMTS family of proteases in several dog breeds, including Beagles.1,3–7

Elevated intraocular pressure (IOP) is considered just one of many risk factors in people with glaucoma but is a relatively consistent finding in dogs with clinically apparent canine PACG (CPACG) and Beagles with later-stage open-angle glaucoma stemming from a mutation in the ADAMTS10 gene (ADAMTS10-OAG).1 Trabecular meshwork (TM) extracellular matrix (ECM) is thought to be a key regulator of IOP in the healthy eye, with increases in IOP triggering matrix metalloproteinase (MMP) production and downregulation of tissue inhibitors of metalloproteinases (TIMPs), which subsequently leads to ECM remodeling and the maintenance of adequate aqueous humor (AH) outflow.8–12 In human and canine eyes with POAG or PACG, TM ECM accumulates and its biomechanical properties change, eventually resulting in elevated IOP.1,9,13,14 However, given the temporal disconnect between gonioscopically apparent iridocorneal angle abnormalities and the onset of clinical glaucoma in many dogs with CPACG, and the ability of many dogs with complete angle closure to maintain normal IOP for some time with medical management, changes to TM ECM provide an incomplete explanation for IOP increases and eventual treatment failure in CPACG.1,14–20 ECM changes likely affect not only the conventional AH outflow pathway via the TM, but also the uveoscleral outflow pathway, and it may be that it is the final loss of the uveoscleral pathway that ultimately renders an eye with CPACG refractory to medical management.21–24 Importantly, latanoprost and other prostaglandin analogues, the most potent ocular antihypertensives in dogs, are believed to exert their ocular hypotensive effect mostly by modulating ECM in the uveoscleral outflow pathway.1,23,25–27

While an extensive body of literature exists regarding ECM changes in the human glaucomas and, to a certain extent, ADAMTS10-OAG, less is known about the relevance or etiopathogenesis of ECM changes in CPACG.1,13,21,22,28–32 Recent work has linked CPACG in Border Collies to a mutation in OLFML3, a gene coding for an ECM glycoprotein.33 Myocilin, an ECM glycoprotein implicated in human glaucomas, also seems to be increased in the TM and AH of dogs with CPACG.31,32,34 Most importantly, MMPs and TIMPs are believed to be the primary regulators of ECM modification in people, and alterations in MMPs and TIMPs in AH have been shown in people and dogs with PACG.9,35–41 We recently found that multiple MMPs and TIMPs were significantly increased in AH from dogs with CPACG when compared with AH from companion dogs without overt evidence of CPACG, with disproportionate increases in MMP-8 and TIMP-2.42

The functional implication of alterations in MMPs and TIMPs in AH from dogs with CPACG remains unclear, particularly as the most recent study found both MMPs and TIMPs to be increased, which would seem contradictory.42 In addition, the previous study did not seek to differentiate between latent and active AH MMPs, and past work has suggested that increased MMPs in glaucomatous eyes primarily exist in their latent forms.9,38,42–44 Therefore, one goal of the current study was to examine whether AH from dogs with CPACG differs from AH from companion dogs without overt evidence of CPACG in its ability to either catalyze or inhibit proteolysis. Because the degree of similarity in pathogenesis between CPACG and ADAMTS10-OAG is also uncertain, we additionally sought to determine whether AH from Beagle dogs with early ADAMTS10-OAG differs from AH from wild-type and ADAMTS10-heterozygous Beagles or from dogs with CPACG in its ability to catalyze or inhibit proteolysis.

Materials and methods

This study was conducted in accordance with the Guidelines for Ethical Research in Veterinary Ophthalmology (GERVO) and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

CPACG samples

Samples of AH from dogs with CPACG were obtained as part of routine clinical procedures at the Foster Hospital for Small Animals at the Cummings School of Veterinary Medicine at Tufts University. Consent for retention and use of such materials is included in the general hospital treatment consent forms that are reviewed and signed by pet owners. Samples from normal dogs were obtained through a separately consented body donation program at the Foster Hospital, the consent form for which also specifically stipulates that tissue samples may be used for research.

Aqueous humor was obtained from dogs with CPACG during therapeutic interventions, including aqueocentesis, glaucoma valve placement, intravitreal gentamicin injection, or enucleation. Eyes were excluded that had previously undergone aqueocentesis, intravitreal gentamicin injection, valve placement, or transscleral or endoscopic laser cyclophotocoagulation. All dogs diagnosed with CPACG were examined by a board-certified veterinary ophthalmologist or ophthalmology resident and received a clinical diagnosis of CPACG based on IOP >25 mmHg and iridocorneal angle narrowing or closure in the absence of signs of other potentially causative ocular disease. Angle morphology was evaluated via gonioscopy in the contralateral normotensive eye (when present) and/or via histopathology (with the understanding that histopathologic assessment of angle morphology in chronic glaucoma can be unreliable).45 Aqueous humor was obtained and stored in accordance with previously described protocols.42 Patient data collected included age, sex, breed, IOP, and current medication regimen, with particular attention paid to topical prostaglandin analogues and topical or systemic anti-inflammatory medications.

Aqueous humor was also obtained from euthanized dogs whose owners elected body and tissue donation for research and teaching purposes. Dogs selected for AH collection had grossly normal eyes aside from age-related changes, no history of cancer or systemic infectious or inflammatory diseases, and no recent history of administration of anti-inflammatory or immunomodulatory medications. A board-certified veterinary ophthalmologist performed a brief gross ocular examination immediately following euthanasia. Gonioscopy and IOP measurement were not performed. Aqueous humor collection and handling were performed as for dogs with CPACG and occurred within 30 minutes of euthanasia. Data collected included patient age, sex, and breed.

ADAMTS10-OAG

Aqueous humor was additionally obtained from purpose-bred Beagles kept at Michigan State University. This sampling was approved by the Michigan State University Institutional Animal Care and Use Committee (IACUC). The antiemetic ondansetron (0.5–1.0 mg/kg; Aurobindo Pharma USA, Inc.) was given orally 2–4 hours before the procedure. The dogs were sedated intravenously with a combination of butorphanol tartrate (0.2–0.4 mg/kg; Bayer HealthCare LLC), midazolam (0.2–0.4 mg/kg; Avet Pharmaceuticals, Inc.), and dexmedetomidine HCl (0.5 μg/kg; Zoetis, Inc.). The ocular surface was prepped aseptically with diluted povidone-iodine solution (0.2%; Medline Industries, Inc.) and anesthetized with proparacaine HCl 0.5% ophthalmic solution (Akorn, Inc.). Sampling (100–200 μL) was performed via limbal aqueous paracentesis by tunneling a 30G needle of an insulin syringe (BD Insulin Syringes with BD Ultra-Fine™ needle) through the sclera and cornea. Following sample collection, neomycin-polymyxin B-dexamethasone 0.1% ophthalmic ointment was applied (Bausch & Lomb). The nonsteroidal anti-inflammatory carprofen was administered subcutaneously (4.4 mg/kg; Zoetis, Inc.), and dexmedetomidine HCl sedation was reversed by intramuscular administration of atipamezole HCl (0.5 mg/kg; Zoetis, Inc.). Samples were flash-frozen in liquid nitrogen, kept frozen at −80C, and then shipped on dry ice.

Aqueous humor was obtained from Beagles with ADAMTS10-OAG (homozygotes), ADAMTS10 carrier Beagles (heterozygotes), and wild-type Beagles. Genotypes were confirmed based on ADAMTS10 gene sequence: ADAMTS10-OAG affected dogs were homozygous and carriers were heterozygous for the G661R missense mutation responsible for OAG in Beagles, whereas the normal dogs were homozygous for the wild-type allele.3,46 Data collected included age at the time of sampling, sex, and most recent IOP measurement.

Evaluation of proteolysis and proteolysis inhibition

Global AH protease activity was measured using a commercially available assay (EnzChek Gelatinase/Collagenase Assay Kit, Thermo Fisher Scientific). In this assay, quenched fluorescein-conjugated gelatin releases detectable fluorescent peptides only after protease digestion. The assay was performed according to manufacturer instructions. Aqueous humor samples were diluted 1:5 in the provided reaction buffer, while fluorescein-conjugated gelatin was diluted to 20 μg/mL. Diluted AH (100 μL per well) and gelatin (20 μL per well) were loaded in microplate wells along with reaction buffer (80 μL per well), and the plate was allowed to incubate at room temperature for 24 hours. Clostridial collagenase at a concentration of 0.2 U/mL (100 μL per well plus 80 μL of reaction buffer and 20 μL of diluted gelatin) served as a positive control and was allowed to incubate for 30 minutes. Fluorescence was measured in a microplate reader with absorption and emission parameters set at 485 nm and 538 nm, respectively. All samples were run in duplicate due to small sample volumes, and mean values were used for further analysis. Mean fluorescence from wells loaded with 180 μL of reaction buffer and 20 μL of gelatin but no AH (i.e., blanks) was subtracted from sample and control values to account for background fluorescence, and fluorescence was then expressed as a percentage of that produced by the positive control.

Protease inhibitor activity was assessed using the same assay kit following the manufacturer’s recommended protocol for measurement of protease inhibitors. Purified active recombinant MMP-2 (Abcam) was used in place of the clostridial collagenase supplied with the kit, as preliminary work showed that AH did not inhibit the latter but did inhibit the former, which appears to be a primary target for IOP-triggered upregulation in nonglaucomatous eyes.9,11 Aqueous humor and gelatin were diluted as for the previous assay, and samples were again run in duplicate. Each well was loaded with 80 μL diluted AH, 20 μL diluted gelatin, and 100 μL MMP-2 diluted to 2.5 μg/mL. MMP-2 without added AH (100 μL diluted MMP-2, 80 μL reaction buffer, and 20 μL diluted gelatin per well) was used as a control. The plate was incubated for 15 minutes, and fluorescence was measured in a microplate reader as described above. Similarly, background fluorescence was subtracted, and fluorescence was expressed as a percentage of that produced by the control.

Data were assessed for normality, and a Welch’s t-test was used to compare relative fluorescence for each assay for dogs with and without CPACG. Results were also compared for each assay between Beagles with ADAMTS10-OAG, ADAMTS10-heterozygous Beagles, and wild-type Beagles using an ANOVA, and between Beagles with ADAMTS10-OAG and a pooled grouping of ADAMTS10-heterozygous and wild-type Beagles using a t-test. Finally, results were compared for each assay between dogs with CPACG and Beagles with ADAMTS10-OAG, and between clinically normal dogs and pooled ADAMTS10-heterozygote and wild-type Beagles, again using a t-test. Differences between groups were considered significant if P<0.05. All analyses were conducted using SAS Enterprise Guide software, Version 8.3 Update 3 (SAS Institute Inc., Cary, NC).

Results

CPACG samples

Aqueous humor was obtained from 27 eyes with CPACG of 27 dogs. Median age was 10.3 years (range 3.6–16.3 years). Mean IOP was 54.9 +/− 15.5 mmHg. The population included 15 spayed female dogs and 12 castrated male dogs. Breeds represented by multiple individuals included Shih Tzu (4), Cocker Spaniel (4), Shiba Inu (2), and Siberian Husky (2). Eighteen of 27 (66%) eyes were being treated with latanoprost at the time of sampling, while 11 of 27 (41%) eyes were being treated with a topical steroid (either 1% prednisolone acetate or dexamethasone/neomycin/polymyxin b) and 8 of 27 (30%) eyes were being treated with topical 0.5% ketorolac tromethamine (drug manufacturers varied over time for all medications). Only 5 dogs with CPACG were not receiving any topical medication at the time of sampling.

Aqueous humor was also obtained from 17 eyes of 17 dogs without overt evidence of CPACG (one eye was chosen at random from each dog). Median age was 10 years (range 3–16 years). This population included 7 spayed female dogs, 9 castrated male dogs, and one intact male dog. Ten different breeds were represented, including 2 Labrador Retrievers and 2 French Bulldogs, and 5 dogs were listed as mixed breed.

ADAMTS10-OAG samples

Aqueous humor was obtained from 37 eyes of 19 Beagles with ADAMTS10-OAG. Median age was 1 year (range 0.9–4.3 years). Mean IOP was 19.7 +/−3.6 mmHg. This population included 7 intact female dogs and 12 intact male dogs. Aqueous humor was also obtained from 3 eyes of 2 ADAMTS10-heterozygous Beagles (aged 1.8 years and 5.3 years, both intact females) and 4 eyes of 2 wild-type Beagles (both aged 0.9 years, both intact males). No dogs were receiving topical glaucoma medications or anti-inflammatories prior to sampling.

Additional detail on the study populations is provided in Supplemental Table 1.

Assay results - CPACG

Mean relative fluorescence for the proteolysis assay was 17.47 +/− 4.67% for AH from eyes with CPACG and 13.28 +/− 2.25% for AH from eyes without overt evidence of CPACG. (Figure 1) As increased fluorescence indicates increased proteolysis, this finding shows that more proteolysis occurs in the presence of AH from dogs with CPACG than in the presence of AH from normal dogs. The difference between groups was statistically significant (P=0.0004).

Figure 1.

Figure 1.

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Proteolytic ability of AH samples incubated with gelatin substrate. Proteolytic ability is expressed as a percentage of the fluorescence measured from control wells containing clostridial collagenase without AH. Increased fluorescence indicates increased breakdown of substrate (i.e. more protease activity). Significant differences were found between normal dogs and dogs with CPACG (*, P=0.0004), between dogs with CPACG and Beagles with and without ADAMTS10-OAG (‡,∫, both P<0.0001), and between normal pet dogs and Beagles with and without ADAMTS10-OAG (†, ˄, both P<0.0001). No difference was found between Beagles with and without ADAMTS10-OAG.

Mean relative fluorescence for the MMP-2 inhibition assay was 16.69 +/− 7.95% for AH from eyes with CPACG and 34.96 +/− 15.04% for AH from normal eyes (Figure 2). For this assay, increased fluorescence again indicates increased proteolysis, but in the presence of activated MMP-2 less fluorescence is seen in samples that included AH from dogs with CPACG than in samples that included AH from normal dogs. These findings show that AH from both normal dogs and dogs with CPACG inhibits MMP-2, but that AH from dogs with CPACG can do so to a greater degree. The difference between groups was again statistically significant (P=0.0001).

Figure 2.

Figure 2.

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MMP-2 inhibitory ability of AH samples incubated with gelatin substrate and MMP-2. MMP-2 inhibitory ability is expressed as a percentage of the fluorescence measured from control wells containing MMP-2 without AH. Decreased fluorescence indicates decreased breakdown of substrate (i.e. more inhibition of MMP-2). Significant differences were found between normal dogs and dogs with CPACG (*, P=0.0001), between dogs with CPACG and Beagles with and without ADAMTS10-OAG (‡,∫, both P<0.0001), and between normal pet dogs and Beagles with and without ADAMTS10-OAG (†, ˄, both P<0.0001). No difference was found between Beagles with and without ADAMTS10-OAG.

Assay results – ADAMTS10-OAG

Mean relative fluorescence for the proteolysis assay was 8.57 +/− 1.72% for Beagles with ADAMTS10-OAG, 7.59 +/− 1.26% for ADAMTS10-heterozygous Beagles, 8.29 +/− 1.15% for wild-type Beagles, and 7.99 +/− 1.15% for pooled heterozygotes and wild-type Beagles (Figure 1). The difference between groups was not statistically significant (P=0.60 for three-group comparison; P=0.40 for pooled comparison).

Mean relative fluorescence for the MMP-2 inhibition assay was 85.85 +/− 13.23% for Beagles with ADAMTS10-OAG, 90.38 +/− 10.01% for ADAMTS10-heterozygous Beagles, 97.61 +/− 6.56% for wild-type Beagles, and 94.51 +/− 8.36% for pooled heterozygotes and wild-type Beagles (Figure 2). The difference between groups was not statistically significant (P=0.21 for three-group comparison; P=0.10 for pooled comparison).

Comparative results – CPACG and ADAMTS10-OAG

Aqueous humor from dogs with CPACG was significantly more able to catalyze proteolysis and significantly more able to inhibit MMP-2 than aqueous humor from dogs with ADAMTS10-OAG (P<0.0001). Aqueous humor from pet dogs without overt evidence of CPACG was also significantly more able to catalyze proteolysis and inhibit MMP-2 than AH from ADAMTS10-heterozygote and wild-type Beagles (P<0.0001).

Discussion

Results of the present study show that AH from dogs with CPACG differs from AH from pet dogs without overt evidence of CPACG in its ability to catalyze proteolysis and also in its ability to inhibit MMP-2. Aqueous humor from dogs with CPACG induced more breakdown of a gelatin substrate than AH from normal dogs, but it also significantly inhibited MMP-2-mediated breakdown of the same substrate. Although both findings were statistically significant, the latter was much greater in magnitude. Aqueous humor from Beagles with ADAMTS10-OAG did not exhibit the same properties.

These findings appear somewhat contradictory but may suggest that proteolysis inhibition is an important process in the context of CPACG and could be a worthy target for further investigation and potential interventions. In particular, it seems possible that the proteolytic capacity of AH from dogs with CPACG should be higher than what was measured in this study based solely on the increases in AH MMPs documented in the previous study and elsewhere in the literature. However, in the assay used here, the inhibitors of MMPs (i.e., TIMPs) are inherently present in the samples, and their effect overrides and obscures what would otherwise be a much greater MMP effect. Because MMP enhancement has received more attention than TIMP suppression as a therapeutic strategy for glaucoma, these findings, which suggest that TIMP dysregulation might be the more powerful force in TM ECM changes in CPACG, could have important implications for future work.23 Regulation of TIMPs is poorly understood, but several candidate drugs, including caffeine, omeprazole, statins, and Rho kinase inhibitors, have been shown to inhibit TIMP function or decrease TIMP expression in cell cultures and other models.47–52

Two significant limitations of this in vitro study are the use of a simple gelatin substrate to stand in for the complex arrangement of the ECM and the use of MMP-2 as a sole representative protease for inhibition. Both of these factors may have influenced the findings. In the eye, TM ECM plays multiple roles, from providing structural support to facilitating cell signaling. It is comprised of a diverse array of molecules, including not only collagen but proteoglycans, elastin, laminin, fibronectin, fibrillin, and hyaluronic acid.13 Other components of TM ECM may differ in their susceptibility to AH proteases or MMP-2, and thus the results of the current study, which used only gelatin (degraded collagen) as a substrate, may not accurately reflect these processes in vivo.

Similarly, MMP-2 is not the only protease present in the AH, and it may not be the most relevant protease in the glaucomatous eye. MMP-2 was selected for this study as it is a ubiquitous protease and has previously been shown to be upregulated in the glaucomatous eye in both people and dogs with PACG.38–40,42,53,54 It is possible that other proteases (such as MMP-8, which showed the greatest increase in glaucoma in the previous study, or the ADAMTS family of proteases) are differentially upregulated in CPACG to compensate for inhibition or insufficient upregulation of MMP-2.3–7,42 TIMP-2 is considered a broad-spectrum protease inhibitor, but it is possible that the magnitude of its effects varies across proteases.56 The use of MMP-2 as a representative protease in this study may therefore overstate the inhibitory capacity of AH from eyes with CPACG. In addition, both the use of a gelatin substrate and the choice of MMP-2 as a representative protease could account for some portion of the differences between AH from eyes with CPACG and AH from eyes with ADAMTS10-OAG, as different ECM components and different proteases may be involved in the pathogenesis of the latter disease.

Drug effects may have also influenced findings for eyes with CPACG. The majority of eyes with CPACG sampled for this study were being treated with both latanoprost and a topical anti-inflammatory. Prostaglandin analogues such as latanoprost are theorized to produce their hypotensive effect by upregulating MMPs and inducing greater uveoscleral outflow.23,25–27 In cell culture models, these compounds increase both MMPs and TIMPs, but in glaucomatous eyes, the same effect is not seen, with some studies instead reporting decreases in MMPs and TIMPs in treated eyes.23,25,26,57–60 Similarly, topical steroids are thought to induce TM ECM deposition by decreasing MMP expression, but the linkage between steroid use and increased IOP appears more tenuous in dogs than in people, and no information exists regarding the effects of steroids on MMPs in the canine eye.61–66 Very little has been published regarding the effects of steroids on TIMP expression, with most studies noting minimal effects, and the literature is almost devoid of information on the impact of systemic or topical NSAIDs on MMPs or TIMPs on components of the eye other than the ocular surface.64,67–69 Nevertheless, it seems probable that drug exposure in eyes with CPACG had some effect on results, and more work is needed to evaluate the effects of commonly used topical glaucoma medications and anti-inflammatories on MMPs and TIMPs in dogs.

Perhaps most importantly, AH from eyes with CPACG was obtained from eyes with late-stage disease. In most cases, interventions that involved AH retrieval were elected because the eye was failing medical management and/or was considered permanently nonvisual. Therefore, it is unclear whether this study’s findings represent part of the pathogenesis of disease or the consequences of chronic IOP elevation, vascular dysregulation, inflammation, or other changes accompanying CPACG.1 Interestingly, however, recent work in people has demonstrated that AH from individuals with both acute and chronic angle closure contains similar levels of MMPs and TIMPs (and that these levels are elevated in comparison with normal eyes or eyes with POAG).70 Regardless of their cause, findings of the current study may provide justification for sampling of dogs in earlier stages of disease and may also contribute to the development of new therapies for dogs with CPACG in later stages of disease. For example, elucidating changes in AH in later stages of disease could lead to improved outcomes for glaucoma filtration surgeries in later-stage patients, since failure of filtering blebs may be linked to constant exposure to profibrotic mediators like TIMPs in glaucomatous AH.13,41,71,72

Eyes with ADAMTS10-OAG, on the other hand, were sampled relatively early in the course of disease (mean IOP in the group was 19.7 mmHg, as compared with 54.9 mmHg for dogs with CPACG). These dogs, along with heterozygote and wild-type counterpart Beagles, were markedly younger than the pet dogs with and without CPACG sampled for the other arm of the study. It is possible that sampling dogs with ADAMTS10-OAG at later stages of disease (ie. higher IOP) may yield different results, particularly as changes in biomechanical properties of the eye have previously been correlated with age and duration of disease in dogs with ADAMTS10-OAG.73 Breed-specific factors may also have influenced results, since all ADAMTS-OAG dogs were Beagles as were their wild-type and homozygote comparators. However, it is also possible that differences in pathogenesis between CPACG and ADAMTS10-OAG are the cause of the disparity in results, particularly since changes in AH MMPs and TIMPs do not seem to be consistent across different types of glaucoma in people.39,70 If eyes with ADAMTS10-OAG do not undergo the same MMP- and TIMP-modulated ECM modification as eyes with CPACG or glaucomatous human eyes, this could partially explain the lack of effect of netarsudil on IOP in dogs with ADAMTS10-OAG.74 Netarsudil, a Rho kinase inhibitor, is a potent antihypertensive drug in human eyes and is thought to work via multiple mechanisms, one of which involves blunting transforming growth factor beta-induced changes in TM ECM mediated by MMPs and TIMPs.50 The effect of netarsudil on IOP in eyes with CPACG has not been studied and may still be worth investigating despite the apparent failure of this drug in eyes with ADAMTS10-OAG.

The difference in age between groups may also explain the differences noted between pet dogs without overt evidence of CPACG and ADAMTS10-heterozygote and wild-type Beagles. Age is thought to affect ECM via multiple mechanisms.13,24,75 Some of the changes in proteolysis and MMP-2 inhibition seen in the older normal pet dogs relative to their younger Beagle counterparts could reflect normal homeostatic responses to aging ECM.75 It is also possible that the postmortem status of the normal pet dogs could have influenced AH parameters, although every effort was made to sample AH as quickly as possible following euthanasia. Reagents were from the same lot number across assays, assays were performed by a single investigator, and relative fluorescence of samples was calculated using within-plate blank and control wells to limit interassay variability. Laboratory error or differences in reagents were therefore considered less likely sources for the differences between pet dogs and purpose-bred Beagles.

Areas for further investigation include evaluation of the interaction between AH in dogs with CPACG and other MMPs such as MMP-8, observation of the effects of AH from dogs with CPACG and ADAMTS10-OAG in a more holistic environment like a perfused anterior chamber model, and sampling of the lamina cribrosa in dogs with and without CPACG and ADAMTS10-OAG, to determine whether similar alterations in proteolysis may also affect the optic nerve head and retina. Additional study is also needed to ascertain the effects of commonly used drugs such as latanoprost and corticosteroids on MMPs, TIMPs, and other ECM-related parameters in normal and glaucomatous canine eyes. Finally, because processes affecting ECM likely affect not only the TM but also other portions of the anterior segment, more work should be done to improve our understanding of uveoscleral and other unconventional aqueous humor outflow pathways in the canine eye.

Results of this study suggest that AH from dogs with CPACG has a mildly enhanced ability to catalyze proteolysis of gelatin substrate and a more pronounced ability to inhibit MMP-2-induced breakdown of gelatin substrate when compared to AH from unaffected pet dogs. When coupled with previous work showing disproportionate increases in TIMP-2 relative to other MMPs and TIMPs in eyes with CPACG compared to normal eyes,42 these results support continued investigation of inhibition of ECM proteolysis as a component of CPACG. The same changes were not seen in AH from dogs with ADAMTS10-OAG, suggesting that different types of canine primary glaucoma might not share a common pathogenesis. Further comparative work is needed to elucidate the similarities and differences between CPACG and ADAMTS10-OAG.

Supplementary Material

Supinfo

Acknowledgements

The authors thank the staff of Michigan State University Campus Animal Resources for their technical assistance, Dr. Vicky Yang for assistance with assay optimization, and Kady Marino and the emergency and critical care doctors and nursing staff at the Cummings School of Veterinary Medicine at Tufts University for their assistance with obtaining samples from donated dogs. Parts of this study were supported by NIH grants R01-EY025752 and R01-EY032478, and BrightFocus grant G2022007S, and by funds from the Department of Clinical Sciences at the Cummings School of Veterinary Medicine at Tufts University.

Footnotes

Conflict of interest statement

The authors declare no conflicts related to this study. Dr. Komáromy received research funding from PolyActiva Pty. Ltd. and CRISPR Therapeutics while the presented work was conducted. He also serves as a consultant for Reichert Technologies and Editor-in-Chief of Veterinary Ophthalmology.

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