Increased Matrix Metalloproteinases 1, 2, and 3 in the Monkey Uveoscleral Outflow Pathway After Topical Prostaglandin F2α–Isopropyl Ester Treatment FREE

TOPICAL TREATMENT with prostaglandin F_2α–isopropyl ester (PGF_2α-IE) lowers intraocular pressure (IOP) in monkey eyes by increasing uveoscleral outflow.^{1 - 3} Similarly, IOP reduction in human eyes treated with the PGF_2αanalog latanoprost also is due to increased uveoscleral outflow.⁴ Early experiments^{5 - 6} in monkeys revealed that topical PGF_2α-IE leads to a widening of the interstitial spaces among ciliary muscle fiber bundles. These interstitial spaces usually contain aqueous humor⁷ and an extracellular matrix composed of collagen types I and III, fibronectin, elastin, and other constituents.^{8 - 9} The boundary of these spaces is defined by basement membrane surrounding the smooth muscle cells and containing collagen type IV and laminin.^{8 ,10} Studies^{9 ,11} have shown reductions of collagen types I, III, and IV in the ciliary muscle of PGF_2α-IE–treated monkey eyes. These observations support the view that increased uveoscleral outflow with PGs is caused by decreased hydraulic resistance resulting from an opening of the interstitial spaces of the ciliary muscle due to reduction of collagens in and surrounding these spaces.

A mechanism for these changes is suggested by several studies with cultured human ciliary smooth muscle cells. Initially, results of these studies^{8 ,12} confirmed that ciliary muscle cells in vitro produce collagen types I, III, and IV; laminin; and fibronectin, which become incorporated into an extracellular matrix surrounding the cell layer. In addition, treatment of the cultures with PGF_2αor latanoprost acid, the biologically active form of latanoprost, induces remodeling of collagens, laminin, and fibronectin in this extracellular matrix.¹³ In additional experiments in the same study, enzyme-linked immunosorbent assay measurements confirmed reduction of collagen types I and III in the cell layer. Further studies with cultured ciliary smooth muscle cells found that these extracellular matrix changes were accompanied by increased secretion of matrix metalloproteinases (MMPs),^{14 - 15} a family of neutral proteinases that can initiate the degradation of collagens and other extracellular matrix components.^{16 - 17} Zymographic findings indicated that the predominant MMPs secreted included MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), and MMP-3 (stromelysin-1).¹⁵ These results suggest that the collagen remodeling observed in the ciliary muscle after topical PGF_2α-IE treatment may be due to stimulation of increased MMP secretion by ciliary smooth muscle cells.

Recently, it has been shown¹⁸ that MMP-1 is expressed in normal human ciliary muscle, the iris root, and the sclera adjacent to the ciliary muscle. These structures are components of the uveoscleral outflow pathway. As such, it is possible that increased MMPs in all 3 structures may contribute to increased uveoscleral outflow associated with use of topical PGs. Hence, the present study was undertaken to investigate MMP changes in the iris root, ciliary muscle, and adjacent sclera of monkey eyes after multiple treatments with topical PGF_2α-IE.

Tissue sections analyzed in the present study were cut from the same tissue blocks as sections analyzed in a previous study¹¹ of PGF_2α-IE effects on collagen types I, III, and IV. The young adult cynomolgus monkeys used had been evaluated by slitlamp biomicroscopy on 2 occasions before initiation of treatment to confirm the absence of signs of ocular inflammation. In addition, blood-aqueous barrier integrity was confirmed by measuring the appearance and disappearance of fluorescence in the anterior chamber after intravenous fluorescein administration. To qualify for further study, anterior chamber fluorescence levels and time course of appearance and decay in both eyes had to be similar and within the range of values obtained for control eyes in previous studies.³ Monkeys meeting these conditions were presumed to have an intact blood-aqueous barrier.³

The following week, each qualifying monkey received 2 µg of PGF_2α-IE (in 5 µL) twice daily (morning and afternoon, approximately 7 hours apart) in one eye and 5 µL of vehicle in the other eye for 5 days as previously described.¹¹ On the fourth day of treatment, IOP was measured 3, 3½, and 4 hours after the morning treatment. On the fifth day, slitlamp biomicroscopy was performed and IOP was measured at similar times. Eight eyes from 4 monkeys (code numbers K91, K344, K453, and K454) responding with IOP reduction greater than 5 mm Hg were obtained. Anterior chamber cells or flare were not observed during treatment of these monkeys. Before treatment, IOP ranged from 12.0 to 16.7 mm Hg (mean ± SD, 14.3 ± 1.1 mm Hg; baseline 1 plus baseline 2 measurements). The difference between measurements taken for baselines 1 and 2 also was nonsignificant(14.1 ± 0.7 and 14.4 ± 1.5 mm Hg, respectively; P = .59). When measurements from baselines 1 and 2 were combined, the starting mean ± SD IOP in treated eyes was 14.7 ± 1.1 mm Hg and in control eyes was 13.8 ± 0.6 mm Hg. This difference was nonsignificant(P = .21). Also, IOP measurements from day 4 were not significantly different from measurements taken on day 5 in treated and control eyes (P = .60 and .16, respectively), suggesting that the treatment effect had stabilized by day 4. However, comparing the mean of IOPs collected for baselines 1 and 2 with the mean of IOPs collected on days 4 and 5 revealed that topical treatment with PGF_2α-IE reduced IOP by 9.0 ± 2.2 mm Hg (P<.001). In contrast, there was no significant IOP change in the contralateral eyes exposed to vehicle control (P = .29).

The animals were humanely killed after the IOP reading on day 5 as previously described.¹¹ The vascular bed was perfused with lactated Ringers solution to remove circulating MMPs from the ocular tissues. The anterior segments were dissected and immediately fixed in methacarn(60% methanol, 30% chloroform, and 10% glacial acetic acid) for 3 hours. Increased sensitivity of immunohistochemical staining of various antibodies has been demonstrated for many antigens after methacarn fixation.^{19 - 20} Fixed anterior segments were transferred to cold 100% ethanol and shipped overnight to the University of California, San Diego, La Jolla. The tissues were embedded in paraffin and sections were collected from the midsagittal region of each eye on slides coated with a tissue section adhesive (Vectabond; Vector Laboratories, Burlingame, Calif). For histopathological analysis, 3 to 4 sections from each eye were stained with hematoxylin-eosin. All procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement on the Use of Animals in Ophthalmic and Vision Research.

Anterior segment tissues were immunostained using a standardized protocol. Each step of the protocol was optimized as previously described.²¹ For example, concentrations and incubation times of solutions used to remove intrinsic melanin, described in the following paragraph, were optimized to eliminate melanin while preserving structural integrity of the tissue. Also, the concentration of each solution containing antibodies or horseradish peroxidase–conjugated streptavidin was optimized to obtain submaximal (nonsaturating) staining intensity as determined by imaging densitometry (described in the following paragraph). Finally, the incubation time with diaminobenzidine was optimized for each primary antibody to obtain the strongest signal (staining intensity) that still was increasing linearly with time. The elimination of saturating binding or development variables from the protocol support the position that the observed changes in staining intensity reflected differences in tissue content of target antigen. A result of this optimization is that the intensity of immunostaining was moderate in appearance.

Sections from treated and control eyes were stained at the same time. Five serial midsagittal sections, 10-µm thick, from each eye were heated to 56°C for 20 minutes, washed in 3 xylene changes to remove paraffin, and rehydrated through graded ethanols. The sections were treated with antigen retrieval solution (AR-10; Biogenex, San Ramon, Calif) at 95°C for 5 minutes. After cooling, the sections were exposed to 3% hydrogen peroxide for 10 minutes to suppress endogenous peroxidase activity. To remove intrinsic melanin, sections were treated successively with aqueous potassium permanganate (2.5 g/L) for 10 minutes and oxalic acid (5.0 g/L) for 3 minutes.^{22 - 24} After rinsing, the sections were blocked for 30 minutes with 0.1% bovine serum albumin (Sigma-Aldrich Corp, St Louis, Mo) and incubated for 2 hours with affinity-purified polyclonal sheep anti–porcine MMP-1 (dilution, 1:25)(AB772; Chemicon, Temecula, Calif), polyclonal rabbit anti–human MMP-2(dilution, 1:100) (AB809; Chemicon), or rabbit anti–human MMP-3 (dilution, 1:1000) (AB810; Chemicon). These concentrations had been optimized in previous pilot studies for quantitative analysis as previously described.²¹ Specificity of these antibodies has been previously confirmed.^{18 ,25} After rinsing, the sections treated with antibody to MMP-1 were exposed to biotinylated donkey anti–sheep IgG for 30 minutes (Biotin-SP-Conjugated Affinipure; Jackson Immunoreasearch Laboratories Inc, West Grove, Pa) (dilution, 1:500). The sections exposed to antibodies to MMP-2 or MMP-3 were exposed to biotinylated goat anti–rabbit immunoglobulin (Biogenex) for 20 minutes. After rinsing, the sections were exposed to horseradish peroxidase–conjugated streptavidin for 20 minutes. Consecutively, each section was rinsed and incubated with 3, 3′-diaminobenzidine chromogen for 10 minutes (HRP-DAB Super Sensitive Immunodetection System; Biogenex). To facilitate comparability, sections from control vehicle– and PG-treated eyes of each monkey were immunostained at the same time.^{26 - 27} To serve as controls for nonspecific staining, sections from each eye were simultaneously processed using the same protocol but without the primary antibody.

Immunohistochemical staining intensity was directly measured using a high-resolution imaging densitometer as previously described.²¹ Measurements from multiple sections stained at the same time facilitated assessment of measurement precision and permitted statistical comparison of differences among control and experimental eyes. Immunostained sections were scanned by placing the slides directly on the platen of an imaging densitometer (model GS-700; Bio-Rad, Hercules, Calif). Resolution of the scans was set to 1200 dpi (50-µm-wide pixels), and the scanning mode was set to transillumination. The optical density (OD) measurements of the immunostained sections all were less than 1.00 OD units. Because the densitometer can accurately measure ODs greater than 3.0 units (Bio-Rad specifications), these measurements were well within the appropriate range for accurate determinations. The scanned digital data were displayed in a masked fashion and analyzed using an image analysis program (Molecular Analyst, version 2.1; Bio-Rad). The OD along 2 line segments positioned perpendicular to the long axis of the ciliary body and near the widest region of the ciliary muscle was measured in each section using 2-dimensional imaging densitometry. The positioning of these line segments over the anterior segment tissues avoided any remaining cluster of pigment granules. A similar line segment was positioned perpendicular to the long axes of the iris root and the sclera adjacent to the ciliary body.

Mean OD scores were determined from the OD volume scores (OD × millimeters) and the corresponding line segments (in millimeters).²¹ For each eye, 10 scores were obtained from 5 midsagittal sections. Background OD was measured along one line segment nearby, but not overlying the tissue. This OD score was defined as baseline and was subtracted from the original OD score. Ciliary and iridial pigmented epithelial cells, which showed a high peak on an image densitometry map, were excluded. The specific OD scores along each line segment over the ciliary muscle, iris, and adjacent sclera were calculated by dividing the OD area score (OD × millimeters, provided by the densitometer) by the length of the line segment(in millimeters) for that score. Mean specific OD scores from the PG-treated eye of each monkey were compared with corresponding scores from the contralateral control eyes using the paired t test. The unpaired t test was used to compare the mean of mean OD scores of all PG-treated and control eyes. In each case, P<.05 was considered significant.

In vehicle-treated monkey eyes, moderate immunoreactivity for MMP-1 was observed in the ciliary muscle, iris root, and sclera adjacent to the ciliary muscle (Figure 1A). More intense MMP-1 staining was observed in the ciliary pigment epithelium and the iris pigment epithelium. The distribution of MMP-2 immunoreactivity in vehicle-treated eyes was similar, with diffuse light staining in the ciliary muscle, iris root, and sclera and moderate immunoreactivity in the ciliary pigment epithelium and iris pigment epithelium (Figure 1C). Light MMP-3 immunoreactivity was present in the ciliary muscle of vehicle-treated eyes (Figure 1E). In contrast, minimal MMP-3 immunoreactivity was present in the iris root and sclera of these eyes. However, there was moderate MMP-3 immunoreactivity in the ciliary pigment epithelium.

In eyes treated with PGF_2α-IE, there was increased MMP-1 and MMP-2 immunoreactivity in the ciliary muscle, iris root, and sclera(Figure 1B, D). Compared with vehicle-treated eyes, there was increased MMP-3 immunoreactivity in the ciliary muscle (Figure 1F). In the iris root, which was minimally stained in vehicle-treated eyes, there was moderate staining of stromal matrix cells. Moderate MMP-3 staining also was observed in the sclera of treated eyes.

Quantitative analysis by imaging densitometry confirmed that compared with vehicle-treated eyes there was increased MMP-1 immunoreactivity in all treated eyes (Figure 2). As shown in Figure 3, combining the MMP-1 immunoreactivity scores for all monkeys shows increased OD scores in the iris root, ciliary muscle, and sclera. Figure 4 and Figure 5 show the combined scores for MMP-2 and MMP-3, respectively. Overall, OD scores for MMP-1 in the iris root, ciliary muscle, and sclera in the treated eyes were increased by a mean ± SD of 89% ± 16%, 61% ± 8%, and 66% ± 57%, respectively. Similarly, OD scores for MMP-2 were increased by 129% ± 53%, 82% ± 27%, and 267% ± 210%, respectively. Matrix metalloproteinase 3 OD scores in treated eyes were increased by 207% ± 84%, 83% ± 49%, and 726% ± 500%, respectively. Table 1 shows that in each case, the increases in the treated eyes were statistically significant compared with vehicle-treated eyes.

The present results show that topical treatment of monkey eyes with PGF_2α-IE increases expression of MMP-1, MMP-2, and MMP-3 in the iris root, ciliary muscle, and adjacent sclera. Observation of this effect in each of the treated monkey eyes further strengthens this result. Because uveoscleral outflow also is increased in monkey eyes after treatment with PGF_2α-IE,^{1 ,3} these results suggest that increased MMP expression may be linked with the increased uveoscleral flow and reduced IOP observed with use of this agent. Only 4 monkeys were studied because this was sufficient to assess the presence of this relationship and was consistent with good utilization and conservation of valuable primate resources. Although this sample was small, regression analysis found a significant correlation between the magnitude of the mean IOP reduction and the intensity of MMP-2 immunoreactivity in the ciliary muscle(P = .04). In view of the small sample, however, the absence of significant correlation between the magnitude of the mean IOP reduction and the intensity of staining in the other regions or with the other MMPs is not conclusive. Thus, the present results establish a link between IOP reduction after PGF_2α-IE treatment and MMP induction. However, a larger sample of monkeys would need to be evaluated to determine the precise relationship between MMP induction and IOP.

A plausible explanation for the effect of the increased MMP observed with topical PGF_2α-IE administration is that it initiates reduction of collagens and other extracellular matrix molecules found in the uveoscleral outflow pathway. This reduction has been previously confirmed¹¹ for collagen types I, III, and IV in the same tissue that was analyzed in the present study. Matrix metalloproteinase 1 is known to hydrolyze a specific site found in collagen types I and III.^{17 ,28} Likewise, MMP-2 is known to hydrolyze specific sites found in collagen type IV and fibronectin. Matrix metalloproteinase 3 is known to hydrolyze specific sites found in collagen types III and IV and in fibronectin and laminin. After these initiating steps, other extracellular hydrolases also participated in extracellular matrix reduction. Matrix metalloproteinases are secreted as inactive proenzymes that are subsequently activated by proteolytic truncation.^{29 - 30} Also, MMP activity is regulated by the presence of tissue inhibitor of MMPs.^{31 - 32} Each of the antibodies used can recognize the proenzyme and the active enzyme. Hence, the magnitude of the increased MMP activity might be less than the magnitude of the increased immunoreactivity.

There might be other anterior segment structures that also increase their production of MMPs after topical PGF_2α-IE treatment. Previous investigation of MMP-1 expression in normal human eyes found immunoreactivity in the ciliary and iris pigment epithelia and in trabecular meshwork.¹⁸ Because these structures are relatively small compared with the section thickness, the intensity of staining in these structures can critically depend on orientation. As shown in Figure 1, the intensity of staining in the ciliary epithelium varies depending on whether it is cross sectioned or cut tangentially. Also, their small size makes it difficult to accurately define their limits for placement of the measurement chords as the resolution of the imaging densitometer is 50 mm. Hence, this study did not investigate the smaller structures. Nevertheless, these structures also might be involved as they both contain MMPs and face compartments containing aqueous humor.

An important consideration is whether the onset of MMP induction is consistent with the rate of IOP reduction observed after topical PGF_2α-IE treatment. Kinetic studies² observed that in anesthetized monkeys, maximal IOP reduction occurred about 3 hours after a single PGF_2α-IE topical treatment. Similarly, in awake monkeys that had received twice-daily treatments with PGF_2α-IE for 3 days, maximal reduction also occurred about 3 hours after the morning treatment on the fourth day.¹ However, detectable MMP-1 activity in human ciliary smooth muscle cultures exposed to PGF_2α was not observed until 12 hours after treatment.¹⁵ It is possible that the zymography assay for MMPs used in the latter study was insufficiently sensitive to observe earlier MMP production. Alternatively, it is possible that increased immunoreactivity of MMPs may only be important after multiple treatments. The possibility of this scenario is supported by the observation that with multiple treatments of monkey eyes with topical PGF_2α, there is an increase in the magnitude of the response that occurs over several days.³³ Also, in patients with glaucoma taking latanoprost for more than 6 months, there is maintenance of reduced IOP after cessation of treatment that persists for 2 days and gradually rises to pretreatment IOP in 2 weeks.³⁴ Hence, MMP induction may be more important for these gradual effects.

In conclusion, our results suggest that increased immunoreactivity of anterior segment MMPs occurring with topical PGF_2α-IE treatment may be involved in increased uveoscleral outflow and reduced IOP. Further studies will be important for understanding the significance of this observation and the mechanism of its regulation.

Accepted for publication March 30, 2001.

This work was prepared in partial fulfillment of requirements for membership by Dr Weinreb in the American Ophthalmological Society.

Corresponding author: Robert N. Weinreb, MD, Glaucoma Center, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0946.