To determine the levels of plasminogen activator inhibitor-1 (PAI-1) and total protein in the aqueous humor of patients with glaucoma vs those without glaucoma.
A total of 125 aqueous humor samples (50-150 μL each) were collected at 3 institutions from patients with glaucoma and a control group of patients with cataract. Fifteen samples were excluded, and the levels of PAI-1 antigen were determined by enzyme-linked immunosorbent assay in 110 samples (36 glaucoma and 74 control). Total protein levels were determined by the Bradford method in 81 samples (28 glaucoma and 53 control), in which the aqueous humor collected was sufficient. Statistical analysis of the results was conducted using the Mann-Whitney U test. The correlation between PAI-1 and protein levels was calculated using the Spearman rank correlation coefficient.
The mean ± SD PAI-1 levels detected in aqueous humor samples of the control and glaucoma groups were 0.44 ± 0.61 and 1.45 ± 1.91 ng/mL, respectively. The mean ± SD levels of total protein were 64.91 ± 89.75 and 86.64 ± 44.16 μg/mL, respectively. For both parameters, the difference between the 2 groups was significant (P< .001). The correlation between PAI-1 and total protein levels was moderate in the glaucoma group (r = 0.43; P = .01) and low in the control group (r = 0.23; P = .04).
The glaucoma group showed in the aqueous humor a 3.3-fold increase in the mean level of PAI-1 compared with the control group, whereas the increase in total protein level was only 1.3-fold. These data are consistent with the possibility that intraocularly produced PAI-1 may contribute to glaucoma pathogenesis.
Reducing the production or activity of PAI-1 in the eye could constitute a new target for the design of drugs to treat glaucoma.
Elevation of the intraocular pressure (IOP) is increasingly recognized as the major causal risk factor for the progression of glaucoma, which is a leading cause of blindness.1-4 It is generally accepted that IOP elevation results from an involutional process occurring in the anterior chamber angle, which obstructs the aqueous outflow and thus impairs the balance between the rates of aqueous inflow and outflow.5-8 The IOP elevation combined with other, yet unknown factors may cause glaucomatous optic nerve injury and lead to blindness. The molecular mechanisms subserving the aqueous flow and IOP are not yet well understood. However, it is conceivable that the IOP could be controlled in part through genes that are expressed in the eye, specifically in the ciliary body and/or trabecular meshwork, the ocular sites involved in aqueous production and outflow. To this group of genes belongs the gene encoding plasminogen activator inhibitor 1 (PAI-1), which as we have previously reported9 is expressed in the rodent eye exclusively in the processes of the ciliary body.
Plasminogen activator inhibitor-1 is an approximately 50-kDa inducible, secreted protein that belongs to the serpin (serine protease inhibitors) superfamily. It constitutes a central component of the fibrinolytic system that maintains the patency of the vascular bed and other tubular structures. Plasminogen activator inhibitor-1 is the principal physiological inhibitor of the tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which specifically convert the inactive zymogen plasminogen into active plasmin.10-13 The latter is a nonspecific trypsin-like protease that degrades fibrin, and it is also involved in extracellular proteolysis by directly degrading matrix components or activating proenzymes of collagen-degrading metalloproteases.13,14 The plasminogen activator/plasmin system, which includes a specific cell surface receptor for uPA, also plays a critical role in events involving tissue remodeling and cell adhesion and migration.15,16 Plasminogen activator inhibitor-1 can interfere with most activities of the plasminogen activator/plasmin system by either inhibiting the plasminogen activator catalytic activity or, in a proteolysis-independent manner, by competing with adhesive components of the cell surface (ie, the uPA/uPA receptor complex and integrins) for binding to extracellular matrix components.15,16
Clinically, elevated levels of plasma PAI-1 have been associated with various thrombotic disorders,12,17-19 as well as disorders linked to atherosclerotic risks such as non–insulin-dependent diabetes,20 and in the acute phase response such as after trauma or bacterial infection.10,19 Plasminogen activator inhibitor-1 gene expression could be enhanced by hormones including glucocorticoids, insulin, angiotensin II,10,18,19 and α- and β-adrenergic agonists21 as well as by the inflammatory agents tumor necrosis factor α, transforming growth factor β, and interleukin 1β.10,19
The present study was prompted by our recent finding showing that in the adult murine eye, the PAI-1 gene is expressed exclusively in the apices of the ciliary body processes, and accordingly, PAI-1 activity was detected in the aqueous humor.9 This distinct localization implies that PAI-1 may play a role in the anterior chamber and trabecular meshwork. Specifically, up-regulation of PAI-1 gene expression in the ciliary epithelium could shift the proteolytic balance toward inhibition and might also interfere with cell adhesion. Thus, increased levels of PAI-1 in the aqueous humor of patients with glaucoma may causally contribute to sclerosis of the juxtacanalicular tissue and thereby to outflow obstruction and glaucoma pathogenesis.
As a preliminary test for a possible linkage between PAI-1 and glaucoma, we compared the levels of PAI-1 in the aqueous humor of patients with glaucoma vs those without glaucoma. To better evaluate the origin of PAI-1 in the human aqueous humor, we also determined the levels of aqueous total protein and analyzed its correlation with PAI-1.
Selection and processing of aqueous humor
The study protocol was reviewed and approved by the institutional review boards of the Assuta Medical Center for the Maccabi Surgical Center, the George Washington University, and the Beilinson Campus, Rabin Medical Center, before beginning the study. All study patients provided informed consent before enrollment. Any patient undergoing trabeculectomy, cataract surgery, or their combination was eligible. Data on age, sex, glaucoma medications, ocular diagnoses, IOP, and diabetes mellitus (DM) were compiled.
Four glaucoma specialists collected 125 samples in a multicenter effort during a 24-month period beginning in October 2001. A total of 110 samples obtained from 110 patients undergoing ocular surgery were included. Fifteen samples were excluded owing to the following: insufficient amounts of aqueous humor for analysis (5), glaucoma suspects (3), patients who had glaucoma with other ocular conditions (2), active uveitis (2), active iritis (1), incomplete data (1), and a second eye of a patient whose first eye had already been included (1). All enrolled patients with glaucoma had open-angle glaucoma, including 14 cases of pseudoexfoliative glaucoma (PSX-G), 11 cases of primary open-angle glaucoma, and 11 cases of normal-tension glaucoma. Of special interest were 15 patients previously untreated, 5 patients treated with 2% pilocarpine hydrochloride, 14 patients with PSX-G, and 21 patients (8 with glaucoma and 13 controls) with type 2 DM.
At the start of each surgical procedure, before infusion of any irrigating solution into the eye, an aqueous humor sample (50-150 μL) was obtained with a 30-gauge insulin syringe through a standard paracentesis incision. The samples were immediately placed on dry ice, transferred to the laboratory, and stored at −70°C until analyzed.
Pai-1 and protein determination
Samples (for most cases, 40 μL, and for some, 20 μL) were tested (mostly in duplicate, according to the amount of aqueous humor available) using a commercial enzyme-linked immunosorbent assay (ELISA) kit (IMUBIND Plasma PAI-1 ELISA kit; American Diagnostica, Inc, Greenwich, Conn) according to the manufacturer’s instructions. This is an ELISA for human PAI-1 that uses monoclonal anti–PAI-1 IgG coated to microtest wells. The ELISA kit used was reported to detect the active and latent forms of PAI-1, as well as PAI-1 complexes with tPA and uPA, with a detection level below 1 ng/mL. The ELISA assay was conducted in a masked manner. Aqueous samples from patients with glaucoma or cataract were numbered in the hospitals according to the order of their collection. They were then delivered to the laboratory as a numbered, mixed group without identifying details and were tested in the same serial order in the 96-multiwell ELISA plates.
When the aqueous humor collected was sufficient (n = 81), samples (10 μL) were tested in a blind manner for total protein levels using the Bradford22 method. This is a widely used sensitive colorimetric method that quantifies proteins based on the principle of protein dye binding.
Descriptive statistics for the levels of PAI-1 and total protein include mean ± SD, median, and a range of values. The mean ± SD levels of PAI-1 and total protein concentration in the aqueous humor in the glaucoma or control group were compared using the 1-sided Mann-Whitney U test, based on the initial assumption that PAI-1 level is higher in glaucoma. The correlation between values (PAI-1 and total protein as well as between PAI-1 and IOP) was calculated using the Spearman rank correlation. This study was performed according to the Declaration of Helsinki.
The 110 eligible aqueous samples were from 2 groups of patients: those with glaucoma (n = 36) and controls without glaucoma (n = 74). There was no significant difference between the 2 groups with respect to age (mean ± SD, 70.44 ± 13.64 years and 68.54 ± 11.75 years, respectively; P = .40). There were generally slightly more women than men in the glaucoma group (1.11 vs 1.00). The mean ± SD preoperative IOP was significantly (P< .001) higher in the glaucoma group (19.17 ± 6.17 mm Hg) than in the control group (15.06 ± 2.34 mm Hg). Fifteen patients (4 with primary open-angle glaucoma, 4 with PSX-G, and 7 with normal-tension glaucoma) were untreated, whereas 21 patients with glaucoma were receiving medical therapy before they underwent surgery (mean ± SD, 2.7 ± 1.1 medicines in compositions of all classes: β-blockers , carbonic anhydrase inhibitors [14 topical and 2 oral], α2-agonists , prostaglandin F2α analogues , and parasympathomimetics ). Three patients with glaucoma had undergone previous uneventful trabeculectomy, and 1 was previously treated with argon laser trabeculoplasty.
With respect to the control group, the aqueous humor extracted from healthy individuals would be the ideal control for patients with glaucoma. Unfortunately, this is practically impossible. Therefore, patients with cataract are usually used as the second-best control.23 As far as we know, there is no information regarding cataract and PAI-1. Our study of the mouse model has shown that the lens does not produce PAI-1.9 Currently, there is no good reason to predict any effect of cataract on human ocular PAI-1, which if similar to the mouse model is produced in the ciliary body.
The mean ± SD PAI-1 level was 0.44 ± 0.61 ng/mL in the control group (median, 0.29; range, 0-4.15 ng/mL) and 1.45 ± 1.91 ng/mL in the glaucoma group (median, 0.83; range, 0-7.6 ng/mL). Values for several percentiles are illustrated in the Figure. The difference between the mean values of the 2 groups was highly significant (P< .001).
It is of interest to compare the PAI-1 values obtained for several subgroups of patients, although the sample sizes may be too small to determine statistical significance. First, the mean ± SD level of PAI-1 in the untreated group of patients with glaucoma was somewhat higher (2.08 ± 2.29 ng/mL) than that of the entire glaucoma group (1.45 ± 1.91 ng/mL); however, these differences were not significant (P = .28 and P=.10, respectively). Second, no correlation was found between the IOP and the PAI-1 values in both groups (r = 0.09, P = .30; r = −0.12, P = .17; respectively). Third, the mean ± SD PAI-1 level of the PSX-G group (1.73 ± 1.93 ng/mL) did not differ significantly from that of the rest of the glaucoma group (ie, PSX-G excluded, 1.22 ± 1.89 ng/mL; P = .24). Fourth, the small group of patients (n = 5) treated with pilocarpine showed a mean ± SD PAI-1 value (0.55 ± 0.37 ng/mL) slightly higher than that of the control group and much lower than in the glaucoma group. Fifth, with respect to PAI-1 levels, the subgroup of patients with DM did not differ from the subgroup of those without DM in the entire patient population (0.89 ± 1.37 vs 0.70 ± 1.24 ng/mL; P = .61) or within the glaucoma group (P = .47). Furthermore, within the DM subgroup, the difference between patients with glaucoma and controls was significant (1.80 ± 1.96 vs 0.37 ± 0.33 ng/mL, respectively; P = .03), similar to that of the non-DM subgroup (1.22 ± 1.83 vs 0.46 ± 0.66 ng/mL, respectively; P = .004).
The mean ± SD protein level found in the glaucoma group was 86.64 ± 44.16 μg/mL (n = 28), only 30% higher than that of the control group, 64.91 ± 89.75 μg/mL (n = 56). This difference, however, was significant (P< .001). The values of both groups were within the known normal range in the aqueous humor (100-200 μg/mL).24 In addition, a moderate correlation was found between the PAI and total protein levels in the entire patient population as well as in the glaucoma group (r = 0.42, P< .001; r = 0.43, P = .01; respectively), whereas in the control group the correlation between these parameters was low (r = 0.23; P = .04). It was of interest to evaluate the protein levels in 3 subgroups of patients with conditions known to lead to breakage of the ocular (aqueous) blood barriers, namely the pilocarpine-treated patients25 (78.25 ± 17.17 μg/mL), those with PSX-G26 (91.64 ± 44.83 μg/mL), and those with DM27 (64.84 ± 54.76 μg/mL). The protein levels in these 3 subgroups did not differ significantly from those in the rest of the patients (P = .60, P = .18, and P = .79, respectively).
In this study we have compared, to our knowledge for the first time, the levels of PAI-1 in the aqueous humor of patients with glaucoma vs those without glaucoma. We found that the mean ± SD PAI-1 level of the glaucoma group was approximately 3 times higher (1.45 ± 1.91 vs 0.44 ± 0.61 ng/mL). The mean values of the 2 groups were significantly different (P < .001) despite the wide distribution of values within each group and the partial overlap between them.
To our knowledge, this is also the first large-scale determination of PAI-1 levels in the human aqueous humor. Previously reported PAI-1 levels (mean ± SD, 2.25 ± 2.54 ng/mL; range, 0.25-8.0 ng/mL) were detected in 11 patients with cataract using a similar commercial ELISA kit.28 This small sample size and the large variability could possibly account for the difference between these and our results. Notably, in the present study the PAI-1 levels detected in the aqueous humor were markedly lower than those previously reported for platelet-poor plasma in healthy individuals (mean±SD, 18 ± 10 ng/mL; range, 4-43 ng/mL; n = 45).29
Notably, tPA and uPA, the target enzymes for PAI-1, were previously detected in the human aqueous humor.28,30,31 Previously it was roughly estimated that most of the tPA was complexed with PAI-1, with only a small free fraction available for proteolysis.28 Thus, the equilibrium may normally be shifted, perhaps delicately, toward fibrinolysis and proteolysis, which could be involved in the maintenance of aqueous outflow. Our results suggest that this situation may be reversed in patients with glaucoma owing to the increase in PAI-1 level in the aqueous humor.
What could be the source of PAI-1 in the human aqueous humor? Previously we reported that in the rodent eye, PAI-1 is synthesized specifically in the apices of the ciliary body processes.9 That this could be possible for the human eye is supported by findings showing that human ciliary body explants and ciliary epithelial cells in vitro could express a group of several genes, including genes encoding protease inhibitors such as α1-antitrypsin.32,33 It is less likely that the bulk of PAI-1 in the aqueous humor traverses from the blood through the epithelial-endothelial ocular blood barrier, which is thought to allow the passage of less than 0.5% of the circulating proteins.24 Thus, the ratio of mean PAI-1/mean total protein in 1 mL of plasma (approximately 18 ng/70 mg) is considerably smaller than that of the aqueous humor in the glaucoma or control group (1.45 ng/86.64 μg and 0.44 ng/64.91 μg, respectively). To account for these ratios in the aqueous humor, a preferential transfer of PAI-1 into the aqueous has to be postulated in both normal and glaucomatous eyes. It is also noteworthy that breakage of the ocular blood barrier is thought to occur in patients with DM27 or PSX-G26 and after pilocarpine treatment,25 yet our patients belonging to each of these subgroups did not show a significantly increased level of aqueous PAI-1 compared with the rest of the glaucoma group. Finally, we considered the possibility that the glaucoma medications, although comprising a chemically and therapeutically heterogeneous group, could contribute to the elevation of PAI-1 in the aqueous humor. In this study, the subgroups of glaucoma-treated (n = 21) vs nontreated (n = 15) patients could be too small to allow drawing a significant conclusion. However, comparing results obtained for these 2 subgroups (1.0 ± 1.5 and 2.08 ± 2.29 ng/mL, respectively; P = .10) suggests that if the medications had any effect on PAI-1 level, it would be reduction rather than enhancement.
The arguments mentioned previously are consistent with the possibility that aqueous PAI-1 is synthesized intraocularly and thus may play an important role in normal ocular physiologic characteristics. As discussed earlier, these roles may be related to the maintenance of aqueous outflow by controlling the fibrinolytic balance and the integrity of the extracellular matrix. The findings presented in this article, showing an increased mean level of PAI-1 in the aqueous humor of the glaucoma group, suggest that enhanced PAI-1 synthesis in the ciliary body may contribute to outflow resistance and IOP elevation, at least in some glaucoma subtypes.
The major caveats of this preliminary study are the variability of glaucoma subtypes and the genetic heterogeneity of the patient population. The positive correlation we found between elevated aqueous PAI-1 levels and glaucoma indicates that further study of this issue is worthwhile. Our data also raise the possibility of using ocular PAI-1 as a target for the design of new glaucoma drugs.
Correspondence: Jacob Dan, MD, PhD; Ruth Miskin, PhD, Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel (email@example.com; firstname.lastname@example.org).
Submitted for Publication: August 2, 2003; final revision received June 21, 2004; accepted June 21, 2004.
Financial Disclosure: None.
Funding/Support: This study was supported by a grant (Drs Miskin and Dan) from the chief scientist of the Israeli Health Ministry, Jerusalem.
Acknowledgment: We thank Edna Schechtman, PhD (Computer Center, The Weizmann Institute of Science), for advice regarding the statistical analysis and Rene Abramovitz for excellent technical assistance.
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