Epithelial–Mesenchymal Transition (EMT)-Related Cytokines in the Aqueous Humor of Phakic and Pseudophakic Fuchs' Dystrophy Eyes

Abstract

Purpose.: The present study aimed to analyze the expression of epithelial–mesenchymal transition (EMT)-related cytokines in the aqueous humor of phakic and pseudophakic Fuchs' endothelial corneal dystrophy (FECD) eyes and their correlation to FECD severity.

Methods.: Aqueous humor samples from phakic FECD eyes (FECD_ph, n = 9), from pseudophakic FECD eyes more than 1 year after cataract surgery (FECD_psph, n = 13), and from cataract controls without FECD (Control_cat, n = 28) were obtained during Descemet membrane endothelial keratoplasty (DMEK) or cataract surgery. Expression of EMT-related cytokines (TGF-β1, TGF-β2, TGF-β3, MCP-1, BFGF, TNF-α, IL-1β) was measured using multiplex bead assay. Corneal central-to-peripheral thickness ratio at 3.5 mm from the center (CPTR_3.5) was determined as an objective metric for FECD severity before surgery by slit-scanning pachymetry.

Results.: Pseudophakic FECD eyes showed significantly elevated expression compared with Control_cat and FECD_ph eyes for TGF-β1 (P < 0.001, respectively), for TGF-β2 (P < 0.05, respectively), and MCP-1 (P < 0.001, respectively). Levels of TGF-β1 (r = 0.6116, P < 0.05) and MCP-1 (r = 0.5934, P < 0.05) were positively correlated with CPTR_3.5. No differences in EMT-associated protein levels were detected comparing FECD_ph eyes and Control_cat eyes.

Conclusions.: Simultaneous elevation of TGF-β1, TGF-β2, and MCP-1 concentrations in FECD_psph eyes confirms that cataract surgery leads to long-term alterations of the intraocular microenvironment. Positive correlation of increased aqueous TGF-β1 and MCP-1 levels with CPTR_3.5 in pseudophakic FECD eyes suggests that changed cytokine levels may be involved in corneal decompensation after cataract surgery. Unchanged aqueous humor levels of EMT-related proteins analyzed in phakic FECD patients indicate that there is no primary role of these aqueous cytokines in FECD pathogenesis.

The corneal endothelium is a cellular monolayer composed of uniform hexagonal cells directly facing the aqueous humor and anterior chamber. Corneal endothelial cells (CECs) regulate stromal hydration through a pump-leak mechanism and thereby maintain optical clarity of the corneal tissue. The normal ultrastructure of the CEC basal membrane, Descemet membrane, consists of an anterior banded layer located posterior to the corneal stroma and a posterior nonbanded layer located anterior to the corneal endothelium.^1,2 The anterior banded layer thickness remains constant (approximately 3 μm) after birth, whereas the posterior nonbanded layer thickness continuously increases (approximately 3 μm at the age of 20 and 10 μm at the age of 80 years) due to permanent extracellular matrix (ECM) secretion by normal CECs throughout lifetime.¹ 

Fuchs' endothelial corneal dystrophy (FECD) is a bilateral disease of the corneal endothelium. It is morphologically characterized by an accelerated decrease in CEC density with at least fractional transformation of CECs to a fibroblast-like cell type and excessive subendothelial deposition of ECM.^3–6 The FECD Descemet membrane ultrastructure is composed of a normal anterior banded layer with either attenuated or absent posterior nonbanded layer and overlying additional layers of ECM.^4–,68 These additional layers comprise a posterior banded layer, accounting for most of the Descemet membrane's characteristic thickening in FECD and exhibiting focal outgrowths presenting as guttae, a border layer, and a subendothelial fibrillar layer.^4,9 The underlying reasons for pathologic accumulation of ECM in FECD need further clarification and may include altered epithelial-to-mesenchymal transition (EMT) as suggested by implication of multiple EMT-related genes in FECD (reviewed in Ref. 10). 

Several proteins have been demonstrated to induce a transformation of hexagonal CECs to a fibroblast-like phenotype in the context of EMT. These include FGF-2,^11,12 IL-1β,¹¹ and members of the TGF-β family.^13,14 Other proteins, including monocyte chemoattractant protein-1 (MCP-1)¹⁵ and TNF-α,^16,17 are expressed in the aqueous humor and have been shown to be associated with EMT and scarring in other cell types. 

The present study aimed to clarify if aqueous humor concentrations of EMT-associated cytokines, including TGF-β1, TGF-β2, TGF-β3, MCP-1, BFGF, TNF-α, and IL-1β, are changed in pseudophakic and phakic FECD eyes compared with cataract controls without FECD and if their aqueous humor levels correlate with severity of the disease.¹⁸ 

Materials and Methods

Human Subjects

Participants with FECD or age-related cataract were recruited at the Department of Ophthalmology, University of Cologne, Cologne, Germany. A complete ophthalmic history was obtained and participants underwent a standard ophthalmic examination, including best corrected visual acuity (BCVA), slit-lamp and fundoscopic examination before surgery. Standard ophthalmic examination also included central corneal thickness (CCT) and peripheral corneal thickness at 3.5 mm from the center (PCT_3.5) measurement with a slit-scanning pachymeter (Orbscan II; Orbtek, Inc., Salt Lake City, UT, USA) for all FECD patients. The central-to-peripheral thickness ratio at 3.5 mm (CPTR_3.5) was calculated as a measure of FECD severity.¹⁸ Previous studies by Repp et al.¹⁸ demonstrated that the central-to-peripheral thickness ratio determined by scanning-slit pachymetry is an objective and repeatable metric of FECD severity. We used peripheral thickness-measurements at 3.5 mm from the corneal center because measurements farther than 3.5 mm could be obtained only inconsistently. 

Patients were eligible for this study if they had FECD and no previous intraocular surgery, if they had FECD and had previous phacoemulsification with posterior chamber intraocular lens implantation for age-related cataract more than 1 year in the past and no other previous intraocular surgery, or if they had age-related cataract without FECD and without previous intraocular surgery. Patients were excluded if they had a known history or signs of glaucoma, pseudoexfoliation syndrome or glaucoma, uveitis, vasculitis or proliferative vitreoretinopathy, neovascular AMD or diabetes, or in the case of ongoing treatment with topical steroids. Only one eye per patient was included. 

Fuchs' endothelial corneal dystrophy was clinically defined as the presence of paracentral or central guttae with or without corneal edema detected by slit-lamp biomicroscopy. Cataract was defined as the presence of age-related cataract detected by slit-lamp biomicroscopy. 

Informed consent was obtained from all patients before surgery. This study was approved by the review board of the University of Cologne, Cologne, Germany. The research adhered to the tenets of the Declaration of Helsinki. 

Collection of Aqueous Humor Samples

Aqueous humor sample collection and analysis were performed under sterile conditions and followed established protocols and previous reports from our institution.^19,20 Descemet membrane endothelial keratoplasty (DMEK) surgeries were performed under general anesthesia, and cataract surgeries were performed under general or local (using 0.4% oxybuprocaine eye drops) anesthesia as described previously.^2,21–24 At the beginning of DMEK or cataract surgery, a limbal paracentesis was made in a very first step, and 0.15 mL aqueous humor was collected using a 30-gauge needle connected to an insulin syringe. Aqueous humor samples were immediately stored at −80°C in sterile polypropylene tubes and kept at −80°C until multiplex bead assay analysis. 

Multiplex Bead Assay Procedure

Aqueous humor concentrations of TGF-β1, TGF-β2, TGF-β3, MCP-1, BFGF, TNF-α, and IL-1β were measured using the TGF-β1, -2, -3 Luminex Performance Assay 3-plex kit (R&D Systems, Wiesbaden, Germany), the Human Luminex Performance Assay Base Kit, Panel A (R&D Systems), and corresponding Luminex Performance Assays (all R&D Systems) on a Luminex 200 dual-laser, flow-based sorting and detection platform (Luminex, Inc., Austin, TX, USA) according to manufacturer's instruction. The multiplex bead assay procedure allows for specific detection of several analytes in a single small sample with increased sensitivity compared with ELISA.²⁵ Fifty microliters per aqueous humor sample was used per reaction. Standard curves were generated using the reference standards supplied with the kits and used to determine respective analyte concentrations for each sample. Minimum detection threshold of individual analytes were TGF-β1: 36.63 pg/mL, TGF-β2: 17.7 pg/mL, TGF-β3: 62.28 pg/mL, MCP-1: 3.36 pg/mL, BFGF: 6.45 pg/mL, TNF-α: 5.42 pg/mL, and IL-1β: 2.81 pg/mL. 

Statistical Analysis

The continuous data from study participants, including age, BCVA, CPTR_3.5, CCT, and PCT_3.5, as well as cytokine concentrations were compared for each pair of groups using two-tailed Mann-Whitney U test. The categorical data, including sex, were compared for each pair of groups using two-tailed Fisher's exact test. Correlations between cytokine concentrations among each other and between cytokine concentrations and age or CPTR_3.5 were calculated using Spearman correlation. All P values were adjusted for multiple testing using the method of Benjamini and Hochberg.²⁶ A P value less than 0.05 was considered statistically significant. Statistical analyses were performed using the PRISM 4 software package (GraphPad Software, San Diego, CA, USA). 

Results

This study included 9 phakic FECD patients without previous intraocular surgery (FECD_ph), 13 pseudophakic FECD patients with previous phacoemulsification with posterior chamber intraocular lens implantation surgery for age-related cataract (FECD_psph), and 28 age-related cataract patients without previous intraocular surgery (Control_cat). Demographic data recorded for all patients including age, sex, BCVA, CPTR_3.5, CCT, and PCT_3.5 are summarized in Table 1. Cataract surgery to DMEK period in the FECD_psph group was 9.1 ± 8.0 years (range, 2–26 years). 

Table 1

 

Demographic Data

 

Cytokine Expression in Aqueous Humor Samples From FECD and Cataract Eyes

The results of the multiplex bead assay are presented in detail in Figure 1. Aqueous humor concentrations above the detection threshold were measured in more than 80% of the samples tested for TGF-β1, TGF-β2, MCP-1, and BFGF. Concentrations below detection threshold were measured in all samples for TGF-β3, IL-1β, and TNF-α. These cytokines were therefore excluded from further analyses. 

Figure 1

 

Cytokine concentrations (expressed in pg/mL). Asterisks on individual graphs indicate that *P < 0.05; ***P < 0.001.

Comparing groups FECD_psph versus Control_cat and FECD_psph versus FECD_ph, significantly elevated protein levels were detected in the FECD_psph group for TGF-β1 (P = 0.0003 and P = 0.0003), TGF-β2 (P = 0.0450 and P = 0.0450), MCP-1 (P = 0.0003 and P = 0.0008). There were no significant changes in concentrations measured for any of the EMT-associated aqueous humor proteins between groups FECD_ph and Control_cat. 

Correlations of Cytokine Expression

The correlations of groups of two respective cytokine concentrations are presented in detail in Table 2. There were positive correlations between TGF-β1 and TGF-β2 (r = 0.6145, P = 0.0003), TGF-β1 and MCP-1 (r = 0.7664, P = 0.0003), and TGF-β2 and MCP-1 (r_s = 0.4679, P = 0.0012). 

Table 2

 

Correlation of Cytokine Concentrations

 

The TGF-β1 (r = 0.6116, P = 0.0487) and MCP-1 (r = 0.5934, P = 0.0487) were positively correlated with CPTR_3.5 in the FECD_psph group. There were no significant correlations between age and any of the respective aqueous humor analyte concentrations. 

Discussion

The present study investigated the differential expression of EMT-associated cytokines in aqueous humor samples from phakic and pseudophakic FECD patients compared with cataract controls using multiplex bead assay analysis. It demonstrates that pseudophakic FECD eyes exhibit increased aqueous TGF-β1, TGF-β2, and MCP-1 concentrations more than 1 year after cataract surgery, whereas aqueous TGF-β1 and MCP-1 levels positively correlate with the corneal CPTR as an objective metric of FECD severity. No changes of EMT-associated cytokine concentrations were observed in FECD eyes without previous intraocular surgery. 

The corneal endothelium represents the innermost cellular layer of the human cornea directly facing the aqueous humor. Changes in aqueous humor composition, including altered expression of cytokines, may therefore be of immediate relevance for CEC function. The present study demonstrates for the first time increased aqueous humor concentrations of EMT-related cytokines TGF-β1, TGF-β2, and MCP-1 in FECD eyes after cataract surgery. These aqueous humor proteins have been extensively studied in other ocular pathologies: members of the TGF-β family affect a variety of cellular functions, such as proliferation and differentiation, apoptosis, immunological processes, and ECM turnover.²⁷ MCP-1 (also referred to as chemokine [cc-motif] ligand 2) is an important proinflammatory cytokine and a potent stimulator of monocyte chemotaxis. Ocular pathologies exhibiting increased aqueous TGF-β and MCP-1 levels include disorders with changes in ECM turnover or inflammatory state–like glaucoma,^28–30 proliferative vitreoretinopathy³¹ and rhegmatogenous retinal detachment,³² central retinal vein occlusion,³³ and branch retinal vein occlusion.^33,34 

A simultaneous increase of aqueous EMT-related cytokine concentrations in pseudophakic FECD eyes more than 1 year after cataract surgery was observed in our study. This novel finding is supported by previous studies demonstrating that cataract surgery causes prolonged changes of the intraocular microenvironment and increase of proinflammatory aqueous humor cytokine concentrations, such as MCP-1 in normal and glaucomatous eyes.^15,30,35 There were no preoperative signs of intraocular inflammation in the pseudophakic FECD eyes included in our study. However, a protracted subclinical intraocular state of inflammation after cataract surgery cannot be excluded. It was proposed that elevated cytokine expression levels in pseudophakic eyes arise from metabolic changes after cataract-tissue removal, changes in aqueous humor dynamics, disruption of the blood–aqueous barrier, or reactive production of proteins by cells of the anterior chamber or by activated inflammatory cells, which may also apply to our findings.³⁰ Cataract surgery affects ocular (patho-) physiology in various conditions and may cause intraocular profibrotic changes in multiple cell types: Pseudophakic bullous keratopathy (PBK), as a very relevant example, is characterized by cataract surgery–related corneal bullous edema, abnormal production of ECM, and formation of a posterior collagenous layer underneath the corneal endothelium³⁶; Jahn et al.³⁷ showed that the prevalence of epiretinal membranes increases after cataract surgery; and Takihara et al.³⁸ reported that trabeculectomy with mitomycin C in pseudophakic eyes is less successful (compared with that in phakic eyes) probably due to increased cicatricial reaction after cataract surgery. Similar mechanisms are very likely to contribute to cataract surgery–related corneal decompensation in FECD in addition to the direct surgery-related endothelial trauma. Van Cleynenbreugel et al.³⁹ showed in a prospective observational study that 35 (39%) of 89 eyes with FECD needed endothelial keratoplasty after cataract surgery. Our study suggests that long-term changes in TGF-β1 and MCP-1 after cataract surgery are particularly involved in FECD disease progression, as indicated by positive correlation of aqueous humor TGF-β1 and MCP-1 concentrations with CPTR_3.5 of pseudophakic FECD eyes (Fig. 2). The CPTR has been shown to serve as an objective metric to assess FECD severity compared with subjective clinical grading, which showed more variability.¹⁸ Interestingly, TGF-β1 and MCP-1 aqueous concentrations also exhibited a high level of correlation (Table 2). Previous in vitro investigations found that TGF-β stimulation induced CEC transformation from a regular hexagonal to a fibroblast-like phenotype; fibroblast-like CECs exhibited increased expression of ECM proteins, including collagen I and IV and fibronectin.⁴⁰ A fibroblast-like phenotype of CECs and collagen I and IV and fibronectin deposition also have been observed in end-stage FECD specimens,^4,41,42 suggesting that in vivo cataract surgery–induced increase in TGF-β levels may expedite the described CEC transformation of the rarified FECD endothelial monolayer and thereby contribute to accelerated ECM deposition and corneal decompensation in the postoperative course. Interestingly, the rate of immune reactions after DMEK does not seem to differ between eyes with significantly elevated TGF-β levels in pseudophakic Fuchs' patients compared with non-Fuchs' or phakic transplant patients.^23,43,44 

Figure 2

 

Correlation between CPTR_3.5 and cytokine concentrations in FECD eyes. Spearman correlation demonstrated that TGF-β1 and MCP-1 levels are positively correlated with CPTR_3.5 in FECD_psph eyes (P < 0.05).

Even though the described results point to an involvement of changes in EMT-associated aqueous humor cytokines in final cornea decompensation of pseudophakic FECD eyes, we did not observe any changes of all cytokines analyzed in FECD eyes without previous cataract surgery. This suggests that there is no primary role of these aqueous humor cytokines in FECD pathogenesis and that the increase in cytokine levels observed in pseudophakic FECD eyes may not be FECD-specific and rather a common response after cataract surgery. 

Moreover, increased expression of cytokines analyzed in the present study are related but not exclusive to the process of EMT or to changes explicitly of the cornea. 

Therefore, future studies need to investigate if a similar reaction paralleling corneal decompensation also may be found in other corneal diseases, such as PBK, and clarify in more detail how these changes of aqueous humor composition affect EMT of CECs in vivo. 

In conclusion, increased concentrations of aqueous TGF-β1, TGF-β2, and MCP-1 in pseudophakic compared with phakic FECD eyes suggest that cataract surgery leads to long-term alterations of the intraocular microenvironment in FECD eyes (as has been previously shown in normal and glaucomatous eyes^15,30,35). Positive correlation of aqueous TGF-β1 and MCP-1 levels with CPTR_3.5 in pseudophakic FECD eyes suggests that changed cytokine levels may be involved in corneal decompensation after cataract surgery. Unchanged aqueous humor levels of EMT-related proteins analyzed in phakic FECD patients indicate that there is no primary role of these aqueous cytokines in FECD pathogenesis. 

Acknowledgments

The authors thank Hans Günter Simons and Sabine Hackbarth for technical support. 

Supported by German Research Foundation Grants FOR 2240 “(Lymph)Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye” (CC, LMH), HE 6743/2-1 and HE 6743/3-1 (LMH); GEROK Program University Hospital of Cologne (LMH); Dr. Gabriele Lederle Foundation, Taufkirchen (LMH); Helmut Lingen Foundation, Cologne (CC); and EU COST BM1302 (Joining Forces in Corneal Regeneration [BOB, CC]). 

Disclosure: M. Matthaei, None; J. Gillessen, None; P.S. Muether, None; R. Hoerster, None; B.O. Bachmann, None; A. Hueber, None; C. Cursiefen, None; L.M. Heindl, None