Journal of Pediatric Ophthalmology and Strabismus

Original Article Supplemental Data

Proliferative Status in the Aqueous Humor of Eyes With Congenital Cataract

Yinglei Zhang, MD; Dan Li, PhD; Qiang Lu, BM; Yu Du, MD; Yi Lu, MD; Xiangjia Zhu, MD

Abstract

Purpose:

To measure the concentrations of growth factors in the aqueous humor of patients with congenital cataract and to investigate the biological effects of a selected cytokine (fibroblast growth factor 4 [FGF4]) on cell proliferation, migration, and transformation.

Methods:

In the aqueous humor obtained from 55 eyes with congenital cataract and 55 eyes with age-related cataract, 40 growth factors were screened and selected cytokines were confirmed with enzyme-linked immunosorbent assays. After the addition of various concentrations of FGF4 (0, 2.5, 15, or 50 ng/mL) to the incubation medium, cellular functions were evaluated.

Results:

The concentration of FGF4 was significantly higher in the aqueous humor of patients with congenital cataract than in that of patients with age-related cataract. The human SRA01/04 lens epithelial cell line was treated with FGF4 and the cell proliferation increased significantly both dose- and time-dependently. The wound healing assay and Transwell migration assay revealed a significant increase in the migration capacity of the SRA01/04 cell line treated with 15 or 50 ng/mL of FGF4 compared with that of control cells. The intensity of immunofluorescent staining for α-smooth muscle actin increased significantly in the SRA01/04 cell line when treated with FGF4. Cytoskeletal protein (F-actin) staining showed that changes of cell morphology were induced in primary lens epithelial cells by FGF4.

Conclusions:

This study provides a comprehensive profile of growth factors in congenital cataract. FGF4 induced cellular changes, and may have utility as a biomarker to predict the formation of visual axis opacification.

[J Pediatr Ophthalmol Strabismus. 2020;57(3):159–168.]

Abstract

Purpose:

To measure the concentrations of growth factors in the aqueous humor of patients with congenital cataract and to investigate the biological effects of a selected cytokine (fibroblast growth factor 4 [FGF4]) on cell proliferation, migration, and transformation.

Methods:

In the aqueous humor obtained from 55 eyes with congenital cataract and 55 eyes with age-related cataract, 40 growth factors were screened and selected cytokines were confirmed with enzyme-linked immunosorbent assays. After the addition of various concentrations of FGF4 (0, 2.5, 15, or 50 ng/mL) to the incubation medium, cellular functions were evaluated.

Results:

The concentration of FGF4 was significantly higher in the aqueous humor of patients with congenital cataract than in that of patients with age-related cataract. The human SRA01/04 lens epithelial cell line was treated with FGF4 and the cell proliferation increased significantly both dose- and time-dependently. The wound healing assay and Transwell migration assay revealed a significant increase in the migration capacity of the SRA01/04 cell line treated with 15 or 50 ng/mL of FGF4 compared with that of control cells. The intensity of immunofluorescent staining for α-smooth muscle actin increased significantly in the SRA01/04 cell line when treated with FGF4. Cytoskeletal protein (F-actin) staining showed that changes of cell morphology were induced in primary lens epithelial cells by FGF4.

Conclusions:

This study provides a comprehensive profile of growth factors in congenital cataract. FGF4 induced cellular changes, and may have utility as a biomarker to predict the formation of visual axis opacification.

[J Pediatr Ophthalmol Strabismus. 2020;57(3):159–168.]

Introduction

Although modern techniques generate better outcomes for children who undergo pediatric cataract surgery, posterior capsule opacification remains the most common postoperative complication.1 In children who undergo primary posterior capsulotomy, the term “visual axis opacification” (VAO) is used more often than posterior capsule opacification2 to describe the aberrant growth of the lens epithelial cells (LECs) that remain on the posterior capsule or anterior vitreous face, and ultimately interrupt the visual axis.2–4 A secondary treatment is usually required for these eyes, causing inevitable damage to the pediatric eye and unexpected surgical expenses to the family. Therefore, a strong drive to produce better outcomes of pediatric cataract surgery has led us to investigate the molecular mechanisms responsible for VAO.

Among the possible hypotheses, it has been suggested that the microenvironment of the aqueous humor is an essential factor in the aberrant growth of LECs. The aqueous humor is a transparent fluid, similar to plasma, that constitutes the microenvironment of the lens. It contains various cytokines, including inflammatory factors and growth factors.5 Zhu et al.6 reported that the proinflammatory status of the aqueous humor in adult myopic eyes is closely related to postoperative proliferative complications, such as capsular contraction syndrome, suggesting that the particular microenvironment of the anterior chamber is extremely important in the biological behavior of LECs. Is it possible that there is also a proliferative microenvironment in pediatric eyes?

Sauer et al.7 found elevated levels of inflammatory markers (interleukin 1β [IL-1β], interferon γ [IFN-γ], IL-6, IL-5, macrophage inflammatory protein 1α [MIP-1α], monocyte chemoattractant protein-1 [MCP-1], and interferon-inducible protein-10 [IP-10]) in the aqueous humor of eyes with congenital cataract, and demonstrated the importance of inflammatory cytokines in initiating the aberrant growth of LECs. However, compared with inflammation, little is known about how other cytokines work. Growth factors typically act as signaling molecules between cells, and play essential roles in regulating cellular proliferation, cell growth, and cellular differentiation, which may be closely related to the pathogenesis of VAO. Several studies have shown that the proliferation of the LECs remaining after surgery and their spread to the center of the visual axis can be mitigated by the inhibition of the epidermal growth factor receptor or the blockage of transforming growth factor β (TGF-β), which significantly alleviate capsular wrinkling, the reduction in vision, and capsular fibrosis. These findings suggest that the proliferative activities of growth factors contribute to the pathogenesis of VAO in children.8,9

In this study, we analyzed the growth factor profiles in aqueous humor samples collected from patients with congenital cataract and their biological functions on LECs to expand our understanding of the molecular mechanism of VAO formation after cataract surgery in pediatric patients.

Patients and Methods

The Institutional Review Board of the Eye and ENT Hospital of Fudan University, Shanghai, China, approved this study. The study was registered at www.clinicaltrials.gov (registration number: NCT03063216). Written informed consent was obtained from each patient or guardian before his or her participation. All procedures adhered to the tenets of the Declaration of Helsinki and were conducted in accordance with the approved research protocol.

Patients

Fifty-five eyes of 55 patients with congenital cataract who were scheduled for phacoemulsification, primary posterior capsulotomy, anterior vitrectomy, and intraocular lens (IOL) implantation at the Eye and ENT Hospital of Fudan University between November 2017 and April 2018 were prospectively recruited. Patients with other ocular diseases (eg, congenital glaucoma and persistent hyperplastic primary vitreous) or systemic metabolic disorders were excluded from the study. In the control group, 55 eyes of 55 patients with age-related cataract who underwent phacoemulsification with IOL implantation were recruited.

Aqueous Humor Acquisition

Aqueous humor samples (80 to 100 µL) were obtained through the site of paracentesis before the injection of viscoelastics during cataract surgery, by an experienced surgeon. The samples were immediately stored at −80°C until cytokine analysis.

Measurement of Aqueous Cytokine Concentrations

An antibody-based protein array (QAH-GF-1, Raybio; RayBiotech, Peachtree Corners, GA) was used for the simultaneous analysis of 40 specific growth factors in aqueous humor samples obtained from 15 patients with congenital cataract and 15 patients with age-related cataract. The assay was based on the sandwich immunoassay principle, and was performed according to the manufacturer's instructions. An aqueous humor sample was applied to each block and immobilized at different locations on the surface of the array membrane. The growth factors in the samples were captured by the corresponding antibodies and detected with a GenePix 4000B system (Axon Instruments, Foster City, CA) after the addition of biotinylated antibodies. After the primary screening, three growth factors (fibroblast growth factor 4 [FGF4], platelet-derived growth factor-AA [PDGF-AA], and neurotrophin 4 [NT-4]) were selected. To verify their concentrations, the aqueous humor samples obtained from 40 patients with congenital cataract and 40 patients with age-related cataract were tested with enzyme-linked immunosorbent assays (ELISAs) (RayBiotech).

Primary LEC Culture

Lens capsules were obtained from children with congenital cataract during continuous curvilinear capsulorhexis of cataract surgery. The capsular membranes were then cultured in complete medium using the primary culture method, as previously described.10 The culture medium was replaced every other day. After the cells reached 70% to 90% confluence, the cells were digested with pancreatin and stored at −80°C or in liquid nitrogen until further analysis.

Culture of Human LEC Cell Line

The human LEC cell line SRA01/04 (abbreviated to SRA in this study) was authenticated with short-tandem-repeat profiling (Shanghai Biowing Applied Biotechnology, Shanghai, China). The SRA cells were cultured in antibiotic-free Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) in the presence of 10% fetal bovine serum (FBS; Gibco) in an incubator at 37°C under 5% carbon dioxide.

Cell Counting Kit-8 (CCK8) Assay

To determine the influence of FGF4 treatment on the proliferation of LECs, Cell Counting Kit-8 assays (CK04; Dojindo Molecular Technologies, Rockville, MD) were conducted. The cells were maintained in culture medium containing 0, 2.5, 15, or 50 ng/mL of FGF4. After 24, 48, 72, 96, or 120 hours, the CCK8 analysis was performed in accordance with the manufacturer's instructions.

EdU Labeling

5-Ethynyl-2′-deoxyuridine (EdU) is a thymi-dine analogue that can be inserted into duplicating DNA to quantify proliferating cells. Cells were seeded in a 96-well plate at a density of 1 × 104 cells per well, and treated with 0, 2.5, 15, or 50 ng/mL FGF4 in triplicate samples. The EdU experiment was performed with an EdU assay kit (C10310-1; RiboBio, Guangzhou, China), according to the manufacturer's instructions. Briefly, the EdU solution was added to each well and incubated for 2 hours. The cells were washed, immobilized, decolorized, and stained with Apollo staining solution, and then with Hoechst 33342. For each group, three replicate wells were observed under a microscope and the cell images were captured immediately.

Transwell Assay

Primary LECs were seeded in the upper chamber on Transwell inserts (#3422; Corning, Kennebunk, ME) with a pore size of 8 µm in 100 µL of serum-free medium, and the lower chamber was filled with 800 µL of culture medium containing 10% fetal bovine serum (FBS). After incubation for 24 hours, the cells invading the lower chamber were fixed with 4% paraformaldehyde and stained with crystal violet. The numbers of cells in three random fields per chamber were counted under microscopy.

Wound Healing Assay

SRA cells were seeded in six-well plates. After treatment with FGF4 (0, 2.5, 15, or 50 ng/mL) for 24 hours, the cells formed a confluent monolayer. A linear scratch was made with a 10 µL pipette tip. The wounded monolayers were washed with phosphate-buffered saline (PBS) to remove any detached cells and debris, and complete medium containing 2% FBS was then added. The ability of the SRA cells to close the wounded space was used to assess their migration ability. The width of the “wound,” determined with light microscopy, was recorded at 0 and 24 hours. Three replicate wells were analyzed for each group.

Fluorescent Immunohistochemical Analysis of the Effects of FGF4 on α-smooth Muscle actin (α-SMA) Expression

SRA cells were cultured for 24 hours with different doses of FGF4 (0, 2.5, 15, or 50 ng/mL), and then fixed with 4% paraformaldehyde. The pretreated cells were blocked with 5% bovine serum albumin and incubated with a rabbit anti-α-SMA monoclonal antibody (1:100; #19245; CST, Framingham, MA) at 4°C overnight, and then with an Alexa-Fluor-555-labeled goat anti-rabbit IgG antibody (1:1,000; #A31572; Invitrogen, Carlsbad, CA) at 37°C for 60 minutes. The α-SMA staining was assessed with a fluorescence microscope. Six to eight fields were selected in each sample, and at least six cells in each field were analyzed. The ImageJ software (National Institutes of Health, Bethesda, MD) was used to quantify the fluorescence intensity of α-SMA in the cells.

Phalloidin Staining

Phalloidin is a highly selective bicyclic peptide that binds all variants of actin filaments, and is therefore used to stain F-actin filaments. In this experiment, phalloidin conjugated to the fluorescent dye iFluor 555 (40737ES75; Yeasen Biotechnology, Shanghai, China) was used to label F-actin. The working solution of iFluor 555 phalloidin was prepared as a 1:1000 dilution with dimethyl sulf-oxide. The primary LECs were obtained from capsulorhexis in patients with both congenital cataract and age-related cataract during cataract surgery. After the primary LECs reached 60% confluence, the cells were washed twice for 10 minutes each with prewarmed (37°C) PBS. The cells were then incubated with PBS containing 0.1% Triton X-100 for 10 minutes to increase their permeability. The cells were washed twice with PBS and incubated in a working solution of phalloidin for 60 minutes. After the cells were washed three times in PBS for 10 minutes each, they were observed under a microscope (DMI 3000B; Leica, Heidelberg, Germany).

Statistical Analysis

The SPSS software (v.21.0; SPSS, Inc., Chicago, IL) was used for all data analysis. All data are expressed as means ± standard deviations. All comparisons of growth factors in the congenital cataract and age-related cataract groups were made with the Student's t test, and the nonparametric Wilcoxon ranked-sum test was used to examine the results of the Transwell assay. The correlation between age at congenital cataract surgery and FGF4 levels was determined with Pearson's bivariate correlation test. All P values were two-sided, and values of less than .05 were considered statistically significant. The cytokine antibody array data were also controlled with the Bonferroni correction. Because there were 40 different comparisons, a P value of .00125 (ie, P = .05/40) was considered statistically significant after the Bonferroni correction.

Results

Baseline Characteristics

In total, 55 patients with congenital cataract and 55 patients with age-related cataract were enrolled in the study. As shown in Table 1, the mean age was 3.61 ± 2.18 years (range: 6 months to 8 years) in the congenital cataract group and 63.62 ± 12.1 years (range: 49 to 77 years) in the age-related cataract group (P < .001). The mean axial length in the congenital cataract group was significantly shorter than that in the age-related cataract group (P = .002).

Patient Baseline Characteristics

Table 1:

Patient Baseline Characteristics

Growth Factor Profiles in the Aqueous Humor

The anterior segment photograph of a 2-year-old child was obtained during the secondary IOL implantation (Figure 1A). Phacoemulsification combined with anterior vitrectomy was performed 12 months previously, and obvious capsular fibrosis, proliferation, and VAO were shown. Compared with the anterior segment photograph of the surgically treated eye at 12 months after cataract surgery in a 63-year-old adult (Figure 1B), a proliferative status in eyes with congenital cataract was suggested.

(A) Anterior segment photograph of a 2-year-old child who underwent phacoemulsification with anterior vitrectomy 12 months ago showed proliferation (asterisk) and capsular fibrosis (black arrows). (B) Anterior segment photograph at the 12-month follow-up in a 63-year-old adult. (C) Comparison of fibroblast growth factor 4 (FGF4) levels in the aqueous humor of 40 patients with congenital cataract (CC) and 40 patients with age-related cataract (ARC). (D) Scatterplot of FGF4 concentrations in the aqueous humor of 40 patients with CC (correlation: r = −0.58, P < .001, r2 = 0.331). Pearson's bivariate correlation analysis was used to determine the relationship between the age at primary cataract surgery and FGF4 level. **P < .001.

Figure 1.

(A) Anterior segment photograph of a 2-year-old child who underwent phacoemulsification with anterior vitrectomy 12 months ago showed proliferation (asterisk) and capsular fibrosis (black arrows). (B) Anterior segment photograph at the 12-month follow-up in a 63-year-old adult. (C) Comparison of fibroblast growth factor 4 (FGF4) levels in the aqueous humor of 40 patients with congenital cataract (CC) and 40 patients with age-related cataract (ARC). (D) Scatterplot of FGF4 concentrations in the aqueous humor of 40 patients with CC (correlation: r = −0.58, P < .001, r2 = 0.331). Pearson's bivariate correlation analysis was used to determine the relationship between the age at primary cataract surgery and FGF4 level. **P < .001.

In the screening stage, the growth factor profiles in the aqueous humor samples were compared between the age-related cataract and congenital cataract groups using an antibody array technique. The levels of FGF4, NT-4, and PDGF-AA in the aqueous humor were significantly higher in the congenital cataract group than in the age-related cataract group (all P < .0012, Student's t test with the Bonferroni correction; Table A, available in the online version of this article).

Protein Levels in Aqueous Humor Samples Determined With the RayBio Human Cytokine Antibody Array (Primary Screening), Mean ± Standard Deviationa

Table A:

Protein Levels in Aqueous Humor Samples Determined With the RayBio Human Cytokine Antibody Array (Primary Screening), Mean ± Standard Deviation

To confirm the screening results, the concentrations of several growth factors (FGF4, NT-4, and PDGF-AA) in the aqueous humor were measured with ELISAs. In Figure 1C, FGF4 expression was significantly greater in the congenital cataract group than in the age-related cataract group (2,270.90 ± 1,982.21 and 415.34 ± 275.37 pg/mL, respectively; P < .001, Student's t test). The concentrations of NT-4 and PDGF-AA were below the detection range of the ELISA kits. The FGF4 expression levels across all 40 patients with congenital cataract correlated negatively with the age at primary cataract surgery, with a correlation coefficient of r = −0.58 (P < .001, r2 = 0.331; Figure 1D), suggesting that the microenvironment in the aqueous humor changes with age.

Cell Proliferation (CCK8, EdU)

To clarify the effects of FGF4 on LEC proliferation, the SRA cell line was treated with FGF4 at a concentration of 0 (defined as the control group), 2.5, 15, or 50 ng/mL for 24, 48, 72, 96, or 120 hours. The absorbance values increased in a dose-dependent manner, as shown by the results of the CCK-8 assay (Figure 2A). The CCK-8 results did not differ markedly between the SRA cells treated with 2.5 ng/mL of FGF4 and the control cells for up to 5 days in culture (both P > .05), whereas between days 3 and 5, the values increased significantly for cells treated with 15 or 50 ng/mL of FGF4 (all P < .05). The absorbance values of the CCK-8 assay were also time-dependent (Figure 2A). The results of the EdU experiment are illustrated in Figures 2B–2C. The proportion of EdU-positive cells increased significantly and dose-dependently in the SRA cells treated with FGF4 compared with the proportion in the control cells. The results of these two assays indicate that the addition of 15 or 50 ng/mL FGF4 to the culture medium signifiantly stimulated the proliferation of SRA cells.

Effects of different concentrations of fibroblast growth factor 4 (FGF4) (2.5, 15, or 50 ng/mL) on the proliferation of human LEC cell line SRA01/04 (SRA) cells in vitro. (A) Compared with the control (0 ng/mL), cell viability increased significantly in cells treated with 15 or 50 ng/mL of FGF4, when measured with the Cell Counting Kit-8 (CCK8) assay (P < .05 from days 3 to 5). (B, C) SRA cell proliferation was measured with 5-ethynyl-2′-deoxyuridine (EdU). All cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and EdU labeling indicated replicating cells. The proportion of proliferating cells increased as the FGF4 concentration increased. Bar = 100 µm. OD = optical density. *P < .05, **P < .001.

Figure 2.

Effects of different concentrations of fibroblast growth factor 4 (FGF4) (2.5, 15, or 50 ng/mL) on the proliferation of human LEC cell line SRA01/04 (SRA) cells in vitro. (A) Compared with the control (0 ng/mL), cell viability increased significantly in cells treated with 15 or 50 ng/mL of FGF4, when measured with the Cell Counting Kit-8 (CCK8) assay (P < .05 from days 3 to 5). (B, C) SRA cell proliferation was measured with 5-ethynyl-2′-deoxyuridine (EdU). All cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and EdU labeling indicated replicating cells. The proportion of proliferating cells increased as the FGF4 concentration increased. Bar = 100 µm. OD = optical density. *P < .05, **P < .001.

Cell Migration

In the wound healing assay, the migration rates of SRAs treated with 15 ng/mL of FGF4 (70.5% ± 3.1%) or 50 ng/mL of FGF-4 (69.6% ± 0.6%) were significantly higher than the control rate (42.1% ± 2.0%, both P < .001; Figures 3A–3B). Contrary to the results of the Transwell assay, treatment with a low dose of FGF4 (2.5 ng/mL) also induced significant cell migration (64.4% ± 3.4%, P < .001; Figures 3A–3B). In the Transwell migration assay, treatment with 15 or 50 ng/mL of FGF4 significantly accelerated SRA migration (115 ± 12 invading cells per field or 118% ± 13% invading cells per field, respectively) relative to that in the control group (77% ± 2% invading cells per field, both P < .001; Figures 3C–3D).

Human LEC cell line SRA01/04 (SRA) cells were treated with different doses of fibroblast growth factor 4 (FGF4) (2.5, 15, or 50 ng/mL) for 24 hours. (A) Effect on cell migration was measured with a wound-healing assay. (B) Migration rates of SRA cells are presented as means ± standard deviation (SD), and the migration of untreated control cells and cells treated with different levels of FGF4 differed significantly (**P < .001). (C) Representative images of SRA cells during Transwell migration. After migration and staining with crystal violet, images of the migrated cells (purple stained) were taken under a microscope. Bar = 100 µm. (D) Quantification of cell migration is presented as mean ± SD (of 3 microscopic fields at 100× total magnification; ** P < .001).

Figure 3.

Human LEC cell line SRA01/04 (SRA) cells were treated with different doses of fibroblast growth factor 4 (FGF4) (2.5, 15, or 50 ng/mL) for 24 hours. (A) Effect on cell migration was measured with a wound-healing assay. (B) Migration rates of SRA cells are presented as means ± standard deviation (SD), and the migration of untreated control cells and cells treated with different levels of FGF4 differed significantly (**P < .001). (C) Representative images of SRA cells during Transwell migration. After migration and staining with crystal violet, images of the migrated cells (purple stained) were taken under a microscope. Bar = 100 µm. (D) Quantification of cell migration is presented as mean ± SD (of 3 microscopic fields at 100× total magnification; ** P < .001).

Cytoskeletal and Cell Transformation in SRA Cells

The α-SMA is a marker of fibroblast differentiation, and was measured to investigate the role of FGF4 in cell transformation. As shown in Figure 4, stimulation with 2.5, 15, or 50 ng/mL of FGF4 significantly increased the intensity of α-SMA staining compared with the control level (P < .05). Supplementation with 15 ng/mL of FGF4 increased the expression of α-SMA more than the other two concentrations.

Fibroblast growth factor 4 (FGF4)–induced fibroblast transformation. (A) Fluorescent immunohistochemistry using a specific anti-α-smooth muscle actin (SMA) primary antibody and an Alexa-Fluor-555-conjugated secondary antibody demonstrated the fibroblast transformation induced by different concentrations of FGF4 (2.5, 15, or 50 ng/mL). Nuclei were stained with DAPI. Bar = 20 µm. (B) The induction effect was quantified and α-SMA fluorescence values were calculated as fold changes relative to the control. Data presented are means ± standard deviation (*P < .05, **P < .001).

Figure 4.

Fibroblast growth factor 4 (FGF4)–induced fibroblast transformation. (A) Fluorescent immunohistochemistry using a specific anti-α-smooth muscle actin (SMA) primary antibody and an Alexa-Fluor-555-conjugated secondary antibody demonstrated the fibroblast transformation induced by different concentrations of FGF4 (2.5, 15, or 50 ng/mL). Nuclei were stained with DAPI. Bar = 20 µm. (B) The induction effect was quantified and α-SMA fluorescence values were calculated as fold changes relative to the control. Data presented are means ± standard deviation (*P < .05, **P < .001).

To explore the effects of FGF4 on cytoskeletal assembly, we examined the expression of F-actin in primary LECs cultured from the anterior lens capsules of patients with congenital cataract and age-related cataract using FITC–phalloidin staining. Based on the values for α-SMA fluorescence, we selected 15 ng/mL of FGF4 to investigate cytoskeletal protein staining. Representative micrographs obtained with confocal immunofluorescence microscopy are shown in Figure 5. Treatment with FGF4 induced the reorganization of the actin cytoskeleton and thus changed the morphology of primary LECs from both congenital cataract and age-related cataract (Figure 5).

Immunolocalization of cytoskeletal protein (F-actin) in the fibroblast growth factor 4 (FGF4) treatment group and control group. (A) Addition of FGF4-induced shape changes in lens epithelial cells (LECs) from children with congenital cataract. (B) Addition of FGF4 induced shape changes in LECs from patients with age-related cataract. White arrows indicate elongated cells. Scale bars = 100 µm.

Figure 5.

Immunolocalization of cytoskeletal protein (F-actin) in the fibroblast growth factor 4 (FGF4) treatment group and control group. (A) Addition of FGF4-induced shape changes in lens epithelial cells (LECs) from children with congenital cataract. (B) Addition of FGF4 induced shape changes in LECs from patients with age-related cataract. White arrows indicate elongated cells. Scale bars = 100 µm.

Discussion

Congenital cataract is an important cause of lifelong visual impairment and remains a significant health care burden in 0.01% to 0.15% of children worldwide.11,12 The physiological and anatomic features of children's eyes, which are still growing, make the management of congenital cataract specific. Although primary posterior capsulectomy and anterior vitrectomy are now performed during primary congenital cataract surgery, VAO still occurs in 40% to 60% of the treated eyes. VAO is the most common postoperative complication, and prevents light entering the eye and thus affects visual development. The lens is an avascular structure surrounded by a microenvironment composed of the aqueous humor and vitreous. The cytokine levels in this microenvironment vary significantly with different eye conditions, such as high myopia, glaucoma, uveitis, and diabetic retinopathy.6,13–15 After cataract surgery, LECs are no longer protected by the capsular membrane and are exposed to the aqueous humor. Therefore, the aqueous humor microenvironment may correlate with VAO development. In the current study, significantly higher levels of FGF4 were detected in the aqueous humor of patients with congenital cataract than in those with age-related cataract. Therefore, FGF4, which stimulates cell proliferation, migration, and fibroblast transformation (Figures 25), may be a crucial factor in postoperative VAO formation.

VAO is a fibrotic condition initiated by an inflammatory response that results from the tissue damage caused by cataract surgery, combined with a foreign matter reaction toward the implanted IOL.16,17 The development of VAO has been analyzed with regard to intraocular inflammation and the uveal and capsular biocompatibility of the intraocular lens.18 Previous studies have shown elevated concentrations of inflammatory cytokines (IL-1β, tumor necrosis factor β, and IL-6) in the aqueous humor of patients with congenital cataract, which correlated with posterior capsular opacification.7,19 The levels of other proinflammatory cytokines, such as IL-1, IL-2, IL-4, IL-18, and IFN-γ, were also considerably higher in children than in adults.20 Therefore, an inflammatory state in eyes with congenital cataract was implied, which could stimulate the proliferation and epithelial-mesenchymal transition of LECs, leading to the development of VAO.1

The VAO response is a consequence of the proliferation, migration, and transdifferentiation of the residual LECs on the capsule or anterior vitreous membrane, which may grow and ultimately encroach on the visual axis. Light scattering changes are induced, giving rise to secondary visual loss.21 It has been demonstrated that primary congenital cataract surgery performed at an earlier age is a greater risk factor for subsequent VAO formation. Considering the growing status of young eyes, we suggest that the high prevalence of postoperative VAO is associated with increased levels of growth factors in the anterior chamber. We screened the aqueous humor for 40 growth factors for the first time, and found significantly higher levels of FGF4 in patients with congenital cataract than in those with age-related cataract. Moreover, the FGF4 levels correlated negatively with the age at primary cataract surgery, indicating that it may be a cytokine associated with young developing eyes. Previous studies have reported the overexpression of TGF-β1 and TGF-β2 during the development of VAO, and the signaling proteins downstream from the TGF-β receptors have been identified as druggable targets for VAO.5 TGF-β regulates many aspects of cellular function, including cell growth, differentiation, inflammation, and wound healing.22–24 However, according to our data, TGF-β1 levels did not differ significantly between the congenital cataract and age-related cataract groups (1,693.5 ± 431.9 and 1,167.8 ± 316.7 pg/mL, respectively, P = .15). On the contrary, FGF4 may be a growth factor that correlates with the proliferation of residual LECs and postoperative VAO in young children. Comparison between congenital and age-related cataract groups cannot exclude the influence of age-related factors on the FGF4 levels. Indeed, the network of cytokines in the aqueous humor of a healthy infant is not known. For ethical reasons, it might be difficult to obtain samples from age-matched infants.

In this study, FGF4 treatment significantly accelerated LEC proliferation and migration, both dose- and time-dependently, in vitro. Therefore, we propose that the higher levels of FGF4 in the aqueous humor of young children stimulate cell proliferation and migration, which are crucial in the postoperative development of VAO. The FGF family consists of at least 23 members, which are involved in numerous physiological processes.25 FGF4 is an autocrine and/or paracrine growth factor that is essential in many cellular events, including cellular proliferation, differentiation, and survival. It has been reported that FGF4 increases the proliferation of neural progenitor cells, bone marrow mesenchymal stem cells, and embryonic stem cells, which indicates that FGF4 plays a major role in stimulating cell proliferation.26,27

Previous studies have suggested that FGF4 stimulates the proliferation of various types of cells in different ways, depending on the cell type.26,28 The current study demonstrates, for the first time, the effects of FGF4 on LEC proliferation and migration. Numerous studies have provided compelling evidence that FGF1 and FGF2 induce cell proliferation and migration in the eyes. The binding of FGF1 and FGF2 to their receptors leads to ligand-induced receptor dimerization, activating membrane-bound tyrosine kinase receptors. This leads to the activation of the PI3-kinase/AKT pathway and the mitogen-activated protein kinase pathway, which promote cell growth.29–32 However, the cellular mechanisms by which FGF4 affects the proliferation and migration of SRA cells requires further study.

We also found that the expression of α-SMA, an important marker of fibroblast transformation, increased significantly in SRA cells treated with FGF4, especially at concentrations of 15 and 50 ng/mL. Because cytoskeletal reorganization is thought to be critical for lens fiber cell elongation and migration, we examined the effects of FGF4 on actin cytoskeletal organization in primary LECs. The confocal microscopic analysis indicated that FGF4 markedly altered the cell shape. The addition of FGF4 to the culture medium induces cell fibrosis and disorganized F-actin alignment. In addition, similar morphological changes were observed in LECs from patients with both congenital cataract and age-related cataract. A common feature of fibrotic VAO is the loss of LEC integrity, associated with aberrant cellular proliferation and migration, and, most significantly, a change in the cellular morphology.33,34 This biological process, known as the epithelial-to-mesenchymal transition, has been shown to occur with the addition of FGF4 to LECs. Therefore, FGF4 may have utility as a biomarker for VAO formation. The characteristics of the epithelial-mesenchymal transition include the acquisition of a spindle-shaped cellular morphology, which is accompanied by the accumulation of α-SMA, the redistribution of actin stress fibers, and the loss of cell polarity.35

Our results are also consistent with the function of FGF4 in cancer. FGF4 treatment was shown to induce tumor metastasis in mouse experiments, and immunohistochemical staining of human and mouse tissue samples indicated that FGF4 induced an epithelialmesenchymal transition phenotype.26,36,37 Among the tested concentrations of FGF4, 2.5 ng/mL is closest to the physiologic condition, whereas 15 or 50 ng/mL induced a much more significant increase in cell proliferation and migration, as illustrated in Figures 23. However, a smaller increment from 2.5 ng/mL should provide more information on the progress of these cellular changes, and we will set a denser gradient in future studies.

In this study, we detected higher FGF4 levels in the aqueous humor of patients with congenital cataract than in those with age-related cataract and demonstrated the effects of FGF4 on LECs in the ocular microenvironment for the first time. FGF4 induces the proliferation, migration, fibroblast transformation, and cytoskeletal reorganization of LECs and morphologic changes in them, which suggests that FGF4 is crucial in the postoperative formation of VAO in patients with congenital cataract.

References

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Patient Baseline Characteristics

CharacteristicCongenital Cataract GroupAge-Related Cataract GroupP
Sex (M/F)30/2532/23.848
Age (years)3.61 ± 2.1863.62 ± 12.1< .001a
Axial length (mm)22.15 ± 1.4923.55 ± 0.77.002a

Protein Levels in Aqueous Humor Samples Determined With the RayBio Human Cytokine Antibody Array (Primary Screening), Mean ± Standard Deviationa

Growth FactorsCongenital Cataract (n = 15)Age-Related Cataract (n = 15)P
FGF-41,504.5 ± 325.2332.2 ± 72.1< .001b
NT-431.7 ± 0.215.2 ± 2.1< .001b
PDGF-AA129.8 ± 13.370.6 ± 21.2< .001b
AR26.1 ± 2.418.8 ± 5.2.053
BDNF4.3 ± 0.32.7 ± 1.3.044
bFGF697.4 ± 64.5699.2 ± 1.4.969
BMP-482.1 ± 30.644.6 ± 20.1.083
BMP-53,315.5 ± 9,898.4303.9 ± 308.3.326
BMP-7307.2 ± 726.860.9 ± 17.8.292
b-NGF0.1 ± 0.10.3 ± 0.9.549
EGF0.1 ± 0.10.1 ± 0.0.249
EGF R220.6 ± 7.6229.2 ± 27.3.753
EG-VEGF23.3 ± 21.59.2 ± 7.5.080
FGF-718.4 ± 2.813.8 ± 0.8.148
GDF-15104.4 ± 121.9142.5 ± 40.0.404
GDNF12.1 ± 4.46.2 ± 7.8.060
GH73.6 ± 13.640.5 ± 19.1.013
HB-EGF0.1 ± 0.00.0 ± 0.0.075
HGF296.3 ± 227.1289.5 ± 23.6.949
IGFBP-1448.7 ± 268.8241.3 ± 194.6.197
IGFBP-210,680.7 ± 1,167.48,834.4 ± 234.1.095
IGFBP-31,703.2 ± 1,313.12,391.6 ± 144.9.406
IGFBP-4254.7 ± 123.0171.8 ± 100.6.317
IGFBP-613,002.5 ± 946.112,295.2 ± 123.0.449
IGF-I350.0 ± 127.9304.3 ± 80.3.499
Insulin527.2 ± 1,042.9182.5 ± 26.8.290
MCF R1,038.3 ± 622.8786.5 ± 66.0.317
NGF R103.5 ± 147.440.2 ± 8.7.191
NT-389.4 ± 0.952.1 ± 2.0.015
OPG38.6 ± 25.556.6 ± 22.6.258
PIGF4.9 ± 3.51.5 ± 0.2.026
SCF5.3 ± 1.114.5 ± 1.4.305
SCF R376.9 ± 32.2366.2 ± 5.5.821
TGFa0.0 ± 0.00.0 ± 0.0.979
TGFb11,693.5 ± 431.91,167.8 ± 316.7.151
TGFb313.1 ± 42.34.7 ± 9.1.513
VEGF147.4 ± 98.8165.0 ± 133.6.711
VEGF R2171.8 ± 58.9148.6 ± 67.9.557
VEGF R315.1 ± 22.314.9 ± 9.3.988
VEGF-D23.4 ± 6.512.4 ± 2.5.055
Authors

From the Department of Ophthalmology, Eye Institute, and NHC Key Laboratory of Myopia, Eye and ENT Hospital, Fudan University, Shanghai, People's Republic of China; Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, People's Republic of China; and Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, People's Republic of China.

The authors have no financial or proprietary interest in the materials presented herein.

Drs. Zhang, Li, and Lu contributed equally to this work and should be considered as equal first authors.

Supported by research grants from the National Natural Science Foundation of China (Grant Nos. 81870642, 81970780, 81470613, and 81670835), the Shanghai High Myopia Study Group, the Shanghai Talent Development Fund (Grant No. 201604), the Shanghai Youth Doctor Support Program (Grant No. 2014118), and the Outstanding Youth Medical Talents Program of the Shanghai Health and Family Planning Commission (Grant No. 2017YQ011).

Correspondence: Xiangjia Zhu, MD, Department of Ophthalmology, Eye and ENT Hospital, Fudan University, 83 Fenyang Road, Shanghai 200031, People's Republic of China. E-mail: zhuxiangjia1982@126.com

Received: October 07, 2019
Accepted: February 17, 2020

10.3928/01913913-20200224-01

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