CCL1, IL-22, and IL-36β Have Higher Expression Levels in Aqueous Humor of Neovascular Age-Related Macular Degeneration Patients in Comparison to Cataract Patients CURRENT

Purpose: To investigate the aqueous humor cytokine expression profile in neovascular age-related macular degeneration (nAMD) patients before and ranibizumab treatments in comparison to cataract patients. Methods : This prospective study included 20 treatment-naïve nAMD eyes of 20 patients who received three consecutive monthly injections of ranibizumab. Aqueous humor samples were collected before the first (baseline), second (1 month later), and third (2 months later) injections. Controls were 20 age- and gender-matched cataract patients without any other ocular disease. The aqueous concentrations of 28 cytokines were measured using a multiplex bead assay. Central macular thickness (CMT) and maximum retinal thickness (MRT)-3mm were measured by spectral domain optical coherence tomography (SD-OCT). The greatest linear diameter (GLD) was measured by fundus fluorescein angiography (FA). Results: Nine cytokines in aqueous humor, including angiogenin, bone morphogenetic protein-9 (BMP-9), C-C motif chemokine ligand 1 (CCL1), CCL13, interleukin-8 (IL-8), fibroblast growth factor-acidic (FGF-α), IL-22, IL-36β, and vascular endothelial growth factor-C (VEGF-C) were significantly higher in nAMD patients in comparison to cataract patients, whether before or after two consecutive monthly ranibizumab injections. Compared with the nAMD patients’ basal levels, two consecutive monthly ranibizumab injections effectively reduced the aqueous concentration of VEGF-A, improved best corrected visual acuity (BCVA), as well as reduced the values of CMT, MRT-3mm, and GLD. Conclusions: Nine cytokines have higher expression levels in nAMD patients in comparison to cataract patients, whether before or after 2 months of ranibizumab therapy. These cytokines may have correlations with the pathogenesis of nAMD.


Introduction
Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly population.
Subfoveal geographic atrophy and choroidal neovascularization (CNV) cause severe visual loss in AMD patients. 1 CNV is the main feature of neovascular AMD (nAMD), in which the formation of new aberrant blood vessels leads to macular edema, hemorrhage, fibrosis, and visual impairment. 2 Although nAMD accounts for only approximately 20% of the overall incidence of AMD, this subtype of patients is most likely to have severe visual loss among AMD patients. 3 Polypoidal choroidal vasculopathy (PCV) is a common subtype of nAMD, which is characterized by branching choroidal networks with polyp-like aneurysmal dilation. 4 Drusen deposition, RPE hypertrophy, pigment extrusion, and photoreceptor degeneration followed by the invasion of choroidal blood vessels through the Bruch membrane into the retina all lead to nAMD. 5 Angiogenesis and the development of local inflammatory responses are two major known contributors to nAMD pathogenesis. 2,6 Vascular endothelial growth factor (VEGF) and many other cytokines are important mediators of inflammatory responses in nAMD patients. 2,7 Ranibizumab is a kind of anti-VEGF drug that can block VEGF to suppress both excessive angiogenesis and inflammation, improving the clinical appearance of nAMD patients. 8 However, recurrent or persistent CNV is still very common, despite repeated application of anti-VEGF agents, and the efficacy of only using anti-VEGF drugs is still debatable. 9,10 It is reported that activated VEGF receptors shed into the vitreous in eyes with nAMD. 11 The aqueous levels of various cytokines (such as VEGF) are significantly correlated with their vitreous fluid levels. 12 Studying cytokines in the aqueous humor of patients with nAMD is feasible and necessary to understand the pathogenesis of nAMD, and could provide ideas to develop new antibody drugs.
Previous research has reported that in the aqueous humor of eyes with nAMD, many cytokines are found at higher concentrations than in the eyes of cataract patients. These cytokines include angiogenin, 13 monocyte chemoattractant protein 1 (MCP-1), 14 interleukin-8 (IL-8), 14 macrophage inflammatory protein (MIP)-1α, 15 macrophage-derived chemokine (MDC), 15 and IFN-γ-inducible protein 10 (IP-10/CXCL10). 15 A previous study has reported that cytokines, including inflammatory factors, are involved in the pathogenesis of nAMD, and that these inflammatory factors respond to anti-VEGF agents differently. 14 However, many cytokines that have different expression in the eyes of nAMD remain unrecognized.
For example, there are few studies about the concentration of C-C motif chemokine ligand (CCL) in aqueous humor of nAMD patients. To fill these gaps in knowledge, we conducted the current analysis.
The purpose of the present study was to compare various cytokine levels in the aqueous humor of eyes with nAMD before and after treatments with ranibizumab, in comparison to cataract controls.
The differences in aqueous cytokines and clinical appearances caused by ranibizumab treatments were also studied.

Study design and approval
This study was a prospective observational study. Approval for the collection and research of human aqueous humor was permitted by the Ethics Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine. All patients were treated in accordance with the Declaration of Helsinki, and informed consents were obtained from all participants prior to their participation in the study.

Inclusion/Exclusion criteria
In the present study, 20 eyes of 20 consecutive patients with nAMD and 20 eyes of 20 consecutive patients with age-related cataract were included. All eyes were treatment-naïve.
All patients with nAMD or cataract were examined in a full ophthalmological examination, including best corrected visual acuity (BCVA) determined as the logarithm of the minimum angle of resolution (logMAR), slit-lamp biomicroscopy, intraocular pressure (IOP) measurement, dilated fundus examination, color fundus photography, and spectral domain optical coherence tomography (SD-OCT). In addition to the above examinations, participates in nAMD group were examined by fundus fluorescein angiography (FA) and fundus indocyanine green angiography (ICGA). SD-OCT was used to exclude patients with AMD in the cataract group.
The diagnosis of nAMD and the discrimination of type I and II CNV were confirmed by FA. The The inclusion criteria were: (1) older than 50 years; (2) treatment-naïve nAMD or cataract; (3) absence of concurrent ocular diseases. The exclusion criteria were: (1) pathologic myopia; (2) a history of treatment for nAMD, including intravitreal drug injection, photodynamic therapy, and steroid therapy; (3) previous intraocular surgery, except for cataract surgery (for nAMD patients, the cataract surgery had to have been performed at least 12 months prior to inclusion); (4) active inflammation, diabetes mellitus, use of immunosuppressive drugs and corticosteroids, localized and systemic malignant tumors were all excluded from this study.

Aqueous humor sample collection
All of the nAMD patients received three consecutive monthly intravitreal injections of 0.5 mg ranibizumab. Aqueous samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before of the third injection). At each injection, approximately 0.15 mL of aqueous humor was collected by anterior chamber limbal paracentesis with a 30-gauge needle, and then the injections of ranibizumab were performed through the pars plana. Immediately after collection, each aqueous humor sample was transferred to sterile plastic tubes and stored at -80°C until analysis.

Measurement of cytokines using multiplex analysis
Twenty-eight cytokines in aqueous humor samples were detected with a multiplex cytokine assay kit (R&D Systems, Minneapolis, MN, USA) using Luminex technology on a Bio-Plex MAGPIX system (Bio-Rad, Waltham, MA, USA) according to manufacturers' instructions. Standard curves for each cytokine were generated (in duplicate) by using the reference set of cytokine concentrations supplied in each kit. To avoid between-run imprecision, we measured cytokines in the samples from all patients in a single run. The sample concentration was calculated using a multi-parameter standard curve for each cytokine.
Statistical Analysis SPSS software (version 18.0) and R statistical language (version 3.6.0) were used to perform statistical analysis. P < 0.05 was deemed to be statistically significant. The statistics were presented by GraphPad Prism 5.
Patients' age were compared by student's t-tests, other quantitative data were analyzed by Mann-Whitney U tests or Wilcoxon signed rank tests. Qualitative data were compared using 2 tests. In the analysis of aqueous humor cytokines concentrations, Mann-Whitney U tests were used to compare the aqueous humor levels of cytokines between nAMD and cataract groups. Wilcoxon signed rank tests were used to compare aqueous humor levels of cytokines before the first ranibizumab treatment and before the third ranibizumab treatment in nAMD group. Multiple testing was corrected using the method of Benjamini and Hochberg.
12 of the 20 nAMD cases (60%) and 10 of the 20 control cases (50%) were men (P = 0.525). In the nAMD group, PCV was found in 4 eyes (20%), type I CNV was found in 6 eyes (30%), type II CNV was found in 10 eyes (50%), and there was no retinal angiomatous proliferation. The concentrations were presented as mean ± SD. †The concentrations of aqueous cytokines were tested by Mann-Whitney U tests (or Wilcoxon signed rank tests for nAMD at baseline vs. at month 3) which were used for comparison between two groups, multiple testing was corrected using the method of Benjamini and Hochberg. Before and after the treatments with ranibizumab, aqueous levels of 9 cytokines in nAMD patients were significantly higher than in cataract patients (   Table   S1). After two consecutive monthly ranibizumab injections but before the third ranibizumab injection, In the current study, higher levels of inflammatory cytokines IL-8, IL-22, and IL-36β in the aqueous humor in eyes with nAMD were measured, which reflects the inflammation-related parhogenesis of nAMD. Prolonged inflammation is usually detrimental and participates in nAMD. 7 For example, IL-1β and TNF-α secreted by macrophages promote angiogenesis of CNV at least in part by stimulating VEGF production in RPE cells. 16  VEGF-dependent or VEGF-independent angiogenesis, and IL-8 can also increase endothelial permeability. 23,24 Higher aqueous humor IL-8 levels in nAMD patients than in cataract patients were reported previously. 15,25,26 One previous study reported that IL-22 levels were significantly elevated in the serum of exudative AMD patients compared with controls, and C5a induced IL-22 expression in human CD4 + T cells. 27 Elevated expression of IL-36β was found in the aqueous humor of acute uveitis, involved in autoreactive T-cell immune response. 28 Angiogenin, FGF-α, and BMP-9 take part in angiogenesis. A previous study reported that aqueous angiogenin levels were significantly higher in the exudative AMD group than in the cataract group, 13 but another study found there was no difference in aqueous angiogenin concentrations between cataract patients and AMD patients at different stages. 29 Angiogenin may activate vessel endothelial and smooth muscle cells to promote angiogenesis, and may also facilitate cell invasion, proliferation, and formation of tubular structures. 30 Human choroid and retina cells synthesize and internalize angiogenin, which is localized to normal and pathologic vasculature in eyes with AMD. 31 In conclusion, studies have shown that angiogenin may play an important role in pathologic angiogenesis in nAMD.
Our study also confirms the high expression of angiogenin in the aqueous humor of nAMD patients.
FGF-α takes part in angiogenesis and inflammation. 32, 33 FGF receptor signaling pathway in endothelial cell plays critical role in diseases associated with aberrant vascular proliferation, such as age-related macular degeneration. 34 The FGF/FGF receptor system could be a target for the development of anti-angiogenic therapies. 33 In a previous study, there was no significant difference in the aqueous concentration of FGF-α between nAMD group and control group, but FGF-α concentration increased significantly after intravitreal injection of bevacizumab. 35 In our study, FGF-α concentration was significantly higher in nAMD patients before and after two consecutive monthly ranibizumab injections, in comparison to cataract patients, which deserves further study.
BMP-9 can suppress β-FGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis through ALK1. 36 In a research of spontaneous metastatic breast cancer mouse model, activating BMP-9 pathway could normalize tumor vessel in breast cancer. 37 Another study reported that both VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis. 38 In a research based on mouse xenograft model of human pancreatic cancer, simultaneous blockade of VEGF and BMP-9/10 signals significantly inhibited tumor angiogenesis, leading to delay of tumor growth. 39 From the above, the role of BMP-9 in angiogenesis is still undefined. In a previous study, BMP-9 did not show any significant difference between nAMD and cataract group, and there was no difference after intravitreal injection of bevacizumab in nAMD patients. 35 Our study found higher expression of BMP-9 in the aqueous humor of nAMD patients, compared with cataract patients, which needs further research in the future.
CCL1 and CCL 13 belong to a subgroup of chemotactic cytokines (chemokines) characterized by the presence of the CXC motif. CC chemokine receptors are critical mediators of chronic inflammatory response which are expressed by T cells and monocyte-macrophages, predominantly. CC chemokine receptors are potential pharmacological targets of chronic inflammation. 40 The present study found CCL1 and CCL 13 had higher aqueous concentrations in eyes with nAMD compared to cataract, significantly. Until now, there was only one previous research measured the aqueous concentration of CCL13 in wet AMD patients, but the concentrations were below the detection threshold, and so could not be measured. 41 In the present study, VEGF-A, VEGF-C, and PIGF in VEGF family were tested. VEGF-A, also called VEGF, is the most important and potent stimulator of angiogenesis 42 , which exhibits strong pro-angiogenic effect through binding to VEGFR-1 and VEGFR-2. [43][44][45] VEGF-A is also the predominant cytokine controlling the formation of neovascularization. VEGF-A could be secreted not only by endothelial cells 43,44,46 , but also by RPE cells 47 and Müller cells in the retina 48 , in response to oxygen deprivation, which is one of the reasons leading to the formation of CNV. Anti-VEGF-A medication for neovascular eyes has revolutionized treatment for nAMD patients and preserved their vision. 49 Some previous studies reported higher VEGF-A levels in the aqueous humor of patients with nAMD than in those with cataract. 14,50 However, some other studies found no significant difference in the aqueous VEGF-A concentration between cataract patients and active nAMD patients, or between cataract patients and early nAMD patients. 29,51,52 In our study, VEGF-A in aqueous humor had no significant difference between nAMD and cataract patients. There were several explications, first, from our data of FA, ICGA, and SD-OCT which were not shown, the activity of CNV in the nAMD group was low, and most of the cases were in inactive chronic state. Active CNV was defined as leakage seen on FA, as well as new macular intraretinal, subretinal hemorrhage, or fluid on SD-OCT. The amounts of visual recovery and retinal thickness improvement were not large, also suggesting that the patients were in prolonged state. Aqueous VEGF-A levels are positively related to the activity of CNV. 29 On the other hand, in our study, the concentration of VEGF-C in aqueous humor was significantly higher in nAMD than in cataract group. VEGF-C has a high affinity for VEGFR-3, which is expressed on endothelial lymphatic cells, promoting lymphangiogenesis. 45 VEGF-C also have weak affinity for VEGFR-2, which explicates its poor implication in angiogenesis. 44,45 Otani et al. reported that strong VEGF-C staining was found in most pigment-containing cells in all CNV specimens. 56 VEGF-C was confirmed to be markedly positive in the surgically removed RPE of a nAMD patient. 57 Until now, only one study reported the aqueous levels of VEGF-C in nAMD patients, although there was no significant difference in baseline VEGF-C levels between nAMD and cataract groups, VEGF-C showed a statistically significant increase after intravitreal injection of bevacizumab. 35 In a study of biomarkers related to cancer resistance to bevacizumab, VEGF-C was detected had higher serum concentration in nonresponders compared with responders to bevacizumab. 58 In renal and peritoneal fibrosis processes, TGF-β promotes lymphangiogenesis through the TGF-β/VEGF-C pathway. 59 As previously mentioned, in our study, the activity of CNV in the nAMD group was low, and most of the cases were in chronic state, in which circumstance, elevated VEGF-C may take part in the prolonged fibrotic processes of nAMD.
In conclusion, the present study investigated aqueous humor concentraions of 28 cytokines in eyes with nAMD and cataract controls. Aqueous angiogenin, BMP-9, CCL1, CCL13, IL-8, FGF-α, IL-22, IL-36β, and VEGF-C levels in nAMD eyes were significantly higher than in cataract eyes, both before and after injections of ranibizumab. These results may further confirm the inflammation-related pathogenesis of nAMD. Therefore, these nine cytokines could be novel therapeutic targets for nAMD.

Declarations
The authors declare that there is no competing interests regarding the publication of the present article. The datasets analyzed in the current study are available from the corresponding author for