Angiogenin, FGF-α, and IL-36β have higher expression levels in aqueous humor of nAMD patients in comparison to cataract patients

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 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 monoclonal antibody fragment which binds all isoforms of VEGF-A 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].

Intraocular inflammatory condition of nAMD could be reflected by aqueous humor and vitreous fluid more directly and accurately than serum. Collecting aqueous humor is easier and safer than vitreous fluid. And it is reported that the aqueous levels of various cytokines (such as VEGF) are significantly correlated with their vitreous fluid levels [11]. Studying aqueous cytokines in nAMD patients 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 [12], monocyte chemoattractant protein-1 (MCP-1) [13], interleukin-8 (IL-8) [13], macrophage inflammatory protein (MIP)-1α [14], macrophage-derived chemokine (MDC) [14], and IFN-γ–inducible protein-10 (IP-10/CXCL10) [14]. 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 [13].

However, many cytokines that have different expressions 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 28 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. The 28 cytokines were chosen because they take part in the processes of inflammation, angiogenesis, or chemotaxis, and most of them have not been deeply studied in nAMD patients, except for VEGF-A, which was selected as a positive indicator control and is known to decrease after treatment of ranibizumab.

Pathologic circumstances, such as ischemia, hypoxia, or inflammation, were proangiogenic factors and could lead to the formation of CNV, corresponding cytokines take part in these processes. The present study investigated 28 relevant aqueous cytokines in nAMD patients and cataract patients. Before and after two consecutive monthly ranibizumab injections, the aqueous concentrations of three cytokines, including angiogenin, FGF-α, and IL-36β were significantly higher in eyes with nAMD compared to cataract eyes. As far as we know, it is the first time that the aqueous levels of IL-36β in nAMD patients have been studied. These consequences need to be confirmed and further studied.

Two consecutive monthly ranibizumab injections effectively reduced the aqueous concentrations of VEGF-A and PlGF in nAMD patients, compared with their basal levels. Consistent with the majority of previous researches [8], our study also found that ranibizumab injections reduced CMT, MRT-3 mm, and GLD. The high aqueous levels of the three cytokines in nAMD patients didn’t decrease with the injection of ranibizumab, because they are not the target of ranibizumab, it also reflects that these factors are steadily increased in nAMD eyes, not a coincidence.

In the current study, higher aqueous levels of inflammatory cytokine IL-36β in eyes with nAMD than cataract eyes were detected. Prolonged inflammation is usually detrimental and participates in nAMD [7]. Elevated expression of IL-36β was found in the aqueous humor of acute uveitis, which was involved in the autoreactive T-cell immune response [15]. The aqueous levels of IL-36β in nAMD patients were studied for the first time in the present study.

It is known that angiogenin and FGF-α participate in the process of angiogenesis. A previous study reported that aqueous angiogenin levels were significantly higher in the exudative AMD group than the cataract group [12], but another study found there was no difference in aqueous angiogenin levels between cataract and AMD patients [16]. Angiogenin could activate vessel endothelial cells and smooth muscle cells to facilitate angiogenesis, cell invasion, proliferation, and the formation of tubular structures [17]. Human choroid and retina cells synthesize and internalize angiogenin, which is localized to normal and pathologic vasculature in eyes with AMD [18]. Our study confirms the high expression of angiogenin in the aqueous humor of nAMD patients. FGF-α promotes angiogenesis and inflammation [19, 20]. FGF receptor signaling pathway in endothelial cells plays a critical role in diseases associated with aberrant vascular proliferation including nAMD [21]. The FGF/FGF receptor system could be a target for the development of anti-angiogenic therapies [20]. In a previous study, there was no significant difference in the aqueous FGF-α levels between the nAMD group and control group, but FGF-α concentrations in nAMD patients increased significantly after the intravitreal injection of bevacizumab [22]. In our study, FGF-α concentrations were significantly higher in nAMD patients before and after two consecutive monthly ranibizumab injections, in comparison to cataract patients, which deserves further study.

In the present study, VEGF-A, VEGF-C, and PlGF in the VEGF family were tested. VEGF-A, also called VEGF, exhibits a strong pro-angiogenic effect through binding to VEGFR-1 and VEGFR-2 [23‐25]. VEGF-A could be secreted not only by endothelial cells [23, 24, 26] but also by RPE cells [27] and Müller cells in the retina [28], in response to oxygen deprivation, which is one of the most likely reasons leading to the formation of CNV. Anti-VEGF-A medication for neovascular eyes has revolutionized treatment for nAMD patients and preserved their vision [29]. Some previous studies reported higher VEGF-A levels in the aqueous humor of patients with nAMD than in those with cataract [13, 30]. However, some other studies found no significant difference in the aqueous VEGF-A levels between cataract patients and active nAMD patients, or between cataract patients and early nAMD patients [16, 31, 32]. We found aqueous VEGF-A levels had no significant difference between nAMD patients and cataract patients in the present study. There were several reasons, first, in the present study, most of the nAMD cases were in inactive chronic states. Aqueous VEGF-A levels are positively related to the activity of CNV [16, 33]. Consistent with our conclusion, Muether et al. reported that the recurrence of CNV activity shown by SD-OCT is always preceded by an increase of aqueous VEGF and usually followed by loss of visual acuity in the further course [34]. Second, in the present study, half of our patients were type I CNV and PCV. Tong et al. found that VEGF levels in eyes with PCV were significantly lower than those in eyes with CNV [35]. It may reflect that PCV and other nAMD subtypes have different angiogenesis courses. Type I CNV, located below the RPE layer, may lead to the low aqueous VEGF level. However, whether VEGF-A is elevated in the aqueous humor of nAMD patients still needs further investigation.

The main limitation of the current study is the sample size was too small to perform subgroup analysis for differences in VEGF-A or other cytokine levels at baseline, and how these change with time. Second, the observation period was short. Thus, more patients are needed to be followed up for a longer time to confirm the roles of these cytokines in the pathogenesis of nAMD. Collecting aqueous humor of cataract patients as controls instead of healthy volunteers was another limitation of the current study.

The present study suggests that aqueous angiogenin, FGF-α, and IL-36β 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 three cytokines could be novel therapeutic targets for nAMD.