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 compared differences in 28 relevant aqueous cytokines between nAMD patients and cataract patients. Changes in the 28 intraocular cytokines concentrations after the use of ranibizumab were also studied. Before and after two consecutive monthly ranibizumab injections, the aqueous concentrations of nine cytokines, including angiogenin, BMP–9, CCL1, CCL13, IL–8, FGF-α, IL–22, IL–36β, and VEGF-C were significantly higher in eyes with nAMD compared to cataract control eyes. As far as we know, it is the first time that the aqueous concentrations of CCL1, IL–22, and 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 in nAMD patients, compared with their basal levels. Consistent with most previous studies,8 our study also found that ranibizumab injections improved BCVA, as well as reduced CMT, MRT–3mm, and GLD.
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 IL–8 is a potent inflammatory cytokine, a chemokine for migratory immune cells, an activator for neutrophilic granulocytes, and an effective proangiogenic factor. Younger age of nAMD onset is associated with the single nucleotide polymorphism (SNP) rs4073 in the IL–8 promoter region.17 Secretion of IL–8 is upregulated in response to various stimuli, including inflammatory marker CRP,18 complement pathways,19 and oxidative stress.20, 21 Activation of inflammation, complement pathways, and oxidative stress are all pathogenic factors of nAMD.6 RPE cells could produce higher levels of IL–8 under chronic oxidative stress, and IL–8 promotes both inflammation and angiogenesis, potentially leading to the development of CNV.22 IL–8 can induce 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 angiogenesis42, which exhibits strong pro-angiogenic effect through binding to VEGFR–1 and VEGFR–2.43–45 VEGF-A is also the predominant cytokine controlling the formation of neovascularization. VEGF-A could be secreted not only by endothelial cells43, 44, 46, but also by RPE cells47 and Müller cells in the retina48, 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, 53 Consistent with our conclusion, Muether et al. reported that monthly intravitreal injection of ranibizumab effectively reduces VEGF levels in aqueous humor of nAMD patients, the recurrence of CNV activity shown by SD-OCT are always preceded by increase of aqueous VEGF and usually followed by loss of visual acuity in the further course.54 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 nAMD.55 It may reflect that PCV and nAMD are distinct clinical entities and have different angiogenesis courses. Type I CNV, located below the RPE layer, may give rise to the low aqueous VEGF concentration.
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.