In the present study, we investigated the levels of 34 cytokines in aqueous humor from treatment-naïve eyes with nAMD, PCV and controls, and explored whether there were differences among the three groups. Our results revealed that the IL-8 and IL-12p70 were significantly higher in nAMD than in PCV, and that there were 9 cytokines levels increased associated with AMD and PCV. IL-8, IL-18, IL-21, IL-31, LIF, SDF1-α, FGF-basic, VEGF-A, VEGF-D levels were significantly higher in eyes with nAMD or PCV compared with control eyes, while BDNF, HGF, IP-10, MCP-1, IL-13 were significantly lower in nAMD and PCV in contrast with controls. nAMD group showed significantly difference with PCV group in higher aqueous concentrations of IL-8 and IL-12p70, which was different from previous studies [13, 14]. Sakurada et al. [13] reported biomarkers of inflammation, elevated CRP and IP-10, and suggested chronic inflammation might be involved in the pathogenesis of nAMD or PCV. However, Agrawal et al. [14] supported that there were no significant differences in cytokine levels observed between nAMD and PCV patients for aqueous humor, which was inconsistent with our findings. IL-8 are the chemokines produced by macrophages and other cells, which are primarily involved in the regulation of inflammatory responses. This result was consistent with previous study which revealed that elevated intraocular levels of IL‐8 and IL‐8 gene polymorphisms were associated with angiogenesis [15]. Balne et al. and Mimura et al. [10, 16] reported that aqueous humor cytokines IL-8 levels was significantly higher in nAMD and PCV patients than controls which was consistent with our findings. They may be the key cytokines to explain the differences of nAMD and PCV, and further studies are necessary to elucidate the pathogenetic role of IL-8 and IL-12p70.
We found that some cytokines levels in the aqueous humor of nAMD and PCV patients had seldom been reported in the past. Some pro-inflammatory and pro-angiogenic cytokines were increased in nAMD and PCV groups compared with controls, such as SDF1-α, IL-31, LIF, VEGF-D. SDF1-α, a potent stimulator of VEGF expression, acting as an angiogenic agent increased during inflammation, is the main candidate factor of neovascularization and vascular permeability [17, 18]. IL-31, an immunoregulatory protein belonging to the pro-inflammatory IL-6 cytokine family [19], might inhibit angiogenesis [19]. LIF, a member of the IL-6 family of cytokines, upregulated with inflammation having the function of both anti-angiogenic and pro-angiogenic, was reported to have the ability to protect the integrity of the vasculature and retinal away from degenerations [20, 21]. The function of these cytokines on the progress of the eyes of nAMD and PCV need further research in the future, which may offer alternative therapeutic approaches to treat visual defects associated with nAMD and PCV. VEGF-D and VEGF-A had been identified to play an important role in angiogenesis. The previous study reported that the high expression of VEGF-D, in addition to VEGF-A, exacerbated the angiogenic response of retinal pigment epithelium(RPE) cell in nAMD [22]. However, as far as we know, there was few research on the concentration of VEGF-D in PCV, let alone the comparison of aqueous humor levels of VEGF-D in three groups, even they seldomly distinguished VEGF family such as detecting VEGF-A and VEGF-D respectively [13, 16]. Our study reported that the mean aqueous humor concentration of not only VEGF-A but also VEGF-D tended to be higher in both nAMD and PCV than that in controls. According to these findings, we had reasons to suspect the possibility that VEGF-D expression in eye besides VEGF-A modify the ocular angiogenesis as angiogenic stimulators in both nAMD and PCV. Therefore, agent combined with anti-VEGFs such as OPT-302 (to inhibit VEGF-C and VEGF-D) may be one of the emerging anti-VEGF treatments in the future [23]. Nowadays, we should pay more attention to the function and concentration of VEGF-D in aqueous humor of nAMD and PCV. Thus, we may infer that development of procedures to neutralize elevated cytokines in the eye with nAMD or PCV may potentially be therapeutic targets, as anti-VEGF drugs to neutralize VEGF.
Some pro-inflammatory and pro-angiogenic cytokines had no change in nAMD and PCV groups compared with controls like IL-6. IL-6 may cause a breakdown of the blood-ocular barrier by inducing an increase of endothelial permeability, and promote CNV. While our study showed no differences between nAMD, PCV and control group, which is in agreement with prior research [24]. However, IL-6 was significantly higher in the AMD group than in the cataract group [10].
Some pro-inflammatory and pro-angiogenic cytokines were dramatically decreased in nAMD and PCV groups compared with controls, such as MCP-1, HGF, IL-13, IP-10. MCP-1, a potent factor that regulated the migration and infiltration of monocytes and macrophages, enhanced by pro-inflammatory molecules, and facilitated angiogenesis [25]. Previous studies have reported inconsistent results on the level of MCP-1. Some suggested that MCP-1 levels in the aqueous humor in eyes with nAMD or PCV were not significantly different from controls [10, 26, 27]. However, some investigators observed that MCP-1 levels in the aqueous humor showed statistically significant elevation in the AMD group compared with control group and then put forward that MCP-1 may be one of the future targets for the treatment of AMD [28-30]. Therefore, more research is still needed for the function and concentration of MCP-1. HGF was identified to stimulate the proliferation, migration, neovascularization and differentiation, which played an important role in ocular angiogenesis and increases leakage from retinal vessels [31]. Dramatically, in our study, aqueous humor concentration of HGF did show a significantly lower trend in nAMD and PCV patients compared with controls. IL-13 played pathophysiological roles in allergic inflammation and fibrosis formation. In the present study, IL-13 was significantly lower in the nAMD and PCV group than in the cataract group, while reports of aqueous humor IL-13 level in nAMD or PCV are still controversial according to recent reports. Fu and colleagues found that IL-13 of aqueous humor was significantly upregulated during AMD development, suggesting that IL-13 could have potent effects on the pathological process of this disease [12]. However, according to another research, aqueous humor levels of IL-13 showed no significant difference between AMD group and the cataract group [10], while there are few reports about IL-13 level in aqueous humor of PCV patient. Therefore, whether IL-13 makes sense in the pathogenic processes of nAMD and PCV or not still needs to be clarified in further research. IP-10 concentrations were reduced in the aqueous humor in eyes with nAMD and PCV in contrast to control group in the current study. However, some previous reports demonstrated that IP-10 expression was increased in AMD and PCV patients, highlighting the involvement of inflammatory pathways [13, 16, 24, 26, 32]. On the other hand, Funk et al. [33] reported that IP-10 concentrations in aqueous humor with AMD were similar to those in controls. This indicates that further studies are necessary to illuminate the role of IP-10 in the pathogenic process of nAMD and PCV.
Limitations of our study should be mentioned. Firstly, the main limitation of this study was the small number of subjects, which meant that our findings should be confirmed in a larger number of patients. Moreover, another limitation was that we did not compare the relationships between structure, function and aqueous humor cytokines concentrations, and further relative research is necessary to better understand the role of cytokines in the aqueous humor of nAMD and PCV. Besides, we didn’t take into account of the exact typing of the classic AMD or atypical AMD. Furthermore, as we reported above that inflammation and angiogenesis may have a common influence on the pathogenesis of nAMD and PCV, a treatment response targeting angiogenesis and inflammation should raise concern in the future study. Last but not least, there are maybe some other cytokines related to nAMD and PCV that was not covered in the present study, such that exploring new and more related-cytokines is needed in the future.