We investigated various cytokine and angiogenic factors associated with aqueous humor. The vitreous sample would be more appropriate than the aqueous sample because it better reflects the state of the retina in AMD patients. Nevertheless, obtaining samples from the vitreous is dangerous and may cause adverse effects, such as vitreous hemorrhage, retinal detachment, endophthalmitis, among others. There may be differences in the concentration of immune mediators between the aqueous and the vitreous humor. However, the collection of samples from the aqueous humor is safer and easier, and similar levels have been reported in both the aqueous and vitreous humors.[10–12] Immune mediators in the aqueous humor likely explain the presence of immune mediators in the vitreous humor. Thus, the results of our study suggest that the eyes of patients with neovascular AMD showed increased MCP-1, MIP-1α, MIP-1β, and VEGF concentrations in the vitreous.
MCP-1 is a potent chemotactic factor for monocytes and macrophages, and plays a critical role in angiogenesis and inflammatory processes.[13] MCP-1 attracts macrophages into the CNV lesion and assists with digestion of the RPE and Bruch’s membrane in the experimental models.[14, 15] MCP-1 also induces angiogenesis by recruiting other cells, such as tumor-associated macrophages. These cells, in turn, release growth and angiogenic factors, such as VEGF.[16] Our results showed significantly elevated aqueous humor levels of MCP-1 in patients with CNV versus controls and were in agreement with those of previous studies. MIP-1α and MIP-1β have been implicated in retinal inflammation, particularly in early T-cell-dependent stages.[17–19] Their production by T lymphocytes entering the tissue is advantageous in many inflammatory situations because they amplify inflammatory cell recruitment. However, this cascade effect is likely to exacerbate predisposing factors in susceptible individuals.[20] Consequently, T cell regulation of MIP-1α and MIP-1β production is important. An in vitro study has shown that RPE cells downregulate the levels of CCL3(MIP-1α) and CCL4(MIP-1β) production by T lymphocytes using the soluble mediators sCD54 and prostaglandin E2 (PGE2).[20] Yang et al. found that MIP-1a expression increased in the corneal neovascularization tissue of mice, suggesting that MCP-1 and MIP-1a is related to inflammation and neovascularization.[21] In addition, it was confirmed that MIP-1a was significantly increased in a study targeting the laser-induced CNV mouse model. MIP-1a was significantly correlated with the CNV lesion area, suggesting that the migration of MIP-1a-mediated macrophages could induce an inflammatory response and affect CNV formation.[22] Our novel results showed elevated aqueous humor concentrations of MIP-1α and MIP-1β in eyes with neovascular AMD. We believe that these findings are attributed to a disrupted RPE cell regulation because of degeneration and suggest that inflammatory factors may influence the pathogenesis of AMD.[23]
Quantitative analysis of OCT images is useful for the treatment strategy selection and for monitoring the biological response to treatment. It is possible to characterize individual CNV lesions according to the presence or number of sub-components. This could help investigate CNV heterogeneous lesions. Unfortunately, there is a lack of a reliable correlation between the role of intraocular cytokines and OCT-based parameters. We analyzed the differences in cytokine levels between wet AMD patients with SRF or PED and those without SRF or PED.
As described in the results, patients with CNV and SRF showed low concentrations of proinflammatory cytokines, such as IL-1α and GM-CSF. IL-1α is a proinflammatory cytokine that derives macrophages and induces acute inflammation.[24] Pathologically, IL-1α can stimulate RPE cells to secrete GM-CSF, which is a potent chemoattractant that recruits macrophages to the retina. IL-1α is also implicated in the pathogenesis of CNV. Blocking IL-1α receptors could inhibit the development of CNV in an experimental animal model.[25] Therefore, lower intraocular concentrations of IL-1α indicate lower levels of inflammation in CNV pathogenesis. Our results indicate that SRF may be associated with a positive prognosis because we found relatively low levels of proinflammatory cytokines in the SRF-positive group. Other studies have found that SRF may be associated with a benign disease course in patients with CNV lesions.[5] Moreover, in the SRF-positive group, we found a negative correlation between the concentration of IL-1α and the volume of SRF (Fig. 2).[26] A potential explanation for this correlation is that SRF could be suggestive of a functional providing RPE and photoreceptor survival, in contrast to vascular atrophy in the sub-RPE space. The presence of SRF may be suggestive of a less aggressive, perhaps even supportive, stage of CNV, rather than advanced destructive neurosensory ingrowth associated with intraretinal exudation. This is supported by the fact that treatment-refractory SRF was not detrimental to the vision outcome in a recent study.[5] Interestingly, positive effects of SRF on the visual outcome have also been reported in diabetic macular edema and retinal vein occlusion.[27, 28]
In the presence of PED on OCT, it was confirmed that IFN-γ and TNF-α values were significantly lower than those of the group without PED. In other animal studies, it has been argued that they may help maintain the outer blood-retinal barrier by providing fluid transport in RPE cells.[29, 30] Therefore, through the results of this study, it can be considered that PED may occur due to a decrease in these cytokines. Significantly smaller values were also measured in the type 1 CNV group than in the type 2 CNV group, and it is thought that additional research is needed on this result.
Our findings suggest that angiographic classification may be an important clue to determine the treatment strategy. Patients with type 1 CNV showed increased levels of MCP-1, MIP-1α, and MIP-1β, but not VEGF, compared to those with type 2 CNV. These results suggest that in patients with type 1 CNV, inhibition of VEGF alone may not be a sufficient treatment. The levels of VEGF and angiogenic inflammatory cytokines were not significantly increased in patients with type 1 CNV compared with the control group. Inhibition of the MCP-1, MIP-1α, and MIP-1β related cascade may be a promising advance in CNV treatment, especially for patients with type 1 CNV. Considering that platelet-derived growth factor (PDGF) is the main growth factor that stimulates the secretion of MCP-1,[31] PDGF inhibition could be a promising treatment for CNV, particularly in patients with type 1 CNV.
Moreover, we confirmed that the VEGF concentration of aqueous humor in type 2 CNV was higher than that of type 1 CNV or in the control group. It is thought that type 2 CNV patients have a higher VEGF concentration in the vitreous cavity and anterior chamber due to the growth of neovascularization in the subretina, and that their prognosis is worse. In fact, several studies also found that, after anti-VEGF injection treatment, type 2 CNV patients showed worse visual acuity and a poor responsiveness to injection when compared to type 1 CNV patients.[32, 33]
A limitation of this study is that the sample size was small; therefore, a larger multicenter prospective randomized study is required to clarify the effects of inflammatory factors in AMD. In addition, it would be good to analyze the sample size larger to confirm the correlation between quantitative data of OCT and aqueous humor cytokine. Moreover, since factors such as SRF and PED may be temporary, additional studies are needed to confirm the change in cytokine concentration according to changes in these factors after injection treatment in the same patient.