Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss in developed countries. AMD may be classified as dry or wet, with geographic atrophy commonly seen with the former type and choroidal neovascularization (CNV) commonly seen in the latter type.1 Wet or neovascular AMD (nAMD) is a result of CNV, which is defined by the growth of new blood vessels from the choroid that extend into the subretinal or subretinal pigment epithelium space and eventually causing vision loss.2 Polypoidal choroidal vasculopathy (PCV) is frequently found in the Asian population, characterized by the presence of multiple polyp-like aneurysmal dilations of choroidal origin and a branching vascular network.2
Compared to nAMD, PCV is dissimilar in epidemiology, genetic heterogeneity, pathogenesis, natural history, and response to treatment.3 It remains unclear whether PCV and nAMD constitute 2 clinically different diseases or PCV is a peculiar phenotype of nAMD. Therefore, we aimed to further understand the disease mechanisms and the key differences by studying cytokines in nAMD and PCV patients, which may in turn facilitate the identification of disease-specific diagnostic markers for nAMD and PCV. Although the measurement of cytokine profiles in aqueous humor is expected to best reflect cytokine levels in ocular, the procedure is invasive. Thus, for purposes of a more accessible clinical tool for diagnosis and progression, measurement of cytokine levels in plasma is preferable.
Previous research reported that both PCV and nAMD were associated with a number of systemic immunological and angiogenesis alterations.4–6 Some increased cytokine levels in plasma with a link of PCV or nAMD exclusively, suggesting that the 2 disorders may have different molecular mechanisms.7,8 However, most existing studies on plasma markers of inflammation and angiogenesis in AMD rarely distinguish nAMD from PCV, even some kinds of cytokines in PCV or nAMD do not have altered levels of plasma compared to healthy controls;9–11 while a recent study reported differences between plasma proinflammatory cytokines possessed a positive but weak ability in discriminating nAMD from PCV, indicating that PCV differed from nAMD in terms of plasma inflammation profile.8
Therefore, the aim of this study was to further investigate markers of inflammation and angiogenesis in plasma of patients with PCV and nAMD and to identify differences in cytokine profiles between these diseases comprehensively.
Study Design and Ethics
The study was designed to sample fresh venous blood from nAMD, PCV, and cataract controls and isolate plasma to analysis the concentrations of 34 angiogenic and inflammatory cytokines by Luminex bead-based multiplex array, and to compare plasma levels of cytokines in patients with nAMD and PCV and in controls. This prospective study follows the tenets of the Declaration of Helsinki and was approved by the institutional review board. Oral and written informed consent was obtained from all participants after explaining the nature of the study and before participation.
All participants were examined using slitlamp biomicroscopy and digital fundus photography. PCV and nAMD were diagnosed with retinal angiography using fluorescein and indocyanine green. nAMD in the presence of active CNV membrane on fluorescence fundus angiography but without the evidence of polypoidal lesions in indocyanine green angiography was diagnosed. PCV was diagnosed in the presence of at least 1 polypoidal dilation of the vessel in indocyanine green angiography, with or without the presence of a branching vascular network, polyp pulsation, orange-red focal polyp-like structures, and a dome-shaped choroid protrusion elevating the retinal pigment epithelium. Controls were selected among the patients undergoing cataract surgery.
Participants were invited for participation if they met inclusion criteria, which had no immune dysfunction, nondiabetes, no use of systemic antimetabolite or anti-inflammatory therapy for any reason, previously untreated, and no unrelated ocular diseases such as any types of retinal diseases, pathological myopia, glaucoma, uveitis, vitreous hemorrhage, choroiditis, hereditary diseases, ocular trauma, and previous intraocular surgery.
Sample Extraction and Preparation
Peripheral venous blood (5 mL) was collected for estimation of plasma cytokines. All centrifugation and plasma isolation were completed within 4 hours. Lithium heparin stabilized blood samples were centrifuged at 3000 revolutions per minute (rpm) for 10 minutes at 4°C after which plasma was isolated and stored at −80°C for final quantification of plasma cytokines.
Measurement of Cytokines
The Luminex platform was applied for the analysis of 34 different cytokines associated with inflammation and angiogenesis in the clinical assays as per the manufacturer’s instructors and recommendations. Plasma levels of interleukin (IL)-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, vascular endothelial growth factor (VEGF)-A, VEGF-D, interferon (IFN)-α, IFN-γ, interferon inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1α, MIP-1β, monocyte chemotactic protein (MCP)-1, leukemia inhibitory factor (LIF), stromal-derived factor (SDF)-1-α, brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), placental growth factor (PIGF), basic fibroblast growth factor (FGF-basic), granulocyte-macrophage colony-stimulating factor (GMCSF), epidermal growth factor (EGF), eotaxin, regulated upon the activation of normal T-cell expressed and secreted (RANTES), platelet-derived growth factor-BB (PDGF-BB), nerve growth factor b (NGFb), stem cell factor (SCF), growth-related gene product-α (GROα), tumor necrosis factor (TNF)-α, and TNF-β were quantified. The Luminex plates were read with Magpix system (Luminex Corp.) following the manufacturer’s guidelines, and Bio-Plex manager 6.1 software (Bio-Rad Laboratories) with a 5-parameter curve-fitting algorithm was used to analyze the data.
All analyses were performed using the Statistical Package for the Social Sciences statistical software for Windows, version 16.0 (SPSS Inc). Each pair of group differences among PCV and nAMD patients and controls was analyzed using 1-way analysis of variance or nonparametric Mann-Whitney U test, depending on normality assumptions for continuous variables. Differences in baseline categorical variables among cases and controls were assessed using χ2 tests for proportions. Multivariate logistic regression analysis was performed to confirm the association of cytokines with the occurrence of disease (nAMD or PCV) after adjusting the age and gender. For all tests, P values <0.05 were considered statistically significant, except Mann-Whitney U test in which P<0.0167 was deemed statistically significant.
Among the 49 cases included in the analysis, 11 cases were diagnosed with nAMD, 24 cases had PCV, and 14 cases were controls. No statistical difference in the average age among all groups [nAMD=75.55±6.83 (mean±SD), PCV=67.46±8.79, controls=70.07±12.65 years old, respectively, all P>0.05]. The male and female ratios were 5:6, 21:3, and 4:10 in the nAMD, PCV, and control groups, respectively. Gender in nAMD and control groups showed difference with PCV group (P<0.05), while there was no statistical significance between the nAMD and control groups.
Table 1 shows the concentrations of the 34 cytokines in the plasma samples. Figure 1 shows concentrations of significantly differences expressed cytokines in the plasma among 3 groups with bar graphs. The plasma levels of LIF in the nAMD group were significantly higher than the PCV group (P=0.016). The levels of EGF, Eotaxin, GMCSF, MCP-1, MIP-1β, IL-21, IL-31, LIF, SDF1-α, VEGF-A, and VEGF-D in the plasma were markedly elevated in the nAMD and PCV groups in comparison with the control group (nAMD vs. control, P<0.0001, 0.001, <0.0001, 0.001, <0.0001, 0.012, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, respectively; PCV vs. control, P=0.002, <0.0001, 0.001, <0.0001, 0.003, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, respectively). In contrast, the level of PDGF-BB in the plasma was significantly lower in the nAMD and PCV groups than in the control group (nAMD vs. control, P=0.038; PCV vs. control, P=0.007). FGF-basic had an obvious increase in nAMD but not in PCV (P=0.006). HGF and IL-5 had a significant decrease in nAMD, while there was no obvious change in PCV (P=0.007, 0.033, respectively). IL-18 had an obvious decrease in PCV rather than nAMD (P=0.005).
TABLE 1 -
Levels of 34 Cytokines (Means±SD) in the Plasma Among 3 Groups
||(nAMD vs Control)
||(PCV vs Control)
||(nAMD vs PCV)
Bold values indicate P value with statistical significance (when data meet normal distribution, we use analysis of variance test which P<0.05 was deemed statistically significant, while we use * to represent un-normal distribution using Mann-Whitney U test which P<0.0167 was deemed statistically significant).
BDNF indicates brain-derived neurotrophic factor; EGF, epidermal growth factor; FGF-basic, basic fibroblast growth factor; GMCSF, granulocyte-macrophage colony stimulating factor; GROα, growth-related gene product α; HGF, hepatocyte growth factor; IFNα, interferon-α; IFNγ, interferon-γ; IL, interleukin; IP-10, interferon inducible protein-10; IL-12p70, interleukin-12p70; LIF, leukemia inhibitory factor; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α; MIP-1β, macrophage inflammatory protein-1β; nAMD, neovascular age-related macular degeneration; NGFb, nerve growth factor b; PCV, polypoidal choroidal vasculopathy; PDGF-BB, platelet-derived growth factor-BB; PIGF, placental growth factor; RANTES, regulated upon the activation of normal T-cell expressed and secreted; SCF, stem cell factor; SD, standard deviation; SDF1-α, stromal-derived factor 1-α; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.
Table 2 showed the association of plasma cytokines with the occurrence of nAMD or PCV. After adjusting for gender and age by multivariate logistic analysis, concentrations of MCP-1, VEGF-A, and VEGF-D were significantly higher in plasma of nAMD and PCV patients compared to controls (all P<0.05, times in nAMD: 3.5, 4.3, and 13.8, respectively, times in PCV: 4.1, 4.0, and 11.5, respectively), and concentration of PDGF-BB was significantly lower in plasma of nAMD and PCV patients than in control cases (all P<0.05, times in nAMD: 1.6, times in PCV: 1.7).
TABLE 2 -
Association of Plasma Cytokines With the Occurrence of nAMD or PCV (Multivariate Logistic Analysis)
|nAMD (vs. control)
|PCV (vs. control)
P values are adjusted for age and gender.
Figures in parentheses indicate 95% CIs.
MCP-1, monocyte chemoattractant protein-1; nAMD, neovascular age-related macular degeneration; OR, odds ratio; PCV, polypoidal choroidal vasculopathy; PDGF-BB, platelet-derived growth factor-BB; VEGF, vascular endothelial growth factor.
Figure 2 showed significantly differences in concentrations of MCP-1, PDGF-BB, VEGF-A, and VEGF-D between disease (nAMD/PCV) and controls in the plasma with dot graphs.
The major findings of the present study were as follows: (1) high levels of MCP-1, VEGF-A, and VEGF-D and low level of PDGF-BB were the characteristic profile of angiogenic and inflammatory cytokines in serum of nAMD and PCV patients; (2) concentration of LIF in the nAMD group differed from the PCV group. These results suggested that PCV and nAMD may have independent pathophysiological features.
It is widely accepted that the activation of proinflammatory cytokines and subsequent upregulation of angiogenic factors in aqueous humor played an important role in the development of nAMD and PCV.12 Many reports have described the analysis of cytokines in aqueous humor, inflammatory cytokines such as MCP-1, angiogenic factors such as VEGF, macrophage-derived chemokines, mediator of inflammation and angiogenesis such as GRO and MIP-α, had been shown to be related to nAMD and PCV when compared with control subjects.12–14 However, aqueous humor cytokine profiles did not show any significant differences among patients with nAMD and PCV.
In terms of plasma cytokine levels, some reports found no significant differences among nAMD, PCV, and control groups, supporting that local rather than systemic deregulation of cytokine level was present in nAMD and PCV.5,13 However, in our study, we evaluated a large panel of cytokine profiles in blood samples from nAMD and PCV patients, demonstrating that significant differences existed in plasma cytokine levels in patients with nAMD (EGF, Eotaxin, GMCSF, MCP-1, MIP-1β, IL-21, IL-31, LIF, SDF1-α, VEGF-A, VEGF-D, PDGF-BB, FGF-basic, HGF, and IL-5) and PCV (EGF, Eotaxin, GMCSF, MCP-1, MIP-1β, IL-21, IL-31, LIF, SDF1-α, VEGF-A, VEGF-D, PDGF-BB, and IL-18) compared to the control subjects. Although after adjusting for gender and age by multivariate logistic analysis, only remained concentrations of MCP-1, VEGF-A, and VEGF-D were significantly higher in plasma with nAMD and PCV compared to control eyes, and concentration of PDGF-BB was significantly lower in plasma with nAMD and PCV than in controls. We also found that nAMD and PCV were distinguished by upregulation LIF in plasma. It suggested that not only local but also systemic deregulation of cytokines levels were present in nAMD and PCV.
MCP-1, which was produced by macrophages, was also demonstrated to be a mediator of inflammation and angiogenesis in various ocular and systemic diseases, which was significantly higher in plasma of PCV and nAMD patients in our study.13 In previous studies of similar design, MCP-1 was similar in plasma of nAMD and PCV patients in contrast to that of the controls, and some reported decreased and increased levels.4,13
Whereas the reason for MCP-1 playing different roles in the development of PCV and nAMD remained unknown, one possibility was that MCP-1 was upregulated in both PCV and nAMD conditions but with different levels and time window which may further triggered different cell signaling pathways to activate different abnormal angiogenesis process. However, the increased expression of MCP-1 in PCV and nAMD patients raised a possibility that the inflammation could be involved in these diseases. Previous study concluded controversial conclusions on the role of inflammation in nAMD.4 Some concluded that inflammasome was harmful and some concluded that it was beneficial. Inflammation may play a dual role in nAMD. The role of inflammation and this hypothesis need to be verified in further studies.
The VEGF family comprises 7 members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PIGF). Among them, VEGF-A plays an especially important role in the pathogenesis of neovascularization (NV), and several anti-VEGF drugs had been demonstrated to provide benefit in patients with CNV due to nAMD and PCV.12,14 VEGF-D plays a role in angiogenesis, vascular permeability, and lymphangiogenesis.15 Research have shown that VEGF-D was a critical mediator of angiogenesis and increased in response to inhibition of VEGF-A, thus they reported a combination therapy with OPT-302 and a VEGF-A inhibitor may provide additive inhibition of CNV growth and vascular leakage in nAMD compared to selective VEGF-A blockade alone.15,16 Previous studies found the levels of VEGF-A and VEGF-D in aqueous humor elevated in nAMD and PCV patients compared to controls, plasma concentrations of VEGF in nAMD patients raised significantly, and VEGF-D was detected in human vitreous and higher expression in the retinal pigment epithelium of nAMD patients, but there were few research on plasma levels of VEGF-D in PCV patients.15,17,18 In the current study, we found significant elevations in plasma VEGF-A and VEGF-D in both nAMD and PCV patients, supporting the view that angiogenesis processes in the systemic circulation contribute to the initiation and progression of nAMD and PCV. However, studies on plasma VEGF levels have yielded inconsistent results. Although increased plasma VEGF levels were reported in a number of studies involving both nAMD and PCV patients, a similar study did not show a significant difference in plasma cytokine levels of VEGF in either nAMD or PCV patients that were supposed to be upregulated.13 Future studies need to validate whether upregulation of systemic VEGF levels present in nAMD and PCV patients.
PDGF-BB, one of the isoforms of PDGF, is a proinflammatory and proangiogenic cytokine, which has been implicated as a second contributor to subretinal NV.19 Dong et al20 found antagonism of PDGF-BB suppressed subretinal NV and enhanced the effects of blocking VEGF-A. Thus, novel dual-targeting antibody fragment that potently neutralized both VEGF-A and PDGF-BB was a strong rationale.21,22 PDGF-BB may be a second validated molecular target for treatment of ocular NV when the concentration increased. However, conclusions as to whether PDGF-BB concentration in peripheral blood elevated or not were not inconsistent. The levels of PDGF-BB had been showed to be highly expressed during angiogenesis in the plasma of patients with nAMD,21 while the concentrations of PDGF-BB in PCV had not been reported. However, a recent study reported that plasma levels of another isoform of PDGF, PDGF-AA, were downregulated in patients with nAMD but not PCV compared to healthy controls.13 It validated that the deregulation of systemic inflammatory markers were present in nAMD patients, which was consistent with our study. Further research on the plasma concentrations of PDGF-BB in nAMD and PCV are needed in the future.
LIF is a member of the IL-6 family of cytokines, expressed during inflammation. It has been reported to upregulate in response to different types of retinal diseases and possess various pharmacological effects, including promoting and inhibiting retinal vascular development, protecting the integrity of the vasculature, preventing retinal injury, and displaying neuroprotective properties.23–25 There were few studies on the levels of LIF in PCV and nAMD. The LIF levels in aqueous humor as we reported before increased in accordance with plasma LIF levels in our present study.17 However, the concentrations of LIF in plasma showed significant differences between nAMD and PCV, while the differences were not found in aqueous humor and other similar research.10,13,17 From the above, plasma LIF levels may be a novel predictive biomarker to distinguish PCV and nAMD. Further research will be needed to detect the LIF level.
There were several limitations of our study. First, the sample size was relatively small. Our study serves as a starting point for a possible relationship between diseases and cytokines, and an expanded sample size is needed to further investigate in the future. Moreover, we expect a research of larger sample size may reveal various immunological characteristics for different phenotypes of nAMD and PCV. Second, we disregarded gender match, leading to gender bias when compared the differences among groups. Third, the period of our study was short, and the alteration of plasma cytokines after intravitreal injection of anti-VEGF therapy need further observation in the future.
In conclusion, increased levels of systemic immune cytokine factors MCP-1, VEGF-A, and VEGF-D and decreased cytokine levels of PDGF-BB suggest an important role of inflammation and angiogenesis in the pathogenesis of nAMD and PCV. The dysregulation of LIF levels may be a systemic potential biomarker which can help unravel the differences between the 2 diseases. Moreover, understanding the changes in cytokine profile may contribute to the identification of new therapeutic targets.
The authors are thankful for the financial support of NSFC Grant No:81670879. The authors would like to express the gratitude to Yanchun Zhai for his help on guiding on statistics.
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