Purpose To investigate the association of posterior vitreous detachment (PVD) with aqueous levels of vascular endothelial growth factor (VEGF) and other inflammatory cytokines.
Methods These are prospective comparative studies. Subjects comprised 98 eyes for VEGF concentration and 80 eyes for other cytokines, which are normal except for cataract. PVD was examined by B-mode ultrasonography, and the subjects were divided into complete PVD group (PVD group) or the other group (without PVD group). At the beginning of cataract surgery, aqueous humour was collected and the concentrations of VEGF and other inflammatory cytokines were determined using ELISA and a multiplex cytokine assay, respectively. The concentrations of these cytokines were compared between the two groups.
Results Complete PVD was observed in 56 (57%) eyes for VEGF concentration analysis, and 51 (64%) eyes for the other cytokines analysis. The concentrations of VEGF, adjusted for the average age, axial length and gender distribution, was 47 pg/mL in the PVD group and 72 pg/mL in the without PVD group. The concentrations of IP-10, MCP-1, CXCL13 and CCL11 were 53, 450, 3.8 and 6.0 pg/mL in the PVD group, and 100, 560, 7.0 and 8.4 pg/mL in the without PVD group, respectively. Multiple regression analysis revealed that the logarithmic concentration of VEGF, IP-10, MCP-1, CXCL13 and CCL11 were significantly lower in the eyes with PVD than in those without PVD independently of age, sex and axial length (p=0.01, p=0.002, p=0.009, 0.006 and 0.03, respectively).
Conclusions PVD is related to the change in the multiple intraocular inflammatory cytokines.
- Aqueous humour
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The vitreous humour plays multiple roles in retinal diseases. Among its various functions, possible association of the status of vitreomacular interface with intraocular cytokines have drawn the attention of the investigators since it became apparent that vitreomacular attachment is associated with exudative age-related macular degeneration (AMD) and its treatment outcome.
First, it has become generally recognised that posterior vitreous attachment to the posterior pole is more frequently observed in eyes with exudative AMD than those without AMD.1–5 A previous study from our group also demonstrated that complete posterior vitreous detachment (PVD) is less frequently observed in typical AMD than in controls. In this study, the larger lesion size was associated with the absence of PVD in typical AMD.6 Second, some investigators suggest that PVD protects against exudative AMD and argue that intervention to induce PVD may be a treatment option for exudative AMD.7 In support of this idea, several studies have demonstrated that incomplete PVD or vitreomacular adhesion (VMA) may have a significantly adverse effect on the efficacy of intravitreal antivascular endothelial growth factor (anti-VEGF) treatment for exudative AMD by examining the visual and anatomical outcome of patients treated with off-label use of bevacizumab and/or ranibizumab.7–11 Although the relation has been statistically proven in these studies, it is still impossible to predict the prognosis of patients with AMD based on the presence or absence of PVD or VMA.
There are several hypotheses that explain the possible association of vitreomacular attachment with these retinal diseases.12 One possible explanation is that PVD is associated with higher oxygen concentration in the vitreous as demonstrated in laboratory experiments.13 The low oxygen level due to vitreomacular attachment exacerbates exudative AMD, because low oxygen concentration in the retina is a potent stimulus for the production of VEGF.14 Other than VEGF, it is currently accepted that inflammatory cytokines play an important role in AMD.15 However, the association of PVD and inflammatory cytokines has never been reported in humans to the best of our knowledge (PubMed Mesh search items: posterior vitreous detachment, vitreomacular adhesion, human). The current study investigated the association of PVD and the concentration of VEGF in the aqueous humour using ELISA. Furthermore, among the cytokines that have been shown to be associated with AMD,16 concentrations of the following cytokines in the aqueous humour were also determined using a multiplex cytokine assay; interferon-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase 9 (MMP-9), interleukin-6 (IL-6), interleukin-10 (IL-10), C-X-C motif chemokine ligand 1 (CXCL1), C-X-C motif chemokine ligand 12 (CXCL12), C-X-C motif chemokine ligand 13 (CXCL13), and C-C motif chemokine ligand 11 (CCL11).
The subjects consisted of 178 eyes of consecutive patients who visited the outpatient clinic of the Japan Community Health Care Organization, Tokyo Shinjuku Medical Center, between September 2010 and August 2012. Ninety-eight and 80 cataract eyes were for the measurement of VEGF concentration and multiple cytokine assays, respectively. Informed consent was obtained from all patients. Patients were excluded if they had received a treatment for ocular diseases or if they had any retinal diseases. Participants had a refractive error between −2 and +2 dioptres.
Posterior vitreous detachment
PVD was examined by B-mode ultrasonography (UD-6000, TOMEY, Aichi, Japan), similar to the published work of previous investigations.1 ,2 ,4 ,5 Briefly, the mobility of the posterior vitreous during ocular saccades was examined, using the ‘through the lid contact’ technique. If the posterior vitreous was detached from the retinal surface and motile with eye movements, the eyes were categorised as having complete PVD (PVD group), otherwise, the eyes were categorised as without PVD group. Without PVD group includes the eyes with partial PVD and no PVD.
Concentration of VEGF and inflammatory cytokines
At the beginning of cataract surgery, a sample of undiluted aqueous humour (usually, a volume of ca. 0.2 mL) was manually aspirated into a disposable syringe, immediately transferred to a sterile tube and stored at −80°C until assay. The concentration of VEGF was measured by ELISA using a kit (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer's instructions. The VEGF kit permitted the detection of two of the four VEGF isoforms (VEGF121 and VEGF165). Detection limit was 2.2 pg/mL.
The concentrations of the following cytokines were determined using a multiplex cytokine assay (Filgen, Aichi, Japan) according to the manufacturer's instructions; IP-10, MCP-1, MMP-9, CXCL12, IL-6, IL-10, CXCL1, CXCL13, and CCL11. Detection limits were 2.76, 0.95, 0.57, 2.87, 0.41, 0.15, 1.25, 0.76 and 0.43 pg/mL for IP-10, MCP-1, MMP-9, CXCL12, IL-6, IL-10, CXCL1, CXCL13 and CCL11, respectively. The measurement was performed twice for each sample, and an average and coefficient of variance (CV) was calculated.
Statistical analysis was performed using JMP Pro software V.11.0 (SAS Institute, Cary, North Carolina, USA). Categorical data were assessed using the χ2 test, and continuous variables were compared with Student's t test. The associations between baseline characteristic factors, such as age, sex, axial length, and the presence or absence of PVD, were examined by multiple regression analysis. Statistical models using log-transformed concentrations were examined because of the skewed distribution of this variable.
The demographic data of the patients in this assay are shown in table 1.
The values were estimated adjusting for the average age (73 years), axial length (23.5 mm) and gender distribution (male:female = 50:48). The concentration of VEGF was 47 (95% CI 36 to 59) pg/mL and 72 (95% CI 60 to 89) pg/mL in the eyes with PVD and without PVD, respectively (figure 1). Multiple regression analysis revealed that the logarithmic concentration of VEGF was significantly lower in the eyes with PVD than in those without PVD independently of age, sex and axial length (p=0.01).
Concentrations of the other cytokines
The demographic data of the patients are shown in table 2.
The estimated levels for each cytokine, adjusting for the average age (75 years), axial length (23.5 mm) and gender distribution (male:female = 37:43) are illustrated in figure 2. After adjusting for age, gender and axial length, the concentrations of IP-10, MCP-1, CXCL13 and CCL11 were 53, 450, 3.8 and 6.0 pg/mL, respectively, in the PVD group, and 100, 560, 7.0 and 8.4 pg/mL, respectively, in the without PVD group. Multiple regression analysis revealed that the logarithmic concentration of IP-10, MCP-1, CXCL13 and CCL11 were significantly lower in the eyes with PVD than in those without PVD independently of age, sex and axial length (p=0.002, p=0.009, 0.006 and 0.03, respectively). The concentration of MMP-9, IL-6, CXCL12, and CXCL1 was not different between the eyes with PVD and those without PVD. The CV was 5.30, 4.13, 10.9, 4.15, 5.23, 26.2, 7.01, 9.88 and 10.0% for IP-10, MCP-1, MMP-9, CXCL12, IL-6, IL-10, CXCL1, CXCL13 and CCL11, respectively. Because we were able to aspirate 0.2 mL of aqueous humour and 0.1 mL was necessary for ELISA and multiplex cytokine assay, respectively, we measured VEGF in one group of samples and multiple cytokines in another, and therefore, it was impossible to show the relation between VEGF and other inflammatory cytokines. However, we investigated the association of the concentrations of each cytokine and chemokine by multivariate correlation analysis. The results are shown in table 3.
Possible association of PVD on VEGF concentrations, and its effect on AMD
The vitreous gel recently has been found to have the important function of oxygen regulation and distribution within the eye. The preretinal oxygen tension is significantly higher in eyes with PVD and in vitrectomised eyes than in eyes without PVD.13 Incomplete PVD may result in relatively low levels of oxygen in the retina, and theoretically, this has been considered to result in the induction of VEGF. The current study clearly demonstrated that the aqueous VEGF was lower in eyes with PVD than in those without PVD, for the first time, in humans. Because aqueous humour levels of cytokines were reported to correlate with vitreous levels,17 it is rational to speculate the VEGF concentration in vitreous is higher in the eyes without PVD than in the eyes with PVD.
Inflammatory cytokines and PVD
The current study revealed that levels of IP-10, a member of the α chemokine family; MCP-1, a member of the CC chemokine family; CXCL13, a B cell chemoattractant; and CCL11, also a member of CC chemokine family were significantly higher in the eyes without PVD. The precise role of IP-10 in AMD remains to be ascertained. IP-10 has been demonstrated to be upregulated in laser-induced choroidal neovascularization (CNV) model mice, and has been shown to be angiostatic by a previous report from our laboratory.16 Recently, it has also been reported that serum IP-10 and CCL11 levels are elevated in patients with AMD.15 CCR3, a receptor of CCL11, has been reported to have an important role in choroidal neovascular AMD.18 CXCL13 has been reported to be upregulated in laser-induced CNV model mice,16 and in patients with AMD.19 CXCL13 also has an angiostatic effect in in vitro experiments.20 It has been reported that MCP-1 levels in aqueous humour were higher in patients with AMD than in the controls.21 MCP-1 is critically involved in macrophage infiltration into lesion in experimental choroidal vascularisation models.22 Although pathogenic association of PVD and the lower concentration of these cytokines remain to be clarified, of special note is that the concentrations of these inflammatory cytokines, as well as VEGF, are affected by the presence of PVD. It is tempting to speculate that these cytokines have a significant effect on the treatment outcome of exudative AMD, and possibly macular oedema, due to branch retinal vein occlusion.23
Limitations of the current study
There are several limitations to the present study. First, the vitreomacular interface was evaluated by means of B-mode ultrasonography, and the without PVD group was non-uniform, that is, including no PVD, peripapillary partial PVD, and parafoveal partial PVD. Second, the patients are from only one institution, and the current study does not represent Japanese patients generally. Third, we collected samples in two separate groups and measured VEGF in one group and other multiple cytokines in another. By the multiplex cytokine assay used in the current study, the concentration of VEGF could not be measured correctly, and ELISA was for the measurement. We were able to aspirate a maximum volume of 0.2 mL at a time, and 0.1 mL was necessary for ELISA and multiplex cytokine assay, respectively. It was therefore impossible to show the relation between VEGF and other inflammatory cytokines. Fourth, the reduction in viscosity following vitrectomy (and PVD) is known to lead to an increase in the diffusion of molecules in the vitreous cavity,24 ,25 and the concentration of the actual intravitreal concentrations of these cytokines has yet to be clarified. As we have shown, several aqueous humour cytokines are positively correlated, but together with the fact that the levels of cytokines except for IP-10, MCP-1, CXCL13 and CCL11 were not affected by the status of vitreomacular interface, we believe that rather than reduced viscosity of vitreous, the status of vitreomacular interface itself is associated with the concentration of these four cytokines. Lastly, the subjects were not patients with AMD, but those with cataract, and this study has not demonstrated the clinical significance of vitreomacular attachment in AMD, so further investigations in subjects with AMD are needed to elucidate exact association between posterior vitreous attachment and inflammation occurring in exudative AMD.
In conclusion, the current study demonstrated that the concentrations of VEGF, IP-10, MCP-1, CXCL13 and CCL11 were significantly high in the eyes without PVD in patients with cataract. The present study revealed that vitreomacular attachment is associated with changes of intraocular inflammatory cytokines.
HT and YN have contributed equally to this work.
Twitter Follow Hidenori Takahashi at @takaha4
Contributors HT and YF conceived the study and its design, acquired the data, drafted the article, critically revised the paper for important intellectual content, and obtained funding. YN and YY conceived the study and its design; acquired, analysed, and interpreted the data; critically revised the manuscript for important intellectual content; contributed to statistical analysis; obtained funding; provided administrative, technical, or material support; and supervised the whole course of the study. XT conceived the study and its design, acquired the data, obtained funding, and provided administrative, technical or material support. HK conceived the study and its design, analysed and interpreted the data, critically revised the manuscript for important intellectual content, obtained funding, provided administrative, technical or material support, and supervised the study. All authors read and approved the final manuscript.
Funding The study was supported in part by grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This paper is derived from details presented at the Annual Meeting of the Macula Society, 2012.
Competing interests HT: Lecturer's fees from Kowa Pharmaceutical, Novartis Pharmaceuticals, Bayer Yakuhin, and Santen Pharmaceutical, grants from Bayer Yakuhin and Novartis Pharma, outside this work. YF: Lecturer's fees from ALCON JAPAN and Otsuka Pharmaceutical, outside this work. HK: Lecturer's fees from Kowa Pharmaceutical, Novartis Pharmaceuticals, and Santen Pharmaceutical, outside this work. YY: Lecturer's fees from Novartis Pharmaceuticals, Bayer Yakuhin, MSD, and Santen Pharmaceutical, grants from Bayer Yakuhin, Santen Pharmaceutical, and Novartis Pharma, outside this work. Advisory board member for Novartis Pharmaceuticals and Bayer Yakuhin.
Patient consent Obtained.
Ethics approval Japan Community Health Care Organization Tokyo Shinjuku Medical Center.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement The authors have full control of all primary data, which will be made available for the journal to review upon request.
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