Inflammation-Related Cytokines of Aqueous Humor in Acute Primary Angle-Closure Eyes

Abstract

Purpose.: To measure levels of various inflammation-related cytokines in the aqueous humor of patients with acute primary angle-closure (APAC) and senile cataract.

Methods.: Aqueous humor samples were prospectively collected from 23 eyes (12 eyes with current APAC and 11 eyes with previous APAC) of 23 APAC patients and 15 eyes of 15 cataract patients. The levels of 15 inflammation-related cytokines in the aqueous humor of APAC and cataract subjects were measured by using the multiplex bead immunoassay technique. Data on patient demographics and preoperative intraocular pressure (IOP) were also collected for correlation analysis.

Results.: Compared with the group with previous APAC and the cataract group, the group with current APAC showed clear and significantly elevated concentrations of interleukin (IL)-6, IL-8, granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein (MCP)-1, MCP-3, and vascular endothelial growth factor (VEGF) (all P < 0.0167). The group with previous APAC and the cataract group had similar levels of cytokines. Intraocular pressure was positively correlated with IL-8 (P = 0.001), G-CSF (P = 0.002), MCP-3 (P < 0.001), and VEGF (P < 0.001).

Conclusions.: An early “acute inflammatory” condition occurs in eyes with current APAC. In addition to controlling IOP, anti-inflammatory treatments are necessary for eyes suffering from APAC.

Introduction

Acute primary angle-closure (APAC) is a well-known ophthalmic emergency, with typical symptoms and clinical signs such as sudden onset of ocular discomfort or pain, eye redness, frontal headache, nausea, vomiting, subjective blurring of vision, and sudden and excessive increases in intraocular pressure (IOP). ¹ The acute elevation of IOP found in APAC can also lead to corneal edema, atrophy of structures in the anterior segment, and inflammation in the anterior chamber. The aqueous humor is one of the major factors that influences IOP. It is important for the maintenance of both physiological function and metabolic homeostasis of anterior chamber structures including the corneal endothelium, trabecular meshwork, iris, and lens. ²  

Previous studies have described alterations in the cytokine and chemokine compositions of the aqueous humor in various ocular diseases, such as uveitis, ³ branch retinal vein occlusion, ⁴ and age-related macular degeneration. ⁵ Increased levels of tumor necrosis factor (TNF)-α, ⁶ transforming growth factor (TGF)-β2, ⁷ interleukin (IL)-6, ⁸ IL-8, ^8,9 serum amyloid A (SAA), ¹⁰ and vascular endothelial growth factor (VEGF) ¹¹ have also been reported in glaucomatous aqueous humor. The evidence from these previous studies indicates that aqueous humor cytokines may have a great influence on the pathogenesis of these diseases. 

To date, the profile of aqueous cytokines in APAC has not been described. Therefore, the aim of the present study was to investigate the possible roles of aqueous cytokines in the pathologic mechanism of APAC. Our approach was to use a multiplex bead immunoassay technique to measure multiple inflammation-related cytokines in the aqueous humor of eyes with APAC and cataract. 

Methods

Subjects and Enrollment Criteria

All participants in this study received a detailed explanation about the study and signed an informed consent form in accordance with the principles embodied in the Declaration of Helsinki. This study was approved by the Ethical Review Committee of Zhongshan Ophthalmic Center. Participants were recruited prospectively and consecutively for this study, between January 2013 and July 2013. All subjects were from the Chinese Han population. 

Participants were selected as a representative sample of patients in the Glaucoma Department of Zhongshan Ophthalmic Center. One eye of each subject was included. All enrolled glaucoma patients fulfilled the following inclusion criteria: subjects aged >18 years; one eye was experiencing APAC for the first time (current APAC) or had first experienced APAC within the previous month (previous APAC). APAC was defined according to the following criteria ¹² : (1) presence of at least two of the following symptoms: ocular or peri-ocular pain, nausea, and/or vomiting, and an antecedent history of intermittent blurring of vision with halos; (2) presenting an IOP of at least 22 mm Hg (as measured by Goldmann applanation tonometry); (3) the presence of at least three of the following signs of conjunctival injection: corneal epithelial edema, mid-dilated unreactive pupil, and a shallow anterior chamber; and (4) the presence of an occluded angle in the affected eye, verified by gonioscopy. All eyes underwent an ultrasound biomicroscopy (UBM) examination to confirm the existence of a narrow-angle pupillary block component. All APAC eyes were scheduled for anterior chamber paracentesis, peripheral iridectomy, or trabeculectomy; and their fellow primary angle-closure suspect eyes were scheduled for prophylactic peripheral iridectomy. 

APAC patients enrolled in this study were divided into two groups according to disease history: a group with current APAC, and a group with previous APAC. The current APAC group met all the following inclusion criteria: (1) suffering an APAC attack; and (2) agreeable to the procedure of anterior chamber paracentesis (this procedure can reduce IOP and quickly restore corneal clarity, which in turn allows the immediate treatment of APAC by prophylactic peripheral iridotomy or further trabeculectomy if IOP cannot be controlled). The previous APAC group met all the following inclusion criteria: (1) had an APAC attack within 1 month before admission to our hospital; (2) had uncontrolled IOP after standardized maximum antiglaucomatous medications for APAC; (3) had at least 180° of peripheral anterior synechiae in the affected eye, as determined with gonioscopy and UBM; and (4) required trabeculectomy. 

The antiglaucomatous medication treatment for APAC was standardized as follows: topical pilocarpine 1% four times daily; topical β-blocker (timolol 0.5%) twice daily and/or brinzolamide (Azopt; Alcon Laboratories, Elkridge, MD), and/or topical α-2 agonists (Alphagan; Allergan, Inc., Irvine, CA); topical steroids; oral acetazolamide 250 mg three times daily and intravenous mannitol 20% at 1 to 2 g/kg 4 hours after the initiation of the treatment in cases where the IOP was not reduced by 20% from the initial IOP, unless contraindicated by systemic disease (e.g., congestive heart failure). 

The control group consisted of cataract patients without glaucomatous optic neuropathy and no history of IOP exceeding 21 mm Hg. These patients underwent routine phacoemulsification surgery with an intraocular lens implant. 

Patients with any of the following criteria were excluded: a secondary acute attack because of lens subluxation, uveitis, iris neovascularization, trauma, tumor, or any obvious cataract leading to an intumescent lens; a known systemic inflammatory, autoimmune, or immunosuppressive disease; a pre-existing ocular disease (retinal vein occlusion, retinal artery occlusion, diabetic retinopathy, age-related macular degeneration); or a history of previous ocular surgery. 

All eyes of the subjects underwent a thorough ophthalmic evaluation, including slit-lamp biomicroscopy, IOP measurement (Goldmann applanation tonometry), gonioscopy, fundus examination, UBM, and B-scanning. Intraocular pressure was measured preoperatively, mainly in the afternoon on the day before the aqueous humor sampling for all studied eyes. We believe that this minimized the influences of diurnal variation of IOP and closely reflected the IOP at the time of aqueous humor sampling. 

Aqueous Humor Collection

Aqueous humor samples (50–100 μL) were collected by a procedure described in our previous study. ¹³ The samples were collected as follows: (1) for the current APAC group, samples were collected during an anterior chamber paracentesis procedure; (2) for the previous APAC group, samples were collected at the beginning of trabeculectomy; (3) for the cataract control group, samples were collected at the beginning of cataract surgery. All samples were obtained before any conjunctival or intraocular manipulation to avoid breakdown of the blood–aqueous barrier associated with surgical trauma. All samples were immediately frozen and stored at −80°C until the analyses were performed. 

Cytokine Analysis

Cytokine concentrations were analyzed by using a multiplex bead immunoassay system (Milliplex Human Cytokine kit; Millipore Corp., Billerica, MA). The assays were performed according to the manufacturer's instructions and analyzed by using the Bio-Plex suspension array system (Bio-Plex200; Bio-Rad, Hercules, CA). The following cytokines related to the inflammatory process were analyzed simultaneously: IL-1β, IL-6, IL-8, IL-10, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, monocyte chemotactic protein (MCP)-1, MCP-3, macrophage-derived chemokine (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β, soluble CD40 ligand (sCD40L), TNF-β, and VEGF. A 25-μL volume of aqueous humor sample was used in each reaction. From the information provided by the manufacturer, the multiplex assay kit can quantitatively measure multiple cytokines from as little as 25 μL of bodily fluids. The detection limit for any analyte was 1 pg/mL, with a dynamic range up to 10,000 pg/mL (according to the manufacturer). 

Statistical Analysis

The data were processed and analyzed statistically by using SPSS (version 13.0; SPSS, Chicago, IL). For categorical variables, the frequency distribution and percentages were calculated and compared by the χ ² test. For numerical variables in parametric distribution, two-samples independent t-test was performed. Statistical significance was accepted at P < 0.05. For differences in IOP, duration before surgery, and cytokine concentrations, the Mann-Whitney U test was performed. Correlations between cytokine concentrations and subjects' demographic data, including age and IOP, were calculated by using Spearman's correlation test. For the correction of multigroup comparisons, P values of 0.0167 for the Mann-Whitney U test and 0.0071 for Spearman's correlation test were considered statistically significant, with significance levels of 0.05 based on Bonferroni's methods. 

Results

Demographic and Clinical Characteristics of the Patients

A total of 38 subjects (eyes) who fulfilled the inclusion criteria were enrolled in the study: 15 were nonglaucomatous cataract controls and 23 were APAC patients (12 who were experiencing current APAC and 11 who had experienced previous APAC). The demographic and clinical data of APAC and cataract patients are summarized in Table 1. The mean ages of the APAC patients and the cataract control individuals were 63.8 and 65.0 years, respectively (P = 0.625). As would be expected, the current APAC group had the highest IOP (48.5 mm Hg), followed by the previous APAC group (26.0 mm Hg), and the cataract group (13.3 mm Hg) (all P < 0.0167). The median duration before surgery of the current APAC group was 3.0 days (range, 1–7 days), and that of the previous APAC group was 20.0 days (range, 11–30 days; P < 0.001). 

Table 1

 

Demographic and Clinical Data of APAC and Cataract Control Patients

Characteristics	APAC Patients	Cataract	P Value*
Total no.	23	15
APAC subgroups	12 current APAC
	11 previous APAC
Age, mean (SD), y	63.8 (8.7)	65.0 (5.2)	0.625
Sex, no. (%)
Male	7 (30)	7 (46)	0.311
Female	16 (70)	8 (54)
IOP, median (IQ), mm Hg	Current APAC: 48.5 (18.2)	13.3 (7.0)	0.001†‡
	Previous APAC: 26.0 (8.0)		<0.001§‡
			<0.001‖‡
Duration before surgery, median (IQ), d	Current APAC: 3.0 (4.2)	-	<0.001†
	Previous APAC: 20.0 (13.0)

 SD, standard deviation; IQ, interquartile range.

*  Significance of differences between groups: χ ² test, two-samples independent t-test, or Mann-Whitney U test.

†  Between the current APAC group and the previous APAC group.

‡  Significance level at 5% (P < 0.0167) by Bonferroni correction for multiple comparisons.

§  Between the current APAC group and the cataract group.

‖  Between the previous APAC group and the cataract group.

Aqueous Cytokines

The aqueous concentrations of 15 cytokines were measured in both the cataract and APAC groups (Table 2). Compared to the cataract group, the APAC groups showed a significantly higher proportion of samples with measurable concentrations of the following cytokines: IL-8 (P = 0.014), G-CSF (P = 0.023), MCP-3 (P = 0.008), and VEGF (P = 0.001) (Fig. 1). 

Figure 1

 

The aqueous humor levels of IL-8, G-CSF, MCP-3, and VEGF in APAC eyes were significantly higher than in cataract control eyes. (*P < 0.05).

Table 2

 

Aqueous Humor Concentrations (pg/mL) of Cytokines in APAC Patients and Cataract Control Patients

Cytokine	APAC Patients	Cataract	P Value*
IL-1β	0	0	1.000
IL-6	672 ± 1291	25.3 ± 44.6	0.148
IL-8	139 ± 253	5.24 ± 4.13	0.014
IL-10	0.19 ± 0.89	0.19 ± 0.76	0.787
G-CSF	1944 ± 4226	1.70 ± 2.84	0.023
GM-CSF	0.56 ± 2.58	0	0.419
IFN-γ	0	0	1.000
MCP-1	2613 ± 2042	1170 ± 597	0.256
MCP-3	10.3 ± 15.0	1.28 ± 2.65	0.008
MDC	4.89 ± 3.96	2.87 ± 3.53	0.139
MIP-1α	20.2 ± 56.7	2.88 ± 6.23	0.198
MIP-1β	23.0 ± 22.5	11.7 ± 9.0	0.175
sCD40L	8.45 ± 23.9	3.39 ± 9.15	0.742
TNF-β	0	0	1.000
VEGF	1160 ± 1800	43.2 ± 45.3	0.001

 Data are expressed as the mean ± SD (pg/mL). Bold values represent significance.

*  Significance of differences between groups: Mann-Whitney U test.

Interleukin 1β, IL-10, GM-CSF, IFN-γ, MIP-1α, sCD40L, and TNF β were detected in fewer than 50% of samples in both control and APAC groups and therefore were not included in further analysis. Further separation of the APAC group into current and previous APAC groups showed that the current APAC group had significantly elevated concentrations of IL-6 (P = 0.002), IL-8 (P < 0.001), G-CSF (P = 0.001), MCP-1 (P = 0.002), MCP-3 (P < 0.001), MIP-1β (P = 0.010), and VEGF (P < 0.001) when compared with the cataract group, and significantly elevated concentrations of IL-6 (P = 0.003), IL-8 (P = 0.010), G-CSF (P = 0.003), MCP-1 (P = 0.004), MCP-3 (P = 0.005), and VEGF (P = 0.005) when compared with the previous APAC group (Fig. 2). The previous APAC group and the cataract group had similar concentrations of cytokines (Table 3). 

Figure 2

 

Scatterplots showing distribution levels of IL-6, IL-8, G-CSF, MCP-1, MCP-3, MDC, MIP-1β, and VEGF in aqueous humor from current APAC, previous APAC, and cataract control patients. A Mann-Whitney U test was performed between groups, and a significant difference was accepted at P < 0.0167 by Bonferroni correction. The solid lines indicate median concentrations. The current APAC group had significantly elevated concentrations of IL-6 (P = 0.002), IL-8 (P < 0.001), G-CSF (P = 0.001), MCP-1 (P = 0.002), MCP-3 (P < 0.001), MIP-1β (P = 0.010), and VEGF (P < 0.001) when compared with the cataract group, and significantly elevated concentrations of IL-6 (P = 0.003), IL-8 (P = 0.010), G-CSF (P = 0.003), MCP-1 (P = 0.004), MCP-3 (P = 0.005), and VEGF (P = 0.005) when compared with the previous APAC group.

Table 3

 

Levels (pg/mL) of IL-6, IL-8, G-CSF, MCP-1, MCP-3, MDC, MIP-1β, and VEGF in Aqueous Humor From Current APAC, Previous APAC, and Cataract Control Patients

Cytokine	Current APAC	Previous APAC	Cataract	P Value*	P Value*	P Value*
IL-6	1228 ± 1598	4.90 ± 6.10	25.3 ± 44.6	0.003†	0.002‡	0.397§
IL-8	258 ± 310	10.0 ± 10.8	5.24 ± 4.13	0.010†	<0.001‡	0.620§
G-CSF	3559 ± 5398	5.35 ± 9.45	1.70 ± 2.84	0.003†	0.001‡	0.874§
MCP-1	3796 ± 1937	1161 ± 1032	1170 ± 597	0.004†	0.002‡	0.276§
MCP-3	16.5 ± 18.3	2.57 ± 4.16	1.28 ± 2.65	0.005†	<0.001‡	0.387§
MDC	6.16 ± 4.39	3.40 ± 2.98	2.87 ± 3.53	0.107†	0.066‡	0.530§
MIP-1β	32.8 ± 23.7	11.3 ± 15.0	11.7 ± 9.0	0.018†	0.010‡	0.691§
VEGF	2043 ± 2118	90.9 ± 59.6	43.2 ± 45.3	0.005†	<0.001‡	0.047§

 Data are expressed as the mean ± SD (pg/mL). Bold values represent significance.

*  Significance of differences among groups: Mann-Whitney U test, and a significant difference was accepted at P < 0.0167 by Bonferroni correction for multiple comparisons.

†  Between the current APAC group and the previous APAC group.

‡  Between the current APAC group and the cataract group.

§  Between the previous APAC group and the cataract group.

Correlation Analysis

The correlations between each cytokine and age and IOP are shown in Table 4. No correlation was found between the aqueous cytokine levels and age. However, IOP was found to correlate positively with IL-8 (ρ = 0.512, P = 0.001), G-CSF (ρ = 0.492, P = 0.002), MCP-3 (ρ = 0.593, P < 0.001), and VEGF (ρ = 0.646, P < 0.001) (Fig. 3). 

Figure 3

 

Spearman's correlations between IOP and IL-8 (ρ = 0.512, P = 0.001), G-CSF (ρ = 0.492, P = 0.003), MCP-3 (ρ = 0.593, P < 0.001), and VEGF (ρ = 0.646, P < 0.001).

Table 4

 

Correlations Between Cytokines and IOP

	Age, y		IOP, mm Hg
	ρ	P Value	ρ	P Value
IL-6	−0.064	0.701	0.352	0.030
IL-8	−0.065	0.697	0.512	0.001
G-CSF	−0.017	0.920	0.492	0.002
MCP-1	0.085	0.611	0.337	0.038
MCP-3	−0.061	0.718	0.593	<0.001
MDC	−0.121	0.468	0.362	0.025
MIP-1β	−0.041	0.806	0.343	0.035
VEGF	−0.068	0.685	0.646	<0.001

 Correlation coefficient (ρ) and P values are calculated by Spearman's correlation, and a significant difference was accepted at P < 0.0071 by Bonferroni correction. Bold values represent significance.

Discussion

A major limitation of aqueous humor testing is that only small sample volumes (typically 50–150 μL of fluid) can be obtained from human eyes. These amounts are barely sufficient to test more than a few cytokines with traditional enzyme-linked immunosorbent assay techniques. Multiplex bead immunoassays, which allow for simultaneous detection of multiple cytokines in small volume clinical samples, ¹⁴ have been used to determine cytokine expression profiles in aqueous humor. Using multiplex bead immunoassays, previous studies have detected that when compared with the cataract group, the aqueous humor levels of TGF-β1, SAA, IL-8, IL-12, IFN-γ, and monokine induced by gamma interferon (MIG) were significantly higher in primary open-angle glaucoma, ^8,10,15 and the levels of IL-8 and MIG were significantly higher in primary angle-closure glaucoma. ¹⁵ However, to the best of our knowledge, this is the first study to provide a comprehensive evaluation of cytokines in the aqueous humor of APAC eyes. 

The bead immunoassay revealed clear elevations of cytokines related to an immune reaction or inflammation in eyes currently experiencing APAC when compared with eyes that had previously experienced APAC and control cataract eyes; that is, IL-6, IL-8, G-CSF, MCP-1, MCP-3, MIP-1β, and VEGF. However, interestingly, no significant differences were found between the aqueous cytokine profiles in the previous APAC eyes and the control cataract eyes. 

Interleukin 6 is an interleukin that acts as a pro-inflammatory cytokine. It is secreted by T cells and macrophages to stimulate an immune response during infection or after trauma, burns, or other tissue damage. Previous studies have demonstrated that IL-6 stimulates the inflammatory and auto-immune processes in many diseases such as diabetes, ¹⁶ Behçet's disease, ¹⁷ and pseudoexfoliation glaucoma. ⁸ Interleukin 6 is an important in vitro and in vivo mediator of increased vascular permeability and endothelial barrier dysfunction. ^18,19 This suggests a possible involvement of elevated IL-6 levels in the breakdown of the blood–aqueous barrier that is characteristic of APAC. Interleukin 8, MCP-1, MCP-3, and MIP-1β are typical inflammatory chemokines. These are formed under pathologic conditions (in response to pro-inflammatory stimuli) and actively participate in the inflammatory response by attracting immune cells to the site of inflammation. Inflammatory chemokines are produced in high concentrations during infection or injury and determine the migration of inflammatory leukocytes into the damaged area. G-CSF is used to stimulate the survival, proliferation, differentiation, and function of granulocytes, ²⁰ which play an important role in inflammation. Previous studies of inflammatory eye disease ^3,21,22 have reported that increased IL-6, IL-8, and MCP-1 levels, in addition to the shifting of the granulocyte stimulating factor from primary GM-CSF to G-CSF, were commonly associated with intraocular inflammation, regardless of disease etiology. We found similar changes among these cytokines in the samples from current APAC patients in the present study. VEGF, one of the most important inducers of angiogenesis and vascular permeability, also has a strong link with inflammation and immunity. ^23,24 Taken together, the evidence suggests that evaluation of the composition of various inflammatory cytokines in the aqueous humor may expand our understanding of APAC pathophysiology at its early stages. There is an early “acute inflammatory” condition in current APAC patients, which is consistent with clinical findings such as ocular pain, eye redness, and excessive increases in IOP. 

No significantly higher concentration of inflammatory cytokines was found between the previous APAC group and the cataract group. This may be due to the decreases in IOP and the subsidence of inflammation following medication treatment in the previous APAC group; these differences may explain the discrepancy between the previous APAC group and the current APAC group. During acute ocular inflammation, inflammatory cytokine levels are elevated by several orders of magnitude; these decrease to moderately elevated levels during remission. ^3,25  

In the present study, the IOP was positively correlated with the levels of IL-8, G-CSF, MCP-3, and VEGF in the anterior chamber even after Bonferroni correction. The significant correlations between these cytokines and the IOP levels suggested a strong link between inflammatory cytokine networks and IOP elevation, although the exact mechanism of this interaction is unclear. Recently, Freedman and Iserovich ²⁶ have proposed that the increase in cytokine levels that parallels the increase in IOP is highly suggestive of IOP as a potential stimulus for cytokine production. In the present study, the highest levels of cytokines were found in the aqueous humor taken from eyes with the highest IOP (i.e., the current APAC group). This finding, once again, suggests that the IOP itself may be responsible for the production of cytokines. Elevated cytokine levels may in turn influence aqueous humor dynamics and thus IOP elevation. 

The relationship between IOP and inflammation is complex. Excessive increases in IOP may cause disruption of the blood–aqueous barrier, thereby allowing the entry of inflammatory cells and mediators into the aqueous humor, resulting in increases of aqueous protein concentrations in the aqueous outflow system. ²⁷ Inflammation in the anterior chamber can in turn alter intraocular circulation of the aqueous humor. The inflammatory cells and proteins in the aqueous humor can form adhesions between the iris and lens, resulting in the formation of posterior synechiae leading to pupillary block, iris bombé, and peripheral anterior synechiae. ²⁸ These are features that can be seen in APAC eyes. 

In a study involving the simultaneous analysis of multiple factors from each sample, several factors, such as sample size and method of data analysis, can affect the outcome. The Bonferroni correction was applied in the present study to determine the significance of changes in cytokine levels. This is a conservative statistical adjustment used for the verification of data observations but it may mask potential changes in exploratory studies. In fact, more cytokines showed significantly positive correlations with IOP elevation without Bonferroni correction. Although we endeavored to present the data in a conservative way, further studies with a larger sample size would be needed to obtain a confirmative aqueous cytokine profile in both the APAC and cataract groups. 

Some potential limitations in our study should be mentioned. The use of topical antiglaucoma medications may influence the aqueous immune milieu. ¹⁰ In this study, the duration of medications use was longer in the previous APAC group owing to the longer duration before surgery, which may be one of the factors causing the difference in cytokine levels. Another limitation of the study was its sample size, as the number of patients enrolled was relatively low. Nevertheless, the results were statistically significant, so the relatively small number of patients may serve to strengthen the results and conclusions of the study. 

Conclusions

In summary, various cytokines related to inflammation showed elevated levels in the aqueous humor of eyes currently experiencing APAC when compared with eyes that had previously experienced APAC or control eyes with cataract. There is an early acute inflammatory condition in current APAC patients. In addition to controlling IOP, anti-inflammatory treatments are necessary for eyes suffering from APAC. Further studies will be necessary to identify the relationship between the profile of aqueous inflammation-related cytokines and its role in postoperative wound healing responses following filtration surgery. 

Acknowledgments

Supported in whole or in part by the National Natural Science Foundation of China (81371008, 81200670) and Guangdong Natural Science Foundation (S2013040016405). 

Disclosure: W. Huang, None; S. Chen, None; X. Gao, None; M. Yang, None; J. Zhang, None; X. Li, None; W. Wang, None; M. Zhou, None; Xin. Zhang, None; Xiu. Zhang, None