Low expression of TGF‐β2 and matrilin2 in human aqueous humour with acute primary angle closure

Abstract Primary angle‐closure glaucoma (PACG) is the leading cause of irreversible blindness in the world. Angle closure induced by pupil block and secondary iris synechia is the fundamental pathology of the PACG. The molecular mechanisms of angle closure have not yet been clearly illustrated. This study was designed to investigate the protein difference in the aqueous humour and explore new biomarker of the PACG. Aqueous humour (AH) was collected from patients with acute primary angle closure (APAC) and cataract (n = 10 in APAC group) and patients with cataract only (n = 10 in control group). Samples were pooled and measured using label‐free proteome technology. Then, the differentially expressed proteins (DEPs) were verified by ELISA using independent AH samples (n = 20 each group). More than 400 proteins were revealed in both groups through proteomics. Comparing the two groups, there were 91DEPs. These proteins participate in biological activities such as inflammation, fibrosis, nerve growth and degeneration and metabolism. We found that the expression of transforming growth factor‐β2 and matrilin2 was downregulated in the APAC group. The two proteins are related to inflammation and extracellular matrix formation, which might be involved in angle closure. This study characterized DEPs in AH of the APAC and found a downregulated protein matrilin2.


| INTRODUC TI ON
Acute primary angle closure (APAC) is an ophthalmic emergency comprising complaints of sudden headache, nausea or vomiting, eye pain and redness and blurry vision.Eyes with APAC usually have a narrow anterior chamber angle and forward bowing of the peripheral iris.2][3][4] Without timely treatment, it can lead to iris synechia, angle dysfunction, higher IOP and irreversible optic nerve damage.][10] However, these treatments are insufficient for the protection of optic nerves.Multiple studies have been conducted on neuroprotection, [11][12][13] but research on the micro-environment of the anterior chamber is limited.
The AH is a fluid in the eyes that maintains IOP, regulates the metabolism of anterior segment tissues and reflects ophthalmic pathophysiology. 14AH extraction is minimally invasive and frequently indicated for the diagnosis and treatment of eye diseases. 15,168][19] However, no clear method has yet been developed.Mass spectrometry-based proteomic and bioinformatic methods have enabled the comparison of proteins in a variety of biological samples with a high level of resolution and accuracy. 20[26] In this study, we initially hypothesized that angle closure is related to AH change in primary angle-closure glaucoma (PACG) and some proteins in AH could reflect the anterior chamber angle before neuropathy.The aim of this study was to analyse the characteristics of differentially expressed proteins (DEPs) based on the AH proteomes of APAC patients and to discover valuable information for treatment in the early stage of PACG.

| Patients
This study was approved by the Tianjin Medical University Eye Hospital Ethics Committee [2016KY(L)-26] and complied with the standards of the Declaration of Helsinki for human experiments.
Patients provided signed informed consent.Sixty patients were recruited for the study, including 30 patients with APAC and cataracts (APAC group) and 30 patients with cataracts (control group).
Samples from 10 patients in each group were used for the proteome experiment, and which from 20 patients in each group were used for the enzyme-linked immunosorbent assay (ELISA).All patients underwent a series of ophthalmic examinations by glaucoma specialists, including IOP, visual acuity, slit lamp, gonioscopy, B-mode ultrasound, ultrasound biomicroscopy, optical coherence tomography, fundus photography and Humphrey visual field examination.
Inclusion criteria for APAC and cataract patients were as follows: IOP > 30 mmHg (1 mmHg = 0.133 kPa), as well as symptoms including conjunctival congestion, corneal oedema, dilated pupil and shallow anterior chamber observed through the slit lamp.The angle closure (>180°) was confirmed through angioscopy or ultrasound biomicroscopy. 10Patients with APAC also had a cataract diagnosis based on the slit lamp.The diagnostic criteria for cataracts were opacity of the lens and gradual loss of vision.The control subjects had no history of glaucoma.Premedication: patients with APAC took eye drops including decreasing IOP and antibiotic, patients with cataract only dropped antibiotic.Medicines decreasing IOP include carteolo hydrochloride, brinzolamide and brimonidine tartrate.Antibiotic is levofloxacin.Patients were excluded from the study if they had other ophthalmic diseases, such as trauma, inflammation or secondary glaucoma, or they have been were received eye surgery.AH samples (50-100 μL) were collected through a 1-mL syringe during surgery.
The samples in a sterile tube were placed on ice, then centrifuged at 4°C (2000 rpm, ~400 g, ×5 min) and finally stored in the freezer (−80°C).

Bradford's method
The Bradford method has been applied for protein quantification for many years. 27Total protein concentrations in AH samples were determined using Bradford's Assay Kit (Thermo Fisher Scientific, USA).
Taking 10 μL of diluted sample or different concentration BSA (bovine serum albumin) solution into a 96-well plate, mixed with 300 μL of protein quantification dye and then reacted for 20 min in the dark.
The absorbance under 595 nm was measured by a microplate reader.
The standard curve was generated according to the absorbance value of different concentration BSA.According to the standard curve formula, the protein concentration of each sample could be calculated.

| Proteolysis
The method of proteolysis was based on sodium dodecyl sulphate polyacrylamide gel electrophoresis as performed previously. 26e SDS-PAGE was performed with 5 μg of protein per sample; the gel was stained and then destained until the background was was added [50 mM NH 4 HCO 3 and acetonitrile (ACN) (1:1)], and the gel was destained for 15 min before washing with ultra-pure water.This was repeated three times until detaining completely.
After that, 100 μL of ACN was added to dehydrate the gel particles until they were white.Subsequently, 200 μL of 10 mM C 4 H 10 O 2 S 2 (DTT) was added to a warm water bath for 60 min before adding 100 μL of ACN to dehydrate gel particles.Then, 200 μL of 55 mM indole-3-acetic acid was added before placing it in the dark for 30 min and adding 100 μL of ACN to dehydrate the gel particles.
The following solutions were used to carry out the suspension: ultra-pure water (once), ACN (once), ultra-pure water (once) and ACN (once), each for 10 min.Then, 100 μL of 0.01 μg/μL trypsin was added to each tube, allowing the enzyme solution and gel particles to completely contact for 30 min at 4°C; this was stopped when the trypsin was absorbed by the gel, and 100 μL of 25 mM NH 4 HCO 3 was then added.The mixture was incubated at 37°C overnight.The next day, the supernatant was collected and transferred into a clean Eppendorf tube.After adding 5% CF 3 COOH (TFA) and 95% double distilled water (ddH 2 O) extraction buffer to the pellets for 1 h, the collected supernatants were transferred into the corresponding Eppendorf tube.The pellets were treated with extraction buffer (2.5% TFA, 50% ACN, 47.5% ddH 2 O) for 1 h, and the supernatant was placed in the previous Eppendorf tubes.
Finally, the peptides were vacuum dried and stored at −20°C.unipr ot.org, http:// www.Genome.jp/ kegg and the STRING database.

| Statistical analysis
MaxQuant significances A was used to assess the differential proteins in the proteome experiment.Differences were considered statistically significant when the fold-change was greater than 1.5 and the p-value was less than 0.05.Subject information and ELISA results were tested by performing an unpaired t-test followed by the Mann-Whitney U test using GraphPad® Prism 7 software.Data were expressed as the mean ± SD, and p < 0.05 was regarded as statistically significant.

| RE SULTS
In this study, samples of AH were collected from patients with APAC and cataract (APAC group) and cataract only (control group).
Through proteome method, we obtained and analysed the DEPs in the APAC group (n = 10, pooled) compared with the control group (n = 10, pooled).The result demonstrates that these proteins are involved in inflammation, metabolism, nerve, adhesion and fibrosis, as illustrated by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI).Based on the biological function, we chose four proteins and verified them in the APAC and control group (n = 20/group, independent sample) by ELISA.Finally, we agree that matrilin2 (MATN2)/transforming growth factor-β2 (TGF-β2) expression was downregulated in the APAC group based on both methods.

| Subjects
According to the guidelines of the fifth European Glaucoma Society and American Academy of Ophthalmology, patients in APAC group had high IOP (>21 mmHg), closed anterior chamber angle and opacified lens, but still in the normal thickness of the retinal nerve fibre layer (Figure 1).Patients in the control group only manifest opacified lens.Table 1 shows the subjects' information in the proteome experiment.The average age has no obviously different between the APAC group (n = 10) and the control group (n = 10), which are 68.00 ± 1.86 and 71.70 ± 2.51 years, respectively (p = 0.25).There is no significant difference in the sex distribution, cup/disc ratio and visual acuity between the groups (p > 0.05); however, there is statistical difference in the mean IOP, anterior chamber depth, axial length and corneal thickness when comparing the two groups (p < 0.05).Table 2 shows the subjects' information in the ELISA test.(p > 0.05) and there is no significant difference in the sex distribution and visual acuity (p > 0.05), however, there is significant difference in the mean cup/disc ratio, IOP, axial length, corneal thickness and anterior chamber depth (p < 0.05).

| Data acquisition
The workflow of the label-free proteome test includes four key steps as follows: protein digestion, peptide abundance recognition, spectral counting and data analysis (Figure 2A).Each group was tested twice (A1A2 and C1C2).In the heatmap, protein levels showed noticeable differences between the groups.The colours present upregulated and downregulated proteins, marked with red and blue, respectively (Figure 2B).

| Data analysis: GO, KEGG, PPI
Approximately 400 proteins were identified through proteome technology.Table 3 lists 91 significant DEPs (fold-change >1.5 and p < 0.05).There were 50 upregulated and 41 downregulated proteins in the APAC group compared with levels in the control group.The interesting proteins are labelled with yellow in Table 3, which are related to inflammation, metabolism, cellular adhesion and nerve growth and degeneration in terms of biological function (Figure 3).

GO analysis shows functions of differentially expressed protein
involving biological process, molecular function and cellular component (Figure 4).With respect to biological function, these proteins are determined to be involved in transport, cell adhesion, cell prolif-    KEGG analysis shows that the DEPs are enriched in multiple pathways, such as the lysosome, axon, glycolysis/gluconeogenesis, extracellular matrix (ECM)-receptor interaction and biosynthesis of amino acids signalling pathways.Additionally, some proteins are also determined to be involved in inflammation pathways, such as the toll-like receptor, mitogen-activated protein kinase (MAPK) and interleukin-17 (IL-17) signalling pathways (Figure 5).
The PPI map displays interactions between proteins (Figure 6).

| Verification of DEPs by ELISA
To further verify protein expression in the AH, samples from patients with APAC and cataract and patients with cataract only were collected again with the same standards as before in proteomic experiment.Patient information is summarized in Table 2. TGF-β2, MATN2 and ANXA1 are downregulated in the APAC group compared to the control.The CDH4 does not show statistical difference between the groups.The trend of TGF-β2 and MATN2 in the APAC group is consistent with the result in proteomic experiment.
However, in comparison with control group, the ANXA1 in APAC group upregulate in the proteomic data and downregulate in the ELISA test (Figure 7).

| DISCUSS ION
APAC can obstruct the outflow of AH, causing higher IOP, which can lead to irreversible optic neuropathy and blindness. 28Previous research usually focused on neuroprotection less noticing the microenvironment of anterior chamber.AH not only provides nutrition support but also reflects the state of anterior chamber angle.Our research aimed to explore the differential proteins in the AH between the APAC group and the control group via proteomic technology and ELISA test and to seek proteins with underlying therapy value for APCG in future.
We measured the amount of protein in every one sample by the way of Bradford and found the concentration of protein was too little to carry out the proteomic test in control group.For solving the problem, we pooled 10 samples as a group for further test.We also analysed the reasons why protein concentration in the control group is lower than the APAC group.First, while collecting the samples, the condition of anterior chamber still be in an inflammatory state, the permeability of vessel was increased, more proteins flowed into the AH.Second, the iris and ciliary body secreted directly more proteins into anterior chamber in APAC patients.Third, the closure of anterior chamber angle slowed down the flow-out of proteins in AH of the APAC.
F I G U R E 3 Classification of significantly DEPs.According to their function, the DEPs are determined to be mainly involved in inflammation, metabolism, the nerve system, adhesion and fibrosis.Arrows represent protein increase or decrease in the APAC aqueous humour (AH) in comparison to control group.
More than 400 proteins were measured by proteome test, there were 91DEPs in comparison with control.In the 91 DEPs, though some proteins have been reported in glaucoma, like TGF-β2, however, it is fewer about the report of the change in AH of early stage of PACG.The anterior chamber angle closure starts from a narrow angle to 360-degree peripheral iris synechia accompanied with inflammatory reaction in the AH, ECM deposition in the trabecular meshwork, tissue adhesion and fibrous union in the anterior chamber angle. 29Based on the protein function, involved signalling pathways and ever reported glaucoma pathologic mechanisms, we chose some interesting proteins (marked yellow in Table 3).Besides, one sample only be enough for verification of four proteins in ELISA test.
Among of those interesting proteins, four proteins were chosen, namely Matrilin2, AnnexinA1, TGF-β2 and CDH4.Summarized from the uniport database and previous reported studies, the four proteins involved in matrix assembly or innate and adaptive immune response or cell adhesion or retinal development and so on.Therefore, their biological process is related with pathology of anterior chamber angle closure.We again measured their expression in AH via ELISA test with multiple independent samples.
The result of ELISA shows that the TGF-β2 and MATN2 express at a lower level in the APAC group than in the control group.This F I G U R E 4 Gene Ontology (GO) analysis of genes of DEPs in the acute primary angle-closure (APAC) group compared with the cataract group.Illustrated in the histogram, GO analysis includes the biological processes (BPs: green), cellular components (CCs: orange) and molecular functions (MFs: blue).The protein-encoding genes are mainly enriched in transport, cell motility, cell adhesion, cell proliferation and cell death processes.They are determined to be located in the extracellular region, membrane, cytoplasm, nucleus and cytoskeleton and to participate in metal ion binding, receptor binding and transmembrane transporter activity.
result is consistent with data of the proteome.However, the expression of ANXA1 and CDH4 in ELISA is inconsistent with the proteome result.ANXA1 is upregulated in the APAC group compared with the control group in the proteomic experiment, whereas it is downregulated in the ELISA test.The CDH4 is downregulated in the APAC group compared with the control group in the proteomic experiment, whereas it is not significantly different in the ELISA.
The MATN2 is downregulated in the APAC group in both methods.1][32] The Uniprot database shows the MATN2 is involved in the ECM composition and acts as an adaptor protein in the ECM.MATN2 also paly roles in inflammation reactions, extracellular filamentous network formation and neurite growth.In terms of inflammation, one study showed that soluble MATN2 promotes inflammatory activity via activation of the TLR/PKR/NFKB signalling pathway in calcific aortic valve disease. 33other study also revealed that elevated MATN2 induced inflammatory responses in post-burn patient serum and the mechanism might be partly mediated by TLR4 in animal experiments. 34

MATN2
can activate innate immune cells and could be involved in early axon injury in inflammatory diseases of the central nervous system. 35reover, MATN2 is also a poly-adhesive connector protein, which cooperates with other proteins to participate in nerve growth and migration, as well as neuromuscular connection. 30,36In our data, the expression of MATN2 was lower in the AH of the APAC group than in controls.However, levels of inflammatory factors, such as CRP, IL6 and CD163 are higher.This is contrary to the results of previous studies on the proinflammatory effect of MATN2.One reason for this difference might be that the level of MATN2 has been decreased after the attack and another reason is it does not act in acute response.The function of MATN2 in the glaucoma is unclear, especially in inflammation, ECM formation and nerve growth.Thus, the function of MATN2 is worthy of further research in the issues of anterior chamber angle, such as iris and trabecular meshwork.
The TGF-β2 is downregulated based on the proteomic experiment and ELISA data.TGF-β2 is an important factor in inflammation and fibrosis.1][42] Studies have clear.The gel was cut into pieces of approximately 1 mm 3 , placed in a 1.5 mL Eppendorf tube and washed with 200 μL of ultra-pure water twice for 10 min.Coomassie brilliant blue destaining buffer | 3 of 16 WANG et al.
The mean age in the APAC (n = 20) and control (n = 20) group show 67.70 ± 1.60 and 72.20 ± 1.63 years, which has no obvious difference F I G U R E 1 Examination of the acute primary angle-closure (APAC) patients.Corneal oedema, shallow anterior chamber, dilated pupil and local iris atrophy were observed via slit-lamp microscopy in the eye of APAC patient (A).The closed angle and bulging iris are shown based on ultrasound biomicroscopy in the eye of the APAC patient (B).The optic disc had clear boundaries, close-to-normal colour and a piece of flame-like bleeding beside the optic disc, representing an attack sign in the fundus image (C).The thickness of the nerve fibre layer was within the normal range in optical coherence tomography (D).TA B L E 1 Demographic and clinical characteristics of cataract and APAC subjects for the proteome.

F I G U R E 2
Workflow of proteomic technology and heatmap of the aqueous humour proteins in acute primary angle-closure (APAC) group and controls.The workflow shows aqueous humour (AH) collection, protein digestion, peptide abundance recognition, spectral count with label-free proteomic technology, data analysis and enzyme-linked immunosorbent assay (ELISA) verification (A).The heatmap shows protein expression in the AH of the APAC group (A1, A2) and controls (C1, C2).Red represents upregulated proteins and blue represents downregulated proteins (B).

267 0.0000 − 1 TA B L E 3
(Continued)  they are found to participate in receptor binding, metal ion binding and transmembrane activity.In cellular components, the proteins are enriched in the extracellular matrix, cytoplasm, nucleus and cytoskeleton regions.

44 F I G U R E 5 4
shown that TGF-β2 could contribute to ECM deposition and inhibit ECM transformation in the AH or trabecular meshwork of glaucoma patients.The ECM deposition decreases drainage in the anterior chamber angle, increases IOP and exacerbates nerve damage.43,Analysis of the signalling pathways of significantly DEPs in APAC group compared with control by the Kyoto Encyclopedia of Genes and Genomes (KEGG).Based on the p-value, the DEPs were mainly enriched in the lysosome, axon, glycolysis/gluconeogenesis, extracellular matrix (ECM)-receptor interaction and biosynthesis of amino acids signalling pathways.Those proteins participate in some inflammation signalling pathways, such as the toll-like receptor signalling pathway, MAPK signalling pathway and IL-17 signalling pathway.F I G U R E 6 Protein-protein interaction (PPI) network of all DEPs.The PPI reflects the interactions among all significantly DEPs between the APAC group and control group.Proteins marked with red rectangles are associated with the PACG based on their biological function.F I G U R E 7 Verification of DEPs by ELISA.The MATN2, ANXA1 and TGF-β2 in the aqueous humour (AH) are downregulated in the APAC group compared to the control.The expression of CDH4 in the AH is not significantly difference between the APAC and control group.Summary of proteomics studies of PACG.
All differential proteins between APAC and cataract based on proteome.
TA B L E 3