Skip to main content
Molecular Vision logoLink to Molecular Vision
. 2011 Jul 14;17:1891–1900.

Alterations in the aqueous humor proteome in patients with a glaucoma shunt device

Arundhati Anshu 1,2, Marianne O Price 2,, Matthew R Richardson 3, Zaneer M Segu 4, Xianyin Lai 5, Mervin C Yoder 3, Francis W Price Jr 1,
PMCID: PMC3144728  PMID: 21850163

Abstract

Purpose

To investigate whether implantation of a glaucoma shunt device leads to inappropriate accumulation of plasma derived proteins in the aqueous humor.

Methods

Aqueous humor samples were collected from 11 patients (study group) with a glaucoma shunt device undergoing either cataract surgery or a corneal transplant and 11 patients (control) with senile cataract undergoing routine cataract extraction. Of the study group, 9 had an Ahmed valve implant and 2 eyes had a Baerveldt implant. Tryptic digests of the mixture of proteins in aqueous humor (AH) were analyzed using Liquid Chromatography/Mass Spectrometry (LC-MS/MS). Proteins were identified with high confidence using stringent criteria and compared quantitatively using a label-free platform (IdentiQuantXLTM).

Results

We identified 135 proteins in the albumin-depleted fraction in both the study and control group AH. Using stringent criteria, 13 proteins were detected at a significantly higher level compared to controls. These proteins are known to play a role in oxidative stress, apoptosis, inflammation and/or immunity and include gelsolin (p=0.00005), plasminogen (p=0.00009), angiotensinogen (p=0.0001), apolipoprotein A-II (p=0.0002), beta-2-microglobulin (p=0.0002), dickkopf-3 (DKK-3; p=0.0002), pigment epithelium-derived factor (p=0.0002), RIG-like 7–1 (p=0.0002), afamin (p=0.0003), fibronectin 1 (FN1; p=0.0003), apolipoprotein A-I (p=0.0004), activated complement C4 protein (C4a; p=0.0004) and prothrombin (p=0.0004). Many of the identified proteins were novel proteins that have not been associated with glaucoma in prior studies. All but C4a (complement C4 is a plasma protein but not in an activated form) are known plasma proteins and the elevated levels of these proteins in the aqueous humor suggests a breach in the blood-aqueous barrier with passive influx into the anterior chamber of the eye.

Conclusions

The presence of these proteins in the aqueous humor suggests that glaucoma shunt device causes either a breach in blood-aqueous barrier or chronic trauma, increasing influx of oxidative, apoptotic and inflammatory proteins that could potentially cause corneal endothelial damage.

Introduction

Glaucoma is an optic neuropathy characterized by progressive loss of retinal ganglion cells that lead to structural changes at the optic nerve head and functional visual loss. It is often, but not always, associated with increased intra-ocular pressure (IOP). According to the World Health Organization, it is the second leading cause of blindness in the world and accounts for 9%–12% of cases of blindness in the US. Management strategies include medical therapy usually in the form of topical anti-glaucoma medications to lower IOP. If IOP is resistant to medical therapy and/or there is progressive optic nerve damage, surgery is considered, usually in the form of trabeculectomy or a glaucoma shunt device.

Glaucoma has long been recognized as an important factor influencing corneal graft survival [1-5]. In our published series of over 4,000 full thickness penetrating keratoplasties, we identified pre-existing glaucoma as a risk factor for graft failure and other authors have reported similar outcomes [2,6,7]. High intra-ocular pressure (IOP) is not only detrimental to optic nerve function, it can also lead to corneal endothelial cell attrition. Glaucoma filtration surgery, although essential for preservation of visual function, has also been known to affect corneal graft survival adversely with several series citing poor longer-term graft survival especially in eyes with a glaucoma shunt device [8-10]. More recently, in eyes undergoing endothelial keratoplasty, glaucoma filtration surgery also had a significantly adverse effect on graft survival [11].

The mechanisms of corneal endothelial damage in eyes with a glaucoma shunt device are not fully understood. Glaucoma shunt devices can damage the corneal endothelium by mechanical means or by permitting retrograde entrance of inflammatory cells into the anterior chamber [12-15]. In addition, glaucoma shunt devices disrupt the blood–aqueous barrier and this could further increase influx of inflammatory mediators that could potentially cause corneal endothelial damage.

Proteomics is one of the emerging techniques for biomarker discovery. Aqueous humor (AH) is the biologic fluid in the eye that has the task of protecting and supplying nutrition to the cornea, lens and trabecular meshwork (TM). A balance between production and drainage of AH is critical to maintaining normal IOP. The protein composition of AH has been shown to change dramatically in various ocular conditions such as corneal graft rejection [16], myopia [17], corneal dystrophies [18-20], and glaucoma [21-24]. Although the exact pathogenesis of glaucoma remains unclear, it is likely that alterations in the AH protein composition trigger signaling molecules that could modify the TM, increasing resistance to outflow and hence glaucoma [25,26].

With this background in mind, we decided to explore the AH proteomics in eyes with a pre-existing glaucoma shunt device to characterize the proteins that could potentially serve as biomarkers for not only glaucoma but also for corneal endothelial damage. The results of this study could potentially influence therapeutic strategies designed to improve longer-term graft survival in these high-risk eyes.

Methods

Sample collection

Patients were selected and samples collected as previously described [27]. Briefly, study subjects were either patients scheduled to undergo routine cataract surgery (controls) or patients with previous glaucoma shunt device scheduled to undergo corneal transplant or cataract surgery at a tertiary referral center, Price Vision Group (Indianapolis, IN). Exclusion criteria were as follows: history of conjunctivitis or any ocular infection within the previous 3 months and ongoing intraocular inflammation. An independent review board (IRB) approved the study and all subjects signed a written Informed Consent document. Before surgery, the patient's eye was anesthetized topically with proparacaine. A stab incision was made in the peripheral cornea, and 0.1 to 0.2 ml of anterior chamber fluid was aspirated using a 30-gauge needle. AH samples were stored frozen in liquid nitrogen until analysis. Any sample suspected of being contaminated with blood or iris pigment was discarded. Samples from 22 subjects were analyzed (11 cataract patients and 11 patients with glaucoma shunt device) as shown in Table 1.

Table 1. Clinical data on study patients and normal controls.

Age Sex Type of surgery during aqueous humor tap
Study patients
69
Female
Cataract
56
Male
Corneal transplant
31
Male
Cataract
56
Male
Corneal transplant
23
Male
Corneal transplant
74
Male
Corneal transplant
45
Male
Cataract
90
Female
Corneal transplant
49
Male
Corneal transplant
89
Male
Corneal transplant
71
Female
Repeat glaucoma shunt
Normal controls
52
Male
Cataract
73
Male
Cataract
76
Female
Cataract
72
Male
Cataract
70
Male
Cataract
73
Male
Cataract
63
Female
Cataract
43
Female
Cataract
60
Female
Cataract
66
Female
Cataract
56 Female Cataract

Materials

Acetonitrile and ammonium bicarbonate were purchased from Fisher Scientific (Fair Lawn, NJ). Dithiothreitol (DTT) and iodoacetamide (IAA) were obtained from Bio-Rad Laboratories (Hercules, CA). Trypsin was purchased from Promega (Madison, WI). ProteoPrep immunoaffinity depletion kit was purchased from Sigma (St. Louis, MO). The following sample preparation and mass spectrometric analyses were performed at MetaCyt Biochemical Analysis Center (Bloomington, IN).

Depletion and protein assay

Depletion of albumin and IgG was performed using the ProteoPrep immunoaffinity depletion kit (Sigma) as described in instruction manual with some modification. As the depletion kit is designed for plasma samples and the protein content in AH is significantly lower, preliminary studies were performed to develop a protocol for optimal AH depletion, which resulted in enhanced protein identification (data not shown). Briefly, an estimation of material to be used to deplete albumin and IgG from AH was made using a BCA protein assay and quantification of albumin and IgG in AH samples relative to plasma assuming total protein content of 80 µg/µl and 75% albumin and IgG in plasma. The estimated amount of material by weight was measured from the ProteoPrep immunoaffinity column and transferred to an empty spin column, and depletion was performed as described in the instruction manual.

Trypsin digestion

Protein samples were subjected to tryptic digestion before analysis as follows: after thermal denaturation at 95 °C for 5 min, samples were reduced through the addition of DTT to a final concentration of 5 mM and incubated at 60 °C for 45 min. Alkylation was then followed by an addition of IAA to a final concentration of 20 mM for 45 min in the dark at room temperature. A second aliquot of DTT was then added, increasing the final concentration of DTT to about 10 mM. The samples were then incubated at room temperature for 30 min to quench the alkylation reaction. Next, trypsin was added (1:30 w/w) and microwave-assisted enzymatic digestion was performed at 45 °C for 15 min at the power of 50 W. Finally enzymatic digestion was quenched through the addition of 0.5 µl of neat formic acid.

Instrumentation

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses of the tryptic digests were performed using a Dionex 3000 Ultimate nano-LC system (Dionex, Sunnyvale, CA) interfaced to an LTQ Orbitrap hybrid mass spectrometer (Thermo Scientific, San Jose, CA). Prior to separation, a 4-µl aliquot of trypsin digestion (1 µg protein equivalent) was loaded onto a PepMap300 C18 cartridge (5 µm, 300 Å; Dionex) and eluted through the analytical column (150 mm×100 µm i.d, 200 Å pores) packed with C18 magic (Michrom Bioresources, Auburn, CA). Peptides originating from protein tryptic digests were separated using a reversed-phase gradient from 10%–55% B, 99.9% acetonitrile with 0.1% formic acid over 50 min for proteins isolated from the aqueous humor, at 500 nl/min flow rate and passed through an ADVANCE ionization source (Michrom Bioresources). The mass spectrometer was operated in an automated data-dependent mode that was switching between MS scan and CID-MS. In this mode, eluted LC products undergo an initial full-spectrum MS scan from m/z 300 to 2,000 in the Orbitrap at 15,000 mass resolutions, and subsequently CID-MS (at 35% normalized collision energy) was performed in the ion trap. The precursor ion was isolated using the data-dependent acquisition mode with a 2 m/z isolation width to select automatically and sequentially five most intense ions (starting with the most intense) from the survey scan. The total cycle (6 scans) was continuously repeated for the entire LC-MS run under data-dependent conditions with dynamic exclusion set to 60 s. Performing MS scanning in the Orbitrap offers high mass accuracy and accurate charge state assignment of the selected precursor ions.

Protein identification and label-free quantification

Peptide and protein identification

The acquired data were searched against the International Protein Index (IPI) human database (ipi.HUMAN.v3.69.fasta) using SEQUEST (v. Twenty-eight rev. 12) algorithms in Bioworks (v. 3.3). General parameters were set to: peptide tolerance 2.0 amu, fragment ion tolerance 1.0 amu, enzyme limits set as “fully enzymatic - cleaves at both ends,” and missed cleavage sites set at 2. The searched peptides and proteins were validated by PeptideProphet [28] and ProteinProphet [29] in the Trans-Proteomic Pipeline (TPP, v. 3.3.0). Only proteins with probability ≥0.9000 and peptides with probability ≥0.8000 were reported.

Protein quantification

Protein quantification was performed using an in-house software package, IdentiQuantXLTM. The retention time of peptide for its intensity extraction was performed with an experiment-based algorithm RetentionTimeXLTM. The intensity of validated peptide was extracted and the protein quantification was calculated from peptide intensity.

Statistical analysis

The relative quantity of each protein was determined in each individual AH sample, and the results for the two groups were compared using the Student’s t-test. A p-value <0.05 was considered to be statistically significant but then post-hoc adjustment of the p-value was performed using Holm-Sidak test to correct for multiple comparisons.

Results

We performed label-free quantitative mass spectrometry on AH samples derived from patients with and without prior glaucoma shunt surgery. AH samples were depleted of interfering abundant proteins such as albumin before LC-MSMS was used for protein identification and quantification. We identified 135 proteins in the albumin-depleted fraction (Table 2) with high confidence in the study as well as control group AH. After using stringent detection criteria, the AH in eyes with a prior glaucoma shunt device showed significantly increased levels of 13 proteins as shown in bold in Table 2. These proteins were pro-inflammatory (plasminogen [p=0.00009], angiotensinogen [p=0.0001], prothrombin [p=0.0004] and C4a protein [p=0.0004]); anti-oxidative/anti-apoptotic (gelsolin [p=0.00005], afamin [p=0.0003] pigment epithelium-derived factor [PEDF; p=0.0002], and dickkopf-3 [DKK-3; p=0.0002]); anti-inflammatory (apolipoprotein A-I [apo A-1; p=0.0004], and apolipoprotein A-II [apo A-II; p=0.0002]); or other roles (fibronectin 1 [FN1; p=0.0003], RIG-like 7–1 [p=0.0002], and beta-2-microglobulin [p=0.0002]). The percent of protein sequence covered by the peptides identified with high confidence is listed for each protein along with the number of unique sequences as well as the fold-change compared to the protein level in control patients.

Table 2. Proteins identified in the albumin-depleted fraction of aqueous humor in patients with glaucoma shunt device using Liquid Chromatography/Mass Spectrometry (LC-MS/MS).

Protein ID Protein name Protein coverage (%) Number of unique sequences CV (%) normal CV (%) shunt Fold change in shunt patients p-value (shunt versus normal) Plasma protein
IPI00026314
Gelsolin
5.12
2
97.38
42.91
4.7
0.000050
Yes
IPI00019580
Plasminogen
21.98
10
66.75
47.85
6.2
0.000090
Yes
IPI00032220
Angiotensinogen
16.49
5
50.43
46.49
4.4
0.000100
Yes
IPI00021854
Apolipoprotein A-II
64
6
71.48
58.76
18.5
0.000200
Yes
IPI00004656
Beta-2-microglobulin
18.49
1
33.81
49.13
5.7
0.000200
Yes
IPI00383937
cDNA FLJ33633 fis, clone BRAMY2022786, highly similar to Homo sapiens dickkopf-3 (DKK-3) mRNA
30.7
4
43.93
45.25
4
0.000200
Yes
IPI00002714
cDNA FLJ52545, highly similar to Dickkopf-related protein 3
18.13
4
43.93
45.25
4
0.000200
Yes
IPI00006114
Pigment epithelium-derived factor
38.28
12
54.02
48.22
4.6
0.000200
Yes
IPI00386812
RIG-like 7–1
23.39
2
35.69
52.57
7.2
0.000200
Yes
IPI00019943
Afamin
13.86
5
53.94
45.3
3.3
0.000300
Yes
IPI00922213
cDNA FLJ53292, highly similar to Homo sapiens fibronectin 1 (FN1), transcript variant 5, mRNA
2.17
1
53.67
53.43
6.2
0.000300
Yes
IPI00021841
Apolipoprotein A-I
54.31
17
49.82
55.18
6.4
0.000400
Yes
IPI00889723
C4A protein
18.43
18
60.51
50.69
4.1
0.000400
No
IPI00019568
Prothrombin (Fragment)
18.81
7
59.4
43.88
2.9
0.000400
Yes
IPI00937598
similar to C4A protein isoform 2
15.9
15
59.85
45.91
3.3
0.000400
No
IPI00017601
Ceruloplasmin
32.68
24
51.22
52.5
4.5
0.000500
 
IPI00022488
Hemopexin
53.9
17
73.75
56.72
6.2
0.000500
 
IPI00215894
Kininogen-1, Isoform LMW
26.93
8
49.09
47.4
3.2
0.000600
 
IPI00789376
KNG1 protein
34.36
7
48.72
47.54
3.1
0.000700
 
IPI00163207
N-acetylmuramoyl-L-alanine amidase
4.69
2
64.87
53.66
4.2
0.000700
 
IPI00909649
Protein
49.53
3
52.92
61.2
7.2
0.000800
 
IPI00893864
Complement factor B
6.45
2
24.5
55.96
4.4
0.000900
 
IPI00022420
Retinol-binding protein 4
33.33
4
63.72
53.93
3.9
0.000900
 
IPI00022463
Serotransferrin
67.62
50
66.88
50.61
3.4
0.000900
 
IPI00032179
Antithrombin-III
43.1
14
62.13
59.34
4.7
0.001000
 
IPI00922058
cDNA FLJ59472, highly similar to Tripeptidyl-peptidase 1
7.8
1
28.97
41.42
2.3
0.001000
 
IPI00783987
Complement C3 (Fragment)
29.95
34
57.03
54.67
3.6
0.001000
 
IPI00892547
Complement component 4A
20.3
20
59.62
59.21
5
0.001000
 
IPI00027482
Corticosteroid-binding globulin
10.12
2
45.96
61.64
5.4
0.001000
 
IPI00556287
Putative uncharacterized protein
17.99
2
34.29
56.42
4.3
0.001000
 
IPI00924948
Putative uncharacterized protein AZGP1
26.87
4
66.28
55.65
4
0.001000
 
IPI00643525
Putative uncharacterized protein C4A
21.33
21
58.55
59.2
5.2
0.001000
 
IPI00739237
similar to complement component 3
4.87
1
42.7
58.4
4.7
0.001000
 
IPI00935601
similar to complement component 4B (Childo blood group), partial
2.85
1
92.62
61.48
6
0.001000
 
IPI00298971
Vitronectin
13.6
4
56.8
59.77
5.1
0.001000
 
IPI00020091
Alpha-1-acid glycoprotein 2
39.8
8
74.23
60.87
4.2
0.002000
 
IPI00022895
Alpha-1B-glycoprotein
35.96
11
64.92
68.72
6.5
0.002000
 
IPI00166729
alpha-2-glycoprotein 1, zinc precursor
40.6
9
55.39
51.01
2.9
0.002000
 
IPI00478003
Alpha-2-macroglobulin
11.47
11
56.79
54.7
3.3
0.002000
 
IPI00218748
Beta-crystallin B2
19.51
4
95.69
65.53
5.7
0.002000
 
IPI00019591
cDNA FLJ55673, highly similar to Complement factor B
5.45
4
44.43
58.6
4.2
0.002000
 
IPI00032258
Complement C4-A
20.07
20
58
62.3
5
0.002000
 
IPI00654875
Complement C4-B
18.18
18
59.41
55.54
3.5
0.002000
 
IPI00218192
Inter-alpha-trypsin inhibitor heavy chain H4
7.33
4
66.74
67.97
7.6
0.002000
 
IPI00032328
Kininogen-1, Isoform HMW
15.84
7
50.67
51.24
2.9
0.002000
 
IPI00384938
Putative uncharacterized protein DKFZp686N02209
29.88
9
68.21
57.76
3.6
0.002000
 
IPI00014048
RNase pancreatic
13.46
1
52.72
61.62
4.8
0.002000
 
IPI00553177
Alpha-1-antitrypsin
66.27
30
75.69
68.19
5.9
0.003000
 
IPI00947307
cDNA FLJ58075, highly similar to Ceruloplasmin
15.75
9
75.54
71.62
7.1
0.003000
 
IPI00892604
Complement component C4B (Childo blood group) 2
26.09
27
57.45
64.62
4.5
0.003000
 
IPI00013179
Prostaglandin-H2 D-isomerase
32.63
3
59.4
52.56
2.7
0.003000
 
IPI00930124
Putative uncharacterized protein DKFZp686C11235
24.1
7
68.59
60.18
3.6
0.003000
 
IPI00947496
124 kDa protein
6.46
5
72.67
68.22
4.4
0.004000
 
IPI00939333
86 kDa protein
11.13
5
57.33
57.59
3
0.004000
 
IPI00477597
Haptoglobin-related protein
20.98
7
48.71
67.78
4.6
0.004000
 
IPI00745872
Serum albumin
56.98
31
62.39
65.64
4
0.004000
 
IPI00796888
26 kDa protein
7.76
1
28.92
68.46
4.1
0.005000
 
IPI00942927
cDNA FLJ57339, highly similar to Complement C3
5.28
3
74.79
75.8
6.2
0.005000
 
IPI00796830
13 kDa protein
10.53
1
63.45
55.98
2.6
0.006000
 
IPI00304273
Apolipoprotein A-IV
37.37
14
62.49
69.08
4.1
0.006000
 
IPI00924859
kininogen-1, Isoform 3
26.6
7
53.65
61.14
3
0.006000
 
IPI00555812
Vitamin D-binding protein
42.62
17
62.88
76.48
5.7
0.006000
 
IPI00022426
Protein AMBP
17.61
4
78.76
46.73
2.4
0.007000
 
IPI00550991
cDNA FLJ35730 fis, clone TESTI2003131, highly similar to ALPHA-1-ANTICHYMOTRYPSIN
43.53
16
51.65
68.56
3.6
0.008000
 
IPI00908876
cDNA FLJ50830, highly similar to Serum albumin
7.27
2
126.5
89.61
13
0.009000
 
IPI00879709
Complement component 6 precursor
2.65
1
137.14
82.89
6.9
0.009000
 
IPI00884251
ENV polyprotein (coat polyprotein) family protein
5.29
1
101.32
85.96
8.3
0.009000
 
IPI00291866
Plasma protease C1 inhibitor
20.6
7
50.85
68
3.3
0.009000
 
IPI00022434
Putative uncharacterized protein ALB
33.17
15
84.91
83.54
7
0.009000
 
IPI00877967
Putative uncharacterized protein F2
16.67
3
38.9
70.48
3.7
0.009000
 
IPI00298828
Beta-2-glycoprotein 1
32.75
8
50.08
58.28
2.4
0.010000
 
IPI00794184
cDNA FLJ37971 fis, clone CTONG2009958, highly similar to CERULOPLASMIN
5.2
2
43.15
54.38
2.2
0.010000
 
IPI00910625
cDNA FLJ51265, moderately similar to Beta-2-glycoprotein 1
31.75
6
57.7
53.85
2.3
0.010000
 
IPI00291262
Clusterin
21.83
8
64.87
81.71
4.9
0.010000
 
IPI00019690
GRAM domain-containing protein 4
1.56
1
42.31
68.53
3.1
0.010000
 
IPI00887739
similar to complement component C3, partial
3.01
2
99.17
85.98
6.6
0.010000
 
IPI00556036
16 kDa protein
9.35
1
46.68
101.59
22.4
0.020000
 
IPI00940791
20 kDa protein
26.49
4
68.87
66.04
2.5
0.020000
 
IPI00022429
Alpha-1-acid glycoprotein 1
41.29
10
92.7
78.72
4
0.020000
 
IPI00022395
Complement component C9
6.26
3
83.12
58.44
2.4
0.020000
 
IPI00291867
Complement factor I
8.92
4
66.9
89.51
5.8
0.020000
 
IPI00022371
Histidine-rich glycoprotein
18.48
6
52.49
65.69
2.7
0.020000
 
IPI00016915
Insulin-like growth factor-binding protein 7
4.61
1
43.16
87.43
5.3
0.020000
 
IPI00830047
Putative uncharacterized protein ENSP00000374858 (Fragment)
14.15
1
86.52
93.61
6.7
0.020000
 
IPI00736860
Putative uncharacterized protein ENSP00000374988 (Fragment)
5.76
1
55.66
81.96
3.7
0.020000
 
IPI00942787
42 kDa protein
34.82
13
61.83
91.49
4.4
0.030000
 
IPI00922262
cDNA FLJ56822, highly similar to Alpha-2-HS-glycoprotein
6.94
2
82.08
90.01
4.1
0.030000
 
IPI00641737
Haptoglobin
37.86
14
60.67
91.2
4.3
0.030000
 
IPI00305461
Inter-alpha-trypsin inhibitor heavy chain H2
3.59
2
67.05
67.19
2.4
0.030000
 
IPI00019038
Lysozyme C
35.14
3
99.3
100.08
6.8
0.030000
 
IPI00855916
Transthyretin
55.43
12
56.66
51.02
1.9
0.030000
 
IPI00910636
cDNA FLJ53848, highly similar to Inter-alpha-trypsin inhibitor heavy chain H2
2.76
1
105.83
80.27
3.2
0.040000
 
IPI00032293
Cystatin-C
11.64
2
37.2
65.84
2.2
0.040000
 
IPI00426060
Putative uncharacterized protein DKFZp686J11235 (Fragment)
24.11
8
50.51
70.47
2.4
0.040000
 
IPI00218413
Biotinidase
3.13
1
152.06
59.58
2.5
0.050000
 
IPI00296165
cDNA FLJ54471, highly similar to Complement C1r subcomponent
1.95
1
38.2
100.53
4.6
0.050000
 
IPI00022418
Fibronectin
1.63
2
49.85
82.97
2.8
0.050000
 
IPI00478493
Haptoglobin isoform 2 preproprotein
34.01
10
64.09
96.65
4.2
0.050000
 
IPI00431645
HP protein
25.62
6
72.49
101.65
4.9
0.050000
 
IPI00910432
cDNA FLJ57921, highly similar to Apolipoprotein D
10.94
1
32.12
115.55
6.7
0.060000
 
IPI00156171
Ectonucleotide pyrophosphatase/phosphodiesterase family member 2
4.06
2
154.37
118.59
9.6
0.060000
 
IPI00026199
Glutathione peroxidase 3
29.2
6
62.58
52.9
1.9
0.060000
 
IPI00292150
Latent-transforming growth factor beta-binding protein 2
1.04
1
35.63
129.41
22.1
0.060000
 
IPI00002678
Opticin
14.46
2
44.87
57.55
1.8
0.060000
 
IPI00852577
HCG2040025
33.02
2
79.85
92.66
3.2
0.070000
 
IPI00021000
Osteopontin
10.19
2
73.71
115.05
6.2
0.070000
 
IPI00514159
Inter-alpha (Globulin) inhibitor H2
8.02
1
61.82
59.15
1.8
0.090000
 
IPI00029863
55 kDa protein (Alpha-2-antiplasmin precursor)
8.69
2
55.95
78.97
−1.8
0.100000
 
IPI00002147
Chitinase-3-like protein 1
9.92
2
56.83
128.11
4.3
0.100000
 
IPI00009650
Lipocalin-1
6.25
1
66.59
61.11
1.8
0.100000
 
IPI00844156
SERPINC1 protein
3.86
1
77.33
80.74
2.3
0.100000
 
IPI00383164
SNC66 protein
19.72
7
64.5
79.48
2.1
0.100000
 
IPI00794403
23 kDa protein
17.82
3
80.99
159.25
9.8
0.200000
 
IPI00922298
cDNA FLJ51445, highly similar to AMBP protein
7.04
1
64.26
81.95
2
0.200000
 
IPI00022431
cDNA FLJ55606, highly similar to Alpha-2-HS-glycoprotein
13.39
4
49.95
144.5
5.1
0.200000
 
IPI00029739
Complement factor H
1.46
1
48.36
116.78
2.6
0.200000
 
IPI00021891
Fibrinogen gamma chain
7.51
2
86.57
136.01
3.5
0.200000
 
IPI00292530
Inter-alpha-trypsin inhibitor heavy chain H1
2.09
1
66.21
40.03
−1.7
0.200000
 
IPI00020986
Lumican
16.86
4
72.95
148.83
7.5
0.200000
 
IPI00514285
Prostaglandin D2 synthase 21 kDa
14.73
2
62.05
70.38
1.7
0.200000
 
IPI00658053
Putative uncharacterized protein CTSD
8.54
1
46.63
73.25
1.7
0.200000
 
IPI00877925
Putative uncharacterized protein SERPINF2
6.44
1
74.76
66.92
−1.5
0.200000
 
IPI00218732
Serum paraoxonase/arylesterase 1
6.2
1
41.72
55.4
1.5
0.200000
 
IPI00006662
Apolipoprotein D
20.11
3
201.07
113.63
2.7
0.300000
 
IPI00165972
Complement factor D preproprotein
15
2
63.48
59
1.5
0.300000
 
IPI00930442
Putative uncharacterized protein DKFZp686M24218
5.67
1
99.15
71.25
1.7
0.300000
 
IPI00935408
CFI protein
4.24
1
81.47
78.69
1.3
0.400000
 
IPI00022417
Leucine-rich alpha-2-glycoprotein
29.97
5
59.22
42.95
1.2
0.400000
 
IPI00334282
Protein FAM3C
7.05
1
38.27
61.04
−1.1
0.500000
 
IPI00012503
Proactivator polypeptide
2.86
1
262.28
79.65
1.8
0.600000
 
IPI00807428
Putative uncharacterized protein
22.98
3
168.41
135.85
2.1
0.600000
 
IPI00011229
Cathepsin D
8.5
2
106.86
100.28
−1.5
0.700000
 
IPI00423460
Putative uncharacterized protein DKFZp686G21220 (Fragment)
9.88
3
104.19
116.55
1.6
0.700000
 
IPI00022337
Retinol-binding protein 3
5.93
3
71.53
69.31
1.1
0.800000
 
IPI00301579 cDNA FLJ59142, highly similar to Epididymal secretory protein E1 7.96 1 39.81 61.76 1.2 0.900000  

Differentially expressed proteins detected using stringent filtering criteria are highlighted in bold.

Discussion

This study highlights for the first time the differential expression of AH proteins in eyes with a glaucoma shunt device compared to normal controls. Many of the identified proteins except fibronectin [30] and PEDF [31] are novel proteins that to our knowledge have not been detected in the AH of glaucoma patients. Interestingly, all of the identified proteins except C4a are known plasma proteins and increased expression in the AH suggests a breach in the blood aqueous barrier caused by a glaucoma drainage device. AH proteome changes identified in this study may not only help elucidate glaucoma pathogenesis but also shed light on the possible mechanisms that result in corneal endothelial damage and hence accelerated corneal transplant failure in eyes with glaucoma surgery.

We found a significant upregulation of complement C4a (fold change, 5; p=0.0004), an activated fragment of complement component C4. Complement activation is under the tight control of complement inhibitors; uncontrolled complement activation can cause cell lysis and inflammation while a balanced activation is necessary for clearing tissue debris and in healing. Imbalance in complement regulation has also been suggested to contribute to the neurodegenerative damage characteristic of glaucoma [32]. Activated complement in the AH, as seen in this study, could possibly cause corneal endothelial damage via direct cell lysis and inflammation.

There was evidence of enhanced fibrinolytic and coagulative activity in the AH as suggested by differential expression of prothrombin (fold change, 2.9; p=0.0004), angiotensinogen (fold change, 4.4; p=0.0001) and plasminogen (fold change, 6.2; p=0.00005). O’Brien et al. [33] have reported elevated prothrombin levels in the plasma of patients with primary open angle glaucoma and have implicated this hypercoagulable state in glaucoma pathophysiology. Angiotensinogen is a component of the renin-angiotensin (RAS) system and plays a role in the regulation of AH dynamics [34]. Plasminogen is a component of the plasmin system, and the main physiologic inhibitor of the plasmin system is plasminogen activator inhibitor-1 (PAI-1). Elevated levels of PAI-1 have been reported in the AH of glaucoma patients thus contributing to glaucoma pathogenesis by reducing proteolysis of the extracellular matrix in the TM and increasing resistance to outflow [35]. Prothrombin is also recognized as a biomarker of systemic sepsis and inflammation [36] and elevated levels in the AH suggest increased inflammation due to a breach in the blood-aqueous barrier caused by the glaucoma shunt device. Increased inflammation has been shown to stimulate increased synthesis of pro-inflammatory cytokines like interleukin-1 and tumor necrosis factor by the corneal endothelium [37,38] leading to corneal endothelial damage.

Afamin (fold change, 3.3; p=0.0003) and gelsolin (fold change, 4.7; p=0.00005) were the 2 extracellular chaperones found to be differentially expressed in the AH of patients with glaucoma shunt device compared to normals. Afamin is a member of the albumin multigene family with vitamin E-binding properties. It plays a crucial role in protecting against oxidative damage and displays neuroprotective activity not only by virtue of binding and transporting vitamin E but also on its own [39]. Gelsolin is an anti-oxidant and anti-apoptotic protein that has been implicated as a therapeutic target in Alzheimer disease since it has been shown to reduce amyloid load by inhibiting Abeta fibrillization in animal studies [40]. A decreased level of gelsolin has been observed in patients with sepsis, myocardial infarction and inflammation while an increased level has been noted in amyloidosis [41,42], so it could be a secondary response to increased amyloid load The upregulation of these extracellular chaperones is likely a response to the increased oxidative stress in the aqueous secondary to glaucoma. Oxidative stress has been recognized as the main pathogenic factor underlying open angle glaucoma [43-45]. It is likely that this may also contribute to corneal endothelial damage. Increased expression of these proteins may reflect the inability of these extracellular chaperones to completely inhibit oxidative and apoptotic damage both in the TM as well as the corneal endothelium.

Pigment epithelium-derived factor (PEDF), a member of the serpin family of proteins and expressed in all ocular tissues of the human eye, was significantly upregulated in eyes with a glaucoma shunt device (fold change, 4.6; p=0.0002). It is neuroprotective and anti-angiogenic and recently recognized as an endogenous anti-inflammatory factor [46,47]. Significantly reduced levels have been reported in advanced glaucoma AH compared to normal controls [31]. Additionally in animal models, PEDF has been shown to protect retinal ganglion cells from pressure-induced ischemia [48]. Our finding of significantly increased expression of PEDF is intriguing and leads us to speculate that perhaps this protein serves a protective role in the AH.

The function of Dickkopf-3 (Dkk3) is unclear; however, Jung et al. [49] suggest that it may acts as an anti-apoptotic molecule by decreasing intracellular levels of reactive oxygen species. Recently Nakamura et al. [50] have demonstrated that it may play a cytoprotective role in the retina by reducing caspase activity and hence protecting against apoptosis. Its role in glaucoma and corneal endothelial damage needs further evaluation.

Increased expression of apolipoprotein A-I (fold change, 6.4; p=0.0004) and A-II (fold change, 18.4; p=0.0002) was observed in this study. Apo A-1 and – II, by virtue of their association with high density lipoprotein (HDL), have anti-inflammatory properties [51]. However, its specific role in glaucoma and corneal endothelial damage is unclear and requires further investigation.

Fibronectin, an extracellular matrix glycoprotein, was increased sixfold in this study compared to normal controls (p=0.0003). This suggests disruption of the blood aqueous barrier that occurs in eyes with a glaucoma shunt device. Vesaluoma et al. [30] have demonstrated increased expression of this protein in eyes with pseudoexfoliation glaucoma. Increased fibronectin can transform TM cells and decrease the breakdown of extracellular matrix material, allowing excess to accumulate. This could ultimately reduce trabecular outflow and raise IOP.

RIG-like 7–1 constitute a family of pattern recognition receptors (PRRs). They mediate the initial sensing of microbial and endogenous danger-associated molecules that are released by tissue damage. By activating transcription of inflammatory genes they are known to control the immediate innate immune response as well as the subsequent adaptive immune response [52]. Increased levels of PRR suggests an alteration in the immunologic milieu of the AH secondary to a breach in the blood aqueous barrier.

Beta-2 microglobulin is a protein associated with major histocompatibility complex class I antigens and has value as a marker for immunologic monitoring with increased levels associated with renal and cardiac allograft rejection [53,54]. Elevated levels as seen in this study (fold change, 5.7; p=0.0002) suggest a potential role for this protein as a biomarker of increased immune mediated corneal endothelial damage in eyes with a glaucoma shunt device.

Based on the protein profile detected in this study we have hypothesized the likely mechanisms underlying corneal endothelial damage in eyes with a shunt device as well as new insights into glaucoma pathophysiology. Glaucoma per se also causes corneal endothelial damage and in the presence of glaucoma shunt device there is likely to be an exaggerated stress response leading to corneal endothelial damage and endothelial failure. This has important implications especially in the setting of corneal transplants. Corneal grafts have significantly poor long-term survival in the presence of a shunt device and future work should be targeted at identifying the specific role for these proteins so that they could potentially serve as therapeutic targets to improve graft outcomes.

This study has several strengths that need to be highlighted. The AH samples were not pooled but analyzed individually to determine proteins associated with a shunt device. We used conservative criteria for determining which proteins were differentially expressed between groups and were able to identify highly significant proteins with a large fold change compared to normals. It has been shown that the proteomic profile in glaucoma patients can vary depending on the severity of visual field defects [22]. The study patients had advanced glaucoma and this could partly explain the identification of novel proteins. The limitations of this study include the small sample size, although it is comparable to previous studies evaluating AH in eyes with glaucoma [21,22]. A useful control group would have been glaucoma patients without a shunt device who were undergoing intra-ocular surgery. A proteomic study with glaucoma as a control group is currently ongoing and should provide more insight into the pathogenic mechanism of corneal endothelial damage specific to glaucoma shunt device. This study reports on differential expression of proteins compared to normal controls but does not provide absolute quantitative data on protein levels.

Lastly, the majority of study patients had an Ahmed glaucoma shunt, which is a valved implant designed to prevent retrograde flow of fluid from the filtering bleb into the eye, so it would be interesting to determine the mechanism of increased inflammation and/or immunologic alterations seen in these eyes. Future work should be directed at evaluating the AH proteomic expression in the presence of valved and non-valved glaucoma shunts to shed light on the possible mechanisms of corneal endothelial damage with different types of shunts.

Conclusion

We demonstrated significantly altered expression of 13 proteins in AH of eyes with a glaucoma shunt device. Many of these proteins play a role in oxidative and apoptotic damage. The findings of this study seem to suggest similar mechanisms underlying both glaucoma and corneal endothelial damage. Future work should be targeted at identifying aqueous proteins that could potentially serve as markers for corneal endothelial damage in eyes with glaucoma shunt device.

Acknowledgments

This study was supported by a grant from the Cornea Research Foundation of America. Authors have no commercial interest in the subject matter discussed in the manuscript. The data from this manuscript has not been presented at meetings/conferences.

AUTHOR QUERIES

Please spell out full name the first time mentioned in the text.

References

  • 1.Wilson SE, Kaufman HE. Graft failure after penetrating keratoplasty. Surv Ophthalmol. 1990;34:325–56. doi: 10.1016/0039-6257(90)90110-h. [DOI] [PubMed] [Google Scholar]
  • 2.Thompson RW, Jr, Price MO, Bowers PJ, Price FW., Jr Long-term graft survival after penetrating keratoplasty. Ophthalmology. 2003;110:1396–402. doi: 10.1016/S0161-6420(03)00463-9. [DOI] [PubMed] [Google Scholar]
  • 3.Ayyala RS. Penetrating keratoplasty and glaucoma. Surv Ophthalmol. 2000;45:91–105. doi: 10.1016/s0039-6257(00)00141-7. [DOI] [PubMed] [Google Scholar]
  • 4.Seitz B, Langenbucher A, Nguyen NX, Kuchle M, Naumann GO. Long-term follow-up of intraocular pressure after penetrating keratoplasty for keratoconus and Fuchs' dystrophy: comparison of mechanical and Excimer laser trephination. Cornea. 2002;21:368–73. doi: 10.1097/00003226-200205000-00008. [DOI] [PubMed] [Google Scholar]
  • 5.Greenlee EC, Kwon YH. Graft failure: III. Glaucoma escalation after penetrating keratoplasty. Int Ophthalmol. 2008;28:191–207. doi: 10.1007/s10792-008-9223-5. [DOI] [PubMed] [Google Scholar]
  • 6.Williams KA, Lowe M, Bartlett C, Kelly TL, Coster DJ. Risk factors for human corneal graft failure within the Australian corneal graft registry. Transplantation. 2008;86:1720–4. doi: 10.1097/TP.0b013e3181903b0a. [DOI] [PubMed] [Google Scholar]
  • 7.Sugar A, Tanner JP, Dontchev M, Tennant B, Schultze RL, Dunn SP, Lindquist TD, Gal RL, Beck RW, Kollman C, Mannis MJ, Holland EJ. Recipient risk factors for graft failure in the cornea donor study. Ophthalmology. 2009;116:1023–8. doi: 10.1016/j.ophtha.2008.12.050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kwon YH, Taylor JM, Hong S, Honkanen RA, Zimmerman MB, Alward WL, Sutphin JE. Long-term results of eyes with penetrating keratoplasty and glaucoma drainage tube implant. Ophthalmology. 2001;108:272–8. doi: 10.1016/s0161-6420(00)00496-6. [DOI] [PubMed] [Google Scholar]
  • 9.Alvarenga LS, Mannis MJ, Brandt JD, Lee WB, Schwab IR, Lim MC. The long-term results of keratoplasty in eyes with a glaucoma drainage device. Am J Ophthalmol. 2004;138:200–5. doi: 10.1016/j.ajo.2004.02.058. [DOI] [PubMed] [Google Scholar]
  • 10.Al-Torbak A. Graft survival and glaucoma outcome after simultaneous penetrating keratoplasty and ahmed glaucoma valve implant. Cornea. 2003;22:194–7. doi: 10.1097/00003226-200304000-00002. [DOI] [PubMed] [Google Scholar]
  • 11.Price MO, Fairchild KM, Price DA, Price FW., Jr Descemet's Stripping Endothelial Keratoplasty Five-Year Graft Survival and Endothelial Cell Loss. Ophthalmology. 2011;118:725–9. doi: 10.1016/j.ophtha.2010.08.012. [DOI] [PubMed] [Google Scholar]
  • 12.Kim CS, Yim JH, Lee EK, Lee NH. Changes in corneal endothelial cell density and morphology after Ahmed glaucoma valve implantation during the first year of follow up. Clin Experiment Ophthalmol. 2008;36:142–7. doi: 10.1111/j.1442-9071.2008.01683.x. [DOI] [PubMed] [Google Scholar]
  • 13.Kirkness CM. Penetrating keratoplasty, glaucoma and silicone drainage tubing. Dev Ophthalmol. 1987;14:161–5. doi: 10.1159/000414385. [DOI] [PubMed] [Google Scholar]
  • 14.McDonnell PJ, Robin JB, Schanzlin DJ, Minckler D, Baerveldt G, Smith RE, Heuer D. Molteno implant for control of glaucoma in eyes after penetrating keratoplasty. Ophthalmology. 1988;95:364–9. doi: 10.1016/s0161-6420(88)33187-8. [DOI] [PubMed] [Google Scholar]
  • 15.Topouzis F, Coleman AL, Choplin N, Bethlem MM, Hill R, Yu F, Panek WC, Wilson MR. Follow-up of the original cohort with the Ahmed glaucoma valve implant. Am J Ophthalmol. 1999;128:198–204. doi: 10.1016/s0002-9394(99)00080-x. [DOI] [PubMed] [Google Scholar]
  • 16.Funding M, Vorum H, Honore B, Nexo E, Ehlers N. Proteomic analysis of aqueous humour from patients with acute corneal rejection. Acta Ophthalmol Scand. 2005;83:31–9. doi: 10.1111/j.1600-0420.2005.00381.x. [DOI] [PubMed] [Google Scholar]
  • 17.Duan X, Lu Q, Xue P, Zhang H, Dong Z, Yang F, Wang N. Proteomic analysis of aqueous humor from patients with myopia. Mol Vis. 2008;14:370–7. [PMC free article] [PubMed] [Google Scholar]
  • 18.Richardson MR, Segu ZM, Price MO, Lai X, Witzmann FA, Mechref Y, Yoder MC, Price FW. Alterations in the aqueous humor proteome in patients with Fuchs endothelial corneal dystrophy. Mol Vis. 2010;16:2376–83. [PMC free article] [PubMed] [Google Scholar]
  • 19.Bramsen T, Stenbjerg S. Fibrinolytic factors in aqueous humour and serum from patients with Fuchs' dystrophy and patients with cataract. Acta Ophthalmol (Copenh) 1979;57:470–6. doi: 10.1111/j.1755-3768.1979.tb01831.x. [DOI] [PubMed] [Google Scholar]
  • 20.Wilson SE, Bourne WM, Maguire LJ, Rahhal FM, Ribaudo RK, Kreutzer DL, O'Rourke J. Aqueous humor composition in Fuchs' dystrophy. Invest Ophthalmol Vis Sci. 1989;30:449–53. [PubMed] [Google Scholar]
  • 21.Bouhenni RA, Al Shahwan S, Morales J, Wakim BT, Chomyk AM, Alkuraya FS, Edward DP. Identification of differentially expressed proteins in the aqueous humor of primary congenital glaucoma. Exp Eye Res. 2011;92:67–75. doi: 10.1016/j.exer.2010.11.004. [DOI] [PubMed] [Google Scholar]
  • 22.Izzotti A, Longobardi M, Cartiglia C, Sacca SC. Proteome alterations in primary open angle glaucoma aqueous humor. J Proteome Res. 2010;9:4831–8. doi: 10.1021/pr1005372. [DOI] [PubMed] [Google Scholar]
  • 23.Grus FH, Joachim SC, Sandmann S, Thiel U, Bruns K, Lackner KJ, Pfeiffer N. Transthyretin and complex protein pattern in aqueous humor of patients with primary open-angle glaucoma. Mol Vis. 2008;14:1437–45. [PMC free article] [PubMed] [Google Scholar]
  • 24.Duan X, Xue P, Wang N, Dong Z, Lu Q, Yang F. Proteomic analysis of aqueous humor from patients with primary open angle glaucoma. Mol Vis. 2010;16:2839–46. [PMC free article] [PubMed] [Google Scholar]
  • 25.Fuchshofer R, Tamm ER. Modulation of extracellular matrix turnover in the trabecular meshwork. Exp Eye Res. 2009;88:683–8. doi: 10.1016/j.exer.2009.01.005. [DOI] [PubMed] [Google Scholar]
  • 26.Alvarado JA, Yeh RF, Franse-Carman L, Marcellino G, Brownstein MJ. Interactions between endothelia of the trabecular meshwork and of Schlemm's canal: a new insight into the regulation of aqueous outflow in the eye. Trans Am Ophthalmol Soc. 2005;103:148–62. [PMC free article] [PubMed] [Google Scholar]
  • 27.Richardson MR, Price MO, Price FW, Pardo JC, Grandin JC, You J, Wang M, Yoder MC. Proteomic analysis of human aqueous humor using multidimensional protein identification technology. Mol Vis. 2009;15:2740–50. [PMC free article] [PubMed] [Google Scholar]
  • 28.Keller A, Nesvizhskii AI, Kolker E, Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem. 2002;74:5383–92. doi: 10.1021/ac025747h. [DOI] [PubMed] [Google Scholar]
  • 29.Nesvizhskii AI, Keller A, Kolker E, Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem. 2003;75:4646–58. doi: 10.1021/ac0341261. [DOI] [PubMed] [Google Scholar]
  • 30.Vesaluoma M, Mertaniemi P, Mannonen S, Lehto I, Uusitalo R, Sarna S, Tarkkanen A, Tervo T. Cellular and plasma fibronectin in the aqueous humour of primary open-angle glaucoma, exfoliative glaucoma and cataract patients. Eye (Lond) 1998;12:886–90. doi: 10.1038/eye.1998.224. [DOI] [PubMed] [Google Scholar]
  • 31.Ogata N, Matsuoka M, Imaizumi M, Arichi M, Matsumura M. Decrease of pigment epithelium-derived factor in aqueous humor with increasing age. Am J Ophthalmol. 2004;137:935–6. doi: 10.1016/j.ajo.2003.08.058. [DOI] [PubMed] [Google Scholar]
  • 32.Tezel G, Yang X, Luo C, Kain AD, Powell DW, Kuehn MH, Kaplan HJ. Oxidative stress and the regulation of complement activation in human glaucoma. Invest Ophthalmol Vis Sci. 2010;51:5071–82. doi: 10.1167/iovs.10-5289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.O'Brien C, Butt Z, Ludlam C, Detkova P. Activation of the coagulation cascade in untreated primary open-angle glaucoma. Ophthalmology. 1997;104:725–9. doi: 10.1016/s0161-6420(97)30245-0. [DOI] [PubMed] [Google Scholar]
  • 34.Cullinane AB, Leung PS, Ortego J, Coca-Prados M, Harvey BJ. Renin-angiotensin system expression and secretory function in cultured human ciliary body non-pigmented epithelium. Br J Ophthalmol. 2002;86:676–83. doi: 10.1136/bjo.86.6.676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Dan J, Belyea D, Gertner G, Leshem I, Lusky M, Miskin R. Plasminogen activator inhibitor-1 in the aqueous humor of patients with and without glaucoma. Arch Ophthalmol. 2005;123:220–4. doi: 10.1001/archopht.123.2.220. [DOI] [PubMed] [Google Scholar]
  • 36.Kinasewitz GT, Yan SB, Basson B, Comp P, Russell JA, Cariou A, Um SL, Utterback B, Laterre PF, Dhainaut JF. Universal changes in biomarkers of coagulation and inflammation occur in patients with severe sepsis, regardless of causative micro-organism. Crit Care. 2004;8:R82–90. doi: 10.1186/cc2459. ISRCTN74215569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Wilson SE, Schultz GS, Chegini N, Weng J, He YG. Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res. 1994;59:63–71. doi: 10.1006/exer.1994.1081. [DOI] [PubMed] [Google Scholar]
  • 38.Sekine-Okano M, Lucas R, Rungger D, De Kesel T, Grau GE, Leuenberger PM, Rungger-Brandle E. Expression and release of tumor necrosis factor-alpha by explants of mouse cornea. Invest Ophthalmol Vis Sci. 1996;37:1302–10. [PubMed] [Google Scholar]
  • 39.Heiser M, Hutter-Paier B, Jerkovic L, Pfragner R, Windisch M, Becker-Andre M, Dieplinger H. Vitamin E binding protein afamin protects neuronal cells in vitro. J Neural Transm Suppl. 2002;(62):337–45. doi: 10.1007/978-3-7091-6139-5_32. [DOI] [PubMed] [Google Scholar]
  • 40.Chauhan V, Ji L, Chauhan A. Anti-amyloidogenic, anti-oxidant and anti-apoptotic role of gelsolin in Alzheimer's disease. Biogerontology. 2008;9:381–9. doi: 10.1007/s10522-008-9169-z. [DOI] [PubMed] [Google Scholar]
  • 41.Paunio T, Kangas H, Kalkkinen N, Haltia M, Palo J, Peltonen L. Toward understanding the pathogenic mechanisms in gelsolin-related amyloidosis: in vitro expression reveals an abnormal gelsolin fragment. Hum Mol Genet. 1994;3:2223–9. doi: 10.1093/hmg/3.12.2223. [DOI] [PubMed] [Google Scholar]
  • 42.Bucki R, Levental I, Kulakowska A, Janmey PA. Plasma gelsolin: function, prognostic value, and potential therapeutic use. Curr Protein Pept Sci. 2008;9:541–51. doi: 10.2174/138920308786733912. [DOI] [PubMed] [Google Scholar]
  • 43.Saccà SC, Pascotto A, Camicione P, Capris P, Izzotti A. Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open-angle glaucoma. Arch Ophthalmol. 2005;123:458–63. doi: 10.1001/archopht.123.4.458. [DOI] [PubMed] [Google Scholar]
  • 44.Tezel G. Oxidative stress in glaucomatous neurodegeneration: mechanisms and consequences. Prog Retin Eye Res. 2006;25:490–513. doi: 10.1016/j.preteyeres.2006.07.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ferreira SM, Lerner SF, Brunzini R, Evelson PA, Llesuy SF. Oxidative stress markers in aqueous humor of glaucoma patients. Am J Ophthalmol. 2004;137:62–9. doi: 10.1016/s0002-9394(03)00788-8. [DOI] [PubMed] [Google Scholar]
  • 46.Zhang SX, Wang JJ, Gao G, Shao C, Mott R, Ma JX. Pigment epithelium-derived factor (PEDF) is an endogenous antiinflammatory factor. FASEB J. 2006;20:323–5. doi: 10.1096/fj.05-4313fje. [DOI] [PubMed] [Google Scholar]
  • 47.Zhou X, Li F, Kong L, Chodosh J, Cao W. Anti-inflammatory effect of pigment epithelium-derived factor in DBA/2J mice. Mol Vis. 2009;15:438–50. [PMC free article] [PubMed] [Google Scholar]
  • 48.Takita H, Yoneya S, Gehlbach PL, Duh EJ, Wei LL, Mori K. Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2003;44:4497–504. doi: 10.1167/iovs.03-0052. [DOI] [PubMed] [Google Scholar]
  • 49.Jung IL, Kang HJ, Kim KC, Kim IG. Knockdown of the Dickkopf 3 gene induces apoptosis in a lung adenocarcinoma. Int J Mol Med. 2010;26:33–8. doi: 10.3892/ijmm_00000431. [DOI] [PubMed] [Google Scholar]
  • 50.Nakamura RE, Hunter DD, Yi H, Brunken WJ, Hackam AS. Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina. BMC Cell Biol. 2007;8:52. doi: 10.1186/1471-2121-8-52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Burger D, Dayer JM. High-density lipoprotein-associated apolipoprotein A-I: the missing link between infection and chronic inflammation? Autoimmun Rev. 2002;1:111–7. doi: 10.1016/s1568-9972(01)00018-0. [DOI] [PubMed] [Google Scholar]
  • 52.Opitz B, Eitel J, Meixenberger K, Suttorp N. Role of Toll-like receptors, NOD-like receptors and RIG-I-like receptors in endothelial cells and systemic infections. Thromb Haemost. 2009;102:1103–9. doi: 10.1160/TH09-05-0323. [DOI] [PubMed] [Google Scholar]
  • 53.Erez E, Aravot D, Erman A, Sharoni E, Raanani E, Abramov D, Dijk DV, Sahar G, Vidne BA. Beta-2 microglobulin in heart transplanted patients. Transplant Proc. 1997;29:2706–7. doi: 10.1016/s0041-1345(97)00564-2. [DOI] [PubMed] [Google Scholar]
  • 54.Roxe DM, Siddiqui F, Santhanam S, del Greco F, Wolf J. Rationale and application of beta-2-microglobulin measurements to detect acute transplant rejection. Nephron. 1981;27:260–4. doi: 10.1159/000182064. [DOI] [PubMed] [Google Scholar]

Articles from Molecular Vision are provided here courtesy of Emory University and the Zhongshan Ophthalmic Center, Sun Yat-sen University, P.R. China

RESOURCES