SUPPLEMENT
Metabolomics/Proteomics Strategies Used to Identify Biomarkers for Exfoliation Glaucoma Sara McNally, PhD* and Colm J. O’Brien, MD, FRCS*w
Abstract: It is currently estimated that 60 to 70 million people worldwide are affected by open-angle glaucoma and the majority of patients who present to clinic have raised intraocular pressure, visual field loss, and cupping of the optic nerve. Although exfoliation glaucoma (XFG) correlates with age, it is the most common cause of secondary open-angle glaucoma in the world and, with elevated intraocular pressure at onset, this disease runs an aggressive clinical course. XFG differs from primary open-angle glaucoma, in that patients have a diminished response to medication, show accelerated rates of disease progression, and therefore have a higher need for surgery. Here we highlight some major findings in the literature, which relate to the search for biomarkers of XFG by metabolomics and proteomics strategies. Key Words: exfoliation syndrome, metabolomics, proteomics
(J Glaucoma 2014;23:S51–S54)
BACKGROUND In exfoliation syndrome (XFS), fibrillar extracellular matrix material is produced by cells in the anterior segment, possibly in response to oxidative stress. Pertinent to glaucoma, excessive production and progressive accumulation of a fibrillar material occurs in various tissues including the anterior segment and outflow pathways.1 In addition, abnormal cross-linking of elastic microfibrils into fibrillar protein aggregates is a stress-induced fibrotic event in the pathogenesis of open-angle glaucoma.1 Central to these assertions was a comprehensive gene expression analysis performed in 2005, which attributes the overall pathophysiology of XFS to differential expression of genes associated with production of elastic microfibrils, cross-linking, matrix metalloproteinase action, and oxidative stress.2 As early diagnosis is key to preventing visual impairment in glaucoma, much recent work has focused on glaucoma biomarker discovery where a biomarker is a molecule/gene/clinical characteristic that allows the identification of a disease. Accepted characteristics for the “ideal” biomarker denote an agent of measureable entity with high sensitivity and specificity, which accurately predicts the presence, progression, or absence of a disease. For XFS, it is critical that a biomarker will predict progression from ocular hypertension to glaucoma and identify those at high risk of progressive damage. The role of “omic” techniques in the elucidation of glaucomatous biomarkers has recently Received for publication August 4, 2014; accepted August 11, 2014. From the *Department of Ophthalmology, Mater Misericordiae University Hospital; and wUCD School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. Disclosure: The authors declare no conflict of interest. Reprints: Sara McNally, PhD, Catherine McAuley Clinical Research Centre, 21 Nelson Street, Mater Misericordiae University hospital, Dublin 7, Ireland (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins DOI: 10.1097/IJG.0000000000000117
J Glaucoma
been reviewed.3 Here we highlight some important findings based on proteomic and metabolomic techniques used to define possible biomarkers from the anterior lens capsule, blood or aqueous humor of exfoliation glaucoma (XFG) patients; these results are summarized in Table 1.
Anterior Lens Exfoliative material isolated from the surgically removed anterior lens capsule has been analyzed to characterize resident protein constituents. Mass-spectrometry (LC-MS/MS) analysis has identified lysyl oxidase-like 1 (LOXL1) (involved in elastin cross-linking) to be genetically associated with the risk of XFS, as well as other proteins [apolipoproteinE (ApoE), latent-transforming growth factor b-binding protein-2 (LTBP-2), complement 3, and clusterin proteins] detected in the exfoliative material.4 An earlier study from 2007 utilized anterior lens capsules from patients with and without XFS which were homogenized, and protein fragments compared by SDS-PAGE after silver staining and liquid chromatography coupled to tandem MS.5 In addition to fibrillin-1, fibronectin, vitronectin, laminin, and amyloid Pcomponent, proteomic approaches identified clusterin and TIMP-3 as well as novel molecules [among them fibulin-2, desmocollin-2, the glycosaminoglycans syndecan-3, and versican, membrane metalloproteases of the ADAM family (a disintegrin and metalloprotease), and the initiation component of the classic complement activation pathway C1q].
Blood Spectrophotometric analysis of blood samples from primary open-angle glaucoma (POAG) and XFG patients reveals evidence of elevated oxidative stress when compared with control serum.6 To date, the literature reports that XFG patients may be characterized by an impaired oxidative-antioxidative balance in the anterior segment.13 In addition, raised levels of oxidative stress indicators nitric oxide and malondialdehyde have been detected in serum from POAG and XFG patients compared with controls. In our laboratory, high-density protein array screening has been used previously to analyze and compare the serum autoantibody profiles of patients with and without XFG to identify glaucoma disease–associated autoantibodies.7 Protein arrays containing >10,000 different human proteins derived from a human fetal cDNA library identify unique antibody profiles that may discriminate between patients and controls.14 High-density colony protein macroarrays were used to characterize the IgG autoantibody profiles from patient sera samples. Comparison of the autoantibody profiles between XFG and control groups identified 7 proteins that were significantly more prevalent in the XFG group, including a previously uncharacterized protein C6orf129 that contains a transmembrane domain; current work is ongoing to further characterize C6orf129 and its role in XFG pathology.
Volume 23, Number 8 Suppl 1, October/November 2014
www.glaucomajournal.com |
S51
McNally and O’Brien
J Glaucoma
Volume 23, Number 8 Suppl 1, October/November 2014
TABLE 1. Efforts to Identify Biomarkers for PXFG by Proteomic/Metabolomic Means
Marker of Interest
Technique
Model
LOXL1 and ApoE; detected LTBP-2, complement 3, and clusterin proteins in sample subset Fibrillin-1, fibronectin, vitronectin, laminin, amyloid P, clusterin, TIMP-3, fibulin2, desmocollin-2, syndecan-3, versican, C1q, and ADAM family membrane metalloproteases Nitric oxide, protein carbonyl, malondialdehyde
Digested peptides analyzed with Thermo LTQ XL linear ion trap mass spectrometer fitted with nanospray source Quadrupole time-of-flight mass spectrometry (MS) Liquid chromatography coupled to tandem MS
PXF material deposited on anterior lens capsule of affected individuals collected at time of cataract surgery
Sharma et al4
Anterior lens capsules from patients with and without exfoliation syndrome (XFS)
Ovodenko et al5
Spectrophotometry
Erdurmus¸ et al6
EEF2, EHD1, CLPTM1, FGFR3, TMEM9B, STMN4, C6orf129 CTGF
High-density protein macroarray Reverse ELISA ELISA, SDS-PAGE
Blood from forearm vein; serumseparated samples of patients with POAG, PXFG, and control subjects (n = 19) Serum samples of patients with PXFG and control subjects
Hydrogen peroxide, catalase
Goth’s colorimetric method, peroxidase activity assay using TMB Microplate assay method 8-IPGF measured with commercial immunoassay kit (Assay Designs Inc., Ann Arbor, MI)
8-isoprostaglandin F2a, ascorbic acid
IL-6, IL8
Multiplex bead analysis, immunoassays
TGF-b1, IL-8, and SAA
Multiplex bead immunoassay technique
References
Dervan et al7
Aqueous humor samples obtained from patients undergoing routine cataract surgery or trabeculectomy Aqueous humor and serum samples from XFS, XFG, and controls
Browne et al8
Aqueous humor aspirated at beginning of phacoemulsification cataract surgery from eyes of cataract patients with XFS and eyes of matched cataract patients without XFS Aqueous humor and anterior segment tissues; eyes with early and late stages of PEX syndrome/glaucoma; normal and glaucomatous control eyes Aqueous humor samples obtained from 64 eyes of 64 Japanese subjects; POAG, PXFG, cataract, control
Koliakos et al10
Koliakos et al9
Zenkel et al11
Takai et al12
ADAM indicates a disintegrin and metalloproteinase domain containing proteins; ApoE, apolipoprotein E; CLPTM1, cleft lip and palate-associated transmembrane protein 1; CTGF, connective tissue growth factor; EEF2, eukaryotic elongation factor 2; EHD1,EH domain-containing protein 1; ELISA, enzyme-linked immunosorbent assay; FGFR3, fibroblast growth factor receptor 3; IL-6/8, interleukin-6/8; IPGF, isoprostaglandin F; LOXL1, lysyl oxidaselike1; LTBP-2, latent transforming growth factor b-binding protein 2; STMN4, stathmin-like 4; TGF, transforming growth factor; SAA, serum amyloid A; TIMP-3, tissue inhibitor of metalloproteinase 3; TMEM9B, TMEM9 domain family, member B.
A recent study from 2014 has analyzed proteins from blood serum of patients of POAG, XFG, and healthy controls to identify a candidate panel of glaucoma biomarkers.15 Comparative differential proteomic analysis has elucidated enrichment of immune and inflammatory network proteins (including members of the complement C pathway) from glaucoma patients.
Aqueous Humor Proteomic analysis of human aqueous humor has come to the fore in recent times as a mechanism of identifying proteins responsible for maintenance of homeostasis in the anterior chamber. A complex mixture of growth factors, cytokines, electrolytes, and organic solutes in human aqueous humor sustain cellular metabolism in the avascular tissues of the anterior segment. We have previously shown that levels of connective tissue growth factor (a matricellular protein that interacts with the profibrotic cytokine TGF-b) are significantly higher in aqueous humor
S52 | www.glaucomajournal.com
of patients with PXFG than in both POAG and normal control subjects undergoing cataract surgery.8 Improved detection technologies have enabled investigations which, to date, have been hindered by the reality that the protein component of human aqueous humor is relatively low, containing between 120 and 500 ng/mL of protein.8,14 Antibody protein arrays and nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS) have been used to characterize the human aqueous humor proteome as a resevoir of 676 nonredundant proteins (cytokines, receptors, and chemokines with catalytic, enzymatic, and structural properties).16 Other groups have highlighted the aqueous humor as a pivotal site of oxidant-antioxidant balance in the distinction between XFS and XFG.9 In addition, the concentration of hydrogen peroxide has been found to be higher in both the serum and aqueous of XFG patients compared with controls.9 A 2003 study utilized commercially available in vitro assays to highlight elevated r
2014 Lippincott Williams & Wilkins
J Glaucoma
Volume 23, Number 8 Suppl 1, October/November 2014
Clinical
Gene
Biomarker panel
PXF material
LOX-L1
IOP
Tissue mRNA
Strategies Used to Identify Biomarkers for XFG
Cytokines
Oxidative stress
Anterior segment tissues
TGFβ1
Blood
LTBP
CTGF
Aqueous
IL-6
Tears
TG2 Discs
TIMP2
RNFL
TIMP1
Proteomics/ Lipid
Clusterin Perimetry
Oxidation: ApoE (MALDI
Omics cascade
SAA1 Genomics
MS)
Transcriptomics
Proteomics Lipidomics
Gene
Glycomics
message
protein
Metabolomics activity
Phenotype FIGURE 1. Biomarker discovery in pseudoexfoliation glaucoma.
concentration of 8-isoprostaglandin F(2a) (an oxidative stress marker) versus decreased ascorbic acid concentration (a protectant of oxidative damage) in the aqueous humor of XFS patients compared with age-matched cataract controls.10 Inflammatory biomarkers may also pose attractive targets for the investigation of disease mechanisms given that aqueous humor from XFS patients versus controls is characterized by an elevated interleukin-6 (IL-6) and interleukin-8 (IL-8) signature when assessed by multibead immunoassay.11 A separate study from Japan also using the multiplex bead immunoassay technique found that aqueous humor from POAG and XFG patients display elevated TGF-b1, IL-8, and serum amyloid A levels when compared with cataract controls.12
Areas to “Keep an Eye on” New approaches to elucidating disease mechanisms are ever evolving and rely heavily on improved proteomic and metabolomic strategies. Interestingly, novel “shot-gun” proteomics approaches (label-free proteomics analysis by nLC-MS) emerging in the field have identified differential expression of inflammatory and radical-scavenging proteins in the tears of POAG patients.17 Separately, metobolomics (which allows the study of metabolites in body fluids or tissues, usually by nuclear magnetic resonance or mass spectrometry) analyses in relation to human ocular disease has been reviewed by Young and Wallace.18 The power of metabolic profiling of vitreous fluid by NMR is described and highlights differential profiles of urea, oxaloacetate, and glucose in patients with lens-induced uveitis versus patients with chronic uveitis. It is clear that validated biomarkers that emerge from ongoing studies have long-term r
2014 Lippincott Williams & Wilkins
potential to highlight specific molecular pathways, which direct drug development and ultimately improve patient outcome. Figure 1 details how efforts from the clinic and the laboratory can yield insight into glaucoma disease phenotype and also demonstrates that there is an abundance of data to be mined from the proteomics/metabolomics spectrum of the “omics cascade” with relevance to XFG. The need to identify biomarkers that enhance our understanding of XFG pathogenesis is matched only by the need for elucidation of biomarkers that correlate with disease progression.
REFERENCES 1. Schlo¨tzer-Schrehardt U. Oxidative stress and pseudoexfoliation glaucoma. Klin Monbl Augenheilkd. 2010;227:108–113. 2. Zenkel M, Poschl E, Von der Mark K, et al. Differential gene expression in pseudoexfoliation syndrome. Invest Ophthalmol Vis Sci. 2005;46:3742–3752. 3. Tezel G. A proteomics view of the molecular mechanisms and biomarkers of glaucomatous neurodegeneration. Prog Retin Eye Res. 2013;35:18–43. 4. Sharma S, Chataway T, Burdon KP, et al. Identification of LOXL1 protein and Apolipoprotein E as components of surgically isolated pseudoexfoliation material by direct mass spectrometry. Exp Eye Res. 2009;89:479–485. 5. Ovodenko B, Rostagno A, Neubert TA, et al. Proteomic analysis of exfoliation deposits. Invest Ophthalmol Vis Sci. 2007;48:1447–1457. 6. Erdurmus¸ M, Yag˘cı R, Atıs¸ O¨, et al. Antioxidant status and oxidative stress in primary open angle glaucoma and pseudoexfoliative glaucoma. Curr Eye Res. 2011;36: 713–718.
www.glaucomajournal.com |
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McNally and O’Brien
J Glaucoma
7. Dervan EW, Chen H, Ho SL, et al. Protein macroarray profiling of serum autoantibodies in pseudoexfoliation glaucoma. Invest Ophthalmol Vis Sci. 2010;51: 2968–2975. 8. Browne J, Ho SL, Kane R, et al. Connective tissue growth factor is increased in pseudoexfoliation glaucoma. Invest Ophthalmol Vis Sci. 2011;52:3660–3666. 9. Koliakos G, Befani C, Mikropoulos D, et al. Prooxidant– antioxidant balance, peroxide and catalase activity in the aqueous humour and serum of patients with exfoliation syndrome or exfoliative glaucoma. Graefes Arch Clin Exp Ophthalmol. 2008;246:1477–1483. 10. Koliakos G, Konstas A, Schlo¨tzer-Schrehardt U, et al. 8Isoprostaglandin F2a and ascorbic acid concentration in the aqueous humour of patients with exfoliation syndrome. Br J Ophthalmol. 2003;87:353–356. 11. Zenkel M, Lewczuk P, Junemann A, et al. Proinflammatory cytokines are involved in the initiation of the abnormal matrix process in pseudoexfoliation syndrome/glaucoma. Am J Pathol. 2010;176:2868–2879. 12. Takai Y, Ito M, Ohira A. Multiplex cytokine analysis of aqueous humor in eyes with primary open-angle glaucoma,
S54 | www.glaucomajournal.com
13. 14. 15.
16. 17.
18.
Volume 23, Number 8 Suppl 1, October/November 2014
exfoliation glaucoma, and cataract. Invest Ophthalmol Vis Sci. 2012;53:241–247. Schlo¨tzer-Schrehardt U. Genetics and genomics of pseudoexfoliation syndrome/glaucoma. Middle East Afr J Ophthalmol. 2011;18:30–36. Bussow K, Nordhoff E, Lubbert C, et al. A human cDNA library for high-throughput protein expression screening. Genomics. 2000;65:1–8. Gonza´lez-Iglesias H, A´lvarez L, Garcı´ a M, et al. Comparative proteomic study in serum of patients with primary open-angle glaucoma and pseudoexfoliation glaucoma. J Proteomics. 2014;98:65–78. Chowdhury U, Madden B, Charlesworth M, et al. Proteome analysis of human aqueous humor. Invest Ophthalmol Vis Sci. 2010;51:4921–4931. Pieragostino D, Agnifili L, Fasanella V, et al. Shotgun proteomics reveals specific modulated protein patterns in tears of patients with primary open angle glaucoma naı¨ ve to therapy. Mol Biosyst. 2013;9:1108–1116. Young S, Wallace G. Metabolomic analysis of human disease and its application to the eye. J Ocul Biol Dis Inform. 2009;2:235–242.
r
2014 Lippincott Williams & Wilkins
Metabolomics/Proteomics Strategies Used to Identify Biomarkers for Exfoliation Glaucoma Sara McNally, PhD* and Colm J. O’Brien, MD, FRCS*w
Abstract: It is currently estimated that 60 to 70 million people worldwide are affected by open-angle glaucoma and the majority of patients who present to clinic have raised intraocular pressure, visual field loss, and cupping of the optic nerve. Although exfoliation glaucoma (XFG) correlates with age, it is the most common cause of secondary open-angle glaucoma in the world and, with elevated intraocular pressure at onset, this disease runs an aggressive clinical course. XFG differs from primary open-angle glaucoma, in that patients have a diminished response to medication, show accelerated rates of disease progression, and therefore have a higher need for surgery. Here we highlight some major findings in the literature, which relate to the search for biomarkers of XFG by metabolomics and proteomics strategies. Key Words: exfoliation syndrome, metabolomics, proteomics
(J Glaucoma 2014;23:S51–S54)
BACKGROUND In exfoliation syndrome (XFS), fibrillar extracellular matrix material is produced by cells in the anterior segment, possibly in response to oxidative stress. Pertinent to glaucoma, excessive production and progressive accumulation of a fibrillar material occurs in various tissues including the anterior segment and outflow pathways.1 In addition, abnormal cross-linking of elastic microfibrils into fibrillar protein aggregates is a stress-induced fibrotic event in the pathogenesis of open-angle glaucoma.1 Central to these assertions was a comprehensive gene expression analysis performed in 2005, which attributes the overall pathophysiology of XFS to differential expression of genes associated with production of elastic microfibrils, cross-linking, matrix metalloproteinase action, and oxidative stress.2 As early diagnosis is key to preventing visual impairment in glaucoma, much recent work has focused on glaucoma biomarker discovery where a biomarker is a molecule/gene/clinical characteristic that allows the identification of a disease. Accepted characteristics for the “ideal” biomarker denote an agent of measureable entity with high sensitivity and specificity, which accurately predicts the presence, progression, or absence of a disease. For XFS, it is critical that a biomarker will predict progression from ocular hypertension to glaucoma and identify those at high risk of progressive damage. The role of “omic” techniques in the elucidation of glaucomatous biomarkers has recently Received for publication August 4, 2014; accepted August 11, 2014. From the *Department of Ophthalmology, Mater Misericordiae University Hospital; and wUCD School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. Disclosure: The authors declare no conflict of interest. Reprints: Sara McNally, PhD, Catherine McAuley Clinical Research Centre, 21 Nelson Street, Mater Misericordiae University hospital, Dublin 7, Ireland (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins DOI: 10.1097/IJG.0000000000000117
J Glaucoma
been reviewed.3 Here we highlight some important findings based on proteomic and metabolomic techniques used to define possible biomarkers from the anterior lens capsule, blood or aqueous humor of exfoliation glaucoma (XFG) patients; these results are summarized in Table 1.
Anterior Lens Exfoliative material isolated from the surgically removed anterior lens capsule has been analyzed to characterize resident protein constituents. Mass-spectrometry (LC-MS/MS) analysis has identified lysyl oxidase-like 1 (LOXL1) (involved in elastin cross-linking) to be genetically associated with the risk of XFS, as well as other proteins [apolipoproteinE (ApoE), latent-transforming growth factor b-binding protein-2 (LTBP-2), complement 3, and clusterin proteins] detected in the exfoliative material.4 An earlier study from 2007 utilized anterior lens capsules from patients with and without XFS which were homogenized, and protein fragments compared by SDS-PAGE after silver staining and liquid chromatography coupled to tandem MS.5 In addition to fibrillin-1, fibronectin, vitronectin, laminin, and amyloid Pcomponent, proteomic approaches identified clusterin and TIMP-3 as well as novel molecules [among them fibulin-2, desmocollin-2, the glycosaminoglycans syndecan-3, and versican, membrane metalloproteases of the ADAM family (a disintegrin and metalloprotease), and the initiation component of the classic complement activation pathway C1q].
Blood Spectrophotometric analysis of blood samples from primary open-angle glaucoma (POAG) and XFG patients reveals evidence of elevated oxidative stress when compared with control serum.6 To date, the literature reports that XFG patients may be characterized by an impaired oxidative-antioxidative balance in the anterior segment.13 In addition, raised levels of oxidative stress indicators nitric oxide and malondialdehyde have been detected in serum from POAG and XFG patients compared with controls. In our laboratory, high-density protein array screening has been used previously to analyze and compare the serum autoantibody profiles of patients with and without XFG to identify glaucoma disease–associated autoantibodies.7 Protein arrays containing >10,000 different human proteins derived from a human fetal cDNA library identify unique antibody profiles that may discriminate between patients and controls.14 High-density colony protein macroarrays were used to characterize the IgG autoantibody profiles from patient sera samples. Comparison of the autoantibody profiles between XFG and control groups identified 7 proteins that were significantly more prevalent in the XFG group, including a previously uncharacterized protein C6orf129 that contains a transmembrane domain; current work is ongoing to further characterize C6orf129 and its role in XFG pathology.
Volume 23, Number 8 Suppl 1, October/November 2014
www.glaucomajournal.com |
S51
McNally and O’Brien
J Glaucoma
Volume 23, Number 8 Suppl 1, October/November 2014
TABLE 1. Efforts to Identify Biomarkers for PXFG by Proteomic/Metabolomic Means
Marker of Interest
Technique
Model
LOXL1 and ApoE; detected LTBP-2, complement 3, and clusterin proteins in sample subset Fibrillin-1, fibronectin, vitronectin, laminin, amyloid P, clusterin, TIMP-3, fibulin2, desmocollin-2, syndecan-3, versican, C1q, and ADAM family membrane metalloproteases Nitric oxide, protein carbonyl, malondialdehyde
Digested peptides analyzed with Thermo LTQ XL linear ion trap mass spectrometer fitted with nanospray source Quadrupole time-of-flight mass spectrometry (MS) Liquid chromatography coupled to tandem MS
PXF material deposited on anterior lens capsule of affected individuals collected at time of cataract surgery
Sharma et al4
Anterior lens capsules from patients with and without exfoliation syndrome (XFS)
Ovodenko et al5
Spectrophotometry
Erdurmus¸ et al6
EEF2, EHD1, CLPTM1, FGFR3, TMEM9B, STMN4, C6orf129 CTGF
High-density protein macroarray Reverse ELISA ELISA, SDS-PAGE
Blood from forearm vein; serumseparated samples of patients with POAG, PXFG, and control subjects (n = 19) Serum samples of patients with PXFG and control subjects
Hydrogen peroxide, catalase
Goth’s colorimetric method, peroxidase activity assay using TMB Microplate assay method 8-IPGF measured with commercial immunoassay kit (Assay Designs Inc., Ann Arbor, MI)
8-isoprostaglandin F2a, ascorbic acid
IL-6, IL8
Multiplex bead analysis, immunoassays
TGF-b1, IL-8, and SAA
Multiplex bead immunoassay technique
References
Dervan et al7
Aqueous humor samples obtained from patients undergoing routine cataract surgery or trabeculectomy Aqueous humor and serum samples from XFS, XFG, and controls
Browne et al8
Aqueous humor aspirated at beginning of phacoemulsification cataract surgery from eyes of cataract patients with XFS and eyes of matched cataract patients without XFS Aqueous humor and anterior segment tissues; eyes with early and late stages of PEX syndrome/glaucoma; normal and glaucomatous control eyes Aqueous humor samples obtained from 64 eyes of 64 Japanese subjects; POAG, PXFG, cataract, control
Koliakos et al10
Koliakos et al9
Zenkel et al11
Takai et al12
ADAM indicates a disintegrin and metalloproteinase domain containing proteins; ApoE, apolipoprotein E; CLPTM1, cleft lip and palate-associated transmembrane protein 1; CTGF, connective tissue growth factor; EEF2, eukaryotic elongation factor 2; EHD1,EH domain-containing protein 1; ELISA, enzyme-linked immunosorbent assay; FGFR3, fibroblast growth factor receptor 3; IL-6/8, interleukin-6/8; IPGF, isoprostaglandin F; LOXL1, lysyl oxidaselike1; LTBP-2, latent transforming growth factor b-binding protein 2; STMN4, stathmin-like 4; TGF, transforming growth factor; SAA, serum amyloid A; TIMP-3, tissue inhibitor of metalloproteinase 3; TMEM9B, TMEM9 domain family, member B.
A recent study from 2014 has analyzed proteins from blood serum of patients of POAG, XFG, and healthy controls to identify a candidate panel of glaucoma biomarkers.15 Comparative differential proteomic analysis has elucidated enrichment of immune and inflammatory network proteins (including members of the complement C pathway) from glaucoma patients.
Aqueous Humor Proteomic analysis of human aqueous humor has come to the fore in recent times as a mechanism of identifying proteins responsible for maintenance of homeostasis in the anterior chamber. A complex mixture of growth factors, cytokines, electrolytes, and organic solutes in human aqueous humor sustain cellular metabolism in the avascular tissues of the anterior segment. We have previously shown that levels of connective tissue growth factor (a matricellular protein that interacts with the profibrotic cytokine TGF-b) are significantly higher in aqueous humor
S52 | www.glaucomajournal.com
of patients with PXFG than in both POAG and normal control subjects undergoing cataract surgery.8 Improved detection technologies have enabled investigations which, to date, have been hindered by the reality that the protein component of human aqueous humor is relatively low, containing between 120 and 500 ng/mL of protein.8,14 Antibody protein arrays and nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS) have been used to characterize the human aqueous humor proteome as a resevoir of 676 nonredundant proteins (cytokines, receptors, and chemokines with catalytic, enzymatic, and structural properties).16 Other groups have highlighted the aqueous humor as a pivotal site of oxidant-antioxidant balance in the distinction between XFS and XFG.9 In addition, the concentration of hydrogen peroxide has been found to be higher in both the serum and aqueous of XFG patients compared with controls.9 A 2003 study utilized commercially available in vitro assays to highlight elevated r
2014 Lippincott Williams & Wilkins
J Glaucoma
Volume 23, Number 8 Suppl 1, October/November 2014
Clinical
Gene
Biomarker panel
PXF material
LOX-L1
IOP
Tissue mRNA
Strategies Used to Identify Biomarkers for XFG
Cytokines
Oxidative stress
Anterior segment tissues
TGFβ1
Blood
LTBP
CTGF
Aqueous
IL-6
Tears
TG2 Discs
TIMP2
RNFL
TIMP1
Proteomics/ Lipid
Clusterin Perimetry
Oxidation: ApoE (MALDI
Omics cascade
SAA1 Genomics
MS)
Transcriptomics
Proteomics Lipidomics
Gene
Glycomics
message
protein
Metabolomics activity
Phenotype FIGURE 1. Biomarker discovery in pseudoexfoliation glaucoma.
concentration of 8-isoprostaglandin F(2a) (an oxidative stress marker) versus decreased ascorbic acid concentration (a protectant of oxidative damage) in the aqueous humor of XFS patients compared with age-matched cataract controls.10 Inflammatory biomarkers may also pose attractive targets for the investigation of disease mechanisms given that aqueous humor from XFS patients versus controls is characterized by an elevated interleukin-6 (IL-6) and interleukin-8 (IL-8) signature when assessed by multibead immunoassay.11 A separate study from Japan also using the multiplex bead immunoassay technique found that aqueous humor from POAG and XFG patients display elevated TGF-b1, IL-8, and serum amyloid A levels when compared with cataract controls.12
Areas to “Keep an Eye on” New approaches to elucidating disease mechanisms are ever evolving and rely heavily on improved proteomic and metabolomic strategies. Interestingly, novel “shot-gun” proteomics approaches (label-free proteomics analysis by nLC-MS) emerging in the field have identified differential expression of inflammatory and radical-scavenging proteins in the tears of POAG patients.17 Separately, metobolomics (which allows the study of metabolites in body fluids or tissues, usually by nuclear magnetic resonance or mass spectrometry) analyses in relation to human ocular disease has been reviewed by Young and Wallace.18 The power of metabolic profiling of vitreous fluid by NMR is described and highlights differential profiles of urea, oxaloacetate, and glucose in patients with lens-induced uveitis versus patients with chronic uveitis. It is clear that validated biomarkers that emerge from ongoing studies have long-term r
2014 Lippincott Williams & Wilkins
potential to highlight specific molecular pathways, which direct drug development and ultimately improve patient outcome. Figure 1 details how efforts from the clinic and the laboratory can yield insight into glaucoma disease phenotype and also demonstrates that there is an abundance of data to be mined from the proteomics/metabolomics spectrum of the “omics cascade” with relevance to XFG. The need to identify biomarkers that enhance our understanding of XFG pathogenesis is matched only by the need for elucidation of biomarkers that correlate with disease progression.
REFERENCES 1. Schlo¨tzer-Schrehardt U. Oxidative stress and pseudoexfoliation glaucoma. Klin Monbl Augenheilkd. 2010;227:108–113. 2. Zenkel M, Poschl E, Von der Mark K, et al. Differential gene expression in pseudoexfoliation syndrome. Invest Ophthalmol Vis Sci. 2005;46:3742–3752. 3. Tezel G. A proteomics view of the molecular mechanisms and biomarkers of glaucomatous neurodegeneration. Prog Retin Eye Res. 2013;35:18–43. 4. Sharma S, Chataway T, Burdon KP, et al. Identification of LOXL1 protein and Apolipoprotein E as components of surgically isolated pseudoexfoliation material by direct mass spectrometry. Exp Eye Res. 2009;89:479–485. 5. Ovodenko B, Rostagno A, Neubert TA, et al. Proteomic analysis of exfoliation deposits. Invest Ophthalmol Vis Sci. 2007;48:1447–1457. 6. Erdurmus¸ M, Yag˘cı R, Atıs¸ O¨, et al. Antioxidant status and oxidative stress in primary open angle glaucoma and pseudoexfoliative glaucoma. Curr Eye Res. 2011;36: 713–718.
www.glaucomajournal.com |
S53
McNally and O’Brien
J Glaucoma
7. Dervan EW, Chen H, Ho SL, et al. Protein macroarray profiling of serum autoantibodies in pseudoexfoliation glaucoma. Invest Ophthalmol Vis Sci. 2010;51: 2968–2975. 8. Browne J, Ho SL, Kane R, et al. Connective tissue growth factor is increased in pseudoexfoliation glaucoma. Invest Ophthalmol Vis Sci. 2011;52:3660–3666. 9. Koliakos G, Befani C, Mikropoulos D, et al. Prooxidant– antioxidant balance, peroxide and catalase activity in the aqueous humour and serum of patients with exfoliation syndrome or exfoliative glaucoma. Graefes Arch Clin Exp Ophthalmol. 2008;246:1477–1483. 10. Koliakos G, Konstas A, Schlo¨tzer-Schrehardt U, et al. 8Isoprostaglandin F2a and ascorbic acid concentration in the aqueous humour of patients with exfoliation syndrome. Br J Ophthalmol. 2003;87:353–356. 11. Zenkel M, Lewczuk P, Junemann A, et al. Proinflammatory cytokines are involved in the initiation of the abnormal matrix process in pseudoexfoliation syndrome/glaucoma. Am J Pathol. 2010;176:2868–2879. 12. Takai Y, Ito M, Ohira A. Multiplex cytokine analysis of aqueous humor in eyes with primary open-angle glaucoma,
S54 | www.glaucomajournal.com
13. 14. 15.
16. 17.
18.
Volume 23, Number 8 Suppl 1, October/November 2014
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