Proteome Alterations in Primary Open Angle Glaucoma Aqueous Humor A. Izzotti,† M. Longobardi,† C. Cartiglia,† and S. C. Sacca`*,‡ Department of Health Sciences, Faculty of Medicine, University of Genoa, Italy, and Department of Head/Neck Pathologies, St. Martino Hospital, Ophthalmology Unit, Genoa, Italy Received May 30, 2010
As the only nourishment and scavenging source for most of the anterior and posterior chamber tissues in the eye, the aqueous humor represents one of the target for glaucoma. The aim of this study is to investigate the yet unexplored relationship between aqueous humor protein content and open-angle glaucoma (POAG) pathogenesis. Aqueous humor was collected from 10 POAG patients (cases) and 14 senile cataract patients (controls), matched for age and gender, undergoing surgery for trabeculectomy and cataract, respectively. Protein samples were cyanine-labeled and hybridized with antibody microarrays. Microarray signals were revealed by laser scanner, quantified, and compared by statistical analyses. Total protein amounts were not significantly different in patients versus controls. Conversely, a proteome cluster significantly modified in patients as compared to controls was identified as highly predictive for disease status. Selected proteins underwent dramatic variation, which was correlated to pathogenetic events characterizing POAG, including oxidative damage, mitochondrial damage, neural degeneration, and apoptosis. The results obtained indicate that proteomic analysis of aqueous humor is a new tool for POAG diagnosis in the case of otherwise uncertain disease recognition. Furthermore, this study allows a better understanding of mechanisms involved in the pathogenesis of POAG, the main cause of irreversible blindness worldwide. Keywords: trabecular meshwork • aqueous humor • proteome • mitochondria • glaucoma
Introduction Primary open angle glaucoma (POAG), the main cause of irreversible blindness worldwide,1 is a neurodegenerative multifactorial disease affecting three target tissues: the lateral geniculate nucleus in the central nervous system,2 the optic nerve head in the retina, and the trabecular meshwork (TM) in the anterior chamber (AC) of the eye. The exact pathogenesis of glaucoma is still unclear. However, it is established that TM plays a primary role in the etiopathogenesis of this disease.3 Aqueous humor (AH), the biological fluid filling both the anterior and the posterior chambers of the eye, plays an important pathogenic role in glaucoma.4 Alteration of AH homeostasis results in increased intraocular pressure (IOP). Furthermore, this liquid has the task of protecting and supplying nutrients and antioxidants to the cornea, lens, and TM.5,6 A number of tissue growth factors have been detected in this fluid.7 AH also promotes regulatory T-cell immunity8 and stimulates immune cell function.9-11 AH may play a primary role in POAG pathogenesis by facilitating the migration of cytokines that stimulate the activity of TM cells.12 Furthermore, AH contains several signaling * Corresponding author. Address: Department of Head/Neck Pathologies, St. Martino Hospital, Ophthalmology Unit, Viale Benedetto XV, 16132 Genoa, Italy. Tel: +39 010 555 2443. Fax: +39 010 555 6585. E-mail: sergio.sacca@ hsanmartino.it. † University of Genoa. ‡ St. Martino Hospital. 10.1021/pr1005372
2010 American Chemical Society
molecules promoting synthesis, degradation, and modification of the extracellular TM matrix.6 The composition of AH proteins changes dramatically with different ocular conditions, such as inflammation and glaucoma.13 However, the relationship between TM and AH proteins as related to POAG pathogenesis has not yet been explored. “Omic” techniques represent a major breakthrough in molecular medicine. Array-based methods allow the detection of thousands of biomarkers in a single-hit experiment. Proteome analysis by antibody microarrays has been proposed to evaluate the expression of hundreds of proteins that are detectable not only in tissues14,15 but also in biological fluids. AH represents a protein-containing biological fluid fundamental for eye pathophysiology16 that can be collected by corneal puncture, a mildly invasive method. Accordingly, we set up the study presented herein to evaluate protein alterations occurring in AH during glaucoma in order to elucidate the pathogenesis of this disease and to develop new methods for its study, as well as patient monitoring.
Experimental Section Study Design. The study has a case-control design. Both patients and controls, all Caucasian, underwent ocular surgery for therapeutic purposes. AH samples were obtained from clinically uncontrolled POAG patients (cases) and senile cataract patients (controls) prior to trabeculectomy and cataract surgery, respectively. Journal of Proteome Research 2010, 9, 4831–4838 4831 Published on Web 07/28/2010
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Table 1. Characteristics of POAG Patients Whose AH Proteome Has Been Tested by Antibody Microarray in the Present Study code
gender
age (years)
IOPa mean (24 h)
IOP min
IOP max
IOP delta
VFDb
UA07 UA12 UA34 UA43 UA52 UA56 UA61 UA72 UA76 UA80
M F F F M F M F M M
65 81 62 87 81 69 82 91 82 75
18.4 19.3 25.1 20.1 22.1 20.3 18.6 19.1 19.3 20.1
12 17 21 15 20 17 15 15 17 17
24 22 34 27 25 25 26 23 24 24
12 5 13 12 5 8 11 8 7 7
4 1 3 1 5 3 2 4 3 5
a IOP values are expressed as mm Hg. b Visual field defect (VFD) is expressed as Glaucomatous Staging System (GSS).
Inclusions Criteria and Patient Enrollment. Case samples were collected from 10 POAG patients (5 males, 5 females) with no tonometric compensation established by clinical and instrumental examinations as previously reported.17,18 The main elements for POAG diagnosis were papilla morphology, IOP values, and visual field analysis. Exclusion criteria were the presence of any other ocular, systemic, or neurological diseases other than POAG-related optic-nerve damage. An additional exclusion criterion was the presence of any glaucoma type other than POAG. The age of POAG patients was 74.9 ( 3.10 years (means ( SE). All patients underwent a Humphrey 30-2 computerized visual field examination (750 Humphrey Field Analyzer II; Humphrey Ind., San Leandro, California) 14-32 days before surgery. To evaluate visual field damage, the Glaucoma Staging System (GSS 2) was used.19 All patients underwent daily tonometry curves (every 2 h between 8:00 a.m. and 8:00 p.m.) the week before surgery. This procedure was performed by the same physician using Goldmann tonometry. The average IOP value recorded 94 was 20.2 ( 3.5 (mean ( SD). Clinical characteristics of glaucomatous patients are reported in Table 1. Sample collection from 14 age- (72.9 ( 3.54 years) and gender- (7 males, 7 females) matched controls was performed before surgical interventions for cataract. IOP was measured in control subjects by standard tonometry and detected mean values of 14.1 ( 2.0 (mean ( SD). The inclusion criteria for controls were an open anterior chamber angle; no history of previous filtration surgery; pupil size >5 mm after dilatation; absence of pseudoexfoliation syndrome, diabetes, uveitis, systemic collagenopathy; objective neurological signs; no history of use of systemic antihypertensive drugs; and no administration of corticosteroids or systemic or topical antiglaucomatous drugs for at least 38 days before enrolment in the study.18 In both groups of examined patients (controls and glaucoma), a certain degree of cataracts was present. For the sake of comparison, we did not include in the control group patients with severe cataract determining blindness or unacceptable vision. In the glaucoma group, even if visual acuity decline was determined more by optic nerve alterations than lens opacities, a moderate cataract was present in all patients. All the enrolled subjects provided informed written consent and were treated in accordance with the Declaration of Helsinki. 4832
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Aqueous Humor Sampling. For both cases and controls, a 100 mL intravenous injection of mannitol was administered before surgery, and eyes were treated with peribulbar anesthesia (bupivacain hydrochloride 5%). Pupils were dilated with phenylephrine 10% and tropicamide 1%. Eyelids and surrounding skin were swabbed with disinfectant, and AH (100-300 µL) was aspirated by corneal puncture, which involved inserting a 26-gauge needle into the AC just before surgery. AH sample aliquots (100-300 µL) were immediately stored in a deep freezer (-80 °C) until proteome analysis was performed, which occurred within 3 months after sample collection. Proteome Assay by Antibody Microarray. The principal aim of our study was to investigate the proteomic profile of each sample in order to identify dysregulated pathways. The proteomic analysis workflow included AH protein fluorescent labeling and purification by column chromatography, protein hybridization on antibody microarrays, detection of fluorescent signals by laser scanning, and data analysis. The proteome analysis of 1264 proteins was carried out using Clontech Ab Microarray TM 500 (Clontech, CA, USA) and Explorer Antibody Microarray (Full Moon BioSystems, Inc., Sunnyvale, CA, USA). Two aliquots of 20 µg of protein (quantified by bicinchoninic acid method) for each AH sample were diluted to 1.1 µg/µL in Extraction/Labeling Buffer (Clontech, CA, USA), then labeled by incubation at 4 °C for 2 h with 9.1 µg of either Cy3 or Cy5 monofunctional protein reactive dyes (GE Healthcare, Little Chalfont Buckinghamshire, UK). Labeled proteins were purified by elution with 1× Desalting Buffer (100 µL) in Protein Desalting Spin Columns (Pierce Biotechnology, Rockford, USA). Purified labeled proteins were quantified by BCA methods using a nanospectrophotometer, NanoDrop ND1000 (Nanodrop Technologies, Wilmington, USA). Labeled proteins were hybridized on two glass microarrays for each sample; the first one was coated with 508 antibodies spotted in duplicate (Ab Microarray TM 500, lot. no. 7030444, Clontech, CA, USA), and the second one was coated with 656 proteins (Explorer Antibody Microarray, Full Moon BioSystems, Inc., Sunnyvale, CA, USA). These antibody microarrays were selected to avoid overlaps in antibody signal. The hybridization was performed at room temperature for 40 min with continuous shaking in hybridization ovens (Bibby Stuart Scientific, Beacon Road, Stone, Staffordshire ST15 0SA, UK). The slides were washed in three different washing buffers (Clontech) for 5 min each at room temperature with gentle rocking. Samples were then dried by centrifugation and analyzed by laser scanning and fluorescence detection using a gas laser microarray scanner (ScanArray Lite, Packard Bioscience, Meriden, CA, USA). Quantification of signals was performed by QuantArray software (GSI group, Lumonics, Bedford, MD, USA), subtracting local background from signal intensities. The entire lists of the examined proteins are available at the Clontech Web site (http://bioinfo.clontech.com/abinfo/initialize.do) and at the Full Moon BioSystems, Inc. Web site (http:// www.fullmoonbiosystems.com/DataSheets/AntibodyArrays/ ASB600-AbList- B2.xls). Statistical Analyses. Gene expression profiles were analyzed by Genespring software version 7.3 (Agilent Technologies, Santa Clara, CA, USA). Background subtracted raw data were log transformed and normalized both per chip and per gene by median centering. Changes in global gene expression profiles were evaluated by unsupervised hierarchical cluster analysis and by principal component analysis of variance. Identification of genes whose expression changed according to disease status
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Figure 1. (A) AH proteome in POAG patients (right panel) as compared to matched controls (left panel). Each dot represents the amount of one protein colored in red for POAG upregulated proteins, or green for POAG downregulated proteins. (B) Box-plot distribution highlighting differences in proteome profile between POAG and controls. Red color, upregulated proteins, green color, downregulated proteins.
and clinical variables was performed by k-nearest neighbor algorithm and volcano plot filtering, taking into account 2-fold variation and ANOVA (P < 0.05) calculated after Bonferroni multiple-testing correction. Differences in AH total protein concentrations as related to nominal clinical variables were tested by ANOVA and Mann-Whitney nonparametric test. The correlation of AH total protein with continuous clinical variables was tested by linear regression analysis (Statview software, Abacus Concept, Berkeley, CA, USA).
Results The AH proteome profile undergoes dramatic changes in POAG patients as compared to matched controls, as reported in Figure 1. Many proteins expressed at high levels in controls are reduced in POAG patients, while other proteins detected at low levels in controls are increased in POAG patients (Figure 1A). This situation accounts for the dramatic differences between POAG and controls in the box-plot distribution, where many proteins expressed at low levels in controls are upregulated in POAG patients (Figure 1B). Hierarchical cluster analysis discriminates sharply between POAG patients (left side of the dendrogram) and controls (right side of the dendrogram) (Figure 2A). Hierarchical protein clustering identified a POAGrelated signature characterized by the upregulation of certain proteins, located in the lower part of the columns, each one corresponding to one patient. Conversely downregulated pro-
teins are represented in the upper part of the columns as POAG compared to controls. Two POAG patients are located far away (right side of the dendrogram) from other POAG patients (left side); nonetheless, even these outliers were easily distinguished from controls. These two patients, coded as UA12 and UA43, are characterized by minimal visual field damage among the POAG patients tested (Table 1). Principal component analysis 168 of variance, which takes into account the global proteome expression profile of each subject, sharply discriminates controls (green dots located in the upper part) and POAG patients (red dots located in the lower part) (Figure 2B). The dramatic differences observed in the AH proteome collected from POAG patients yielded robust diagnostic results when antibody microarray data were processed by support vector machine analysis. Using this algorithm and labeling samples as unknown with regard to their pathological status, all 24 samples were correctly classified as controls or POAG affected. Conversely, total protein in AH was not a good indicator of POAG. Total protein was slightly increased in POAG as compared to controls, but not to a statistically significant extent (2.20 ( 0.54 vs 1.65 ( 0.34 mg/mL, mean ( SE, NS). Linear regression analysis indicated that total protein concentration was not significantly affected by age in either controls (r ) 0.026, NS) or POAG patients (r ) -0.306, NS). Proteins whose altered expression was most remarkably varied in POAG as compared to controls were identified by k-nearest neighborhood algorithm, which Journal of Proteome Research • Vol. 9, No. 9, 2010 4833
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Figure 2. (A) Hierarchical clustering analysis classifying POAG patients (G) or controls (C) accordingly to their AH proteome expression. Red color, POAG upregulated proteins, green-blue color, POAG downregulated proteins. Each column corresponds to one patient. The two (G) patients on the right were characterized by a low visual field damage as compared to other glaucomatous patients located in the left part of the hierarchical tree. (B) Principal component analysis of variance taking into account the global proteome expression profile of each patient. Controls (green dots) are sharply discriminated from POAG patients (red dots). The two (G red circles) patients located in the low left quadrant were characterized by a low visual field damage (two right columns in panel A).
allows the identification of proteins that predict disease status, that is, POAG. These proteins were further filtered by Volcano plot selection, taking into account only proteins with more than 2-fold significant variance in expression between POAG and controls (P < 0.05). The list of these 31 proteins undergoing significant variations in AH of POAG patients as compared to controls is reported in Supplementary Table 1, Supporting Information with a description of their biological functions. Six are mitochondrial proteins involved in the electron transport chain, trans-membrane transport, protein repair, and mitochondrial integrity maintenance. Under physiological conditions, these proteins are segregated inside cells into functional mitochondria. Their presence in AH indicates that severe damage occurs in cells in tight contact with AH and that this 4834
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damage primarily affects mitochondria. Imbalances in energy and ATP production in POAG are demonstrated by the presence in the AH of increased levels of ATPase and Na+/K+ transporting beta-3 polypeptide. This is a subunit component of the membrane; thus, its presence in AH reflects the occurrence of cell membrane degradation resulting from cytolysis. Five proteins are directly involved in apoptosis induction, through the intrinsic, that is, mitochondrial-dependent, pathway. These include several caspases, such as BAX and BIK. However, at least in part, apoptosis is also activated through the extrinsic, that is, mitochondrial-independent, activation pathway, as demonstrated by the POAG-related increase of Fas and TNFrelated factors in AH. The activation of these two proteins results from the occurrence of inflammatory processes caused
research articles by the activation of four protein kinases C (Supplementary Table 1, Supporting Information). Six proteins are components of the intercellular junction and contribute to tissue integrity and maintenance of cell-cell adhesion. These include catenins, junctional plaque protein, dynein, and cadherins. Their presence in AH indicates that these functions are severely damaged in tissues located in the anterior chamber specifically targeted by POAG pathogenesis, that is, the TM. Protein repair appears to be overloaded in POAG patients, as demonstrated by the induction of mitochondrial and cytoplasmic chaperones including calnexin. This cytoplasmic chaperone is particularly abundant in melanosomes, intracellular organelles abundant in TM. Four proteins are typically located in neurons and are involved in various neuronal functions, including neuron survival, neuroglycan production, NMDA receptor activation, glutamate detoxification, and neural protein repair. Several proteome alterations indicate the occurrence of oxidative stress in the anterior chamber of POAG patients. The presence of oxidative stress is demonstrated by the increased presence of nitric oxide synthase 2. In addition, other antioxidant proteins undergo downregulation in the AH of POAG patients. These include total glutathione S-transferases and superoxide dismutase. In POAG patients, the correlations between the clinical variables examined were as follows. Age was not significantly correlated with any clinical variable including IOP and visual field defects. This is likely because age does not reflect the disease time span. AH protein concentration was significantly correlated with IOP mean (r ) 0.916, P ) 0.01) and IOP min (r ) 0.808, P < 0.05). No protein was differentially represented, as evaluated by volcano plot filtering, in patients having high vs low (i.e., over or below the 50th percentile) IOP max, IOP delta, or visual field defect (expressed according to GSS).
Discussion Our study highlights that AH protein examination may be a suitable tool to study POAG pathogenesis. AH proteome changes reflect molecular and cellular damage in POAG target tissues, that is, TM and optic nerve head. AH sampling is a mildly invasive technique thus comparing favorably to TM sampling, which can be performed only in cases of therapeutic surgical trabeculectomy performed in severe POAG patients who have not sufficiently responded to drug therapy. The detection of neural proteins in AH during POAG provides evidence that this analysis allows indirect examination of the functional and molecular activity in neural tissue. Therefore, these results indicate that AH may be a surrogate body fluid that can be explored using a mildly invasive technique to determine the pathophysiology of POAG target tissues. AH proteome alterations have been related to ocular diseases such as uveitis,20 myopia,21 dry eye,22 proliferative diabetic retinopathy,23 pseudoexfoliative glaucoma,24 and possibly cataract.25 The AH proteome alterations detected in POAG patients appear to reflect damage occurring in both anterior and posterior chambers of the eye. Furthermore, the severity of visual filed damage is reflected in the proteome profile. In fact, the two patients having the lowest visual field damage are characterized by a proteome profile different from those observed in other glaucomatous patients. The main glaucomatous pathogenic pathways activated, as indicated by the alterations detected in the corresponding AH proteins, are oxidative stress, mitochondrial alterations, apoptosis, tissue disaggregation, and neuronal damage. Oxidative
stress has been recognized as a main pathogenic factor for POAG,26 as demonstrated by a body of evidence including the detection of high levels of oxidative DNA damage in TM,17 which is correlated with IOP increase and visual field defects.27 Additional evidence has accumulated to support the important role of oxidative stress in POAG.3,28 Antioxidant defects during the course of POAG have been reported not only in AH,29 but also in blood serum.30,31 The source of such oxidative damage is still uncertain. Because POAG is not associated with any major environmental risk factor, attention has been focused on the main endogenous source of reactive oxygen species, that is, mitochondria. A body of evidence has been accumulating in support of the role of mitochondrial alterations during POAG32 in both TM33 and the optic nerve.34 Recently, it has been demonstrated that TM mitochondria in POAG patients undergo severe molecular damage in terms of mitochondrial DNA deletions, which results in mitochondrial loss.35 The important role of mitochondrial damage in POAG is supported by the finding that mutant myocilin impairs mitochondrial functions in human trabecular meshwork cells.36 Oxidative damage and AH antioxidant defects target TM, the anterior chamber tissue most sensitive to oxidative damage,37 thus triggering the POAG pathogenic cascade. Under physiological conditions, mitochondrial components segregate inside the cell and inside functional mitochondria. The results presented here demonstrate that, under POAG pathological conditions, mitochondrial proteins can be detected in AH by highly sensitive antibody microarray, thus demonstrating the occurrence of cell and mitochondrial destruction. Mitochondrial damage triggers intracellular calcium release and activation of apoptosis through the intrinsic activation pathway,38 as demonstrated by increased levels of a variety of mitochondria-related pro-apoptotic proteins detected in the AH of POAG patients. However, proteins involved in the activation of apoptosis through the extra-mitochondrial pathway have also been detected. This finding indicates that apoptosis occurring in ocular tissues during POAG is induced by a variety of mechanisms including primarily mitochondrial damage, but also inflammation, vascular dysregulation, and hypoxia.39,40 Apoptosis induces cell loss and tissue degeneration, as indicated by the increased levels in POAG AH of proteins involved in the maintenance of intercellular adhesion, such as catenins, junction proteins, cadherins, etc. These findings explain at molecular levels the observation that TM undergoes progressive cell loss and cell disaggregation during POAG progression.41 The discovery of these proteins in glaucomatous AH also justifies the decreased cell adhesion of optic-nerve head astrocytes during the course of glaucoma42 as well as their damage in response to elevated intraocular pressure.43 Therefore, apoptosis induces similar tissue degeneration in the two main POAG target tissues, that is, TM and optic nerve head. Evidence of this selective effect may be observed in the AH proteome. In POAG AH, we observed an increase of calnexin, a typical molecular chaperone activated by oxidative stress-induced protein damage. An increase in calnexin has been detected in TM and specifically in melanosomes, intracellular organelles particularly abundant in TM.44 Proteins involved in the maintenance of tissue integrity are altered in POAG AH. These proteins can have a role in pathogenic mechanisms of myocilin-associated glaucoma.45 When myocilin expression was increased more than 3-fold, transfected tissue showed a dramatic decrease in actin fibers and intercellular focal adhesions. Cell adhesion to fibronectin and spreading were also compromised.46 Accordingly, it is Journal of Proteome Research • Vol. 9, No. 9, 2010 4835
research articles established that extracellular myocilin elicits antiadhesive and counter-migratory effects in TM cells.47 A significantly higher amount of neural proteins was detected in the AH of POAG patients as compared to control AH, where neural proteins were almost undetectable. We propose two possible interpretations to explain the presence of these proteins in AH. The first one is that these proteins are released locally in the anterior chamber during POAG. The production of these proteins could reflect the occurrence of a similar pathological condition affecting both TM and optic nerve head, resulting in cell loss and degeneration. Accordingly, these proteins could be released from the optic nerve head, but they could also be released in the anterior chamber by TM cells. TM cells have a neuroectodermic origin, expressing, at least in part, a neural-like phenotype.48 TM cells derive from mesenchymal cells of the neural crest.49 Thus, their neural embryological origin could help explain the presence of neural proteins during POAG in the AH surrounding damaged TM. The second hypothesis is that these neural proteins detectable in POAG AH are imported to the anterior chamber from other eye compartments. The death of neural cells in the optic nerve releases these proteins into the posterior chamber. Hence, proteins released by neural death reach the anterior chamber and AH through the physiological exchanges occurring between these two ocular compartments.50 The diffusion rate from vitreous humor to AH has been estimated to be less than 10-15% for vitreally injected fluids. Thus, the finding of neural proteins in AH indicates that neural damage occurring in POAG is dramatic and is likely to be almost 10 times higher in the posterior chamber than in AH. Accordingly, a highly sensitive method such as protein detection by antibody microarray is needed to detect POAGrelated neural damage by analyzing the AH proteome. A peculiar neural protein detected in AH in POAG patients was optineurin (OPTN). The gene that codes for this protein is associated with normal-tension glaucoma and primary open angle glaucoma and is a pivotal protein for survival and homeostasis of optic nerve head neurons. Rare mutations of OPTN are typically related to the appearance of early onset glaucomas.51 The expression of OPTN is regulated by various cytokines, particularly NF-κB.52 The oxidative stress occurring in AC is a key mediator of the observed activation of NF-κB and the induction of inflammatory mediators. OPTN has the potential to contribute to pathophysiological changes in the outflow pathway by increasing the levels of oxidative damage in TM cells.53 OPTN is also present in TM,54 and its expression is increased in TM by IOP increase.55 Accordingly, it is evident that pathogenic mechanisms inducing optic nerve damage are reflected in AH protein content. This is the case for NOS2 and glutamate-ammonia ligase. NOS2 is an inducible NOS involved in NO production, contributing to the vascular dysregulation observed in optic nerve vascularization during POAG.56 Nitric oxide synthase 2 (NOS2) contributes to neural death in some settings, but its role in glaucoma remains controversial. Further studies are required to assess the general importance of NOS2 in glaucoma.57 Nitric oxide from iNOS appears to be a key mediator of glial-induced neuronal death, while NO also plays an important role in the physiological modulation of small blood vessels.58 Therefore, the tracing of this protein could elucidate the functional impairment of TM. The increase in glutamate-ammonia ligase that we detected may be linked with both an increase in melatonin59 and an accumulation of cytotoxic glutamate in neurons,60 as well as endothelin release as one of the final steps of the POAG pathogenic cascade2. 4836
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Izzotti et al. Endothelin-1 (ET-1) is known to induce Ca(2+)-independent contraction of the trabecular meshwork. This contraction involves RhoA and its kinases as intracellular mediators.61 Protein kinase C (PKC) levels are significantly increased in POAG AH. PKC is a family of enzymes involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. The PKC family consists of ∼10 isozymes that make up three subfamilies. PKC plays an important role in the regulation of myosin light chain phosphorylation, which induces cellular contraction62 and cytoskeletal dynamics within the TM. Accordingly, PKC could influence AH outflow, affecting cellular relaxation, contraction, and morphological changes in TM and sclerocorneal cells.63 Thus, it is likely that activation of PKC triggers other regulatory mechanisms involved in cytoskeletal reorganization and cell adhesive characteristics, which in turn influence cell shape in TM cells.63 PKC have been also implicated in transcriptional regulation of matrix metalloproteinases, which maintain normal AH outflow rates.64 Elevated matrix metalloproteinase levels in the AH of glaucomatous eyes may be produced by inflammatory cells and by TM cells.65 Considering global proteome expression, these results provide evidence that it is possible to use AH analysis to identify POAG-affected patients. This issue bears particular relevance as POAG diagnosis is often difficult and cannot be performed using only a single test such as the simple evaluation of intraocular pressure. In fact, intraocular pressure undergoes remarkable variations in the same subject due to a variety of factors including circadian rhythm.18 Furthermore, IOP increase can be masked by increased corneal thickness hampering reliable IOP determination by standard tonometry.66 In addition, POAG may occur in the absence of an evident IOP increase; thus, the continued monitoring of visual fields and reassessment of target IOP levels are fundamental in the management of glaucoma.67 In the absence of other evidence, the detection of retinal nerve fiber layer abnormalities represents the first hint of POAG.68 Recently, osteopontin increases have been detected in the AH of a glaucoma mouse model as related to optic nerve damage.69 Our results, obtained by antibody microarray in human AH, detected a nonsignificant 14% increase in POAG vs control; osteopontin was increased in >75-year-old subjects as compared to younger subjects both in POAG (13%) and controls (10%) without any statistical significance. Our finding indicate that the situation observed in human is more complex than those observed in the animal model. Indeed, in humans osteopontin presence in AH, confirmed by our findings, may be affected by a variety of other factors, such as aging, vascular defects, and neuronal damages.
Conclusion The aqueous humor molecular alterations reflect glaucoma pathogenesis: in the anterior chamber occur the same events that occur both at the level of optic nerve and central nervous system. Rather, our research provides evidence that the depletion of the trabecular cell population is the basis of glaucoma pathogenesis and confirm the central role played by oxidative stress. Many other questions remain currently without an answer, that is, if there is a molecule that translates these anterior chamber molecular happenings in a signal for the optic nerve and the central nervous system. Furthermore, the application of microarray proteome analysis to AH may be a new diagnostic tool for POAG and could be
research articles useful to elucidate uncertain diagnoses. Further studies in this direction will reveal the potential of this analysis for diagnostic purposes. Conversely, evidence has already been provided that the proteomic approach can lead to a better understanding of the mechanisms involved in the pathogenesis of POAG, the main cause of irreversible blindness worldwide. Abbreviations: TM, trabecular meshwork; IOP, intraocular pressure, POAG, primary open angle glaucoma; anterior chamber, AC; aqueous humor, AH.
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