Association of tear matrix metalloproteinase 9 immunoassay with signs and symptoms of dry eye disease: A cross-sectional study using qualitative, semiquantitative, and quantitative strategies.

PURPOSE: This study aimed to analyze the association of tear matrix metalloproteinase 9 (MMP-9) immunoassay with the severity of dry eye (DE) signs and symptoms through qualitative, semiquantitative, and quantitative evaluations of immunoassay band. MATERIALS AND METHODS: This cross-sectional study enrolled 320 eyes of 320 patients. The clinical signs of DE were assessed using the Ocular Surface Disorder Index (OSDI) score, visual analogue scale (VAS), tear breakup time (tBUT), tear volume evaluation by tear meniscometry, and staining scores of the cornea and conjunctiva by the Oxford grading scheme. The tear MMP-9 immunoassay results were interpreted using qualitative (positive or negative), semi-quantitative (reagent band density on a four-point scale: 0 = negative; 1 = weakly positive; 2 = moderately positive; 3 = strongly positive), and quantitative (ratio of reagent band density to control band density) indicators. RESULTS: Positive MMP-9 immunoassay results were significantly related to shorter tBUT, tBUT ≤3 seconds, higher corneal staining score, corneal staining score ≥2, and conjunctival staining score ≥2. The semi-quantitative results of the MMP-9 immunoassay were positively correlated with higher corneal staining score (r = 0.122, p = 0.029) and negatively correlated with tBUT (r = -0.125, p = 0.025). However, in the quantitative analysis, none of the DE signs or symptoms were correlated to the band density of the MMP-9 immunoassay. CONCLUSIONS: The positive MMP-9 immunoassay results were related to the severity of ocular signs of DE. However, using quantitative measures of the MMP-9 immunoassay to assess the clinical severity of DE requires further investigation.

ACE2 and TMPRSS2 are expressed on the human ocular surface, suggesting susceptibility to SARS-CoV-2 infection.

PURPOSE: Conjunctival signs and symptoms are observed in a subset of patients with COVID-19, and SARS-CoV-2 has been detected in tears, raising concerns regarding the eye both as a portal of entry and carrier of the virus. The purpose of this study was to determine whether ocular surface cells possess the key factors required for cellular susceptibility to SARS-CoV-2 entry/infection. METHODS: We analyzed human post-mortem eyes as well as surgical specimens for the expression of ACE2 (the receptor for SARS-CoV-2) and TMPRSS2, a cell surface-associated protease that facilitates viral entry following binding of the viral spike protein to ACE2. RESULTS: Across all eye specimens, immunohistochemical analysis revealed expression of ACE2 in the conjunctiva, limbus, and cornea, with especially prominent staining in the superficial conjunctival and corneal epithelial surface. Surgical conjunctival specimens also showed expression of ACE2 in the conjunctival epithelium, especially prominent in the superficial epithelium, as well as weak or focal expression in the substantia propria. All eye and conjunctival specimens also expressed TMPRSS2. Finally, Western blot analysis of protein lysates from human corneal epithelium obtained during refractive surgery confirmed expression of ACE2 and TMPRSS2. CONCLUSIONS: Together, these results suggest that ocular surface cells including conjunctiva are susceptible to infection by SARS-CoV-2, and could therefore serve as a portal of entry as well as a reservoir for person-to-person transmission of this virus. This highlights the importance of safety practices including face masks and ocular contact precautions in preventing the spread of COVID-19 disease.

The presence of kynurenine aminotransferases in the human cornea: Evidence from bioinformatics analysis of gene expression and immunohistochemical staining.

PURPOSE: Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea. METHODS: Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma. RESULTS: Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva. CONCLUSIONS: The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.

Investigation of Glutathione S-Transferase Isoenzyme Protein Expression in Patients With Pterygium.

PURPOSE: We investigated glutathione S-transferase (GST) enzymes in terms of their potential effects on the pathogenesis of pterygium. METHODS: Twenty-six pterygium specimens and 15 normal conjunctival specimens of 15 control subjects were investigated. Expressions of GST (alpha, mu, pi, and theta) enzymes were assessed by immunohistochemical staining. A brown color in the cytoplasm and/or nuclei of epithelial cells was evaluated as positive staining for GST enzymes. For each antibody, the intensity of the reaction [negative (-), weak (1+), moderate (2+), or strong (3+)] was determined to describe the immunoreactions. RESULTS: The median age was 52 years in the both groups. There was no significant difference between the groups in terms of age, sex, and intraocular pressure measurements (P > 0.05 for all). Of the 26 pterygium specimens, 15 (57.7%) (8 weak, 4 moderate, and 3 strong staining) were identified with GST pi-1 (GSTP1) expression and 20 (76.9%) (12 weak, 7 moderate, and 1 strong staining) with GST theta-1 (GSTT1) expression. Of the 15 control specimens, 4 (26.7%) (4 weak staining) were identified with the GSTP1 expression, and 1 (6.7%) with GSTT1 expression. GSTP1 and GSTT1 expressions were significantly higher in the pterygium specimens than in the controls (P = 0.043, P < 0.001; respectively). None of tissue specimens had positive staining for GST mu-1 or GST alpha-1 in both groups (both; P = 1.00). CONCLUSIONS: The significant increase of GSTP1 and GSTT1 expressions in pterygium may be because of the increased activation of GST in response to excessive free radical formation from ultraviolet exposure to maintain antioxidant capacity in pterygium.

The presence of lysyl oxidase-like enzymes in human control and keratoconic corneas.

PURPOSE: Lysyl oxidases, a family comprising lysyl oxidase (LOX) and four LOX-like enzymes (LOXL1-4), catalyse the cross-linking of elastin and collagen fibrils. Keratoconus (KC) is characterized by progressive thinning leading to irregular astigmatism, resulting in significant visual impairment. Although the pathogenesis of KC remains unclear, one of the current hypotheses is based on alterations in the organization and structure of collagen fibrils. To extend existing general knowledge about cross-linking enzymes in the human cornea, in the present study we have focused on the detection of LOXL enzymes. METHOD: The localization and distribution of LOXL1-4 were assessed in cryosections of 7 control donors (three males and three females; 25-68 years; mean age 46±17.6 years) and 8 KC corneas (5 males and 3 females; 25-46 years; mean age 31.3±7.5 years) using indirect fluorescent immunohistochemistry (IHC). The specimens were examined using an Olympus BX51 microscope (Olympus Co., Tokyo, Japan) at a magnification of 200-1000x. Western blot analysis of 4 control and 4 KC corneas was performed for all tested enzymes. RESULTS: All four LOX-like enzymes were present in all layers of control corneas as well as in the limbus and conjunctiva. Almost no differences between control and pathological specimens were found for LOXL1. A lower staining intensity of LOXL2 was found using IHC and Western blot analysis in KC specimens. Decreases of the signal and small irregularities in the staining were found in the epithelium, keratocytes and extracellular matrix, where a gradual anterior-posterior weakening of the signal was observed. LOXL3 IHC staining was lower in the corneal stromal extracellular matrix and keratocytes of KC samples. No prominent differences were detected using IHC for LOXL4, but a slight decrease was observed in KC corneas using Western blot analysis. CONCLUSION: We presume that the decrease of LOXL2 in KC corneas is more likely a consequence of the associated pathological processes (activation of stromal cells due to tissue weakening and consequent structural changes) than a direct cause leading to KC development. At this time, we are unable to provide a coherent explanation for the observed decrease of LOXL3 and LOXL4 in KC corneas.

Prolidase Enzyme Activity in Conjunctiva and Pterygium Tissues.

BACKGROUND: The aim of this study was to determine prolidase activity in conjunctival tissue and its relationship with pterygium. MATERIAL AND METHODS: Prolidase activity was measured in 23 pterygium and 25 healthy conjunctival tissues and the 2 groups were compared. RESULTS: Prolidase enzyme activity could not be measured in either the healthy conjunctival or in pterygium tissues. The mean serum prolidase levels of the control and pterygium groups were 967.46±353.64 and 858.29±301.83, respectively. Statistically, there was no significant difference between the groups with regard to serum prolidase levels (p>0.05). CONCLUSIONS: In conclusion, absence of prolidase activity in pterygium tissue indicates that there is no collagen turnover in this tissue. We may explain this finding with the elastin-rich structure of the conjunctiva.

Role of glutathione peroxidase 4 in conjunctival epithelial cells.

PURPOSE: The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells. METHODS: An immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity. RESULTS: Small interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA-treated cells. Moreover, cell death in GPx4 siRNA-treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide. CONCLUSION: These results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.

Matrix metalloproteinase 9 and transglutaminase 2 expression at the ocular surface in patients with different forms of dry eye disease.

OBJECTIVE: To evaluate the expression of matrix metalloproteinase 9 (MMP9) and transglutaminase 2 (TG2) in different forms of dry eye. DESIGN: Case control study. PARTICIPANTS: Seventy-five female subjects divided into 3 groups: group 1, 15 healthy controls; group 2, 30 subjects with Sjögren syndrome (SS); and group 3, 30 subjects with Meibomian gland dysfunction (MGD). METHODS: A clinical assessment was carried out and impression cytologic specimens were processed for immunoperoxidase staining for MMP9 and TG2 and real-time polymerase chain reaction analyses were carried out for MMP9, TG2, interleukin-6, interferon-γ, B-cell lymphoma 2, and caspase 3. To study MMP9 and TG2 expression after anti-inflammatory treatment, patients were divided into 2 subgroups, one treated with saline and the other treated with saline plus topical corticosteroid eye drops (0.5% loteprednol etabonate) 4 times daily for 15 days. For statistical analysis, Student t test, Mann-Whitney U test, and Spearman's correlation coefficient were used as appropriate. MAIN OUTCOME MEASURES: Conjunctival expression of MMP9 and TG2. RESULTS: MMP9 and TG2 expression were higher in both patient groups than in controls (P < 0.0001). Group 2 patients showed higher expression than group 3 (P < 0.0001). The Spearman's correlation coefficient showed in group 2 a positive correlation between MMP9 and TG2 expression (ρ = 0.437; P = 0.01), but no correlation in group 3 (ρ = 0.143; P = 0.45). Corticosteroid treatment significantly reduced MMP9 and TG2 expression in both groups, ameliorating symptoms and signs. A much higher percentage reduction was observed in SS. CONCLUSIONS: The pathogenic mechanisms of the 2 forms of dry eye give an account for the different MMP9 and TG2 expressions in the 2 groups of patients. The higher expression in SS is determined by the direct autoimmune insult to the ocular surface epithelia, whereas in MGD patients, with an epithelial damage due to an unbalanced tear secretion, the molecules expression is significantly lower, although higher than in controls. The corticosteroid treatment induced a reduction of both molecules, although higher in SS than in MGD, because of its direct inhibitory effect on inflammation.

Conjunctival matrix metalloproteinases and their inhibitors in glaucoma patients.

PURPOSE: Chronic conjunctival inflammation, caused by various reasons, for example long-term use of topical drugs and/or their preservatives, affects the outcome of glaucoma surgery by interfering with wound healing. Matrix metalloproteinases (MMPs) remodel extracellular matrix (ECM) and are involved in the wound healing process. This study was designed to evaluate the conjunctival expression of MMPs and their tissue inhibitors (TIMPs) in the normal eye, primary open-angle glaucoma (POAG) and exfoliation glaucoma (ExG) and whether there is an association between staining intensities and deep sclerectomy outcome. METHODS: Immunohistochemical procedures were performed on conjunctival samples which were obtained from POAG (n=11) and ExG (n=14) patients as well as normal (n=7) subjects. Antibodies against MMPs (MMP-1, -2, -3 and -9) and TIMPs (TIMP-1, -2 and -3) were used. RESULTS: In conjunctival stroma, expression levels of MMP-2 (p=0.047), MMP-3 (p=0.009), MMP-9 (p<0.001), TIMP-1 (p=0.003), TIMP-2 (p<0.001) and TIMP-3 (p<0.001) in ExG and MMP-9 (p=0.008), TIMP-2 (p=0.02) and TIMP-3 (p=0.002) in POAG were significantly increased compared to control. We further found correlations between expression of MMP-1 and MMP-3 and the length of pilocarpine treatment. CONCLUSION: The expression of MMPs and TIMPs is increased in the conjunctiva of POAG and ExG patients having a long history of topical antiglaucoma drops. Antiglaucoma agents and/or their preservatives alter the remodelling balance of ECM in conjunctiva of POAG and ExG eyes. The balance between MMPs and TIMPs may play a crucial role in the conjunctival wound healing process and the outcome of glaucoma surgery.

Increased extracellular matrix metalloproteinase inducer (EMMPRIN) expression in the conjunctival epithelium exposed to antiglaucoma treatments.

OBJECTIVE: To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells. METHODS: A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs). RESULTS: Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p < 0.001) and LE groups (2.55 ± 0.40 RFI, p = 0.029). Similar increase was observed in HLA-DR expression in the HE (4.58 ± 1.38 RFI, p < 0.001) and LE groups (2.52 ± 0.47 RFI, p = 0.046) as compared to control subjects (1.75 ± 0.27 RFI). Across all subjects enrolled in the study, there was a significant correlation between HLA-DR and EMMPRIN (R(2) = 0.875, p < 0.0001). IOBA-NHC cells exposed to BAK presented a significant increase in EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA. CONCLUSIONS: The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN.

DNA methyl transferases are differentially expressed in the human anterior eye segment.

PURPOSE: DNA methylation is an epigenetic mark involved in the control of genes expression. Abnormal epigenetic events have been reported in human pathologies but weakly documented in eye diseases. The purpose of this study was to establish DNMT mRNA and protein expression levels in the anterior eye segment tissues and their related (primary or immortalized) cell cultures as a first step towards future in vivo and in vitro methylomic studies. METHODS: Total mRNA was extracted from human cornea, conjunctiva, anterior lens capsule, trabeculum and related cell cultures (cornea epithelial, trabecular meshwork, keratocytes for primary cells; and HCE, Chang, B-3 for immortalized cells). cDNA was quantified by real-time PCR using specific primers for DNMT1, 2, 3A, 3B and 3L. Immunolocalization assays were carried out on human cornea using specific primary antibodies for DNMT1, 2 and 3A, 3B and 3L. RESULTS: All DNMT transcripts were detected in human cornea, conjunctiva, anterior lens capsule, trabeculum and related cells but showed statistically different expression patterns between tissues and cells. DNMT2 protein presented a specific and singular expression pattern in corneal endothelium. CONCLUSIONS: This study produced the first inventory of the expression patterns of DNMTs in human adult anterior eye segment. Our research highlights that DNA methylation cannot be ruled out as a way to bring new insights into well-known ocular diseases. In addition, future DNA methylation studies using various cells as experimental models need to be conducted with attention to approach the results analysis from a global tissue perspective.

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

PURPOSE: This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding. METHODS: Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs. RESULTS: The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining. CONCLUSIONS: Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.

Enzymes of urea synthesis are expressed at the ocular surface, and decreased urea in the tear fluid is associated with dry-eye syndrome.

PURPOSE: The present study aims at determining whether enzymes of urea synthesis are expressed in the human lacrimal gland and in tissues of ocular surface (conjunctiva, cornea), to give evidence for the hypothesis that urea can be locally formed from ocular tissues and is important for the composition of the tear fluid. METHODS: The presences of enzymes (arginase 1, 2 and agmatinase) that directly contribute to the formation of urea were investigated in the lacrimal gland and tissues of ocular surface by RT-PCR and immunohistochemistry. We collected tear fluid, aqueous humour, and blood samples from a total of 38 subjects, and tear fluid samples from a total of 78 subjects, with and without dry-eye syndrome (DES, keratoconjunctivitis sicca), and determined the urea concentration. RESULTS: The enzymes arginase 1, 2 and agmatinase were expressed in all tissues examined except for arginase 1, which was not expressed in the cornea. There was no correlation of urea concentration in tear fluid with aqueous humour and blood plasma (r = 0.13, p = 0.58 and r = 0.45, p = 0.05 respectively). However, correlation of urea concentration between aqueous humour and blood plasma was highly significant (r = 0.7, p = 0.0001). The concentration of urea in the tear fluid of patients with DES compared to healthy control group was significantly reduced (p < 0.0001). CONCLUSION: Enzymes that are directly involved in the formation of urea are expressed in ocular tissues. This may imply that in the ocular surface is a well-coordinated system of enzymes that can produce urea which might be independent of external urea supply.

Overexpression of peroxiredoxin 2 in pterygium. A proteomic approach.

Pterygium is one of the most frequent pathologies in ophthalmology, and is a benign, fibrovascular lesion originating from the bulbar conjunctiva. It is composed of an epithelium and highly vascular, subepithelial, loose connective tissue. The etiology of pterygium is not clearly understood; the most widely recognized originating factor is ultraviolet radiation. It has been proposed that pterygium and neoplasia have common features, raising the possibility that pterygium is a neoplastic-like growth disorder. In this study, proteomic analysis was performed to show that peroxiredoxin 2 is overexpressed in pterygia compared to healthy conjunctivas. Twelve pterygium specimens were obtained together with healthy conjunctival tissue from the same eyes. Total proteins of pterygia and healthy conjunctivas were analyzed in SDS-PAGE. This analysis showed protein bands expressed exclusively in pterygium samples at the range of 20-25 kDa. After this, 2D electrophoresis was performed for the separation of total proteins; differential spots expressed in pterygium were excised and sequenced. Mass spectrometry (MS) data were searched in the NCBInr and EST databases using the MASCOT program. The spot was identified as peroxiredoxin 2. Real-time PCR, western blot and immunohistochemistry showed that peroxiredoxin 2 was increased in pterygium compared to healthy conjunctiva. Although, these results suggest that overexpression of peroxiredoxin 2 in pterygium could protect the cell against oxidative stress-induced apoptosis, further studies are required to establish the functional role of peroxiredoxin 2 in pterygium to determine its role in peroxidation and apoptosis in this pathology.

Immunohistochemical analysis of angiotensin converting enzyme in Sardinian pterygium.

Pterygium is a common ocular surface disorder characterized by excessive cell proliferation, inflammation, fibrosis, angiogenesis and extracellular matrix remodeling. The Angiotensin converting enzyme (ACE or ACE I) is the major component of the Renin-angiotensin system (RAS) converting the inactive decapeptide Angiotensin I (Ang I) to the active octapeptide Angiotensin II (Ang II). Besides this 'classical role', it can act as transcriptional regulator in response to external stimuli that may lead to cell damage and tissue remodeling. Due to this role, it can be internalized into the nuclear compartment to act as transcriptional factor for proteins involved in the inflammatory response. The aim of the present study was to determine ACE expression and localization in pterygium and culture pterygium cells by immunohistochemistry. Our results are the first to demonstrate nuclear immunolocalization of ACE, more so in pterygium compared to conjunctiva epithelial cells in histological sections. ACE was not detected in the nuclei of subcultivated pterygium epithelial cells. The nuclear localization of ACE may be correlated with an anti-inflammatory path mediated by activation of its transcriptional role.

Changes in lysyl oxidase (LOX) distribution and its decreased activity in keratoconus corneas.

Inadequate cross-linking between collagen lamellae is a characteristic feature of keratoconus corneas. The formation of covalent bonds between collagen and elastin fibrils, which maintain the biomechanical properties of the cornea, is mediated by the cuproenzyme lysyl oxidase and four lysyl oxidase-like enzymes. The aim of this study was to determine the distribution of lysyl oxidase and the total lysyl oxidase activity (lysyl oxidase and the four lysyl oxidase-like enzymes) in control and keratoconic corneas. Seven control and eight keratoconic corneas were used for the imunohistochemical detection of lysyl oxidase in corneal cryosections using two different antibodies. The total lysyl oxidase activity in the culture medium of corneal fibroblasts from six explanted keratoconic and four control corneas was measured using a fluorometric assay in the presence and absence of the lysyl oxidase inhibitor beta-aminopropionitrile and determined as the production of H(2)O(2) in nM per µg of total protein. In the control tissue, the most intense signal for lysyl oxidase was present in the corneal epithelium, in which perinuclear dots brightly projecting from more or less homogenous cytoplasmic staining may represent the lysyl oxidase propeptide. Less intense staining was present in keratocytes, the extracellular matrix and in the corneal endothelium. The epithelium of the limbus and the perilimbal conjunctiva showed intense to very intense staining. The distribution of lysyl oxidase was clearly decreased in at least five of the eight keratoconic specimens. The most marked signal reduction was observed in the stromal matrix and in keratocytes. Moreover, the signal in pathological specimens revealed a more irregular pattern, including the presence of intra- and extracellular clumps in the epithelium. Interestingly, endothelial cells showed no or very weak staining in areas just beneath negative stromal tissue. The mean activity of total lysyl oxidase in the keratoconic samples (2.60 ± 2.23 nM H(2)O(2)/µg of total protein) was more than 2.5-fold lower than in control tissue (6.83 ± 2.53 nM H(2)O(2)/µg of total protein), and the decrease was statistically significant (p = 0.0178). The location of lysyl oxidase in the healthy cornea, limbus and perilimbal conjunctiva was described. We hypothesize that the restricted lysyl oxidase distribution in keratoconic corneas, and particularly the decrease of total lysyl oxidase activity in cultured keratoconic fibroblasts, is one potential reason for the inadequate collagen cross-linking that is a hallmark of this disease.

Activation of MAPK signaling pathway and NF-kappaB activation in pterygium and ipsilateral pterygium-free conjunctival specimens.

PURPOSE: To evaluate mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways in pterygium and pterygium-free conjunctivas. METHODS: Primary pterygia (n = 21), ipsilateral superior-temporal bulbar conjunctivas (n = 8), and healthy conjunctival (n = 5) biopsy specimens were analyzed. Total and phosphorylated (phospho) levels of extracellular-regulated 1/2 (ERK1/2), p38, and c-jun N-terminal (JNK) MAPKs and NF-κB inhibitor-alpha (IκΒ-α) were analyzed by immunobead-based assay. Tissue phospho-, total protein, and activation values determined by phospho/total ratios were compared. Correlation among those values and clinical parameters were determined. Average-linkage hierarchical cluster analysis identified patients with similar protein activation values. The k-nearest neighbor classifier predicted the origin of specimens based on protein levels. RESULTS: Pterygium samples had significantly lower total JNK and IκΒ-α levels than did healthy conjunctivas. Decreased total JNK and IκΒ-α and increased phospho-IκΒ-α levels and phospho/total ratio of JNK and IκΒ-α were present in ipsilateral conjunctivas compared with healthy conjunctivas. Protein levels were correlated among them in pterygium, ipsilateral, and healthy conjunctivas and with sun exposure, pterygium grade, and pterygium measurements. Cluster analysis of activation values and ratios in pterygium and ipsilateral-conjunctiva revealed different groups of patients with similar values. Prediction accuracy was 70% to 80% for the classifiers phospho- and total protein levels and phospho/total ratio. CONCLUSIONS: Pterygium and pterygium-free ipsilateral conjunctivas had alterations in MAPK and NF-κB pathways not present in healthy conjunctivas. The high prediction accuracy based on phospho- and total protein levels and phospho/total ratio of ERK1/2, p38, JNK, and IκB-α suggests these molecules as potential biomarkers of inflammation in pterygia.

Increased conjunctival expression of protease activated receptor 2 (PAR-2) in seasonal allergic conjunctivitis: a role for abnormal conjunctival epithelial permeability in disease pathogenesis?

AIMS: Aeroallergen exposure to the conjunctival epithelium in seasonal allergic conjunctivitis (SAC) may induce a cellular stress response that disrupts the barrier properties of the conjunctival epithelium, resulting in allergic disease. Whether such changes occur in SAC is unknown. Epithelial permeability is known to be increased when protease activated receptor 2 (PAR-2) is activated. We evaluated the expression of PAR-2 in patients with SAC-in-season (SACS) and compared it with control non-atopic subjects or those with out-of-season allergic conjunctivitis (OSAC). METHODS: Six SACS, eight normal and four OSAC specimens were examined immunohistochemically for PAR-2 and quantified in a masked fashion for the percentage of epithelia stained for each marker using Image-J software. Conjunctival epithelial heights were measured in all groups to confirm the presence of allergic eye disease. RESULTS: Mean percentage staining of PAR-2 was significantly greater in SACS that in normal specimens (73.4 ± 15.4% vs 32.8 ± 30.0%, p=0.038) or in OSAC (73.4 ± 15.4% vs 1.4 ± 2.2%, p=0.01). Mean conjunctival epithelial height was significantly raised in SACS (63.8 ± 9.0 µm) versus controls (44.7 ± 11.2 µm) (p=0.003, unpaired t test). CONCLUSIONS: Conjunctival epithelial PAR-2 is significantly upregulated in SAC. This supports the view that disruption of the barrier properties of the conjunctival epithelium is an important event in SAC pathogenesis.

Antioxidant enzyme mRNA expression in conjunctival epithelium of healthy human subjects.

OBJECTIVE: To determine the profile, relative quantitation, and correlation of gene expression of the antioxidant enzymes copper-zinc superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD), extracellular superoxide dismutase (EC-SOD), catalase (CAT), glutathione synthetase (GSS), and glutathione reductase (GSR) in conjunctival samples from healthy subjects. DESIGN: Descriptive study. PARTICIPANTS: Sixteen healthy donors (32 eyes) were included in this study. METHODS: Conventional and real-time polymerase chain reactions (PCR) were performed using total RNA isolated from conjunctival impression cytology taken from bulbar superior conjunctiva from both eyes. RESULTS: Products amplified by conventional PCR had only 1 band with the expected size for each target gene. Melt-curve analysis of real-time PCR products also identified a single amplified product for each gene. Log-transformed antioxidant enzyme mRNA expression levels, relative to glyceraldehyde-3-phosphate dehydrogenase expression (mean ± standard error of the mean [SEM]), were CuZn-SOD, 1.52 ± 0.13; Mn-SOD, 1.72 ± 0.08; EC-SOD, 0.35 ± 0.08; GSS, 2.43 ± 0.20; GSR, 2.52 ± 0.16; and CAT, 0.90 ± 0.08. The mRNA levels for CuZn-SOD were strongly correlated with GSS, GSR, Mn-SOD, and EC-SOD. Similarly, the levels of mRNA of GSS and GSR were strongly correlated with each other and with Mn-SOD and EC-SOD. CONCLUSIONS: Normal human conjunctiva expresses the antioxidant enzymes genes CuZn-SOD, Mn-SOD, EC-SOD, CAT, GSS, and GSR. The relative quantitation of these genes expressed in conjunctivas of normal eyes will allow further comparisons in pathological circumstances. Knowledge of correlated gene expression will provide a better understanding of the antioxidant balance in the ocular surface.