Aqueous Humor Of Patients Research Articles

Tissue inhibitor of metalloproteinase 4 in aqueous humor of patients with primary open angle glaucoma, pseudoexfoliation syndrome and pseudoexfoliative glaucoma and its role in proteolysis imbalance

BackgroundTo quantify the levels of tissue inhibitor of metalloproteinase 4 (TIMP4) and its ratios with free metalloproteinases (MMP) in the aqueous humor of patients with primary open angle glaucoma (POAG), pseudoexfoliation syndrome (PXS) and pseudoexfoliative glaucoma (PXG) and to evaluate a possible imbalance between MMPs and TIMPs in these samples.MethodsFree MMP2, MMP3, MMP9, TIMP1, TIMP2, TIMP4 concentrations and active levels of MMP2 and MMP3 were determined with immunoassay ELISA and activity assay kits in 168 aqueous samples.ResultsTIMP4 was elevated in glaucoma patients(POAG: 0.95 ± 0.49 PXG: 1.28 ± 1.38 pg/ml. p < 0.001). POAG, PXS and PXG samples demonstrated higher MMP2, TIMP1 and TIMP2 concentrations (p < 0.001). Samples from the PXS and PXG groups had a lower total/active MMP2 ratio (p < 0.004 and p < 0.008 respectively). Stoichiometric analysis showed an overbalance of TIMPsover MMPs in both POAG & PXG groups,especially of TIMP4.ConclusionTIMP4 elevation is a novel finding in glaucomatous eyes. A disregulation of extracellular matrix homeostasis is suggested in POAG, PXS and PXG.

Elevated Levels of Multiple Biomarkers of Alzheimer's Disease in the Aqueous Humor of Eyes With Open-Angle Glaucoma

To investigate levels of biomarkers of Alzheimer's disease in the aqueous humor in patients with open-angle glaucoma (OAG). Aqueous humor samples were collected from 38 patients with cataracts, 20 patients with POAG, and 32 patients with exfoliation glaucoma. Aqueous levels of apolipoprotein (Apo) AI, ApoCIII, ApoE, transthyretin (TTR), complement factor H, complement C3, and α2-macroglobulin (α2M) were determined by multiplex bead immunoassay. Clinical data were obtained from medical charts. Age and sex did not differ significantly among groups. POAG patients had significantly higher aqueous levels of Apo AI, ApoCIII, ApoE, TTR, and α2M compared with cataract patients (P < 0.001, P = 0.036, P < 0.001, P < 0.001, and P < 0.001, respectively). Corresponding values for exfoliation glaucoma patients were also significantly higher compared with those for cataract patients (P = 0.003, P = 0.009, P < 0.001, P < 0.001, and P < 0.001), and those levels correlated positively with each other in eyes with OAG (P < 0.001 for all combinations). Complement factor H and complement C3 levels did not differ among groups. Mean deviation values for the Humphrey visual field test correlated positively with levels of Apo AI, ApoE, TTR, and complement factor H in OAG patients (P = 0.026, 0.012, 0.008, and 0.027, respectively), but age and IOP values were not correlated with protein levels. OAG patients had elevated levels of multiple biomarkers of Alzheimer's disease in the aqueous humor.

Proteomic analysis of the aqueous humor in patients with wet age‐related macular degeneration

A number of studies have shown that the levels of some proteins in the aqueous humor (AH) are altered and correlate with the mechanisms or prognosis of many eye diseases. To identify the possible mechanisms that lead to the development of wet age-related macular degeneration (AMD), a proteomic analysis of the AH composition from wet AMD patients was performed and compared with that from non-AMD cataract patients. Six wet AMD and six non-AMD cataract patients were enrolled. A proteomic approach which included two-dimensional electrophoresis coupled with MS and bioinformatics methods were used to identify AH proteins with altered expression in wet AMD compared with non-AMD patients. An ELISA was used to validate the proteomic results. We separated 78 protein spots and identified 68 that were differently expressed in the wet AMD group and controls. Numerous proteins identified in this study are implicated in inflammation, apoptosis, angiogenesis, and oxidative stress. The AH protein composition was significantly different between wet AMD and non-AMD patients. The proteins identified in this study may be potential biomarkers of wet AMD development and might play a role in the mechanisms of wet AMD.

Searching for viral antibodies and genome in intraocular fluids of patients with Fuchs uveitis and non-infectious uveitis

To characterise the polyspecific intraocular antibody synthesis in aqueous humor of patients with Fuchs uveitis and other types of non-infectious uveitis. Aqueous and serum samples collected from 24 patients with Fuchs uveitis, 21 patients with non-infectious uveitis, and 27 healthy subjects undergoing elective cataract surgery (control group) were analysed. In addition, vitreous samples, collected from seven uveitis patients (five Fuchs and two panuveitis) during retinal surgery, were examined. Specific immunoglobulin G antibodies against cytomegalovirus (CMV), rubella virus, herpes simplex virus (HSV), and varicella zoster virus (VZV) were investigated, and Goldmann-Witmer coefficients (GWCs) were calculated. Real-time PCR was performed to detect viral genome for HSV, VZV, and CMV, while nested PCR was conducted to detect rubella RNA. None of the control samples tested positive for any of the viral antibodies investigated. Intraocular antibody production was found in eight samples of patients affected by Fuchs uveitis (6/8 positive for rubella virus and 2/8 positive for herpes virus). Among patients with non-infectious uveitis, three tested positive for intraocular antibody production (one RV, one HSV and one for VZV). PCR was positive for RV in two patients with Fuchs uveitis, in three patients with non-infectious uveitis (one for RV and two for HSV), and in three control subjects (one for CMV and one for HSV). Our series confirmed the presence of specific viral antibodies, especially against rubella virus, in the subgroup of patients affected by Fuchs uveitis, suggesting that this virus may be responsible for this chronic inflammatory condition. Rubella virus is probably the main causative agent of Fuchs uveitis, but other viruses may also be involved in the pathogenesis of this disease.

Aqueous humor levels of vascular endothelial growth factor and adiponectin in patients with type 2 diabetes before and after intravitreal bevacizumab injection

To determine the levels of vascular endothelial growth factor (VEGF) and adiponectin (APN) in the aqueous humor of patients with type 2 diabetes before and after injection of bevacizumab (IVB). Twenty eyes of twenty consecutive patients with type 2 diabetes with PDR and clinically significant macular edema were enrolled in this study. Aqueous samples were collected at baseline and one month after IVB to evaluate VEGF and APN levels. Twenty age-matched patients undergoing cataract surgery were used as control. Best-corrected visual acuity (BCVA) and foveal thickness (FT) changes after IVB were also measured. Safety was assessed by recording the incidence of ocular and non-ocular adverse events. At baseline APN and VEGF levels were significantly lower in controls than in PDR patients (APN: 3.6 ± 1.1 vs 18.7 ± 4.5 ng/ml; VEGF: 22.6 ± 16.1 vs 146.2 ± 38.71 pg/ml). After IVB, both compounds significantly decreased. FT and BCVA at baseline were significantly different between controls and patients (FT: 215.6 ± 34.8 vs 532.7 ± 112.4 μm; BCVA: 23.6 ± 4.2 vs 18.4 ± 7.3 letters). After IVB a significant decrease of FT with a concomitant improvement of BCVA occurred. Neither ocular nor systemic adverse events were reported. Our findings demonstrate that patients with type 2 diabetes, PDR and macular edema show VEGF and APN levels in aqueous humor higher than those found in control subjects. IVB significantly reduced the levels of both compounds, which remained anyway at concentrations higher than those recorded in control subjects.

Levels of Aqueous Humor Trace Elements in Patients with Non-Exsudative Age-related Macular Degeneration: A Case-control Study

Trace elements might play a role in the complex multifactorial pathogenesis of age-related macular degeneration (AMD). The aim of this study was to measure alterations of trace elements levels in aqueous humor of patients with non-exsudative (dry) AMD.For this pilot study, aqueous humor samples were collected from patients undergoing cataract surgery. 12 patients with dry AMD (age 77.9±6.62, female 8, male 4) and 11 patients without AMD (age 66.6±16.7, female 7, male 4) were included. Aqueous levels of cadmium, cobalt, copper, iron, manganese, selenium, and zinc were measured by use of Flow-Injection-Inductively-Coupled-Plasma-Mass-Spectrometry (FI-ICP-MS), quality controlled with certified standards.Patients with AMD had significantly higher aqueous humor levels of cadmium (median: 0.70 µmol/L, IQR: 0.40–0.84 vs. 0.06 µmol/L; IQR: 0.01–.018; p = 0.002), cobalt (median: 3.1 µmol/L, IQR: 2.62–3.15 vs. 1.17 µmol/L; IQR: 0.95–1.27; p<0.001), iron (median: 311 µmol/L, IQR: 289–329 vs. 129 µmol/L; IQR: 111–145; p<0.001) and zinc (median: 23.1 µmol/L, IQR: 12.9–32.6 vs. 5.1 µmol/L; IQR: 4.4–9.4; p = 0.020) when compared with patients without AMD. Copper levels were significantly reduced in patients with AMD (median: 16.2 µmol/L, IQR: 11.4–31.3 vs. 49.9 µmol/L; IQR: 32.0–.142.0; p = 0.022) when compared to those without. No significant differences were observed in aqueous humor levels of manganese and selenium between patients with and without AMD. After an adjustment for multiple testing, cadmium, cobalt, copper and iron remained a significant factor in GLM models (adjusted for age and gender of the patients) for AMD.Alterations of trace element levels support the hypothesis that cadmium, cobalt, iron, and copper are involved in the pathogenesis of AMD.

Asymmetric Dimethyloarginin (ADMA) Concentration in the Aqueous Humor of Patients with Exfoliation Syndrome or Exfoliative Glaucoma

Background: Asymmetric dimethyloarginin or dimethylarginin (ADMA) is a marker and maker of oxidative stress. It is elevated in several pathological conditions, such as hyperhomocysteinemia and endothelial dysfunction, which have also been reported in patients with exfoliation syndrome (XFS), or exfoliative glaucoma (XFG). We evaluated ADMA levels in the aqueous humor of XFS and XFG patients.Methods: This study included 48 aqueous samples; 16 from cataract patients with XFS, 16 from cataract patients with XFG and 16 from age-matched cataract control patients. ADMA levels were determined employing a commercial ELISA kit. Results: ADMA concentration was significantly greater in XFG patients (0.398 ± 0.026 μM) compared to either XFS (0.168 ± 0.028 μM; p < 0.0001) or normal cataract controls (0.245 ± 0.025 μM; p = 0.0002). In contrast, no significant difference was detected for ADMA levels in the aqueous of XFS patients as compared to normal controls (p = 0.0477).Conclusions: Aqueous humor ADMA concentration is significantly elevated in XFG patients supporting the view that oxidative stress plays a key role in XFG pathobiology. The lower level of this marker in XFS suggests that the development of XFG is a prerequisite for ADMA elevation.

Expression of CXCL9, -10, and -11 in the Aqueous Humor of Patients With Herpetic Endotheliitis

To evaluate the expression of CXCL9, -10, and -11 chemokines in the aqueous humor of patients with herpetic endotheliitis. Aqueous humor was collected from 26 patients with herpetic endotheliitis and 21 control subjects. The concentrations of CXCL9, -10, and -11 in the aqueous humor were measured with enzyme-linked immunosorbent assays. The correlation between chemokine levels and clinical parameters of disease severity was analyzed. Flow cytometry was performed to count CXCR3 cells and CXCR3CD4 cells in the aqueous humor. The concentrations of CXCL9, -10, and -11 were 429.08 ± 297.41 pg/mL, 23,102.49 ± 15,964.95 pg/mL, and 258.25 ± 103.25 pg/mL in patients with herpetic endotheliitis and 16.84 ± 16.73 pg/mL (P < 0.01), 188.45 ± 183.43 pg/mL (P < 0.01), and 7.32 ± 6.45 pg/mL (P < 0.01) in control subjects, respectively. Aqueous chemokine levels correlated significantly with keratitic precipitates and corneal edema in patients with herpetic endotheliitis. The mean percentages of CXCR3 and CXCR3CD4 cells were higher in herpetic endotheliitis patients compared with the controls. Expression of CXCL9, -10, and -11 chemokines and their receptor CXCR3 increases in the aqueous humor of patients with herpetic endotheliitis. Chemokine levels are associated with the clinical severity of the disease.

Therapeutic Effect of Intravitreal Injection of Triamcinolone in the Treatment of Endothelial Graft Rejection

To investigate the efficacy of intravitreal triamcinolone acetonide (IVTA) injection in the treatment of endothelial graft rejection and the expression of T-helper type 1 (Th-1) chemokines in the aqueous humor. Eleven patients (11 eyes) with acute endothelial graft rejection underwent IVTA (4 mg/0.1 mL) injection in addition to conventional treatment, including systemic and topical steroids and cyclosporine A (group A); 13 patients (13 eyes) received conventional treatment only (group B). Clinical characteristics were analyzed. In group A, Th-1 chemokines (CXCL-9, -10, and -11) in aqueous humor were measured by enzyme-linked immunosorbent assay and CXCR3(+) cells were counted by flow cytometry before treatment and at the period of improvement. There were no significant differences in visual acuity, intraocular pressure, and recurrence rate between the 2 groups. The mean time to improvement was 9.1 ± 3.9 days in group A and 15.4 ± 5.5 days in group B (P = 0.04). The concentrations of CXCL-9, -10, and -11 were 562.7 ± 109.4, 30293.1 ± 12537.1, and 228.3 ± 80.8 pg/mL before treatment and 6.7 ± 6.8 (P < 0.01), 207.5 ± 209.8 (P < 0.01), and 18.9 ± 19.5 (P < 0.01) pg/mL after treatment, respectively. The CXCL-10 level correlated with the time to improvement of graft rejection (P = 0.02). Flow cytometry demonstrated a significantly decreased number of CXCR3(+) and CXCR3(+)CD4(+) cells in the aqueous humor after treatment. IVTA injection was effective in reducing the time to improvement of endothelial graft rejection. Increased expression of CXCL-9, -10, and -11, and their receptors in the aqueous humor of patients with endothelial graft rejection decreased after treatment.

Aqueous humor oxidative stress proteomic levels in primary open angle glaucoma

The purpose of this work was to investigate the expression of glutamine synthase (GS), nitric oxide synthase (NOS) superoxide dismutase (SOD) and glutathione transferase (GST) in the aqueous humor of patients with primary open angle glaucoma and controls. Aqueous humor proteome was analyzed by antibody microarray. The expression of tested proteins was detected by protein Cy3/Cy5 labeling, column purification and hybridization on antibody-spotted glass microarray. Fluorescent signals were detected by fluorescence laser scanning. Aqueous humor levels of SOD as well as of GST were significantly lower (2.0- and 2.2-fold, p < 0.01) among patients than controls; both NOS and GS expression were significantly higher (2.2- and 2.6 fold, p < 0.01) among patients than controls. Our data showed substantial differences of GS, NOS2, SOD and GST aqueous humor levels between glaucomatous patients and controls as measured by antibody microarray technology. The overproduction of NO through inducible NOS can form toxic products and change the metabolic conditions of the TM. The GS over-expression might be related to neuronal injury or to the potential role of glutamate as a modulator in the ciliary body signaling. The reduced expression of the antioxidant enzymes SOD and GST could aggravate the unbalance between both oxygen- and nitrogen-derived free radicals production and detoxification. Based on our results, GS, NOS2, SOD and GST as measured by antibody microarray technology may be useful oxidative markers in aqueous humor of glaucomatous patients.

Simultaneous increases in multiple proinflammatory cytokines in the aqueous humor in pseudophakic glaucomatous eyes

To evaluate cytokine and growth factor levels in the aqueous humor in patients with open-angle glaucoma (OAG). Kumamoto University Hospital, Kumamoto, Japan. Cross-sectional cohort study. Aqueous humor samples were collected from cataract cases and OAG cases. Aqueous levels of cytokines and growth factors were determined by multiplex immunoassay. The data were analyzed using the Tukey-Kramer honestly-significant-difference test and multiple regression analyses. The study evaluated 52 cataract cases and 73 OAG cases. In the cataract cases, the mean interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor-α, epidermal growth factor, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, and VEGF levels (all pg/mL) were 21.4, 4.6, 829.4, 0.8, 0.5, 33.8, 1.6, and 77.9, respectively. In 23 phakic primary OAG (POAG) and 26 phakic exfoliation glaucoma cases, the corresponding values were 15.1 and 8.3, 15.2 and 12.3, 1142.5 and 1253.9, 0.3 and 0.5, 1.5 and 1.4, 57.7 and 58.0, 2.5 and 3.3, 37.4 and 59.7, respectively. In pseudophakic OAG cases, the IL-8 levels for exfoliation glaucoma (P=.0002) and MCP-1 for POAG (P=.0008) and exfoliation glaucoma (P<.0001) were significantly different compared with phakic OAG. Interleukin-6, IL-8, and MCP-1 levels were correlated (P<.0001). Multiple regression analyses showed the highest association of pseudophakic status with IL-8 and MCP-1 levels (P=.0002 and P<.0001, respectively). The OAG patients, especially those with pseudophakic eyes, had simultaneous cytokine level increases, suggesting the aqueous humor microenvironment is altered in pseudophakic glaucomatous eyes. No author has a financial or proprietary interest in any material or method mentioned.

Cytokine and CXC chemokine expression patterns in aqueous humor of patients with presumed tuberculous uveitis

Aqueous humor (AH) samples from 14 patients with presumed tuberculous uveitis (PTU), and 30 control patients were assayed for the proinflammatory cytokines interleukin IL-4, IL-12, IL-15, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, the immunosuppressive cytokine IL-10, and the chemokines GRO-α/CXCL1, IL-8/CXCL8, MIG/CXCL9, IP-10/CXCL10 and SDF-1/CXCL12 with the use of a multiplex assay. Among cytokines, IL-4 and IL-12 were not detected. IL-15, IL-17, IFN-γ, TNF-α and IL-10 levels in AH were significantly higher in patients than in controls (p<0.001; p=0.004; p<0.001; p<0.001; p<0.001, respectively). Among chemokines, SDF-1 levels did not differ significantly between patients and controls, whereas GRO-α, IL-8, MIG and IP-10 levels were significantly higher in patients than in controls (p=0.001; p<0.001; p<0.001; p<0.001, respectively). Mean GRO-α levels in AH of PTU patients were 6-fold higher than IL-8 levels and mean IP-10 levels were 15-fold higher than MIG levels. Clinical disease activity correlated significantly with the levels of IL-15, IFN-γ, TNF-α and IP-10. Logistic regression analysis demonstrated a significant positive association between PTU and high levels of IFN-γ, IL-8, MIG and IP-10. These data suggest that both T helper (Th) Th1 and Th17 cells are involved in PTU and that the cytokine profile is polarized toward a Th1 response. GRO-α and IP-10 might be involved in neutrophil and activated T lymphocyte chemoattraction in PTU, respectively.

Connective Tissue Growth Factor Causes Glaucoma by Modifying the Actin Cytoskeleton of the Trabecular Meshwork

The most critical risk factor for optic nerve damage in cases of primary open-angle glaucoma (POAG) is an increased intraocular pressure (IOP) caused by a resistance to aqueous humor outflow in the trabecular meshwork (TM). The molecular pathogenesis of this increase in outflow resistance in POAG has not yet been identified, but it may involve transforming growth factor TGF-β2, which is found in higher amounts in the aqueous humor of patients with POAG. Connective tissue growth factor (CTGF) is a TGF-β2 target gene with high constitutive TM expression. In this study, we show that either adenoviral-mediated or transgenic CTGF overexpression in the mouse eye increases IOP and leads to optic nerve damage. CTGF induces TM fibronectin and α-SMA in animals, whereas actin stress fibers and contractility are both induced in cultured TM cells. Depletion of CTGF by RNA interference leads to a marked attenuation of the actin cytoskeleton. Rho kinase inhibitors cause a reversible decline in the IOP of CTGF-overexpressing mice to levels seen in control littermates. Overall, the effects of CTGF on IOP appear to be caused by a modification of the TM actin cytoskeleton. CTGF-overexpressing mice provide a model that mimics the essential functional and structural aspects of POAG and offer a molecular mechanism to explain the increase of its most critical risk factor.

Cytokine concentration in aqueous humour of eyes with exudative age‐related macular degeneration

To measure the concentration of cytokines in the aqueous humour of eyes with exudative age-related macular degeneration (AMD). The clinical interventional study included a study group of 18 patients with exudative AMD and a control group of 20 patients undergoing routine cataract surgery. Age did not vary significantly (p = 0.36) between study group (80.8 ± 6.4 years) and control group (77.0 ± 9.9 years), nor did gender (p = 0.75). During the interventions, aqueous humour samples were obtained, in which the concentration of cytokines was measured using a solid-phase chemiluminescence immunoassay. Macular thickness was measured by optical coherence tomography (OCT). In the study group as compared to the control group, significantly higher concentrations were measured for epithelial growth factor (EGF) (p = 0.017), human growth factor (HGF) (p= 0.048), intercellular adhesion molecule-1 (ICAM1) (p = 0.028), interleukin 12p40 (IL12p40) (p = 0.009), interleukin 1a2 (IL1a2) (p = 0.01), interleukin 3 (IL3) (p = 0.02), interleukin 6 (IL6) (p = 0.006), interleukin 8 (IL8) (p = 0.02), monocyte chemoattractant protein-1 (MCP-1) (p = 0.048), monokine induced by interferon gamma (MIG) (p = 0.016), matrix metalloproteinase 9 (MMP9) (p = 0.004) and plasminogen activator inhibitor 1 (PAI1) (p = 0.006). Macular thickness was significantly associated with the concentrations of EGF (p = 0.001), HGF (p = 0.02), ICAM1 (p = 0.001), interleukin 12p40 (p = 0.006), IL 1a2 (p = 0.002), MIG (p = 0.001), MMP9 (p < 0.001) and PAI1 (p = 0.01). Interleukin 6 and MCP-1 showed significant associations with the height of retinal pigment epithelium detachment. Numerous cytokines are associated with the presence and the amount of exudative AMD.

Monocyte chemoattractant protein‐1 in the aqueous humour of patients with age‐related macular degeneration

To investigate the role of inflammation in age-related macular degeneration by measuring the levels of cytokines in the aqueous humour. Samples of aqueous humour were collected from 34 patients with age-related macular degeneration and 16 age-matched control subjects undergoing cataract surgery. Age-related macular degeneration stage was determined clinically, before surgery. Levels of cytokines were measured using Luminex X-MAP technology, and positive results were verified by Western blot. Age-related macular degeneration was moderate in 18 patients and advanced in 16. The advanced age-related macular degeneration group was further divided into patients with active choroidal neovascularization (n = 7), disciform scar (n = 7) or central geographic atrophy (n = 2). Higher-than-normal levels of monocyte chemoattractant protein-1 in the aqueous humour were associated with advanced age-related macular degeneration (200 ± 140 pg/mL vs. 100 ± 61 pg/mL; P = 0.03), especially active choroidal neovascularization (255 ± 155 pg/mL; P = 0.02), Western blot analysis verified the monocyte chemoattractant protein-1 findings. Patients with disciform scar showed a trend of abnormally high levels of interleukin-12 (p70) (1.7 ± 2.4 pg/mL vs. 0.2 ± 1 pg/mL; P = 0.07), tumour necrosis factor-α (1.8 ± 2.4 pg/mL vs. 0.3 ± 1 pg/mL; P = 0.06) and interleukin-12 (4.7 ± 6.4 pg/mL vs. 1.2 ± 2.1 pg/mL; P = 0.08). Elevated levels of inflammation-related cytokines in the aqueous humour in various stages of age-related macular degeneration may suggest a pathogenic role of inflammation. Monocyte chemoattractant protein-1 may be indicative of the angiogenic phase. Further corroborative studies are required.

Multiplex Cytokine Analysis of Aqueous Humor in Eyes with Primary Open-Angle Glaucoma, Exfoliation Glaucoma, and Cataract

To measure levels of various inflammatory cytokines in the aqueous humor of patients with primary open-angle glaucoma (POAG), exfoliation glaucoma (EXG), and senile cataract. Aqueous humor samples were obtained from 64 eyes of 64 Japanese subjects (POAG, 20 eyes; EXG, 23 eyes; and cataract, 21 control eyes). The levels of eight cytokines including interleukin (IL)1-β, IL-6, IL-8, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, serum amyloid A (SAA), migration inhibitory factor (MIF), and vascular endothelial growth factor (VEGF)-A were estimated using the multiplex bead immunoassay technique. Compared with the cataract group, the levels of TGF-β1, IL-8, and SAA were significantly higher in aqueous humor samples from the POAG (5.0-fold, 2.3-fold, and 11.9-fold, respectively) and EXG (12.5-fold, 4.0-fold, and 18.3-fold, respectively) groups. Except for a significant decrease in the IL-6 level in the POAG (0.23-fold) group, no other cytokine levels differed in the POAG and EXG groups compared with the cataract group. The levels of TGF-β1, IL-8, and SAA were positively correlated with each other (ρ = 0.723-0.786; P < 0.0001), the intraocular pressure (IOP) (ρ = 0.392-0.662; P < 0.0001-0.0019), and the number of glaucoma medications (ρ = 0.478-0.659; P < 0.0001-0.0001). Cytokine networks including TGF-β1, IL-8, and SAA in aqueous humor may have critical roles in IOP elevations in patients with open-angle glaucoma.

Increased Nitric Oxide and Vascular Endothelial Growth Factor Levels in the Aqueous Humor of Patients with Coats' Disease

To determine the aqueous humor levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the eyes of patients with Coats' disease and study the correlation between these levels. Samples of aqueous humor were obtained from 7 patients with Coats' disease and 15 age-matched patients with congenital cataracts as controls. Nitrite and nitrate (NOx), the stable end products of NO, were detected by the Griess reaction, and VEGF levels were assessed by enzyme-linked immunosorbent assay. The aqueous humor NOx and VEGF levels were elevated in the eyes of patients with Coats' disease compared with those of controls (P=0.001 and P<0.001, respectively). The median NOx level was 55.2 μM (range, 23.0-75.3 μM) in the Coats' disease group and 18.8 μM (range, 8.7-36.2 μM) in the control group. The median VEGF level was 731.7 pg/mL (range, 288.3-1364.3 pg/mL) in the Coats' disease group and 33.3 pg/mL (range, 9.0-96.8 pg/mL) in the control group. No correlation was observed between the aqueous humor concentrations of NOx and VEGF. NOx and VEGF are increased but not related in the aqueous humor samples of patients with Coats' disease. NO and VEGF may play roles in the pathogenesis of Coats' disease. Further studies are needed to clearly elucidate the relationship among VEGF, NO, and other cytokines in Coats' disease.

8-Isoprostaglandin F2a Levels in Aqueous Humor of Senile and Diabetic Cataract Patients

We investigated the concentrations of 8-isoprostaglandin F2a, a marker of oxidative stress in aqueous humor of patient's of senile cataract and compared the results with diabetic cataract to determine whether diabetic cataract is associated with increased oxidative stress. Aqueous humor was aspirated at the beginning of phacoemulsification cataract surgery from 25 eyes of 25 patients with senile cataract and 25 eyes of age matched diabetic cataract patients. 8-Isoprostaglandin F2a (8-IPGF) concentration in the aqueous was determined with an immunoassay method. The mean concentration of 8-isoprostaglandin F2a in the aqueous from patients with diabetic cataract i.e.624 ± 95.7 pg/ml (range 400-750 pg/ml), was significantly higher than that measured in the aqueous of age matched senile cataract i.e.190 ± 41.2 pg/ml ((range 100-290 pg/ml).8- ISoprostaglandin F2awas significantly increased in the aqueous of patients with diabetes, confirming the evidence of a role for free radical induced oxidative damage in the pathophysiology of diabetic cataract.. The antioxidative capacity of aqueous humor should next be investigated in these two types of cataracts.

CXC Chemokine Expression Profiles in Aqueous Humor of Patients with Different

AbstractPurpose To investigate the CXC chemokine expression profiles in the aqueous humor from patients with active uveitis associated with Behçet’s disease, VKH disease, and HLA‐B27‐related intraocular inflammation and to correlate.Methods Aqueous humor (AH) samples from patients with Behçet’s disease (BD) (n=29), Vogt‐Koyanagi‐Harada (VKH) disease (n=21), and HLA‐B27‐associated uveitis (n=8), and 42 control patients were assayed for the neutrophil chemoattractants CXCL1/GRO‐α and CXCL8/IL‐8 and the lymphocyte chemoattractants CXCL9/MIG, CXCL10/IP‐10 and CXCL12/SDF‐1 with the use of a multiplex chemokine assay.Results Chemokine levels except CXCL12/SDF‐1 were significantly higher in the 3 disease groups than in normal controls. Considering all patients, mean CXCL1/GRO‐α levels were 15‐fold higher than CXCL8/IL‐8 levels and mean CXCL10/IP‐10 levels were 22‐fold higher than CXCL9/MIG levels. In patients with the same disease activity, AH levels of CXCL1/GRO‐α and CXCL10/IP‐10 were significantly higher in patients with BD than in patients with VKH disease and HLA‐B27‐associated uveitis (p=0.0474; p&lt;0.001, respectively).Conclusion These data suggest that CXCL1/GRO‐α and CXCL10/IP‐10 are the predominant CXC chemokines involved in neutrophil and activated T lymphocyte chemoattraction in endogenous uveitis, particularly in BD.

Transforming growth factor-beta2 utilizes the canonical Smad-signaling pathway to regulate tissue transglutaminase expression in human trabecular meshwork cells

Transforming growth factor-beta2 (TGF-β2) is elevated in the aqueous humor of patients with glaucoma. This growth factor is known to increase extracellular matrix (ECM) deposition in the trabecular meshwork (TM) as well as increase intraocular pressure (IOP) in perfused human cultured anterior eye segments. In addition overexpression of TGF-β2 in the mouse TM leads to elevated IOP. Exogenous TGF-β2 also increases tissue transglutaminase (TGM2) protein levels and enzyme activity in TM cells. TGM2 is a calcium-dependent enzyme that mediates cross-linking of ECM proteins, thus making ECM proteins resistant to enzymatic degradation and physical breakdown. We have investigated the signaling pathway by which TGF-β2 induces TGM2 in human TM cells. Primary cultures of human TM cells ( N = 6) were treated for 48 h with TGF-β2 (0–10 ng/ml) in serum-free medium. TGM2 enzyme activity differences between non-treated and TGF-β2 treated TM cells were studied using a biotin cadaverine assay. Endogenous TGF-β2 protein levels were examined in normal trabecular meshwork (NTM) and glaucomatous trabecular meshwork (GTM) cell strains. Immunohistochemistry was used to evaluate the expression and co-localization of TGF-β2 and TGM2 in NTM and GTM tissues. Activation of Smad3 signaling pathway was evaluated by western immunoblot analysis using phospho-specific antibodies following exogenous TGF-β2 treatment. Pharmacological specific inhibitor of Smad3 (SIS3) and short interfering (si)RNAs were used to suppress Smad3 activity and CTGF gene expression respectively. Endogenous TGF-β2 levels were significantly elevated in cultured GTM cells ( p < 0.05) when compared to NTM cells. Immunohistochemistry studies also demonstrated elevated expression and co-localization of both TGF-β2 and TGM2 in glaucoma human TM tissues. Exogenous TGF-β2 increased both TGM2 protein levels and enzyme activity in TM cells. Phosphorylation of Smad3 was stimulated in TM cell strains by exogenous TGF-β2. TGF-β2 induction of TGM2 was not inhibited with selective siRNA knockdown of CTGF. In contrast, a specific inhibitor of Smad3 (SIS3) and siRNA knockdown of Smad3 ( p < 0.05) suppressed TGF-β2 induction of TGM2. This study demonstrated that TGF-β2 induction of TGM2 can be mediated via the canonical Smad-signaling pathway but does not appear to involve CTGF as a downstream mediator. Regulation of the Smad-signaling pathway in the TM may be useful in the therapy for glaucoma associated with aberrant TGF-β2 signaling.