Human Aqueous Humor Research Articles

Liquid chromatography–tandem mass spectrometry for rapid and selective simultaneous determination of fluoroquinolones level in human aqueous humor

Endophthalmitis, an intraocular infection, may lead to irreversible loss of vision. Antimicrobial chemotherapy is prescribed prior to ocular surgical procedures to avoid endophthalmitis. Fluoroquinolones are the most commonly prescribed and used antibiotics during such procedures. However, the selection of a single entity and proper regimen is not specified in medical guidelines. The objective of this study is to develop a rapid and selective simultaneous high-performance liquid chromatography-tandem mass spectroscopic method to explore the bioavailability of 4 fluoroquinolones, including 0.5% moxifloxacin hydrochloride, 0.3% gatifloxacin, 0.3% ciprofloxacin hydrochloride, and 0.3% ofloxacin, in human aqueous humor after antibiotic topical administration using gemifloxacin as Internal Standard according to the European Medicines Agency (EMA) and US Food and Drug Administration (FDA) guidelines. The newly validated method was capable of accurately and precisely quantifying each antibiotic at the lowest reported lower limit of quantification of 10 ng/mL and operating on a very low pipetting volume of 15 μL, indicating a reliable quantitation of all analytes simultaneously using a very small quantity of aqueous humor with total chromatographic run time of 2.5 min. Sixty-seven patients were divided into 4 groups for each antibiotic. Before the cataract surgery, each group received 4 drops of one of the antibiotics over 1 h, separated by 15 min time interval. After 15–20 min from the last drop, approximately 50–100 μL of aqueous humor was collected during surgery for analysis. The average concentrations of moxifloxacin, gatifloxacin, ofloxacin and ciprofloxacin in aqueous humor samples were 891.8, 271.7, 191.4 and 69.5 ng/mL, respectively. Only moxifloxacin average concentration was higher than the minimum inhibitory concentration for the common endophthalmitis pathogens.

Intraocular Oxygen and Antioxidant Status: New Insights on the Effect of Vitrectomy and Glaucoma Pathogenesis

The purpose of this study was to investigate correlations of partial pressure of oxygen (pO2) in the ocular anterior segment of human eyes and aqueous humor antioxidant levels of ascorbate (AsA) and total reactive antioxidant potential (TRAP) with glaucoma and vitreous status. This prospective, cross-sectional study stratified patients (n= 288 eyes) by lens and vitreous status and the presence of primary open-angle glaucoma for statistical analyses. Intraocular pO2 concentrations were measured using a fiberoptic probe in patients at the beginning of planned glaucoma and/or cataract surgery. Aqueous humor specimens were obtained for antioxidant analysis of AsA and TRAP. Following prior pars plana vitrectomy, pO2 levels were significantly higher than in the reference group of cataract surgery in the anterior chamber angle (16.2 ± 5.0 vs. 13.0 ± 3.9mm Hg; P= .0171) and in the posterior chamber (7.6 ± 3.1 vs. 3.9 ± 2.7mm Hg; P < .0001). AsA and TRAP levels were significantly lower (1.1 ± 0.4 vs. 1.4 ± 0.5mM, respectively; 403.3 ±116.5 vs. 479.0 ± 146.7 Trolox units, respectively; P= .004 and P= .024, respectively) in patients after vitrectomy. In patients with an intact vitreous, neither pO2 nor antioxidant status correlated with lens status or glaucoma. Increased pO2 and antioxidant depletion following vitrectomy suggests an alteration of the intraocular oxidant-antioxidant balance. Our study links physiologic factors such as increased pO2 in the anterior chamber angle and the posterior chamber to decreased antioxidant levels in aqueous humor following vitrectomy. Oxidative stress/damage to the trabecular meshwork in such post-vitrectomy cases may contribute to intraocular pressure elevation and increased risk of glaucoma. NOTE: Publication of this article is sponsored bythe American Ophthalmological Society.

Effects of astaxanthin on VEGF level and antioxidation in human aqueous humor: difference by sex.

In our previous report, we showed the effect of astaxanthin intake on VEGF level in the aqueous humor and the relationship between VEGF level and reactive oxygen species-related parameters and other relevant factors. VEGF level is associated with total hydroperoxide level, and a multivariate analysis identified sex as a secondary factor affecting these relationships. Here, we analyzed the effects of astaxanthin on the relationship between VEGF level and reactive oxygen species-related parameters by sex. Patients (16 males and 19 females, aged 71.3 and 70.6, respectively) underwent bilateral cataract surgery on one side before and the other side after astaxanthin treatment (6 mg/day for 2 weeks). Levels of VEGF, hydrogen peroxide, and total hydroperoxide, and O2•− scavenging activity, were measured in the aqueous humor. In females only, VEGF level was negatively correlated with O2•− scavenging activity before the astaxanthin intake (r = −0.6, p<0.01) and positively correlated with total hydroperoxide level before and after the astaxanthin intake (r = 0.7 and 0.8, respectively, p<0.01). In conclusion, astaxanthin appears to affect O2•− scavenging activity in the aqueous humor in females, and is likely to be involved in the control of VEGF levels in the anterior eye.

Liquid chromatography-tandem mass spectrometry for monitoring vitamin D hydroxymetabolites in human aqueous humor

A stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 25-hydroxyvitamin D3/D2 (25(OH)D3/D2) in human aqueous humor samples.

In Vivo Stability Profiles of Anti-factor D Molecules Support Long-Acting Delivery Approaches.

The collection of aqueous humor (phase 1 b/2 Mahalo study) from patients dosed intravitreally with anti-factor D (AFD; FCFD4514S, lampalizumab), a humanized antibody fragment previously under investigation to treat geographic atrophy (GA) secondary to age-related macular degeneration, presented a unique opportunity to examine AFD properties in clinical samples. We investigated AFD stability and target-binding characteristics to set up strategies for engineering and evaluating optimized molecules that enable less frequent dosing. Two variants, AFD.v8 and AFD.v14, were evaluated as alternatives to AFD for longer-acting treatments. Mass spectrometry, surface plasmon resonance, and immunoassay were used to assess AFD stability and binding activity in aqueous humor samples from Mahalo patients. In vitro stability and binding activity of AFD, AFD.v8, and AFD.v14 were assessed in human vitreous humor versus buffer at 37 °C over 16 weeks and in vivo in rabbits over 28 days along with pharmacokinetic determinations. In human aqueous humor, AFD specific binding was >85% through 30 days, and deamidation was <3% through 60 days, consistent with the AFD stability and binding activity in vitreous humor from humans in vitro and rabbits in vivo. Target binding, stability, and rabbit pharmacokinetic parameters of AFD.v8 and AFD.v14 were similar to those of AFD. Physiological stability and activity of AFD translated across in vitro and in vivo studies in humans and rabbits. The two variants AFD.v8 and AFD.v14 demonstrated comparable potency and pharmacokinetics. These findings, along with previously demonstrated improved solubility of AFD.v8 and AFD.v14, provide proof-of-concept for developing other similar long-acting therapeutic variants.

Bioequivalence Study Methods with Pharmacokinetic Endpoints for Topical Ophthalmic Corticosteroid Suspensions and Effects of Subject Demographics.

To establish bioequivalence for topical ophthalmic corticosteroid suspensions, some of U.S. product-specific guidances (PSGs) for generic drug products recommend evaluation of aqueous humor (AH) pharmacokinetics (PK). However, the AH PK study is complex because the relationships among AH PK, subject demographics, ocular anatomy, physiology and the compounds' physicochemical characteristics are not well understood. The objective of this research is to provide an overview of the in vivo human AH studies submitted to the U.S. Food and Drug Administration (FDA) for ophthalmic corticosteroid suspensions and to investigate the impact of subject demographics on the human AH PK. We summarized demographic data, sampling time points, sample size per time point and PK parameters to investigate correlations in the studies submitted to the FDA. In the evaluation of subject-specific covariates, the area under the concentration-time curves (AUC) and maximum concentrations (Cmax) were significantly different among ethnicities and age groups. Gender was not primarily associated with differences in AH PK. Our results suggest that the difference in ethnicity and age of the study population play an important role in the AH PK profiles of topical ophthalmic corticosteroid suspensions. Considering the subject-specific covariate effects in designing bioequivalence studies with AH PK endpoints could reduce bias from covariate imbalance and help identify true effects of formulation differences.

Analytical convergence in surrogate control matrices.

Analytical convergence in surrogate control matrices.

Validation of direct method to quantify dexamethasone in human aqueous humor by LC-MS/MS.

Dexamethasone (Dex) has been used for the treatment of ocular diseases, presenting concentrations up to 150ng/ml in the biological matrix. Drug sampling has been performed from aqueous humor (AH), which the volume varies from 50 to 200μl, becoming a challenge for analytical analyses. We developed and validated a direct and sensitive method by LC-MS/MS for Dex measurement in human AH, using water as surrogate matrix to reduce the sample volume applied in some validation assays. With accuracies of 99.6% and precision within 15% in the range of 0.1-150ng/ml, Dex had an 87-day stability. Our method is robust and sensitive enough to be applied in bioequivalence studies with human AH using only 20μl of biological sample.

Induction of oxidative stress in human aqueous and vitreous humors by Nd:YAG laser posterior capsulotomy.

To evaluate whether the Q-switched Nd:YAG laser treatment applied in routine capsulotomy elicits oxidative stress in aqueous and vitreous humors. Thirty-six patients who had to undergo a 25 gauge pars plana vitrectomy due to vitreoretinal disorders were enrolled, 15 of them underwent a Q-switched Nd:YAG laser capsulotomy 7d before vitrectomy due to posterior capsule opacification (PCO) (Nd:YAG laser group) while the remaining 21 patients were not laser treated before vitrectomy (no Nd:YAG laser group). Samples of the aqueous and vitreous humors were collected during vitrectomy from all patients for the assessment of oxidative parameters which were compared between the Nd:YAG laser group and no Nd:YAG laser group. Thiobarbituric acid reactive substances (TBARS), a product of membrane lipid peroxidation, nitrite levels, the antioxidative activities of SOD and catalase, the 4-HNE-protein conjugate formation, indicating structural modifications in proteins due to lipoperoxidation, were assessed in aqueous and vitreous samples. In the human vitreous humor TBARS levels are significantly higher in the Nd:YAG laser group compared to the no Nd:YAG laser group and importantly, there is a significant correlation between the TBARS levels and the total energy of Nd:YAG laser used during capsulotomy. Moreover the anti-oxidative activities of SOD and catalase were significantly decreased by Nd:YAG laser treatment, both in aqueous and vitreous humors. In accordance with the TBARS data and anti-oxidative enzyme activities, significantly higher levels of proteins were conjugated with the lipoperoxidation product 4-HNE in the aqueous and vitreous humors in the Nd:YAG laser-treated group in comparison to no Nd:YAG laser group. These data, clearly suggest that any change that Q-switched Nd:YAG photo disruption may cause in the aqueous and vitreous compartments, resulting in a higher level of oxidative damage might be of considerable clinical significance particularly by accelerating the aging of the anterior and posterior segments of the eye and by worsening the intraocular pressure, the uveal, the retinal (especially macular) pathologies.

Leucine-rich G Protein-coupled Receptor-5 Is Significantly Increased in the Aqueous Humor of Human Eye with Proliferative Diabetic Retinopathy.

Leucine-rich G protein-coupled receptor-5 (LGR5) is known to be a stem cell marker in many organs. LGR5 may have important roles in proliferative diabetic retinopathy (PDR) because LGR5 potentiate the Wnt/β-catenin pathway, which plays crucial roles in pathologic neovascularization in the retina. The association between LGR5 and retinal pathologic neovascularization has not yet been reported. In the present study, LGR5 was compared in human aqueous humor (AH) between normal control and patients with PDR to confirm the relationship between LGR5 and PDR. AH was collected from 7 naïve PDR patients and 3 control subjects before intravitreal injection and cataract surgery, respectively. LGR5 and key members of Wnt/β-catenin were assessed by western blotting. In the present study, it was confirmed for the first time that LGR5 is detected in AH and it increases in PDR patients. Key members of Wnt/β-catenin pathway were also increased in AH of PDR patients compared to control. These findings might support the hypothesis that LGR5 has important roles in PDR especially considering the roles of the Wnt/β-catenin pathway, which is activated by LGR5, contributing to retinal pathologic neovascularization.

Activation of human macrophages by human corneal allogen in vitro.

PurposeTo study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection.MethodsHuman monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFNγ), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively.ResultsCorneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1α and MIP-1β, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation.ConclusionsHuman macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.

Metabolic characterization of human aqueous humor in the cataract progression after pars plana vitrectomy

BackgroundWhile pars plana vitrectomy (PPV) has become the third most commonly performed surgery in the world, it can also induce multiple post complications easily. Among them, cataract progression is the most frequent one that can lead to blindness eventually.MethodsTo understand the underlying mechanisms of post PPV cataract progression, we performed comprehensive metabolic characterization of aqueous humor (AH) samples from 20 cataract patients (10 post PPV complication and 10 none PPV cataract) by a non-targeted metabolomic analysis using gas chromatography combined with time-of-flight mass spectrometer (GC/TOF MS).ResultsA total of 263 metabolites were identified and eight of them are determined to be significantly different (VIP ≥ 1 and p ≤ 0.05) between post PPV group and none PPV control group. The significantly changed metabolites included glutaric acid and pelargonic acid that play key roles in the regulation of oxidative stress and inflammatory responses. Furthermore, we constructed a metabolic regulatory network in each group based on metabolite-metabolite correlations, which reveals key metabolic pathways and regulatory elements including amino acids and lipids metabolisms that are related to cataract progression.ConclusionsAltogether, this work discovered some potential metabolite biomarkers for post PPV cataract diagnostics, as well as casted some novel insights into the underlying mechanisms of cataract progression after PPV.

Full-length myocilin protein is purified from mammalian cells as a dimer

Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15 mg/L) while the second method involved a purification approach from 293 cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293 cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293 cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.

Age-related pro-inflammatory and pro-angiogenic changes in human aqueous humor.

To reveal age-related aqueous cytokine changes in human aqueous humor. Aqueous humor was collected from 12 young children (3-6.5 years old) and 71 healthy adults (22-106 years old) with cataract but without other systemic or ocular disorders. Levels of 22 cytokines, chemokines and vascular endothelial growth factor (VEGF) were measured and analyzed. The following proteins showed significant increase from childhood to adult: interferon-gamma (IFN-γ), interleukin (IL)-13, IL-6, IL-12(p70), IL-10, CCL2, CCL3, CCL4, CXCL8, CXCL9, CXCL10, IFN-α2 and VEGF (all P<0.05). IFN-γ, IL-13, IL-12(p70), IL-10, CCL3, CXCL9 and VEGF also showed moderate strength age-related increase in the adult group (r>0.5). The strength of correlation between aging and CCL4 were fair (r=0.398). The concentrations of IL-2, IL-4, IL-5, IL-1β and TNF-α were low in both groups. From childhood to adult, the immunological milieu of the anterior chamber become more pro-inflammatory and pro-angiogenic. Such changes may represent the parainflammation state of the human eye.

Gender differences in mRNA expression of aquaporin 8 and glutathione peroxidase in cataractous lens following intake of an antioxidant supplement

Antioxidants can decrease oxidative damage and prevent age-related ocular disease. Our previous investigation on human aqueous humor following intake of a lutein-containing antioxidant supplement reported an increase in the scavenging activity of superoxide in both genders and an increase in the amount of hydrogen peroxide (H2O2) in females. Aquaporin 8 (AQP8) is a diffusion facilitator of H2O2 and glutathione peroxidase (Gpx) is a H2O2 scavenging enzyme. The correlation between AQP8 and Gpx may be the key to determining how oxidative stress in the aqueous humor affects the lens after intake of antioxidant supplements. In this study, 24 patients with the same grade of binocular cataract were included. Anterior capsule samples, including lens epithelial cells (LECs), were collected during cataract surgery before (as pre-intake samples) and after 6 weeks of oral intake of Ocuvite Lutein ® (as post-intake samples). The mRNA expression of APQ8 and Gpx was measured using real-time polymerase chain reaction. Among males, AQP8 expression decreased significantly after the supplementation (P = .03), while there was no statistical change among females. AQP8 expression was significantly correlated to that of Gpx in post-intake samples among females (R = 0.69, P = .02), while no correlation was evident among males. The results suggest antioxidant supplementation may work by different mechanisms on LECs between genders. After supplementation, a decrease in AQP8 in LECs may inhibit the influx of H2O2 from the aqueous humor in males. In females however, the correlation between AQP8 and Gpx in LECs may indicate an increase in Gpx activity following the influx of H2O2 from the aqueous humor and further scavenging of H2O2.

A novel multiplex detection array revealed systemic complement activation in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common tumors within the oral cavity. Early diagnosis and prognosis tools are urgently needed. This study aimed to investigate the activation of the complement system in OSCC patients as potential biomarker. Therefore, an innovative complement activation array was developed.Characterized antibodies detecting the complement activation specific epitopes C3a, C5a and sC5b-9 along with control antibodies were implemented into a suspension bead array. Human serum from a healthy (n = 46) and OSCC patient (n = 57) cohort were used to investigate the role of complement activation in oral tumor progression. The novel multiplex assay detected C3a, C5a and sC5b-9 from a minimal sample volume of human tears, aqueous humor and blood samples. Limits of detection were 0.04 ng/mL for C3a, 0.03 ng/mL for C5a and 18.9 ng/mL for sC5b-9, respectively. Biological cut-off levels guaranteed specific detections from serum. The mean serum concentration of a healthy control cohort was 680 ng/mL C3a, 70 ng/mL C5a and 2247 ng/mL sC5b-9, respectively. The assay showed an intra-assay precision of 2.9–6.4% and an inter-assay precision of 9.2–18.2%.Increased systemic C5a (p < 0.0001) and sC5b-9 (p = 0.01) concentrations in OSCC patients were determined using the validated multiplex complement assay. Higher C5a concentrations correlated with tumor differentiation and OSCC extension state. Systemic sC5b-9 determination provided a novel biomarker for infiltrating tumor growth and C3a levels were associated with local tumor spreading.Our study suggests that systemic complement activation levels in OSCC patients may be useful to assess disease progression.

Quantitative Proteomic Analysis of Human Aqueous Humor Using iTRAQ 4plex Labeling.

iTRAQ 4plex method enables multiplexing of up to four samples for simultaneous quantitation to improve sensitivity and scope of proteomic analysis. Here, we describe iTRAQ 4plex labeling of human aqueous humor specimens followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and data analysis for peptide identification and quantitation using Proteome Discoverer software. The protocol can be applied for other animals as well; however, pooling of specimens may be required to obtain sufficient amount of protein for labeling.

Clinical implication of triamcinolone in aqueous humor after depository sub-Tenon application

To determine the triamcinolone concentration in the aqueous humor after anterior sub-Tenon injection versus posterior sub-Tenon injection and the possible association between the levels of triamcinolone in the aqueous and the elevation in intraocular pressure (IOP). University teaching hospitals at Wenzhou and Shanghai, China. Prospective case series. The aqueous humor was sampled and triamcinolone quantitated after anterior sub-Tenon injection or posterior sub-Tenon injection in a cohort of patients and in experimental rabbits. Patients' IOPs were measured before and up to 3months after triamcinolone acetonide injection. The study comprised 46 patients and 18 rabbits. The mean aqueous humor triamcinolone concentration in rabbit eyes and human eyes was significantly higher after an anterior sub-Tenon injection than that after a posterior sub-Tenon injection (rabbit eye: anterior sub-Tenon injection, 2.67ng/mL±0.78 [SD] and posterior sub-Tenon injection 1.38±0.48ng/mL; human eye: 133.8±149.2ng/mL and 10.2±10.9ng/mL, respectively) (both P<.0001). After anterior sub-Tenon injection, the mean triamcinolone concentration in the aqueous humor was significantly higher in human eyes than in rabbit eyes (133.8±149.2ng/mL versus 11.0±8.1ng/mL) (P=.0003). The levels of triamcinolone in the human aqueous humor had a significant positive association with the elevation in IOP (β=0.12, P=.026). The triamcinolone level in aqueous humor was associated with IOP elevation after sub-Tenon application. Posterior sub-Tenon injection with a small injection volume ofconcentrated suspension offered a better targeted delivery and might significantly reduce steroid-associated IOP elevation.

Quantitative metabolomic analysis of the human cornea and aqueous humor

Introduction Cornea is the outermost part of the eye supplied mostly by aqueous humor (AH). Therefore, the comparison of the metabolomic compositions of AH and cornea may help to determine which compounds are produced inside the cornea, and which penetrate into cornea from AH for intra-corneal consumption. Keratoconus (KC) is the most common form of the cornea dystrophy, and the analysis of KC corneas can unravel the metabolomic changes occurring in AH and cornea of KC patients.

Human aqueous humor levels of transforming growth factor-β2: Association with matrix metalloproteinases/tissue inhibitors of matrix metalloproteinases

The present study aims to investigate the association of transforming growth factor-β2 (TGF-β2) and matrix metalloproteinases (MMPs), MMP-2 and MMP-3, and tissue inhibitors of matrix metalloproteinases (TIMPs), TIMP-1, TIMP-2 and TIMP-3 in the aqueous humor of patients with high myopia or cataracts. The levels of TGF-β2 and MMPs/TIMPs were measured with the Luminex xMAP Technology using commercially available Milliplex xMAP kits. The association between TGF-β2 and MMPs/TIMPs levels was analyzed using the Spearmans correlation test. The levels of TGF-β2 were identified to be positively correlated with the levels of TIMP-1 and TIMP-3 (TIMP-1: r=0.334; P=0.007; TIMP-3: r=0.309; P=0.012). The levels of MMP-2, MMP-3 and TIMP-2 did not significantly correlate with TGF-β2 levels (P>0.05). A positive correlation was identified between TGF-β2 and TIMPs in the aqueous humor of human eyes with elongated axial length. It appears that TGF-β2 stimulates the expression of TIMPs as a compensatory reaction to the development of high myopia.