Aqueous Humor Levels Research Articles

α-Klotho prevents diabetic retinopathy by reversing the senescence of macrophages

BackgroundDiabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus (DM) and a significant cause of acquired blindness in the working-age population worldwide. Aging is considered as an important risk factor for DR development. Macrophages in aged mice bear typical M2 marker proteins but simultaneously express a pro-inflammatory factor profile. This may explain why the level of intraocular inflammation does not decrease during proliferative diabetic retinopathy (PDR) despite the occurrence of neovascularization and fibrosis (M2 activation). α-Klotho (KL) was originally discovered as a soluble anti-aging factor, which is mainly expressed in kidney tubular epithelium, choroid plexus in the brain and secreted in the blood. However, the role of KL in DR pathophysiology has not been previously reported.MethodsType 1 (streptozotocin [STZ]-induced) and type 2 (a high-fat diet along with a low dose of STZ) diabetic mouse models were established and injected with or without KL adenovirus via the tail vein for 12 weeks. Vldlr−/− mice were injected intravitreally with or without soluble KL protein from P8 to P15. The retinal structure and function were analyzed by electroretinogram and optical coherence tomography. The neovascular lesions were analyzed by retinal flat mount and RPE flat mount. The senescence markers, macrophage morphology, and KL expression levels were detected by immunofluorescence staining. A cell model was constructed using RAW264.7 cells stimulated by 4-hydroxynonenal (4HNE) and transfected with or without KL adenovirus. The senescence-associated secretory phenotypes were detected by qRT-PCR. Senescence was detected by SA-β-Gal staining. Serum, aqueous humor, and vitreous humor KL levels of proliferative diabetic retinopathy (PDR) patients were measured by enzyme‐linked immunosorbent assay. Quantitative proteomics and bioinformatics were applied to predict the change of proteins and biological function after overexpression of KL in macrophages. The effects of KL on the HECTD1 binding to IRS1 were analyzed by bioinformatics, molecular docking, and Western Blot.ResultsSerum, aqueous humor, and vitreous humor KL levels were lower in patients with PDR than in those with cataracts. KL relieved the retinal structure damage, improved retina function, and inhibited retinal senescence in diabetic mice. KL administration attenuated the neovascular lesions in VLDLR−/− mice by decreasing the secretion of VEGFA and FGF2 from macrophages. KL also protected RAW264.7 cells from 4HNE-induced senescence. Additionally, it inhibited E3 ubiquitin ligase HECTD1 expression in both diabetic mouse peripheral blood mononuclear cells and 4HNE-treated RAW264.7 cells. KL inhibited HECTD1 binding to IRS1 and reduced the ubiquitination of IRS1.ConclusionsMacrophage aging is involved in DM-induced retinopathy. KL alleviates DM-induced retinal macrophage senescence by downregulating HECTD1 and decreasing IRS1 ubiquitination and degradation. Meanwhile, KL administration attenuated the neovascular lesions by altering the activation state of macrophages and decreasing the expression of VEGFA and FGF2.

Anterior Chamber Flare as a Non-Invasive Assessment of Intraocular Immune Status and Ocular Complications in Proliferative Diabetic Retinopathy.

Proliferative diabetic retinopathy (PDR) is a vision-threatening complication of diabetes mellitus (DM). Anterior chamber (AC) flare and intraocular cytokines are potent biomarkers reflecting the intraocular immune status in PDR. This study aimed to elucidate the complex interrelationship between AC flare and intraocular cytokines in PDR eyes. A retrospective observational study was conducted on 19 PDR eyes of 19 patients with type 2 DM, and on 19 eyes of 19 patients with idiopathic macular hole or epiretinal membrane as controls. AC flare was measured before pars plana vitrectomy (PPV). Aqueous humor (AH) and vitreous fluid (VF) samples were collected at the time of PPV, and the quantities of 27 cytokines in both intraocular fluids were analyzed. In the PDR and control groups, Spearman's rank correlation analysis revealed a positive correlation between AC flare and IL-8 level in both AH and VF. Additionally, IL-8 levels in AH correlated positively with IL-8 levels in VF. In the PDR group, receiver operating characteristic curve analysis identified IL-8 level in AH as a significant predictor for both diabetic macular edema (DME) and vitreous hemorrhage (VH) complications. The cut-off values of IL-8 were established at ≥26.6 pg/mL for DME and ≥7.96 pg/mL for VH. Given the positive correlation between AC flare and AH IL-8 level, the present findings suggest that AC flare value may potentially be a non-invasive biomarker for predicting DME.

Proteomic Analysis of Aqueous Humor in Central Retinal Artery Occlusion: Unveiling Novel Insights Into Disease Pathophysiology.

Central retinal artery occlusion (CRAO) is an ocular emergency that results from acute blockage of the blood supply to the retina and leads to a sudden vision loss. Other forms of ischemic retinopathies include diabetic retinopathy (DR), which involves chronic retinal ischemia and remains the leading cause of blindness in working-age adults. This study is the first to conduct a proteomic analysis of aqueous humor (AH) from patients with CRAO with a comparative analysis using vitreous humor (VH) samples from patients with DR. AH samples were collected from 10 patients with CRAO undergoing paracentesis and 10 controls undergoing cataract surgery. VH samples were collected from 10 patients with DR and 10 non-diabetic controls undergoing pars plana vitrectomy (PPV). Samples were analyzed using mass spectrometry. Compared with controls, AH levels of 36 differentially expressed proteins (DEPs) were identified in patients with CRAO. Qiagen Ingenuity Pathway Analysis (IPA) revealed 11 proteins linked to ophthalmic diseases. Notably, enolase 2, a glycolysis enzyme isoform primarily expressed in neurons, was upregulated, suggesting neuronal injury and enzyme release. Additionally, clusterin, a protective glycoprotein, was downregulated. ELISA was conducted to confirm proteomics data. VH samples from patients with DR exhibited changes in a distinct set of proteins, including ones previously reported in the literature. The study provides novel insights into CRAO pathophysiology with multiple hits identified. Proteomic results differed between DR and CRAO studies, likely due to the different pathophysiology and disease duration. This is the first proteomic analysis of CRAO AH, with the potential to identify future therapeutic targets.

Cytokine analysis of aqueous humor in patients with cytomegalovirus corneal endotheliitis.

To evaluate cytokine levels of aqueous humor in patients with cytomegalovirus (CMV) corneal endotheliitis and their relationships with CMV DNA load. 44 aqueous humor samples were obtained from 26 patients with CMV corneal endotheliitis at various stages of treatment. 33 samples obtained from cataract patients during the same period were selected as a control group. Each sample was used to measure the concentration of the CMV DNA load using real-time quantitative polymerase chain reaction, and to examine the levels of IL-6, IL-8, IL-10, MCP-1, VCAM-1, VEGF, IP-10, G-CSF, ICAM-1 and IFN-γ using a cytometric bead array. All 10 cytokines were found to have statistically significant differences between the CMV endotheliitis and cataract groups. The Spearman correlation test showed that the concentration of CMV DNA load was significantly associated with the levels of IL-6 (P = 0.005, r = 0.417), IL-8 (P < 0.001, r = 0.514), IL-10 (P < 0.001, r = 0.700), MCP-1 (P = 0.001, r = 0.487), VEGF (P < 0.001, r = 0.690), IP-10 (P = 0.001, r = 0.469), G-CSF (P < 0.001, r = 0.554) and ICAM-1 (P < 0.001, r = 0.635), but not significantly associated with VCAM-1 (P = 0.056) and IFN-γ (P = 0.219). There was a combined innate and adaptive immune response in aqueous humor in patients with CMV endotheliitis. Levels of multiple cytokines were significantly correlated with viral particle. Cytokines are potential indicators to help diagnose CMV endotheliitis, evaluate disease activity and assess treatment response.

Pigment Dispersion Contributes to Ocular Immune Privilege in a DBA/2J Mouse Model of Pigmentary Glaucoma.

To investigate the effects of anterior chamber pigment dispersion on ocular immune privilege and the possible mechanisms involved in a DBA/2J mouse model of pigmentary glaucoma. DBA/2J mice were utilized as a pigment dispersion model, and age-matched C57BL/6J mice were used as the control group in this study. Proteins in the aqueous humor (AH) and serum were quantified using the bicinchoninic acid assay. Immune cells in the AH were detected using hematoxylin and eosin staining and immunocytochemistry. The expression of TGF-β2 in the AH and cytokine levels (IL-10, IFN-γ) in serum were measured using ELISA. Anterior chamber-associated immune deviation (ACAID) was induced in DBA/2J mice by injecting antigens into the anterior chamber. Delayed-type hypersensitivity (DTH) assays were used to assess the induction of ACAID. In DBA/2J mice, before and after pigment dispersion, following anterior chamber injection of pigment particles, and after ACAID modeling, the expression of regulatory T cells (Tregs) was detected using flow cytometry. Compared to C57BL/6J mice, the protein concentration, immune cell count, and TGF-β2 levels in the AH were elevated in DBA/2J mice. Protein concentration and IL-10 levels in serum were increased, while IFN-γ levels were decreased in DBA/2J. Additionally, the expression of Treg cells in the spleen of DBA/2J mice was significantly increased after pigment dispersion and anterior chamber injection of pigment particles. At 3 and 6 months, DTH responses in DBA/2J mice were not inhibited, thus preventing ACAID induction. However, the opposite was observed at 9 months in DBA/2J mice. Furthermore, the ACAID group exhibited an augmented expression of Treg cells. Dispersion of pigment particles in the anterior chamber of the eye enhances the state of ocular immune privilege by influencing the immunosuppressive microenvironment and inducing more Treg cells to reestablish ACAID.

Levels of asymmetric dimethylarginine in plasma and aqueous humor: a key risk factor for the severity of fibrovascular proliferation in proliferative diabetic retinopathy.

Proliferative diabetic retinopathy (PDR) is a common diabetes complication, significantly impacting vision and quality of life. Previous studies have suggested a potential link between arginine pathway metabolites and diabetic retinopathy (DR). Connective tissue growth factor (CTGF) plays a role in the occurrence and development of fibrovascular proliferation (FVP) in PDR patients. However, the relationship between arginine pathway metabolites and FVP in PDR remains undefined. This study aimed to explore the correlation between four arginine pathway metabolites (arginine, asymmetric dimethylarginine[ADMA], ornithine, and citrulline) and the severity of FVP in PDR patients. In this study, plasma and aqueous humor samples were respectively collected from 30 patients with age-related cataracts without diabetes mellitus (DM) and from 85 PDR patients. The PDR patients were categorized as mild-to-moderate or severe based on the severity of fundal FVP. The study used Kruskal-Wallis test to compare arginine, ADMA, ornithine, and citrulline levels across three groups. Binary logistic regression identified risk factors for severe PDR. Spearman correlation analysis assessed associations between plasma and aqueous humor metabolite levels, and between ADMA and CTGF levels in aqueous humor among PDR patients. ADMA levels in the aqueous humor were significantly greater in patients with severe PDR than in those with mild-to-moderate PDR(P=0.0004). However, the plasma and aqueous humor levels of arginine, ornithine, and citrulline did not significantly differ between mild-to-moderate PDR patients and severe PDR patients (P>0.05). Binary logistic regression analysis indicated that the plasma (P=0.01) and aqueous humor (P=0.006) ADMA levels in PDR patients were risk factors for severe PDR. Furthermore, significant correlations were found between plasma and aqueous humor ADMA levels (r=0.263, P=0.015) and between aqueous humor ADMA and CTGF levels (r=0.837, P<0.001). Elevated ADMA levels in plasma and aqueous humor positively correlate with the severity of FVP in PDR, indicating ADMA as a risk factor for severe PDR.

The Consistency of Anti-Toxocara IgG Between the Aqueous Humor and Vitreous of Patients With Clinically Suspected Ocular Toxocariasis

To assess the consistencies of anti-Toxocara IgG (T-IgG) and Goldmann-Witmer coefficient (GWC) between paired aqueous humor (AH) and vitreous samples from patients with clinically-suspected ocular toxocariasis (OT). Inter-test reliability assessment. A total of 47 patients with clinically-suspected OT who underwent vitrectomy were included. AH, vitreous and serum samples from each patient were collected and levels of specific T-IgG in them were detected. The association and agreement of T-IgG and GWC between AH and vitreous were evaluated. The area under the receiver operating characteristic curve (AUROC) was generated to assess the diagnostic performance of AH. The T-IgG levels and GWC values in vitreous were higher than those in AH (P = 0.023 and P = 0.029, respectively), but similar positivity rates in the T-IgG (P = 1.000) and GWC>3 (P = 1.000) were apparent between vitreous and AH. In addition, there was a positive correlations between the AH and vitreous T-IgG levels (rs = 0.944, P < 0.001) and the GWC values (rs = 0.455, P = 0.022). Moreover, the consistencies between AH and vitreous samples in their T-IgG and GWC positivity rates were almost perfect (both, κ = 0.915, 95% CI, 0.799-1.000) in both. The AUROC reached 0.991, with a 95% confidence interval of 0.971-1.000. The best cut-off value for accurate OT diagnosis was found at 1.434, yielding 96% sensitivity and 100% specificity. Our findings demonstrate that AH and vitreous samples had significant correlations and perfect agreements for both T-IgG and GWC, suggesting that the AH may serve as a proxy for vitreous to provide a safer, earlier, and more convenient screening of OT.

Changes of aqueous humor cytokine profiles of patients with high intraocular pressure after PPV for retinal detachment

High intraocular pressure (IOP) is one of the early complications after pars plana vitrectomy (PPV), which may cause glaucoma and poor visual prognosis secondary to surgery. Proliferative vitreoretinopathy (PVR) is one of the complications of retinal detachment (RD) and is the main reason for the poor prognosis, which is related to different kinds of cytokines. It’s essential for the basic mechanism to analyze the relative aqueous humor cytokine profiles with IOP after PPV for RD. In this study, we have collected the aqueous humor of 16 patients and qualified 27 cytokines using Luminex and compared biomarkers with the high IOP group and the normal group. As a result, the concentrations of VEGF, IL-6, FGF2, and G-CSF upregulated significantly (P < 0.05), while VEGFR2 downregulated significantly (P < 0.05) in the high IOP group. IL-6 was positively correlated with high IOP (r = 0.561, P = 0.041). Meanwhile, the concentrations of IL-6 (r = 0.543, P = 0.03), IL-5 (r = 0.576, P = 0.019), IL-15 (r = 0.614, P = 0.011), IL-4 (r = 0.517, P = 0.04), ICAM-1 (r = 0.611, P = 0.012), and G-CSF (r = 0.636, P = 0.008) were significantly associated with preoperative PVR classification, and the aqueous humor levels of IL-4 (r = 0.567, P = 0.022), HGF (r = 0.701, P = 0.005), and MCP-1 (r = 0.565, P = 0.035) are significant relative to laser points. Hence, cytokines might potentially be the therapeutic target of high IOP after PPV.

Association between primary angle closure glaucoma and uric acid levels in serum and aqueous humor

PurposeTo evaluate abnormalities in serum and aqueous humor uric acid (UA) levels in primary angle closure glaucoma (PACG). MethodsPatients with PACG and age-similar and gender-similar controls (patients scheduled for cataract extraction) were enrolled prospectively. Serum UA levels were determined by enzymatic colorimetry; aqueous humor UA levels by Enzyme-Linked ImmunoSorbent Assay. A t-test was used to compare UA levels between PACG patients and controls, with one-way ANOVA used to compare levels across PACG subgroups with differing disease severity. Comparisons between PACG patients and controls were adjusted for systemic and ocular confounding factors using binary logistic regression. ResultsIn all, 131 PACG patients and 112 controls were included. The serum UA level was 266 ± 69 μmol/L in the PACG group and 269 ± 73 μmol/L in the control group (p = 0.71). The aqueous humor UA level was 35.4 ± 8.2 μmol/L in the PACG group and 53.9 ± 18.6 μmol/L in the control group (p < 0.001). This difference remained significant after adjusting for age, gender, systolic blood pressure, diastolic blood pressure, body mass index, axial length, central corneal thickness, anterior chamber depth, lens thickness, white-to-white distance, corneal endothelial cell density, and serum UA level (odds ratio: 0.88, 95 % confidence interval: 0.83–0.93, p < 0.001). ConclusionAqueous humor UA levels differ between PACG patients and controls, but serum UA levels do not. This indicates that local UA plays a role in the pathogenesis of PACG, but systemic UA does not.

MicroRNA expression profiling in tears and blood as predictive biomarkers for anti-VEGF treatment response in wet age-related macular degeneration.

This study aimed to investigate the potential of microRNAs (miRNAs) in tears, blood, and aqueous humor as biomarkers for predicting treatment response in wet age-related macular degeneration (AMD) patients undergoing anti-vascular endothelial growth factor (anti-VEGF) therapy. In a single-center prospective cohort study, treatment-naïve wet AMD patients and age-matched controls were enrolled. Clinical data and miRNA levels (miR-199a-3p, miR-365-3p, miR-200b-3p, miR-195-5p, miR-335-5p, and miR-185-5p) in tears, blood, and aqueous humor were collected. Treatment response was categorized into responders and non-responders based on visual acuity and central subfield thickness. MiRNA levels were quantified using reverse-transcription PCR. Statistical analyses were performed, including ROC analysis, to evaluate predictive accuracy. Dysregulated miRNA profiles were observed in wet AMD tears and blood compared to controls. Specifically, miR-199a-3p, miR-195-5p, and miR-185-5p were upregulated, while miR-200b-3p was downregulated in tears. All six miRNAs were elevated in wet AMD blood samples. Notably, responders showed higher tear expression of miR-195-5p and miR-185-5p. Combining these miRNAs yielded the highest predictive power (AUC = 0.878, p = 0.006) for anti-VEGF responders. Dysregulated miRNA profiles in tears and blood suggest their potential as biomarkers for wet AMD. MiR-195-5p and miR-185-5p in tears demonstrate predictive value for anti-VEGF treatment responders. This study underscores the non-invasive prediction potential of miRNA tear analysis in wet AMD treatment responses.

A highly sensitive fluorescent probe RN-NA reveals peroxynitrite as a novel biomarker for primary open angle glaucoma.

To directly quantify peroxynitrite (ONOO-) using a highly sensitive fluorescence resonance energy transfer probe RN-NA, investigate the association between ONOO- and primary open angle glaucoma (POAG), and clarify whether RN-NA could be used as a potential tool for POAG diagnosis. Plasma and aqueous humor (AH) samples were collected from POAG patients (n=100, age: 59.70±6.87y) and age-related cataract (ARC) patients (n=100, age: 61.15±4.60y) admitted to our hospital. Next, RN-NA was used to detect ONOO- in plasma and AH samples, and the relationship between ONOO- level and POAG was analyzed using binary logistic regression. Besides, Pearson correlation analysis was applied to characterize the correlation of the levels of ONOO- with the patients' age, intraocular pressure (IOP), and mean deviation of visual field testing. The ONOO- scavenger MnTMPyP was employed to treat the 3-morpholinosyndnomine (SIN-1)-induced ocular hypertension in mice (n=7, 6-8wk). Finally, the IOP and ONOO- in both eyes were measured 30min after the last drug treatment. ONOO- levels of AH and plasma were significantly higher in the POAG group than in the ARC group (P<0.01). Additionally, ONOO- levels were closely correlated with POAG in a binary logistic regression analysis [odds ratio (OR)=1.008, 95% confidence interval (CI): 1.002-1.013, P<0.01 for AH; OR=1.004, 95%CI: 1.002-1.006, P<0.001 for plasma]. Pearson correlation analysis showed that ONOO- levels in AH or plasma were positively associated with visual field defects (R=0.51, P<0.01 for AH; R=0.45, P<0.001 for plasma), and ONOO- levels in plasma and AH were correlated in the POAG group (R=0.69, P<0.001). However, administering MnTMPyP to mouse eyes reversed the elevated IOP caused by SIN-1 (P<0.05). ONOO- levels in AH and plasma, detected by RN-NA, are significantly related to POAG and positively correlated with visual field defects in POAG patients. Hence, ONOO- is a potential biomarker of POAG, especially advanced POAG. Besides, anti-nitration compounds may be novel ocular hypotensive agents based on the animal study.

Effect of aflibercept combined with triamcinolone acetonide on aqueous humor growth factor and inflammatory mediators in diabetic macular edema.

To investigate the efficacy of aflibercept combined with sub-tenon injection of triamcinolone acetonide (TA) in treating diabetic macular edema (DME) and to examine changes in growth factors and inflammatory mediator levels in aqueous humor after injection. Totally 67 DME patients (67 eyes) and 30 cataract patients (32 eyes) were enrolled as the DME group and the control group, respectively. The DME group was divided into the aflibercept group (34 cases) and the aflibercept combined with TA group (combined group, 33 cases). The aqueous humor of both groups was collected during the study period. The aqueous levels of vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1β (IL-1β) were detected using a microsphere suspension array technology (Luminex 200TM). Aqueous cytokines, best-corrected visual acuity (BCVA), central macular thickness (CMT), and complications before and after treatment were compared between the aflibercept group and combined group. The concentrations of VEGF, MCP-1, IL-6, and IL-8 in the aqueous humor were significantly higher in the DME group than those of the control group (all P<0.01). After 1mo of surgery, the concentrations of VEGF, MCP-1, IL-6, and IL-8 in the aqueous humor were significantly lower in the combined group than those of the aflibercept group (all P<0.01). The BCVA and CMT values of the two groups were statistically different after 1 and 2mo of treatment (P<0.01). However, the difference was not statistically significant after 3mo of treatment (P>0.05). The cytokines VEGF, MCP-1, IL-6, and IL-8 in the aqueous humor of DME patients are significantly increased. Aflibercept and aflibercept combined with TA have good efficacy in DME patients, can effectively reduce CMT, improve the patient's vision, and have high safety. Aflibercept combined with TA can quickly down-regulate the aqueous humor cytokines and help to relieve macular edema rapidly. However, the long-term efficacy is comparable to that of aflibercept alone.

Expression of microRNAs related to apoptosis in the aqueous humor and lens capsule of patients with glaucoma.

The aim of this study is to investigate the expression profiles of microRNAs (miRNAs) related to apoptosis in the aqueous humor (AH) and lens capsule (LC) of patients with glaucoma. AH and LC samples were collected from patients with open-angle glaucoma and control participants who were scheduled for cataract surgery. A miRNA PCR array comprising 84 miRNAs was used to analyze the AH (glaucoma, n = 3; control, n = 3) and LC samples (glaucoma, n = 3; control, n = 4). Additionally, the AH and LC samples (glaucoma, n = 3; control, n = 4) were subjected to quantitative real-time PCR to validate the differentially expressed miRNAs determined using the PCR array. Bioinformatics analysis was performed to identify the interactions between miRNAs and diseases. Additionally, the differential expression of these miRNAs and the target gene was validated through in vitro experiments using a retinal ganglion cell (RGC) model. Expression levels of 19 and 3 miRNAs were significantly upregulated in the AH and LC samples of the glaucoma group, respectively (p < 0.05). Of these, the expression levels of hsa-miR-193a-5p and hsa-miR-222-3p showed significant differences in both AH and LC samples. Bioinformatics analysis showed experimentally validated 8 miRNA:gene pairs. Among them, PTEN was selected to analyze the expression level in AH and LC from separate cohort (glaucoma, n = 5; control, n = 4). The result showed downregulation of PTEN concurrent with upregulation of the two miRNAs in LC samples of glaucoma group. In vitro experiments validated that the expression levels of hsa-miR-193a-5p and hsa-miR-222-3p were significantly upregulated, and that of PTEN was significantly downregulated in the H2O2-treated RGC, while the level of PTEN was recovered through co-treatment with miR-193a inhibitor or miR-222 inhibitor. This is the first study to investigate the differential expression of apoptosis-related miRNAs in the AH and LC of patients with glaucoma. Hsa-miR-193a-5p and hsa-miR-222-3p, which were upregulated in both AH and LC, may be considered potential biomarkers for glaucoma.

Aqueous humor TGFβ and fibrillin-1 in Tsk mice reveal clues to POAG pathogenesis

Aqueous humor (AH) and blood levels of transforming growth factor β (TGFβ) are elevated in idiopathic primary open angle glaucoma (POAG) representing a disease biomarker of unclear status and function. Tsk mice display a POAG phenotype and harbor a mutation of fibrillin-1, an important regulator of TGFβ bioavailability. AH TGFβ2 was higher in Tsk than wild-type (WT) mice (by 34%; p = 0.002; ELISA); similarly, AH TGFβ2 was higher in human POAG than controls (2.7-fold; p = 0.00005). As in POAG, TGFβ1 was elevated in Tsk serum (p = 0.01). Fibrillin-1 was detected in AH from POAG subjects and Tsk mice where both had similar levels relative to controls (p = 0.45). 350 kDa immunoblot bands representing WT full-length fibrillin-1 were present in human and mouse AH. A 418 kDa band representing mutant full-length fibrillin-1 was present only in Tsk mice. Lower molecular weight fibrillin-1 antibody-reactive bands were present in similar patterns in humans and mice. Certain bands (130 and 32 kDa) were elevated only in human POAG and Tsk mice (p ≤ 0.04 relative to controls) indicating discrete isoforms relevant to disease. In addition to sharing a phenotype, Tsk mice and human POAG subjects had common TGFβ and fibrillin-1 features in AH and also blood that are pertinent to understanding glaucoma pathogenesis.

Fuch's Endothelial Corneal Dystrophy in Cataract Patients Is Associated with Elevated Levels of Inflammatory Chemokines, but Not Growth Factors, in the Aqueous Humor.

The study investigated a profile of chemokines and growth factors in the aqueous humor (AH) of eyes with Fuch's endothelial corneal dystrophy (FECD) and cataracts in comparison with cataract patients as a control group. A total of 52 AH samples (26 FECD + cataract and 26 cataract/control) were collected before cataract surgery. None of the patients had any clinically apparent inflammation at the time of AH collection. The AH levels of MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β(CCL4), RANTES (CCL5), eotaxin (CCL11), IP-10 (CXCL10), FGF basic, G-CSF, GM-CSF, PDGF-bb, and VEGF were compared between the groups. The analyses were performed using the Bio-Plex 200 System from Bio-Rad. Among the studied parameters, the AH levels of RANTES, eotaxin, and IP-10 significantly increased in the FECD + cataract eyes, compared with the cataract controls (p < 0.05). Elevated levels of the RANTES, Eotaxin, and IP-10 indicate more intense inflammation in the eyes of patients in the FECD + cataract group. Moreover, these factors exhibit potential as predictive biomarkers for early detection of FECD in cataract patients. The discovery of elevated concentrations of biochemical markers in a patient, who has not yet received a clinical diagnosis, may suggest the need for heightened observation of the other eye to monitor the potential development of FECD.

Changes in PEDF, MMP-2, and TGF-β2 levels in the aqueous humor of cataract patients and their correlation with disease severity

ObjectiveTo explore the changes of Pigment Epithelium-Derived Factor (PEDF), Matrix Metalloproteinase-2 (MMP-2), and Transforming Growth Factor-β2 (TGF-β2) levels in the aqueous humor of cataract patients and their correlation with disease severity. Methods93 cataract patients and 56 healthy subjects were study objects. PEDF, MMP-2, and TGF-β2 levels of aqueous humor were compared, and the correlation between each index and Lens Opacity Classification System (LOCS) III classification was analyzed. ROC curve was used to analyze the evaluation value of the combined detection of each index on cataract development, and logistic regression to analyze the influence of the changes of each index on cataract development. ResultsPEDF levels were lower and MMP-2 and TGF-β2 levels were higher in the aqueous humor of cataract patients than in healthy subjects. PEDF levels in the aqueous humor were negatively correlated with LOCS III classification, while MMP-2 and TGF-β2 levels were positively correlated with LOCS III classification. The AUC value of combined detection was higher than that of PEDF, MMP-2, and TGF-β2 in the aqueous humor alone. MMP-2 ≥ 15.13 pg/mL, TGF-β2 ≥ 385.91 pg/mL and PEDF < 198.85 ng/mL were risk factors for cataract development. ConclusionThe changes in PEDF, MMP-2, and TGF-β2 levels in the aqueous humor of cataract patients are related to LOCS III classification. The combined detection is valuable in evaluating cataract development.

Role of Novel Inflammatory Factors in Central Retinal Vein Occlusion with Macular Edema.

Background and Objectives: To investigate associations among the aqueous humor levels of novel inflammatory factors, including FMS-related tyrosine kinase 3 ligand (Flt-3L), fractalkine, CXC chemokine ligand 16 (CXCL-16), and endocan-1; the severity of macular edema in central retinal vein occlusion (CRVO); and the prognosis of CRVO with macular edema after antivascular endothelial growth factor (VEGF) therapy. Materials and Methods: Aqueous humor was obtained during anti-VEGF treatment with intravitreal ranibizumab injection (IRI) in patients with CRVO and macular edema (n = 19) and during cataract surgery in patients with cataracts (controls, n = 20), and the levels of VEGF and novel inflammatory factors were measured. Macular edema was evaluated by central macular thickness (CMT) and neurosensory retinal thickness (TNeuro), and improvement was evaluated by calculating the percentage change in CMT and TNeuro from before to 1 month after IRI. Results: The levels of VEGF and the novel inflammatory factors were significantly higher in the CRVO group, and the levels of Flt-3L, CXCL-16, and endocan-1 were significantly correlated with each other and with the aqueous flare value. Baseline levels of Flt-3L, CXCL-16, and endocan-1 had a significantly negative correlation with the change in CMT, and the baseline level of CXCL-16 was significantly negatively correlated with the change in TNeuro. Conclusions: Relations among novel inflammatory factors should be further investigated. These findings may help improve understanding of macular edema in CRVO patients and aid the development of new treatments targeting novel inflammatory factors.

Efficacy and Safety of BTK Inhibitors in Vitreoretinal Lymphoma: A Single-Center, Retrospective Analysis of 24 Patients

Background: Vitreoretinal lymphoma (VRL) is a special subtype of rare central nervous system lymphoma (PCNSL). Most of the pathologic types are diffuse large B-cell lymphoma of non-germinal central origin. Currently, the optimal treatment strategy for VRL is not clear, and the traditional treatment usually includes about 20 repeated intravitreal injections of low doses of methotrexate or intraocular local radiotherapy. Eventually, however, 60-90% of VRL will progress to PCNSL. Therefore, CNS prophylaxis of VRL patients is very important. A previous prospective clinical study of our research group (including 10 VRL patients) confirmed that single-drug BTK inhibitors can replace repeated intravitreal injection of low doses of methotrexate, with rapid effect, high safety, and certain value in preventing CNS progression. Methods: In order to further clarify the efficacy and safety of BTK inhibitors in VRL patients, we included 24 VRL patients who were consecutively admitted to the Hematology Department of Beijing Tongren Hospital from May 2020 to December 2022, and retrospectively analyzed their clinical features, treatment methods, treatment outcomes, survival status and treatment-related adverse reactions. Results: Of the 24 patients, 14 were female and 10 were male, with a median age of 62 years old (36-75). There were 18 patients with primary VRL (PVRL) and 6 patients with secondary VRL (SVRL) to PCNSL. The IL-10 in aqueous humor/vitreous fluid at baseline was significantly increased in all patients (362.1±89.5pg/mL), and the IL-10/IL-6 ratio was greater than 1. Except for one patient with both uncontrolled PCNSL and VRL, the other 23 VRL patients showed no evidence of CNS involvement by brain MRI and cerebrospinal fluid detection. All 24 patients were treated with a BTK inhibitor (Zanubrutinib 160mg bid, or Orelabrutinib 150mg qd), of whom, 9 VRL patients were also treated with intravitreal methotrexate injection (0.4mg/ time, weekly ×12 times → monthly ×9 times) concurrently, and the remaining 15 patients did not receive any antitumor therapy other than BTK inhibitors. After 1 month of treatment with BTK inhibitors, the efficacy was evaluated by slit lamp microscope, optical coherence tomography (OCT), and aqueous humor cytokines. 21 patients achieved CR (87.5%), 2 PR (8.3%), and 1 PD (progression of CNS lesion but improvement of intraocular disease). The IL-10/IL-6 levels in aqueous humor were lower than 1 in all 24 patients 1 month after treatment, and the IL-10 levels in aqueous humor were slightly higher than the normal range in two patients. The median duration of BTK inhibitors use was 10 months (1.5-23.0 months). At a median follow-up of 17 months (5.4-31.5 months), 15 patients (62.5%) developed disease progression, including 11 patients (45.8%) with CNS progression and 4 patients (16.7%) with intraocular recurrence. The median progression-free survival (PFS) time was 11.9 months, and the median overall survival (OS) time was not reached. The 1-year and 2-year PFS rates were 34% and 26%, and the 1-year and 2-year OS rates were 96% and 82%, respectively. The recurrence rate was 33.3% in the combined treatment group and 80.0% in the BTK inhibitors monotherapy group. A total of three patients died, all due to CNS progression. BTK inhibitors were generally well tolerated in VRL patients, with no adverse reactions that led to treatment discontinuation. 4 patients had grade 1-2 hypertension (16.7%), 1 patient had grade 1 hypotension (4.2%), 2 patients had grade 1 joint pain (8.5%), and 6 patients had grade 1 subcutaneous hemorrhage and ecchymosis (25.0%). Four patients developed COVID-19 infection while taking the drug and the drug was suspended. All four patients recovered from COVID-19 infection within 2 weeks and did not develop significant pneumonia. Conclusion: This is the largest report regarding the use of BTK inhibitors in VRL patients. BTK inhibitor has a rapid local control effect on VRL and is well tolerated. However, BTK inhibitors monotherapy has unsatisfied preventive effect on the CNS, and it is still necessary to explore the value of combinational therapy, such as combined BTK inhibitors and high-dose methotrexate.

Association between inflammatory cytokines and oxidative stress levels in aqueous humor with axial length in human myopia

This study investigated the content of inflammatory cytokines and oxidative stress levels in the aqueous humor (AH) of patients with high myopia (HM) and explored the relationship between these factors and the axial length (AL) of the eye, to explore the roles of mild intraocular inflammation and oxidative stress imbalance in the occurrence and development of myopia. AH samples from 40 patients (70 eyes) were collected during implantable collamer lens (ICL-V4c) surgery. The subjects were divided into three groups according to AL: group A (AL ≤ 26 mm), group B (26 < AL ≤ 28 mm), and group C (AL ≥ 28 mm). The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-2 (MMP-2), and interleukin-1β (IL-1β) in the AH of the three groups were measured using the Luminex system. Oxidative stress levels were measured using reagent kits targeting total antioxidant capacity (T-AOC), catalase (CAT), and nitric oxide (NO) and malonaldehyde (MDA) content. The results showed compared with group A, IL-1β, MMP-2, and IL-6 concentrations were significantly higher and T-AOC levels were significantly lower in group C. There were no significant differences in CAT, NO, MDA, or TNF-α levels among the groups. The concentrations of IL-6 (r = 0.379, p = 0.016), MMP-2 (r = 0.469, p = 0.002), and MDA (r = 0.354, p = 0.025) in AH were positively correlated with the AL, whereas T-AOC (r = −0.678, p = 0.000) was negatively correlated with AL. These results suggest that mild intraocular inflammation and oxidative stress imbalance may be associated with myopia. Further experiments are needed to confirm the role of mild intraocular inflammation and oxidative stress imbalance in the occurrence and development of myopia.

Trefoil Family Factor Peptide 1-A New Biomarker in Liquid Biopsies of Retinoblastoma under Therapy.

Effective management of retinoblastoma (RB), the most prevalent childhood eye cancer, depends on reliable monitoring and diagnosis. A promising candidate in this context is the secreted trefoil family factor peptide 1 (TFF1), recently discovered as a promising new biomarker in patients with a more advanced subtype of retinoblastoma. The present study investigated TFF1 expression within aqueous humor (AH) of enucleated eyes and compared TFF1 levels in AH and corresponding blood serum samples from RB patients undergoing intravitreal chemotherapy (IVC). TFF1 was consistently detectable in AH, confirming its potential as a biomarker. Crucially, our data confirmed that TFF1-secreting cells within the tumor mass originate from RB tumor cells, not from surrounding stromal cells. IVC-therapy-responsive patients exhibited remarkably reduced TFF1 levels post-therapy. By contrast, RB patients' blood serum displayed low-to-undetectable levels of TFF1 even after sample concentration and no therapy-dependent changes were observed. Our findings suggest that compared with blood serum, AH represents the more reliable source of TFF1 if used for liquid biopsy RB marker analysis in RB patients. Thus, analysis of TFF1 in AH of RB patients potentially provides a minimally invasive tool for monitoring RB therapy efficacy, suggesting its importance for effective treatment regimens.