Effects of galectin-3 protein on UVA-induced damage in retinal pigment epithelial cells.

Several inflammatory molecules have been suggested as biomarkers of age-related macular degeneration (AMD). Galectin-3 (Gal-3), which has been shown to have a protective role in corneal injury by promoting epithelial cells adhesion and migration to the extracellular matrix, is also highly expressed in the retinal pigment epithelium (RPE) of patients with AMD. This study evaluated the role of Gal-3 in an in vitro model of UVA-induced RPE damage, as a proof-of-concept. ARPE-19 cells (human RPE cell line), were incubated with Gal-3 at 0.5-2.5 µg/mL concentrations prior to UVA irradiation for 15, 30, and 45 min, which resulted in accumulated doses of 2.5, 5, and 7.5 J/cm2, respectively. After 24 h incubation, MTT and LDH assays, immunofluorescence, and ELISA were performed. UVA irradiation for 15, 30, and 45 min proved to reduce viability in 83%, 46%, and 11%, respectively. Based on the latter results, we chose the intermediate dose (5-J/cm2) for further analysis. Pretreatment with Gal-3 at concentrations > 1.5 µg/mL showed to increase the viability of UVA-irradiated cells (~ 75%) compared to untreated cells (64%). Increased levels of cleaved caspase 3, a marker of cell death, were detected in the ARPE cells after UVA irradiation with or without addition of exogenous Gal-3. The inhibitory effect of Gal-3 on UVA-induced cell damage was characterized by decreased ROS levels and increased p38 activation, as detected by fluorescence analysis. In conclusion, our study suggests a photoprotective effect of Gal-3 on RPE by reducing oxidative stress and increasing p38 activation.

Deletion of Annexin A1 in Mice Upregulates the Expression of Its Receptor, Fpr2/3, and Reactivity to the AnxA1 Mimetic Peptide in Platelets.

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.

Involvement of the annexin A1-Fpr anti-inflammatory system in the ocular allergy.

Annexin A1 (ANXA1)-formyl peptide receptor (Fpr) system is potent effective mediators in the control of the inflammatory response. In this study, we evaluate the potential involvement of the Fpr family in the protective effect of the mimetic peptide of ANXA1 (ANXA12-26) using an experimental allergic conjunctivitis (AC) model in mice. Ovalbumin (OVA)/Alum-immunized wild-type (WT) and ANXA1-null (ANXA1-/-) Balb/c mice (days 0 and 7) were challenged by eye drops containing OVA on days 14-16, and two groups received ANXA12-26 alone or with Fpr antagonist Boc2 intraperitoneally during challenged days. As expected, plasma IgE anti-OVA levels increased significantly in the OVA-immunized WT and ANXA1-/- mice, supporting the efficacy of AC model. AC increased Fpr1 and Fpr2 levels in the conjunctiva and the lack of endogenous ANXA1 exacerbated Fpr2 expression only. In contrast, administering ANXA12-26 in the WT mice diminished Fpr2 levels in the conjunctiva, and the effect was reverted by Boc2. Ultrastructural analysis showed the co-localization of Fpr2 and ANXA1 in the plasma membrane of mast cells (MCs), eosinophils and neutrophils, supporting this system as being operative in the AC. Boc2 abrogated the ANXA12-26 effect by increasing the MC degranulation and the eosinophil influx in the conjunctiva, and these findings were supported by peroxidase eosinophil, eotaxin and MC protease levels. Additionally, the ANXA12-26-Fpr system in the AC was associated with the activation of ERK and JNK. Collectively, the data provided in vivo supports the anti-allergic effects of the ANXA1-Fpr system and may serve as a therapeutic target in this ocular disorder.

Galectin-3: role in ocular allergy and potential as a predictive biomarker.

AIMS: To evaluate galectin-3 (Gal-3), a ß-galactoside binding protein, as a possible biomarker in ocular allergy and further investigated the role of endogenous Gal-3 in a murine model of ovalbumin (OVA)-induced allergic conjunctivitis (AC). METHODS: Conjunctival impression cytology specimens from control and patients with severe vernal keratoconjunctivitis, treated or untreated, were used to evaluate Gal-3 expression by immunocytochemistry. To investigate the mechanism of action of Gal-3, OVA-immunised BALB/c male wild-type (WT) and Gal-3 null (Gal-3-/-) mice were challenged with eye drops containing OVA on days 14-16 with a subset of animals pretreated with 0.03% tacrolimus (TC) or dexamethasone (Dex). RESULTS: Patients with AC and OVA-sensitised WT mice exhibited increased levels of Gal-3 in the conjunctiva compared with control, an effect reverted by the action of Dex and TC therapy. Twenty-four hours after the final OVA challenge, total and anti-OVA IgE levels increased significantly in the blood of OVA-sensitised WT and Gal-3-/- mice compared with controls, supporting the efficacy of the AC model. The lack of endogenous Gal-3 exacerbated the local inflammatory response, increasing the influx of eosinophils and mast cell activation. Additionally, OVA-sensitised Gal-3-/- animals exhibited increased CD4+ expression in the eyes as well as eotaxin, IL-4, IL-13 and interferon-γ levels in the tear fluid compared with WT animals. CONCLUSION: Gal-3 contributes to the pathogenesis of ocular allergy and represents a relevant therapeutic target.

Evaluation of galectin-1 and galectin-3 as prospective biomarkers in keratoconus.

AIMS: To evaluate the expression of ß-galactoside-binding proteins galectin (Gal)-1 and Gal-3 in patients with keratoconus (KC) and postcorneal collagen cross-linking (CXL) treatment in vitro. METHODS: Tear fluid, cornea samples and conjunctival impression cytology specimens from control and KC patients were used to evaluate Gal-1 and Gal-3 expressions. Primary keratocytes were isolated by collagenase digestion from surgically removed corneas of five normal or KC human corneal buttons and cultured in Dulbecco's modified eagle medium/Ham's F12 medium supplemented with 2% fetal bovine serum. These cells were evaluated under two experimental conditions: control and submitted to the application of ultraviolet A light and riboflavin 0.1% (CXL) for 30 min. RESULTS: Patients with KC displayed increased levels of Gal-1 and Gal-3 in conjunctival epithelial cells compared with control. Furthermore, KC corneas were associated with intense expression of Gal-1 in the stroma, released by keratocytes. Ultrastructural analysis of keratocytes showed a marked increase of endogenous Gal-3 levels, but not Gal-1, in KC. In vitro, CXL induced significant release of Gal-1 in keratocyte supernatants (116±18 ng/mL, P<0.05) and decreased inflammatory biomarkers as interleukin (IL)-6, IL-8, matrix metalloproteinase (MMP)-2 and MMP-9. Gal-3 levels were not detected in the keratocyte supernatants. CONCLUSION: Gal-1 and Gal-3 represent new interesting KC biomarkers as revealed by their different expression patterns in KC and control corneal samples. CXL has an immunosuppressive effect on keratocytes by reducing the release of cytokines and MMPs and increased expression of anti-inflammatory protein Gal-1.

Anti-inflammatory effect of galectin-1 in a murine model of atopic dermatitis.

Atopic dermatitis (AD) is caused by both dysregulated immune responses and an impaired skin barrier. Although beta-galactoside-binding protein galectin-1 (Gal-1) has immunomodulatory effects in several inflammatory disorders, therapeutic strategies based on its anti-inflammatory properties have not been explored in AD. Thus, we evaluate pharmacological treatment with Gal-1 in the progression of an ovalbumin (OVA)-induced AD-like skin lesions. The skin of OVA-immunized male BALB/c mice was challenged with drops containing OVA on days 11, 14-18 and 21-24. Additionally, in the last week, a subset of animals was treated intraperitoneally with recombinant Gal-1 (rGal-1) or dexamethasone (Dex). Treatment with rGal-1 decreased the clinical signs of dermatitis in BALB/c mice and diminished local eotaxin and IFN-γ levels. The treatment also suppressed the infiltration of eosinophils and mast cells, which was verified by reduced expression of mouse mast cell protease 6 (mMCP6) and eosinophil peroxidase (EPX). These localized effects are associated with extracellular signal-regulated kinase (ERK) activation and downregulation of endogenous Gal-1. The inhibition of disease progression induced by rGal-1 was also correlated with reduced plasma IL-17 levels. Our results demonstrate that rGal-1 is an effective treatment for allergic skin inflammation in AD and may impact the development of novel strategies for skin inflammatory diseases. KEY MESSAGES: Pharmacological treatment with rGal-1 reduces clinical signs of atopic dermatitis. Systemic treatment with rGal-1 inhibits eosinophil and mast cell influx in the skin of AD animals. rGal-1 reduced local eotaxin levels and systemic IL-17 levels. The inhibition of disease progression induced by rGal-1 was correlated with upregulation of phosphorylated ERK.