Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands.

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((ß-D-galactopyranosyl)-(1→4)-ß-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-ß-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3' of each of the two sugars, resulting in a 3'-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl ß-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3' position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series.

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.

MG-Pe: A Novel Galectin-3 Ligand with Antimelanoma Properties and Adjuvant Effects to Dacarbazine.

Melanoma is a highly metastatic and rapidly progressing cancer, a leading cause of mortality among skin cancers. The melanoma microenvironment, formed from the activity of malignant cells on the extracellular matrix and the recruitment of immune cells, plays an active role in the development of drug resistance and tumor recurrence, which are clinical challenges in cancer treatment. These tumoral metabolic processes are affected by proteins, including Galectin-3 (Gal-3), which is extensively involved in cancer development. Previously, we characterized a partially methylated mannogalactan (MG-Pe) with antimelanoma activities. In vivo models of melanoma were used to observe MG-Pe effects in survival, spontaneous, and experimental metastases and in tissue oxidative stress. Analytical assays for the molecular interaction of MG-Pe and Gal-3 were performed using a quartz crystal microbalance, atomic force microscopy, and contact angle tensiometer. MG-Pe exhibits an additive effect when administered together with the chemotherapeutic agent dacarbazine, leading to increased survival of treated mice, metastases reduction, and the modulation of oxidative stress. MG-Pe binds to galectin-3. Furthermore, MG-Pe antitumor effects were substantially reduced in Gal-3/KO mice. Our results showed that the novel Gal-3 ligand, MG-Pe, has both antitumor and antimetastatic effects, alone or in combination with chemotherapy.

Combining inhibition of galectin-3 with and before a therapeutic vaccination is critical for the prostate-tumor-free outcome.

BACKGROUND: Prostate cancer (PCa) is a major health problem worldwide. Taxol derivatives-based chemotherapies or immunotherapies are usually proposed depending on the symptomatic status of the patient. In the case of immunotherapy, tumors develop robust immune escape mechanisms that abolish any protective response, and to date why prostate cancer is one of the most resistant diseases remains unresolved. METHODS: By using a combination of clinical data to study the transcriptome of metastasis samples from patients with castration-refractory prostate cancer, and state of the art cellular and molecular biology assays in samples from tumor-bearing mice that have been submitted to surgical resection of the tumor before receiving a vaccination, we answered several essential questions in the field of immunotherapy for prostate cancer. We also used two different methods to inhibit the expression of galectin-3 (Gal-3) in tumor cells: a stable RNA interference method to control the expression of this galectin efficiently only in tumor cells, and low and non-cytotoxic doses of docetaxel to easily transfer our findings to clinical settings. RESULTS: Herein, we show for the first time that Gal-3 expressed by prostate tumor cells is the main immune checkpoint responsible for the failure of vaccine-based immunotherapy. Our results show that low and non-cytotoxic doses of docetaxel lead to the inhibition of Gal-3 expression in PCa cells as well as in clinical samples of patients with metastatic and castration-resistant PCa promoting a Th1 response. We thus optimized a prostate cancer animal model that undergoes surgical resection of the tumor to mimic prostatectomy usually performed in patients. Importantly, using Gal-3-knocked down-PCa cells or low and non-cytotoxic doses of taxane before vaccination, we were able to highly control tumor recurrence through a direct impact on the proliferation and infiltration of CD8+ cytotoxic T. CONCLUSIONS: Thus, Gal-3 expression by PCa cells is a crucial inhibitor for the success of immunotherapy, and low doses of docetaxel with non-cytotoxic effect on leukocyte survival could be used before immunotherapy for all patients with PCa to reduce the expression of this critical negative immune checkpoint, pre-conditioning the tumor-microenvironment to activate an antitumor immune response and promote tumor-free outcome.

Galectin-3 Stimulates Tyro3 Receptor Tyrosine Kinase and Erk Signalling, Cell Survival and Migration in Human Cancer Cells.

The TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and protein S (ProS1), are implicated in tumorigenesis and chemoresistance in various cancers. The ß-galactoside binding protein galectin-3 (Gal-3), which is also implicated in oncogenesis, has previously been shown to be a ligand for MerTK. However, the selectivity of Gal-3 for the other TAM receptors, and its TAM-mediated signalling and functional properties in cancer cells, remain to be explored. The present study was aimed at determining these, including through direct comparison of Gal-3 with the two canonical TAM ligands. Exogenous Gal-3 rapidly stimulated Tyro3 receptor phosphorylation to the same extent as the Tyro3 ligand ProS1, but not Axl, in the cultured human cancer cell lines SCC-25 (express both Tyro3 and Axl) and MGH-U3 (express Tyro3 only). Gal-3 also activated intracellular Erk and Akt kinases in both cell lines and furthermore protected cells from acute apoptosis induced by staurosporine but not from serum-starvation induced apoptosis. In addition, Gal-3 significantly stimulated cancer cell migration rate in the presence of the Axl blocker BGB324. Therefore, these results have shown Gal-3 to be a novel agonist for Tyro3 RTK, activating a Tyro3-Erk signalling axis, as well as Akt signalling, in cancer cells that promotes cell survival, cell cycle progression and cell migration. These data therefore reveal a novel mechanism of Tyro3 RTK activation through the action of Gal-3 that contrasts with those of the known TAM ligands Gas6 and ProS1.

Galectin-3 Induces Atrial Fibrosis by Activating the TGF-ß1/Smad Pathway in Patients with Atrial Fibrillation.

BACKGROUND: Atrial fibrosis plays a critical role in the occurrence and maintenance of atrial fibrillation. The role of TGF-ß1 in mediating atrial fibrosis is well documented. The ß-galactoside-binding lectin galectin-3 (Gal-3) is mainly produced by macrophages in biological events such as inflammation and angiogenesis. Previous studies have shown that Gal-3 is associated with atrial fibrosis, but the relationship between TGF-ß1 and Gal-3 in atrial fibrosis remains unclear. OBJECTIVE: To determine whether Gal-3 induces atrial fibrosis and atrial fibrillation by activating the TGF-ß1/Smad pathway and whether the expression of Gal-3 is mediated by TGF-ß1, which can enable assessing the relationship between Gal-3 and TGF-ß1 in atrial fibrosis. METHODS: In this study, 30 patients' right atrial appendages were collected and divided into 3 groups: congenital heart disease sinus rhythm group (n = 10, as a control group), rheumatic heart disease sinus rhythm group (n = 10), and rheumatic heart disease atrial fibrillation group (n = 10). Rat atrial fibroblasts were cultured in vitro, and recombinant Gal-3 and recombinant TGF-ß1 proteins were added to the cell culture. The expression of Gal-3, TGF-ß1, Smad2, and collagen I was detected by Western blotting and quantitative real-time PCR. Atrial tissues were stained with Masson's trichrome stain to evaluate the extent of atrial fibrosis. The expression of Gal-3 and TGF-ß1 was detected by immunohistochemical staining and immunofluorescence staining. Gal-3 and TGF-ß1 interaction was demonstrated by immunoprecipitation. RESULTS: The expression levels of Gal-3, TGF-ß1, Smad2, and collagen I were elevated in the rheumatic heart disease atrial fibrillation group compared with the congenital heart disease sinus rhythm group and the rheumatic heart disease sinus rhythm group. In cultured atrial fibroblasts, there is a synergistic interaction between Gal-3 and TGF-ß1. Gal-3 stimulated the TGF-ß1/Smad pathway, and overexpression of TGF-ß1 induced Gal-3 expression. CONCLUSIONS: Gal-3 and TGF-ß1 interact with each other and stimulate the downstream TGF-ß1/Smad pathway. This finding suggests that Gal-3 could be an important factor in TGF-ß1-induced fibrosis in atrial fibrillation.

Galectin-3 initiates epithelial-stromal paracrine signaling to shape the proteolytic microenvironment during corneal repair.

Paracrine interactions between epithelial cells and stromal fibroblasts occur during tissue repair, development, and cancer. Crucial to these processes is the production of matrix metalloproteinases (MMPs) that modify the microenvironment. Here, we demonstrated that the carbohydrate-binding protein galectin-3 stimulated microenvironment remodeling in the cornea by promoting the paracrine action of secreted interleukin-1ß (IL-1ß). Through live cell imaging in vitro, we observed rapid activation of the MMP9 promoter in clusters of cultured human epithelial cells after direct heterotypic contact with single primary human fibroblasts. Soluble recombinant galectin-3 and endogenous galectin-3 of epithelial origin both stimulated MMP9 activity through the induction of IL-1ß secretion by fibroblasts. In vivo, mechanical disruption of the basement membrane in wounded corneas prompted an increase in the abundance of IL-1ß in the stroma and increased the amount of gelatinase activity in the epithelium. Moreover, corneas of galectin-3-deficient mice failed to stimulate IL-1ß after wounding. This mechanism of paracrine control has broad importance for our understanding of how the proteolytic microenvironment is modified in epithelial-stromal interactions.

Galectin-3 enhances monocyte-derived macrophage efferocytosis of apoptotic granulocytes in asthma.

BACKGROUND: Galectin-3 is a 32 kDa protein secreted by macrophages involved in processes such as cell activation, chemotaxis and phagocytosis. Galectin-3 has previously been shown to improve the ability of airway macrophages to ingest apoptotic cells (efferocytosis) in chronic obstructive pulmonary disease (COPD) and may be of interest in non-eosinophilic asthma (NEA) which is also characterised by impaired efferocytosis. It was hypothesised that the addition of exogenous galectin-3 to monocyte-derived macrophages (MDMs) derived from donors with NEA would enhance their ability to engulf apoptotic granulocytes. METHODS: Eligible non-smoking adults with asthma (n = 19), including 7 with NEA and healthy controls (n = 10) underwent a clinical assessment, venepuncture and sputum induction. MDMs were co-cultured with apoptotic granulocytes isolated from healthy donors with or without exogenous recombinant galectin-3 (50 µg/mL) and efferocytosis was assessed by flow cytometry. Galectin-3 expression and localisation in MDMs was visualised by immunofluorescence staining and fluorescence microscopy. Galectin-3, interleukin (IL)-6 and CXCL8 secretion were measured in cell culture supernatants by ELISA and cytometric bead array. RESULTS: Baseline efferocytosis (mean (±standard deviation)) was lower in participants with asthma (33.2 (±17.7)%) compared with healthy controls (45.3 (±15.9)%; p = 0.081). Efferocytosis did not differ between the participants with eosinophilic asthma (EA) (31.4 (±19.2)%) and NEA (28.7 (±21.5)%; p = 0.748). Addition of galectin-3 significantly improved efferocytosis in asthma, particularly in NEA (37.8 (±18.1)%) compared with baseline (30.4 (±19.7)%; p = 0.012). Efferocytosis was not associated with any of the clinical outcomes but was negatively correlated with sputum macrophage numbers (Spearman r = - 0.671; p = 0.017). Galectin-3 was diffusely distributed in most MDMs but formed punctate structures in 5% of MDMs. MDM galectin-3 secretion was lower in asthma (9.99 (2.67, 15.48) ng/mL) compared with the healthy controls (20.72 (11.28, 27.89) ng/mL; p = 0.044) while IL-6 and CXCL8 levels were similar. CONCLUSIONS: Galectin-3 modulates macrophage function in asthma, indicating a potential role for galectin-3 to reverse impaired efferocytosis in NEA.

Extracellular Galectin-3 Induces Accelerated Oligodendroglial Differentiation Through Changes in Signaling Pathways and Cytoskeleton Dynamics.

Galectin-3 (Gal-3) is a chimeric protein structurally composed of unusual tandem repeats of proline and short glycine-rich segments fused onto a carbohydrate recognition domain. Our studies have previously demonstrated that Gal-3 drives oligodendrocyte (OLG) differentiation to control myelin integrity and function. The cytoskeleton plays a key role in OLG maturation: the initial stage of OLG process extension requires dynamic actin filament assembly, while subsequent myelin wrapping coincides with the upregulation of actin disassembly proteins which are dependent on myelin basic protein (MPB) expression. In this context, the aim of the present work was to elucidate the mechanism by which recombinant Gal-3 (rGal-3) induces OLG maturation, giving special attention to the actin cytoskeleton. Our results show that rGal-3 induced early actin filament assembly accompanied by Erk signaling deactivation, which led to a decrease in the number of platelet-derived growth factor receptor α (PDGFRα)+ cells concomitantly with an increase in the number of 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase)+ cells at 1 day of treatment (TD1), and Akt signaling activation at TD1 and TD3. Strikingly, rGal-3 induced an accelerated shift from polymerized to depolymerized actin between TD3 and TD5, accompanied by a significant increase in MBP, gelsolin, Rac1, Rac1-GTP, and ß-catenin expression at TD5. These results were strongly supported by assays using Erk 1/2 and Akt inhibitors, indicating that both pathways are key to rGal-3-mediated effects. Erk 1/2 inhibition in control-treated cells resembled an rGal-3 like state characterized by an increase in MBP, ß-catenin, and gelsolin expression. In contrast, Akt inhibition in rGal-3-treated cells reduced MBP, ß-catenin, and gelsolin expression, indicating a blockade of rGal-3 effects. Taken together, these results indicate that rGal-3 accelerates OLG maturation by modulating signaling pathways and protein expression which lead to changes in actin cytoskeleton dynamics.

Galectin-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin ß1-RhoA-JNK signaling activation.

Oxidized low-density lipoprotein (Ox-LDL)-induced endothelial cell injury plays a crucial role in the pathogenesis of atherosclerosis (AS). Plasma galectin-3 (Gal-3) is elevated inside and drives diverse systemic inflammatory disorders, including cardiovascular diseases. However, the exact role of Gal-3 in ox-LDL-mediated endothelial injury remains unclear. This study explores the effects of Gal-3 on ox-LDL-induced endothelial dysfunction and the underlying molecular mechanisms. In this study, Gal-3, integrin ß1, and GTP-RhoA in the blood and plaques of AS patients were examined by ELISA and western blot respectively. Their levels were found to be obviously upregulated compared with non-AS control group. CCK8 assay and flow cytometry analysis showed that Gal-3 significantly decreased cell viability and promoted apoptosis in ox-LDL-treated human umbilical vascular endothelial cells (HUVECs). The upregulation of integrinß1, GTP-RhoA, p-JNK, p-p65, p-IKKα, and p-IKKß induced by ox-LDL was further enhanced by treatment with Gal-3. Pretreatment with Gal-3 increased expression of inflammatory factors (interleukin [IL]-6, IL-8, and IL-1ß), chemokines(CXCL-1 and CCL-2) and adhesion molecules (VCAM-1 and ICAM-1). Furthermore, the promotional effects of Gal-3 on NF-κB activation and inflammatory factors in ox-LDL-treated HUVECs were reversed by the treatments with integrinß1-siRNA or the JNK inhibitor. We also found that integrinß1-siRNA decreased the protein expression of GTP-RhoA and p-JNK, while RhoA inhibitor partially reduced the upregulated expression of p-JNK induced by Gal-3. In conclusion, our finding suggests that Gal-3 exacerbates ox-LDL-mediated endothelial injury by inducing inflammation via integrin ß1-RhoA-JNK signaling activation.

[Galectin-3 induces differentiation of rat bone marrow mesenchymal stem cells into hepatocyte-like cells].

OBJECTIVE: To investigate the effect of galectin-3 in inducing the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocyte- like cells and explore the involvement of the signaling pathways in the induced cell differentiation. METHODS: The third passage of cultured rat femoral BMSCs were treated with 0.5 µg/mL galectin-3, 20 ng/mL hepatocyte growth factor (HGF) or both to induce their differentiation, with untreated rat BMSCs and hepatocytes as controls. At 7, 14, 21 and 28 days of induction, the cells were examined for morphological changes followed by glycogen staining, quantitative real-time PCR and Western blotting. Gene microarray technique was used to examine the mRNA expression profile of the BMSCs induced with galectin-3. The BMSCs were also induced with galectin-3 in combination with XMU-MP-1, a Hippo signaling pathway inhibitor, after which Western blotting was performed to detect the expressions of YAP, P-YAP, ALB, AFP and CK-18 in the cells. RESULTS: The cells isolated from the femoral bone marrow of SD rats showed a consistent surface marker phenotype with the BMSCs. Induction with galectin-3, HGF, or both all resulted in gradual morphological changes of the BMSCs into hepatocyte-like cells, and the cells with a combined induction for 28 days showed the highest morphological similarity with hepatocytes. The cells induced with galectin-3, HGF, or their combination for 28 days all showed increased positivity rate of glycogen staining, which was the highest in the cells with combined induction (P < 0.05) without significant difference between the cells induced with galectin-3 and HGF alone (P > 0.05). Induction with galectin-3 and HGF alone both increased the expressions of AFP, ALB and CK-18 mRNAs in the cells, and their expression levels were similar between the cells at 28 days (P > 0.05). Galectin-3 and HGF did not show an interactive effect on the mRNA expressions of AFP (F=0.236, P=0.640) or ALB (F=50.639, P=0.000), but had a synergistic effect on CK-18 mRNA expression (F=50.639, P=0.000). The protein expressions of AFP, ALB and CK18 were also increased in the induced cells but not detected in the cells without induction. Gene microarray results revealed 27 up-regulated genes and 62 down-regulated genes in galectin-3-induced BMSCs involving TGF-ß, PI3K-Akt and Hippo signal pathways. Induction with galectin-3 and galectin-3+XMU-MP-1 increased YAP expression in the cells, and galectin-3+XMU-MP-1 was more efficient to induce the differentiation of the BMSCs. CONCLUSIONS: Galectin-3 can induce the differentiation of rat BMSCs into hepatocyte-like cells, and the combination with HGF increases the efficiency of induced differentiation of the cells. TGF-ß, PI3K-Akt and Hippo pathways are involved in the induced differentiation of the BMSCs, and inhibiting Hippo pathway can improve the induction efficiency.

Galectin-3 down-regulates antioxidant peroxiredoxin-4 in human cardiac fibroblasts: a new pathway to induce cardiac damage.

Galectin-3 (Gal-3) is increased in heart failure (HF) and promotes cardiac fibrosis and inflammation. We investigated whether Gal-3 modulates oxidative stress in human cardiac fibroblasts, in experimental animal models and in human aortic stenosis (AS). Using proteomics and immunodetection approaches, we have identified that Gal-3 down-regulated the antioxidant peroxiredoxin-4 (Prx-4) in cardiac fibroblasts. In parallel, Gal-3 increased peroxide, nitrotyrosine, malondialdehyde, and N-carboxymethyl-lysine levels and decreased total antioxidant capacity. Gal-3 decreased prohibitin-2 expression without modifying other mitochondrial proteins. Prx-4 silencing increased oxidative stress markers. In Gal-3-silenced cells and in heart from Gal-3 knockout mice, Prx-4 was increased and oxidative stress markers were decreased. Pharmacological inhibition of Gal-3 with modified citrus pectin restored cardiac Prx-4 as well as prohibitin-2 levels and improved oxidative status in spontaneously hypertensive rats. In serum from 87 patients with AS, Gal-3 negatively correlated with total antioxidant capacity and positively correlated with peroxide. In myocardial biopsies from 26 AS patients, Gal-3 up-regulation paralleled a decrease in Prx-4 and in prohibitin-2. Cardiac Gal-3 inversely correlated with Prx-4 levels in myocardial biopsies. These data suggest that Gal-3 decreased Prx-4 antioxidant system in cardiac fibroblasts, increasing oxidative stress. In pathological models presenting enhanced cardiac Gal-3, the decrease in Prx-4 expression paralleled increased oxidative stress. Gal-3 blockade restored Prx-4 expression and improved oxidative stress status. In AS, circulating levels of Gal-3 could reflect oxidative stress. The alteration of the balance between antioxidant systems and reactive oxygen species production could be a new pathogenic mechanism by which Gal-3 induces cardiac damage in HF.

Galectin-3 impacts Cryptococcus neoformans infection through direct antifungal effects.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes cryptococcosis, which is a major opportunistic infection in immunosuppressed individuals. Mammalian ß-galactoside-binding protein Galectin-3 (Gal-3) modulates the host innate and adaptive immunity, and plays significant roles during microbial infections including some fungal diseases. Here we show that this protein plays a role also in C. neoformans infection. We find augmented Gal-3 serum levels in human and experimental infections, as well as in spleen, lung, and brain tissues of infected mice. Gal-3-deficient mice are more susceptible to cryptococcosis than WT animals, as demonstrated by the higher fungal burden and lower animal survival. In vitro experiments show that Gal-3 inhibits fungal growth and exerts a direct lytic effect on C. neoformans extracellular vesicles (EVs). Our results indicate a direct role for Gal-3 in antifungal immunity whereby this molecule affects the outcome of C. neoformans infection by inhibiting fungal growth and reducing EV stability, which in turn could benefit the host.

Regulation of RAW264.7 macrophage polarization on smooth and rough surface topographies by galectin-3.

Recognition of topographical features induces phenotypic changes in macrophages although the receptors and signaling pathways are not completely characterized. As integrin molecules in focal adhesions/podosomes are in intimate contact with topography and topography modulates the NFkB pathway through cholesterol enriched raft-associated adhesive signaling structures we hypothesized that a cell-surface signaling complex comprised of galectin-3 together with its ligand CD98 and integrinß1 is important for topography-directed lineage determination. This study used polished, sand blasted and acid etched (SLA) surfaces and two novel grooved topographies (G1 and G2) produced by anisotropic etching of Si <1 1 0> to evaluate the role of galectin-3 in macrophage polarization in RAW 264.7 macrophages, as determined by gene expression and morphology. In the presence of the galectin-3 inhibitor, lactose, the M2 marker (mannose receptor) was down-regulated while the M1 marker (iNOS) was up-regulated on smooth and rough surfaces. This skewing of phenotype suggests a role for galectin-3 in macrophage polarization towards the M2 phenotype. Additionally, we evaluated the role of PI3K on polarization using PI3K inhibitor LY294002. We found that the M2 marker was down-regulated on both PO (surface polished) and G1 surfaces implicating PI3K in lineage determination. We also found that surface topography altered cell morphology; macrophages had a larger area on G2 surfaces. Lactose treatment significantly reduced the cell area on all topographies suggesting that the galectin-3 is also involved in signaling complexes triggering the rearrangement of the actin cytoskeleton. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2499-2509, 2017.

Galectin­3 induces the phenotype transformation of human vascular smooth muscle cells via the canonical Wnt signaling.

Galectin­3, a galactoside­binding protein, is highly expressed in carotid plaques and plays an important role in the atherosclerotic lesions. The phenotype transformation of vascular smooth muscle cells is the basic pathological change of atherosclerosis. This study investigated the effects of exogenous galectin­3 on the function and phenotype transformation of human umbilical vascular smooth muscle cells (HUSMC). In this study, we treated vascular smooth muscle cells with recombinant galectin­3 and tested its effect on cell proliferation, migration, and phenotype transformation. Our results showed that exogenous galectin­3 promoted human umbilical vascular smooth muscle cells (HUSMC) proliferation and migration. Exogenous galectin­3 enhanced the expression of the smooth muscle synthetic protein osteopontin, smooth muscle contractile proteins calponin and smooth muscle α­actin. The galectin­3­induced change in cell phenotype was associated with the activation of canonical Wnt signaling, as measured by ß­catenin axin2 and cyclin D1 expression. ß­catenin inhibition by small interfering RNA reduced cell proliferation, decreased cell motility, and blocked galectin­3­induced phenotype transformation of human umbilical vascular smooth muscle cells (HUSMC). Our data suggest galectin­3 promotes the phenotype transformation of human umbilical vascular smooth muscle cells (HUSMC) by activating Wnt/ß­catenin signaling pathway.

Galectin-3 promotes caspase-independent cell death of HIV-1-infected macrophages.

HIV-1-infected macrophages are a key contributor to the formation of a viral reservoir and new treatment strategies focus on eliminating this pool of virus. Galectin-3 is a potent apoptosis-inducing protein that regulates diverse cellular activities. In the present study, we investigated whether galectin-3 could induce cell death in HIV-1-infected macrophages using HIV-1-infected THP1 monocytes (THP1-MNs) and THP1-derived macrophages (THP1-MΦs) as in vitro cellular models. We found that THP1-MΦs were more resistant than the THP1-MNs to HIV-1 infection-induced death, and that HIV-1 infection of the THP1-MΦs increased expression of the anti-apoptotic proteins Mcl-1, Bcl-2 and Bcl-xL. Additionally, galectin-3 but not FasL, tumor necrosis factor (TNF)-related apoptosis-inducing ligand or TNF-α, could induce cell death in HIV-1-infected THP1-MΦs. A similar result was shown for primary human monocyte-derived macrophages. Galectin-3-induced cell death was also significantly increased in macrophages obtained from SIVmac251-infected macaques compared to that of macrophages from healthy macaques. Furthermore, galectin-3-induced cell death in HIV-1-infected THP1-MΦs was caspase independent. Interestingly, endonuclease G (Endo G) was increased in the nucleus and decreased in the cytoplasm of galectin-3-treated cells; thus, galectin-3-induced cell death in HIV-1-infected THP1-MΦs is most likely related to the translocation of Endo G from the cytoplasm to the nucleus. These findings suggest that galectin-3 may potentially aid in the eradication of HIV-1/SIV-infected macrophages.

Role for Galectin-3 in Calcific Aortic Valve Stenosis.

BACKGROUND: Aortic stenosis (AS) is a chronic inflammatory disease, and calcification plays an important role in the progression of the disease. Galectin-3 (Gal-3) is a proinflammatory molecule involved in vascular osteogenesis in atherosclerosis. Therefore, we hypothesized that Gal-3 could mediate valve calcification in AS. METHODS AND RESULTS: Blood samples and aortic valves (AVs) from 77 patients undergoing AV replacement were analyzed. As controls, noncalcified human AVs were obtained at autopsy (n=11). Gal-3 was spontaneously expressed in valvular interstitial cells (VICs) from AVs and increased in AS as compared to control AVs. Positive correlations were found between circulating and valvular Gal-3 levels. Valvular Gal-3 colocalized with the VICs markers, alpha-smooth muscle actin and vimentin, and with the osteogenic markers, osteopontin, bone morphogenetic protein 2, runt-related transcription factor 2, and SRY (sex-determining region Y)-box 9. Gal-3 also colocalized with the inflammatory markers cd68, cd80 and tumor necrosis factor alpha. In vitro, in VICs isolated from AVs, Gal-3 induced expression of inflammatory, fibrotic, and osteogenic markers through the extracellular signal-regulated kinase 1 and 2 pathway. Gal-3 expression was blocked in VICs undergoing osteoblastic differentiation using its pharmacological inhibitor, modified citrus pectin, or the clustered regularly interspaced short palindromic repeats/Cas9 knockout system. Gal-3 blockade and knockdown decreased the expression of inflammatory, fibrotic, and osteogenic markers in differentiated VICs. CONCLUSIONS: Gal-3, which is overexpressed in AVs from AS patients, appears to play a central role in calcification in AS. Gal-3 could be a new therapeutic approach to delay the progression of AV calcification in AS.

Galectin-3 induces ovarian cancer cell survival and chemoresistance via TLR4 signaling activation.

Paclitaxel resistance becomes common in patients with aggressive ovarian cancer and results in recurrence after conventional therapy. Galectin-3 is a multifunctional lectin associated with cell migration, cell proliferation, cell adhesion, and cell-cell interaction in tumor cells. Whether circulating galectin-3 is involved in paclitaxel resistance in ovarian cancer remains unknown. The current study investigated the effect of galectin-3 on toll-like receptor 4 (TLR4) signaling and thus paclitaxel resistance. With blood and cancer tissue samples obtained from 102 patients, we identified associations between serum galectin-3 level or TLR4 expression and paclitaxel resistance phenotype. In vitro, treatment with exogenous galectin-3 restored cell survival and migration of SKOV-3 and ES-2 cells was decreased by galectin-3 silencing and paclitaxel treatment. Furthermore, exogenous galectin-3 boosted expression of TLR4, MyD88, and p-p65, as well as interleukin (IL)-6, IL-8, and vascular endothelial growth factor (VEGF) release induced by paclitaxel. Moreover, galectin-3 inhibited the interaction between TLR4 and caveolin-1 (Cav-1) in SKOV-3 and ES-2 cells. In addition, overexpression of Cav-1 dampened the expression of MyD88 and p-p65 stimulated by galectin-3 and enhanced apoptosis in SKOV-3 cells under paclitaxel exposure. In summary, our study elucidated that exogenous galectin-3 might induce paclitaxel resistance through TLR4 signaling activation by inhibiting TLR4-Cav-1 interaction, revealing a novel insight into paclitaxel resistance induction.

Galectin-3 modulates the EGFR signalling-mediated regulation of Sox2 expression via c-Myc in lung cancer.

Galectin-3 is a ubiquitous lectin exerting multiple cellular functions such as RNA splicing, protein trafficking and apoptosis. Its expression is positively correlated with the poor prognosis in lung cancer patients. Galectin-3 can promote cancer progression through its effects on cell proliferation, cell survival or cancer metastasis. However, the role of galectin-3 in the regulation of cancer stem-like cells (CSCs) is still unclear. Here, we investigated the hypothesis that galectin-3 might regulate lung CSCs via the EGF receptor (EGFR) signaling pathway. In our study, galectin-3 facilitated EGFR activation and enhanced the sphere formation activity of lung cancer cells. Furthermore, galectin-3 promoted Sox2 expression in an EGFR activation-dependent manner; importantly, forced expression of Sox2 blunted the effect of galectin-3 knockdown on lung cancer sphere formation ability. These results suggest that galectin-3 promotes EGFR activation leading to the upregulation of Sox2 expression and lung CSCs properties. Moreover, we showed that the carbohydrate-binding activity of galectin-3 was important for the regulation of EGFR activation, Sox2 expression and sphere formation. We have recently reported that c-Myc is a transcriptional activator of Sox2. We further found that galectin-3 enhanced c-Myc protein stability leading to increased c-Myc binding to the Sox2 gene promoter. We also examined the effect of the stemness factors, Oct4, Nanog and Sox2 on the expression of galectin-3. We found that Oct4 enhanced galectin-3 expression. Our results together suggest that galectin-3 enhances lung cancer stemness through the EGFR/c-Myc/Sox2 axis; Oct4, in turn, promotes galectin-3 expression, forming a positive regulatory loop in lung CSCs.

Galectins regulate the inflammatory response in airway epithelial cells exposed to microbial neuraminidase by modulating the expression of SOCS1 and RIG1.

Influenza patients frequently display increased susceptibility to Streptococcus pneumoniae co-infection and sepsis, the prevalent cause of mortality during influenza pandemics. However, the detailed mechanisms by which an influenza infection predisposes patients to suffer pneumococcal pneumonia are not fully understood. A murine model for influenza infection closely reflects the observations in human patients, since if the animals that have recovered from influenza A virus (IAV) sublethal infection are challenged with S. pneumoniae, they undergo a usually fatal uncontrolled cytokine response. We have previously demonstrated both in vitro and in vivo that the expression and secretion of galectin-1 (Gal1) and galectin-3 (Gal3) are modulated during IAV infection, and that the viral neuraminidase unmasks galactosyl moieties in the airway epithelia. In this study we demonstrate in vitro that the binding of secreted Gal1 and Gal3 to the epithelial cell surface modulates the expression of SOCS1 and RIG1, and activation of ERK, AKT or JAK/STAT1 signaling pathways, leading to a disregulated expression and release of pro-inflammatory cytokines. Our results suggest that the activity of the viral and pneumococcal neuraminidases on the surface of the airway epithelial cells function as a "danger signal" that leads to rapid upregulation of SOCS1 expression to prevent an uncontrolled inflammatory response. The binding of extracellular Gal1 or Gal3 to the galactosyl moieties unmasked on the surface of airway epithelial cells can either "fine-tune" or severely disregulate this process, respectively, the latter potentially leading to hypercytokinemia.