Clinical relevance of galectin-1 and galectin-3 in rheumatoid arthritis patients: Differential regulation and correlation with disease activity

Galectins, a family of animal lectins, play central roles in immune system regulation, shaping both innate and adaptive responses in physiological and pathological processes. These include rheumatoid arthritis (RA), a chronic multifactorial autoimmune disease characterized by inflammatory responses that affects both articular and extra-articular tissues. Galectins have been reported to play central roles in RA and its experimental animal models. In this perspective article we present new data highlighting the regulated expression of galectin-1 (Gal-1) and galectin-3 (Gal-3) in sera from RA patients under disease-modifying anti-rheumatic drugs (DMARDs) and/or corticoid treatment in the context of a more comprehensive discussion that summarizes the roles of galectins in joint inflammation. We found that Gal-1 levels markedly increase in sera from RA patients and positively correlate with erythrocyte sedimentation rate (ERS) and disease activity score 28 (DAS-28) parameters. On the other hand, Gal-3 is downregulated in RA patients, but positively correlates with health assessment questionnaire parameter (HAQ). Finally, by generating receiver-operator characteristic (ROC) curves, we found that Gal-1 and Gal-3 serum levels constitute good parameters to discriminate patients with RA from healthy individuals. Our findings uncover a differential regulation of Gal-1 and Gal-3 which might contribute to the anti-inflammatory effects elicited by DMARDs and corticoid treatment in RA patients.Fil: Mendez Huergo, Santiago Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Hockl, Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Stupirski, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Maller, Sebastian Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Morosi, Luciano Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pinto, Nicolás Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Berón, Ana M.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Musuruana, Jorge L.. Provincia de Santa Fe. Ministerio de Salud. Hospital J. B. Iturraspe; ArgentinaFil: Nasswetter, Gustavo Guillermo. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Cavallasca, Javier A.. Provincia de Santa Fe. Ministerio de Salud. Hospital J. B. Iturraspe; ArgentinaFil: Rabinovich, Gabriel Adrián. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Circulating galectin-1 delineates response to bevacizumab in melanoma patients and reprograms endothelial cell biology

Blockade of vascular endothelial growth factor (VEGF) signaling with bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), or with receptor tyrosine kinase inhibitors, has improved progression-free survival and, in some indications, overall survival across several types of cancers by interrupting tumor angiogenesis. However, the clinical benefit conferred by these therapies is variable, and tumors from treated patients eventually reinitiate growth. Previously we demonstrated, in mouse tumor models, that galectin-1 (Gal1), an endogenous glycan-binding protein, preserves angiogenesis in anti-VEGF–resistant tumors by co-opting the VEGF receptor (VEGFR)2 signaling pathway in the absence of VEGF. However, the relevance of these findings in clinical settings is uncertain. Here, we explored, in a cohort of melanoma patients from AVAST-M, a multicenter, open-label, randomized controlled phase 3 trial of adjuvant bevacizumab versus standard surveillance, the role of circulating plasma Gal1 as part of a compensatory mechanism that orchestrates endothelial cell programs in bevacizumab-treated melanoma patients. We found that increasing Gal1 levels over time in patients in the bevacizumab arm, but not in the observation arm, significantly increased their risks of recurrence and death. Remarkably, plasma Gal1 was functionally active as it was able to reprogram endothelial cell biology, promoting migration, tubulogenesis, and VEGFR2 phosphorylation. These effects were prevented by blockade of Gal1 using a newly developed fully human anti-Gal1 neutralizing mAb. Thus, using samples from a large-scale clinical trial from stage II and III melanoma patients, we validated the clinical relevance of Gal1 as a potential mechanism of resistance to bevacizumab treatment

Histopathological findings in <i>Lgals1</i>^<i>-/-</i> and WT mice at 19 dpi with <i>T</i>. <i>cruzi</i> Tulahuén strain.

<p>A) Microphotographs representative of heart and skeletal muscle histopathological abnormalities (H&E). Parasite density (B) and Inflammation Index (C) were calculated as indicated in the Methods section. Bars represent mean ± SEM of 5–7 mice per group. Statistical analysis was performed using Mann-Whitney U test. *<i>p</i><0.05. F: Female mice; M: Male mice.</p

Expression and release of Gal–1 in cultures of HL–1 cells infected with <i>T</i>. <i>cruzi</i>.

<p>Cells were infected with trypomastigotes of Tulahuén or Brazil strains, in a parasite:cell ratio of 5:1, and incubated for additional 2 or 5 days. A) Immunoblot analysis of Gal–1 expression in lysates from non-infected (a) and infected (b) HL–1 cells. Immunoreactive protein bands were semiquantified by densitometry. Results are expressed as Arbitrary Units (AU) relative to β-actin. B) RT-qPCR analysis of Gal–1 mRNA expression of non-infected and infected HL–1 cells. Results are expressed as relative to GAPDH mRNA. C) Detection of Gal–1 in the supernatant of non-infected and infected HL–1 using trypomastigotes of the Tulahuén and Brazil strains, as measured by ELISA. D) Detection of LDH activity in the supernatants of non-infected and infected HL–1 cells by using the LDH-UP kit (Weiner Lab, Argentina), following the manufacturer’s instructions. Results are expressed as Units/ml (U/ml). Data represent the mean ± SEM of three (A and B) and two (C and D) independent experiments. Statistical analysis was performed using Student’s <i>t</i> test for data shown in A (a <i>vs</i> b) and using one-way ANOVA followed by Tukey test in the remaining experiments. *<i>p</i><0.05; ***<i>p</i><0.001.</p

Effect of exogenous rGal–1 in <i>T</i>. <i>cruzi</i> infection.

<p>HL–1 cells were incubated with rGal–1 (10 and 50 μg/ml) for 24 h and then infected with trypomastigotes of both strains. After 4 dpi with <i>T</i>. <i>cruzi</i> Tulahuén (A) or Brazil strain (D), cells were fixed and stained with an anti-<i>T</i>. <i>cruzi</i> mouse serum. Representative images are shown in (B) and (E). Similar experiments were performed after 2 dpi with <i>T</i>. <i>cruzi</i> of the Tulahuén (C) or Brazil strains (F). In this case, some wells were treated with 100 mM lactose, added simultaneously with rGal–1. G) HL–1 cells transfected with pcDNA3-Gal–1 vector or empty vector (mock) were infected with trypomastigotes of both strains, in the presence or absence of 100 mM lactose. Cells were fixed and stained after 2 dpi, with an anti-<i>T</i>. <i>cruzi</i> mouse serum. In all cases, the percentage of infected cells was determined by counting an average of 3,500 cells in each slide on 3–5 distinct coverslips in randomly selected fields. Results are expressed as mean ± SEM of triplicates determinations from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001.</p

Parasitemia levels (A) and survival rate (B) of WT and <i>Lgals1</i>^<i>-/-</i> mice acutely infected with <i>T</i>. <i>cruzi</i> Tulahuén strain, via the intraperitoneal route.

<p>For parasitemia levels, each point represents the mean ± SEM of 5–15 animals per group, and statistical analysis was performed using Mann-Whitney U test. *<i>p</i><0.05, **<i>p</i><0.01 <i>vs</i>. WT mice; ^$<i>p</i><0.05, ^$$<i>p</i><0.01 <i>vs</i>. male mice. For survival rate, statistical analysis was achieved with Log-rank test.</p

Effect of Gal–1 on phosphatidylserine exposure in <i>T</i>. <i>cruzi</i> infected HL–1 cells.

<p>Cells were incubated with rGal–1 (10 and 50 μg/ml) for 18 h and, then infected with <i>T</i>. <i>cruzi</i>, Tulahuén (A) or Brazil (B) strains. Annexin V assay was performed at 3 dpi. Results expressed as mean ± SEM are representative of two independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey test. *<i>p</i><0.05; **<i>p</i><0.01. Only comparisons between infected groups were shown.</p

Binding of rGal–1 to <i>T</i>. <i>cruzi</i> trypomastigotes.

<p>A) Fluorescence assay of trypomastigotes incubated with rGal–1 (25 μg/ml) for 1 h, followed by incubation with a mouse anti-Gal–1 Ab labeled with Alexa Fluor 488. Staining with a rabbit polyclonal serum anti-Tc13, a surface protein presented in trypomastigotes, was used as positive control. B) Representative histograms of trypomastigotes of the Tulahuén or Brazil strain incubated with Gal-1-FITC (25 μg/ml). Red lines correspond to parasites treated with Gal-1-FITC, black lines to parasites incubated with streptavidin-FITC used as negative control.</p