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. 2023 Oct 24;14(1):72.
doi: 10.1186/s13293-023-00556-1.

Sex-specific role of galectin-3 in aortic stenosis

Lara Matilla #  1 , Ernesto Martín-Núñez #  1 , Mattie Garaikoetxea  1 , Adela Navarro  1 , Ibai Tamayo  2 , Amaya Fernández-Celis  1 , Alicia Gainza  1 , Joaquín Fernández-Irigoyen  3 , Enrique Santamaría  3 , Pieter Muntendam  4 , Virginia Álvarez  1 , Rafael Sádaba  1 , Eva Jover #  5 , Natalia López-Andrés #  6
Affiliations

Sex-specific role of galectin-3 in aortic stenosis

Lara Matilla et al. Biol Sex Differ. .

Abstract

Background: Aortic stenosis (AS) is characterized by inflammation, fibrosis, osteogenesis and angiogenesis. Men and women develop these mechanisms differently. Galectin-3 (Gal-3) is a pro-inflammatory and pro-osteogenic lectin in AS. In this work, we aim to analyse a potential sex-differential role of Gal-3 in AS.

Methods: 226 patients (61.50% men) with severe AS undergoing surgical aortic valve (AV) replacement were recruited. In AVs, Gal-3 expression and its relationship with inflammatory, osteogenic and angiogenic markers was assessed. Valve interstitial cells (VICs) were primary cultured to perform in vitro experiments.

Results: Proteomic analysis revealed that intracellular Gal-3 was over-expressed in VICs of male AS patients. Gal-3 secretion was also higher in men's VICs as compared to women's. In human AVs, Gal-3 protein levels were significantly higher in men, with stronger immunostaining in VICs with myofibroblastic phenotype and valve endothelial cells. Gal-3 levels in AVs were positively correlated with inflammatory markers in both sexes. Gal-3 expression was also positively correlated with osteogenic markers mainly in men AVs, and with angiogenic molecules only in this sex. In vitro, Gal-3 treatment induced expression of inflammatory, osteogenic and angiogenic markers in male's VICs, while it only upregulated inflammatory and osteogenic molecules in women-derived cells. Gal-3 blockade with pharmacological inhibitors (modified citrus pectin and G3P-01) prevented the upregulation of inflammatory, osteogenic and angiogenic molecules.

Conclusions: Gal-3 plays a sex-differential role in the setting of AS, and it could be a new sex-specific therapeutic target controlling pathological features of AS in VICs.

Keywords: Angiogenesis; Aortic stenosis; Calcification; Galectin-3; Inflammation; Sex differences; Valve interstitial cell.

Plain language summary

Aortic stenosis (AS) is a condition that affects the aortic valves (AVs) of the heart and leads to death if untreated. Males and females show clear differences in the onset of AS, both clinically and in valve deterioration. In this study we identified galectin-3 (Gal-3) as a molecule involved in the development of AS alterations with different effects in men and women. We analyzed AVs of 226 patients (139 male and 87 female) with severe AS who underwent surgical AV replacement to study the association of Gal-3 with markers of mechanisms related to AS, such as inflammation, calcification and blood vessels formation. We performed experiments in valvular interstitial cells (VICs) to evaluate the impact of Gal-3 in these cells and its potential use as a therapeutic target. Our results showed that Gal-3 was more expressed in AVs and VICs of men over women. In AVs, Gal-3 levels were associated with inflammatory markers either in male and female, while they correlated with osteogenic markers mainly in men and with angiogenic only in male. The treatment of VICs with Gal-3 produced increased levels of inflammatory and osteogenic molecules by cells of both sexes, but of angiogenic markers only in male’s. Pharmacological inhibition of Gal-3 prevented the increase of these pathological markers in VICs. Overall, our study indicates that Gal-3 is a molecule implicated in the setting of AS in a sex-differential way and its targeting may lead to a new sex-specific therapeutic option for AS treatment.

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Conflict of interest statement

NLA and PM report shareholder interest in G3-Pharmaceuticals. PM reports having received remuneration from G3-Pharmaceuticals.

Figures

Fig. 1
Fig. 1
Gal-3 is up-regulated in male VICs and AVs. Proteomic heatmap representing in red up-regulated proteins in men vs. women-derived VICs and in green down-regulated proteins (A). Levels of Gal-3 validated in VICs isolated from men and women AVs measured by WB (B) and ELISA (C). Levels of Gal-3 validated by ELISA in human AVs samples from males compared to females (D). Representative microphotographs immunostained for Gal-3 in AVs from men and women (E). Representative microphotographs double immunostained for Gal-3 and α-SMA (F), vimentin (G), CD31 (H) and VE-cadherin (I). Gal-3 galectin-3, CD31 cluster differentiation 31, SMA smooth muscle actin. N = 108 for men and N = 83 for women for AVs and N = 20 for men and N = 24 for women for VICs. *p < 0.05 vs men, **p < 0.01 vs men
Fig. 2
Fig. 2
Gal-3 is associated with inflammation in men and women AVs. Positive correlations between Gal-3 and IL-6 (A), CCL-2 (B), RANTES (C), ICAM-1 (D), CD44 (E) and CD68 (F) in AVs from AS patients. Representative microphotographs of AV sections from AS patients immunostained for Gal-3 along with CD68 (G), CD206 (H), CD45 (I) and CD80 (J). Protein levels of IL-6 (K) and CCL-2 (L) measured by ELISA after stimulation in vitro with Gal-3 and with MCP or G3P-01. IL interleukin, CCL-2 C–C motif chemolike ligand 2, ICAM-1 Intercellular Adhesion Molecule 1, CD cluster of differentiation, Gal-3 galectin-3. N = 108 AVs from men and N = 83 AVs from women. In vitro: number of biological replicates: 12 men and 12 women; number of technical replicates: 3–6
Fig. 3
Fig. 3
Gal-3 sex-differential effects on osteogenic markers. Positive correlations between Gal-3 and BMP-2 (A), BMP-9 (B), osteopontin (C) and osteocalcin (D) in AVs from men and women. Representative microphotographs of AV sections from AS patients immunostained for Gal-3 in combination with osteopontin (E), Runx2 (F), Sox-9 (G) and BMP-2 (H). Protein levels of osteopontin (I) and Sox-9 (J) measured by ELISA and WB respectively, after stimulation in vitro with Gal-3 and with MCP or G3P-01. BMP bone morphogenetic protein, Gal-3 galectin-3, Runx2 Runt-related transcription factor 2, Sox-9 SRY-Box Transcription Factor 9. N = 108 AVs from men and N = 83 AVs from women. In vitro: number of biological replicates: 12 men and 12 women; number of technical replicates: 3–6
Fig. 4
Fig. 4
Gal-3 exerts pro-angiogenic effects only in men. Positive correlations between Gal-3 and VEGF-A (A), FGF-7 (B), VEGFR3 (C) and Chemerin (D) in AVs from men and women. Representative microphotographs of AV sections from AS patients immunostained for Gal-3 in combination with VEGF-A (E) and VEGFR3 (F). Protein levels of VEGF-A (G) and FGF-7 (H) measured by ELISA after stimulation in vitro with Gal-3 and with MCP or G3P-01. Gal-3 galectin-3, VEGF vascular endothelial growth factor, FGF-7 fibroblast growth factor-7, VEGFR receptor of vascular endothelial growth factor. N = 108 AVs from men and N = 83 AVs from women. In vitro: number of biological replicates: 12 men and 12 women; number of technical replicates: 3–6
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