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Observational Study
. 2016 Nov 4;5(11):e004360.
doi: 10.1161/JAHA.116.004360.

Role for Galectin-3 in Calcific Aortic Valve Stenosis

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Free PMC article
Observational Study

Role for Galectin-3 in Calcific Aortic Valve Stenosis

J Rafael Sádaba et al. J Am Heart Assoc. .
Free PMC article

Abstract

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.

Keywords: aortic stenosis; calcification; galectin‐3; inflammation; valve; valvular interstitial cells.

Figures

Figure 1
Figure 1
Fibrosis, inflammation, and calcification in AS valves compared to controls. ECM components in AVs at mRNA (A) and protein (B) levels. mRNA levels of MMPs and its inhibitors (TIMPs) in AVs (C). MMPs activities in controls and AS valves (D). Inflammatory markers were measured in stenotic and control AVs at mRNA (E) and protein (F) levels. mRNA levels of calcification markers in AVs (G) and protein expressions of calcification markers in AVs (H). All conditions were performed at least in triplicate. Histogram bars represent the mean±SEM of each group of subjects (control n=11 and patients with severe calcific AS n=77) in arbitrary units (AU) normalized to HPRT and stain‐free gel for cDNA and protein, respectively. *P<0.05 versus control group. AS indicates aortic stenosis; AVs, aortic valves; BMP, bone morphogenetic protein; CCL2, C‐C motif chemokine ligand 2; Col1a1, collagen type I alpha 1 chain; Col3a1, collagen type III alpha 1 chain; CTGF, connective tissue growth factor; ECM, extracellular matrix; HPRT, hypoxanthine‐guanine phosphoribosyltransferase; IL, interleukin; MMPs, matrix metalloproteinases; Runx2, runt‐related transcription factor 2; Sox‐9, SRY (sex‐determining region Y)‐box 9; α‐SMA, alpha‐smooth muscle actin; TGF‐β, transforming growth factor beta; TIMPs, tissue inhibitors of matrix metalloproteinases; TNF‐α, tumor necrosis factor alpha.
Figure 2
Figure 2
Gal‐3 is associated with osteogenic markers in AS patients. Hematoxilin/eosin staining in AVs from controls and AS patients (A). Representative picture of an AV section from controls and AS patients stained with Gal‐3 at low (×10) and high (×40) magnification (B). Gal‐3 mRNA and protein expressions in AVs from patients (C) and correlation between valvular Gal‐3 protein levels and serum Gal‐3 levels (D). Representative pictures of AV sections stained with Gal‐3 (red) and α‐SMA, vimentin, osteopontin, BMP‐2, Runx2, Sox‐9, cd68, cd80, and TNF‐α (brown). Magnification, ×40 (E). All conditions were performed at least in triplicate. Histogram bars represent the mean±SEM of each group of patients (control n=11 and patients with severe calcific AS n=77) in arbitrary units (AU) normalized to HPRT and β‐actin for cDNA and protein respectively. *P<0.05 versus control group. AS indicates aortic stenosis; AVs, aortic valves; BMP, bone morphogenetic protein; Gal‐3, galectin‐3; HPRT, hypoxanthine‐guanine phosphoribosyltransferase; Runx2, runt‐related transcription factor 2; Sox‐9, SRY (sex‐determining region Y)‐box 9; α‐SMA, alpha‐smooth muscle actin; TGF‐β, transforming growth factor beta; TNF‐α, tumor necrosis factor alpha.
Figure 3
Figure 3
Gal‐3 induces inflammation, ECM components, and calcification markers in VICs. Effects of Gal‐3 on inflammatory markers, ECM components (A), and calcification markers (B) in VICs. Effects of Gal‐3 on phosphorylation of intracellular pathways in VICs (C). Effects of cell signaling chemical inhibitors on the proinflammatory and profibrotic effect of Gal‐3 in VICs (D). Effects of cell signaling chemical inhibitors on the pro‐osteogenic effect of Gal‐3 in VICs (E). All conditions were performed at least in triplicate. Histogram bars represent the mean±SEM of 6 assays in arbitrary units (AU) normalized to stain free for protein and to β‐actin and HPRT for cDNA. *P<0.05 versus control; $ P<0.05 vs Gal‐3. Akt indicates protein kinase B; BMP, bone morphogenetic protein; CCL2, C‐C motif chemokine ligand 2; Col1a1, collagen type I alpha 1 chain; ECM, extracellular matrix; ERK1/2, extracellular signal‐regulated kinase 1 and 2; Gal‐3, galectin‐3; HPRT, hypoxanthine‐guanine phosphoribosyltransferase; IL, interleukin; MAPK, mitogen‐activated protein kinase; NFκB, nuclear factor kappa B; Runx2, runt‐related transcription factor 2; Sox‐9, SRY (sex‐determining region Y)‐box 9; α‐SMA, alpha‐smooth muscle actin; TNF‐α, tumor necrosis factor alpha; VICs, valvular interstitial cells.
Figure 4
Figure 4
Gal‐3 inhibition reduces inflammation, ECM components, and calcification markers in VICs. Effects of the pharmacological inhibitor of Gal‐3, MCP, on inflammation, Gal‐3, fibronectin protein levels (A), and calcification markers (B) in VICs cultured in osteogenic medium. Effects of the Gal‐3 knockout (KO) on inflammation, Gal‐3, fibronectin protein levels (C), and calcification markers (D) in VICs cultured in osteogenic medium. All conditions were performed at least in triplicate. Histogram bars represent the mean±SEM of 4 assays in arbitrary units (AU) normalized to stain free for protein. *P<0.05 versus control. BMP, bone morphogenetic protein; ECM, extracellular matrix; Gal‐3, galectin‐3; IL, interleukin; MCP, modified citrus pectin; Runx2, runt‐related transcription factor 2; Sox‐9, SRY (sex‐determining region Y)‐box 9; VICs, valvular interstitial cells.

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References

    1. Iung B, Baron G, Butchart EG, Delahaye F, Gohlke‐Barwolf C, Levang OW, Tornos P, Vanoverschelde JL, Vermeer F, Boersma E, Ravaud P, Vahanian A. A prospective survey of patients with valvular heart disease in Europe: the Euro Heart Survey on Valvular Heart Disease. Eur Heart J. 2003;24:1231–1243. - PubMed
    1. Danielsen R, Aspelund T, Harris TB, Gudnason V. The prevalence of aortic stenosis in the elderly in iceland and predictions for the coming decades: the AGES‐Reykjavik study. Int J Cardiol. 2014;176:916–922. - PMC - PubMed
    1. Otto CM, Burwash IG, Legget ME, Munt BI, Fujioka M, Healy NL, Kraft CD, Miyake‐Hull CY, Schwaegler RG. Prospective study of asymptomatic valvular aortic stenosis. Clinical, echocardiographic, and exercise predictors of outcome. Circulation. 1997;95:2262–2270. - PubMed
    1. Bosse Y, Miqdad A, Fournier D, Pepin A, Pibarot P, Mathieu P. Refining molecular pathways leading to calcific aortic valve stenosis by studying gene expression profile of normal and calcified stenotic human aortic valves. Circ Cardiovasc Genet. 2009;2:489–498. - PubMed
    1. Mohler ER III, Gannon F, Reynolds C, Zimmerman R, Keane MG, Kaplan FS. Bone formation and inflammation in cardiac valves. Circulation. 2001;103:1522–1528. - PubMed

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