Galectin-1 prevents pathological vascular remodeling in atherosclerosis and abdominal aortic aneurysm

Abstract

Pathological vascular remodeling is the underlying cause of atherosclerosis and abdominal aortic aneurysm (AAA). Here, we analyzed the role of galectin-1 (Gal-1), a β-galactoside-binding protein, as a therapeutic target for atherosclerosis and AAA. Mice lacking Gal-1 (Lgals1^-/-) developed severe atherosclerosis induced by pAAV/D377Y-mPCSK9 adenovirus and displayed higher lipid levels and lower expression of contractile markers of vascular smooth muscle cells (VSMCs) in plaques than wild-type mice. Proteomic analysis of Lgals1^-/- aortas showed changes in markers of VSMC phenotypic switch and altered composition of mitochondrial proteins. Mechanistically, Gal-1 silencing resulted in increased foam cell formation and mitochondrial dysfunction in VSMCs, while treatment with recombinant Gal-1 (rGal-1) prevented these effects. Furthermore, rGal-1 treatment attenuated atherosclerosis and elastase-induced AAA, leading to higher contractile VSMCs in aortic tissues. Gal-1 expression decreased in human atheroma and AAA compared to control tissue. Thus, Gal-1-driven circuits emerge as potential therapeutic strategies in atherosclerosis and AAA.

Fig. 1.. Endogenous Gal-1 controls development of atherosclerosis.

(A) Representative immunohistochemistry of GAL-1 in atherosclerotic plaques and healthy aortas. Scale bars, 100 μm. (B) Determination of LGALS1 and (C) α-SMA mRNA in atherosclerotic plaques (intima layer, n = 7) versus healthy human control aortic samples (media layer, n = 7). (D) Western blot analysis of GAL-1 in atherosclerotic plaques (intima layer, n = 14) and healthy aortic (media layer, n = 14) tissue-conditioned medium. (E) Representative ORO staining in the whole aorta of WT (n = 5) and Lgals1^−/− (n = 5) mice harvested at 16 weeks of AAV-PCSK9 infection and high-fat diet. (F) Representative ORO/hematoxylin staining in aortic root of WT (n = 10) and Lgals1^−/− (n = 12) mice at 16-week harvest after AAV-PCSK9 infection and high-fat diet. Scale bars, 200 μm. Data are means ± SEM. *P < 0.05 or **P < 0.01, Mann-Whitney U test. D.A.U., densitometric arbitrary units.

Fig. 2.. Gal-1 deletion increases atherosclerotic plaque instability.

Representative photographs and quantification of ORO (A), Sirius red [bright field (B) and polarized (C)], α-SMA (D), and CD68 (E) staining in the aortic root from WT (n = 10) and Lgals1^−/− (n = 12) mice. Values are means ± SEM. *P < 0.05, Mann-Whitney U test. m, media layer. Scale bars, 100 μm. (F) Representative images and quantification of peritoneal macrophages isolated from WT or Lgals1^−/− mice and incubated with Dil-oxidized LDL (10 μg/ml) for 4 hours with or without rGal-1 (1 μg/μl; 4-hour preincubation). Data are means ± SEM of three independent experiments. *P < 0.05, WT versus WT + rGal-1 or Lgals1^−/− versus Lgals1^−/− + rGal-1; **P < 0.01, Lgals1^−/− versus WT, ANOVA test followed by Tukey’s test. Scale bars, 50 μm.

Fig. 3.. Altered mitochondria and phenotypic switch in VSMCs from Gal-1–deficient mice.

(A) Proteomic analysis of aortic homogenates from Gal-1–deficient versus WT mice. Enrichment analysis of proteins differentially expressed in aorta from WT (n = 9) or Lgals1^−/− [knockout (KO); n = 7] mice showing biological processes significantly altered (FDR < 5%). The heatmap shows the average of protein abundance changes (Zq) in WT and KO animals in relation to the value in WT mice. Increased (red) or decreased (blue) expression is expressed according to the indicated Zq scale. Zq is measured in SD units, and ΔZq is calculated as the difference between the average of Zq in KO and WT samples. Statistical significance of changes (P value) is calculated using two-tailed Student’s t test. (B) Relative Nampt, mt-Nd6, and mt-Nd1 mRNA expression normalized to 18S/Gapdh, as well as complex 1 activity, in VSMCs transfected with Gal-1–siRNA or scrambled siRNA. (C) Relative Fn1, Vcam-1, α-Sma, and Klf4 mRNA expression normalized to 18S/Gapdh mRNA of VSMCs transfected with Gal-1–siRNA or scrambled siRNA for 48 hours and treated with TNF (100 ng/ml) for 24 hours. Data are means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test.

Fig. 4.. Gal-1 alters mitochondrial fitness in VSMCs.

(A) Relative Co-1, Sdh, Tfam, and Ppar-α mRNA expression normalized to 18S/Gapdh in VSMCs transfected with Gal-1–siRNA or scrambled siRNA. (B) Graphs depicting the OCR and extracellular lactate levels in VSMCs transfected with scrambled siRNA or Gal-1–siRNA exposed or not to rGal-1 (1 μg/μl, 48 hours). (C) Graphs depicting the OCR and extracellular lactate levels in VSMCs incubated with oxLDL (0.1 mg/ml, 48 hours) with or without rGal-1 (1 μg/μl, 48 hours). Data are means ± SEM of five to six independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test [for (A)] and ANOVA test followed by Tukey’s test [for (B) and (C)].

Fig. 5.. Treatment with recombinant Gal-1 limits the severity of atherosclerosis and enhances atherosclerotic plaque stability in mice.

(A) Representative ORO staining in whole aorta of saline-treated (n = 5) or rGal-1–treated (n = 10) mice at 16-week harvest after AAV-PCSK9 infection and high-fat diet. (B) Representative ORO/hematoxylin staining in the aortic root of WT mice treated with rGal-1 (n = 16) compared with saline-treated WT mice (n = 12) at 16-week harvest after AAV-PCSK9 infection and high-fat diet. Scale bars, 200 μm. (C to G) Representative photographs and quantification of ORO (C), Sirius red [bright field (D) and polarized (E)], α-Sma (F), and CD68 (G) staining in the aortic root of WT mice treated with rGal-1 (n = 16) compared with WT mice saline-treated (n = 12) at 16-week harvest after AAV-PCSK9 infection and high-fat diet. Scale bars, 100 μm. Values are means ± SEM. *P < 0.05 or **P < 0.01, Mann-Whitney U test.

Fig. 6.. Treatment with recombinant Gal-1 mitigates AAA development.

(A) Representative immunohistochemistry of GAL-1 expression in the wall of human AAA patients and control aorta. Scale bar, 100 μm. (B) GAL-1 expression was determined by Western blot in conditioned medium from the AAA wall (n = 11) and healthy aortic tissue (n = 9). (C and D) Determination of LGALS1 mRNA (C) and α-SMA (D) mRNA in the wall of human AAA tissues (n = 8) versus healthy human control aortic samples (n = 9). (E) Determination of Lgals1 mRNA in the wall of elastase-induced AAA at day 3 (n = 8) and at day 14 (n = 10) versus healthy aortic samples (n = 6). (F) Representative Masson’s trichrome staining and quantification of the increment of aortic diameter at 14 days after perfusion of elastase in saline-treated (n = 9) and Gal-1–treated (rGal-1, n = 7) mice. Scale bars, 200 μm. Representative α-Sma and CD68 staining and quantification at 14 days after perfusion of elastase in saline-treated (n = 9) and Gal-1–treated (rGal-1, n = 7) mice. Scale bars, 200 μm. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Mann-Whitney U test.

Fig. 7.. Proposed model: Role of Gal-1 in pathological vascular remodeling.

The development of atherosclerosis and AAA is associated with down-regulation of Gal-1 expression. Treatment with rGal-1 attenuated both PCSK9-AAV–induced atherosclerosis and elastase-induced AAA. The mechanisms underlying these protective effects involve modulation of foam cell formation and control of mitochondria functionality in VSMCs, preventing the loss of contractile VSMC phenotype.