SCIENCE ADVANCES | RESEARCH ARTICLE DISEASES AND DISORDERS Copyright © 2022 The Authors, some Galectin-1 prevents pathological vascular remodeling rights reserved; exclusive licensee in atherosclerosis and abdominal aortic aneurysm American Association for the Advancement of Science. No claim to Raquel Roldán-Montero1,2, Juan M. Pérez-Sáez3, Isabel Cerro-Pardo1, Jorge Oller4,5, original U.S. Government Diego Martinez-Lopez1, Estefania Nuñez6, Sebastian M. Maller3, Carmen Gutierrez-Muñoz1, Works. Distributed Nerea Mendez-Barbero1,2, Joan C. Escola-Gil7,8, Jean-Baptiste Michel9, Maria Mittelbrunn4,5, under a Creative Jesús Vázquez2,6, Luis M. Blanco-Colio1,2, Gabriel A. Rabinovich3,10*†, Jose L. Martin-Ventura1,2*† Commons Attribution NonCommercial Pathological vascular remodeling is the underlying cause of atherosclerosis and abdominal aortic aneurysm (AAA). License 4.0 (CC BY-NC). Here, we analyzed the role of galectin-1 (Gal-1), a -galactoside–binding protein, as a therapeutic target for athero- sclerosis 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. INTRODUCTION and other ECM proteins, which, along with VSMC loss, favor aortic Cardiovascular (CV) diseases are the leading cause of global mortal- dilation and potential rupture (5). ity and a major contributor to disability (1). Among them, athero- Galectins, a family of ancient -galactoside–binding proteins, can sclerosis and aortic aneurysms are characterized by a pathological reprogram immune and vascular programs through intracellular remodeling of the vascular wall that could progress to plaque and/ and extracellular mechanisms (6). Intracellularly, galectins can reg- or aortic wall rupture and eventually to CV events and/or deaths. ulate signaling pathways, innate defense mechanisms, and splicing. Treatment of both atherosclerosis and aneurysms is only based on Extracellularly, they can elicit various cellular responses through the control of different CV risk factors, highlighting the need to find glycan-dependent circuits, including modulation of cytokine pro- potential therapeutic targets to prevent the devastating effects of duction, cell adhesion, angiogenesis, and cell death (7). Through these vascular rupture (2). mechanisms, galectins can control inflammation, wound healing, The main driver of atherosclerosis is lipid retention and modifi- antimicrobial immunity, and tumor-immune escape (6–8). Specifi- cation, mainly by oxidation, leading to foam cell formation, which cally, galectin-1 (Gal-1), a prototype member of the galectin family, characterizes the early atherosclerotic lesions. Progression to inter- promotes resolution of acute and chronic inflammation by recali- mediate fibrolipidic lesions is fueled by different processes, including brating innate and adaptive immune responses via glycosylation-­ phenotypic modulation of vascular smooth muscle cells (VSMCs) dependent pathways (9, 10). However, despite considerable progress characterized by decreased contractile function, higher proliferative in elucidating the role of Gal-1 in immunopathology (7), its contri- activity, and increased extracellular matrix (ECM) production (3). bution to CV diseases is just emerging (11). Mice lacking Gal-1 Last, failure of resolution programs, engagement of cell death path- showed enhanced cardiac inflammation during acute myocardial ways leading to necrotic core formation, and degradation of ECM infarction (AMI), and administration of recombinant Gal-1 (rGal-1) favor the instability of atherosclerotic plaques and facilitate their attenuated AMI-associated inflammatory responses (12). rupture (4). Abdominal aortic aneurysm (AAA) shares many of In this study, we identify a central role of Gal-1 in atherosclerotic these pathological processes, with a predominant role of proteolysis. plaque size, burden, and stability in vivo through mechanisms Proteases released by infiltrating or resident cells may degrade elastin involving modulation of foam cell formation and mitochondrial functionality associated to VSMC phenotypic switch. Moreover, we demonstrate the potential therapeutic role of rGal-1 in both athero- 1 sclerosis and AAA. In addition, we validate the relevance of Gal-1 IIS-Fundación Jiménez-Díaz-Autonoma University of Madrid (UAM). Madrid, Spain. 2 CIBER de Enfermedades Cardiovasculares (CIBERCV), Madrid, Spain. 3Laboratorio in human vascular pathologies by investigating its regulated expres- de Glicomedicina, Instituto de Biología y Medicina Experimental (IBYME), Consejo sion in human atherosclerotic plaques and AAA lesions. Nacional de Investigaciones Científicas y Técnicas (CONICET), C1428ADN Buenos Aires, Argentina. 4Centro de Biología Molecular Severo Ochoa, Centro Superior de Investigaciones Científicas-UAM, Madrid, Spain. 5Instituto de Investigación del Hospital 12 de Octubre, Madrid, Spain. 6Centro Nacional de Investigaciones Cardio- RESULTS vasculares Carlos III, Madrid, Spain. 7IIB Sant Pau, Barcelona, Spain. 8CIBERDEM, Gal-1 limits development of atherosclerosis Madrid, Spain. 9Inserm U1148, Paris, France. 10Facultad de Ciencias Exactas y Naturales, To study the relevance of Gal-1 in chronic vascular pathologies, we Universidad de Buenos Aires, C1428AGE Buenos Aires, Argentina. *Corresponding author. Email: jlmartin@fjd.es (J.L.M.-V.); gabyrabi@gmail.com (G.A.R.) first analyzed Gal-1 expression, at both the mRNA and protein levels, †These authors contributed equally to this work as co-senior authors. in human atherosclerotic plaques compared with healthy aortic Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 1 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE wall samples. Gal-1 staining was prominent in the tunica media To further study the effects of Gal-1 deletion in serum cholesterol of control aortas mainly composed of -smooth muscle actin levels, we analyzed mRNA expression of the main receptors involved (-SMA)–positive VSMCs, while it was markedly decreased in in LDL and VLDL clearance. No significant differences in mRNA human atherosclerotic plaques (Fig. 1A). Decreased LGALS1 and expression of both receptors (Ldlr or Vldlr) could be observed in -SMA mRNA was verified by quantitative polymerase chain liver homogenates of different mouse strains (fig. S1). To gain fur- reaction (qPCR) analysis of human atherosclerotic plaques (Fig. 1, ther mechanistic insights, we next performed an unbiased proteomic B and C). In addition, secreted Gal-1 was also decreased in condi- study of liver homogenates from WT and Lgals1−/− mice. Enrich- tioned medium from atherosclerotic plaques when compared to ment analysis of the biological processes that were significantly control aortas (Fig. 1D). altered [false discovery rate (FDR) < 5%] between these genotypes To investigate the role of endogenous Gal-1 in atherosclerosis, revealed subtle changes in heme-binding proteins (e.g., cytochrome c we analyzed the effect of Gal-1 deletion in a hyperlipidemic model and hemopexin); however, we did not observe any major alteration of advanced atherosclerosis induced by pAAV/D377Y-mPCSK9 in key proteins involved in cholesterol biosynthetic pathways (fig. S1). adenovirus injection along with high-fat diet for 16 weeks (13). We To further elucidate possible mechanisms underlying the role of first performed a pilot study in wild-type (WT) versus Lgals1−/− endogenous Gal-1 in CV pathology, we compared atherosclerotic le- mice (n = 5 each group) and analyzed the atherosclerotic burden by sion size in the aortic root in Lgals1−/− versus WT mice. Gal-1–deficient enface analysis of the whole aorta. As observed by oil red O (ORO) mice exhibited significantly increased plaque size compared with their staining, Lgals1−/− mice showed a 164% increase of lesion area of the WT counterpart (Fig. 1F). Moreover, as plaque composition is a main aorta as compared to WT mice (Fig. 1E). We then performed a larger determinant of atherosclerosis progression and rupture, we then experiment in WT (n = 10) and Lgals1−/− (n = 12) mice under the analyzed different markers of plaque instability including lipids and same experimental conditions. No differences were observed in collagen content, necrotic core size, as well as -SMA and CD68 ex- body weights between both genotypes at the end of the experiment pression in atherosclerotic plaques. Mice lacking Gal-1 exhibited higher [WT = 33.9 ± 1.0 g versus Lgals1−/− = 35.8 ± 0.9 g, P = not significant lipid content in lesions compared with WT mice (Fig. 2A), while no (NS)]; however, increased levels of total cholesterol and very low significant changes were observed in the necrotic core area (Fig. 2B) density lipoprotein (VLDL) and low-density lipoprotein (LDL) or collagen content (Fig. 2C) between both genotypes. Expression cholesterol were observed in sera from mice lacking Gal-1 (fig. S1). of -SMA and calponin (two markers of contractile VSMCs) was A Control Atherosclerosis B C 20 200 -SMA/GADPH A.U. GAL-1/GADPH A.U. 15 150 10 100 5 50 0 0 Control Atherosclerosis Control Atherosclerosis D E F Control Atherosclerosis kDa 20 1,000,000 60 15 800,000 Lesion size (µm2) GAL-1 (D.A.U.) 40 600,000 10 % Area 400,000 5 20 200,000 0 0 0 WT Lgals1−/− Control Atherosclerosis WT Lgals1−/− Fig. 1. Endogenous Gal-1 controls development of atherosclerosis. (A) Representative immunohistochemistry of GAL-1 in atherosclerotic plaques and healthy aortas. Scale bars, 100 m. (B) Determination of LGALS1 and (C) -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 m. Data are means ± SEM. *P < 0.05 or **P < 0.01, Mann-Whitney U test. D.A.U., densitometric arbitrary units. Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 2 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE A F B C D 15 10 % -Sma 5 0 WT Lgals1−/− E Fig. 2. Gal-1 deletion increases atherosclerotic plaque instability. Representative photographs and quantification of ORO (A), Sirius red [bright field (B) and polarized (C)], -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 m. (F) Representative images and quantification of peritoneal macrophages isolated from WT or Lgals1−/− mice and incubated with Dil-oxidized LDL (10 g/ml) for 4 hours with or without rGal-1 (1 g/l; 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 m. substantially reduced in Lgals1−/− compared with control mice (Fig. 2D Enrichment analysis of the biological processes that were significantly and fig. S2), while CD68 staining was similar in both mouse genotypes changed (FDR < 5%) revealed two major pathways differentially regu- (Fig. 2E). To gain further insights into the mechanisms underlying lated in aorta of WT versus Lgals1−/− mice, including up-regulation higher lipid retention inside the vascular wall of atherosclerotic Gal-1– of cell adhesion pathways and down-regulation of lipid metabolic pro- deficient mice, and given the association of ORO–positive cells with cesses (Fig. 3A). In addition, enrichment analysis of cellular compo- CD68+ cells (Fig. 2, A and E), we analyzed the ability of isolated peri- nents revealed up-regulation of extracellular space mediators and toneal macrophages from Lgals1−/− and WT mice to take up fluo- down-regulation of mitochondrial proteins (fig. S3). Integrated anal- rescently labeled Dil-oxidized LDL (10 g/ml, 4 hours). An increased ysis of biological processes and cellular components revealed a de- number of Gal-1–deficient macrophages accumulated greater lipid crease of several proteins implicated in mitochondrial structure and content as compared to WT macrophages (Fig. 2F). Moreover, treat- function. To further investigate a possible direct effect of Gal-1 deletion ment with rGal-1 (1 g/ml; 4-hour preincubation) prevented in- in the expression of these genes, primary culture VSMCs were trans- creased Dil-oxidized LDL uptake elicited by either Gal-1–deficient fected with Gal-1 small interfering RNA (siRNA) (~50 to 70% of or WT macrophages (Fig. 2F). Thus, Gal-1 controls the severity of Lgals1 mRNA reduction). Consistent with the proteomic data, mt-Nd6 atherosclerosis and influences lipid content and foam cell formation. and Nampt mRNA expression was down-regulated in Gal-1–silenced VSMCs (Fig. 3B). Notably, MT-ND6 together with MT-ND1-5 are Gal-1 controls mitochondrial function and phenotypic subunits of the NADH (reduced form of nicotinamide adenine di- switch of VSMCs nucleotide) dehydrogenase (ubiquinone), an enzyme also known as Given the atheroprotective effect of Gal-1, we performed a global pro- complex I (14). In agreement, both mt-Nd1 mRNA expression and teomic approach in aorta homogenates from Lgals1−/− and WT mice. complex I activity were also decreased in Gal-1–silenced VSMCs (Fig. 3B). Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 3 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE A B Complex I 0.00060 0.00055 0.00050 mOD/min 0.00045 0.00040 0.00035 0.00030 siRNAc siRNA Gal-1 C 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 signifi- cantly 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 Zq 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 ex- pression 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, -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. When we analyzed proteins that were significantly up-regulated VSMC phenotypic modulation, such as -SMA and KLF4 (indicating in biological processes, fibronectin (Fn1) and vascular cell adhesion contractile and synthetic phenotypes, respectively). -Sma mRNA molecule-1 (Vcam-1) showed the highest differences between both expression was substantially down-regulated, while Klf4 mRNA genotypes. Consistent with the proteomic data, both Fn1 and Vcam1 was up-regulated in Gal-1–silenced VSMCs (Fig. 3C). mRNA expression were increased in Gal-1–silenced VSMCs after To gain further insights into the role of Gal-1 in regulating mito- tumor necrosis factor (TNF) stimulation (Fig. 3C). Because Fn1 and chondrial fitness in VSMCs, we tested the effect of Gal-1 on mito- VCAM-1 have been previously associated with a VSMC phenotype chondrial genes and function. We found that Gal-1–silenced VSMCs switch in atherosclerosis (3, 15–19) and loss of VSMC contractile displayed a decrease in the transcription of genes encoding mito- phenotype has been associated with altered mitochondrial biology chondrial complexes (mt-CO1 and mt-SDH) and those implicated and a metabolic shift to glycolysis (20), we further analyzed the in mitochondrial function (TFAM and PPAR) (Fig. 4A). When we impact of Gal-1 deletion in the regulation of markers associated to analyzed cellular metabolism, we found that oxygen consumption Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 4 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE A B 60 OCR (pmol/min/norm. unit) siRNAc siRNA Gal-1 Basal respiration Maximal respiration 40 siRNA Gal-1 + rGal-1 40 60 2.5 OCR (pmol/min/norm. unit) OCR (pmol/min/norm. unit) 2.0 30 Lactate levels 40 1.5 20 20 1.0 20 10 0.5 0 0 0.0 0 siRNAc siRNA Gal-1 siRNA Gal-1 + rGal-1 siRNAc siRNA Gal-1 siRNA Gal-1 + rGal-1 siRNAc siRNA Gal-1 siRNA Gal-1 + rGal-1 0 20 40 60 Time (min) C 100 OCR (pmol/min/norm. unit) Control 80 oxLDL Maximal respiration Basal respiration oxLDL + rGal-1 60 150 2.0 OCR (pmol/min/norm. unit) OCR (pmol/min/norm. unit) 60 1.5 Lactate levels 40 100 40 1.0 20 50 20 0.5 0 0 Control OxLDL OxLDL + rGal-1 0 Control OxLDL OxLDL + rGal-1 0.0 Control OxLDL OxLDL + rGal-1 0 10 20 30 40 50 Time (min) Fig. 4. Gal-1 alters mitochondrial fitness in VSMCs. (A) Relative Co-1, Sdh, Tfam, and Ppar- 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 g/l, 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 g/l, 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)]. rate (OCR) was significantly decreased in Gal-1–silenced VSMCs vehicle-treated and rGal-1–treated mice, respectively. Moreover, when (Fig. 4B). Moreover, heightened glycolysis, as measured by the we compared atherosclerotic lesion size in the aortic root, mice extracellular lactate levels, was increased in Gal-1–silenced VSMCs treated with rGal-1 exhibited significantly reduced plaque size com- (Fig. 4B). Given these findings, we then investigated whether rGal-1 pared with mice treated with vehicle control (Fig. 5B). We then could alleviate mitochondrial dysfunctionality in Gal-1–silenced measured different markers of plaque stability in both groups. VSMCs or in VSMCs incubated with oxidized LDL (oxLDL; 0.1 mg/ml No changes were observed for lesional lipid or collagen content for 48 hours) (Fig. 4, B and C). Of note, exposure to rGal-1 or its (Fig. 5, C and E), while a reduction in necrotic core area was evident vehicle -mercaptoethanol (contained in Gal-1 preparation) did not in mice treated with rGal-1 (Fig. 5D).-SMA and calponin staining alter LDL oxidation (fig. S4). Treatment with rGal-1 (1 g/ml) pre- was greater in mice treated with rGal-1 compared with those treated vented both the decreased OCR and the increased extracellular lactate with vehicle control (Fig. 5F and fig. S2), while CD68 staining was production, induced by Gal-1 siRNA or oxLDL (Fig. 4, B and C). similar in both mouse groups (Fig. 5G). Thus, Gal-1 controls vascular remodeling through mechanisms To investigate whether the protective effects of Gal-1 in advanced involving modulation of mitochondrial function and phenotypic atherosclerosis were also verified in other conditions involving switch of VSMCs. pathological vascular remodeling, we further explored the role of this lectin in AAA. We first analyzed Gal-1 expression in human Treatment with rGal-1 mitigates atherosclerotic plaque AAA tissue samples. Our findings revealed a reduction of Gal-1, formation and AAA development along with -SMA staining, when comparing the media layer of Given the deleterious effect of Gal-1 deletion in the stability of AAA with that of healthy aorta (Fig. 6A). Soluble Gal-1 was also advanced atherosclerotic plaques, and the beneficial effect of rGal-1 decreased in AAA wall tissue-conditioned medium compared to on foam cell formation and VSMC mitochondrial function, we then control aortas (Fig. 6B). Further analysis of mRNA expression con- studied the potential therapeutic effects of rGal-1 administration firmed down-regulation of LGALS1 and -SMA mRNA expression during the development of vascular pathology. As observed by ORO in human AAA tissues (Fig. 6, C and D). Lgals1 mRNA was also staining, in vivo supplementation with rGal-1 resulted in a 34% re- decreased in aortic walls obtained from elastase-induced AAA as duction of lesion area in the aorta as compared to administration of compared with control aortas (Fig. 6E). Moreover, treatment with vehicle control (Fig. 5A). However, no differences were observed in rGal-1 (100 g/3 days a week) led to smaller aortic dilation and body weights (44.1 ± 0.8 versus 43.5 ± 0.9 g, P = NS) or lipid content higher contractile VSMC content, as shown by -SMA staining and (total cholesterol = 38.6 ± 6.3 versus 43.6 ± 8.5 mM; VLDL-LDL calponin, compared with vehicle-treated mice (Fig. 6F and fig. S2), cholesterol = 36.5 ± 6.2 versus 42.1 ± 8.4 mM, P = NS) between while no significant changes were observed in CD68 expression Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 5 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE A Vehicle rGal-1 50 40 ** 30 C D Sirius E Sirius F G % Area 20 Oil red O (bright field) (polarized) Sma CD68 10 0 Vehicle rGal-1 Vehicle B m Vehicle rGal-1 m rGal-1 800,000 ** ** Lesion size (um2) 600,000 400,000 ** 200,000 0 Vehicle rGal-1 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 infec- tion and high-fat diet. Scale bars, 200 m. (C to G) Representative photographs and quantification of ORO (C), Sirius red [bright field (D) and polarized (E)], -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 m. Values are means ± SEM. *P < 0.05 or **P < 0.01, Mann-Whitney U test. (Fig. 6F). These results highlight the therapeutic value of rGal-1 supple- expression not only varies between human and mouse AAA but also mentation in chronic pathologies involving vascular remodeling. fluctuates among different AAA experimental models (27). It is well known that atherosclerosis and AAA progression are linked to a decrease in VSMC content and/or by loss of contractile markers of DISCUSSION VSMCs (15–18, 28). Thus, whether the overall decreased expression The dual role of Gal-1 in modulating inflammatory and vascular of Gal-1 in human atherosclerotic tissue or AAA is associated to a responses (6, 10, 21, 22) prompted us to investigate the contribution decreased content of VSMC and -SMA expression or whether it is of this lectin to pathological vascular remodeling in atherosclerosis due to a protective effect of this lectin in pathological vascular re- and AAA. We demonstrated through a global loss-of-function modeling is an intriguing question. approach that Gal-1 deficiency results in increased atherosclerotic To evaluate the relevance of Gal-1 in advanced atherosclerosis, burden and instability. Treatment with rGal-1 protein prevents de- we used an atherosclerotic model based on pAAV/D377Y-mPCSK9 velopment and instability of atherosclerotic plaques and AAA. Last, injection along with high-fat diet for 16 weeks, which increases Gal-1 expression was down-regulated in human atherosclerotic circulating lipid levels by altering LDLr recycling and, consequently, plaques and AAA. generates lipid-rich atherosclerotic plaques (29). Atherosclerosis is Expression of Gal-1 was initially observed in human coronary a systemic diffuse disease, which affects different territories, sup- atherosclerotic lesions associated to intimal proliferating VSMCs porting the importance of analyzing the global atherosclerotic burden (23) and later detected in murine atherosclerotic lesions, in both the (30). We measured the atherosclerotic plaques along the whole aorta media and intima layers (24). More recently, expression of this lectin by enface analysis, showing a higher atherosclerotic burden in Gal-1– was identified in endothelial cells and VSMCs in normal vessels of deficient mice as compared to WT mice. Unexpectedly, Lgals1−/− mice control mice and also in intimal VSMCs of injured vessels obtained displayed higher systemic cholesterol levels under hyperlipidemic after angioplasty in a mice carotid artery ligation model (25). Here, conditions. Thus, we studied whether main receptors involved in we identified high Gal-1 expression in healthy aortas, mainly com- liver lipoprotein clearance could be altered under our experimental posed of VSMCs, while atherosclerotic plaques and AAA showed conditions; however, no differences were found on LDLr or VLDLr lower expression. Although previous studies have not analyzed Gal-1 mRNA expression. Furthermore, nontargeted proteomic analysis expression in human AAA tissues, it was recently reported that this of liver samples of Lgals1−/− and WT mice showed no major changes lectin is significantly elevated in aortic tissues of angiotensin II– in lipid metabolic processes. Thus, further analysis should be per- infused AAA in mice (26). In contrast, we have shown a decrease formed to evaluate the potential role of Gal-1 in liver VLDL assembly in Gal-1 expression in elastase-induced AAA in mice. Thus, Gal-1 and secretion under hyperlipidemic conditions. Last, we also found Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 6 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE A C D E Control AAA 1.5 * Gal-1/Gadph A.U. GAL-1 * 1.0 0.5 ** 0.0 Healthy AAA day 3 AAA day 14 α-SMA F Masson α-Sma CD68 Vehicle B Control AAA 14.5 kDa (Gal-1) rGal-1 *** 400 15 % Aortic diameter increase * 300 * 10 % CD68 200 5 100 0 0 Vehicle rGal-1 Vehicle rGal-1 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 m. (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 -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 m. Representative -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 m. Values are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Mann-Whitney U test. an increase in maximal lesion size in Gal-1–deficient mice compared atherosclerosis progression (31–33). In addition, we have recently to WT mice in the aortic root, in agreement with the enface analysis observed that VSMCs from patients with aneurysm also display on the whole aorta. Moreover, we found a higher content of lipids dysfunctional mitochondria associated to a synthetic phenotypic inside atherosclerotic plaques in Gal-1–deficient mice, while we could switch (34). In this regard, lineage-tracing studies revealed that the not find significant changes in the necrotic core area and collagen progeny of VSMCs has a remarkable plasticity in the vascular wall content. We also observed a greater uptake of oxLDL by peritoneal and can assume a wide variety of phenotypes during vascular re- macrophages from Lgals1−/− versus WT mice, which favored LDL modeling, including loss of contractile markers, while adopting retention inside the arterial wall; this effect was prevented by expo- alternative phenotypes characteristic of foam cells, osteochondro- sure to rGal-1, suggesting an important role of Gal-1 in limiting cytes, or myofibroblasts, among others (17, 35–39). In this regard, foam cell formation. Last, the percentage of -SMA and calponin aortic proteomics analysis also identified a clear up-regulation of Fn staining was decreased in Gal-1–deficient mice, while no changes in and VCAM-1, which are commonly associated with phenotypic CD68 expression were detected. Collectively, these data support a modulation of VSMCs, being Fn linked with a profibrotic state protective role of Gal-1 in atherosclerosis by modulating critical and VCAM-1 expression connected with a proinflammatory VSMC determinants of plaque progression and instability. profile (15–18). In agreement, we found that Gal-1 silencing resulted We then performed an unbiased proteomic analysis of whole in enhanced Fn1 and Vcam-1 mRNA expression in TNF-stimulated aorta homogenates to elucidate possible mechanisms associated with VSMCs in vitro. Our proteomics data in whole aorta, and the de- Gal-1 effects. We found that, among biological pathways and cellular crease in -SMA expression in atherosclerotic plaques of Lgals1−/− processes significantly enriched by Database for Annotation, Visu- mice, led us to examine whether Gal-1 silencing has a direct role on alization and Integrated Discovery (DAVID) analysis in the aorta of VSMC phenotypic switch. We also observed increased KLF4 and Lgals1−/− mice, mitochondrial proteins were clearly down-regulated. decreased -SMA expression in Gal-1–silenced VSMCs. Similarly, A decline in the expression of mitochondrial components and/or Gal-1 knockdown in VSMCs incubated with platelet-derived growth factors involved in mitochondrial activity was also observed after factor (PDGF)–BB decreased -SMA expression (40), while Gal-1 Gal-1 silencing in VSMCs. Impaired mitochondrial respiration and induced -SMA expression in fibroblasts (41, 42). In this regard, loss reduced oxidative phosphorylation in VSMCs have been linked to of VSMC contractile phenotype has been associated with a metabolic Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 7 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE shift to glycolysis and altered mitochondrial fitness (20, 32). Syn- supplementation was associated with prevention of -SMA loss but thetic VSMCs demonstrated an increased glycolytic flux, with a did not modify the frequency of CD68 expression, suggesting no decrease in glucose oxidation (43). We found that Gal-1–silenced major changes in the control of macrophage infiltration in this VSMCs showed decreased OCR (an indicator of mitochondrial pathologic setting. In contrast, it has been recently shown that Gal-1 oxidative phosphorylation) but displayed increased lactate produc- deficiency reduced the incidence and severity of angiotensin-induced tion. rGal-1 reversed the increased OCR and decreased lactate in- AAA by decreasing vascular inflammation (26). In that particular duced by Gal-1 siRNA or oxLDL in VSMCs. In this regard, it was study, the authors suggested that the absence of oxidized Gal-1 previously described that, in a lactate-enriched microenvironment, associated to Gal-1 deletion could be responsible for the protective VSMCs acquire a synthetic phenotype (44). Thus, altered lactate effect in AAA. The discrepancies with our study could be related production via Gal-1 modulation might contribute to a loop of to differences between approaches (Gal-1 deletion versus treatment VSMC phenotypic switch. Our data suggest a central role of Gal-1 with rGal-1), reflecting the effects of the endogenous versus exog­ in mitochondrial functionality and phenotypic switch of VSMCs enous lectin, and between experimental models (27). Our data during vascular remodeling. suggest that treatment with rGal-1 is vasculoprotective in both Currently, atherosclerosis is mostly treated with drugs that modu- atherosclerosis and AAA through mechanisms involving modula- late CV risk factors, including statins to prevent CV risk. However, tion of foam cell formation, as well as mitochondrial metabolic fitness residual CV risk persists and new approaches mainly focused on and preservation of VSMC contractile phenotype. Because of the anti-inflammatory agents are being tested (45, 46). In this regard, increasing importance of the diverse VSMC phenotypes in vascular previous studies demonstrated the protective role of rGal-1 treatment remodeling (15–18, 37), molecular targets that interfere with the in the resolution of acute and chronic inflammatory conditions (47), modulation of VSMC fate could be envisioned as alternative ap- including AMI (11), autoimmune neuroinflammation (48), inflam- proaches to treat chronic vascular pathologies (52). We and others matory bowel diseases (49), and diabetes (50, 51). In this study, have previously shown that different members of the galectin family, we investigated the role of Gal-1 as a novel therapeutic strategy in including Gal-3, can also modulate physiologic and pathologic two different chronic vascular diseases, such as atherosclerosis and vascular remodeling (53–55), highlighting the involvement of a AAA. In advanced atherosclerosis, treatment with rGal-1 decreased coordinated galectin-glycan network in controlling chronic vascular atherosclerotic burden, independently of plasma lipid levels. More- processes. over, Gal-1 administration reduced maximal lesion size and necrotic Some limitations of our study should be discussed. The core extension at the aortic root, along with increased -SMA stain- experimental model of atherosclerosis used in this study (AAV-­ ing, but did not alter CD68 expression. Similarly, in AAA, rGal-1 PCSK9) develops extensive lesions in the aortic root, the aorta, Lgals1+/+ Lgals1−/− Macrophages Contractile VSMC Foam cells VSMC phenotypic phenotype switch Functional mitochondria Dysfunctional mitochondria 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. Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 8 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE and its principal branches, similar to most common models Experimental models of atherosclerosis (ApoE and Ldlr ko mice); however, they fail to Gal-1–deficient mice (Lgals1−/−; C57BL6/J) were originally provided develop atherosclerosis in the coronary arteries. Moreover, plaque by F. Poirier (Jacques Monod, Paris, France). The corresponding rupture and thrombotic occlusion rarely occur in the coronary ar- WT control mice (C57BL6/J) were purchased from The Jackson teries in these models. Thus, we only assessed the effect of Gal-1 in Laboratory (Bar Harbor, USA). No differences in size and weight plaque progression and instability using indirect markers, such were detected between WT and Lgals1−/− mice. All animals were as lipid content in the plaques, the presence of a necrotic core, and housed in isolation rooms at the Animal Facilities of the Institute of the frequency of contractile VSMCs. To enhance the translational IIS-Fundación Jiménez Díaz. The Ethics Review Board of the Insti- application of our findings, new models that better resemble hu- tute of IIS-Fundación Jiménez Díaz approved all animal procedures, man conditions should be tested. In this respect, transverse aortic and the project was authorized by the IIS-FJD-Universidad Autónoma constriction performed in ApoE−/− mice can lead to coronary plaque de Madrid (CEI 59-1036-A061) and by the Spanish Authority formation, progression, and myocardial events (56). governing animal experimentation, the Comunidad Autónoma de Our results suggest that Gal-1 affects both macrophage and Madrid (registered approval letter 10/008932.9/15). All animal pro- VSMC functionality and metabolism. Although we did not observe cedures were performed in accordance with the guidelines of Direc- changes in CD68 expression, we cannot rule out a possible anti-­ tive 2010/63/EU of the European Parliament on the protection of inflammatory effect of Gal-1 in macrophage number as CD68 is not animals used for scientific purposes. a specific marker of myeloid cells (57). CD68 and other markers that The experimental model of atherosclerosis was induced in a are frequently used to identify macrophages may also be expressed 10-week-old male as described (13). Mice were maintained on normal in VSMCs (53, 58). Thus, further studies targeting Gal-1 specifically diet before pAAV/D377Y-mPCSK9 injection (a gift of J. Bentzon, in hematopoietic cells and/or VSMCs would contribute to better de- Aarhus University). The same viral preparation was aliquoted before fine the role of this lectin in regulating atherosclerotic lesions in vivo. storage and used for all studies. C57BL6/J WT and Lgals1−/− mice In summary, our findings support a broad protective role of Gal-1 were given retro-orbital injections of 1011 vector genome copies of in pathological vascular disorders through mechanisms involving pAAV/D377Y-mPCSK9. Immediately following the AAV-PCSK9 modulation of lipid uptake by macrophages along with alterations injection, mice were switched to a high-fat diet (TD88137, Envigo) in mitochondrial function and phenotypic switch of VSMCs (Fig. 7). for 16 weeks. Two experimental approaches were performed. First, Treatment with rGal-1 prevented both atherosclerosis and AAA in we analyzed the role of endogenous Gal-1 by comparing WT (n = 10) mice, highlighting the therapeutic potential of this lectin in attenu- and Lgals1−/− (n = 12) mice. An additional group of WT (n = 5) and ating the severity of chronic pathologic vascular remodeling. Last, Lgals1−/− (n = 5) mice was subjected to similar conditions and used decreased expression of Gal-1 in advanced human atherosclerosis for enface analysis. Second, we tested the therapeutic effects of and AAA underlines the clinical relevance of our findings. exogenous Gal-1 in WT mice treated with rGal-1 (100 g/3 days a week, n = 16) compared to vehicle/saline (n = 12) during 16 weeks of high-fat diet. rGal-1 was obtained and purified as described (60) MATERIALS AND METHODS and diluted in PBS containing -mercaptoethanol (0.3 l/ml). Dose Human tissues of rGal-1 was chosen on the basis of our previous studies (48, 49). Aortas were collected from deceased organ donors under the authoriza- All mice were maintained under barrier conditions. Water and tion of the French Biomedicine Agency (PFS 09-007, BBMRI network, western diet were available ad libitum. At the end of the study, BB-0033-00029). Ethical committee advice and patient written 16-hour fasted mice were anesthetized [ketamine (100 mg/kg) and informed consent were obtained before participation (RESAA xylazine (15 mg/kg)], saline-perfused, and euthanized. Blood and AMETHYST studies, CPP Paris-Cochin 2095, 1930, and 1931, samples were collected for biochemistry. Serum total cholesterol INSERM Institutional Review Board, IRB0000388). All human studies and VLDL/LDL cholesterol were measured enzymatically using conformed to the principles outlined in the Declaration of Helsinki. commercial kits adapted for a COBAS 6000 autoanalyzer (Roche After macroscopic examination, the aortas were classified ac- Diagnostics). cording to Stary classification (59) and the Virmani word list (4) into For experimental AAA, mice (C57BL6/J, 12 weeks old) were control aortas (devoid of atheromatous lesions) and aortas with anaesthetized using 2% isoflurane, and a horizontal laparotomy was atherosclerotic plaques. In addition, AAA walls were collected from performed. Using a surgical stereomicroscope, the abdominal aorta patients who were undergoing surgical repair of AAA. A small por- was separated from the level of the left renal vein to the bifurcation tion of tissue from each sample was fixed in 3.7% paraformaldehyde and temporarily ligated between the renal and iliac arteries. An for classical histology and immunohistochemistry assessments. In aortotomy was created with a 30-gauge needle, and the aorta was atherosclerotic aortas, the adventitia and media were carefully re- exsanguinated. A PE-26 polyethylene tube was introduced through moved, and the dissected intimal lesion was processed separately, the aortotomy, and the aorta was infused for 5 min at 100 mm Hg while in AAA, the adventitia was removed and the media layer was with either saline buffer (control) or type 1 porcine pancreatic elastase further processed. Atherosclerotic plaques, as well as control aortas (specific activity, 6 U/mg protein; E1250, Sigma Chemical). The and AAA tissues, were either incubated in culture medium to obtain aortotomy was then repaired, the ligation was eliminated, and the tissue-conditioned medium or placed in liquid N2 for later RNA or restoration of blood flow was visually confirmed. Incisions were protein extraction. For tissue-conditioned medium, samples were closed, and mice were housed under standard conditions. Experimental cut into small pieces (5 mm2) and incubated in RPMI 1640 medium groups included elastase-infused WT mice treated with vehicle/ without fetal bovine serum (FBS) for 24 hours at 37°C (6 ml/g of wet saline (n = 9) or with rGal-1 (100 g/3 days a week, n = 7). On day 14 tissue). The tissue-conditioned medium was collected after centrif- after surgery, all mice were anaesthetized with a mixture of ketamine/ ugation at 3000g for 10 min at 20°C and frozen at −80°C. xylazine (100 and 10 mg/kg body weight, respectively) and euthanized Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 9 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE by cervical dislocation. External pre-perfusion and 14-day post-­ Proteomic analysis perfusion aortic diameters were measured to a resolution of Protein extraction 0.01 mm with a calibrated ocular grid while mice were under anes- Samples from liver and aorta from WT and Lgals1−/− mice were thesia and physiological blood pressure. Aortic diameter expan- processed for proteomic analysis. Protein extracts were obtained sion of ≥100% compared with that observed before perfusion by tissue homogenization with ceramic beads (MagNaLyser Green defined AAA. Aortic tissue samples were obtained for histological Beads apparatus, Roche, Germany) in extraction buffer [100 mM analysis. An additional cohort of elastase-infused WT mice (n = 8 at tris-HCl (pH 6.8) and 2% SDS]. Supernatants were collected, and day 3 and n = 10 at day 14, after elastase perfusion) and untreated protein concentration was measured by RC/DC Protein Assay healthy WT mice (n = 6) were subjected to mRNA isolation from (Bio-Rad). Protein extracts were stored at −80°C for further analysis. aortic tissues. Protein digestion Protein extracts (250 g) were subjected to filter-aided digestion Enface analysis of aorta (Nanosep Centrifugal Devices with Omega Membrane-30K, PALL) Atherosclerotic burden was quantified by enface analysis of the according to the manufacturer’s instructions. Briefly, cysteine resi- whole aorta. For enface preparations, the aorta was opened longitu- dues were blocked with 50 mM iodoacetamide buffer and pro- dinally from the heart to the iliac arteries while still attached to teins were digested with trypsin (1:40 trypsin/protein; Promega) in the heart and major branching arteries in the body. The aorta (from 100 mM ammonium bicarbonate (pH 8.8) overnight at 37°C. The the heart to the iliac bifurcation) was then removed and pinned out resulting peptides were desalted with Oasis cartridges (Waters Cor- on a white wax surface in a dissecting pan, using stainless steel poration, Milford, MA, USA) following the manufacturer’s instruc- pins 0.2 mm in diameter. After overnight fixation with 4% para- tions, dried with SpeedVac, and stored at −20°C. formaldehyde and a rinse in phosphate-buffered saline (PBS), the TMT labeling aortas were immersed for 1 hour in a filtered solution contain- Peptides were subjected to two 9-plex multiplexed isobaric labeling ing 0.2% ORO and unstained in 80% ethanol. The ORO-stained experiments (eight WT and eight Lgals1−/− mice) in the case of liver aortas were photographed and used for quantification of athero- samples and to two 8-plex multiplexed isobaric labeling experiments sclerotic lesions. (nine WT and seven Lgals1−/− mice) in the case of aorta samples, using TMT reagents (Thermo Fisher Scientific) and following the Morphometric analysis manufacturer’s instructions. In each TMT experiment, one channel Hearts containing aortic roots were carefully dissected and frozen was reserved for a reference internal standard sample created by in optimal cutting temperature (OCT) compound. Aortic roots were pooling control samples. Labeled peptides were desalted with Oasis sectioned at 5 m thickness beginning proximally at the first evi- cartridges (Waters Corporation, Milford, MA, USA), vacuum-dried, dence of the aortic valves at their attachment site of aorta. Sections and stored at −20°C for further mass spectrometry (MS) analysis. were treated with ORO/hematoxylin for lipid staining at 100-m LC-MS analysis intervals from 0 to 900 M distal to the proximal site. Maximal Labeled peptide samples were analyzed using an Ultimate 3000 HPLC lesion area was calculated for each mouse by averaging the values system (Thermo Fisher Scientific) coupled via a nanoelectrospray for three sections. The individual maximal lesion areas were further ion source (Thermo Fisher Scientific, Bremen, Germany) to a Q averaged to determine the maximal lesion area for each mouse group. Exactive HF mass spectrometer (Thermo Fisher Scientific). C18-based Lipid content was defined as the percentage of ORO staining area to reversed-phase separation was used with PepMap 100 C18 5UM total plaque area. Picrosirius red staining was performed for analysis 0.3x5MM as trapping column (Thermo Fisher Scientific) and a of collagen content by measuring birefringence to plane-polarized 50-cm analytical column (EASY-Column, Thermo Fisher Scientific). light. Necrotic core was analyzed in the bright-field sections ob- Peptides were loaded in buffer A [0.1% formic acid in water (v/v)] tained after picrosirius red by delimitating the acellular areas with and eluted with a 338-min linear gradient of buffer B [100% acetonitrile cholesterol clefts. and 0.1% formic acid (v/v)] at 200 nl/min. Mass spectra were ac- For AAA lesions, aortic tissue cross sections (3 to 4 m) were quired in a data-dependent manner, with an automatic switch be­ stained with Masson’s trichrome and then evaluated (53). Changes tween MS and MS/MS using a top-15 method. MS spectra were acquired in aortic diameter were calculated as percentage of dilation over in an Orbitrap analyzer with a mass range of 400 to 1500 m/z (mass/ baseline at day 14. charge ratio) and 70,000-pixel resolution. HCD fragmentation was per­ formed at 30 of normalized collision energy, and MS/MS spectra were Immunohistochemistry analyzed at 60,000-pixel resolution in the Orbitrap. Primary antibodies were anti–Gal-1 (AF1152, R&D Systems), Protein identification CD68 (Ab53444, Abcam), and -SMA (clone 1A4, Sigma-Aldrich). For peptide identification, MS/MS spectra were searched with the Rabbit anti-goat horseradish peroxidase (HRP), donkey anti-rat HRP SEQUEST HT algorithm implemented in Proteome Discoverer 2.1 (The Jackson Laboratory), and Alexa Fluor 488 donkey anti-rat (Thermo Fisher Scientific) against a UniProt database composed of (Invitrogen) were used as secondary antibodies. HRP was then added, mouse protein sequences (July 2020), using trypsin digestion with a and sections were stained with 3,3′-diaminobenzidine (DAB) maximum of two missed cleavages, using Cys carbamidomethylation substrate-chromogen (DAKO) and counterstained with hematoxylin. (57.021464 Da) and TMT labeling at the N-terminal end as fixed Computer-assisted morphometric analysis was performed with modifications and Lys (229.162932 Da) and Met oxidation (15.994915) Image-Pro Plus software (version 1.0 for Windows). The threshold as dynamic modifications. Precursor mass tolerance was set at setting for area measurement was equal for all images. Samples from 800 parts per million (ppm), and fragment mass tolerance was set each animal were examined in a blinded manner. Results were ex- at 0.03 Da; precursor charge range was set to 2 to 4. Results were pressed as % positive area versus total area (CD68 and -SMA). analyzed using the probability ratio method (61), and the FDR was Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 10 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE calculated based on the search of results against the corresponding housekeeping transcript [18S/glyceraldehyde-3-phosphate dehydro- decoy database using a refined method with an additional filter for genase (GAPDH)]. The following PCR primers for human [LGALS1, precursor mass tolerance of 15 ppm (62). One percent FDR was used 5′-CTGCAACACTTCCAGGCTGG-3′ (reverse) and 5′-CCTGGA- as criterion for peptide identification. GAGTGCCTTCGAGTG-3′ (forward); ACTA2, 5′-GCTCAGCAGTAG- Protein quantification and statistical analysis TAACGAAGGA-3′ (reverse) and 5′-CTATGAGGGCTATGCCTTGCC-3′ Protein quantification and statistical analysis were performed using (forward)] and for mouse [Lgals1, 5′-TCAGCCTGGTCAAAGGT- the models previously developed in our laboratory (63–65) with the GAT-3′ (reverse) and 5′-TGAACCTGGGAAAAGACAGC-3′ SanXoT software package (66). Quantitative information was extracted (forward); Acta2, 5′-TTAGCATAGAGATCCTTCCTG-3′ (reverse) from the MS/MS spectra of TMT-labeled peptides. Peptide quantifica- and 5′-CATCTTTCATTGGGATGGAG-3′ (forward); mt-Nd6, tion was analyzed using the Weighted Spectrum, Peptide and Protein 5′-GGTTAGCATTAAAGCCTTCACCT-3′ (reverse) and model, which uses raw quantifications as input data and computes 5′-CATCAACCAATCTCCCAAACCAT-3′ (forward); Namp, 5′-TG- the protein log2 fold changes for each individual with respect to the val- CCGTGAAAAGAAGACAGA-3′ (reverse) and 5′-ACTTCTTTGGCCTC­ ue of the reference internal standard sample. In this model, protein CTGGAT-3′ (forward); mt-Co1, 5′-CTCGCCTAATTTATTCCACTTCA-3′ log2 ratios are expressed as standardized variables in units of SD ac- (reverse) and 5′-GGGGCTAGGGGTAGGGTTAT-3′ (forward); cording to their estimated variances (Zq values). For functional analysis, mt-Nd1, 5′-CTAGCAGAAACAAACCGGGC-3′ (reverse) and proteins were annotated using DAVID (67). MS proteomics data were 5′-CCGGCTGCGTATTCTACGTT-3′ (forward); Tfam, 5′-CAG- deposited at the ProteomeXchange Consortium (68) via the PRIDE GAGGCAAAGGATGATTC-3′ (reverse) and 5′-CCAAGACTTCA­ partner repository with the dataset identifier PXD024637. TTTCATTGTCG-3′ (forward); Sdh, 5′-CTTGAATCCCTGCTCTGTGG-3′ (reverse) and 5′-AAAGCTGAGAGTGCCAAGAG-3′ (forward); In vitro experiments in VSMCs and macrophages Ppar-, 5′-TCGAGGAAGGCACTACACCT-3′ (reverse) and Mouse VSMCs were obtained directly from primary culture of aorta 5′-TCTTCCCAAAGCTCCTTCAA-3′ (forward); Gapdh, 5′-GG- tissue of WT mice. The aorta was carefully removed from the iliac CATGGACTGTGGTCATGAG-3′ (reverse) and 5′-TGCACCAC- arteries to the heart and immersed in PBS. To remove undesirable CAACTGCTTAGC-3′ (forward)] and TaqMan probes (18s: leftovers, aortas were cleansed physically, cut into small pieces, and 4310875; Klf4: Mm00516104_m1; Fn1: Mm01256744_m1; Vldlr: enzymatically treated with 4 mg of collagenase per aorta (Sigma Life Mm00443298_m1; Ldlr: Mm00440169_m1; Vcam-1: Mm01320970_m1) Science) in dulbecco´s minimum esssential medium (DMEM)– were purchased from Applied Biosystems and optimized according F12 medium (Thermo Fisher Scientific) plus 20% FBS and were further to the manufacturer’s protocol. seeded in p60 plaques and incubated at 37°C for 5 days. Cells were harvested for passaging at 2- to 3-day intervals and used between Western blot the second and seventh passages. For experiments except those of For tissue-conditioned medium, the procedure was normalized seahorse, cells were FBS-depleted for 24 hours and, in some cases, by incubating each gram of wet tissue in 6 ml of RPMI 1640 alone stimulated for 24 hours with TNF (100 ng/ml) before cells were (without FBS) and equal amounts of proteins (20 g) were subjected pelleted and resuspended for RNA analysis. LDLs from a pool of to SDS–polyacrylamide gel electrophoresis. Polyvinylidene difluoride serum of healthy donors were obtained by ultracentrifugation and membranes were incubated with an anti–Gal-1 monoclonal antibody oxidized by incubation with 5 mM CuSO4 for 18 hours at 37°C. (R&D Systems) and processed as previously described (70). Mem- Peritoneal macrophages were collected from WT or Lgals1−/− branes were subjected to densitometry, and values were normalized mice by peritoneal lavage 4 days after intraperitoneal injection of against Ponceau Red S staining. 3% (w/v) thioglycolate. Cells were cultured for 24 hours in RPMI 1640 supplemented with 10% FBS, l-glutamine, and antibiotics. Then, cells Mitochondrial functional studies were incubated with Dil-oxidized LDL (10 g/ml) for 4 hours. Cells Complex I activity were fixed in 4% paraformaldehyde for 15 min and stained with Complex I activity was measured using the Complex I Enzyme 4′,6-diamidino-2-phenylindole (DAPI). Percentage of foam cells was Activity Microplate Assay Kit (ab109721, Abcam) following the analyzed in 5 to 10 random fields per cell culture as described (69). manufacturer’s instructions. Cells were pelleted after siRNA trans- fection, and whole-cell lysates were analyzed. siRNA-mediated knockdown assays Extracellular flux analysis siRNA specific for mouse Gal-1 was obtained from Santa Cruz Bio- OCRs were measured in XF-96 Extracellular Flux Analyzers technology (sc-37259). Transfection of siRNA was performed using (Seahorse Bioscience) as described (71) in mouse aortic VSMCs. Lipofectamine as described (70). Experiments were also performed Briefly, mouse aortic VSMCs were seeded in nonbuffered DMEM with a negative control available from Ambion (AM4636). containing either 25 mM glucose or 1 mM CaCl2. Three measure- ments were obtained under basal conditions and on addition of Real-time PCR oligomycin (1 mM), fluoro carbonyl cyanide phenylhydrazone Human atherosclerotic or AAA tissues, as well as aortic wall tissues (1.5 mM), and rotenone (100 nM) + antimycin A (1 mM). from healthy human controls, and mouse aortic and liver homogenates Extracellular l-lactate determination were snap-frozen in liquid nitrogen; homogenates (0.2 g) were re- Extracellular lactate determination was performed in conditioned suspended in TRIzol buffer (Life Technologies); and total RNA was medium (20 l) after 48 hours for both siRNA-transfected cells and purified. Similarly, lysates from VSMCs were resuspended in TRIzol, cells treated with oxLDL with single-use reagent strips for Accutrend and total RNA was purified. Duplicate samples were quantified by Plus Lactate Pro (Roche), based on an enzymatic spectrophotometry determining absorbance at 260 nm, and real-time PCR was performed system by lactate oxidase layer. OCR measurements and l-lactate as described (53). The expression of target genes was normalized to were normalized to total protein cell extracts. Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 11 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE Statistical analysis 16. S. Allahverdian, C. Chaabane, K. Boukais, G. A. Francis, M. L. Bochaton-Piallat, Smooth Results are expressed as means ± SE. For analysis of data from human muscle cell fate and plasticity in atherosclerosis. Cardiovasc. Res. 114, 540–550 (2018). samples or from the experimental models, groups were compared 17. R. C. Wirka, D. Wagh, D. T. Paik, M. 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Sethi, Inhibition (Complemento II-CM, S2017/BMD-3673), Fondo de Investigaciones Sanitarias ISCiii-FEDER of galectin-3 reduces atherosclerosis in apolipoprotein E-deficient mice. Glycobiology 23, [Biobancos RD09/0076/00101 and PRB3 (IPT17/0019, ProteoRed)], and FEDER “Una manera de 654–663 (2013). hacer Europa.” CIBERCV and CIBERDEM are Instituto de Salud Carlos III projects. This work also 56. A. Marino, Y. Zhang, L. Rubinelli, M. A. Riemma, J. E. Ip, A. Di Lorenzo, Pressure overload received funding from “La Caixa” Banking Foundation under the project code HR17-00247. The leads to coronary plaque formation, progression, and myocardial events in ApoE−/− mice. CNIC is supported by Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y JCI Insight 4, e128220 (2019). Universidades (MCNU), and Pro CNIC Foundation and is a Severo Ochoa Center of Excellence 57. E. Gottfried, L. A. Kunz-Schughart, A. Weber, M. Rehli, A. Peuker, A. Müller, (SEV-2015-0505). Further support was provided by Santander-UAM, Agencia Nacional de M. Kastenberger, G. Brockhoff, R. Andreesen, M. Kreutz, Expression of CD68 in Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (PICT 2010-870, non-myeloid cell types. Scand. J. Immunol. 67, 453–463 (2008). Argentina), Fundación Sales, and Fundación Bunge y Born (Argentina). We thank the support 58. S. Allahverdian, A. C. Chehroudi, B. M. McManus, T. Abraham, G. A. Francis, Contribution of Ferioli, Ostry, and Caraballo families. Author contributions: R.R.-M. performed in vivo of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells experiments with models of atherosclerosis, immunohistochemistry, and in vitro studies. in human atherosclerosis. Circulation 129, 1551–1559 (2014). J.M.P.-S. produced and purified rGal-1 protein and contributed to analysis and discussion of 59. H. C. Stary, A. B. Chandler, S. Glagov, J. R. Guyton, W. Insull, Jr., M. E. Rosenfeld, the results. I.C.-P. performed proteomic studies and participated in the in vivo studies. S. A. Schaffer, C. J. Schwartz, W. D. Wagner, R. W. Wissler, A definition of initial, fatty J.O. performed in vitro experiments, analyzed data, and discussed results. D.M.-L. performed streak, and intermediate lesions of atherosclerosis. A report from the Committee in vivo experiments with the AAA model. S.M.M., C.G.-M., and N.M.-B. contributed to in vivo Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 13 of 14 SCIENCE ADVANCES | RESEARCH ARTICLE and in vitro studies. E.N. analyzed proteomics data. J.C.E.-G., J.-B.M., J.V., and M.M. analyzed at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset data and discussed results. L.M.B.-C. performed experiments in models of atherosclerosis, identifier PXD024637. analyzed data, and discussed results. G.A.R. and J.L.M.-V. conceived, designed, and supervised the study; analyzed data; and drafted the manuscript. All authors revised the final manuscript. Submitted 7 October 2021 Competing interests: The authors declare that they have no competing interests. Data and Accepted 25 January 2022 materials availability: All data needed to evaluate the conclusions in the paper are present Published 16 March 2022 in the paper and/or the Supplementary Materials. MS proteomics data have been deposited 10.1126/sciadv.abm7322 Roldan-Montero et al., Sci. Adv. 8, eabm7322 (2022) 16 March 2022 14 of 14