Abdominal aortic aneurysm (AAA) evolution is unpredictable and no specific treatment exists for AAA, except surgery to prevent aortic rupture. Galectin-3 has been previously associated with CVD, but its potential role in AAA has not been addressed. Galectin-3 levels were increased in the plasma of AAA patients (n=225) compared with the control group (n=100). In addition, galectin-3 concentrations were associated with the need for surgical repair, independently of potential confounding factors. Galectin-3 mRNA and protein expression were increased in human AAA samples compared with healthy aortas. Experimental AAA in mice was induced via aortic elastase perfusion. Mice were treated intravenously with the galectin-3 inhibitor modified citrus pectin (MCP, 10 mg/kg, every other day) or saline. Similar to humans, galectin-3 serum and aortic mRNA levels were also increased in elastase-induced AAA mice compared with control mice. Mice treated with MCP showed decreased aortic dilation, as well as elastin degradation, vascular smooth muscle cell (VSMC) loss, and macrophage content at day 14 postelastase perfusion compared with control mice. The underlying mechanism(s) of the protective effect of MCP was associated with a decrease in galectin-3 and cytokine (mainly CCL5) mRNA and protein expression. Interestingly, galectin-3 induced CCL5 expression by a mechanism involving STAT3 activation in VSMC. Accordingly, MCP treatment decreased STAT3 phosphorylation in elastase-induced AAA. In conclusion, increased galectin-3 levels are associated with AAA progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest the potential role of galectin-3 as a therapeutic target in AAA.

Introduction

Abdominal aortic aneurysm (AAA) is a major health problem, causing approximately 1–2% of male deaths in economically developed societies. AAA is a permanent focal dilation of the abdominal aorta that exceeds more than 3 cm. Mechanistically, AAA involves proteolytic and oxidative injuries, followed by elastin degradation, vascular smooth muscle cell (VSMC) depletion, and an immune-inflammatory response of the aortic wall [1,2]. Clinically, AAA is usually asymptomatic and is often detected as an incidental finding during the investigation of an unrelated problem or as a consequence of radiological screening. Even in those cases where AAA is diagnosed, the only way to prevent aortic rupture in patients with an AAA >5.5 cm is surgery, while no specific treatment exists for those patients with an aortic diameter of 3–5.5 cm. Moreover, AAA progression to rupture is unpredictable, combining periods of acceleration with others of stability. Understanding the mechanisms and mediators underlying AAA formation and rupture could lead to novel prognostic and/or therapeutic biomarkers [3,4].

Galectins are a family of carbohydrate-binding proteins that regulate different processes, such as cell differentiation and proliferation, immune-inflammatory responses, oxidative stress, apoptosis, angiogenesis, and fibrosis [5]. Galectin-3 is a 30 kDa protein with a unique chimeric structure containing a carbohydrate recognition domain at the C-terminal and a collagen-like domain at the N-terminal [5]. Functions of galectin-3 depend on its localization. Intracellularly, galectin-3 regulates the cell cycle and cell growth via carbohydrate-independent mechanisms [6]. Extracellularly, galectin-3 is involved in the modulation of cell–cell interactions and immune-inflammatory responses, predominately via carbohydrate-binding functions [7]. Extracellular galectin-3 plasma levels are increased in patients with chronic kidney diseases [8] and/or cardiovascular diseases (CVD), mainly heart failure [9]. Moreover, increased circulating galectin-3 predicts total and CV mortality in patients with or without CVD [10,11]. Finally, galectin-3 has been proposed as a therapeutic target in CVD [12], as galectin-3 inhibition using modified citrus pectin (MCP) was shown to reduce cardiac remodeling and atherosclerotic plaque progression [13,14]. MCP, a derivative of a pectin found in the peel and pulp of citrus fruits, contains fragments of the original pectin molecule and binds to the carbohydrate recognition domain of galectin-3 [15]. MCP modulates galectin-3 bioactivity by altering extracellular functions such as cell–cell interactions and inflammation [16].

In the present study, we analyzed the potential role of galectin-3 in AAA development. For that purpose, we have tested the levels of galectin-3 in samples of AAA patients, as well as in the experimental model of AAA induced by elastase perfusion in mice. In addition, the effect of galectin-3 inhibition on the development of experimental AAA was investigated using MCP treatment.

Methods

Human plasma samples

Venus blood samples were collected into heparin tubes and sample tubes were centrifuged for 12 min at 3000 g. Plasma was then aliquoted and immediately frozen at −80°C. A cohort of plasma samples from AAA patients (aortic size >3 cm, n=225) and a control group (aortic size <3 cm, n=100) was obtained for the present study (Table 1).

Table 1
Clinical characteristics of study participants
 Dichotome variables (%)Controls (N=100)AAA (N=225)P value
Familial predisposition 3.5 6.7 0.289 
Current smoker 18.2 40.2 <0.001*** 
Diabetes 13.8 14.2 0.952 
Hypertension 44.8 51.8 0.231 
Dyslipemic 35 50.2 0.019* 
Use of ace-inhibitor 17.0 28.2 0.046* 
Use of β-blocker at baseline 26.1 32.0 0.251 
Use of β-agonist at baseline 8.0 7.8 0.951 
Use of statins at baseline 31.8 48.2 0.003** 
Use of antiplatelets at baseline 22.7 44.2 <0.001*** 
PAD at screening 0.0 26.6 <0.001*** 
Continuous variables Mean (SD) Controls (N=100)  AAA (N=225) P value 
BMI (kg/m226.4 (3.2) 27.4 (3.7) 0.017* 
Systolic blood pressure (mmHg) 148.2 (19.6) 154.7 (22.5) 0.012* 
Diastolic blood pressure (mmHg) 79.8 (9.7) 88.3 (11.9) <0.001*** 
C-reactive protein (mg/l) 5.57 (13.5) 6.03 (8.48) 0.779 
Aortic diameter (mm) 17.8 (2.4) 44.3 (14.3) <0.001*** 
Gal-3 (ng/ml) 6.93 (2.8) 7.78 (3.6) 0.008** 
 Dichotome variables (%)Controls (N=100)AAA (N=225)P value
Familial predisposition 3.5 6.7 0.289 
Current smoker 18.2 40.2 <0.001*** 
Diabetes 13.8 14.2 0.952 
Hypertension 44.8 51.8 0.231 
Dyslipemic 35 50.2 0.019* 
Use of ace-inhibitor 17.0 28.2 0.046* 
Use of β-blocker at baseline 26.1 32.0 0.251 
Use of β-agonist at baseline 8.0 7.8 0.951 
Use of statins at baseline 31.8 48.2 0.003** 
Use of antiplatelets at baseline 22.7 44.2 <0.001*** 
PAD at screening 0.0 26.6 <0.001*** 
Continuous variables Mean (SD) Controls (N=100)  AAA (N=225) P value 
BMI (kg/m226.4 (3.2) 27.4 (3.7) 0.017* 
Systolic blood pressure (mmHg) 148.2 (19.6) 154.7 (22.5) 0.012* 
Diastolic blood pressure (mmHg) 79.8 (9.7) 88.3 (11.9) <0.001*** 
C-reactive protein (mg/l) 5.57 (13.5) 6.03 (8.48) 0.779 
Aortic diameter (mm) 17.8 (2.4) 44.3 (14.3) <0.001*** 
Gal-3 (ng/ml) 6.93 (2.8) 7.78 (3.6) 0.008** 

* p<0.05, ** p<0.01 and *** p<0.001.

AAA was defined as a maximal aortic diameter greater than 3 cm. AAA cases among first-degree relatives were recorded, as well as smoking status, coexisting diabetes mellitus, hypertension, and the use of β-blockers, β-agonists, angiotensin-converting enzyme (ACE) inhibitors, aspirin, and statins. Body mass index, and systolic and diastolic blood pressure were also measured and recorded. Ankle systolic blood pressure was also measured, and the maximal anterior–posterior diameter of the infrarenal aorta was measured at the peak of systole from the inner edge to the outer edge of the aorta. The ankle–brachial index (ABI) was calculated as the mean of the two recorded ankle arterial blood pressures divided by the brachial systolic blood pressure. Peripheral arterial disease (PAD) was defined as an ABI lower than 0.90 or >1.4.

Patients with AAAs of less than 50 mm diameter were offered annual control scans by the screening team. The interobserver variation of aortic diameter measurements was 1.52 mm. Patients with an AAA diameter measuring 50 mm or more were referred for a computed tomography (CT) scan and vascular surgical evaluation. If no repair was indicated, the vascular surgical departments took over the management due to a need for closer surveillance.

Informed consent was obtained from all subjects before participation, and the study was approved by the Local Ethics Committee of the Central Denmark Region, Denmark, and performed in accordance with the Helsinki Declaration.

Human aortic tissue samples

Healthy human aortas (N=9) were sampled from deceased organ donors with the authorization of the French Biomedicine Agency (PFS 09-007). These control aortic samples were macroscopically normal and devoid of early atheromatous lesions. Nine samples of AAA walls were collected from patients that were undergoing surgical repair of AAA and were enrolled in the RESAA protocol [17]. Informed consent was given for the use of the human samples for research purposes. All of our human studies conformed to the principles outlined in the Declaration of Helsinki.

Mice

Wild-type (WT) control mice (C57BL6/J) were purchased from The Jackson Laboratory (Bar Harbor). All animals were housed in isolation rooms in the animal facility of our institute. Water and a chow-fed diet was available ad libitum. The Ethics Review Board of our institute approved all animal procedures, and the project was authorized by the IIS-FJD-Universidad Autónoma de Madrid (CEI 59-1036-A061) and by the Spanish Authority governing animal experimentation, the Comunidad Autónoma de Madrid (registered approval letter 10/008932.9/15). All animal procedures were performed in accordance with the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

Experimental AAA formation

Twelve-week-old mice were anesthetized using 2% isofluorane inhalational anesthesia and a horizontal laparotomy was performed. Using a surgical stereomicroscope, the abdominal aorta was separated from the level of the left renal vein to the bifurcation and temporarily ligated between the renal and iliac arteries. An aortotomy was created with a 30-gauge needle and the aorta was exsanguinated. A PE-26 polyethylene tube was introduced through the aortotomy, and the aorta was infused for 5 min at 100 mmHg with either saline buffer (control) or type I porcine pancreatic elastase (specific activity 6 U/mg protein; E1250; Sigma Chemicals). The aortotomy was then repaired, the ligation was eliminated, and the restoration of blood flow visually confirmed. Incisions were closed and the mice housed under standard conditions. Experimental groups were: elastase-infused WT mice treated intravenously with saline (WT, N=17); elastase-infused WT mice treated intravenously with MCP (10 mg/kg) every other day (MCP, N=14). On day 14 postsurgery, all mice were anesthetized with a mixture of ketamine/xylazine (100 and 10 mg/kg body weight respectively) and killed by cervical dislocation. An additional model involved mice being killed at day 3 (WT, n=7 and MCP, n=7). Aortic tissue samples were obtained for histological analysis. Two additional models were performed to assess the effect of MCP on cytokine mRNA expression at day 3 (WT and MCP, N=8 each one) and at day 14 (WT, N=11 and MCP, N=8). An additional cohort of elastase-infused WT mice and untreated healthy WT mice was subjected to mRNA isolation from aortic tissues, tissues for histology, and serum from circulating blood.

In vitro studies

Human VSMCs were purchased from ATCC (CRL-1999) and maintained in HAMs F12 (BioWhittaker) supplemented with 10% FBS (BioWhittaker), 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). For experiments, cells were preincubated with 0% FBS during 24 h and incubated with galectin-3 recombinant protein (R&D) with or without JAK inhibitor I (sc-204021, Santa Cruz Biotechnology).

mRNA analysis using real-time polymerase chain reaction

Human and mouse aortic wall tissues were snap-frozen in liquid nitrogen and homogenates (0.2 g) were resuspended in TRIzol buffer (Life Technologies) and the total RNA was purified. RNA was also isolated using the TRIzol method from in vitro studies using different experimental conditions. Duplicate samples were quantified by determining the absorbance at 260 nm and real-time PCR was performed. The expression of target genes was normalized to housekeeping transcripts (18S and GADPH). The following PCR primers were used: hGal3: 5′-TTTGCCTGGGGGAGTGGTGCCT-3′ (sense) and 5′-TGGGCTTCACCGTGCCCAGAA-3′ (antisense); 18S: 5′-CCGTCGTAGTTCCGACCATAA-3′ (sense) and 5′-CAGCTTTGCAACCATACTCCC-3′ (antisense); mGal3: 5′-GCTTATCCTGGCTCAACTGC-3′ (sense) and 5′-TTCACTGTGCCCATGATTGT-3′ (antisense). The Taqman primers used were: CCL5 mouse (Mm01302427_m1) and human (Hs00174575); IL-6 mouse (Mm00446190_m1), IL-1β mouse (Mm00434228_m1), TNF-α mouse (Mm00443258_m1), matrix metalloproteinase (MMP)-2 mouse (Mm00439498_m1), MMP-3 mouse (Mm00440295_m1), MMP-9 mouse (Mm00442991), and MMP-13 mouse (Mm00439). All measurements were performed in triplicate. The amount of target mRNA in samples was estimated by the 2ΔCT relative quantification method. The mRNA level in each experimental sample was expressed as fold increase over control values after normalization.

Immunohistochemistry and histomorphometry

Human and mouse AAA or control samples were embedded in paraffin. Serial sections (4 μm) of aortas were cut for histomorphometry and immunohistochemistry. Histomorphometry was performed on Masson trichrome-stained histological sections as previously described [18]. Verhoeff-van Gieson staining was performed and elastin layer degradation was graded as follows: grade 1, intact, well-organized elastic laminae; grade 2, elastic laminae with some interruptions and breaks; grade 3, elastin layers with multiple breaks and interruptions; and grade 4, severe elastin fragmentation and/or loss of whole sections.

Immunohistochemistry in aortic sections was performed using the following primary antibodies: galectin-3 (EP2775Y, Abcam), the smooth muscle cell marker α-actin (Cy3 conjugated, clone 1A4, Sigma), the macrophage marker Iba-1 (Wako), the neutrophil marker Ly6G (clone 1A8, Biolegend), CCL5 (abin1172331, antibodies online), and pSTAT3 (abcam76315). For colocalization studies in human samples, immunohistochemistry for galectin-3 was followed by immunohistochemistry for α-actin (SIGMA) or the macrophage marker CD68 (DAKO) in serial sections. Nonspecific staining was assessed using control polyclonal IgG antiserum. Primary antibodies were visualized by treating sections with the corresponding secondary antibody and ABComplex/HRP. Sections were stained with 3,3′-diaminobenzidine/3-amino-9-ethylcarbazole and mounted in DPX. VSMC loss (α-actin staining) was graded as follows: grade 1, intact, well-organized cells; grade 2, minimal aberrations in VSMCs; grade 3, a few VSMC interruptions and some disorganization; and grade 4, severe loss of VSMCs and marked disorganization. Computer-assisted morphometric analysis was performed with the Image-Pro Plus software (version 1.0 for Windows) as described in [19]. The threshold setting for area measurement was equal for all images. Samples from each animal were examined in a blinded manner. Results were expressed as the percentage positive area compared with total area of macrophages, galectin-3, CCL5, and pSTAT3.

ELISA

Galectin-3 levels in human plasma samples (DGAL30, R&D) and mouse serum samples (ELM-Galectin3, Raybiotech) were analyzed using commercially available ELISA following the manufacturer’s instructions.

Western blot

Human VSMCs from different experimental conditions were collected and pelleted. Western blots of cellular proteins were analyzed as previously described [18]. The membranes were blotted with Abs for phospho-STAT3 and total STAT3 (9134 and 9132 respectively, Cell Signaling Technology).

Statistical analysis

Results are expressed as mean ± standard error. For the analysis of galectin-3 in patients and controls, an unpaired t-test was performed. Multivariate logistic regression analysis was performed to assess the association between galectin-3 with AAA presence adjusting for the potential identified confounders. Potential confounders were defined as having a probability of no association with AAA presence below 10%. These potential confounders were also used in Cox regression analysis concerning an association of the need for later surgical repair and galectin-3 concentration above and below the median. For analysis of the effect of MCP treatment in the experimental model, the Mann–Whitney test was used. In vitro experiments were replicated at least three times. Statistics were performed using SPSS software (23.0; SPSS, Inc., Chicago) and significance was defined as P<0.05.

Results

Galectin-3 plasma levels are associated with AAA progression

We analyzed galectin-3 plasma levels in AAA patients (n=225) and controls (n=100) (Table 1). We observed that galectin-3 was increased in AAA patients when compared with controls (7.8 ± 0.2 vs. 6.9 ± 0.3 ng/ml, P<0.05) (Figure 1A). After adjustment by confounders (risk factors and drug treatments, see Supplementary Table S1), the odds ratio (OR) for presenting AAA in patients according to galectin-3 levels was 1.15 [95% confidence interval (95% CI), 1.02 to 1.30; P<0.05, Supplementary Table S1]. The survival curves of the association with the need for surgical repair with galectin-3 values above or below the median are shown in Figure 1B, suggesting that galectin-3 is associated with AAA evolution. In addition, Cox regression analysis of the association of high galectin-3 levels and the need for later preventive repair showed a significant increased risk, independent of potential confounding factors [Hazard ratio, HR = 1.83 (95% CI: 1.13–2.97), P=0.015] (Supplementary Table S2).

Galectin-3 concentrations in plasma of AAA patients

Figure 1
Galectin-3 concentrations in plasma of AAA patients

(A) Galectin-3 plasma levels in the control group (n=100) and AAA patients (aortic diameter >3 cm, n=225) from Denmark (VIVA study); *P<0.05 for AAA patients vs. controls. (B) Kapplan–Meier curve of the need for surgical repair in AAA patients according to galectin-3 plasma concentrations above or below the median.

Figure 1
Galectin-3 concentrations in plasma of AAA patients

(A) Galectin-3 plasma levels in the control group (n=100) and AAA patients (aortic diameter >3 cm, n=225) from Denmark (VIVA study); *P<0.05 for AAA patients vs. controls. (B) Kapplan–Meier curve of the need for surgical repair in AAA patients according to galectin-3 plasma concentrations above or below the median.

Galectin-3 mRNA and protein are up-regulated in human AAA tissues

We analyzed galectin-3 mRNA and protein expression in human wall aneurysms and compared levels with those in healthy aortic wall samples. We found that galectin-3 mRNA expression in human AAA walls was elevated compared with healthy control aortas (P<0.01, Figure 2A). Galectin-3 protein expression was almost absent from healthy aortic tissue and was highly up-regulated in the human AAA wall (Figure 2B). Moreover, we observed that galectin-3 is expressed in both macrophages and VSMCs in the AAA wall (Supplementary Figure S1).

Galectin-3 expression in human AAA tissue

Figure 2
Galectin-3 expression in human AAA tissue

(A) Quantification of galectin-3 mRNA in the wall of human AAA tissues (n=9) vs. healthy human control aortic samples (n=9). Data are the mean + SEM and are expressed relative to healthy control values, **P=0.01. (B) Representative immunohistochemistry of galectin-3 in the wall of human AAA patients and controls.

Figure 2
Galectin-3 expression in human AAA tissue

(A) Quantification of galectin-3 mRNA in the wall of human AAA tissues (n=9) vs. healthy human control aortic samples (n=9). Data are the mean + SEM and are expressed relative to healthy control values, **P=0.01. (B) Representative immunohistochemistry of galectin-3 in the wall of human AAA patients and controls.

Galectin-3 inhibition decreases elastase-induced aortic dilation

We analyzed the levels of galectin-3 in mouse serum and tissue from elastase-induced experimental AAA and compared them with those in healthy mice samples. Galectin-3 concentrations were significantly increased at day 3 when compared with healthy mice (P<0.01, Figure 3A). Moreover, galectin-3 mRNA expression in the AAA wall was increased compared with healthy control aortas (P<0.01, Figure 3B).

Galectin-3 levels in elastase-induced AAA in mice

Figure 3
Galectin-3 levels in elastase-induced AAA in mice

(A) Galectin-3 serum levels in elastase-induced AAA of WT mice (n=6 at day 3 and n=14 at day 14) compared with untreated healthy WT controls (n=12); **P=0.01 for day 3 vs. controls. (B) Quantification of aortic galectin-3 mRNA at day 14 postelastase infusion in WT mice (n=6) compared with aortic tissues of untreated healthy WT controls (n=10). Data are the mean and SEM and are expressed relative to healthy control values; ** P<0.01

Figure 3
Galectin-3 levels in elastase-induced AAA in mice

(A) Galectin-3 serum levels in elastase-induced AAA of WT mice (n=6 at day 3 and n=14 at day 14) compared with untreated healthy WT controls (n=12); **P=0.01 for day 3 vs. controls. (B) Quantification of aortic galectin-3 mRNA at day 14 postelastase infusion in WT mice (n=6) compared with aortic tissues of untreated healthy WT controls (n=10). Data are the mean and SEM and are expressed relative to healthy control values; ** P<0.01

To assess the potential role of galectin-3 as a mediator of AAA, mice were perfused with elastase and treated intravenously with the galectin-3 inhibitor MCP (n=14) or saline (WT, n=17) the day before surgery and every other day until day 14. As shown in Figure 4A, MCP-treated mice showed a decrease in aortic dilation compared with WT mice (P<0.05).

Galectin-3 inhibition by MCP decreases elastase-induced AAA formation in mice

Figure 4
Galectin-3 inhibition by MCP decreases elastase-induced AAA formation in mice

(A) Representative Masson’s trichrome staining and quantification of increment in aortic diameter at 14 days postperfusion of elastase in saline (WT, n=17) and MCP-treated mice (MCP, n=14) mice. Values shown are aortic diameter increase for individual mice, with mean and SEM indicated; *P<0.05, scale bars = 100 μM. (B) Representative iba-1 staining at 14 days postperfusion in WT, MCP, and healthy mice. Negative control is shown. Quantification of positive staining of macrophage content in AAA lesions from saline (WT, n=17) and MCP-treated (MCP, n=14) mice (**P<0.01); scale bars = 100 µM.

Figure 4
Galectin-3 inhibition by MCP decreases elastase-induced AAA formation in mice

(A) Representative Masson’s trichrome staining and quantification of increment in aortic diameter at 14 days postperfusion of elastase in saline (WT, n=17) and MCP-treated mice (MCP, n=14) mice. Values shown are aortic diameter increase for individual mice, with mean and SEM indicated; *P<0.05, scale bars = 100 μM. (B) Representative iba-1 staining at 14 days postperfusion in WT, MCP, and healthy mice. Negative control is shown. Quantification of positive staining of macrophage content in AAA lesions from saline (WT, n=17) and MCP-treated (MCP, n=14) mice (**P<0.01); scale bars = 100 µM.

The three main characteristics of aneurysmal wall dilation are elastin degradation, VSMC loss, and immune-inflammatory response in the wall. Using Verhoeff-van Gieson staining, we observed that elastin degradation was reduced in MCP-treated mice compared with WT mice on day 14 after elastase perfusion (3 ± 0.2 vs. 3.7 ± 0.1, P<0.05). In addition, α-actin staining revealed that VSMC loss was lower in MCP-treated mice vs. WT mice (2.6 ± 0.2 vs. 3.1 ± 0.1, P<0.05). Finally, we found that macrophage content was reduced in MCP-treated mice compared with WT mice (Figure 4B, P=0.01), whereas no significant differences were observed in neutrophil infiltration (not shown).

Galectin-3 inhibition decreases CCL5 expression in vivo and in vitro

Since galectin-3 plays a role as a proinflammatory mediator in pathological vascular remodeling [20,21], we tested the potential effect of MCP treatment in cytokine expression in two new experimental models where animals were killed at day 3 and 14 to obtain mRNA from AAA tissues. At day 3, MCP treatment decreased mRNA expression of galectin-3 (P<0.05), IL-1β (P<0.05), and CCL5 (P<0.05), but not for IL-6 (P=0.08), TNF-α (P>0.1), or CCL2 (P>0.1). Galectin-3, and CCL5 mRNA and protein expression were increased in AAA mice at day 14 compared with control mice (Figure 5A and B). Finally, MCP-treated mice exhibited a decrease in galectin-3 and CCL5 mRNA and protein expression at day 14 compared with WT mice (Figure 5A and B, P<0.05).

Galectin-3 inhibition by MCP decreases galectin-3 and CCL5 expression in elastase-induced AAA in mice

Figure 5
Galectin-3 inhibition by MCP decreases galectin-3 and CCL5 expression in elastase-induced AAA in mice

(A) Quantification by qPCR of galectin-3 (left) and CCL5 (right) mRNA expression in the vascular wall obtained from healthy mice (n=6) or elastase-induced AAA mice injected with saline (WT, n=11) or treated with MCP (n=8); *P<0.05 vs. healthy, **P<0.05 vs WT. (B) Representative galectin-3 and CCL5 staining at 14 days postperfusion in WT, MCP, and healthy mice. Quantification of % of positive staining of galectin-3 (left) and CCL5 (right) in AAA lesions from saline (WT, n=15) and MCP-treated (MCP, n=14) mice (*P<0.05 for both); scale bars = 100 µM.

Figure 5
Galectin-3 inhibition by MCP decreases galectin-3 and CCL5 expression in elastase-induced AAA in mice

(A) Quantification by qPCR of galectin-3 (left) and CCL5 (right) mRNA expression in the vascular wall obtained from healthy mice (n=6) or elastase-induced AAA mice injected with saline (WT, n=11) or treated with MCP (n=8); *P<0.05 vs. healthy, **P<0.05 vs WT. (B) Representative galectin-3 and CCL5 staining at 14 days postperfusion in WT, MCP, and healthy mice. Quantification of % of positive staining of galectin-3 (left) and CCL5 (right) in AAA lesions from saline (WT, n=15) and MCP-treated (MCP, n=14) mice (*P<0.05 for both); scale bars = 100 µM.

To further analyze the potential mechanisms underlying the effect observed in vivo, we performed in vitro studies in VSMC. First, we observed that incubation with recombinant galectin-3 increased CCL5 mRNA expression at 24 h in VSMC (not shown, P<0.05). To address the potential signaling pathways underlying this effect, we analyzed the Janus kinase/signal transducers and activators of the transcription (JAK–STAT) pathway. Galectin-3 induced pSTAT3 activation at 3 h (Figure 6A, P<0.05) and conversely, inhibition of STAT3 activation by a JAK inhibitor decreased galectin-3-induced CCL5 mRNA expression at 24 h (Figure 6B, P<0.05). Finally, we observed that MCP treatment showed a tendency to decrease pSTAT3 at day 3 compared with WT mice (WT = 10 ± 5 vs. MCP = 7 ± 5% of positive immunostaining, P=0.2) while this decrease reached statistical significance after 14 days of treatment (Figure 6C, P<0.05).

Galectin-3 induces CCL5 mRNA expression through STAT activation in human VSMC

Figure 6
Galectin-3 induces CCL5 mRNA expression through STAT activation in human VSMC

(A) Western blot quantification of pSTAT3 following stimulation with recombinant galectin-3 (80 nM) at different times on VSMC. Data are normalized by total STAT3 levels. (B) Quantification by qPCR of CCL5 expression after incubation with recombinant galectin-3 in the presence or absence of a JAK inhibitor (250 nM). All experiments were performed at least in triplicate; *P <0.05 vs basal; **p<0.05 vs Gal-3 stimulated. (C) Representative pSTAT3 staining at 14 days postperfusion in WT, MCP, and healthy mice. Quantification of % of positive staining of pSTAT3 in AAA lesions from saline (WT, n=16) and MCP-treated (MCP, n=14) mice (*P<0.05); scale bars = 100 µM.

Figure 6
Galectin-3 induces CCL5 mRNA expression through STAT activation in human VSMC

(A) Western blot quantification of pSTAT3 following stimulation with recombinant galectin-3 (80 nM) at different times on VSMC. Data are normalized by total STAT3 levels. (B) Quantification by qPCR of CCL5 expression after incubation with recombinant galectin-3 in the presence or absence of a JAK inhibitor (250 nM). All experiments were performed at least in triplicate; *P <0.05 vs basal; **p<0.05 vs Gal-3 stimulated. (C) Representative pSTAT3 staining at 14 days postperfusion in WT, MCP, and healthy mice. Quantification of % of positive staining of pSTAT3 in AAA lesions from saline (WT, n=16) and MCP-treated (MCP, n=14) mice (*P<0.05); scale bars = 100 µM.

Discussion

In recent years, the potential role of galectin-3 as a circulating biomarker of CVDs has been supported by several studies. Increased galectin-3 plasma levels have been observed both, at the stable but also acute phases of atherothrombosis [22,23]. In the same line of evidence, galectin-3 concentrations are increased in the plasma of patients with heart failure [24]. More importantly, galectin-3 plasma levels predict heart failure and/or CV death in large population studies [2528]. Now, we have shown increased galectin-3 plasma levels in AAA patients compared with controls. More importantly, we also observed a strong positive correlation of galectin-3 with the need for surgical repair, independently of aortic size, among other factors. However, there was no added value of galectin-3 in using of aortic size to predict surgical repair, probably due to the high predictive value of aortic size (area under the curve, 0.87). Despite this, the independent association of galectin-3 with the need for surgical repair highlights the potential etiologic role of galectin-3 in the development of AAA.

Galectin-3 expression is increased in CV tissues under pathological remodeling. Galectin-3 is increased at both transcriptional and translational levels in the heart in the early ischemic period [29], as well as in aortas of hypercholesterolemic rabbits or after balloon injury in rats [30]. With regards to AAA, galectin-3 was identified as a gene whose expression is induced by elastase in an intracranial saccular aneurysm [31]. We have also shown that galectin-3 expression is increased in the wall of both human and elastase-induced experimental AAA. Moreover, galectin-3 staining was observed in both macrophages and VSMC in the AAA wall. We previously observed that macrophage differentiation by phorbol esters is associated with increased galectin-3 expression and release [22]. Moreover, the expression of galectin-3 is enhanced when macrophages or aortic VSMC are loaded with lipids and transformed into foam cells, suggesting that galectin-3 could be a marker of VSMC phenotype switching [32]. Galectin-3 has also been involved in VSMC osteogenic differentiation [33] and in fibrotic responses to aldosterone in vitro and in vivo [21]. The human AAA wall is characterized by the presence of cholesterol crystals and iron deposits, related to a process of phagocytosis, along with a chronic immune-inflammatory response, fibrosis, angiogenesis, and calcification [34]. Whether the increased levels of galectin-3 expression in human and experimental AAA walls reflect the increased recruitment of leukocytes or differentiated VSMC, or are due to the potential role of galectin-3 as a mediator of AAA, cannot as yet be distinguished.

Galectin-3 has been involved in pathological CV remodeling and the inhibition of galectin-3 using different approaches has been successfully employed to prevent CVD at the experimental level [20,21,35,36]. Following binding of neutralizing ligands such as MCP, galectin-3 undergoes conformational changes, forms pentamers, and loses its activity [12]. It has been reported that MCP is rich in β-galactose and one mechanism of action for MCP is by antagonizing a β-galactoside galectin-3-binding protein [37]. MCP is able to bind extracellular galectin-3, but also intracellular galectin-3. Importantly, MCP is also able to inhibit galectin-3 expression in animal models [13,21,36,38,39]. In our mouse model of elastase-induced experimental AAA, MCP treatment decreased galectin-3 expression, along with lower AAA expansion compared with controls. Three main characteristics of aneurysmal wall injury are elastin degradation, VSMC depletion, and an immune-inflammatory response of the aortic wall. Elastin layers are one of the main structural components of the aorta and, consequently, the degradation of these elastin layers is a key initial step that allows vessel dilatation. The observed decrease in aortic diameter in AAA of MCP-treated mice was accompanied by reduced degradation of the elastin layer and increased VSMC content, indicating a better preservation of the aortic wall by galectin-3 inhibition. However, no changes were observed for MMP-2, -3, -9, and -13 mRNA using MCP treatment in AAA (not shown), which could suggest that the decreased elastin degradation observed by MCP treatment could be secondary to the observed decreased inflammatory response; however, we could not discard an effect of MCP on MMP activity that has not been addressed in the present study.

The adventitial immune-inflammatory response that takes place in AAA is characterized by the presence of inflammatory cells, cytokines, and chemokines [40]. Increased cytokine and chemokine expression (including CCL5), as well as proinflammatory transcription factors (including JAK–STAT), has been observed in human and experimental AAA walls [4143]. Galectin-3 is involved in proinflammatory responses and is a potent chemoattractant for monocytes and macrophages, and thus could enhance the recruitment of these cells to the artery wall [44]. We have shown that inhibition of galectin-3 by MCP decreased macrophage infiltration, as well as CCL5 expression, in the AAA wall. Galectin-3 acts in an autocrine and paracrine manner to promote the release of proinflammatory mediators. Galectin-3 induced the expression and release of several cytokines, including CCL5, in fibroblasts [45] and macrophages [46]. Accordingly, we have shown that galectin-3 induced CCL5 mRNA expression in human VSMC. Specifically, galectin-3 is able to induce different proinflammatory signaling pathways, such as JAK–STAT [47], among others. JAK–STAT is involved in the regulation of proinflammatory cytokine expression. In this respect, we observed that treatment with a JAK inhibitor prevented galectin-3-induced CCL5 mRNA expression in VSMCs. Moreover, MCP treatment decreased pSTAT3 in experimental AAA. Collectively, these data suggest that galectin-3 may effectively be targeted to prevent pathological cardiovascular remodeling. Clinical trials are currently being conducted to investigate the potential function of galectin-3 inhibitors in different diseases (e.g. MCP in patients with hypertension, ClinicalTrials.gov identifier NCT01960946).

On the whole, we have shown that increased circulating and aortic galectin-3 levels are associated with AAA presence and progression, while galectin-3 inhibition decreased experimental AAA development. Our data suggest a potential role of galectin-3 as a therapeutic target in AAA.

Clinical perspectives

  • Abdominal aortic aneurysm (AAA) is usually asymptomatic and AAA evolution is unpredictable. Moreover, no specific therapy is available, except surgery for those patients with aortic diameter >5.5 cm.

  • Galectin-3 has been proposed as a prognostic and therapeutic target in different cardiovascular diseases, but no information in AAA is available. Increased galectin-3 levels were observed in plasma and tissue of AAA patients compared with controls; moreover, galectin-3 plasma levels were associated with AAA evolution.

  • Interestingly, increased galectin-3 plasma and tissue levels were also observed in elastase-induced experimental AAA. Conversely, galectin-3 inhibition decreased experimental AAA formation, contributing to vessel structure preservation by decreasing the inflammatory response. Galectin-3 is therefore a potential prognostic and therapeutic target in AAA.

We are grateful to Dr Jean-Baptiste Michel (INSERM, Paris) for kindly supplying human aortic samples.

Author Contribution

Carlos-Ernesto Fernandez performed the analysis of galectin-3 concentration in mice samples, as well as immunohistochemical and PCR analysis in human and animal tissues, and contributed to the writing of the paper. Carlos Tarin performed and designed animal experiments for histological studies. Diego Martinez-Lopez and Monica Torres Fonseca performed animal experiments for mRNA analysis. Raquel Roldan-Montero performed mRNA extraction and PCR analysis of animal tissues. Melina Vega de Ceniga and Jes Sandal Lindholt performed the statistical analysis of Gal-3 concentration in human samples and edited the paper. Jesus Egido and Natalia Lopez de Andres edited and contributed to discussions of the paper. Luis-Miguel Blanco-Colio performed the analysis of galectin-3 concentration in human samples and edited the paper. Jose-Luis Martin-Ventura conceived, designed, and coordinated the research plan and wrote/edited the paper.

Funding

The Spanish MINECO [SAF2016-80843-R], Proyecto de cooperacion Interuniversitaria UAM-Santander con America Latina [CEAL-AL/2017-09], Fondo de Investigaciones Sanitarias ISCiii-FEDER [Biobancos PT13/0010/0012 and PI14/00386], Centro de Investigación Biomédica en Red de Enfermedades cardiovasculares (CIBERCV) y Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) and FRIAT.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • AAA

    abdominal aortic aneurysm

  •  
  • ABI

    Ankle brachial index

  •  
  • ACE

    angiotensin-converting enzyme

  •  
  • CT

    computed tomography

  •  
  • CCL5

    chemokine (C-C motif) ligand 5

  •  
  • CVD

    cardiovascular diseases

  •  
  • GADPH

    Glyceraldehyde-3-phosphate-deshydrogenase

  •  
  • HR

    hazard ratio

  •  
  • IL-1B

    interleukin 1-beta

  •  
  • MCP

    modified citrus pectin

  •  
  • MMP

    matrix metalloproteinase

  •  
  • OR

    odds ratio

  •  
  • PAD

    peripheral arterial disease

  •  
  • STAT3

    Signal transducer and activator of transcription 3

  •  
  • TNF-a

    Tumor necrosis factor -alpha

  •  
  • VSMC

    vascular smooth muscle cell

  •  
  • WT

    wild type

References

References
1
Davis
F.M.
,
Rateri
D.L.
and
Daugherty
A.
(
2015
)
Abdominal aortic aneurysm: novel mechanisms and therapies
.
Curr. Opin. Cardiol.
30
,
566
573
2
Kuivaniemi
H.
,
Ryer
E.J.
,
Elmore
J.R.
and
Tromp
G.
(
2015
)
Understanding the pathogenesis of abdominal aortic aneurysms
.
Expert Rev. Cardiovasc. Ther.
13
,
975
987
3
Golledge
J.
,
Norman
P.E.
,
Murphy
M.P.
and
Dalman
R.L.
(
2016
)
Challenges and opportunities in limiting abdominal aortic aneurysm growth
.
J. Vasc. Surg.
65
,
225
233
4
Wanhainen
A.
,
Mani
K.
and
Golledge
J.
(
2016
)
Surrogate markers of abdominal aortic aneurysm progression
.
Arterioscler. Thromb. Vasc. Biol.
36
,
236
244
5
Yang
R.Y.
,
Rabinovich
G.A.
and
Liu
F.T.
(
2008
)
Galectins: structure, function and therapeutic potential
.
Expert Rev. Mol. Med.
10
,
e17
6
von Mach
T.
,
Carlsson
M. C.
,
Straube
T.
,
Nilsson
U.
,
Leffler
H.
and
Jacob
R.
(
2014
)
Ligand binding and complex formation of galectin-3 is modulated by pH variations
.
Biochem. J.
457
,
107
115
7
Ochieng
J.
,
Furtak
V.
and
Lukyanov
P.
(
2004
)
Extracellular functions of galectin-3
.
Glycoconj. J.
19
,
527
535
8
Desmedt
V.
,
Desmedt
S.
,
Delanghe
J. R.
,
Speeckaert
R.
and
Speeckaert
M.M.
(
2016
)
Galectin-3 in renal pathology: more than just an innocent bystander
.
Am. J. Nephrol.
43
,
305
317
9
Filipe
M.D.
,
Meijers
W.C.
,
Rogier van der Velde
A.
and
de Boer
R.A.
(
2015
)
Galectin-3 and heart failure: prognosis, prediction & clinical utility
.
Clin. Chim. Acta
443
,
48
56
10
Chen
A.
,
Hou
W.
,
Zhang
Y.
,
Chen
Y.
and
He
B.
(
2015
)
Prognostic value of serum galectin-3 in patients with heart failure: a meta-analysis
.
Int. J. Cardiol.
182
,
168
170
11
Daniels
L.B.
,
Clopton
P.
,
Laughlin
G.A.
,
Maisel
A.S.
and
Barrett-Connor
E.
(
2014
)
Galectin-3 is independently associated with cardiovascular mortality in community-dwelling older adults without known cardiovascular disease: The Rancho Bernardo Study
.
Am. Heart J.
167
,
674
682
12
de Boer
R.A.
,
van der Velde
A.R.
,
Mueller
C.
,
van Veldhuisen
D.J.
,
Anker
S.D.
,
Peacock
W.F.
et al
(
2014
)
Galectin-3: a modifiable risk factor in heart failure
.
Cardiovasc. Drugs Ther.
28
,
237
246
13
Calvier
L.
,
Martinez-Martinez
E.
,
Miana
M.
,
Cachofeiro
V.
,
Rousseau
E.
,
Sádaba
J. R.
et al
(
2015
)
The impact of galectin-3 inhibition on aldosterone-induced cardiac and renal injuries
.
JACC Heart Fail.
3
,
59
67
14
MacKinnon
A.C.
,
Liu
X.
,
Hadoke
P.W.
and
Miller
M.R.
(
2013
)
Inhibition of galectin-3 reduces atherosclerosis in apolipoprotein E-deficient mice
.
Glycobiology
23
,
654
663
15
Ridley
B.L.
,
O’Neill
M.A.
and
Mohnen
D.
(
2001
)
Pectins: structure, biosynthesis, and oligogalacturonide-related signaling
.
Phytochemistry
57
,
929
967
16
Gunning
A.P.
,
Bongaerts
R.J.
and
Morris
V.J.
(
2009
)
Recognition of galactan components of pectin by galectin-3
.
FASEB J.
23
,
415
424
17
Caligiuri
G.
,
Rossignol
P.
,
Julia
P.
,
Groyer
E.
,
Mouradian
D.
,
Urbain
D.
et al
(
2006
)
Reduced immunoregulatory CD31+ T cells in patients with atherosclerotic abdominal aortic aneurysm
.
Arterioscler. Thromb. Vasc. Biol.
26
,
618
623
18
Tarín
C.
,
Fernandez-Garcia
C.E.
,
Burillo
E.
,
Pastor-Vargas
C.
,
Llamas-Granda
P.
,
Castejón
B.
et al
(
2016
)
Lipocalin-2 deficiency or blockade protects against aortic abdominal aneurysm development in mice
.
Cardiovasc. Res.
111
,
262
273
19
Burillo
E.
,
Tarin
C.
,
Torres-Fonseca
M.M.
,
Fernandez-García
C. E.
,
Martinez-Pinna
R.
,
Martinez-Lopez
D.
et al
(
2016
)
Paraoxonase-1 overexpression prevents experimental abdominal aortic aneurysm progression
.
Clin. Sci. (Lond.)
130
,
1027
1038
20
Nachtigal
M.
,
Ghaffar
A.
and
Mayer
E.P.
(
2008
)
Galectin-3 gene inactivation reduces atherosclerotic lesions and adventitial inflammation in ApoE-deficient mice
.
Am. J. Pathol.
172
,
247
255
21
Calvier
L.
,
Miana
M.
,
Reboul
P.
and
Cachofeiro
V.
(
2013
)
Galectin-3 mediates aldosterone-induced vascular fibrosis
.
Arterioscler. Thromb. Vasc. Biol.
33
,
67
75
22
Madrigal-Matute
J.
,
Lindholt
J. S.
,
Fernandez-Garcia
C. E.
,
Benito-Martin
A.
,
Burillo
E.
,
Zalba
G.
et al
(
2014
)
Galectin-3, a biomarker linking oxidative stress and inflammation with the clinical outcomes of patients with atherothrombosis
.
J. Am. Heart Assoc.
3
,
pii: e000785 doi:10.1161/JAHA.114.000785
23
Winter
M.P.
,
Wiesbauer
F.
,
Alimohammadi
A.
,
Blessberger
H.
,
Pavo
N.
,
Schillinger
M.
et al
(
2016
)
Soluble galectin-3 is associated with premature myocardial infarction
.
Eur. J. Clin. Invest.
46
,
386
391
24
van Kimmenade
R.R.
,
Januzzi
J.L.J.
,
Ellinor
P.T.
,
Sharma
U.C.
,
Bakker
J.A.
,
Low
A.F.
et al
(
2006
)
Utility of amino-terminal pro-brain natriuretic peptide, galectin-3, and apelin for the evaluation of patients with acute heart failure
.
J. Am. Coll. Cardiol.
48
,
1217
1224
25
van der Velde
A.R.
,
Meijers
W.C
,
Ho
J.E.
,
Brouwers
F.P.
,
Rienstra
M.
,
Bakker
S.J.
et al
(
2016
)
Serial galectin-3 and future cardiovascular disease in the general population
.
Heart
102
,
1134
1141
26
Jagodzinski
A.
,
Havulinna
A.S.
,
Appelbaum
S.
,
Zeller
T.
,
Jousilahti
P.
,
Skytte-Johanssen
S.
et al
(
2015
)
Predictive value of galectin-3 for incident cardiovascular disease and heart failure in the population-based FINRISK 1997 cohort
.
Int. J. Cardiol.
192
,
33
39
27
Maiolino
G.
,
Rossitto
G.
,
Pedon
L.
,
Cesari
M.
,
Frigo
A.C.
,
Azzolini
M.
et al
(
2015
)
Galectin-3 predicts long-term cardiovascular death in high-risk patients with coronary artery disease
.
Arterioscler. Thromb. Vasc. Biol.
35
,
725
732
28
Tuñón
J.
,
Blanco-Colio
L.M.
,
Cristóbal
C.
,
Tarín
N.
,
Higueras
J.
,
Huelmos
A.
et al
(
2014
)
Usefulness of a combination of monocyte chemoattractant protein-1, galectin-3, and N-terminal probrain natriuretic peptide to predict cardiovascular events in patients with coronary artery disease
.
Am. J. Cardiol.
113
,
434
440
29
Hashmi
S.
and
Al-Salam
S.
(
2015
)
Galectin-3 is expressed in the myocardium very early post-myocardial infarction
.
Cardiovasc. Pathol.
24
,
213
223
30
Arar
C.
,
Gaudin
J. C.
,
Capron
L.
and
Legrand
A.
(
1998
)
Galectin-3 gene (LGALS3) expression in experimental atherosclerosis and cultured smooth muscle cells
.
FEBS Lett.
430
,
307
311
31
Kadirvel
R.
,
Ding
Y.H.
,
Dai
D.
,
Lewis
D.A.
and
Kallmes
D.F.
(
2010
)
Differential expression of genes in elastase-induced saccular aneurysms with high and low aspect ratios
.
Neurosurgery
66
,
578
584
,
discussion 84
32
Rong
J.X.
,
Shapiro
M.
,
Trogan
E.
and
Fisher
E.A.
(
2003
)
Transdifferentiation of mouse aortic smooth muscle cells to a macrophage-like state after cholesterol loading
.
Proc. Natl Acad. Sci. U.S.A.
100
,
13531
13536
33
Menini
S.
,
Iacobini
C.
,
Ricci
C.
and
Blasetti Fantauzzi
C.
(
2013
)
The galectin-3/RAGE dyad modulates vascular osteogenesis in atherosclerosis
.
Cardiovasc. Res.
100
,
472
480
34
Michel
J.B.
,
Martin-Ventura
J.L.
,
Egido
J.
,
Sakalihasan
N.
,
Treska
V.
,
Lindholt
J.S.
et al
(
2011
)
Novel aspects of the pathogenesis of aneurysms of the abdominal aorta in humans
.
Cardiovasc. Res.
90
,
18
27
35
Vergaro
G.
,
Prud’homme
M.
,
Fazal
L.
,
Merval
R.
,
Passino
C.
,
Emdin
M.
et al
(
2016
)
Inhibition of galectin-3 pathway prevents isoproterenol-induced left ventricular dysfunction and fibrosis in mice
.
Hypertension
67
,
606
612
36
Martínez-Martínez
E.
,
Calvier
L.
,
Fernández-Celis
A.
,
Rousseau
E.
,
Jurado-López
R.
,
Rossoni
L.V.
et al
(
2015
)
Galectin-3 blockade inhibits cardiac inflammation and fibrosis in experimental hyperaldosteronism and hypertension
.
Hypertension
66
,
767
775
37
Glinsky
V.V.
and
Raz
A.
(
2009
)
Modified citrus pectin anti-metastatic properties: one bullet, multiple targets
.
Carbohydr. Res.
344
,
1788
17891
38
Kolatsi-Joannou
M.
,
Price
K.L.
,
Winyard
P.J.
and
Long
D.A.
(
2011
)
Modified citrus pectin reduces galectin-3 expression and disease severity in experimental acute kidney injury
.
PLoS ONE
6
,
e18683
39
Sádaba
J.R.
,
Martínez-Martínez
E.
,
Arrieta
V.
,
Álvarez
V.
,
Fernández-Celis
A.
,
Ibarrola
J.
et al
(
2016
)
Role for galectin-3 in calcific aortic valve stenosis
.
J. Am. Heart Assoc.
5
,
pii: e004360 doi:
40
Dale
M.A.
,
Ruhlman
M.K.
and
Baxter
B.T.
(
2015
)
Inflammatory cell phenotypes in AAAs: their role and potential as targets for therapy
.
Arterioscler. Thromb. Vasc. Biol.
35
,
1746
17455
41
Sharma
A.K.
,
Lu
G.
,
Jester
A.
,
Johnston
W.F.
,
Zhao
Y.
,
Hajzus
V.A.
et al
(
2012
)
Experimental abdominal aortic aneurysm formation is mediated by IL-17 and attenuated by mesenchymal stem cell treatment
.
Circulation
126
,
S38
S45
42
Iida
Y.
,
Xu
B.
,
Xuan
H.
,
Glover
K.J.
,
Tanaka
H.
,
Hu
X.
et al
(
2013
)
Peptide inhibitor of CXCL4-CCL5 heterodimer formation, MKEY, inhibits experimental aortic aneurysm initiation and progression
.
Arterioscler. Thromb. Vasc. Biol.
33
,
718
726
43
Liao
M.
,
Xu
J.
,
Clair
A.J.
,
Ehrman
B.
,
Graham
L.M.
and
Eagleton
M.J.
(
2012
)
Local and systemic alterations in signal transducers and activators of transcription (STAT) associated with human abdominal aortic aneurysms
.
J. Surg. Res.
176
,
321
328
44
Sano
H.
,
Hsu
D.K.
,
Yu
L.
,
Apgar
J.R.
,
Kuwabara
I.
,
Yamanaka
T.
et al
(
2000
)
Human galectin-3 is a novel chemoattractant for monocytes and macrophages
.
J. Immunol.
165
,
2156
2164
45
Filer
A.
,
Bik
M.
,
Parsonage
G.N.
,
Fitton
J.
,
Trebilcock
E.
,
Howlett
K.
et al
(
2009
)
Galectin 3 induces a distinctive pattern of cytokine and chemokine production in rheumatoid synovial fibroblasts via selective signaling pathways
.
Arthritis Rheum.
60
,
1604
16014
46
Papaspyridonos
M.
,
McNeill
E.
,
de Bono
J.P.
and
Smith
A.
(
2008
)
Galectin-3 is an amplifier of inflammation in atherosclerotic plaque progression through macrophage activation and monocyte chemoattraction
.
Arterioscler. Thromb. Vasc. Biol.
28
,
433
440
47
Jeon
S.B.
,
Yoon
H.J.
,
Chang
C.Y.
,
Koh
H.S.
,
Jeon
S.H.
and
Park
E.J.
(
2010
)
Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway
.
J. Immunol.
185
,
7037
7046