Abstract
OBJECTIVES
Galectin-3 is a cardiac biomarker for heart failure in humans. However, it has not been investigated in dogs with naturally occurring heart disease. This study aimed to compare plasma galectin-3 concentration in healthy dogs and those with myxomatous mitral valve disease (MMVD) and explore the potential association of galectin-3 with other cardiac biomarkers, inflammatory cytokines, echocardiographic estimates, and dog characteristics.
ANIMALS
10 healthy dogs and 30 dogs with MMVD were prospectively recruited.
PROCEDURES
In this case-control study, plasma galectin-3, inflammatory cytokines, echocardiographic estimates, and other cardiac biomarkers were measured, and dog characteristics were recorded.
RESULTS
Plasma galectin-3 concentration was significantly higher in dogs with MMVD (2.94 [interquartile range, 1.61 to 5.20] ng/mL) than in healthy controls (1.56 [0.69 to 1.84] ng/mL, P = .009). Logistic regression analysis revealed that galectin-3 concentration and age predicted the presence of MMVD (predictive accuracy = 90.0%, P < .05). A cut-off value ≥ 1.9 ng/mL for galectin-3 differentiated healthy dogs from dogs with MMVD (70% sensitivity; 90% specificity AUC, 0.77; P = .01).
CLINICAL RELEVANCE
Plasma galectin-3 concentration was higher in dogs with MMVD than in healthy dogs, indicating that it is a novel cardiac biomarker in dogs with MMVD although there was no significant difference between MMVD stages.
Introduction
Myxomatous mitral valve disease (MMVD) is a common cardiac disease in dogs that involves degenerative changes in the mitral valve. These degenerative changes result in mitral regurgitation, volume overload, and left-sided congestive heart failure.1,2 Despite being the most common cardiac disease in dogs, the pathogenesis of MMVD has not yet been elucidated.1,2 Cardiac biomarkers, such as N-terminal pro-brain natriuretic peptide (NT-proBNP), cardiac troponin I (cTnI), and proinflammatory cytokines, have been used to identify cardiac diseases, predict the progression of the disease, and determine medication efficiency.3,4
Galectin-3, a beta-galactoside-binding lectin originally investigated in relation to cancer progression, is upregulated in human patients with heart failure and correlated with cardiac fibrosis.4–10 Several cell types secrete galectin-3, including activated macrophages, triggering resting fibroblasts into displaying a matrix-forming phenotype.4,9,11 In the clinical setting with human patients, galectin-3 is used as a biomarker for cardiac fibrosis and for the evaluation of heart failure progression and prognosis.5,12,13
Upregulation of galectin-3 has been detected in animal models of heart disease.4,13,14 In a murine model, significant myocardial fibrosis was observed after galectin-3 was infused into the pericardial sac of healthy rats.4 Compared to controls, dogs that developed heart failure after aortic banding exhibited elevated levels of cardiomyocyte galectin-3, and echocardiographic diastolic parameters correlated with galectin-3 expression.14 Also, previous studies identified elevated circulating galectin-3 levels in dogs with spontaneous heart diseases, including MMVD.13,15 However, the clinical value of galectin-3 as a biomarker for the diagnosis of MMVD has not been investigated, and factors influencing the plasma concentration of galectin-3 have not been identified thus far.
This study aimed to test the hypothesis that compared to healthy dogs, the plasma galectin-3 level is significantly increased in dogs with MMVD. We also investigated the potential associations of other cardiac biomarkers, inflammatory cytokines, echocardiographic estimates, and canine characteristics with galectin-3 concentration.
Methods
Animals
This case-control study was approved by the Institutional Animal Care and Use Committee of Konkuk University (approval No. KU 20006), Seoul, Republic of Korea. Written informed consent was obtained from the dog owners involved in this study.
The study population consisted of client-owned dogs prospectively recruited at the Konkuk University Veterinary Medicine Teaching Hospital from January 2020 to December 2020. Dogs with MMVD verified by echocardiography were included in the MMVD group and further categorized into 4 stages based on the American College of Veterinary Internal Medicine (ACVIM) classification system.16 Healthy dogs (without physical or echocardiographic evidence of cardiac disease) were included as controls. Dogs with congenital heart disease; significant systemic, organ-related, or endocrine disease; azotemia (creatinine > 2.0 mg/dL) diagnosed by laboratory evaluation and imaging procedures such as radiography and ultrasonography; or taking anti-inflammatory drugs were excluded. Data, including breed, age, sex, medical history, and presence and duration of clinical signs, were collected via client interviews. Dogs with clinical signs were further evaluated for severity based on a modified scoring system by Polizopoulou et al.17
A physical examination was performed on all dogs. Body weight, temperature, heart rate, and respiratory rate were determined, and the chest was auscultated for the presence of abnormal lung sounds and a murmur. The indirect blood pressure of the dogs was measured using an automated oscillometric device (Cardell Insight Diagnostic Monitor; Midmark) while standing. The cuff had a width of approximately 40% of the left forelimb circumference. Five consecutive blood pressure measurements were recorded; the minimum and maximum pressure readings were excluded, and the mean of 3 measurements was calculated.
Galectin-3
For all samples, blood was collected via jugular venipuncture using a 23-gauge needle attached to a syringe. Two milliliters of blood were needed to run the galectin-3 assay. Blood was immediately transferred to EDTA tubes. Plasma was separated by centrifugation at 1,000 X g for 15 min, transferred to microtubes in aliquots, and stored at −80 °C for later analysis. Plasma concentrations of galectin-3 were measured using a canine galectin-3 ELISA kit (Canine Gal-3 ELISA kit; BlueGene Biotech) according to previously described methods.13 The kit manufacturer's detectable range for the assay was 0.1 to 10 ng/mL. For subsequent statistical analysis, galectin-3 concentrations higher than the detection limit of the assay were assumed as 10 ng/mL.
NT-proBNP and cTnI
Blood was immediately placed into serum-separating tubes following collection. The serum was separated by centrifugation at 1,000 X g for 15 min. Serum aliquots were transferred to a commercial laboratory (IDEXX Laboratories) on ice and analyzed for NT-proBNP using species-specific ELISA assay and cTnI using a highly sensitive chemiluminescence immunoassay. For subsequent statistical analysis, cTnI concentrations lower than the detection limit of the assay were assumed as 0 ng/mL.
Inflammatory cytokines
Whole blood was immediately placed into EDTA tubes upon collection. The samples were allocated into RNAse-free microtubes, mixed with RNAlate Stabilization Solution (ThermoFisher Scientific), and stored at −80 °C for later analysis.
RNA isolation was performed using a commercially available kit (RiboPure blood kit; Ambion Ltd) according to the manufacturer's protocol. The frozen mixture was gradually thawed at room temperature (20 °C) and centrifuged for 1 min at 16,000 X g. The supernatant was removed, and the pellet of blood cells was lysed by adding 800 μL lysis solution and 50 μL sodium acetate solution. Acid-phenol-chloroform (500 μL) was then added to the homogenous mixture. After incubating for 5 min at room temperature (20 °C), the mixture was centrifuged for 1 min at 16,000 X g. The upper aqueous phase was separated and transferred into a new microtube, 600 μL 100% ethanol was added, and the mixture was transferred again into a new microtube and thoroughly mixed. The sample (700 μL) was passed through a filter cartridge, followed by washing with 700 μL Wash Solution 1 and Wash Solution 2/3, and the flow-through was discarded. The filter was transferred to a newly labeled collection tube, and 50 μL preheated elution solution was added, and the mixture was incubated for 20 s at room temperature. The tube was centrifuged for 30 seconds at 16,000 X g. The collected RNA was stored at −80 °C for further use.
cDNA was synthesized using a commercially available master mix (High-Capacity RNA-to-cDNA kit; Applied Biosystems) according to the manufacturer's instructions. The primers for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-2, IL-8, IL-33, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used for real-time PCR. The SYBR Green PCR Master Mix (SYBR Green PCR Master Mix; Applied Biosystems) was used for the PCR reaction, performed on a LightCycler 96 system (Roche). Each reaction mixture contained 0.4 μL each 10 µM forward and reverse primers, 1 μL cDNA sample as PCR template, 10 μL SYBR Green PCR Master Mix, and 8.2 μL distilled water to obtain a final volume of 20 μL. cDNA samples were replaced with distilled water for the negative control reaction. The amplification conditions were as follows: 50 °C for 2 min, 90 °C for 10 min, and 40 cycles of PCR (95 °C for 15 s and 60 °C for 1 min), followed by a dissociation step (95 °C for 1 min, followed by a gradual increase of temperature from 55 °C to 95 °C).
Real-time data were evaluated using LightCycler 96 software (version 1.1: Roche). Relative expression levels were normalized to GAPDH expression and calculated using the comparative cycle threshold (2−ΔΔCt) method.
Thoracic radiography
The dogs were manually restrained without sedation in the ventrodorsal and right lateral recumbency on the examination table. Right lateral and ventrodorsal views of thoracic radiographs obtained during maximal inspiration were used to calculate the vertebral heart score and detect abnormalities in the lung field (Titan 2000V; Comed Medical System). The vertebral heart score was calculated as previously described.18
Echocardiography
To diagnose MMVD, assess its severity, and exclude other cardiac diseases, echocardiography was performed and evaluated by an experienced investigator (J-HK). The dogs were placed in the right and left lateral recumbency on the examination table without any sedation. Echocardiography was conducted using an ultrasonographic unit (EPIQ 7; Philips Ultrasound) equipped with 3.0- to 8.5-MHz phased-array transducers and electrocardiographic monitoring. Diagnosis of MMVD was defined as the presence of thickened or prolapsed mitral valve leaflets and mitral regurgitation on the right parasternal long axis and the left apical 4-chamber views. An imaging of the mitral regurgitant jet was obtained using a color Doppler echocardiogram and regurgitation jet area compared to the left atrium area (RJ score) was calculated. The transmitral flow was obtained using the pulsed-wave Doppler method between the mitral leaflet tips during diastole and measurements of peak velocity of transmitral flow during early diastole (E wave), peak velocity of transmitral flow during late diastole (A wave), and transmitral flow E wave velocity to A wave velocity ratio (E/A) were performed. Mitral annulus velocities were measured at the septal annulus by using tissue Doppler imaging and E wave velocity to E’ wave velocity ratio (E/E’) was calculated. Peak velocity of mitral regurgitation (MR velocity) and left atrial to aortic root ratio (LA/Ao) were also measured. M-mode measurements of the left ventricle from the right parasternal short axis view were used to obtain end-diastolic left ventricular internal dimension normalized for body weight, end-systolic left ventricular internal dimension normalized for body weight, end-diastolic interventricular septal dimension normalized for body weight, end-systolic interventricular septal dimension normalized for body weight, end-diastolic left ventricular posterior wall dimension normalized for body weight, end-systolic left ventricular posterior wall dimension normalized for body weight, fractional shortening, and ejection fraction. The measurements were performed over 3 consecutive cardiac cycles, and the mean value was calculated.
Statistical analysis
Statistical analyses were performed using SPSS Statistics version 25.0 (IBM Corporation). To detect a significant difference between each group, with 80% power and a significance level of 5%, a total sample size of 40 was required in this study. Descriptive statistics are presented as the median and IQR. Statistical significance was set at P < .05.
The nonparametric Mann-Whitney U-test and Kruskal-Wallis test were used to compare the indices between the control and MMVD groups and among the ACVIM stages of the MMVD and control groups. Pairwise comparisons were conducted using the Mann-Whitney U-test with Bonferroni adjustment, with P < .01 considered statistically significant. Univariate and multiple regression analyses were used to investigate the potential associations between galectin-3 expression and cardiac biomarkers (NT-proBNP and cTnI), inflammatory cytokines (TNF-α, IL-1β, IL-2, IL-8, and IL-33), echocardiographic parameters, and dog characteristics (age, scores for clinical signs, duration of clinical signs, body weight, heart rate, respiratory rate, body temperature, and systolic blood pressure). In the multiple regression model, a forward stepwise manner was used for analysis with only the main effects. Logistic regression was used to develop models for the detection of MMVD, and receiver operating characteristic curve analysis was used to determine the optimal cut-off value of galectin-3 concentration for discriminating between healthy dogs and dogs with MMVD.
Results
Descriptive statistics
In total, 40 dogs (20 male dogs, 3 intact and 17 castrated; 20 female dogs, 2 intact and 18 spayed) were included in the study, with a median body weight of 4.39 kg (IQR: 3.14 to 5.95 kg) and a median age of 11 years (IQR, 8 to 13 years). Ten dogs were healthy and included in the control group, whereas 30 dogs were diagnosed with MMVD. The median age of the control group and case group was 5.5 years (IQR, 1.68 to 8.75 years) and 12 years (IQR, 10.75 to 13.47 years), respectively. The dogs in the MMVD group were stratified based on the ACVIM staging as follows: stage A (n = 1), stage B1 (9), stage B2 (11), and stage C (9). The dogs participating in the study were of 14 breeds; breeds in the control group were Poodle (n = 2), Dachshund (2), English Cocker Spaniel (1), Beagle (1), Fox Terrier (1), Papillon (1), Miniature Pinscher (1), and mixed (1). Breeds in the MMVD group were Maltese (n = 9), Poodle (6), Shih-Tzu (5), Yorkshire Terrier (2), Pomeranian (2), English Cocker Spaniel (1), Pug (1), Cavalier King Charles Spaniel (CKCS; 1), and mixed (3). Characteristics of dogs at various stages of MMVD and healthy controls are shown (Table 1).
Characteristics of dogs at various stages of MMVD and healthy controls.
| Group | Control | MMVD | MMVD Stage B1 | MMVD Stage B2 | MMVD Stage C |
|---|---|---|---|---|---|
| Number | 10 | 30 | 9 | 11 | 9 |
| Sex (male/female) [IM:CM]/[IF:SF] | 5/5 [1:4]/[1:4] | 15/15 [2:13]/[1:14] | 2/7 [0:2]/[1:6] | 8/3 [1:7]/[0:3] | 4/5 [1:3]/[0:5] |
| Weight (kg) | 5.05 (3.82–6.90) | 4.12 (2.91–5.42) | 5.4 (3.18–6.20) | 3.6 (2.85–4.6) | 4.25 (3.00–4.88) |
| Age (years) | 5.5 (1.68–8.75) | 12 (10.75–13.47)a | 12 (11.35–16.00)a | 12 (10–13)a | 11 (9.5–13.75) |
| HR (beats/min) | 131 (120–138) | 144 (129–171)a | 138 (126–162) | 144 (120–152) | 168 (147.5–189)a |
| Murmur [grade/N] | – | [2/2] [3/6] [4/9] [5/9] [6/2] | [2/1] [3/3] [4/3] [5/1] | [3/2] [4/5] [5/4] | [2/1] [3/1] [4/1] [5/4] [6/2] |
| SAP (mmHg) | 132 (119–150) | 139 (133.5–148) | 145 (138.5–153.5) | 136 (130–147) | 135 (134–149) |
| Clinical scores | 0 (0–0) | 1.5 (0–6)a | 0 (0–5) | 1 (0–3) | 6 (4–10.5)a, b |
| Clinical duration (days) | 0 (0–0) | 0 (0–8.5)a | 0 (0–2.5) | 0 (0–8) | 6 (0.3–25.5)a |
Summary of dog characteristics. The characteristics of 1 dog with MMVD stage A are not listed. Data are presented as medians and IQRs. Mann-Whitney U-test and Kruskal-Wallis test were performed.
CM = Castrated male. HR = Heart rate. IF = Intact female. IM = Intact male. MMVD = Myxomatous mitral valve disease. SAP = Systolic arterial blood pressure. SF = Spayed female.
Values with superscript letters in the same row are significantly different when compared as follows:
Outcomes
Significant differences between control and MMVD groups were detected in terms of age (P = .001), heart rate (P = .011), clinical scores (P = .002), duration of clinical signs (P = .028), and levels of NT-proBNP (P = .000), galectin-3 (P = .009), TNF-α (P = .036), and IL-2 (P = .004) (Figure 1). On echocardiography, significant differences were detected in terms of MR velocity (P = .004) and RJ score (P = .004).
A—Box-whisker plot showing plasma concentrations of galectin-3 in healthy dogs and dogs with myxomatous mitral valve disease (MMVD). Solid horizontal lines indicate the interquartile range (top quartile and bottom quartile) and median; whiskers indicate range. Concentration of galectin-3 significantly increased in dogs with MMVD (P = .009). B—Box-whisker plot showing plasma concentrations of galectin-3 in dogs classified according to the American College Veterinary Internal Medicine stage. Whiskers indicate range. Concentration of galectin-3 was significantly higher in dogs with MMVD stage C (P = .002) than in healthy dogs. There were no significant differences among the control, MMVD stage A (1 dog, not shown), B1, and B2 groups.
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.03.0063
Dogs with MMVD were further classified into different ACVIM stages of MMVD (Figure 1). Significant differences in age (P = .003) and levels of NT-proBNP (P = .000) and IL-2 (P = .004) were detected between the control and stage B1 groups. Significant differences in age (P = .005), level of NT-proBNP (P = .000), MR velocity (P = .005), and RJ score (P = .005) were also noted between the control and stage B2 groups (Tables 1, 2 and 3). Between the control and stage C groups, there were significant differences in heart rate (P = .000), clinical scores (P = .000), clinical duration (P = .000), levels of NT-proBNP (P = .000) and galectin-3 (P = .002), MR velocity (P = .009), and RJ score (P = .009).
Compared to dogs with stage C MMVD, dogs with stage B1 MMVD exhibited significantly different values for end-diastolic left ventricular posterior wall dimension normalized for body weight (P = .008), LA/Ao ratio (P = .001), and E wave velocity (P = .008) (Table 2). Dogs with stage B2 MMVD showed significantly different clinical scores (P = .001) and LA/Ao ratios (P = .01) compared to those with stage C MMVD (Tables 1 and 2).
Echocardiographic measurements of dogs with MMVD and healthy controls.
| Group | Control | MMVD | MMVD Stage B1 | MMVD Stage B2 | MMVD Stage C |
|---|---|---|---|---|---|
| LVIDdN | 1.38 (1.36–1.5) | 1.5 (1.295–1.82) | 1.09 (0.975–1.41) | 1.6 (1.37–1.73) | 1.87 (1.575–2.18) |
| LVIDsN | 0.85 (0.55–0.86) | 0.67 (0.49–0.78) | 0.48 (0.385–0.665) | 0.67 (0.62–0.77) | 0.76 (0.54–1.02) |
| LVPWdN | 0.51 (0.30–0.55) | 0.43 (0.37–0.52) | 0.50 (0.39–0.605)b | 0.45 (0.37–0.52) | 0.41 (0.37–0.445) |
| LVPWsN | 0.67 (0.53–0.79) | 0.78 (0.69–0.835) | 0.76 (0.675–0.090) | 0.79 (0.69–0.83) | 0.78 (0.665–0.845) |
| IVSdN | 0.47 (0.47–0.53) | 0.47 (0.41–0.545) | 0.50 (0.43–0.585) | 0.47 (0.41–0.50) | 0.43 (0.375–0.52) |
| IVSsN | 0.72 (0.64–0.75) | 0.68 (0.615–0.75) | 0.68 (0.60–0.75) | 0.68 (0.62–0.75) | 0.70 (0.595–0.79) |
| FS (%) | 40.07 (34.53–59.47) | 55.76 (50.47–61.89) | 56.16 (46.21–61.08) | 53.63 (48.33–61.73) | 60 (53.5–63.6) |
| EF (%) | 72.27 (66.37–91.20) | 87.61 (83.05–92.06) | 88.10 (79.60–91.71) | 86.30 (81.83–92.16) | 90 (85.51–92.63) |
| MR velocity (m/s) | N | 5.45 (3.81–6.05)a | 4.73 (1.50–5.66) | 6.03 (5.33–6.20)a | 5.43 (4.30–5.58)a |
| LA/Ao | 1.21 (0.91–1.28) | 1.29 (1.06–1.50) | 1.11 (0.94–1.21)b | 1.30 (1.21–1.44)b | 1.64 (1.47–2.49) |
| RJ score (%) | N | 44.23 (27.10–59.50)a | 32.16 (4.45–44.70) | 57.20 (40.16–67.86)a | 58.13 (26.05–66.71)a |
| E wave (cm/s) | 75.56 (69.74–83.79) | 79.67 (65.51–106.64) | 66.02 (43.58–86.66)b | 76.34 (74.31–110.53) | 106.64 (81.51–124.38) |
| A wave (cm/s) | 67.71 (45.20–87.17) | 82.45 (62.20–107.65) | 86.03 (59.58–112.98) | 94.79 (76.34–107.15) | 64.32 (54.82–108.16) |
| E’ wave (cm/s) | 8.47 (7.83–8.85) | 6.64 (4.82–8.77) | 4.84 (4.02–6.24) | 6.76 (5.10–8.12) | 9.05 (5.10–13.03) |
| E/A | 1.11 (0.80–1.87) | 0.85 (0.73–1.11) | 0.74 (0.65–0.83) | 0.85 (0.78–1.10) | 1.39 (0.80–2.32) |
| E/E’ | 9.60 (8.16–9.95) | 12.20 (8.85–14.88) | 11.39 (9.41–13.72) | 12.56 (9.34–15.25) | 12.29 (8.28–14.74) |
Summary of echocardiographic measurements. The data for the 1 dog with MMVD stage A are not shown. Data are presented as medians and IQRs. Mann-Whitney U-test and Kruskal-Wallis test were performed.
A wave = Peak velocity of transmitral flow during late diastole. E wave = Peak velocity of transmitral flow during early diastole. E’ wave = Peak early diastolic annular velocity. E/A = Transmitral flow E wave velocity to A wave velocity ratio. E/E’ = E wave velocity to E’ wave velocity ratio. EF = Ejection fraction. FS = Fractional shortening. IVSdN = End-diastolic interventricular septal dimension normalized for body weight. IVSsN = End-systolic interventricular septal dimension normalized for body weight. LA/Ao = Left atrial to aortic root ratio. LVIDdN = End-diastolic left ventricular internal dimension normalized for body weight. LVIDsN = End-systolic left ventricular internal dimension normalized for body weight. LVPWdN = End-diastolic left ventricular posterior wall dimension normalized for body weight. LVPWsN = End-systolic left ventricular posterior wall dimension normalized for body weight. MR velocity = Peak velocity of mitral regurgitation. N = Not available. RJ score = Regurgitant jet area compared to the left atrium area.
Values with superscript letters in the same row are significantly different when compared as follows:
Galectin-3, NT-proBNP, cTnI concentrations, and inflammatory cytokine expression in dogs at various stages of MMVD and in healthy controls.
| Group | Control | MMVD | MMVD Stage B1 | MMVD Stage B2 | MMVD Stage C |
|---|---|---|---|---|---|
| Gal-3 (ng/mL) | 1.56 (0.69–1.84) | 2.94 (1.61–5.20)a | 3.57 (1.47–6.88) | 2.36 (0.75–3.08) | 3.48 (2.03–5.58)a |
| NT-proBNP (pmol/L) | 130 (102.75–241.75) | 1,128 (707.25–2,649)a | 738 (343.5–1,352)a | 1,224 (527–2,268)a | 2712 (1,158.5–5,267)a |
| cTnI (ng/mL) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–0) | 0 (0–1.05) |
| TNF-α | 0.82 (0.57–1.32) | 1.34 (1.01–2.46)a | 1.99 (1.33–3.77) | 1.36 (0.80–2.96) | 1.18 (1.01–1.81) |
| IL-1β | 1.09 (0.85–1.22) | 0.76 (0.54–1.06) | 0.66 (0.48–1.04) | 1.01 (0.66–1.29) | 0.55 (0.42–0.83) |
| IL-2 | 0.83 (0.51–1.39) | 2 (1.53–4.71)a | 3.23 (1.96–5.83)a | 1.84 (1.56–5.28) | 1.64 (1.10–4.62) |
| IL-8 | 1.07 (0.68–1.71) | 1.1 (0.54–2.96) | 1.28 (0.41–3.18) | 1.05 (0.53–3.17) | 0.95 (0.65–1.22) |
| IL-33 | 1.15 (0.58–1.78) | 1.11 (0.78–1.95) | 1.02 (0.51–1.93) | 1.34 (1.00–2.74) | 1.02 (0.53–1.36) |
Summary of galectin-3, NT-proBNP, cTnI concentrations, and inflammatory cytokine expression calculated using the comparative cycle threshold (2−ΔΔCt) method. The data for the 1 dog with MMVD stage A are not shown. Data are presented as medians and IQRs. Mann-Whitney U-test and Kruskal-Wallis test were performed.
cTnI = Cardiac troponin I; Gal-3 = Galectin-3. IL-1β = Interleukin-1β. IL-2 = Interleukin-2. IL-8 = Interleukin-8. IL-33 = Interleukin-33. NT-proBNP = N-terminal pro-brain natriuretic peptide. TNF-α = Tumor necrosis factor-α.
Values relative to control (P < .05).
Associations with plasma galectin-3 level
In univariate regression analysis, the combined plasma galectin-3 level of the dogs in the MMVD group increased significantly with increasing heart rate (R2 = 0.157, P = .011). However, galectin-3 concentrations did not increase significantly with increasing heart rate at different stages of MMVD.
Setting the level of galectin-3 as the dependent variable and other parameters, namely cardiac biomarkers, inflammatory cytokines, echocardiographic parameters, and dog characteristics, as independent variables, multiple regression analyses confirmed heart rate and body weight as factors influencing galectin-3 concentration (R2 = 0.273, P = .003). However, other cardiac biomarkers, inflammatory cytokines, and echocardiographic parameters were not significantly associated with galectin-3 levels.
Diagnostic accuracy for MMVD prediction
A model for the detection of MMVD in dogs was applied using logistic regression analysis. The model included galectin-3 (odds ratio, 2.9; P = .04) and age (odds ratio 1.48; P = .008) and showed a predictive accuracy of 90% for identifying MMVD (Table 4). The other indices were not included in the model because no significance was found in NT-proBNP (P = .053) and cTnI (P = 0.158). The AUC was 0.77 (Figure 2). The cut-off value of ≥ 1.9 ng/mL for galectin-3 differentiated dogs with MMVD from healthy dogs and conferred 70% sensitivity and 90% specificity (P = .01).
Cut-off value of galectin-3 used to differentiate dogs with MMVD from healthy dogs.
| Cut-off value (ng/mL) | Sensitivity (%) | Specificity (%) | |
|---|---|---|---|
| Galectin-3 (n = 40) | 1.82 | 70 | 70 |
| 1.84 | 70 | 80 | |
| 1.90 | 70 | 90 | |
| 1.98 | 66.7 | 90 | |
| 2.03 | 63.3 | 90 |
Receiver operating characteristic curve of galectin-3 to differentiate dogs with MMVD from healthy dogs. Galectin-3 has an AUC of 0.77 (95% CI, 0.627 to 0.920, P = 0.01). A cut-off value of 1.9 ng/mL showed a sensitivity of 70% and a specificity of 90%.
Citation: American Journal of Veterinary Research 84, 9; 10.2460/ajvr.23.03.0063
Discussion
In this study, we found that dogs with MMVD were significantly older than the healthy controls. Additionally, the concentration of plasma galectin-3 was significantly higher in dogs with MMVD, especially in those with severe MMVD, than that in healthy dogs. Nonetheless, there was no significant correlation between age and the level of plasma galectin-3. Heart rate and body weight were significant factors affecting plasma galectin-3 concentration. Plasma galectin-3 concentration and age successfully predicted MMVD, suggesting that galectin-3 is a cardiac biomarker in dogs with MMVD and may be potentially useful for MMVD screening.
Although cardiac fibrosis is associated with MMVD, its pathogenesis has not been elucidated.13,19,20 Our results showed that the concentration of plasma galectin-3, a marker of fibrosis, was increased in dogs with MMVD, concordant with those of a previous study.13 Increased fibrosis has been identified in the subendocardial and papillary muscles of the heart, which are susceptible to injury due to decreased perfusion.13,19 The pattern of cardiac fibrosis in MMVD is characterized by large areas of replacement fibrosis and differs from that of the aging heart, which exhibits small areas of replacement fibrosis due to a reduction in collagen degradation.13,21,22 In this study, not only was galectin-3 significantly expressed in the fibrotic area but was also elevated in the plasma of dogs with MMVD, indicating that plasma galectin-3 is a marker of fibrosis at the tissue level.13
However, plasma galectin-3 level did not increase in stages A, B1, and B2 of MMVD and was not correlated with echocardiographic parameters. This may be because the sample size was not sufficiently large to be statistically significant, or the disease may not have been a sufficient stimulus to trigger an increase in plasma galectin-3 level. Echocardiography is not a sensitive method to detect cardiac fibrosis,13 which may explain why, in this study, fibrosis could not be detected by echocardiography. Cardiac fibrosis may occur at a certain point in the disease progression and circulating galectin-3 levels might not be correlated linearly with the stages of MMVD and echocardiographic estimates in dogs, unlike in humans.7,23
Regression analyses revealed a significant association between heart rate and galectin-3 concentration. Heart rate is influenced by the autonomic nervous system; therefore, the activation of the sympathetic nervous system may lead to an increase in the fibrosis of cardiac muscles.1,24 Body weight was also significantly associated with plasma galectin-3 concentration. Adipocytes can synthesize galectin-3; hence, elevated levels of galectin-3 have been reported in obese patients.25 Nonetheless, body weight unlikely influences galectin-3 concentration in this study because significant differences between the groups were not detected. Another explanation is that excessive body weight can increase the levels of inflammatory cytokines, thus affecting the progression of cardiac fibrosis.25–28 Although a significant association between inflammatory cytokines level and galectin-3 level was not detected in this study, the concentration of galectin-3 is associated with inflammatory cytokines level in humans.8,29,30 This discrepancy may be attributed to the higher level of galectin-3 in human patients than in dogs; cardiac diseases in humans can result in more extensive areas of cardiac fibrosis than those in dogs.13 In addition, the levels of inflammatory cytokines can be elevated by inflammatory and noninflammatory diseases.31 Medications for cardiac diseases can also affect cytokine expression.31,32 Therefore, although we excluded dogs with systemic or organ-related dysfunction, other factors could still influence inflammatory cytokine levels; thus, stricter inclusion criteria should be implemented in future studies.
In humans, the plasma concentration of galectin-3 predicted the onset of congestive heart failure in asymptomatic patients, and the level of galectin-3 was associated with the incidence and mortality of heart failure.8,33,34 The incidence of heart failure with preserved ejection fraction (HFpEF) has increased in aging human patients, accounting for over one-third of congestive heart failure patients.14 Patients with HFpEF have diastolic dysfunction, normal ejection fraction, and myocardial fibrosis—a pattern similar to the early stage of MMVD in dogs.8,35 NT-proBNP is used as a diagnostic biomarker to screen human patients with HFpEF; however, its concentration tends to fluctuate, and the level is often below the cut-off value.14 Plasma galectin-3 level, but not NT-proBNP level, was increased in patients with HFpEF and associated with quality of life and functional performance.8 Galectin-3 is an independent predictor of all-cause mortality in hospitalized patients with HFpEF.8
In our study, plasma galectin-3 levels > 1.9 ng/mL could differentiate dogs with MMVD from dogs without MMVD, with a predictive accuracy of 90%, whereas other cardiac biomarkers, including NT-proBNP and cTnI, failed to do so. However, its sensitivity was 70% and specificity was 90%, so galectin-3 level may not be used as a sole biomarker for MMVD.
Based on the knowledge that galectin-3 level is elevated in human patients with HFpEF, we expected that galectin-3 level might be associated with echocardiographic indices of diastolic function; however, no such correlation was observed. Following aortic banding in a canine model, a correlation between the galectin-3 level and diastolic parameters was revealed; however, diastolic dysfunction artificially due to aortic banding was different from the one naturally caused by MMVD.14 A study with a larger sample size is required to determine the relationship between plasma galectin-3 levels and diastolic dysfunction in dogs with MMVD.
This study had some limitations. First, the sample size was too small to divide into subgroups, especially in stage A of MMVD. The dog with stage A MMVD was a CKCS, a breed known to develop MMVD at a relatively young age.15 This study showed that galectin-3 has the potential to differentiate dogs with MMVD from healthy dogs; thus, further studies with a larger number of CKCS dogs may provide a deeper insight into the predictive ability of galectin-3 in diagnosing MMVD in asymptomatic CKCS dogs. Second, the heart rate was measured during physical examination in this study. The use of 24-hour electrocardiogram (Holter) monitor may have allowed a more accurate hear rate. Third, control and case groups were not age-matched in this study. However, no significant correlation between age and galectin-3 level was found. Finally, although galectin-3 is a powerful marker for the prediction of the outcome and mortality of cardiac disease in humans, our case-control study was not conducted over a sufficient longitudinal period to determine its prognostic value in dogs. Also, galectin-3 level did not increase in stages A, B1, and B2 of MMVD and did not show a significant difference between MMVD stages. Therefore, these results limit the clinical use as a biomarker for staging MMVD.
In conclusion, plasma galectin-3 levels increased significantly in dogs with MMVD, and galectin-3 can differentiate between dogs with MMVD and healthy dogs with a predictive accuracy of 90%. Heart rate and body weight influenced plasma galectin-3 levels, indicating that demographic characteristics and clinical signs should be considered when assessing galectin-3 levels in canine patients. Further prospective studies with a larger population size are required to investigate the additional prognostic effects of galectin-3.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The authors have nothing to disclose.
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