university of groningen galectin-3 in heart failure … is a carbohydrate-binding protein and has...

University of Groningen

Galectin-3 in heart failurevan der Velde, Allart Rogier

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Chapter 8Galectin-3 and ABO Blood Group: Eff ects on Plasma Level, Prognosis and Binding Properties

AR van der VeldeWC MeijersHH SilljéTM Kole J SuwijnME KleberG DelgadoJJ SchuringaWH van GilstW MärzRA de Boer

Manuscript in preparation

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ABsTRACT

Background: Previous studies reported an association between ABO type blood group, and cardiovascular (CV) events and outcomes. The precise mechanisms underpinning this striking observation remain unknown, although differences in von Willebrand factor (vWF) plasma levels have been proposed as an explanation. Recently, galectin-3 was identified as an endogenous ligand of vWF and red blood cells (RBCs) and therefore we aimed to explore the role of galectin-3 in different blood groups.

methods: We studied the plasma levels of galectin-3 in different blood groups in 2 large cohorts. Galectin-3 was measured at baseline in the Ludwigshafen Risk and Cardiovas-cular Health (LURIC) study (1228 patients hospitalized for coronary angiography) and results were validated in a community-based cohort of the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study (3549 patients). Using two in vitro assays, we assessed the binding capacity of galectin-3 to red blood cells and vWF in different blood groups. Additionally, the prognostic value for all-cause mortality of galectin-3 in different blood groups in 2 different cohorts was determined.

Results: Patients in LURIC with non-O blood groups had substantially and significantly lower median plasma levels of galectin-3 (blood group A: 13.1 [10.5-16.4]; blood group B: 13.0 [10.5-15.8]; blood group AB: 12.4 [8.9-15.1] ng/mL), compared to patients with blood group O (15.1 [12.4-19.0] ng/mL). The independent prognostic value of galectin-3 for all-cause mortality is mostly due to to the effects observed in non-O blood groups. These results were validated in a community-based cohort, and although effects were smaller, the same trends were observed. Mechanistically, we demonstrated that galec-tin-3 binds more strongly to RBCs and vWF in non-O blood groups, compared to blood group O.

Conclusions: Although plasma galectin-3 levels are lower in non-O blood groups, the prognostic value of galectin-3 is primarily present in subjects with non-O blood group. A physical interaction between galectin-3 and blood group epitopes may modulate galectin-3 activity.

181

Galectin-3 and ABO Blood Group: Effects on Plasma Level, Prognosis and Binding Properties

Chap

ter 8

inTRoduCTion

Lifetime risk to experience cardiovascular disease (CV) is estimated around 50 percent at the age of 30 years 1 and CV disease accounts for 32% of all deaths in the US.2 Over the past decades, incidence of mortality in subjects with CV disease have shown to decline.3 However, the prognosis of patients with CV disease and especially in patients with heart failure, remains poor.4 Therefore it is of utmost importance to generate novel thera-peutic strategies or to identify risk factors that can serve as therapeutic targets. Several risk factors have been identified in CV disease. In the Framingham Heart Study, high blood pressure, smoking, high serum LDL cholesterol, low serum HDL cholesterol and presence of diabetes mellitus were identified as most important risk factors.5 However, residual risk remains high and we do not fully understand all factors contributing to CV disease development.

In the past years, the ABO blood group has been identified as a novel and intriguing risk factor of CV disease. Multiple studies have shown an association between non-O blood groups and the risk of coronary heart disease 6, the size of a myocardial infarction after an acute coronary syndrome 7, increased mortality in patients with ischemic heart disease 8, and venous thrombosis.9 The exact mechanisms behind these associations remain unclear to date, but as a possible common mechanism, variable levels and activ-ity of von Willebrand Factor (vWF) have been proposed. vWF is widely acknowledged as a key determinant in CV homeostasis, and has been linked to thrombosis and CV events.10,11

Galectin-3 is a carbohydrate-binding protein and has been shown to be involved in inflammation, cancer and CV disease 12–14 , and recently, has been identified as a novel partner of vWF.15 It was shown that galectin-3 is able to modulate vWF-mediated throm-bus formation via a direct (physical) interaction with vWF.15 A possible link between galectin-3 and blood group has been described previously – a genome wide association study showed that the ABO gene locus was strongly associated with plasma galectin-3 levels.16 This ABO locus appears to be a very pleiotropic locus that associates with several CV traits.17 We hypothesized that ABO, galectin-3 and vWF would interact, and, specifi-cally, that the described associations between galectin-3 and CV outcome 18 can, at least in part, be explained by an interaction with the ABO blood group and vWF levels.

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meTHods

study population

LURICThe Ludwigshafen Risk and Cardiovascular Health (LURIC) study consists of 3,316 pa-tients who were hospitalized for coronary angiography between 1997 and 2000. Indica-tions for coronary angiography were chest pain or a positive non-invasive stress test suggestive of myocardial ischemia. Further methods and results have been described previously.19 All participants provided informed consent. The study was approved by the ethical committee of the Ärztekammer Rheinland-Pfalz. In total, galectin-3 values and blood group information were available for 1,228 patients.

PREVENDThe Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study is a prospec-tive, observational, community-based study and was used to validate our findings.16,20 The PREVEND study enrolled community-dwelling subjects during 1997-1998, and the study was designed to track long-term development of cardiac, renal and peripheral vascular disease. More details of the design of the study have been described previ-ously.21,22 Galectin-3 and blood group were available in 3,549 subjects. In both studies, all participants provided informed consent and the study procedures were conducted in accordance with the 1975 Declaration of Helsinki.

Galectin-3 measurements

In the LURIC study, galectin-3 levels were measured in plasma samples from baseline. These samples were stored at -80°C and were analysed using the ARCHITECT analyser (Abbott Diagnostics, Abbott Park, IL, USA). This automated assay uses the same antibod-ies and conjugates as in the manual assay, and has a lower limit of detection of 1.01 ng/mL and intra- and inter-assay variability of 3.2% and 0.8%, respectively.23 In the PRE-VEND study, blood was drawn at baseline and anticoagulated with EDTA. Samples were stored at -80°C until time of analysis. Galectin-3 concentration was measured in plasma samples from baseline using the BGM Galectin-3 ELISA kit (BG Medicine Inc., Waltham, USA). Intra- and inter-assay coefficients of this assay are 3.2% and 5.6%, respectively. The assay has a lower limit of detection of 1.13 ng/mL and did not show cross-reactivity with other collagens or other members of the galectin family.24

Blood group determination

Blood group in LURIC was determined in daily analyses in the Hemostasis Laboratory of the Ludwigshafen Cardiac Centre using a blood group antisera macroscopic agglutina-tion assay (ABO- and Rh-blood group sera, Loxo GmbH, Dossenheim, Germany).

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In PREVEND, blood groups were determined using 3 different single nucleotide poly-morphisms (SNPs). Participants were genotyped for SNPs rs8176719, rs8176746 and rs8176747. Using a combination of these SNPs, blood group could be determined, as described previously. 25

Clinical endpoints

In LURIC, mortality data was collected from local registries. Two independent and expe-rienced clinicians, who were blinded for patient characteristics, reviewed information from death certificates, medical records from hospitals and data from autopsies. 18,26 In PREVEND, mortality data were collected using the municipal register and cause of death was obtained using the Prismant health care data system or Dutch Central Bureau of Statistics. Follow-up times ranged to last follow-up or were censored on the date of event or last contact, whatever occurred first.

in vitro studies

Isolation of red blood cells (RBCs)Neonatal cord blood was obtained from healthy full-term pregnancies from donors from the obstetrics departments of the Martini Hospital Groningen and University Medical Center Groningen (UMCG) after informed consent was given. All donors were informed about the studies that were performed, as approved by the local Medical Ethical Commit-tee of the UMCG. Furthermore, healthy volunteers from the research lab also provided blood specimen. Blood was collected in 10 mL EDTA tubes. 20µl of blood was used to determine the ABO blood group using a Serafol ABO bedside test (Bio-Rad Laboratories BV, Veenendaal, the Netherlands). The remaining blood was centrifuged at 3500 rpm for 5 minutes. The buffy coat appeared as a dense white layer in the middle between the red blood cells and plasma. Plasma and buffy coat was removed from the tube. RBCs remained in the tube and were resuspended in PBS and again centrifuged at 2000 rpm for 5 minutes at 4º Celsius. This washing step was repeated 3 times. Subsequently, the remaining RBCs were diluted 12.5x in PBS-3% glutaraldehyde in a tube and this was put on a rotating wheel for 1 hour at room temperature. Afterwards the cells were washed 5 times with PBS (0.0025% NaN3) and centrifuged at 2000 rpm for 2 minutes at 4º Celsius and in the last step cells were resuspended at 3-4% in PBS (0.0025% NaN3). Cells were stored at 4º Celsius for several days.

Hemagglutination assayRBCs were counted using a Fuchs-Rosenthal counting chamber. All cells were diluted to the lowest concentration of RBCs. We first calibrated our hemagglutination assay to determine the amount of RBCs that were needed to show hemagglutination and to clearly distinguish between agglutinated and non-agglutinated cells. We tested 3 dif-

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ferent concentrations of RBCs (5 µl/ 10 µl / 15 µl of 2000 cells/µl) and 2 concentrations of galectin-3 (1 µM / 2 µM). Following calibration, we used 15 µl RBCs / 2 µM galectin-3 in the first well of a round-bottom, 96-wells plate (Costar #3799, Corning Inc., USA). 2 µM galectin-3 was serially diluted 1:1 into the next wells and 87,5 µl PBS was added to a total volume of 185 µl. Finally, 15 µl (2000 cells/µl) of RBCs were added to each well. The plate was incubated for 30 minutes at 4º Celsius and pictures were made using the ImageQuant LAS 4000 (GE Healthcare, Europe GmbH, Diegem, Belgium). Hemagglutina-tion was assessed using ImageJ software (National Institutes of Health, USA) and the hemagglutination-index ((surface area of RBCs after incubation/surface area of the total well)*100) (HA-index) was calculated.

Von Willebrand Factor ELISAvWF was measured in human plasma using the vWF ELISA kit (Abcam, Cambridge, UK). This kit was designed for the quantitative measurement of human vWF in plasma, serum and cell culture supernatants. Intra- and inter-assay coefficients of variation of this assay are 5% and 7.1%, respectively. The lower level of detection is 2.5 mU/mL.

Galectin-3 – von willebrand factor binding studyAs described previously15, an immunosorbent assay was performed in which a microti-ter 96-wells plate was coated with galectin-3 (5 µg/well), overnight at 4º Celsius. After washing 3 times with PBS (0.1% Tween-20), was blocked for 2 hours with PBS (0.1% Tween-20/3% BSA) at 37º Celsius. After washing 2 times with PBS (0.1% Tween-20), plasma of different blood groups was incubated in the wells for 1 hour at 37º Celsius. After discarding the plasma, the plate was washed 2 times with PBS (0.1% Tween-20). Bound vWF was detected by adding 50 µL HRP-labeled polyclonal vWF antibody (1:1000; P0226, DAKO, Glostrup, Denmark). 50 µL 3,3’,5,5’-tetramethylbenzidine (TMB) was added to detect HRP activity and after 10 minutes 50 µL of stop solution (H2SO4) was added to stop the reaction. The absorbance was measured at a wavelength of 450nm, using a microplate reader (Biotek Synergy H1, Winooski, USA).

statistical analysis

Normally distributed variables are presented as means ± standard deviation (SD) or standard error of the mean (SEM). Non-normally distributed variables are expressed as medians [interquartile range (IQR)]. To compare normally distributed values across two groups, a two-sample t-test was performed and to compare non-normally distributed values, we used the Wilcoxon rank-sum test. The comparison of categorical values was done using the Pearson’s Chi-square test. Characteristics across four groups were com-pared using the ANOVA for continuous and normally distributed values and the Kruskal-

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Wallis test for continuous, non-normally distributed values. In comparison of >1 group with a control group, we used the ANOVA with a post hoc Dunnett’s test.

Galectin-3 was transformed logarithmically, because of a skewed distribution as as-sessed by the Shapiro-Wilk test. The association of galectin-3 with all-cause mortality was assessed using Cox proportional hazard models. Cox proportional hazards models were multivariable adjusted for age and sex and additionally adjusted for eGFR, smoking, systolic blood pressure, BMI, LDL-cholesterol, diabetes mellitus, lipid lowering therapy, triglycerides and CRP. This model is an established risk model for all-cause mortality in the LURIC study and has been used previously in other studies.18 Results are stratified to blood group and summarized as hazard ratios, with 95% confidence intervals based on robust standard error estimates and presented in forest plots. P-values < 0.05 were considered to be statistically significant. Analyses were performed using STATA/MP (version 13.1).

ResulTs

study population

Baseline characteristics of patients in the LURIC study are presented in Table 1. Mean age (SD) was 62.5±10.5 years and the majority of the population was male (70.5%). Hyperten-sion and smoking was very common is this cohort (72.6% and 62.1%, respectively). To validate our findings in a more healthier cohort, we studied the relation between galec-tin-3 and blood group in a community-based cohort: the PREVEND study. Participants of the PREVEND study were younger (mean age: 49.4±12.7) and gender was almost equally distributed (male sex: 51%). Smoking was common (45.7%), but hypertension was less abundant (27.7%) (Supplemental Table 1).

Galectin-3 plasma levels stratified by blood group

After stratifying the LURIC cohort to blood group, plasma levels of galectin-3 were significantly higher in blood group O, compared to the other blood groups (Table 1; Figure 1). Furthermore, vWF was significantly lower in blood group O, compared to other blood groups. In the general population, galectin-3 levels were also significantly dif-ferent among blood groups and were higher in blood group O, compared to the other blood groups (Table 1; Figure 1). Moreover, subjects with homozygous blood groups had lower plasma levels of galectin-3 compared to subjects with heterozygous blood groups (Supplemental Figure 1).

Binding of galectin-3 and red blood cells

To further characterize a potential interaction between galectin-3, vWF and blood group, different in vitro assays were performed. To examine the interaction between galectin-3

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Tabl

e 1:

Bas

elin

e ch

arac

teris

tics

of th

e LU

RIC

stud

y pa

rtic

ipan

ts, i

n to

tal a

nd s

trat

ified

by

bloo

d gr

oup.

Varia

ble

Tota

lBl

ood

grou

p 0

Bloo

d gr

oup

ABl

ood

grou

p B

Bloo

d gr

oup

AB

P-va

lue

N(N

=122

8)(N

=477

)(N

=567

)(N

=119

)(N

=65)

Age

(yr)

, mea

n (S

D)

62.5

(10.

5)62

.5 (1

0.1)

62.5

(10.

7)62

.8 (1

1.3)

61.1

(11.

2)0.

74

Sex

(mal

e), N

(%)

836

(68.

1%)

325

(68.

1%)

390

(68.

8%)

79 (6

6.4%

)42

(64.

6%)

0.89

Smok

er, N

(%)

762

(62.

1%)

291

(61.

0%)

350

(61.

7%)

80 (6

7.2%

)41

(63.

1%)

0.65

BMI (

kg/m

2 ), m

ean

(SD

)27

.2 (4

.0)

27.4

(4.2

)27

.2 (4

.0)

26.8

(3.8

)27

.1 (3

.6)

0.62

Type

II d

iabe

tes

mel

litus

, N (%

)20

8 (1

6.9%

)86

(18.

0%)

96 (1

6.9%

)17

(14.

3%)

9 (1

3.8%

)0.

70

(His

tory

of)

hyp

erte

nsio

n, N

(%)

883

(71.

9%)

344

(72.

1%)

406

(71.

6%)

87 (7

3.1%

)46

(70.

8%)

0.98

Hea

rt ra

te (b

pm),

med

ian

[IQR]

67.0

[60.

0-76

.0]

68.0

[60.

0-76

.0]

68.0

[60.

0-76

.0]

64.0

[58.

0-72

.0]

66.0

[59.

0-79

.0]

0.04

Syst

olic

blo

od p

ress

ure,

med

ian

[IQR]

141.

0 [1

23.0

-158

.0]

141.

0 [1

23.0

-158

.0]

142.

0 [1

24.0

-158

.0]

140.

0 [1

22.0

-158

.0]

140.

0 [1

20.0

-152

.0]

0.85

Dia

stol

ic b

lood

pre

ssur

e, m

edia

n [IQ

R]80

.0 [7

2.0-

89.0

]80

.0 [7

2.0-

89.0

]80

.0 [7

2.0-

88.0

]80

.0 [7

0.0-

90.0

]80

.0 [7

0.0-

89.0

]0.

98

eGFR

(MD

RD),

med

ian

[IQR]

86.1

[73.

0-96

.1]

83.2

[70.

6-94

.6]

87.7

[73.

9-10

0.5]

85.0

[73.

8-92

.9]

82.7

[74.

0-92

.0]

0.16

Glu

cose

(mm

ol/L

), m

edia

n [IQ

R]5.

6 [5

.2-6

.5]

5.6

[5.1

-6.5

]5.

7 [5

.2-6

.7]

5.7

[5.3

-6.2

]5.

6 [5

.2-6

.1]

0.57

Chol

este

rol (

mm

ol/L

), m

edia

n [IQ

R]5.

3 [4

.6-6

.1]

5.2

[4.6

-6.0

]5.

4 [4

.7-6

.2]

5.2

[4.4

-6.1

]5.

1 [4

.7-5

.8]

0.15

LDL

(mm

ol/L

), m

edia

n [IQ

R]3.

0 [2

.5-3

.6]

2.9

[2.5

-3.5

]3.

1 [2

.5-3

.6]

3.0

[2.4

-3.6

]2.

8 [2

.4-3

.5]

0.20

HD

L (m

mol

/L),

med

ian

[IQR]

1.0

[0.8

-1.2

]0.

9 [0

.8-1

.1]

1.0

[0.8

-1.2

]1.

0 [0

.8-1

.1]

1.0

[0.7

-1.1

]0.

22

Trig

lyce

rides

(mm

ol/L

), m

edia

n [IQ

R]1.

6 [1

.2-2

.2]

1.7

[1.2

-2.3

]1.

6 [1

.2-2

.2]

1.5

[1.2

-2.2

]1.

6 [1

.1-2

.2]

0.70

NT-

proB

NP

(ng/

L), m

edia

n [IQ

R]29

1.0

[108

.0-8

74.0

]28

5.0

[108

.0-9

72.0

]26

3.0

[101

.0-8

39.0

]36

1.5

[122

.0-7

85.0

]30

1.0

[132

.0-8

90.0

]0.

67

Gal

ectin

-3 (n

g/m

L), m

edia

n [IQ

R]13

.8 [1

1.1-

17.3

]15

.1 [1

2.4-

19.0

]13

.1 [1

0.5-

16.4

]13

.0 [1

0.5-

15.8

]12

.4 [8

.9-1

5.1]

<0.0

01

vWF

(U/d

L), m

edia

n [IQ

R]15

2.0

[116

.0-1

96.5

]13

2.0

[99.

0-17

5.0]

160.

0 [1

26.0

-202

.0]

175.

0 [1

38.0

-202

.0]

183.

0 [1

36.0

-220

.0]

<0.0

01

187


Chap

ter 8

and blood group, a hemagglutination assay was performed, as displayed in supplemen-tal figure 2. Galectin-3 mediates the hemagglutination of red blood cells and we tested if there were differences in galectin-3 induced hemagglutination among blood groups. In this assay we showed that binding of galectin-3 with red blood cells was significantly different between blood groups, with RBCs from blood group O binding galectin-3 less strong (Figure 2).

interaction between vwF and galectin-3

Because galectin-3 has been presented as new partner for vWF, we assessed this binding in different blood groups in a vWF-galectin-3 binding assay. Blood plasma with similar levels of vWF, as determined in ELISA, and equalized to similar concentrations with 0.9% NaCl, was incubated in a plate coated with galectin-3. Using vWF antibodies, the galectin-3-vWF binding was detected. This assay showed that the binding for galectin-3 to vWF was stronger in non-O blood groups, compared to blood group O (Figure 3).

Prognostic value of galectin-3

We furthermore studied the prognostic value of galectin-3 in different blood groups in the LURIC study. Median follow-up time was 8.1 [7.8-8.9] years and 318 deaths were observed. Cox regression analyses showed that after adjusting for established risk mark-ers like age, sex, eGFR, smoking, systolic blood pressure, BMI, LDL-cholesterol, diabetes mellitus, lipid lowering therapy, triglycerides and CRP, galectin-3 was an independent predictor for all-cause mortality (Supplemental Tables 2&3). However, this effect was greatly attributed to the effect of galectin-3 in non-O blood groups, although galectin-3 plasma levels in these patients were lower (Figure 3).

Figure 1: Plasma galectin-3 levels in LURIC and PREVEND, stratified by ABO blood group. Data is presented as mean ± SEM. **: P<0.01 compared to blood group O; ***: P<0.001 compared to blood group O.

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We also assessed the prognostic value of galectin-3 among different blood groups in the general population, after adjustment for the same risk markers. In the PREVEND study, median follow-up time was 12.6 [12.3-12.9] years and 353 subjects died in this period. Although the effect was limited, the same trend was observed: galectin-3 was an independent predictor for all-cause mortality, but again this effect depends mainly on the prognostic value of galectin-3 in the non-O blood groups (Figure 3).

Figure 2: Galectin-3-mediated hemagglutination of blood from different blood groups (A) and hemagglu-tination induced by 0.5 µM galectin-3 (B). Galectin-3 binding to vWF in different blood groups, as assessed by measuring absorbance at 450 nm (C). *: P<0.05 compared to blood group O; **: P<0.01 compared to blood group O; ***: P<0.001 compared to blood group O (N=6: blood group O; N=4: blood group A; N= 2: blood group B; N= 2: blood group AB).

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disCussion

We demonstrate that circulating galectin-3 levels in subjects with non-O blood groups are significantly lower compared to levels in subjects with blood group O. However, the prognostic value of galectin-3 is strongest in subjects with non-O blood groups, in other words, in those subjects with lowest circulating levels. As a potential mechanism, we

Figure 3: Graphical illustration of risk estimates for experiencing all-cause mortality in subjects with blood group O and non-O blood group, with increasing levels of plasma galectin-3 in the LURIC (A) and PREVEND (B) studies. The distribution of galectin-3 plasma levels is depicted for both groups and is combined with the association of log-transformed galectin-3 with all-cause mortality, adjusted for age, sex, eGFR, smoking, systolic blood pressure, BMI, LDL-cholesterol, diabetes mellitus, lipid lowering therapy, triglycerides and CRP. Data is presented as hazard ratio (95% CI). A similar increase in galectin-3 has more prognostic value in subjects with non-O blood group compared to subjects with blood group O.

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propose that vWF may mediate this, as circulating vWF and galectin-3 were inversely related. We show that galectin-3 binds stronger to RBCs and vWF of subjects with non-O blood groups, compared to subjects with blood group O.

Accumulating evidence suggests that ABO blood group is involved in the pathogen-esis of cardiovascular disease.17 Previous studies have shown that the presence of non-O blood groups is associated with worse outcomes, compared to blood group O.27–29 In a recent case-control study consisting 165 centenarians and 5063 blood donors from the same geographical region, it was observed that among centenarians the prevalence of blood group O was higher (56.4% vs 43.5%; P=0.001).30

The ABO blood group is the most important blood group system and is determined by complex carbohydrate moieties at the extracellular surface of the red blood cell membrane.31 The A and B alleles encode for different glycosyltransferases that can add N-acetylgalactosamine or D-galactose, respectively. These carbohydrate moieties are coupled to the common precursor backbone, present in subjects with blood group O.32 Next to the expression of RBCs, these blood group epitopes are also expressed by other cells like the vascular endothelium and are also present on molecules like vWF.33

The exact mechanisms underpinning these observations remain unclear, but this effect may be mediated by vWF. Several studies described major effects of ABO blood group on plasma levels of vWF: plasma vWF levels appear to be 25% lower in O blood group, compared to non-O blood groups.6 This implicates that subjects with blood group O may experience a higher incidence of bleeding events and subject with non-O blood groups, experience a higher incidence of thrombotic events.34

The effect of the ABO blood group on plasma levels of vWF, seems to be the result of a direct effect of the ABO blood group.35 The conversion of the blood group O determinant into other antigens of the ABO blood group was correlated with an increased capacity to modify N-linked glycosylation of vWF.36 Therefore, changes in vWF glycan composition do also affect the biological activity of vWF and is not restricted to its plasma levels.37 Carbohydrate structures on the surface of vWF membrane play an important role in the life cycle of vWF. Galectin-3 is a carbohydrate-binding protein and has recently been identified as new partner of vWF.15 Furthermore, galectin-3 also possesses N-glycans on its membrane and the affinity of transmembrane glycoproteins to the galectin-3 molecule is proportional to the number and branching of their N-glycans.38 Therefore we hypothesize that the biological activity of galectin-3 might also be directly regulated by glycosylation of the molecule by the ABO blood group.

In agreement with previous studies, we confirmed that plasma vWF levels are ~25% higher in non-O blood groups. Additionally, we now show in two independent cohorts with different populations, that galectin-3 levels are significantly lower in non-O blood groups. We furthermore show that galectin-3 levels are lower in patients who had a heterozygous blood group. This inverse relationship between galectin-3 and vWF levels

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ter 8

in different blood groups is an interesting phenomenon and may be explained because they are ligands of each other.

Numerous studies have assessed the prognostic value of galectin-3 in various co-horts.39 We again corroborated these findings in the current study and herein confirm that galectin-3 is an independent predictor for all-cause mortality, but particularly in subjects with non-O blood groups. The striking observation that non-O blood groups have lower galectin-3 values, but that this confers strong prognostic value should be explored in further detail. We speculate that the observed discrepancy in our results may be the effect of galectin-3 binding with blood group epitopes and glycosylation might play a role in this. Previously it was suggested that galectin-3 binds more strongly to oli-gosaccharides that bear the blood group A, B or B-like determinants, compared to those bearing blood group O, as shown in erythrocyte binding.40 We have confirmed this in 2 different in vitro assays and hypothesize that stronger binding of galectin-3 with RBCs and vWF in non-O blood groups, could explain lower levels of circulating galectin-3.

The prognostic value and absolute levels of biomarkers may differ between differ-ent subgroups in a study cohort, as was observed previously for other biomarkers. For instance, plasma hemoglobin levels differ between sexes, and also age, renal function and presence of diabetes are important determinants of hemoglobin level.41 Even for the established cardiac marker NT-proBNP, important determinants exist that lead to differences in circulating levels; renal failure tends to increase natriuretic peptide levels, whereas patients with obesity show lower levels of NT-proBNP.42,43 Using a com-bination of biomarkers might improve risk prediction of clinical outcome and therefore healthcare-related costs. This biomarker-guided approach can pave the road to a more personalized treatment of patients.

In conclusion, we postulate that binding of galectin-3 to the A-, B- and AB- blood group epitopes affects the circulating plasma levels and its biological activity, and thereby also its prognostic power for a given concentration. Future studies should provide more de-tailed data on this interaction, and practical information how to deal with this potential confounder.

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Chapter 8

196

supp

lem

enta

l Tab

le 1

: Ba

selin

e ch

arac

teris

tics

of th

e PR

EVEN

D s

tudy

par

ticip

ants

, in

tota

l and

str

atifi

ed b

y bl

ood

grou

p.

Fact

orTo

tal

Bloo

d gr

oup

0Bl

ood

grou

p A

Bloo

d gr

oup

BBl

ood

grou

p A

BP-

valu

e

N(N

=354

9)(N

=154

9)(N

=161

1)(N

=272

)(N

=117

)

Age

(yr)

, med

ian

[IQR]

49.4

(12.

7)49

.5 (1

2.2)

49.6

(12.

7)50

.9 (1

2.4)

48.8

(12.

4)0.

31

Sex

(mal

e), N

(%)

1812

(51.

2%)

789

(51.

1%)

835

(52.

1%)

125

(46.

1%)

63 (5

4.3%

)0.

29

Smok

ed la

st 5

yea

rs, N

(%)

1610

(45.

7%)

718

(46.

5%)

732

(46.

0%)

116

(42.

8%)

44 (3

8.6%

)0.

30

BMI (

kg/m

2 ), m

edia

n [IQ

R]26

.1 (4

.2)

26.0

(4.1

)26

.2 (4

.3)

26.2

(4.4

)26

.4 (4

.0)

0.34

Type

II d

iabe

tes

mel

litus

, N (%

)53

(1.5

%)

25 (1

.6%

)21

(1.3

%)

4 (1

.5%

)3

(2.6

%)

0.70

(His

tory

of)

hyp

erte

nsio

n, N

(%)

984

(27.

7%)

433

(28.

0%)

439

(27.

3%)

81 (2

9.8%

)31

(26.

5%)

0.83

Hyp

erch

oles

tero

lem

ia, N

(%)

926

(26.

4%)

388

(25.

4%)

446

(28.

0%)

57 (2

1.4%

)35

(30.

4%)

0.06

6

Hea

rt ra

te (b

pm),

med

ian

[IQR]

68.0

[62.

0-76

.0]

68.0

[62.

0-75

.0]

68.0

[62.

0-75

.0]

70.0

[64.

0-77

.0]

69.0

[62.

0-78

.0]

0.04

9

Syst

olic

BP

(mm

Hg)

, med

ian

[IQR]

126.

0 [1

14.0

-141

.0]

125.

0 [1

13.0

-141

.0]

126.

0 [1

14.0

-141

.0]

126.

0 [1

13.0

-145

.0]

124.

0 [1

16.0

-140

.0]

0.84

Dia

stol

ic B

P (m

mH

g), m

edia

n [IQ

R]73

.0 [6

7.0-

80.0

]73

.0 [6

7.0-

81.0

]73

.0 [6

7.0-

80.0

]75

.0 [6

7.0-

80.5

]75

.0 [7

0.0-

80.0

]0.

50

eGFR

(MD

RD),

med

ian

[IQR]

80.2

[71.

6-89

.5]

79.8

[71.

3-89

.4]

80.4

[71.

8-89

.5]

81.5

[72.

6-88

.9]

81.4

[70.

4-90

.3]

0.89

Glu

cose

(mm

ol/L

), m

edia

n [IQ

R]4.

7 [4

.3-5

.1]

4.7

[4.3

-5.1

]4.

7 [4

.4-5

.2]

4.7

[4.4

-5.0

]4.

7 [4

.3-5

.1]

0.56

Chol

este

rol (

mm

ol/L

), m

edia

n [IQ

R]5.

6 [4

.9-6

.4]

5.5

[4.9

-6.3

]5.

6 [4

.9-6

.4]

5.6

[4.9

-6.3

]5.

6 [4

.9-6

.5]

0.28

LDL

(mm

ol/L

), m

edia

n [IQ

R]3.

7 [3

.0-4

.3]

3.6

[3.0

-4.3

]3.

7 [3

.0-4

.4]

3.6

[2.9

-4.2

]3.

7 [2

.8-4

.3]

0.22

HD

L (m

mol

/L),

med

ian

[IQR]

1.3

[1.0

-1.5

]1.

3 [1

.0-1

.5]

1.3

[1.0

-1.5

]1.

3 [1

.1-1

.6]

1.3

[1.0

-1.5

]0.

52

Trig

lyce

rides

(mm

ol/L

), m

edia

n [IQ

R]1.

2 [0

.8-1

.7]

1.2

[0.8

-1.7

]1.

2 [0

.9-1

.7]

1.1

[0.8

-1.7

]1.

4 [0

.9-1

.9]

0.12

NT-

proB

NP

(ng/

L), m

edia

n [IQ

R]37

.1 [1

6.3-

73.3

]36

.5 [1

6.1-

74.8

]36

.5 [1

6.1-

71.7

]41

.3 [1

9.0-

75.7

]35

.5 [1

5.9-

63.0

]0.

38

Gal

ectin

-3 (n

g/m

L), m

edia

n [IQ

R]10

.8 [9

.0-1

3.0]

11.3

[9.6

-13.

4]10

.5 [8

.7-1

2.7]

9.9

[8.4

-12.

1]9.

8 [8

.5-1

2.2]

<0.0

01

197


Chap

ter 8

supplemental Table 2: Hazard ratio (95% CI) of log-transformed galectin-3 for all-cause mortality, by blood group, using blood group O as the reference group, in the LURIC and PREVEND study.

LURIC study PREVEND study

HR (95% CI) P-value HR (95% CI) P-value

Galectin-3 only:

Blood group:

O 1.00 1.00

non-O 1.57 [1.24-1.99] <0.001 1.24 [1.00-1.54] 0.046

+ Adjusted for age, sex:

Blood group:

O 1.00 1.00

non-O 1.57 [1.23-1.99] <0.001 1.12 [0.91-1.39] 0.29

+ Fully adjusted:*

Blood group:

O 1.00 1.00

non-O 1.43 [1.13-1.82] 0.003 1.06 [0.85-1.33] 0.60

* Adjusted for age, sex, eGFR, smoking, systolic blood pressure, BMI, LDL-cholesterol, diabetes mellitus, lipid lowering therapy, triglycerides, CRP.

Chapter 8

198

supplemental Table 3: Hazard ratio (95% CI) of log-transformed galectin-3 for all-cause mortality, by blood group in the LURIC and PREVEND study, reporting additional P-values for interaction between the blood groups.

LURIC study O blood group Non-O blood group P-value for interaction

All-cause mortality HR (95% CI) HR (95% CI)

Galectin-3 2.65 (1.57-4.49) 3.83 (2.72-5.40) 0.24

Galectin-3 1.98 (1.12-3.50) 3.40 (2.33-4.97) 0.08

+ sex, age

Galectin-3 1.82 (1.03-3.20) 2.75 (1.88-4.03) 0.14

+ fully adjusted*

PREVEND study O blood group Non-O blood group P-value for interaction

All-cause mortality HR (95% CI) HR (95% CI)

Galectin-3 3.43 (2.06-5.71) 4.02 (2.75-5.88) 0.60

Galectin-3 1.35 (0.75-2.43) 1.85 (1.14-3.00) 0.33

+ sex, age

Galectin-3 1.36 (0.71-2.60) 1.70 (1.02-2.83) 0.73

+ fully adjusted*

* Adjusted for age, sex, eGFR, smoking, systolic blood pressure, BMI, LDL-cholesterol, diabetes mellitus, lipid lowering therapy, triglycerides, CRP.

199


Chap

ter 8

supplemental figure 1: Galectin-3 levels, stratified by ABO genotype and presented as mean ± SEM.

PART 3

Galectin-3 as a modifiable factor in heart failure

university of groningen galectin-3 in heart failure … is a carbohydrate-binding protein and has...

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