# Sex differences in circulating proteins in heart failure with preserved ejection fraction

Susan Stienen, Joao Pedro Ferreira, Masatake Kobayashi, Gregoire Preud'homme, Daniela Dobre, Jean-Loup Machu, Kévin Duarte, Emmanuel Bresso, Marie-Dominique Devignes, Natalia López Andrés, et al.

► To cite this version:

Susan Stienen, Joao Pedro Ferreira, Masatake Kobayashi, Gregoire Preud'homme, Daniela Dobre, et al.. Sex differences in circulating proteins in heart failure with preserved ejection fraction. Biology of Sex Differences, 2020, 11 (1), pp.47. 10.1186/s13293-020-00322-7. hal-02928742

HAL Id: hal-02928742

https://hal.univ-lorraine.fr/hal-02928742

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1 **Sex-differences in circulating proteins in heart failure with preserved ejection fraction**

2 Susan Stienen, MD, PhD¹; João Pedro Ferreira, MD, PhD¹,²; Masatake Kobayashi, MD¹; Gregoire

3 Preud'homme MSc¹; Daniela Dobre¹,³, Jean-Loup Machu, MSc¹; Kevin Duarte PhD¹; Emmanuel Bresso,

4 PhD⁴; Marie-Dominique Devignes, PhD⁴; Natalia López Andrés, PhD⁵; Nicolas Girerd, MD, PhD¹; Svend

5 Aakhus⁶,⁷; Giuseppe Ambrosio⁸; Hans-Peter Brunner-La Rocca, MD, PhD⁹; Ricardo Fontes-Carvalho¹⁰;

6 Alan G. Fraser¹¹; Loek van Heerebeek, MD, PhD¹²; Gilles de Keulenaer¹³; Paolo Marino¹⁴; Kenneth

7 McDonald¹⁵; Alexandre Mebazaa¹⁶; Zoltàn Papp, PhD¹⁷; Riccardo Raddino¹⁸; Carsten Tschöpe¹⁹; Walter

8 J. Paulus, MD, PhD²⁰; Faiez Zannad, MD, PhD¹¥; Patrick Rossignol, MD, PhD¹¥

9 ¹ Université de Lorraine, INSERM, Centre d'Investigation Clinique et Plurithématique 1433, INSERM U1116, CHRU de Nancy, F-CRIN INI-CRCT (Cardiovascular and Renal Clinical Trialists), Nancy, France;

10 ²Department of Physiology and Cardiothoracic Surgery, Cardiovascular Research and Development Unit, Faculty of Medicine, University of Porto, Porto, Portugal;

11 ³Clinical research and Investigation Unit, Psychotherapeutic Center of Nancy, Laxou, France

12 ⁴LORIA (CNRS, Inria NGE, Université de Lorraine), Campus Scientifique, F-54506, Vandœuvre-lès-Nancy, France; ⁵Navarrabiomed, Complejo Hospitalario de Navarra (CHN), Universidad Pública de Navarra (UPNA), IdiSNA.

13 Pamplona. Spain;

14 ⁶Oslo University Hospital, Oslo, Norway

15 ⁷ISB, Norwegian University of Science and Technology, Trondheim, Norway;

16 ⁸Division of Cardiology, University of Perugia School of Medicine, Perugia, Italy;

17 ⁹Department of Cardiology, Maastricht University Medical Center, Maastricht, the Netherlands;

18 ¹⁰Department of Surgery and Physiology, Cardiovascular Research Unit (UnIC), Faculty of Medicine, University of Porto, Porto, Portugal;

19 ¹¹Wales Heart Research Institute, Cardiff University, Cardiff, United Kingdom;

20 ¹²Department of Cardiology, Onze Lieve Vrouwe Gasthuis, Amsterdam, the Netherlands;

21 ¹³Laboratory of Physiopharmacology, Antwerp University, and ZNA Hartcentrum, Antwerp, Belgium;

22 ¹⁴Clinical Cardiology, Università del Piemonte Orientale, Department of Translational Medicine, Azienda

23 Ospedaliero Universitaria "Maggiore della Carità", Novara, Italy;

24 ¹⁵St Michael's Hospital Dun Laoghaire Co. Dublin, Dublin, Ireland;

25 ¹⁶Department of Anaesthesiology and Critical Care Medicine, Saint Louis and Lariboisière University Hospitals and INSERM UMR-S 942, Paris, France;

26 ¹⁷Division of Clinical Physiology, Department of Cardiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary;

27 ¹⁸Department of Cardiology, Spedali Civili di Brescia, Brescia, Italy;

28 ¹⁹Department of Cardiology, Campus Virchow-Klinikum, Charite Universitaetsmedizin Berlin, Berlin Institute of Health - Center for Regenerative Therapies (BIH-BCRT), and the German Center for Cardiovascular Research (DZHK; Berlin partner site), Berlin, Germany;

29 ²⁰Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers, Amsterdam, the Netherlands

30

31 ¥ both authors contributed equally as last author

32

33 Address for correspondence:

34 Susan Stienen

35 Centre d'Investigations Cliniques-INSERM CHU de Nancy,

36 Institut Lorrain du Cœur et des Vaisseaux Louis Mathieu,
1 4 Rue du Morvan, 54500 Vandoeuvre lès Nancy, France.

2 E-mail: s.stienen@amc.uva.nl

3
# 1 Abstract

# 2 Background

3 Many patients with heart failure with preserved ejection fraction (HFpEF) are women. Exploring
4 mechanisms underlying the sex-differences may improve our understanding of the pathophysiology of
5 HFpEF. Studies focusing on sex-differences in circulating proteins in HFpEF patients are scarce.

# 6 Methods

7 A total of 415 proteins were analyzed in 392 HFpEF patients included in The Metabolic Road to Diastolic
8 Heart Failure: Diastolic Heart Failure study (MEDIA-DHF). Sex-differences in these proteins were
9 assessed using adjusted logistic regression analyses. The associations between candidate proteins and
10 cardiovascular (CV) death or CV hospitalization (with sex interaction) were assessed using Cox regression
11 models.

# 12 Results

13 We found 9 proteins to be differentially expressed between female and male patients. Women expressed
14 more LPL and PLIN1, which are markers of lipid metabolism, more LHB, IGFBP3 and IL1RL2 as
15 markers of transcriptional regulation, and more Ep-CAM as marker of hemostasis. Women expressed less
16 MMP-3, which is a marker associated with extracellular matrix organization, less NRP1, which is
17 associated with developmental processes, and less ACE2, which is related to metabolism. Sex was not
18 associated with the study outcomes (adj. HR 1.48, 95%CI 0.83-2.63), p=0.18).

# 19 Conclusion

20 In chronic HFpEF, assessing sex-differences in a wide range of circulating proteins, led to the
21 identification of 9 proteins that were differentially expressed between female and male patients. These
22 findings may help further investigations into potential pathophysiological processes contributing to
23 HFpEF.
# 1 Introduction

2 Heart failure with preserved ejection fraction (HFpEF) presents an important challenge for clinical

3 practice given its rising incidence, poor prognosis and lack of clear evidence from randomized clinical

4 trials of treatment that can reduce mortality (1-4). Consistent epidemiological data demonstrate that

5 women constitute the majority of patients with HFpEF (5). Although to some extent explained by sex-

6 differences in cardiovascular structure and function, immune system biology and the myocardial response

7 to comorbidities (6), the underlying pathophysiological mechanisms of HFpEF remain incompletely

8 understood.

9 Given the sex-differences in HFpEF, we sought differences in biomarker profiles possibly linked

10 to underlying pathophysiology. In this study, we therefore assessed sex-differences in 415 circulating

11 proteins in a prospective observational cohort of HFpEF patients (The Metabolic Road to Diastolic Heart

12 Failure (MEDIA-DHF) study; NCT02446327).

13

14

15
# 1 Methods

## 2 *Study population*

In MEDIA-DHF, a multicenter, multinational, observational study, a total of 626 HFpEF patients were enrolled between 2012 and 2014 in 10 centers (listed in *Supplemental table 1*). A description of this study population and methods has been published previously (7). To summarize, after standardized echocardiography (*Supplemental data*) and/or local natriuretic peptide measurements, eligible patients with a diagnosis of diastolic dysfunction as established by the 2007 ESC recommendations(8) were included. Three clinical modes of presentations were considered: i) acute decompensated HF patients, ii) patients recently discharged after admission for an acute HF episode (<60 days), or iii) ambulatory chronic disease patients. Data on demographics, clinical parameters, laboratory values (including extensive biomarker measurements), electrocardiography and echocardiography were obtained at inclusion in the study. Follow-up visits took place at 3, 6 and 12 months after inclusion in the study.

Only patients in whom the circulating proteins were measured were included in this substudy (N=392; see also flowchart in *Supplemental figure 1*). No protein measurements were performed in acute decompensated HF patients.

## 17 *Biomarker measurements*

Plasma samples taken at inclusion in the study were analyzed for circulating protein (including natriuretic peptides) using the Olink Proseek Multiplex cardiovascular disease (CVD) II, III, Inflammation, Cardiometabolic and Organ damage panels (Olink Proteomics, Uppsala, Sweden). The assay uses a proximity extension assay (PEA) technology where 92 oligonucleotide-labelled antibody probe pairs per panel are allowed to bind to their respective targets in 1 µL plasma sample. The PEA technology has been described previously (9). In brief, when binding to their correct targets, they give rise to new DNA amplicons with each ID barcoding their respective antigens. The amplicons are subsequently quantified using a Fluidigm BioMark HD real-time PCR platform. Data is quality controlled and normalized using an internal extension control and an inter-plate control, to adjust for intra- and inter-run variation. The
extension control is composed of an antibody coupled to a unique pair of DNA-tags that serves as a synthetic control that is added to every sample well. It will adjust for technical variation introduced in the extension step and hence reduce intra-assay variability. The final assay read-out is presented in log2-normalized protein expression (NPX) data where an increase of 1 NPX confers a doubling in concentration of the specific biomarker. This arbitrary unit is therefore a relative quantification of proteins and compares fold changes between groups. All assay validation data for the proteins in the Cardiovascular II, Cardiovascular III, Inflammation, Cardiometabolic and Organ damage panels (detection limits, intra- and inter-assay precision data, accuracy, etc) are available on manufacturer's website (www. olink.com). We excluded proteins that were below the lower limit of detection in more than 50% of the patients (N=45). For the proteins below the LOD in less than 50% of patients, the LOD value was imputed. A total of 415 protein circulating proteins were studied. The abbreviations, full names and respective Olink multiplex panels of all measured proteins are described in the *Supplemental table 2*.

13

## Clinical outcome

The pre-specified endpoint of MEDIA-DHF was a composite of CV death and/or CV hospitalizations (NCT02446327). All endpoints were adjudicated by an independent endpoint committee blinded to the biomarker data.

18

## Network analyses

The FHF-GKB (Fight Heart Failure - Graph Knowledge Box) resource is a customized upgrade of the EdgeBox provided by the EdgeLeap company (available from: https://www.edgeleap.com/edgebox). It extracts data from public data sources hence providing most available public knowledge about human protein-disease, protein-protein and protein-pathway relationships. The FHF-GKB resource enables the study of a total of 20,386 protein nodes imported from Uniprot(10) (including all circulating proteins involved in this study), 28,176 disease nodes from Disease Ontology(11) and DisGenet(12) and 2,222 pathway nodes from Reactome (v65)(13). Protein-protein relationships were retrieved from STRING

26
1

(v10.5) (14)or Reactome or WikiPathways(15) or Mentha(16) or BioGrid(17), protein – disease

2

associations from DisGenet (2018-08-24) and protein – pathway relationships from Reactome.

3

4

Statistical analyses

5

6

Baseline clinical, demographic and echocardiographic characteristics were compared between male and
female patients using chi-square, t-test or Mann-Whitney tests, as appropriate.

7

8

9

10

11

12

13

14

To study whether biomarker patterns differ between male and female patients, we first identified
which clinical variables were associated with female sex using logistic regression analysis (“clinical
model”). All variables listed in table 1 were considered in univariate analyses, apart from dyslipidemia as
a diagnosis, hemoglobin as a continuous variable, NT-proBNP, and use of aspirin. Variables with ≥20%
missing values were excluded from further analyses (pulmonary artery pressure (PASP), left atrial area,
ratio of the early (E) to late (A) ventricular filling velocities (E/A), waist circumference, sodium,
potassium and CRP). Anemia was defined as hemoglobin <12 g/dL in women and <13 g/dL in men,
according to World Health Organization recommendations.

15

16

17

18

19

20

21

22

23

24

25

Missing predictor values with <20% of missing values were imputed using linear regression
analyses (see *Table 1* for the percentage of missing data for each variable). We imputed missing data 10
times, performed the analysis over all the 10 imputations and averaged results using Rubin's rules (18).
Log-linearity of continuous variables was assessed visually by inspecting the shape of the distribution of
the beta-estimates vs. the median by quintiles with regard to the outcome of interest ("female sex"). If
deemed appropriate based on log-linearity, continuous variables were categorized. Variables with
significant p-values (<0.05) in univariate analyses were considered in the multivariate model. The
discrimination of the final multivariate model for estimation of clinical end-points was assessed by
calculating the area under the curve (AUC). Similar beta-estimates of variables in the multivariate model
derived from the multiple imputation datasets were obtained when the pooled mean hemoglobin (N=46),
heart rate (N=12) and total cholesterol levels (N=67) were imputed in the original datasets (data not
1 shown). Further analyses were therefore performed in the original dataset with pooled mean levels for
2 hemoglobin, heart rate and total cholesterol.

3 To identify differences in proteins between sexes, we studied which proteins were significantly
4 associated with female sex on top of the clinical model. Therefore, all proteins were tested individually on
5 top of the clinical model (i.e., fully adjusted on the clinical variables) in a bivariate logistic regression
6 model, correcting for multiple comparisons using a Bonferroni adjusted p-value of <0.00012 (alpha
7 divided by 415 proteins). We subsequently searched for correlated proteins (>0.3). In order to ascertain
8 independence, we excluded the protein with the largest mean absolute correlation from the two correlated
9 proteins. An additional analysis excluding proteins correlated >0.5 was performed.

10 The FHF-GKB complex network was queried in order to explore pathways and proteins that could
11 connect together biomarker (BM) nodes of interest. Queries were expressed according to query patterns
12 defining a path structure between two nodes such as BM-BM, BM-pathway-BM, BM-protein-BM, where
13 the BM nodes are taken from a list of interest. The resulting graphs were merged in a figure illustrating all
14 possible paths not longer than two edges, connecting proteins through pathways and proteins.

15 The association between sex and the composite endpoint of CV death or CV hospitalizations
16 within 1 year after inclusion in the study was studied using Kaplan-Meier estimates and Cox proportional
17 hazards models. In multivariate analyses, adjustment was performed for clinical variables previously
18 found to be independently associated with CV death or CV hospitalizations in MEDIA-DHF (19). These
19 clinical variables were: a history of CAD, pulmonary rales at baseline and age. The proportional hazard
20 assumption was not met and a landmark analysis was performed excluding those patients with an endpoint
21 or being censored within 120 days after inclusion in the study.

22 Statistical analyses were performed using SPSS 24 (IBM inc., Armonk, New York) and R (The R
23 Foundation for Statistical Computing, Vienna, Austria). This is the first report on sex-differences in
24 clinical features, circulating proteins and outcome in MEDIA-DHF.

25

26
# 1 Results

## 2 *Baseline characteristics*

3 There were more female than male patients included in MEDIA-DHF (64% vs. 36%, p<0.001). The distribution of demographic, clinical and laboratory variables according to sex is summarized in *Table 1*.

5 Female patients were older by an average of 2 years, had lower hemoglobin levels (by 4.5%), a higher heart rate (by 3 bpm) and total cholesterol (by 120 mg/dL) when compared to male patients. Male patients were more often current smokers and more likely to have a history of diabetes mellitus, CAD, peripheral arterial disease and prior hospitalizations for HF. Echocardiographic variables are listed in *Table 2*.

9

## 10 *Clinical model: variables associated with female sex*

11 Baseline clinical variables that were independently associated with female sex are depicted in *Table 3*.

12 These variables were: higher heart rate, higher total cholesterol, lower hemoglobin, no CAD, no prior HF hospitalizations and no history of smoking.

14

## 15 *Association between circulating proteins and female sex*

16 For all individual proteins (including NT-proBNP), associations with female sex were assessed. A total of 17 were identified of which 9 proteins were not correlated (r <0.3) with other proteins. Of these 9 proteins, 6 were positively associated with female sex (insulin-like growth factor-binding protein 3 (IGFBP3), interleukin-1 receptor-like 2 (IL1RL2), lipoprotein lipase (LPL), epithelial cell adhesion molecule (Ep-CAM), perilipin-1 (PLIN1) and lutropin subunit beta (LHB)) and 3 were negatively associated (neuropilin-1 (NRP1), matrix metalloproteinase-3 (MMP-3), angiotensin-converting enzyme 2 (ACE2)) with female sex (Table 3; *Supplemental table 2*). Proteins with moderate correlations (r <0.5) are shown in *Supplemental table 3*.
1 *Network analyses*

2 The visualization of the interactions between circulating proteins and pathways is depicted in the *Central*

3 *Figure*. The 6 proteins that were higher expressed in female vs. male patients were linked to pathways
4 involved in lipid metabolism, transcriptional regulation and hemostasis. The proteins that were higher

5 expressed in male vs. female patients pointed to pathways related to extracellular matrix organization and

6 developmental processes. Common pathways between the “female” and “male” proteins were signal
7 transduction cascades, protein metabolism and cytokine signaling.

8

9 *Outcome*

10 There was no difference in the rates of the composite endpoint of CV death or CV hospitalization between

11 female and male patients after 1 year of follow-up (event rates: 41/250 (16.4%) female patients vs. 19/142

12 (13.4%) male patients, log rank test: p=0.46; Fig.1). Crude and adjusted hazard ratios for the composite
13 endpoint are depicted in *Table 4* (adjusted HR 1.48, 95% CI 0.83-2.63, p=0.18).

14

15
# 1 Discussion

In this study, we investigated sex-differences in circulating proteins measured in a cohort of 392 HFpEF patients. From over 400 proteins measured, we found 9 proteins to be significantly and independently associated with female sex and HFpEF. Females expressed more IGFBP3, IL1RL2, LPL, Ep-CAM, PLIN1 and LHB which are markers of lipid metabolism, transcriptional regulation and hemostasis; whereas they expressed less NRP1, MMP-3 and ACE2 which are markers associated with extracellular matrix organization and developmental processes.

# 9 *Sex-differences: clinical features*

We found several clinical variables associated with female subjects with HFpEF: higher heart rate, higher total cholesterol, lower hemoglobin, absence of CAD, no prior HF hospitalizations and not smoking.

These findings are in agreement with previous studies (6, 20, 21). Although previously shown for acute HF patients(22), an association between female sex and higher cholesterol levels in HFpEF has thus far not been reported. Although females were slightly older than male subjects, there was no significant association between age and female sex in this cohort. Interestingly, females were 14% less likely to receive statin therapy at baseline in MEDIA-DHF compared to males. A possible explanation for the higher total cholesterol levels but lower rates of CAD in females compared to males may be that females tend to have microvascular lesions and endothelial dysfunction whereas males are more likely to have obstructive coronary lesions(23), which may potentially lead to an underdiagnosis of coronary artery disease in females.

# 22 *Sex-differences: circulating proteins*
In this study, plasma levels of 9 proteins were significantly differentially expressed between female and male patients. Of these 9 proteins, 6 were higher expressed in female compared to male patients (IGFBP3, IL1RL2, LPL, Ep-CAM, PLIN1 and LHB) whereas the other 3 proteins were higher expressed in males (NRP1, ACE2 and MMP-3). Network analysis revealed that the proteins that were higher expressed in female vs. male patients may be linked to pathways involved in lipid metabolism, transcriptional regulation and hemostasis. The proteins that were higher expressed in males vs. females pointed more towards pathways involved in extracellular matrix organization and developmental processes. Common pathways were signal transduction cascades, protein metabolism and cytokine signaling.

Proposed mechanistic actions and previous studies investigating the differently expressed circulating proteins in HFpEF are described next and summarized in Table 5. IGFBP3 is the most abundant carrier protein for insulin-like growth factor 1 (IGF-1) which is known to regulate proliferation, differentiation, metabolism, and cell survival in various tissues. It has been linked to hypertension, obesity, cardiovascular disease and many cancers (24, 25). After an acute myocardial infarction, IGFBP-3 and IGF-1 levels significantly increased, which was associated with improved outcomes and echocardiographic parameters (LV dimensions, mass and ejection fraction)(26). A recent study in 84 HFpEF patients demonstrated a high prevalence of anabolic hormonal deficiencies (including IGF-1) and an low IGF-1 serum levels were associated with increased left atrial size and volume (27). Although IGFBP-3 levels were numerically higher in female compared to male participants in the Cardiovascular Health Study(28), to our knowledge, there have been no reports, so far, on sex-differences in HFpEF, or other cardiovascular diseases.

IL1RL2 (or IL-36 receptor) activates pro-inflammatory pathways upon binding to IL-36 (29). Accumulating data indicates that IL1RL2 is involved in inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis(29). No previous studies have reported on IL1RL2 in HFpEF or other cardiovascular disease.
The transmembrane glycoprotein expressed in epithelium, Ep-CAM, is involved in various processes such as cell signaling, cell-cell adhesion, proliferation and differentiation, tumorigenesis and metastasis of carcinomas and has been associated with inflammatory bowel disease(30). Ep-CAM was not associated with adverse cardiovascular outcomes in a registry of 263 chronic heart failure patients(31). To our knowledge, no studies have investigated sex-differences in Ep-CAM, in healthy subjects or in patients.

LPL is an enzyme widely expressed in the heart which catalyzes the hydrolysis of triglyceride-rich lipoproteins to fatty acids, as fuel for cardiomyocyte metabolism (32). It plays an important role in atherogenesis and has been associated with coronary heart disease, Alzheimer disease and chronic lymphocytic leukemia(33). LPL deficiency leads to hypertriglyceridemia(34) whereas overexpression of LPL in a mice model resulted in insulin resistance and obesity (35, 36). Influencing cardiac LPL in diabetic mouse models (by either overexpression or downregulation) resulted in impaired cardiac function (37).

Another enzyme involved in lipid metabolism is PLIN1 which is a surface protein of adipocyte lipid droplets that regulates storage and hydrolysis of adipose triglycerides. It has been associated with metabolic diseases such as diabetes, obesity, hepatic steatosis, certain cancers and cardiovascular disease (38). Downregulation of PLIN1 in mice led to excessive cardiac hypertrophy and failure (39). A previous study demonstrated that PLIN1, as a marker of myocardial steatosis, was higher expressed in the right atria of patients with CAD compared to those without (40).

LHB (the beta subunit of luteinizing hormone) promotes spermatogenesis and ovulation by stimulating the testes and ovaria to synthesize steroids. A previous study measured LH in men and postmenopausal women with CAD but also in controls and demonstrated that LH levels were higher in females compared to males but that there was no difference between CAD and control subjects (41). It is therefore possible that our finding of higher LHB levels in female compared to male HFpEF patients may be explained by higher LHB in females in general and that it may not be HFpEF-specific.
1 Proteins with higher expression in male compared to female HFpEF patients were NRP, ACE2 and MMP-

2 3. NRP1 is a transmembrane receptor for class III semaphorins and for members of the vascular

3 endothelial growth factor family(42). It plays a role in neuronal and vascular development during

4 embryogenesis, angiogenesis and maintenance of vascular integrity (43). In a murine model of cardiac

5 pressure overload, animals that were heterozygous for neuropilin showed higher mortality rates (44).

6 Additionally, Tromp *et al*. demonstrated that NRP1 was associated with poor outcome in HFpEF but not
7 in HFrEF patients (45).

8 ACE2 hydrolyses angiotensin I and angiotensin II generating angiotensin[1-9] and angiotensin [1-

9 7], respectively. Both angiotensin[1-9] and angiotensin[1-7] are believed to possess direct protective

10 effects against cardiac remodeling and it is hypothesized that the failing heart overexpresses ACE2 to

11 protect itself against the deleterious effects of angiotensin II (46). There is however conflicting data on sex

12 differences in ACE2 expression. One study demonstrated similar ACE2 levels between healthy male and

13 female subjects (47), while another study demonstrated that male controls had significantly higher ACE2

14 levels compared to females (48). Although still significantly higher in male compared to female patients,

15 the latter study demonstrated also that ACE2 was significantly higher in patients with type 1 diabetes and

16 coronary heart disease compared to controls (48). In line with this, a recent study in heart failure with

17 reduced ejection fraction patients also showed that ACE2 was higher in male than female patients,

18 independent of the use of pharmacological therapies targeting the renin-angiotensin-aldosterone

19 system(49). It is clear that further study to sex-differences in ACE2 is warranted.

20 MMP-3 (or stromelysin-1) is an enzyme involved in the breakdown of extracellular matrix

21 proteins and tissue remodeling in physiological (e.g. embryogenesis) and pathophysiological (e.g. tumor

22 metastasis and atherosclerosis) processes(50). High MMP-3 levels have been described in atherosclerotic

23 plaques (51). In contrast, a common mutation in the MMP-3 promoter (which results in decreased MMP-3

24 expression) was associated with atherosclerosis development (52). Also, in patients with advanced dilated

25 cardiomyopathy, MMP-3 levels were undetectable suggesting that lower levels are associated with worse
1 cardiac function (53). However, in a study of post-MI patients, MMP-3 levels were higher in male
2 compared to female patients and higher MMP-3 levels were associated with poor outcome in MI (54). In
3 our study, we found that MMP-3 levels were higher in male than in female HFpEF patients. It is obvious
4 that the exact role of MMP-3 remains unclear and warrants further investigation.

5 It must be noted that one of the circulating proteins that was excluded from the final selection
6 because of being correlated to a different protein (PLIN1 in this case), was leptin. Leptin is an hormone
7 that is released by adipocytes when they are overfilled with lipids, causing reduced food intake and
8 increased energy expenditure (55). It activates the sympathetic nervous system resulting in unfavorable
9 neurohormonal changes such as an increase in blood pressure and cardiac hypertrophy(56). A role in the
10 pathophysiology of HFpEF was also suggested In HF patients (both HFrEF as HFpEF), leptin levels were
11 higher as compared to healthy controls (57). Interestingly, the actions of leptin on the autonomic nervous
12 system appear to be particularly marked in women(58). In our study, female HFpEF patients had higher
13 leptin levels compared to male patients. Although it has been previously hypothesized that leptin may play
14 a role in the pathophysiology of HFpEF (59), the role of sex-differences herein are not yet fully
15 understood.

16 As can be appreciated above, for most of the circulating proteins that were differentially expressed
17 between female and male HFpEF patients in our study, we could not find previous studies on sex-
18 differences. However, there have been reports on sex-differences for some of the observed pathways.
19 Body fat distribution is different between sexes where women have more subcutaneous and men more
20 visceral fat. Visceral adiposity, as compared to subcutaneous adiposity, is associated with increased rates
21 of lipolysis and inflammation, hence resulting in an increased susceptibility for metabolic complications
22 (as reviewed in (60)). In HFpEF, it is hypothesized comorbidities such as diabetes and obesity induce a
23 low-grade systemic pro-inflammatory state(61). Whether sex-differences in lipid metabolism are of
24 importance in the pathophysiology of HFpEF warrants further investigation.
Moreover, there is an increasing body of evidence suggesting sex-differences in primary and
secondary hemostasis, and fibrinolysis. It has been demonstrated, for example, that women have higher
platelet counts and activity and generate more thrombin compared to men (as reviewed in (62)).

Lastly, sex-differences in extracellular matrix turnover have also been previously reported.

Women demonstrate a smaller fibrotic response compared to men as reflected by a lower expression of
TGF-beta (a highly pro-fibrotic growth-factor) in women (as reviewed in (63)). Moreover, in a mouse
model, matrix metalloproteinase activity was higher in males compared to females after an acute MI,
leading to poorer outcome in males (64). Although cardiac fibrosis has been hypothesized by Paulus et al.
to be one of the key players in the pathophysiology of HFpEF(61), the role of sex-differences herein has
yet to be identified. It is also possible that our finding of more activated extracellular matrix pathways in
male compared to female HFpEF patients simply reflects the presence of more CAD in males (as observed
in our study) and subsequent myocardial injury-induced extracellular matrix alterations.

13

14 *Sex-differences: outcome*

15
16
17
18
19
20
21
22
23
Female sex was not associated with cardiovascular death and/or cardiovascular hospitalizations in our
study. Our findings are in line with a recent post-hoc analysis from PARAGON demonstrating similar
rates of the composite of heart failure hospitalizations or death for cardiovascular reasons(65), but in
conflict with previous other studies. Lam *et al.* observed in a post-hoc analysis of I-PRESERVE that
female sex was associated with better prognosis, although the effect was moderated by 4 common baseline
characteristics which were atrial fibrillation, renal dysfunction, stable angina pectoris and NYHA class
III/IV (21). In the Meta-Analysis Global Group in Chronic Heart Failure (MAGGIC) meta-analysis,
women had lower rates of all-cause mortality over 3 years compared to men, irrespective of ejection
fraction (66). Our findings and those of Lam *et al.* suggest that the relation between sex and outcome in
1 HFpEF is confounded to some extent by comorbidities or other patient characteristics. Future studies need
to determine whether a sex-difference in prognosis exists.

3

4 *Perspectives and Significance*

5 For the majority of the circulating proteins that were differently expressed between female and male
6 HFpEF patients in our study, sex-differences in a healthy population have not been studied. A recent
7 population-based study compared 30 cardiometabolic biomarkers (not including the proteins that were
8 measured in MEDIA-DHF) between female and male participants and found that biomarker profiles
9 significantly differed (67). Whether the sex-differences in circulating proteins in our cohort of HFpEF
10 patients reflect pathophysiological or physiological processes warrants further study. Our findings of
11 different pathways being activated in female compared to male HFpEF patients may be the basis of
12 investigating therapies specifically targeting the identified pathways, potentially remediating the poor
13 track record of past HFpEF large outcome trials (68). In addition, they may be of help in the search for
14 biologically plausible explanations for the sex-differences in treatment response as reported for neprilysin
15 inhibition (69), mineralocorticoid receptor antagonists (70), and cardiac resynchronization therapy (71).

16

17 *Limitations*

18 The most important limitation of this study is that we could not externally validate our findings due to the
19 absence of other HFpEF cohorts in which similar proteins were measured. It is clear that other studies
20 need to confirm our findings. Second, as already mentioned before, sex-differences have not been studied
21 in a general population for the majority of circulating proteins tested in our study. Moreover, in this study
22 only HFpEF patients were included and a 'healthy' control group (including reference values for the
23 measured proteins) was thus absent. It is therefore not clear whether the observed differences in biomarker
24 expression reflects pathophysiological and/or physiological processes. Third, at the time of enrollment,
1 HFpEF was diagnosed following 2007 ESC diagnostic recommendations. Since the 2007 criteria, other
2 criteria have been proposed and it has recently been shown that large variations in prevalence of diastolic
3 dysfunction may be expected according to which criterion is used (72). However, when applying the
4 H₂FPEF score by Reddy and colleagues (73) to the MEDIA-DHF cohort, only 2% of patients had a low
5 probability of HFpEF whereas 58% and 40% of patients had an intermediate or high probability,
6 respectively. Fourth, although the biomarker assay in this study covers a wide variety of disease domains,
7 the possibility exists that other, now unmeasured, proteins may (also) play a role in the pathophysiology of
8 HFpEF. Fifth, the biomarker assay does not provide standard concentration units, making comparisons
9 with clinically applied cut-offs difficult.

10

## Conclusion

12 Assessing sex-differences in >400 circulating proteins analyzed in a large cohort of HFpEF patients led to
13 the identification of 9 uncorrelated sex-specific proteins. Females expressed more markers associated with
14 lipid metabolism, transcriptional regulation and hemostasis, whereas they expressed less proteins
15 associated with extracellular matrix organization and developmental processes. These findings may help
16 further investigations into potential pathophysiological processes contributing to HFpEF.

17

## Declarations

19 *Ethics approval and consent to participate*

20 The study protocol complied with the Declaration of Helsinki and was approved by the respective Ethics

21 Committees of the participating institutions. Written informed consent was provided by all patients.

22 (ClinicalTrials.gov Identifier: NCT02446327)

23 *Consent for publication*

24 Not applicable

25 *Availability of data and material*
1 The data that support the findings of this study are available from the corresponding author upon
2 reasonable request.

### Competing interest and funding

4 This study was supported by a grant from the European Union (FP7-HEALTH-2010-MEDIA), by the
5 French Programme Hospitalier de Recherche Clinique (PHRC) and by the RHU Fight-HF, a public grant
6 overseen by the French National Research Agency (ANR) as part of the second "Investissements
d'Avenir" program (reference: ANR-15-RHU-0004), the GEENAGE (ANR-15-IDEX-04-LUE) program,
8 by the Contrat de Plan Etat Région Lorraine and FEDER IT2MP.) N-LA was supported by a Miguel
9 Servet contract CP13/00221 from the "Instituto de Salud Carlos III-FEDER". WJP and LvH were
10 supported by CVON, Dutch Heart Foundation, The Hague, The Netherlands (RECONNECT and EARLY-HFpEF projects). AM received speaker's honoraria from Orion, Otsuka, Philips, Roche and Servier. AM
11 received fees as member of advisory board and/or Steering Committee and/or research grant from
13 Adrenomed, Epygon, Neurotronik, Roche, Sanofi and Sphyngotec. AM owns shares in S-Form Pharma.
14 SS acknowledges funding received from the European Society of Cardiology in form of an ESC Research
15 Grant (R-2018-18686).

16

### Authors' contributions

18 SS analysed and interpreted the data and drafted the manuscript. JPF made substantial contributions to the
19 design and analysis, and drafting of the manuscript. MK analysed the data and revised the manuscript. DD
20 made substantial contributions to the conception of the data and revised the manuscript. GP, JLM and KD
21 developed the software used for the analysis and revised the manuscript. KD developed the software used
22 for the analysis and revised the manuscript. EB and MDD performed the complex network analyses and
23 revised the manuscript. NLA helped interpreting the data and revised the manuscript. SA, GA, HFBLR,
24 RFC, AF, LvH, GdK, PM, KM, AM, ZP, RR, CT, WJP made substantial contributions to the conception
25 of the data and revised the manuscript. NG, FZ and PR made substantial contributions to the conception of
26 the data, the design of the study, and substantial revisions to the manuscript. All co-authors have approved
1 the submitted version and both to be personally accountable for the author's own contributions and to
2 ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the
3 author was not personally involved, are appropriately investigated, resolved, and the resolution
4 documented in the literature.

5 Acknowledgments

6 We thank other colleagues for their important contributions to the MEDIA study in the participating
7 centres, including the CRB Lorrain for the biobanking, Gabor Kunszt (Oslo University Hospital), Tamas
8 Erdei and Julie Edwards (Cardiff University).

9
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16

17
<caption>Table 1 Baseline characteristics of patients in the MEDIA-DHF cohort according to sex</caption>

3

<table><thead><tr><th></th><th>Global (N=392)</th><th>Male (N=142)</th><th>Female (N=250)</th><th>p-value</th><th>% of missing values</th></tr></thead><tbody><tr><td>Age, years, median (IQR)</td><td>74.0 (67.5 - 80.0)</td><td>73.0 (67.0 - 79.0)</td><td>75.0 (68.0 - 81.0)</td><td>0.028</td><td>0%</td></tr><tr><td>Recently decompensated HF, n (%)</td><td>60 (15.3 %)</td><td>25 (17.6 %)</td><td>35 (14.0 %)</td><td>0.34</td><td>0%</td></tr><tr><td>Smoking status, n (%)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Never</td><td>205 (52.7 %)</td><td>39 (27.9 %)</td><td>166 (66.7 %)</td><td rowspan="3">&lt;0.001</td><td rowspan="3">1%</td></tr><tr><td>Former</td><td>150 (38.6 %)</td><td>84 (60.0 %)</td><td>66 (26.5 %)</td></tr><tr><td>Current</td><td>34 (8.7 %)</td><td>17 (12.1 %)</td><td>17 (6.8 %)</td></tr><tr><td>BMI, kg/m², mean±SD</td><td>30.6 ± 6.2</td><td>30.5 ± 5.6</td><td>30.6 ± 6.5</td><td>0.79</td><td>1%</td></tr><tr><td>Alcohol status, n (%)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Non-consumer</td><td>245 (63.3 %)</td><td>73 (52.1 %)</td><td>172 (69.6 %)</td><td rowspan="3">0.001</td><td rowspan="3">1%</td></tr><tr><td>1-2 drinks/day</td><td>124 (32.0 %)</td><td>56 (40.0 %)</td><td>68 (27.5 %)</td></tr><tr><td>&gt;2 drinks/day</td><td>18 (4.7 %)</td><td>11 (7.9 %)</td><td>7 (2.8 %)</td></tr><tr><td>Waist circumference, cm, mean±SD</td><td>104.8 ± 14.1</td><td>108.2 ± 13.8</td><td>102.9 ± 13.9</td><td>0.001</td><td>21%</td></tr><tr><td>SBP, mmHg, mean±SD</td><td>137.2 ± 23.1</td><td>137.5 ± 22.3</td><td>137.0 ± 23.5</td><td>0.83</td><td>2%</td></tr><tr><td>DBP, mmHg, mean±SD</td><td>74.0 ± 11.6</td><td>74.7 ± 11.1</td><td>73.7 ± 11.9</td><td>0.41</td><td>2%</td></tr><tr><td>Pulmonary rales, n (%)</td><td>89 (23.0 %)</td><td>33 (23.4 %)</td><td>56 (22.8 %)</td><td>0.89</td><td>1%</td></tr><tr><td>NYHA class III/IV, n (%)</td><td>73 (18.7 %)</td><td>23 (16.2 %)</td><td>50 (20.1 %)</td><td>0.34</td><td>0%</td></tr><tr><td>Peripheral edema, n (%)</td><td>178 (45.4 %)</td><td>74 (52.1 %)</td><td>104 (41.6 %)</td><td>0.045</td><td>1%</td></tr><tr><td>Elevated JVP, n (%)</td><td>24 (6.3 %)</td><td>12 (8.7 %)</td><td>12 (4.9 %)</td><td>0.15</td><td>3%</td></tr><tr><td>Hepatomegaly, n (%)</td><td>13 (3.6 %)</td><td>6 (4.5 %)</td><td>7 (3.1 %)</td><td>0.68</td><td>7%</td></tr><tr><td>Fatigue at exertion, n (%)</td><td>305 (79.2 %)</td><td>105 (75.5 %)</td><td>200 (81.3 %)</td><td>0.18</td><td>2%</td></tr><tr><td>Heart rate, bpm, mean±SD</td><td>69.4 ± 14.4</td><td>67.1 ± 12.9</td><td>70.8 ± 15.0</td><td>0.016</td><td>3%</td></tr><tr><td>Hypertension n (%)</td><td>342 (87.7 %)</td><td>121 (86.4 %)</td><td>221 (88.4 %)</td><td>0.57</td><td>1%</td></tr><tr><td>Atrial fibrillation, n (%)</td><td>117 (31 %)</td><td>49 (36 %)</td><td>68 (28 %)</td><td>0.13</td><td>3%</td></tr><tr><td>Diabetes mellitus n (%)</td><td>154 (39.3 %)</td><td>67 (47.2 %)</td><td>87 (34.8 %)</td><td>0.016</td><td>0%</td></tr><tr><td>Dyslipidemia n (%)</td><td>226 (58.2 %)</td><td>75 (53.6 %)</td><td>151 (60.9 %)</td><td>0.16</td><td>1%</td></tr><tr><td>Previous HF hospitalization, n (%)</td><td>137 (35.5 %)</td><td>65 (46.8 %)</td><td>72 (29.1 %)</td><td>&lt;0.001</td><td>2%</td></tr><tr><td>CAD, n (%)</td><td>128 (33.7 %)</td><td>73 (52.9 %)</td><td>55 (22.7 %)</td><td>&lt;0.001</td><td>3%</td></tr><tr><td>Stroke or TIA, n (%)</td><td>44 (11.3 %)</td><td>14 (10.0 %)</td><td>30 (12.0 %)</td><td>0.54</td><td>1%</td></tr><tr><td>Peripheral artery disease, n (%)</td><td>34 (8.9 %)</td><td>18 (13.0 %)</td><td>16 (6.5 %)</td><td>0.030</td><td>2%</td></tr><tr><td>COPD, n (%)</td><td>71 (18.4 %)</td><td>30 (21.4 %)</td><td>41 (16.7 %)</td><td>0.25</td><td>2%</td></tr><tr><td>Laboratory values, mean±SD</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LDL, mg/dL</td><td>99.8 ± 37.1</td><td>89.8 ± 30.2</td><td>104.5 ± 39.1</td><td>&lt;0.001</td><td>20%</td></tr><tr><td>HDL, mg/dL</td><td>53.7 ± 19.7</td><td>47.3 ± 15.5</td><td>56.8 ± 20.8</td><td>&lt;0.001</td><td>19%</td></tr><tr><td>Total cholesterol, mg/dL</td><td>176.1 ± 43.1</td><td>160.2 ± 35.1</td><td>183.9 ± 44.6</td><td>&lt;0.001</td><td>17%</td></tr></tbody></table>
<table><tr><td>Hemoglobin, g/dL</td><td>13.0 ± 1.6</td><td>13.4 ± 1.8</td><td>12.8 ± 1.5</td><td>&lt;0.001</td><td>12%</td></tr><tr><td>Anemia, n (%)</td><td>106 (30.6 %)</td><td>46 (39.0 %)</td><td>60 (26.3 %)</td><td>0.019</td><td>12%</td></tr><tr><td>eGFR, mL/min/1.73m2</td><td>66.4 ± 23.1</td><td>66.4 ± 23.7</td><td>66.4 ± 22.8</td><td>1.00</td><td>6%</td></tr><tr><td>NT-proBNP (in NPX)</td><td>4.2 ± 1.3</td><td>4.4 ± 1.4</td><td>4.1 ± 1.3</td><td>0.042</td><td>0%</td></tr><tr><td>Medication prescription rates, n (%)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>ACEi or ARB</td><td>319 (81.4 %)</td><td>114 (80.3 %)</td><td>205 (82.0 %)</td><td>0.67</td><td>0%</td></tr><tr><td>Betablockers</td><td>286 (73.0 %)</td><td>111 (78.2 %)</td><td>175 (70.0 %)</td><td>0.080</td><td>0%</td></tr><tr><td>Thiazide diuretics</td><td>87 (22.3 %)</td><td>22 (15.5 %)</td><td>65 (26.1 %)</td><td>0.015</td><td>0%</td></tr><tr><td>Loop diuretics</td><td>235 (59.9 %)</td><td>89 (62.7 %)</td><td>146 (58.4 %)</td><td>0.41</td><td>0%</td></tr><tr><td>MRA</td><td>43 (11.0 %)</td><td>16 (11.3 %)</td><td>27 (10.8 %)</td><td>0.87</td><td>0%</td></tr><tr><td>Aspirin</td><td>159 (40.6 %)</td><td>64 (45.1 %)</td><td>95 (38.0 %)</td><td>0.17</td><td>0%</td></tr><tr><td>Insulin</td><td>52 (13.3 %)</td><td>26 (18.3 %)</td><td>26 (10.4 %)</td><td>0.028</td><td>0%</td></tr><tr><td>Statin</td><td>241 (61.5 %)</td><td>96 (67.6 %)</td><td>145 (58.0 %)</td><td>0.060</td><td>0%</td></tr><tr><td>Oral anticoagulants</td><td>161 (41.1 %)</td><td>57 (40.1 %)</td><td>104 (41.6 %)</td><td>0.78</td><td>0%</td></tr></table>

Legend: HF, heart failure; BMI, body mass index; DM, diabetes mellitus; CAD, coronary artery disease; PAD, peripheral artery disease; COPD, chronic obstructive pulmonary disease; OSAS, obstructive sleep apnea syndrome; TIA, transient ischemic attack; DBP, diastolic blood pressure; SBP, systolic blood pressure; NYHA, New York Heart Association; JVP, jugular venous pressure; eGFR, estimated glomerular filtration rate; BNP, brain natriuretic peptide; NT-proBNP, N-terminal pro-brain natriuretic peptide; NPX, normalized protein expression; ASA, acetylsalicylic acid; ACEi, ACE-inhibitor; ARB, angiotensin receptor blocker; BB, betablocker; MRA, mineralocorticoid receptor antagonist; LVEF, left ventricular ejection fraction; E/e', the ratio of mitral inflow velocity and early mitral annulus velocity, PASP, pulmonary artery systolic pressure; TAPSE, Tricuspidal Annular Plane Systolic Excursion; ; E/A, ratio of the early (E) to late (A) ventricular filling velocities; LVEDVi, left ventricular end-diastolic volume index; LVESVi, left ventricular end-systolic volume index; LAVI, left atrial volume index; IQR, interquartile range; SD, standard deviation.

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<table><caption>Table 2 Echocardiography characteristics of patients in the MEDIA-DHF cohort according to sex</caption><thead><tr><th></th><th>Global<br>(N=392)</th><th>Male<br>(N=142)</th><th>Female<br>(N=250)</th><th>p-value</th><th>% of<br>missing<br>values</th></tr></thead><tbody><tr><td>Echocardiographic variables, mean±SD</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>LVEF, %</td><td>60.8 ± 7.0</td><td>60.5 ± 6.9</td><td>61.0 ± 7.0</td><td>0.54</td><td>0%</td></tr><tr><td>E/e'</td><td>13.3 ± 5.2</td><td>12.6 ± 4.6</td><td>13.7 ± 5.4</td><td>0.057</td><td>7%</td></tr><tr><td>PASP, mmHg</td><td>34.8 ± 12.7</td><td>34.9 ± 15.0</td><td>34.8 ± 11.4</td><td>0.97</td><td>25%</td></tr><tr><td>TAPSE, cm</td><td>20.5 ± 4.8</td><td>21.0 ± 5.4</td><td>20.2 ± 4.4</td><td>0.13</td><td>10%</td></tr><tr><td>E/A</td><td>1.3 ± 0.9</td><td>1.4 ± 1.0</td><td>1.2 ± 0.8</td><td>0.033</td><td>30%</td></tr><tr><td>LDEVi</td><td>43.8 ± 14.0</td><td>48.3 ± 14.4</td><td>41.1 ± 13.1</td><td>&lt;0.001</td><td>4%</td></tr><tr><td>LDESi</td><td>17.4 ± 7.3</td><td>19.6 ± 7.5</td><td>16.2 ± 6.7</td><td>&lt;0.001</td><td>6%</td></tr><tr><td>LAVI, ml/m²</td><td>43.8 ± 15.8</td><td>45.0 ± 16.6</td><td>43.1 ± 15.4</td><td>0.27</td><td>3%</td></tr></tbody></table>

Legend: LVEF, left ventricular ejection fraction; E/e', the ratio of mitral inflow velocity and early mitral annulus velocity, PASP, pulmonary artery systolic pressure; TAPSE, Tricuspidal Annular Plane Systolic Excursion; E/A, ratio of the early (E) to late (A) ventricular filling velocities; LVEDVi, left ventricular end-diastolic volume index; LVESVi, left ventricular end-systolic volume index; LAVI, left atrial volume index; IQR, interquartile range; SD, standard deviation.
<caption>Table 3 Adjusted and multiple testing-corrected circulating proteins associated with female sex</caption>

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<table><thead><tr><th rowspan="2"></th><th colspan="2">Clinical model (AUC 0.82)</th><th colspan="2">Proteins</th></tr><tr><th>OR (95% CI) for female sex association</th><th>p-value</th><th>OR (95% CI) for female sex association</th><th>p-value</th></tr></thead><tbody><tr><td>Heart rate, per 10 bpm increase</td><td>1.22 (1.00-1.48)</td><td>0.049</td><td></td><td></td></tr><tr><td>Total cholesterol, per 10 mg/dL increase</td><td>1.13 (1.03-1.02)</td><td>0.010</td><td></td><td></td></tr><tr><td>Smoking status</td><td></td><td></td><td></td><td></td></tr><tr><td>Never</td><td>Ref.</td><td>-</td><td></td><td></td></tr><tr><td>Former</td><td>0.21 (0.12-0.36)</td><td>&lt;0.001</td><td></td><td></td></tr><tr><td>Current</td><td>0.33 (0.14-0.80)</td><td>0.014</td><td></td><td></td></tr><tr><td>CAD</td><td>0.36 (0.21-0.62)</td><td>&lt;0.001</td><td></td><td></td></tr><tr><td>Previous HF hospitalization</td><td>0.47 (0.27-0.82)</td><td>0.008</td><td></td><td></td></tr><tr><td>Hb, per 1 g/dL increase</td><td>0.69 (0.57-0.82)</td><td>&lt;0.001</td><td></td><td></td></tr><tr><td>Higher expression in females vs. males</td><td></td><td></td><td></td><td></td></tr><tr><td>IL1RL2</td><td></td><td></td><td>4.13 (2.22-7.68)</td><td>&lt;0.001</td></tr><tr><td>LPL</td><td></td><td></td><td>4.08 (2.44-6.82)</td><td>&lt;0.001</td></tr><tr><td>LHB</td><td></td><td></td><td>3.15 (2.20-4.52)</td><td>&lt;0.001</td></tr><tr><td>IGFBP3</td><td></td><td></td><td>2.83 (1.74-4.58)</td><td>&lt;0.001</td></tr><tr><td>PLIN1</td><td></td><td></td><td>2.44 (1.62-3.66)</td><td>&lt;0.001</td></tr><tr><td>Ep-CAM</td><td></td><td></td><td>1.87 (1.41-2.48)</td><td>&lt;0.001</td></tr><tr><td>Less expression in females vs. males</td><td></td><td></td><td></td><td></td></tr><tr><td>NRP1</td><td></td><td></td><td>0.05 (0.01-0.19)</td><td>&lt;0.001</td></tr><tr><td>MMP-3</td><td></td><td></td><td>0.22 (0.14-0.35)</td><td>&lt;0.001</td></tr><tr><td>ACE2</td><td></td><td></td><td>0.41 (0.28-0.61)</td><td>&lt;0.001</td></tr></tbody></table>

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Legend: eGFR, estimated glomerular filtration rate; OR, odds ratio; CI, confidence interval; AUC, area under the curve; bpm, beats per minute;

CAD, coronary artery disease; HF, heart failure; Hb, hemoglobin; IGFBP3, insulin-like growth factor-binding protein 3; NRP1, neuropilin 1;

IL1RL2, interleukin-1 receptor-like 2; LPL, lipoprotein lipase; ACE2, angiotensin-converting-enzyme 2; EpCAM, epithelial cell adhesion molecule; CA14, carbonic anhydrase 14; PLIN1, perilipin-1; LHB, lutropin subunit beta.

Each OR unit increase represents doubling in the NPX values

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<caption>Table 4 Crude and adjusted hazard ratios for the prediction of cardiovascular death and/or cardiovascular hospitalization during 1 year of follow-up according to sex</caption>

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<table><thead><tr><th></th><th>Crude HR (95%CI)</th><th>Adj. HR (95%CI)*</th></tr></thead><tbody><tr><td><em>Complete cohort</em></td><td></td><td></td></tr><tr><td>Female vs. Male</td><td>1.26 (0.73-2.16), p=0.41</td><td>1.48 (0.83-2.63), p=0.18</td></tr><tr><td><em>Landmark analysis</em><br>(>120 days)</td><td></td><td></td></tr><tr><td>Female vs. Male</td><td>1.70 (0.72-4.03), p=0.23</td><td>1.89 (0.77-4.65), p=0.17</td></tr></tbody></table>

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*adjusted on coronary artery disease, pulmonary rales at baseline and age. Since our aim was to assess the association of female sex on top of this

clinical risk score and female sex was already incorporated in the prognostic risk score, sex was not considered in the prognostic risk score used

for these analyses.

The proportional hazard assumption was not met and a landmark analysis was performed excluding those patients with an endpoint or being censored within 120 days after inclusion in the study.

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<table><caption>**Table 5** Overview of the proteins differentially expressed between sexes</caption><thead><tr><th rowspan="2">Individual biomarker</th><th rowspan="2">Mechanistic significance</th><th colspan="2">Previous relevant reports</th><th colspan="2">Reports on sex differences</th></tr><tr><th>Basic reports</th><th>Clinical reports</th><th>Healthy population</th><th>Heart disease</th></tr></thead><tbody><tr><td>More highly expressed in females</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>IL1RL2</td><td>Activates pro-inflammatory pathways upon binding of IL-36 (29)</td><td></td><td>Involved in inflammatory diseases such as psoriasis, inflammatory bowel disease and rheumatoid arthritis(29)</td><td>No</td><td>No</td></tr><tr><td>LPL</td><td>Enzyme that catalyzes the hydrolysis of triglycerides</td><td>Overexpression or downregulation of cardiac LPL in diabetic mouse models resulted in impaired left ventricular function (37).</td><td>Associated with coronary heart disease, Alzheimer disease and chronic lymphocytic leukemia(33)</td><td>No</td><td>No</td></tr><tr><td>LHB</td><td>Promotes spermatogenesis and ovulation by stimulating the testes and ovaria to synthesize steroids</td><td></td><td>Levels not different between CAD and control subjects (41).</td><td>Yes (higher in females vs. males (41))</td><td>Yes (higher in female vs. male CAD patients(41))</td></tr><tr><td>IGFBP3</td><td>Most abundant carrier protein for insulin-like growth factor 1 (IGF-1) which is known to play a major role in metabolism</td><td>The IGF system (including IGFBP3) has been previously associated with cardiovascular disease and many cancers (24, 25)</td><td>In HFpEF patients, low IGF-1 and IGFBP3 were associated with increased parameters of left atrial size and volume (27) Higher IGF-1 after an acute MI was associated with improved clinical outcomes and echocardiographic measures (LV dimensions, mass and ejection fraction)(26)</td><td>Yes (numerically higher in female vs. male but no formal comparison made(28))</td><td>No</td></tr><tr><td>PLIN1</td><td>Surface protein of adipocyte lipid droplets that regulates</td><td>Linked to endocrine metabolism disease</td><td>Higher expressed in the right atria of patients with CAD compared to</td><td>No</td><td>No</td></tr></tbody></table>
<table><tr><td></td><td>storage and hydrolysis of adipose triglycerides(74)</td><td>(diabetes, obesity etc.), cancers and cardiovascular disease (38)</td><td>those without (40).</td><td></td><td></td></tr><tr><td>Ep-CAM</td><td>Transmembrane glycoprotein expressed in epithelium</td><td>Involved in various processes such as cell signaling, cell-cell adhesion, proliferation and differentiation, tumorigenesis and metastasis of carcinomas(30)</td><td>Not associated with adverse cardiovascular outcomes in a registry of 263 chronic heart failure patients(31)<br/>Associated with inflammatory bowel disease(30)</td><td>No</td><td>No</td></tr><tr><td>Less expressed in females</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>NRP1</td><td>Transmembrane receptor for class III semaphorins and for members of the vascular endothelial growth factor family(42)</td><td>Neuronal and vascular development during embryogenesis, angiogenesis and maintenance of vascular integrity (43)</td><td>Associated with poor outcome in HFpEF but not in HFrEF patients (45)</td><td>No</td><td>No</td></tr><tr><td>MMP-3</td><td>Enzyme involved in the breakdown of extracellular matrix proteins</td><td>Involved in physiological (e.g. embryogenesis) and pathophysiological (e.g. tumor metastasis and atherosclerosis) processes(50)</td><td>Conflicting data: High levels have been described in atherosclerotic plaques (51) and associated with poor outcome after MI(54). However, a common mutation in the MMP-3 promoter (which results in decreased MMP-3 expression) was associated with atherosclerosis development (52).</td><td>No</td><td>Yes (higher in male vs. female patients post-MI (54))</td></tr><tr><td>ACE2</td><td>Hydrolyses angiotensin I and angiotensin II generating angiotensin[1-9] and angiotensin [1-7], respectively.</td><td></td><td>ACE2 levels were higher in patients with type 1 diabetes and coronary heart disease vs. controls(48).</td><td>Yes (conflicting data: similar levels in male and females (47) or higher in male vs. female (48))</td><td>Yes (higher in males vs. females with type 1 DM and CAD (48))</td></tr></table>
)
<figure><img src="image_2.png" /><figcaption>Figure 1 Primary outcome according to sex</figcaption></figure>

Legend: CV, cardiovascular; No., number.
**Central Figure**

**Network analyses for the visualization of pathways and protein-interactions between circulating proteins associated with sex in MEDIA-DHF.** The proteins that were higher expressed in female vs. male patients may be linked to pathways involved in lipid metabolism, transcriptional regulation and hemostasis. The proteins that were higher expressed in male vs. female patients may be linked to pathways such as extracellular matrix organization and developmental processes. Common pathways between the proteins seem signal transduction, protein metabolism and cytdfauokine signaling.

<figure><img src="image_3.png" /></figure>

The FHF-GKB complex network was queried in order to explore pathways and proteins that could connect together biomarker nodes of interest. Queries were expressed according to query patterns defining a path structure between two nodes such as BM-BM, BM-pathway-BM, and BM-pathway, where the BM nodes are taken from a list of interest. The resulting graphs were merged in a figure illustrating all possible paths not longer than two edges, connecting proteins through pathways and proteins. This graph depicts biomarker-pathway—protein interactions. There were no direct interactions between proteins. Blue: proteins higher expressed in males; Pink: proteins higher expressed in females. Green: pathways.
## Supplemental data

### Echocardiography

All patients underwent echocardiography and images were stored in a digital cine-loop format for off-line analysis according to the American Society of Echocardiography and the European Association of Cardiovascular Imaging relative to the cardiac chamber and right heart measurements<fnref n="1" />

LV structure and function were evaluated from standard 2D views, including apical four-chamber, apical two-chamber and parasternal long- and short-axis views. Ventricular dimensions, wall thickness, mass and geometry were determined from 2D parasternal short- and long-axis views. LV volumes, stroke volume and ejection fraction were calculated using the biplane method of disks summation (modified Simpson's rule). All cardiac chamber volumes and mass measurements were indexed to body surface area.

Left atrial volume was assessed by the biplane area-length method from apical 2- and 4-chamber views at end-systole and was indexed to body surface area (LA volume index, LAVi). Left atrial area (cm²) was estimated from the apical views. If LA volume was not available, LA area was used to evaluate remodeling. Patients with either LAVi>40ml/m² or LA area>20cm² were considered to have dilated LA.

Peak velocity of early (E) and late (A) wave of transmitral flow and E-wave deceleration time (DT) were measured from the pulsed-wave Doppler obtained at the tip of mitral leaflets. The average of septal and lateral annular velocities (e') was obtained by tissue Doppler imaging

<footnote n="1">¹ Lang RM *et al*. Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging. Eur Heart J Cardiovasc Imaging 2015; 16(3):233-70.</footnote>
(TDI). The E/e' ratio was calculated using the peak E-wave velocity and the average of septal and lateral. LV outflow tract (LVOT) time velocity integral, isovolumic relaxation time (IVRT) and color M-mode of early diastolic mitral inflow into the left ventricle (Flow propagation velocity, Vp) were acquired from apical four- and five-chamber views. Pulmonary venous flow (PVF) was sampled using pulsed-wave Doppler at 1 cm into the orifice of the right upper pulmonary vein.

Right ventricular function was estimated by measuring TAPSE (mm) and pulmonary arterial systolic pressure (PASP) was calculated using the peak velocity of tricuspid regurgitation (TR) and the maximum IVC diameter (IVC baseline) and respiratory variation (Ratio IVC inspiration / IVC baseline) measured 3 cm before merger with the right atrium.

Overall, echocardiographic data were complete in >75% of patients, except for the following variables: A (and E/A ratio), S/D, Ard-Ad, E/Vp (which were available in 70%, 61%, 44% and 62% of the population, respectively).
<table><caption>**Supplemental table 1**

**Participating centers MEDIA-DHF**</caption><tr><td>Nancy Clinical Investigation Centre, University of Lorraine, France (and EDDH European Drug Development Hub, Nancy, France as project manager)</td></tr><tr><td>Lariboisière Hospital, Paris, France</td></tr><tr><td>Free University Medical Centre, Amsterdam, The Netherlands</td></tr><tr><td>CHARITE, universitätsmedizin Berlin, Berlin Germany</td></tr><tr><td>University of Porto, Porto, Portugal</td></tr><tr><td>University of Maastricht, Maastricht, The Netherlands</td></tr><tr><td>University of Antwerp, Antwerp, Belgium</td></tr><tr><td>Cardiff University, Cardiff, United Kingdom</td></tr><tr><td>Foundation for Applied Medical Research, Pamplona, Spain</td></tr><tr><td>University of Eastern PIEMONTE Medical School, Novara, Italy</td></tr><tr><td>University of Brescia, Brescia, Italy</td></tr><tr><td>University of Perugia, Perugia, Italy</td></tr><tr><td>University College Dublin, National University of Ireland, Dublin, Ireland</td></tr><tr><td>University Debrecen, Debrecen, Hungary</td></tr><tr><td>Oslo University Hospital</td></tr></table>
<table><caption>Supplemental table 2 Overview of circulating proteins measured at baseline in MEDIA-DHF</caption><thead><tr><th>Organ damage panel</th><th></th></tr></thead><tbody><tr><td>5'-AMP-activated protein kinase subunit beta-1 (PRKAB1) Q9Y478</td><td>Kidney Injury Molecule (KIM1) Q96D42</td></tr><tr><td>Adhesion G-protein coupled receptor G1 (ADGRG1) Q9Y653</td><td>Leukotriene A-4 hydrolase (LTA4H) P09960</td></tr><tr><td>Aldehyde dehydrogenase, dimeric NADP-preferring (ALDH3A1) P30838</td><td>Linker for activation of T-cells family member 2 (LAT2) Q9GZY6</td></tr><tr><td>Anterior gradient protein 2 homolog (AGR2) O95994</td><td>Lutropin subunit beta (LHB) P01229</td></tr><tr><td>Apoptosis-inducing factor 1, mitochondrial (AIFM1) O95831</td><td>Macrophage erythroblast attacher (MAEA) Q7L5Y9</td></tr><tr><td>B-cell scaffold protein with ankyrin repeats (BANK1) Q8NDB2</td><td>Macrophage-capping protein (CAPG) P40121</td></tr><tr><td>BH3-interacting domain death agonist (BID) P55957</td><td>Melanoma-associated antigen D1 (MAGED1) Q9Y5V3</td></tr><tr><td>BMP and activin membrane-bound inhibitor homolog (BAMBI) Q13145</td><td>Methionine aminopeptidase 1 (METAP1) P53582</td></tr><tr><td>Calcitonin (CALCA) P01258</td><td>Mevalonate kinase (MVK) Q03426</td></tr><tr><td>Calreticulin (CALR) P27797</td><td>Mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) Q9Y4K4</td></tr><tr><td>Carbonic anhydrase 12 (CA12) O43570</td><td>Mothers against decapentaplegic homolog 1 (SMAD1) Q15797</td></tr><tr><td>Carbonic anhydrase 14 (CA14) Q9ULX7</td><td>NAD-dependent protein deacylase sirtuin-5, mitochondrial (SIRT5) Q9NXA8</td></tr><tr><td>Casein kinase I isoform delta (CSNK1D) P48730</td><td>NEDD8 ultimate buster 1 (NUB1) Q9Y5A7</td></tr><tr><td>Claspin (CLSPN) Q9HAW4</td><td>Neutrophil cytosol factor 2 (NCF2) P19878</td></tr><tr><td>CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3- sialyltransferase 1 (ST3GAL1)</td><td>Nibrin (NBN) O60934</td></tr><tr><td>Q11201</td><td>Nitric oxide synthase, endothelial (NOS3) P29474</td></tr><tr><td>Cocaine esterase (CES2) O00748</td><td>Nucleobindin-2 (NUCB2) P80303</td></tr><tr><td>Contactin-2 (CNTN2) Q02246</td><td>Parvalbumin alpha (PVALB) P20472</td></tr><tr><td>Corticoliberin (CRH) P06850</td><td>Paxillin (PXA) P49023</td></tr><tr><td>C-type lectin domain family 1 member A (CLEC1A) Q8NC01</td><td>Peptidyl-prolyl cis-trans isomerase FKBP1B (FKBP1B) P68106</td></tr><tr><td>C-type natriuretic peptide (NPPC) P23582</td><td>Perilipin-1 (PLIN1) O60240</td></tr><tr><td>Desmoglein-4 (DSG4) Q86SJ6</td><td>Placenta growth factor (PGF) P49763</td></tr><tr><td>Dipeptidyl aminopeptidase-like protein 6 (DPP6) P42658</td><td>Platelet-derived growth factor C (PDGFC) Q9NRA1</td></tr><tr><td>DNA topoisomerase 2-beta (TOP2B) Q02880</td><td>Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2 (INPPL1) O15357</td></tr><tr><td>Ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) Q9Y5L3</td><td>Plexin domain-containing protein 1 (PLXDC1) Q8IUK5</td></tr><tr><td>Ectonucleoside triphosphate diphosphohydrolase 6 (ENTPD6) O75354</td><td>Polypeptide N-acetylgalactosaminyltransferase 10 (GALNT10) Q86SR1</td></tr></tbody></table>
<table>
  <tr>
    <td>EGF-like repeat and discoidin I-like domain-containing protein 3 (EDIL3) O43854</td>
    <td>Probetacellulin (BTC) P35070</td>
  </tr>
  <tr>
    <td>Enteropeptidase (TMPRSS15) P98073</td>
    <td>Programmed cell death protein 1 (PDCD1) Q15116</td>
  </tr>
  <tr>
    <td>Epidermal growth factor-like protein 7 (EGFL7) Q9UHF1</td>
    <td>Prolow-density lipoprotein receptor-related protein 1 (LRP1) Q07954</td>
  </tr>
  <tr>
    <td>Erbin (ERBIN) Q96RT1</td>
    <td>Proteasome subunit alpha type-1 (PSMA1) P25786</td>
  </tr>
  <tr>
    <td>Erythropoietin (EPO) P01588</td>
    <td>Protein amnionless (AMN) Q9BXJ7</td>
  </tr>
  <tr>
    <td>Fatty acid-binding protein 9 (FABP9) Q0Z7S8</td>
    <td>Protein fosB (FOSB) P53539</td>
  </tr>
  <tr>
    <td>Forkhead box protein O1 (FOXO1) Q12778</td>
    <td>Protein max (MAX) P61244</td>
  </tr>
  <tr>
    <td>Fructose-2,6-bisphosphatase TIGAR (TIGAR) Q9NQ88</td>
    <td>Protein phosphatase 1B (PPM1B) O75688</td>
  </tr>
  <tr>
    <td>Hematopoietic prostaglandin D synthase (HPGDS) O60760</td>
    <td>[Pyruvate dehydrogenase [acetyl-transferring]]-phosphatase 1,</td>
  </tr>
  <tr>
    <td>Inactive tyrosine-protein kinase 7 (PTK7) Q13308</td>
    <td>mitochondrial (PDP1) Q9P0J1</td>
  </tr>
  <tr>
    <td>Integrin beta-1-binding protein 1 (ITGB1BP1) O14713</td>
    <td>Ras association domain-containing protein 2 (RASSF2) P50749</td>
  </tr>
  <tr>
    <td>Interferon-inducible double-stranded RNA-dependent protein</td>
    <td>Ras GTPase-activating protein 1 (RASA1) P20936</td>
  </tr>
  <tr>
    <td>kinase activator A (PRKRA) O75569</td>
    <td>Receptor-type tyrosine-protein phosphatase eta (PTPRJ) Q12913</td>
  </tr>
  <tr>
    <td>Renin receptor (ATP6AP2) O75787</td>
    <td>EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) Q12805</td>
  </tr>
  <tr>
    <td>REST corepressor 1 (RCOR1) Q9UKL0</td>
    <td>Endoglin (ENG) P17813</td>
  </tr>
  <tr>
    <td>Retinoic acid receptor responder protein 1 (RARRES1) P49788</td>
    <td>Fetuin-B (FETUB) Q9UGM5</td>
  </tr>
  <tr>
    <td>Ribonucleoside-diphosphate reductase subunit M2 B (RRM2B) Q7LG56</td>
    <td>Ficolin-2 (FCN2) Q15485</td>
  </tr>
  <tr>
    <td>Serpin A9 (SERPINA9) Q86WD7</td>
    <td>Glutaminyl-peptide cyclotransferase (QPCT) Q16769</td>
  </tr>
  <tr>
    <td>Serum paraoxonase/arylesterase 2 (PON2) Q15165</td>
    <td>Granulysin (GNLY) P22749</td>
  </tr>
  <tr>
    <td>Syntaxin-8 (STX8) Q9UNK0</td>
    <td>Growth arrest-specific protein 6 (GAS6) Q14393</td>
  </tr>
  <tr>
    <td>Syntaxin-binding protein 3 (STXBP3) O00186</td>
    <td>Hepatocyte growth factor receptor (MET) P08581</td>
  </tr>
  <tr>
    <td>Troponin I, cardiac muscle (TNNI3) P19429</td>
    <td>Ig lambda-2 chain C regions (IGLC2) P0CG05</td>
  </tr>
  <tr>
    <td>Tyrosine-protein kinase Fes/Fps (FES) P07332</td>
    <td>Insulin-like growth factor-binding protein 3 (IGFBP3) P17936</td>
  </tr>
  <tr>
    <td>Tyrosine-protein kinase Fgr (FGR) P09769</td>
    <td>Insulin-like growth factor-binding protein 6 (IGFBP6) P24592</td>
  </tr>
  <tr>
    <td>Tyrosine-protein kinase Yes (YES1) P07947</td>
    <td>Integrin alpha-M (ITGAM) P11215</td>
  </tr>
  <tr>
    <td>Vascular endothelial growth factor C (VEGFC) P49767</td>
    <td>Intercellular adhesion molecule 1 (ICAM1) P05362</td>
  </tr>
  <tr>
    <td>Vasohibin-1 (VASH1) Q7L8A9</td>
    <td>Intercellular adhesion molecule 3 (ICAM3) P32942</td>
  </tr>
  <tr>
    <td>Wiskott-Aldrich syndrome protein (WAS) P42768</td>
    <td>Interleukin-7 receptor subunit alpha (IL7R) P16871</td>
  </tr>
  <tr>
    <td></td>
    <td>Latent-transforming growth factor beta-binding protein 2 (LTBP2) Q14767</td>
  </tr>
  <tr>
    <td><strong>Cardiometabolic panel</strong></td>
    <td>Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1) Q8NHL6</td>
  </tr>
</table>
<table>
   <tr>
    <td>Angiogenin (ANG) P03950</td>
    <td>Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2) Q8N423</td>
   </tr>
   <tr>
    <td>Angiopoietin-related protein 3 (ANGPTL3) Q9Y5C1</td>
    <td>Leukocyte immunoglobulin-like receptor subfamily B member 5 (LILRB5) O75023</td>
   </tr>
   <tr>
    <td>Apolipoprotein M (APOM) O95445</td>
    <td>Lithostathine-1-alpha (REG1A) P05451</td>
   </tr>
   <tr>
    <td>Beta-Ala-His dipeptidase (CNDP1) Q96KN2</td>
    <td>Liver carboxylesterase 1 (CES1) P23141</td>
   </tr>
   <tr>
    <td>Beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) P15907</td>
    <td>Low affinity immunoglobulin gamma Fc region receptor II-a (FCGR2A) P12318</td>
   </tr>
   <tr>
    <td>Cadherin-1 (CDH1) P12830</td>
    <td>Low affinity immunoglobulin gamma Fc region receptor III-B (FCGR3B) O75015</td>
   </tr>
   <tr>
    <td>Carbonic anhydrase 1 (CA1) P00915</td>
    <td>L-selectin (SELL) P14151</td>
   </tr>
   <tr>
    <td>Carbonic anhydrase 3 (CA3) P07451</td>
    <td>Lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1) Q9Y5Y7</td>
   </tr>
   <tr>
    <td>Carbonic anhydrase 4 (CA4) P22748</td>
    <td>Lysosomal Pro-X carboxypeptidase (PRCP) P42785</td>
   </tr>
   <tr>
    <td>Cartilage acidic protein 1 (CRTAC1) Q9NQ79</td>
    <td>Mannose-binding protein C (MBL2) P11226</td>
   </tr>
   <tr>
    <td>Cartilage oligomeric matrix protein (COMP) P49747</td>
    <td>Mast/stem cell growth factor receptor Kit (KIT) P10721</td>
   </tr>
   <tr>
    <td>C-C motif chemokine 5 (CCL5) P13501</td>
    <td>Membrane cofactor protein (CD46) P15529</td>
   </tr>
   <tr>
    <td>C-C motif chemokine 14 (CCL14) Q16627</td>
    <td>Membrane primary amine oxidase (AOC3) Q16853</td>
   </tr>
   <tr>
    <td>C-C motif chemokine 18 (CCL18) P55774</td>
    <td>Metalloproteinase inhibitor 1 (TIMP1) P01033</td>
   </tr>
   <tr>
    <td>CD59 glycoprotein (CD59) P13987</td>
    <td>Microfibrillar-associated protein 5 (MFAP5) Q13361</td>
   </tr>
   <tr>
    <td>Coagulation factor VII (F7) P08709</td>
    <td>Multiple epidermal growth factor-like domains protein 9 (MEGF9) Q9H1U4</td>
   </tr>
   <tr>
    <td>Coagulation factor XI (F11) P03951</td>
    <td>Neural cell adhesion molecule 1 (NCAM1) P13591</td>
   </tr>
   <tr>
    <td>Collagen alpha-1(XVIII) chain (COL18A1) P39060</td>
    <td>Neural cell adhesion molecule L1-like protein (CHL1) O00533</td>
   </tr>
   <tr>
    <td>Complement C1q tumor necrosis factor-related protein 1 (C1QTNF1) Q9BXJ1</td>
    <td>Neurogenic locus notch homolog protein 1 (NOTCH1) P46531</td>
   </tr>
   <tr>
    <td>Complement C2 (C2) P06681</td>
    <td>Neuropilin-1 (NRP1) O14786</td>
   </tr>
   <tr>
    <td>Complement factor H-related protein 5 (CFHR5) Q9BXR6</td>
    <td>Neutrophil defensin 1 (DEFA1) P59665</td>
   </tr>
   <tr>
    <td>Complement receptor type 2 (CR2) P20023</td>
    <td>Neutrophil gelatinase-associated lipocalin (LCN2) P80188</td>
   </tr>
   <tr>
    <td>Cystatin-C (CST3) P01034</td>
    <td>Nidogen-1 (NID1) P14543</td>
   </tr>
   <tr>
    <td>Dipeptidyl peptidase 4 (DPP4) P27487</td>
    <td>Oncostatin-M-specific receptor subunit beta (OSMR) Q99650</td>
   </tr>
   <tr>
    <td>Peptidyl-glycine alpha-amidating monooxygenase (PAM) P19021</td>
    <td>C-C motif chemokine 17 (CCL17) Q92583</td>
   </tr>
   <tr>
    <td>Phospholipid transfer protein (PLTP) P55058</td>
    <td>CD40 ligand (CD40-L) P29965</td>
   </tr>
   <tr>
    <td>Plasma serine protease inhibitor (SERPINA5) P05154</td>
    <td>Chymotrypsin C (CTRC) Q99895</td>
   </tr>
   <tr>
    <td>Platelet glycoprotein Ib alpha chain (GP1BA) P07359</td>
    <td>C-X-C motif chemokine 1 (CXCL1) P09341</td>
   </tr>
   <tr>
    <td>Platelet-activating factor acetylhydrolase (PLA2G7) Q13093</td>
    <td>Decorin (DCN) P07585</td>
   </tr>
  </table>
<table>
  <tr>
    <td>Plexin-B2 (PLXNB2) O15031</td>
    <td>Dickkopf-related protein 1 (Dkk-1) O94907</td>
  </tr>
  <tr>
    <td>Procollagen C-endopeptidase enhancer 1 (PCOLCE) Q15113</td>
    <td>Fatty acid-binding protein, intestinal (FABP2) P12104</td>
  </tr>
  <tr>
    <td>Prolyl endopeptidase FAP (FAP) Q12884</td>
    <td>Fibroblast growth factor 21 (FGF-21) Q9NSA1</td>
  </tr>
  <tr>
    <td>Receptor-type tyrosine-protein phosphatase S (PTPRS) Q13332</td>
    <td>Fibroblast growth factor 23 (FGF-23) Q9GZV9</td>
  </tr>
  <tr>
    <td>Regenerating islet-derived protein 3-alpha (REG3A) Q06141</td>
    <td>Follistatin (FS) P19883</td>
  </tr>
  <tr>
    <td>Serum amyloid A-4 protein (SAA4) P35542</td>
    <td>Galectin-9 (Gal-9) O00182</td>
  </tr>
  <tr>
    <td>SPARC-like protein 1 (SPARCL1) Q14515</td>
    <td>Gastric intrinsic factor (GIF) P27352</td>
  </tr>
  <tr>
    <td>Superoxide dismutase [Cu-Zn] (SOD1) P00441</td>
    <td>Gastrotropin (GT) P51161</td>
  </tr>
  <tr>
    <td>T-cell immunoglobulin and mucin domain-containing protein 4 (TIMD4) Q96H15</td>
    <td>Growth hormone (GH) P01241</td>
  </tr>
  <tr>
    <td>Tenascin (TNC) P24821</td>
    <td>Growth/differentiation factor 2 (GDF-2) Q9UK05</td>
  </tr>
  <tr>
    <td>Tenascin-X (TNXB) P22105</td>
    <td>Heat shock 27 kDa protein (HSP 27) P04792</td>
  </tr>
  <tr>
    <td>Thrombospondin-4 (THBS4) P35443</td>
    <td>Heme oxygenase 1 (HO-1) P09601</td>
  </tr>
  <tr>
    <td>Thyroxine-binding globulin (SERPINA7) P05543</td>
    <td>Hydroxyacid oxidase 1 (HAOX1) Q9UJM8</td>
  </tr>
  <tr>
    <td>Transcobalamin-2 (TCN2) P20062</td>
    <td>Interleukin-1 receptor antagonist protein (IL-1ra) P18510</td>
  </tr>
  <tr>
    <td>Transforming growth factor beta receptor type 3 (TGFBR3) Q03167</td>
    <td>Interleukin-1 receptor-like 2 (IL1RL2) Q9HB29</td>
  </tr>
  <tr>
    <td>Transforming growth factor-beta-induced protein ig-h3 (TGFBI) Q15582</td>
    <td>Interleukin-4 receptor subunit alpha (IL-4RA) P24394</td>
  </tr>
  <tr>
    <td>Trypsin-2 (PRSS2) P07478</td>
    <td>Interleukin-6 (IL6) P05231</td>
  </tr>
  <tr>
    <td>Tyrosine-protein kinase receptor Tie-1 (TIE1) P35590</td>
    <td>Interleukin-17D (IL-17D) Q8TAD2</td>
  </tr>
  <tr>
    <td>Uromodulin (UMOD) P07911</td>
    <td>Interleukin-18 (IL-18) Q14116</td>
  </tr>
  <tr>
    <td>Vascular cell adhesion protein 1 (VCAM1) P19320</td>
    <td>Interleukin-27 (IL-27) Q8NEV9,</td>
  </tr>
  <tr>
    <td>Vasorin (VASN) Q6EMK4</td>
    <td>Q14213</td>
  </tr>
  <tr>
    <td>Vitamin K-dependent protein C (PROC) P04070</td>
    <td>Kidney Injury Molecule (KIM1) Q96D42</td>
  </tr>
  <tr>
    <td></td>
    <td>Lactoylglutathione lyase (GLO1) Q04760</td>
  </tr>
  <tr>
    <td><strong>Cardiovascular II panel</strong></td>
    <td>Lectin-like oxidized LDL receptor 1 (LOX-1) P78380</td>
  </tr>
  <tr>
    <td>2,4-dienoyl-CoA reductase, mitochondrial (DECR1) Q16698</td>
    <td>Leptin (LEP) P41159</td>
  </tr>
  <tr>
    <td>A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAM-TS13) Q76LX8</td>
    <td>Lipoprotein lipase (LPL) P06858</td>
  </tr>
  <tr>
    <td>ADM (ADM) P35318</td>
    <td>Low affinity immunoglobulin gamma Fc region receptor II-b (IgG Fc receptor II-b) P31994</td>
  </tr>
  <tr>
    <td>Agouti-related protein (AGRP) O00253</td>
    <td>Lymphotactin (XCL1) P47992</td>
  </tr>
  <tr>
    <td>Alpha-L-iduronidase (IDUA) P35475</td>
    <td>Macrophage receptor MARCO (MARCO) Q9UEW3</td>
  </tr>
  <tr>
    <td>Angiopoietin-1 (ANG-1) Q15389</td>
    <td>Matrix metalloproteinase-7 (MMP-7) P09237</td>
  </tr>
  <tr>
    <td>Angiopoietin-1 receptor (TIE2) Q02763</td>
    <td>Matrix metalloproteinase-12 (MMP-12) P39900</td>
  </tr>
</table>
<table><tr><td>Angiotensin-converting enzyme 2 (ACE2) Q9BYF1</td><td>Melusin (ITGB1BP2) Q9UKP3</td></tr><tr><td>Bone morphogenetic protein 6 (BMP-6) P22004</td><td>Natriuretic peptides B (BNP) P16860</td></tr><tr><td>Brother of CDO (Protein BOC) Q9BWV1</td><td>NF-kappa-B essential modulator (NEMO) Q9Y6K9</td></tr><tr><td>Carbonic anhydrase 5A, mitochondrial (CA5A) P35218</td><td>Osteoclast-associated immunoglobulin-like receptor (hOSCAR) Q8IYS5</td></tr><tr><td>Carcinoembryonic antigen-related cell adhesion molecule 8 (CEACAM8) P31997</td><td>Pappalysin-1 (PAPPA) Q13219</td></tr><tr><td>Cathepsin L1 (CTSL1) P07711</td><td>Pentraxin-related protein PTX3 (PTX3) P26022</td></tr><tr><td>C-C motif chemokine 3 (CCL3) P10147</td><td>Placenta growth factor (PGF) P49763</td></tr><tr><td>Platelet-derived growth factor subunit B (PDGF subunit B) P01127</td><td>Caspase-3 (CASP-3) P42574</td></tr><tr><td>Poly [ADP-ribose] polymerase 1 (PARP-1) P09874</td><td>Cathepsin D (CTSD) P07339</td></tr><tr><td>Polymeric immunoglobulin receptor (PIgR) P01833</td><td>Cathepsin Z (CTSZ) Q9UBR2</td></tr><tr><td>Programmed cell death 1 ligand 2 (PD-L2) Q9BQ51</td><td>C-C motif chemokine 15 (CCL15) Q16663</td></tr><tr><td>Proheparin-binding EGF-like growth factor (HB-EGF) Q99075</td><td>C-C motif chemokine 16 (CCL16) O15467</td></tr><tr><td>Pro-interleukin-16 (IL16) Q14005</td><td>Note: New assay under development N/A</td></tr><tr><td>Prolargin (PRELP) P51888</td><td>C-C motif chemokine 24 (CCL24) O00175</td></tr><tr><td>Prostasin (PRSS8) Q16651</td><td>CD166 antigen (ALCAM) Q13740</td></tr><tr><td>Protein AMBP (AMBP) P02760</td><td>Chitinase-3-like protein 1 (CHI3L1) P36222</td></tr><tr><td>Proteinase-activated receptor 1 (PAR-1) P25116</td><td>Chitotriosidase-1 (CHIT1) Q13231</td></tr><tr><td>Protein-glutamine gamma-glutamyltransferase 2 (TGM2) P21980</td><td>Collagen alpha-1(I) chain (COL1A1) P02452</td></tr><tr><td>Proto-oncogene tyrosine-protein kinase Src (SRC) P12931</td><td>Complement component C1q receptor (CD93) Q9NPY3</td></tr><tr><td>P-selectin glycoprotein ligand 1 (PSGL-1) Q14242</td><td>Contactin-1 (CNTN1) Q12860</td></tr><tr><td>Receptor for advanced glycosylation end products (RAGE) Q15109</td><td>C-X-C motif chemokine 16 (CXCL16) Q9H2A7</td></tr><tr><td>Renin (REN) P00797</td><td>Cystatin-B (CSTB) P04080</td></tr><tr><td>Serine protease 27 (PRSS27) Q9BQR3</td><td>Elafin (PI3) P19957</td></tr><tr><td>Serine/threonine-protein kinase 4 (STK4) Q13043</td><td>Ephrin type-B receptor 4 (EPHB4) P54760</td></tr><tr><td>Serpin A12 (SERPINA12) Q8IW75</td><td>Epidermal growth factor receptor (EGFR) P00533</td></tr><tr><td>SLAM family member 5 (CD84) Q9UIB8</td><td>Epithelial cell adhesion molecule (Ep-CAM) P16422</td></tr><tr><td>SLAM family member 7 (SLAMF7) Q9NQ25</td><td>E-selectin (SELE) P16581</td></tr><tr><td>Sortilin (SORT1) Q99523</td><td>Fatty acid-binding protein, adipocyte (FABP4) P15090</td></tr><tr><td>Spondin-2 (SPON2) Q9BUD6</td><td>Galectin-3 (Gal-3) P17931</td></tr><tr><td>Stem cell factor (SCF) P21583</td><td>Galectin-4 (Gal-4) P56470</td></tr><tr><td>Superoxide dismutase [Mn], mitochondrial (SOD2) P04179</td><td>Granulins (GRN) P28799</td></tr></table>
<table><tr><td>T-cell surface glycoprotein CD4 (CD4) P01730</td><td>Growth/differentiation factor 15 (GDF-15) Q99988</td></tr><tr><td>Thrombomodulin TM P07204</td><td>Insulin-like growth factor-binding protein 1 (IGFBP-1) P08833</td></tr><tr><td>Thrombopoietin (THPO) P40225</td><td>Insulin-like growth factor-binding protein 2 (IGFBP-2) P18065</td></tr><tr><td>Thrombospondin-2 (THBS2) P35442</td><td>Insulin-like growth factor-binding protein 7 (IGFBP-7) Q16270</td></tr><tr><td>Tissue factor (TF) P13726</td><td>Integrin beta-2 (ITGB2) P05107</td></tr><tr><td>TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) O14763</td><td>Intercellular adhesion molecule 2 (ICAM-2) P13598</td></tr><tr><td>Tumor necrosis factor receptor superfamily member 10A (TNFRSF10A) O00220</td><td>Interleukin-1 receptor type 1 (IL-1RT1) P14778</td></tr><tr><td>Tumor necrosis factor receptor superfamily member 11A (TNFRSF11A) Q9Y6Q6</td><td>Interleukin-1 receptor type 2 (IL-1RT2) P27930</td></tr><tr><td>Tumor necrosis factor receptor superfamily member 13B (TNFRSF13B) O14836</td><td>Interleukin-2 receptor subunit alpha (IL2-RA) P01589</td></tr><tr><td>Tyrosine-protein kinase Mer (MERTK) Q12866</td><td>Interleukin-6 receptor subunit alpha (IL-6RA) P08887</td></tr><tr><td>Vascular endothelial growth factor D (VEGFD) O43915</td><td>Interleukin-17 receptor A (IL-17RA) Q96F46</td></tr><tr><td>V-set and immunoglobulin domain-containing protein 2 (VSIG2) Q96IQ7</td><td>Interleukin-18-binding protein (IL-18BP) O95998</td></tr><tr><td><strong>Cardiovascular III panel</strong></td><td>Junctional adhesion molecule A (JAM-A) Q9Y624</td></tr><tr><td>Aminopeptidase N (AP-N) P15144</td><td>Kallikrein-6 (KLK6) Q92876</td></tr><tr><td>Azurocidin (AZU1 P20160</td><td>Low-density lipoprotein receptor (LDL receptor) P01130</td></tr><tr><td>Bleomycin hydrolase (BLM hydrolase) Q13867</td><td>Lymphotoxin-beta receptor (LTBR) P36941</td></tr><tr><td>Cadherin-5 (CDH5) P33151</td><td>Matrix extracellular phosphoglycoprotein (MEPE) Q9NQ76</td></tr><tr><td>Carboxypeptidase A1 (CPA1) P15085</td><td>Matrix metalloproteinase-2 (MMP-2) P08253</td></tr><tr><td>Carboxypeptidase B (CPB1) P15086</td><td>Matrix metalloproteinase-3 (MMP-3) P08254</td></tr><tr><td>Matrix metalloproteinase-9 (MMP-9) P14780</td><td>von Willebrand factor (vWF) P04275</td></tr><tr><td>Metalloproteinase inhibitor 4 (TIMP4) Q99727</td><td></td></tr><tr><td>Monocyte chemotactic protein 1 (MCP-1) P13500</td><td><strong>Inflammation panel</strong></td></tr><tr><td>Myeloblastin (PRTN3) P24158</td><td>Adenosine Deaminase (ADA) P00813</td></tr><tr><td>Myeloperoxidase (MPO) P05164</td><td>Artemin (ARTN) Q5T4W7</td></tr><tr><td>Myoglobin (MB) P02144</td><td>Axin-1 (AXIN1) O15169</td></tr><tr><td>Neurogenic locus notch homolog protein 3 (Notch 3) Q9UM47</td><td>Beta-nerve growth factor (Beta-NGF) P01138</td></tr><tr><td>N-terminal prohormone brain natriuretic peptide (NT-proBNP) NA</td><td>Caspase-8 (CASP-8) Q14790</td></tr><tr><td>Osteopontin (OPN) P10451</td><td>C-C motif chemokine 3 (CCL3) P10147</td></tr><tr><td>Osteoprotegerin (OPG) O00300</td><td>C-C motif chemokine 4 (CCL4) P13236</td></tr><tr><td>Paraoxonase (PON3) Q15166</td><td>C-C motif chemokine 19 (CCL19) Q99731</td></tr></table>
<table><tr><td>Peptidoglycan recognition protein 1 (PGLYRP1) O75594</td><td>C-C motif chemokine 20 (CCL20) P78556</td></tr><tr><td>Perlecan (PLC) P98160</td><td>C-C motif chemokine 23 (CCL23) P55773</td></tr><tr><td>Plasminogen activator inhibitor 1 (PAI) P05121</td><td>C-C motif chemokine 25 (CCL25) O15444</td></tr><tr><td>Platelet endothelial cell adhesion molecule (PECAM-1) P16284</td><td>C-C motif chemokine 28 (CCL28) Q9NRJ3</td></tr><tr><td>Platelet-derived growth factor subunit A (PDGF subunit A) P04085</td><td>CD40L receptor (CD40) P25942</td></tr><tr><td>Proprotein convertase subtilisin/kexin type 9 (PCSK9) Q8NBP7</td><td>CUB domain-containing protein 1 (CDCP1) Q9H5V8</td></tr><tr><td>Protein delta homolog 1 (DLK-1) P80370</td><td>C-X-C motif chemokine 1 (CXCL1) P09341</td></tr><tr><td>P-selectin (SELP) P16109</td><td>C-X-C motif chemokine 5 (CXCL5) P42830</td></tr><tr><td>Pulmonary surfactant-associated protein D (PSP-D) P35247</td><td>C-X-C motif chemokine 6 (CXCL6) P80162</td></tr><tr><td>Resistin (RETN) Q9HD89</td><td>C-X-C motif chemokine 9 (CXCL9) Q07325</td></tr><tr><td>Retinoic acid receptor responder protein 2 (RARRES2) Q99969</td><td>C-X-C motif chemokine 10 (CXCL10) P02778</td></tr><tr><td>Scavenger receptor cysteine-rich type 1 protein M130 (CD163) Q86VB7</td><td>C-X-C motif chemokine 11 (CXCL11) O14625</td></tr><tr><td>Secretoglobin family 3A member 2 (SCGB3A2) Q96PL1</td><td>Cystatin D (CST5) P28325</td></tr><tr><td>Spondin-1 (SPON1) Q9HCB6</td><td>Delta and Notch-like epidermal growth factor-related receptor (DNER) Q8NFT8</td></tr><tr><td>ST2 protein (ST2) Q01638</td><td>Eotaxin (CCL11) P51671</td></tr><tr><td>Tartrate-resistant acid phosphatase type 5 (TR-AP) P13686</td><td>Eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) Q13541</td></tr><tr><td>Tissue factor pathway inhibitor (TFPI) P10646</td><td>Fibroblast growth factor 21 (FGF-21) Q9NSA1</td></tr><tr><td>Tissue-type plasminogen activator (t-PA) P00750</td><td>Fibroblast growth factor 23 (FGF-23) Q9GZV9</td></tr><tr><td>Transferrin receptor protein 1 (TR) P02786</td><td>Fibroblast growth factor 5 (FGF-5) Q8NF90</td></tr><tr><td>Trefoil factor 3 (TFF3) Q07654</td><td>Fibroblast growth factor 19 (FGF-19) O95750</td></tr><tr><td>Trem-like transcript 2 protein (TLT-2) Q5T2D2</td><td>Fms-related tyrosine kinase 3 ligand (Flt3L) P49771</td></tr><tr><td>Tumor necrosis factor ligand superfamily member 13B (TNFSF13B) Q9Y275</td><td>Fractalkine (CX3CL1) P78423</td></tr><tr><td>Tumor necrosis factor receptor 1 (TNF-R1) P19438</td><td>Glial cell line-derived neurotrophic factor (GDNF) P39905</td></tr><tr><td>Tumor necrosis factor receptor 2 (TNF-R2) P20333</td><td>Hepatocyte growth factor (HGF) P14210</td></tr><tr><td>Tumor necrosis factor receptor superfamily member 6 (FAS) P25445</td><td>Interferon gamma (IFN-gamma) P01579</td></tr><tr><td>Tumor necrosis factor receptor superfamily member 10C (TNFRSF10C) O14798</td><td>Interleukin-1 alpha (IL-1 alpha) P01583</td></tr><tr><td>Tumor necrosis factor receptor superfamily member 14 (TNFRSF14) Q92956</td><td>Interleukin-2 (IL-2) P60568</td></tr><tr><td>Tyrosine-protein kinase receptor UFO (AXL) P30530</td><td>Interleukin-2 receptor subunit beta (IL-2RB) P14784</td></tr><tr><td>Tyrosine-protein phosphatase non-receptor type substrate 1 (SHPS-1) P78324</td><td>Interleukin-4 (IL-4) P05112</td></tr></table>
<table><tr><td>Urokinase plasminogen activator surface receptor (U-PAR) Q03405</td><td>Interleukin-5 (IL5) P05113</td></tr><tr><td>Urokinase-type plasminogen activator (uPA) P00749</td><td>Interleukin-6 (IL6) P05231</td></tr><tr><td>Interleukin-7 (IL-7) P13232</td><td>Thymic stromal lymphopoietin (TSLP) Q969D9</td></tr><tr><td>Interleukin-8 (IL-8) P10145</td><td>TNF-related activation-induced cytokine (TRANCE) O14788</td></tr><tr><td>Interleukin-10 (IL10) P22301</td><td>TNF-related apoptosis-inducing ligand (TRAIL) P50591</td></tr><tr><td>Interleukin-10 receptor subunit alpha (IL-10RA) Q13651</td><td>Transforming growth factor alpha (TGF-alpha) P01135</td></tr><tr><td>Interleukin-10 receptor subunit beta (IL-10RB) Q08334</td><td>Tumor necrosis factor (Ligand) superfamily, member 12(TWEAK) O43508</td></tr><tr><td>Interleukin-12 subunit beta (IL-12B) P29460</td><td>Tumor necrosis factor (TNF) P01375</td></tr><tr><td>Interleukin-13 (IL-13) P35225</td><td>Tumor necrosis factor ligand superfamily member 14 (TNFSF14) O43557</td></tr><tr><td>Interleukin-15 receptor subunit alpha (IL-15RA) Q13261</td><td>Tumor necrosis factor receptor superfamily member 9(TNFRSF9) Q07011</td></tr><tr><td>Interleukin-17A (IL-17A) Q16552</td><td>Urokinase-type plasminogen activator (uPA) P00749</td></tr><tr><td>Interleukin-17C (IL-17C) Q9P0M4</td><td>Vascular endothelial growth factor A (VEGF-A) P15692</td></tr><tr><td>Interleukin-18 (IL-18) Q14116</td><td></td></tr><tr><td>Interleukin-18 receptor 1 (IL-18R1) Q13478</td><td></td></tr><tr><td>Interleukin-20 (IL-20) Q9NYY1</td><td></td></tr><tr><td>Interleukin-20 receptor subunit alpha (IL-20RA) Q9UHF4</td><td></td></tr><tr><td>Interleukin-22 receptor subunit alpha-1 (IL-22 RA1) Q8N6P7</td><td></td></tr><tr><td>Interleukin-24 (IL-24) Q13007</td><td></td></tr><tr><td>Interleukin-33 (IL-33) O95760</td><td></td></tr><tr><td>Latency-associated peptide transforming growth factor beta-1 (LAP TGF-beta-1) P01137</td><td></td></tr><tr><td>Leukemia inhibitory factor (LIF) P15018</td><td></td></tr><tr><td>Leukemia inhibitory factor receptor (LIF-R) P42702</td><td></td></tr><tr><td>Macrophage colony-stimulating factor 1 (CSF-1) P09603</td><td></td></tr><tr><td>Matrix metalloproteinase-1 (MMP-1) P03956</td><td></td></tr><tr><td>Matrix metalloproteinase-10 (MMP-10) P09238</td><td></td></tr><tr><td>Monocyte chemotactic protein 1 (MCP-1) P13500</td><td></td></tr><tr><td>Monocyte chemotactic protein 2 (MCP-2) P80075</td><td></td></tr><tr><td>Monocyte chemotactic protein 3 (MCP-3) P80098</td><td></td></tr><tr><td>Monocyte chemotactic protein 4 (MCP-4) Q99616</td><td></td></tr><tr><td>Natural killer cell receptor 2B4 (CD244) Q9BZW8</td><td></td></tr></table>
<table><tr><td>Neurotrophin-3 (NT-3) P20783</td><td></td></tr><tr><td>Neurturin (NRTN) Q99748</td><td></td></tr><tr><td>Oncostatin-M (OSM) P13725</td><td></td></tr><tr><td>Osteoprotegerin (OPG) O00300</td><td></td></tr><tr><td>Programmed cell death 1 ligand 1 (PD-L1) Q9NZQ7</td><td></td></tr><tr><td>Protein S100-A12 (EN-RAGE) P80511</td><td></td></tr><tr><td>Signaling lymphocytic activation molecule (SLAMF1) Q13291</td><td></td></tr><tr><td>SIR2-like protein 2 (SIRT2) Q8IXJ6</td><td></td></tr><tr><td>STAM-binding protein (STAMBP) O95630</td><td></td></tr><tr><td>Stem cell factor (SCF) P21583</td><td></td></tr><tr><td>Sulfotransferase 1A1 (ST1A1) P50225</td><td></td></tr><tr><td>T cell surface glycoprotein CD6 isoform (CD6) Q8WWJ7</td><td></td></tr><tr><td>T-cell surface glycoprotein CD5 (CD5) P06127</td><td></td></tr><tr><td>T-cell surface glycoprotein CD8 alpha chain (CD8A) P01732</td><td></td></tr><tr><td>TNF-beta (TNFB) P01374</td><td></td></tr></table>
<table><caption>Supplemental table 2 Circulating protein concentrations (in NPX) according to sex</caption><thead><tr><th></th><th>Global<br>(N=392)</th><th>Male<br>(N=142)</th><th>Female<br>(N=250)</th><th>p-value</th></tr></thead><tbody><tr><td>IGFBP3</td><td>4.4 ± 0.6</td><td>4.2 ± 0.6</td><td>4.6 ± 0.5</td><td>&lt;0.001</td></tr><tr><td>IL1RL2</td><td>3.2 ± 0.4</td><td>3.1 ± 0.4</td><td>3.3 ± 0.4</td><td>&lt;0.001</td></tr><tr><td>LPL</td><td>9.8 ± 0.6</td><td>9.5 ± 0.6</td><td>9.9 ± 0.5</td><td>&lt;0.001</td></tr><tr><td>Ep-CAM</td><td>4.8 ± 1.0</td><td>4.5 ± 0.8</td><td>5.0 ± 1.0</td><td>&lt;0.001</td></tr><tr><td>PLIN1</td><td>2.2 ± 0.8</td><td>1.9 ± 0.6</td><td>2.3 ± 0.8</td><td>&lt;0.001</td></tr><tr><td>LHB</td><td>1.6 ± 1.1</td><td>1.1 ± 1.1</td><td>2.0 ± 1.0</td><td>&lt;0.001</td></tr><tr><td>NRP1</td><td>2.2 ± 0.2</td><td>2.2 ± 0.2</td><td>2.1 ± 0.2</td><td>&lt;0.001</td></tr><tr><td>MMP_3</td><td>5.9 ± 0.8</td><td>6.3 ± 0.7</td><td>5.6 ± 0.7</td><td>&lt;0.001</td></tr><tr><td>ACE2</td><td>3.8 ± 0.7</td><td>4.1 ± 0.8</td><td>3.7 ± 0.6</td><td>&lt;0.001</td></tr></tbody></table>

Legend: IGFBP3, insulin-like growth factor-binding protein 3; NRP1, neuropilin 1; IL1RL2, interleukin-1 receptor-like 2; LPL, lipoprotein lipase; ACE2, angiotensin-converting-enzyme 2; EpCAM, epithelial cell adhesion molecule; CA14, carbonic anhydrase 14; PLIN1, perilipin-1; LHB, lutropin subunit beta.
<table><caption>**Supplemental Table 3**

**Circulating protein concentrations (in NPX) according to sex (after exclusion of proteins with correlation > 0.5)**</caption><thead><tr><th></th><th>Global<br>(N=392)</th><th>Male<br>(N=142)</th><th>Female<br>(N=250)</th><th>p-value</th></tr></thead><tbody><tr><td>IGFBP3</td><td>4.4 ± 0.6</td><td>4.2 ± 0.6</td><td>4.6 ± 0.5</td><td>&lt;0.0001</td></tr><tr><td>NRP1</td><td>2.2 ± 0.2</td><td>2.2 ± 0.2</td><td>2.1 ± 0.2</td><td>&lt;0.0001</td></tr><tr><td>IL1RL2</td><td>3.2 ± 0.4</td><td>3.1 ± 0.4</td><td>3.3 ± 0.4</td><td>&lt;0.0001</td></tr><tr><td>LPL</td><td>9.8 ± 0.6</td><td>9.5 ± 0.6</td><td>9.9 ± 0.5</td><td>&lt;0.0001</td></tr><tr><td>ACE2</td><td>3.8 ± 0.7</td><td>4.1 ± 0.8</td><td>3.7 ± 0.6</td><td>&lt;0.0001</td></tr><tr><td>LEP</td><td>5.8 ± 1.1</td><td>5.1 ± 1.1</td><td>6.2 ± 0.9</td><td>&lt;0.0001</td></tr><tr><td>Gal_3</td><td>5.2 ± 0.4</td><td>5.0 ± 0.4</td><td>5.2 ± 0.4</td><td>&lt;0.0001</td></tr><tr><td>EpCAM</td><td>4.8 ± 1.0</td><td>4.5 ± 0.8</td><td>5.0 ± 1.0</td><td>&lt;0.0001</td></tr><tr><td>MB</td><td>7.2 ± 0.7</td><td>7.4 ± 0.7</td><td>7.0 ± 0.7</td><td>&lt;0.0001</td></tr><tr><td>MMP_3</td><td>5.9 ± 0.8</td><td>6.3 ± 0.7</td><td>5.6 ± 0.7</td><td>&lt;0.0001</td></tr><tr><td>CA14</td><td>1.2 ± 0.4</td><td>1.3 ± 0.4</td><td>1.1 ± 0.4</td><td>&lt;0.0001</td></tr><tr><td>PLIN1</td><td>2.2 ± 0.8</td><td>1.9 ± 0.6</td><td>2.3 ± 0.8</td><td>&lt;0.0001</td></tr><tr><td>CALCA</td><td>4.7 ± 1.0</td><td>5.3 ± 1.0</td><td>4.4 ± 0.9</td><td>&lt;0.0001</td></tr><tr><td>LHB</td><td>1.6 ± 1.1</td><td>1.1 ± 1.1</td><td>2.0 ± 1.0</td><td>&lt;0.0001</td></tr></tbody></table>

Legend: IGFBP3, insulin-like growth factor-binding protein 3; NRP1, neuropilin 1; IL1RL2, interleukin-1 receptor-like 2; LPL, lipoprotein lipase; ACE2, angiotensin-converting-enzyme 2; LEP, leptin; Gal_3, galectin 3; EpCAM, epithelial cell adhesion molecule; MB, myoglobin; MMP_3, matrix metalloproteinase 3; CA14, carbonic anhydrase 14; PLIN1, perilipin 1; CALCA, calcitonin; LHB, lutropin subunit beta.