USE OF CD146 AS A MARKER OF THE VASCULAR WALL TENSION

Abstract

The present invention concerns the use of CD146 as a marker of the vascular wall tension, especially for diagnosing, predicting and/or prognosticating diseases associated with variations of the vessel wall tension.

Claims

1. (canceled)

2. A method for treating a disease associated with variations of the vascular wall tension in a subject, wherein the method comprises: measuring the quantity of CD 146 in a sample from the subject; and treating the subject with a prophylactic or therapeutic treatment selected from the group consisting of drug treatment and surgical treatment.

3. The method of claim 2, wherein said disease is a mitral valve disease.

4. The method of claim 2, wherein said disease is organ congestion, wherein the subject does not have clinical sign of heart disease.

5-6. (canceled)

7. The method according to claim 2, wherein the sample is blood or a fractional component thereof.

8. The method according to claim 2, wherein the method further comprises measuring in the sample of the subject the presence or absence and/or quantity of one or more other biomarkers selected from the group consisting of B-type natriuretic peptide (BNP), pro-B-type natriuretic peptide (proBNP), amino terminal pro-B-type natriuretic peptide (NTproBNP), mid-regional pro-atrial natriuretic peptide (MR-proANP) and troponin T.

9. The method according to claim 2, wherein the quantity of CD 146 is measured using a binding agent capable of specifically binding to CD 146 and/or to fragments thereof.

10-14. (canceled)

15. The method of claim 9, wherein the measuring comprises an immunoassay technology, a mass spectrometry analysis method, a chromatography method, or a combination of the foregoing.

16. The method of claim 3, wherein the mitral valve disease-is mitral stenosis or mitral regurgitation.

17. The method of claim 2, further comprising: re-measuring the quantity of CD146 in a sample from the subject after the treatment; and discharging the patient if the quantity of CD146 has decreased.

18. The method of claim 2, further comprising: re-measuring the quantity of CD146 in a sample from the subject after the treatment; and providing further treatment if the quantity of CD146 has increased.

19. The method of claim 2, wherein the treatment comprises administering a drug selected from the group consisting of a diuretic drug, an anticoagulant, a beta blocker, an ACE inhibitor and a calcium channel blocker.

20. The method of claim 2, wherein the treatment comprises valve repair.

21. The method of claim 4, wherein the organ congestion is pulmonary or visceral congestion.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0114] FIG. 1: Levels of soluble CD146 and NT-proBNP before and after venous stress test.

[0115] FIG. 2: Prevalence of severe stenosis, atrial fibrillation and pulmonary hypertension and their combination in MS patients.

[0116] FIG. 3: Plasma biomarker levels according to severity of mitral stenosis, pulmonary hypertension and atrial fibrillation

[0117] A/ plasma levels of BNP [pg/ml], MR-proANP [pmol/l] and sCD146 [ng/ml] in patients with moderate or severe stenosis. B/ plasma levels of the 3 biomarkers in MS patients with or without pulmonary hypertension. C/ biomarker levels in presence or absence of atrial fibrillation.

[0118] FIG. 4: Correlation of biomarker values to systolic pulmonary artery pressure. The diagrams illustrate the correlation between systolic pulmonary pressure [sPAP, in mmHg] and the biomarkers BNP [pg/ml], MR-proANP [pmol/l], sCD146 [ng/ml]. Circles denote MS patients, crosses PAH patients.

[0119] FIG. 5: Plasma biomarker levels according to etiology of pulmonary hypertension. The figure illustrates plasma levels of BNP [pg/ml], MR-proANP [pmol/l] and sCD146 [ng/ml] in patients with pulmonary hypertension secondary to mitral stenosis (post-capillary) and pre-capillary pulmonary arterial hypertension.

[0120] FIG. 6: Plasma levels of BNP and sCD146 according to pulmonary congestion. Increasing radiological evidence of pulmonary congestion is associated with higher levels of sCD146. Plasma levels of BNP show higher dispersion and do not correlate with radiological pulmonary congestion.

[0121] FIG. 7: Plasma levels of BNP and sCD146 according to troponin levels. Increasing levels of troponin are associated with higher BNP but similar or lower levels of sCD146. Median and interquartile range are displayed.

I/ EFFECT OF VASCULAR STRESS ON CD146

[0122] The venous stress mechanistic study aims at evaluating the role of peripheral congestion on plasma levels of sCD146 in chronic HF patients.

Methods

Subjects

[0123] A total of 44 chronic HF outpatients of Columbia University Medical Center (New York, USA) with left-ventricular ejection fraction (LVEF)<40%, NYHA functional class II or III without evidence of congestion on physical exam and on stable medical therapy underwent venous stress test according to a previously described protocol (Colombo P C et al., European Heart Journal. 2014; 35:448-454). Inclusion and exclusion criteria are summarized in Table 1:

TABLE-US-00001 TABLE 1 Inclusion and exclusion criteria for the venous stress study Inclusion criteria Exclusion criteria Age >18 years Systolic blood pressure 90 mmHg LVEF <40% Unattainable peripheral venous No evidence of peripheral venous access in either forearm congestion (no peripheral edema, NYHA Class II angina jugular venous distension <6 cm, Heart transplant negative hepatojugular reflex, no Relevant renal dysfunction (Serum ascites) creatinine >2.5 mg/dl) Patient receiving optimal medical Acute infection therapy, including ACEi or ARB History of venous and beta-blockers, as tolerated, thromboembolism for at least 4 weeks Liver cirrhosis Pregnancy

[0124] Peripheral venous stress without ischemia was created by inflating a pressure cuff around the dominant arm (=test arm), increasing venous arm pressure up to 30 mmHg above baseline. Blood was sampled through an indwelling venous catheter from the antecubital or basilic vein of the test arm after 90 minutes and from the control arm (lacking an inflated cuff) at baseline and after 90 minutes.

Blood Samples Storage and Analysis

[0125] All samples were immediately centrifuged, and the aliquot portions of plasma and serum were stored in microcentrifuge tubes at 80 C. until assayed. Deidentified specimens were sent to independent core laboratories for analysis. Measurements of concentrations of NT-proBNP were performed at Lariboisiere University Hospital, Paris, France using a 1-step sandwich chemiluminescent immunoassay (Cobas, Roche Diagnostics, Basel, Switzerland). Concentrations of sCD146 were determined by ELISA (CY-QUANT ELISA sCD1460, Biocytex, France) at Lariboisiere University Hospital, Paris, France.

Statistical Analysis

[0126] Continuous variables are expressed, after testing for normality using the Shapiro-Wilk test, as meanstandard deviation or median [interquartile range], as appropriate. Nominal variables are expressed as frequency (percentages). Wilcoxon signed rank test was used to examine the differences between test and control arm before and after venous congestion. Differences between two independent groups were assessed with the Wilcoxon rank sum test or Fisher's exact test, as appropriate. The null hypothesis was rejected with an adjusted two-sided p-value <0.05. Analyses were performed with the use of IBM SPSS Statistics, Version 21.0. (IBM Corp, Armonk N.Y., USA) and SAS, Version 9.2. (SAS Institute Inc, Cary N.C., USA).

Ethical Considerations

[0127] The study was performed in observance of national laws and in accordance with the ethical standards of the Declaration of Helsinki, and was approved by local Ethical Committees. All patients provided written informed consent.

Results

Baseline Characteristics

[0128] Patients included in this study were clinically stable, predominantly middle-aged men with chronic HF with severely depressed LVEF. Cardiovascular risk factors were highly prevalent and ischemic heart disease accounted for at least a third of HF etiologies. Most patients were treated with disease-modifying therapies according to current guidelines (McMurray J J V et al., ESC Committee for Practice Guidelines. ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC. European Heart Journal. 2012; 33:1787-1847.)

[0129] Baseline characteristics of patients included in this study are summarized in Table 2:

Impact of the Venous Stress on the Biomarker Levels

[0130] As shown in FIG. 1A, the induction of venous stress in stable HF patients was associated with an increase in circulating levels of sCD146 in the congested arm (481 [371-553] ng/ml) when compared to baseline (454 [339-507| ng/ml, p=0.001) and to the control arm (442 [374-527] ng/ml, p=0.02). Of note, sCD146 levels were also slightly higher in the control arm after 90 minutes compared to baseline (442 [374-527] vs. 454 [339-507| ng/ml, p=0.04).

[0131] By contrast, FIG. 1B illustrates plasma levels of NT-proBNP before and after congestion: no difference in levels of NT-proBNP in the congested arm (308 [218-1140] ng/ml) when compared to baseline (341 [191-1147| ng/ml, p=0.51) and to the control arm (327 [152-1137] ng/ml, p=0.29) was found.

Conclusions

[0132] The present study confirmed high circulating sCD146, above normal values (Bardin N et al., Thromb Haemost. 2003; 90:915-920), in HF patients. An increase in pressure in forearm veins raised plasma levels of sCD146, but not of NT-proBNP, in the congested arm. A more modest but significant increase in plasma levels of sCD146 was also found in the contralateral arm (not exposed to congestion), likely due to a spillover from the congested arm into the bloodstream (Colombo P C et al., European Heart Journal. 2014; 35:448-454).

[0133] The present study demonstrates the increased release of sCD146 in presence of venous stress. To the knowledge of the inventors, this is the first demonstration of a biomarker specifically associated with peripheral venous congestion. Hence, in HF, endothelial sCD146 is a marker of congestion of the peripheral vasculature.

[0134] Cuff inflation during venous stress test not only promoted congestion, but necessarily reduced arterial perfusion pressure by impinging on the brachial artery. However, a reduction in perfusion pressure is a typical clinical feature of advanced HF where arterial blood pressure progressively declines (Barlera S, et al., Circ Heart Fail. 2013; 6:31-39), thus making this model even more relevant, from a pathophysiological standpoint. Of note, this human model of venous congestion has previously shown to mimic on a local scale, notable aspects of the phenotype that is typical of acute HF such as inflammation, neurohormonal and endothelial cell activation (Colombo P C et al., European Heart Journal. 2014; 35:448-454).

II/ USE FOR THE ASSESSMENT OF MITRAL STENOSIS

[0135] Mitral stenosis (MS) may cause progressive dyspnea on exertion, pulmonary hypertension (PH), atrial fibrillation and right ventricular (RV) failure. Patients with MS presenting with change in dyspnea severity often require a complete cardiologic assessment, but the use of biomarkers may be an alternative for the initial assessment of MS and its complications. The aims of this study are to evaluate the role of several cardiovascular biomarkers for this purpose.

Methods

Study Population

[0136] Consecutive, clinically stable patients with moderate (valve area between 1.5 and 2 cm.sup.2) or severe (valve area <1.5 cm.sup.2) MS were included in the study between Jan. 1, 2011 and Mar. 31, 2015. This prospective multicenter observational study was performed at University Hospital of Besancon (France), Bichat University Hospital in Paris (France), and Cumhuriyet University Hospital of Sivas (Turkey). Exclusion criteria were: age <18 years; left ventricular ejection fraction <40%; recent acute coronary syndrome or documented coronary disease; combination of MS with severe aortic valve disease or more than mild mitral regurgitation; recent (<1 month) cardiac decompensation. Patients with high heart rate (>110 beats per minute), uncontrolled hypertension (systolic blood pressure >180 mmHg), severe anemia (hemoglobin <9 g/dL) or pregnancy were excluded as well.

[0137] One small cohort (n=16) of patients with pre-capillary PH (PAH) examined at University Hospital of Besancon was included for comparison of biomarker levels in different etiologies of pulmonary hypertension.

Echocardiographic Study

[0138] All patients underwent transthoracic echocardiography at inclusion, and measurements were made in accordance with the recommendations of the European Association for Echocardiography (Baumgartner H, et al. (2009) EAE/ASE recommendations for clinical practice. Eur J Echocardiogr 10:1-25. doi: 10.1093/ejechocard/jen303). Severity of mitral valve stenosis (MS) was assessed by planimetric evaluation in the parasternal short axis view. MS was considered severe if the valve area was 1.5 cm.sup.2 and moderate if the valve area was between 1.5 and 2 cm.sup.2. Extent of mitral regurgitation was estimated by color Doppler and classified depending on the degree of incursion of the flow into the left atrium. Left ventricular (LV) systolic function was evaluated by measurement of the LV ejection fraction by the Simpson's biplane method. The left atrial size was assessed by measuring its area from the apical four chamber view or its volume in both the four and two chamber views. The left atrium (LA) was considered to be dilated if area was >20 cm.sup.2, or volume >60 mL, or diameter >40 mm. Pulmonary pressures were calculated from maximal tricuspid regurgitation velocity on continuous Doppler in the apical four chamber view, taking into account the size and compliance of the inferior vena cava. A diagnosis of PH was retained for a tricuspid regurgitation velocity >2.8 m/sec, corresponding to systolic pulmonary artery pressure (PAP) >30 mmHg. Right ventricular (RV) function was assessed by measuring RV fractional area change (RV fac), tricuspid annular plane systolic excursion (TAPSE) and systolic myocardial velocity at the lateral tricuspid annulus (TAPSE S). RV dysfunction was defined as the alteration of any one of these three criteria (i.e. RV fac <40%, TAPSE <18 mm; TAPSE S<12 cm/sec).

Biomarker Testing

[0139] Samples of venous blood were drawn on the same day as echocardiography was performed. Samples were centrifuged within 10 minutes in a refrigerated centrifuge, and stored at 80 C.

[0140] BNP was measured using the ARCHITECT i2000 system (Abbott Laboratories, Chicago, Ill., USA). MR-proANP was measured using immunoluminometric assay (B.R.A.H.M.S. AG, Hennigsdorf, Germany) and sCD146 using the CY-QUANT ELISA sCD146 kit (Biocytex, Asnieres, France).

Statistical Analysis

[0141] Continuous variables are expressed as median (interquartile range), nominal variables as frequency (percentages). Group characteristics were compared with non-parametric tests: Fisher's exact test or the Mann-Whitney U-test for two groups, as appropriate; Chi-square or the Kruskall-Wallis H-test for three groups, as appropriate. For statistically significant differences between the groups, subsequent pairwise comparisons were performed using Dunn's procedure with Bonferroni correction of the p-value for multiple comparisons. Corrected p-values are reported. Correlation analysis was performed using Spearman's correlation coefficient. The diagnostic performance of all three biomarkers was assessed by receiver operating characteristic (ROC) analysis and expressed as area under the curve (AUC). The null hypothesis was rejected with an adjusted two-sided p-value <0.05. All analyses were performed with the use of IBM SPSS Statistics, Version 21.0. (IBM Corp, Armonk N.Y., USA).

Ethical Considerations

[0142] The study was conducted in accordance with the principles of the Declaration of Helsinki and local national laws and was approved by the local Ethical Committee. All patients provided written informed consent. The study is registered with ClinicalTrials.gov under the number NCT01374880.

Results

Study Population

[0143] A total of 117, predominantly women (n=96, 72%) with mitral stenosis (MS) were included. In 82 patients (70%) the stenosis was classified as severe and moderate in the other 35 patients (30%). The median planimetric area was 1.34 cm.sup.2 (IQR 1.1-1.58 cm.sup.2) and left atria were dilated in the majority of patients (n=104, 89%) with a median diameter of 49 mm (42-54 mm) and a volume of 101 ml (75-135 ml). Pulmonary hypertension (n=83, 71%) and atrial fibrillation (n=49, 42%) were common among MS patients (FIG. 2).

[0144] Table 3 summarizes the baseline characteristics of the MS patients:

TABLE-US-00002 TABLE 3 Baseline characteristics of the studied patients Mitral stenosis PAH (n = 117) (n = 16) p-value Demographics Age |years] 55 (46-67) 64 (53-71) 0.10 Male gender 21 (18%) 8 (50%) 0.008 Weight [kg] 73 (60-82) 61 (49-89) 0.31 Height [cm] 160 (155-166) 168 (158-173) 0.052 BMI [kg/m2] 27.8 (23.5-31.3) 23.6 (19.1-28.2) 0.01 Hypertension 37 (32%) 6 (38%) 0.78 NYHA class I 26 (22%) 3 (19%) II 58 (50%) 5 (31%) III 32 (27%) 2 (12%) IV 1 (1%) 6 (38%) Treatment Betablockers 61 (52%) 2 (13%) 0.003 ACE-inhibitors or AT2 antagonist 15 (29%) 4 (25%) 1.00 Aldosterone antagonist 34 (29%) 4 (25%) 1.00 Diuretics 57 (49%) 13 (81%) 0.017 Digoxin 14 (28%) 0 (0%) 0.016 Vital signs Systolic blood pressure [mmHg] 120 (108-130) 123 (118-132) 0.41 Diastolic blood pressure [mmHg] 71 (65-81) 70 (69-78) 0.76 Heart rate [bpm] 75 (69-86) 78 (66-95) 0.62 Atrial fibrillation 49 (42%) 2 (13%) 0.028 Lab values Hemoglobin [g/dl] 13.7 (12.6-14.5) 14 (12.5-15) 0.54 eGFR [ml/min] 75 (59-90) 65 (57-97) 0.52 BNP [pg/ml] 121 (77-205) 103 (56-165) 0.35 MR-proANP [pmol/l] 96 (39-188) 174 (103-225) 0.054 sCD146 [ng/ml] 320 (263-440) 562 (322-620) 0.003 Echocardiography Left ventricle Septal thickness [mm] 9 (8-10) 11 (9-13) 0.016 PW-thickness [mm] 9 (8-10) 10 (9-11) 0.032 LV-EDD [mm] 47 (43-51) 45 (40-50) 0.11 LV-ESD [mm] 32 (29-35) 26 (19-33) 0.002 LV-EF [%] 60 (57-68) 65 (60-70) 0.11 Right ventricle RV dilation 29 (25%) 14 (87%) <0.001 RV dysfunction 0 (0%) 5 (31%) <0.001 Systolic PAP [mmHg] 37 (30-45) 68 (46-80) <0.001 Mitral valve Area (planimetry) [cm2] 1.34 (1.1-1.58) 2.05 (2-2.1) <0.001 Left atrium LA diameter [mm] 49 (42-54) 39 (32-43) <0.001 LA area |cm2] 33 (28-38) 18 (16-20) <0.001 LA volume [ml] 101 (75-135) . (..)

Impact of Severity of MS, Pulmonary Hypertension and Atrial Fibrillation on Biomarkers

[0145] As shown in FIG. 3A, plasma levels of BNP (p=0.029), MR-proANP (p=0.027) and sCD146 (p=0.011) were higher in patients with severe MS compared to moderate MS.

[0146] As shown in Table 4 below, the area under the curve (AUC) after ROC analysis to discriminate between severe and moderate MS was between 0.63 and 0.65 for all three biomarkers:

TABLE-US-00003 TABLE 4 Receiver operating characteristics (ROC) curves of different biomarkers for the assessment of complications of mitral stenosis Area under the curve (95% confidence interval) Biomarkers Severe stenosis Pulmonary hypertension BNP [pg/ml] 0.633 (0.512-0.754) 0.636 (0.521-0.752) MR-proANP [pmol/l] 0.635 (0.525-0.744) 0.629 (0.516-0.741) sCD146 [ng/ml] 0.653 (0.532-0.775) 0.604 (0.490-0.717)

[0147] FIG. 3B illustrates that MS patients with systolic pulmonary pressure above 30 mmHg had higher levels of BNP (p=0.025) and MR-proANP (p=0.034) but no change of sCD146 levels compared to patients without PH. Systolic pulmonary artery pressure (sPAP) modestly correlated with BNP (p=0.185, p=0.044), MR-proANP (p=0.338, p<0.001), sCD146 (p=0.341, p<0.001), as shown in FIG. 4. The area under the curve (AUC) after ROC analysis to discriminate between sPAP30 mmHg and sPAP>30 mmHg was between 0.60 and 0.64 for all three biomarkers (Table 4).

[0148] As shown in FIG. 3C, plasma levels of BNP (p=0.002), and sCD146 (p<0.001) were higher in MS patients with atrial fibrillation compared to patients in sinus rhythm. However, the presence or not of atrial fibrillation did not influence plasma MR-proANP.

Biomarkers in Pulmonary Hypertension

[0149] Among patients with elevated pulmonary artery pressures (n=99), the median systolic pulmonary artery pressure was 45 mmHg (35-50 mmHg) in MS patients with PH (n=83) and 68 mmHg (46-80 mmHg) in patients with pre-capillary PAH (p<0.001). Baseline characteristics of the cohort of patients with PAH (n=16) are summarized in Table 3.

[0150] FIG. 5 illustrates plasma levels of all three biomarkers in MS patients with PH compared to levels in patients with PAH: PAH patients had higher levels of sCD146 compared to MS patients with PH (p=0.007), whereas no differences in BNP and MR-proANP were observed. Furthermore, among patients with elevated pulmonary artery pressures, RV dysfunction was present in 5 cases (5%) and associated with higher median levels of sCD146 (612 vs. 344 ng/ml, p=0.013) compared to patients with normal RV function, whereas no differences in BNP and MR-proANP levels were observed.

Conclusions

[0151] Plasma levels of all three biomarkers were higher in severe MS compared to moderate MS. PH was associated with levels of BNP and MR-proANP. The presence of atrial fibrillation increased plasma levels of BNP and sCD146, whereas MR-proANP was not affected by atrial fibrillation. PAH patients had higher levels of sCD146 compared to MS patients with PH. RV dysfunction was associated with higher levels of sCD146.

[0152] In conclusion, mitral stenosis (MS) and its complications affect plasma levels of cardiovascular biomarkers. The use of MR-proANP may be useful for monitoring disease progression in MS, in particular for the assessment of severe stenosis and the presence of pulmonary hypertension in the early phase. sCD146 might help identifying patients with more advanced pulmonary hypertension and RV-dysfunction.

III/ USE FOR THE ASSESSMENT OF PULMONARY CONGESTION

[0153] Aim of this study was the evaluation of sCD146 for the assessment of the degree of pulmonary congestion in the early phase of ACS.

Methods

Study Population

[0154] Consecutive patients with ACS admitted to the Coronary Care Unit (CCU) of the Cardiology Department of the University Hospital of Bmo (Czech Republic) from July 2009 to November 2012 were enrolled. The diagnosis of ACS was based on appropriate symptoms in conjunction with consistent changes on electrocardiogram, i.e. ST-segment elevation or depression, new left bundle branch block or negative T wave (Thygesen K, et al. Third universal definition of myocardial infarction. 2012. pp. 2551-67). Exclusion criteria were: age >85 years or estimated life expectancy due to non-cardiovascular reasons <12 months; known or newly diagnosed malignancy, inflammatory disease or connective-tissue disease; distance from the place of residence to the hospital of >100 km; absence of coronary stenosis with reduction of the intraluminal diameter >50% on coronary angiography.

Blood Samples Storage and Analysis

[0155] Samples of venous blood for standard biochemical and hematological analyses as well as sCD146 were drawn immediately upon hospital admission before PCI. Troponin T and brain natriuretic peptide (BNP) were drawn exactly 24 hours after onset of chest pain. Samples were centrifuged within 10 min in a refrigerated centrifuge, and stored at 80 C. Troponin T was analyzed with the high-sensitive assay (Roche Diagnostics, Basel, Switzerland), BNP using the AxSYM BNP-Microparticle Enzyme Immunoassay (Abbott Laboratories, Chicago, Ill., USA), and sCD146 by ELISA (CY-QUANT ELISA sCD1460, Biocytex, France).

Radiological Evaluation of Pulmonary Congestion

[0156] Pulmonary congestion was assessed by conventional chest radiography at admission.

[0157] Images were evaluated by certified radiologists and classified in 3 groups: no or mild congestion, interstitial pulmonary edema, alveolar pulmonary edema.

Ethical Considerations

[0158] Written informed consent was obtained from all subjects before participation in the study. The study was performed in observation of national laws and in accordance with the ethical standards of the Declaration of Helsinki, and was approved by the Ethics Committee of Faculty Hospital Brno (Brno, Czech Republic).

Statistical Analysis

[0159] Values are expressed as median (interquartile range, IQR) or as number (percentage), as appropriate. Three groups were compared with the Chi-square or the Kruskall-Wallis H-test, as appropriate. For statistically significant differences between the groups, subsequent pairwise comparisons were performed using Dunn's procedure with Bonferroni correction of the p-value for multiple comparisons. The null hypothesis was rejected with an adjusted two-sided p-value <0.05. All analyses were performed with the use of IBM SPSS Statistics, Version 21.0. (IBM Corp, Armonk N.Y., USA).

Results

[0160] A total of 1021 patients presenting with ACS were prospectively screened. Patients without chest radiography at admission (n=94; 9%) were excluded from this analysis. Baseline characteristics of the 927 patients included in the study are summarized in Table 5:

TABLE-US-00004 TABLE 5 Baseline characteristics of the studied patients No or mild Interstitial Alveolar Total congestion edema edema p- N = 927 N = 835 N = 72 N = 20 value Age [years] 61 (55-67) 61 (55-67) 63 (55-68) 70 (59-73) 0.010 Male gender 707 (76%) 643 (77%) 50 (69%) 14 (70%) 0.281 Height [cm] 174 (168-179) 174 (168-180) 170 (168-179) 170 (165-174) 0.120 Weight [kg] 85 (75-95) 85 (75-95) 8 (76-90) 83 (74-97) 0.582 Systolic blood 140 (120-160) 140 (120-160) 135 (113-155) 135 (103-151) 0.071 pressure [mmHg] (120-160) Diastolic blood 80 (70-90) 80 (70-90) 80 (70-90) 70 (63-80) 0.008 pressure [mmHg] Heart rate [/min] 76 (66-87) 75 (66-86) 82 (74-105) 87 (73-98) <0.001 Type of acute 0.022 coronary syndrome Unstable angina 36 (4%) 35 (4%) 1 (1%) 0 (0%) NSTEMI 278 (30%) 248 (30%) 18 (25%) 12 (60%) STEMI 613 (66%) 552 (66%) 53 (74%) 8 (40%) Risk factors Hypertension 508 (55%) 459 (55%) 35 (49%) 14 (70%) 0.224 Dyslipidemia 380 (41%) 339 (41%) 31 (43%) 10 (50%) 0.653 Diabetes 206 (22%) 171 (21%) 24 (33%) 11 (55%) <0.001 Active smoking 424 (46%) 384 (46%) 34 (49%) 6 (30%) 0.597 Family history 192 (28%) 179 (29%) 11 (24%) 2 (20%) 0.718 Previous myo- 107 (12%) 99 (12%) 7 (10%) 1 (5%) 0.562 cardial infarction Previous PCI 83 (9%) 80 (10%) 2 (3%) 1 (5%) 0.125 Previous CABG 22 (2%) 18 (2%) 4 (6%) 0 (0%) 0.149 Previous stroke 49 (5%) 38 (5%) 9 (13%) 2 (10%) 0.010 Peripheral artery 53 (6%) 41 (5%) 7 (10%) 5 (25%) <0.001 disease COPD 39 (4%) 35 (4%) 2 (3%) 2 (10%) 0.362 Atrial fibrillation 25 (3%) 19 (2%) 3 (4%) 3 (15%) 0.002 Laboratory values at admission Hemoglobin 143 (133-153) 143 (133-153) 142 (129-157) 137 (113.5-149) 0.170 [g/L] Leucocytes 10.9 (8.7-13.7) 10.7 (8.6-13.3) 12.4 (9.7-16.5) 13.4 (10.9 17.8) <0.001 [G/L] Sodium 140 (137-141) 140 (137-141) 139 (137-141) 138 (137-142) 0.196 [mmol/L] Potassium 4 (3.7-4.4) 4 (3.7-4.4) 4.1 (3.8-4.4) 4.5 (4.1-5.0) 0.002 [mmol/L] Glucose 7.6 (6.3-10) 7.5 (6.2-9.7) 9 (7.8-12.1) 13 (7.5-17.1) <0.001 [mmol/L] Creatinine 82 (71-97) 82 (70-96) 84 (75-98) 103 (81-131) 0.002 [mol/L] Troponin T* 1.41 (0.372-3.78) 1.34 (0.33-3.52) 3.47 (1.22-7.00) 1.16 (0.52-3.66) <0.001 [g/L] CRP* 18 (6-62) 15 (6-51) 70 (24-175) 66 (39-163) <0.001 [mg/L] Length of stay 5 (4-7) 5 (4-7) 6 (4-9) 7 (5-12) 0.007 [days] In-hospital 10 (1.1%) 4 (0.5%) 4 (5.6%) 2 (10.0%) <0.001 mortality Legend: BNP brain natriuretic peptide - CABG coronary artery bypass graft surgery - COPD chronic obstructive pulmonary disease - CRP C-reactive protein - NSTEMI non-ST-elevation myocardial infarction - PCI percutaneous coronary intervention - STEMI ST-elevation myocardial infarction. *Reported BNP and Troponin T values are at 24 hours after admission, CRP values are at 48 h after admission

[0161] Patients were subsequently classified in 3 groups according to the degree of pulmonary congestion on chest radiography at admission: No or mild congestion (n=835), interstitial edema (n=72), alveolar edema (n=20). Ninety-two (10%) patients showed signs of pulmonary edema on chest radiography.

[0162] Patients with severe pulmonary congestion presented more often with NSTEMI, were older, had higher prevalence of diabetes, atrial fibrillation and peripheral artery disease, had higher creatinine levels and lower diastolic blood pressure at admission compared to the other subgroups. Patients with no or mild congestion had lower heart rate, lower inflammation parameters and lower glucose compared to patients with severe congestion. Of note, interstitial edema was associated with higher troponin levels compared to the other subgroups.

[0163] Patients with pulmonary congestion (interstitial or alveolar edema) had increased in-hospital mortality compared to patients without or with mild pulmonary congestion (5.6% and 10.0% vs. 0.5%, p<0.001). There was also a trend toward longer hospital stay with increasing pulmonary congestion, although pairwise comparisons were not statistically significant.

[0164] For the overall population, median level of sCD146 was 320 ng/mL (IQR 251-398 ng/mL, range 95-2866 ng/mL) and the median level of BNP was 263 pg/mL (IQR 125-473 pg/mL, range 10-11567 pg/mL).

[0165] FIG. 6 shows that median plasma levels of BNP were higher in patients with interstitial (679 pg/mL, IQR 355-1097 pg/mL) or alveolar (665 pg/mL, IQR 267-1214 pg/mL) pulmonary edema compared to patients without or with mild signs of congestion (251 pg/mL, IQR 119-430 pg/mL). No difference between patients with interstitial and alveolar pulmonary was found (p-values shown in FIG. 6).

[0166] FIG. 6 further shows that plasma levels of sCD146 were better associated with radiological evidence of pulmonary congestion than BNP with stepwise increase in circulating sCD146 with increasing degree of pulmonary congestion: median plasma levels of sCD146 in patients without or with mild signs of congestion, interstitial edema and alveolar edema were 316 ng/mL (IQR 249-388 ng/mL), 348 ng/mL (IQR 267-478 ng/mL) and 438 ng/mL (IQR 346-690 ng/mL), respectively. Moreover, plasma levels of sCD146 showed a lower variability compared to BNP.

[0167] FIG. 7 shows the relationship of BNP, sCD146 and myocardial necrosis. BNP levels were associated with the level of troponin. Splitting the population in 3 groups according to troponin levels, BNP values were higher in the second and third tertiles of troponin compared to the first tertile (p<0.001). In contrast to BNP, sCD146 levels were not increased, and even slightly decreased, in the second and third tertiles of troponin compared to baseline.

Conclusions

[0168] Plasma levels of sCD146 well reflected radiological evidence of pulmonary congestion: the higher plasma levels of sCD146, the worse the degree of pulmonary congestion. Interestingly, in contrast to BNP, sCD146 levels were not affected by the level of troponin. In other words, the novel endothelial biomarker sCD146 correlates with radiological evidence of pulmonary congestion in the early phase of ACS and, in contrast to BNP, is not affected by the amount of myocardial cell necrosis.

[0169] Since relevant pulmonary congestion complicates the course of one of ten patients presenting with ACS and, as confirmed in our study, negatively influences short-term outcome, sCD146 may help emergency physicians, cardiologists and intensivists to assess and monitor pulmonary congestion in patients with ongoing ACS.