CT measured pulmonary artery to ascending aorta ratio stratified by echocardiographically obtained systolic pulmonary artery pressure values for noninvasive detection of pulmonary hypertension in patients with severe aortic valve stenosis

Background Transthoracic echocardiography (TTE) offers a measurement method for the determination of pulmonary hypertension (PH) in patients with severe aortic valve stenosis (AS) with determination of maximal tricuspid regurgitation velocity (TRVmax) and systolic pulmonary artery pressure (sPAP). Radiological parameters for noninvasive detection of PH, most importantly computed tomography (CT) based PA/AA-ratio = ratio of pulmonary artery diameter (PA) and ascending aorta diameter (AA), are also included in the latest ESC guidelines. The aim of the present study was to define cut-off values for PA/AA-ratio taking also into account cardiovascular biomarkers to determine criteria for noninvasive diagnosis of PH. Methods 194 patients with severe AS undergoing transcatheter aortic valve replacement (TAVR) underwent pre-procedural TTE and CT with measurement of PA/AA-ratio. Additionally, common cardiovascular biomarkers were determined. Results TAVR patients with an sPAP ≥ 40 mmHg or a TRVmax ≥ 2.9 m/s had a PA/AA-ratio ≥ 0.80 in an AUROC analysis. The cut-off value of ≥ 0.80 resulted in a significantly higher mortality rate (log-rank test: p = 0.034) in these patients in a Kaplan–Meier analysis regarding 1-year survival after TAVR. Significant differences in biomarker expression between patients with a PA/AA-ratio ≥ 0.80 or < 0.80 occurred for BNP (p = 0.001), cTnI (p = 0.032), GDF-15 (p = 0.002) and H-FABP (p = 0.015). Conclusion PA/AA-ratio ≥ 0.80 is a promising radiological parameter that can provide information about mortality in patients with severe AS undergoing TAVR; combined with biomarkers it may contribute to noninvasive detection of PH in patients with severe AS. Graphical abstract


Introduction
Pulmonary hypertension (PH) due to severe aortic valve stenosis (AS) is a frequently diagnosed sequelae in the clinical setting, which occurs pathophysiologically due to chronic pressure load initially on the left ventricle and subsequently on the left atrium and pulmonary circulation [1].Clinically, the gold standard of right heart catheterization as a preoperative diagnostic tool has been abandoned and transthoracic echocardiography (TTE) is used as the tool of choice to assess the potential presence of PH in severe AS [2].The systolic pulmonary artery pressure (sPAP) can be determined and estimated to differentiate between the presence and absence of PH in clinical setting, although various thresholds are used and discussed controversially in the literature.In most cases, a present sPAP ≥ 40 mmHg is considered as relevant pulmonary hypertension [3][4][5]; however, there are other studies that refer to an sPAP threshold of 45 mmHg [6,7] or even 50 mmHg [8,9] in their work.In recent years, however, maximal tricuspid regurgitation velocity (TRVmax) has become increasingly important in addition to sPAP, especially in research setting.A cut-off value of 2.9-3.4 m/s indicates an intermediate probability and > 3.4 m/s indicates a high probability of pulmonary hypertension [10].Also, TTE is highly dependent on the experience of the corresponding examiner or also on the available sound quality of the subject, so that erroneous measurements or non-conclusive data may occur.
In order to still provide patients with noninvasive options for PH detection, imaging plays an increasingly crucial role.It was already shown in a previous work [11] that the isolated use of a cut-off value of ≥ 29 mm for the main pulmonary artery diameter as defined by the European Society for Cardiology (ESC) guidelines of 2015 [12] is only an estimation and less a solid basis for the presence of PH.In addition, the currently valid ESC guidelines of 2022 [13] describe the ratio of pulmonary artery diameter (PAA) and ascending aorta diameter (AA)-so-called PA/AA-ratio-with a cutoff value of 0.9 as a potentially more accurate and valuable methodology of noninvasive radiological PH determination.
Therefore, the aim of the present study was to calculate a cut-off value for PA/AA-ratio based on echocardiographic sPAP data, to further investigate this value with respect to mortality, and finally to further underline PH determination based on this radiological criterion with serum biomarker analyses of various common cardiovascular biomarkers.

Study population
Originally, this study population included 221 patients just before transcatheter aortic valve replacement (TAVR) procedure between 2016 and 2018 at Paracelsus Medical University Hospital Salzburg and Kepler University Hospital Linz.27 patients had to be excluded due to missing weight or height data, missing CT data or inadaquate CT quality.At last, 194 patients were recommended for inclusion in the study.Relevant exclusion criteria even before study recruitment were patients with a bicuspid aortic valve, acute cardiac decompensation at the time of TTE or at the time of TAVR, as well as patients with any history that might indicate a pre-capillary component of pulmonary hypertension [chronic thrombembolic pulmonary hypertension (CTEPH), idiopathic pumonary arterial hypertension, intersitial lung disease or underlying rheumatologic diseases with pulmonary involvement such as scleroderma, lupus erythematosus, etc.].Therefore, in this selected patient population, a postcapillary cause [severe AS and possibly limited left ventricular ejection fraction (LVEF)] is assumed for the pulmonary hypertension.
The study protocol was approved by the local ethics committees of Paracelsus Medical University Salzburg (415-E/1969/5-2016) and Johannes Kepler University Linz (E-41-16) and conducted in accordance to principles of the Declaration of Helsinki and Good Clinical Practice.Written informed consent to participate in the study was available from all patients before study inclusion.

Transthoracic echocardiography
Common ultrasound devices (iE33 and Epiq 5; Philips Healthcare, Hamburg, Germany) were used for performing TTE as routinely diagnostic on average 1-4 weeks before TAVR.These examinations were each conducted by experienced clinicians with more than 4 years of training in echocardiography.Severe AS was classified according to current guidelines of European Society for Cardiology measuring using an AV Vmax (maximal velocity over aortic valve) of 4.0 m/s, an AV dpmean (mean pressure gradient over aortic valve) ≥ 40 mmHg and an aortic valve area ≤ 1.0 cm 2 for definition of severe AS.Patients with low-flow, lowgradient AS situation were excluded.Via Simpson's method LVEF was calculated.To graduate mitral, aortic and tricuspid valve regurgitation in minimal, mild (I), moderate (II) and severe (III) spectral and color-Doppler images were used.Maximum tricuspid regurgitant jet velocity (TRVmax) was obtained by continuous wave Doppler over the tricuspid valve.The probability of the presence of PH was considered medium at a threshold of ≥ 2.9 m/s and high at a threshold of ≥ 3.4 m/s.Pulmonary artery pressure (PAP) was calculated using the formula 4 × TRVmax 2 and adding the estimated right atrial pressure (RAP).The latter corresponds to the central venous pressure and was determined by the diameter of the inferior vena cava (IVC).With an IVC diameter ≥ 21 mm and a respiratory caliber fluctuation < 50%, a RAP of 15 mmHg was assumed.For an IVC diameter < 21 mm as well as a respiratory caliber fluctuation ≥ 50%, a RAP of 3 mmHg was calculated.Other scenarios not corresponding to these constellations were provided with an intermediate value of 8 mmHg [14].Finally, the simplified Bernoulli Eq. (4 × TRVmax 2 ) + RAP was applied to obtain a sPAP result.Different TRVmax (≥ 2.9 m/s and ≥ 3.4 m/s) values and sPAP (40, 45 and 50 mmHg) values were used to determine PH in accordance with the current literature [15][16][17][18][19].

CTA protocol and measurement of MPA diameter for PH assessment
The included study patients at both centers routinely received a pre-interventional, ECG triggered CTA of the whole aorta and femoral arteries to asses, among others, the aortic annulus size, the aortic anatomy and vascular access.Scans were performed on multidetector CT scanners (Somatom Definition AS+, Siemens Healthcare, Erlangen, Germany; Brilliance 64, Philips Healthcare, Hamburg, Germany) with a patient size-adapted tube voltage (80-120 kVp) and active tube current modulation.A bolus-tracking technique was applied with a 100 mL bolus of non-ionic iodinated contrast media followed by 70 mL saline solution injected at a flow rate of 3.5-5 mL/s.This imaging, as well as TTE, was completed in a separate inpatient stay approximately 1-4 weeks before the TAVR procedure.
A stationary workstation (Impax, Agfa-Gevaert, Mortsel, Belgium) was used for image analysis.Two experienced radiologists-one board certified with nine years of experience in vascular imaging (radiologist 1), one in the fourth year of training (radiologist 2)-independently performed the following measurements on axial sections in mediastinal window settings on the end-diastolic phase: (1) Main pulmonary artery (PA) diameter was measured perpendicular to the vessel axis at the widest point within 3 cm of the bifurcation of the pulmonary trunk.( 2) At the same level as the main PA measurement, the widest diameter of the ascending aorta (AA) was measured (Fig. 1) as previously described [20,21].The quotient of main pulmonary artery diameter and ascending aortic diameter formed the PA/AAratio as the basis for the CT-based, radiological definition of PH.The results of both observers were compared in terms of inter-observer variability, and the mean value was used for further analysis.50 randomly selected cases were reassessed by radiologist 2 after a 4-week interval to evaluate intra-observer variability.The radiologists were blinded to all clinical, hemodynamic and laboratory data.

Biomarker analysis
Blood samples were taken from the patients in a fasting state and in an upright position using a vacuum-containing system on the day of hospitalization and thus one day before the TAVR procedure.By centrifugation of the collection tubes the plasma was separated from the blood components.Afterwards, plasma was frozen at − 80 °C.All 194 samples were measured at similar time points under same conditions.

Statistical analysis
Statistical analysis was performed using SPSS (Version 25.0, SPSSS Inc., USA).Graphical representations were created Fig. 1 Measurement of diameters of interest on axial CT.AA: ascending aorta; PA: main pulmonary artery; red double headed arrow: distance between bifurcation of the pulmonary trunk and level of measurement within main pulmonary artery using GraphPad Prism (Version 8.0.0,GraphPad Software, San Diego, California, USA) in addition to SPSS.
Kolmogorow-Smirnow-Lilliefors test was carried out to test variables for normal distribution.Normally distributed metric data was expressed as mean ± standard deviation (SD) and analyzed using an unpaired student's t-test.Notnormally distributed metric data was expressed as median and interquartile range (IQR); Mann-Whitney-U-test was applied for statistical analysis here.Frequencies/percentages were used for categorial data and compared using the chisquare test.
Area under the receiver operator characteristics (AUROC) curves with area under the curve (AUC) and separate analysis of Youden Index (YI) were performed using different sPAP values (sPAP 40-45-50 mmHg) and TRVmax values (TRVmax ≥ 2.9 m/s and ≥ 3.4 m/s) to determine the respective cut-off for the PA/AA-ratio.
For the analysis of inter-and intra-observer variability, the Pearson correlation coefficient with 95% confidence interval (CI) was reported for two independent investigators (radiologist 1 and 2, inter-observer variability) or two measurements taken 4 weeks apart by one investigator (radiologist 2; intraobserver variability).
Correlation analyses were absolved using Pearson correlation coefficient (metric data) or Spearman's rank-correlation coefficient (nominal/ordinal data) to determine the strength between PA/AA or PA/AA ≥ 0.80 to further variables (age, gender, height, weight etc.).
To detect possible influencing factors regarding the presence of a potential PH with a PA/AA-ratio ≥ 0.80, a univariate, binary logistic regression analysis was figured out.For better comparability, a z-transformation was absolved for metric data.Multivariate, binary logistic regression was performed to assess independent factors regarding the prediction of a PA/AA-ratio ≥ 0.80.Therefore, covariates with p < 0.100 in the univariate analysis were entered and a backward variable elimination was carried out.
Univariate Cox proportional hazard regression model was used to calculate hazard ratio (HR) and 95% CI for several influencing factors associated with 1-year-mortality.Again, the z-transform was applied for metric data.Afterwards, multivariate Cox regression was performed to assess independent predictors of mortality.Therefore, again, covariates associated with mortality in the univariate analysis (p < 0.100) were entered and a backward variable elimination was performed.
After establishing a generalized cut-off value for the detection of a PA/AA-ratio ≥ 0.80, different expressions of biomarker plasma levels were statistically compared based on the two groups ("No PH": PA/AA-ratio < 0.80 and "PH": PA/AA-ratio ≥ 0.80).
Subsequently, AUROC analyses were carried out to determine an optimal cut-off value of examined cardiovascular biomarkers according to a prediction of PA/AA-ratio ≥ 0.80.
In order to investigate not only the effect of a singular biomarker, biomarkers were examined in combinations of two or three.For this purpose, a binary logistic regression was completed and the obtained values were again submitted to an AUROC analysis.
A p-value < 0.050 was considered statistically significant.
Therefore, a value of 0.80 was used for the PA/AA-ratio in the following.

Study cohort
194 patients with primary, severe AS from the Paracelsus Medical University Hospital Salzburg and Kepler University Hospital Linz were included in the study.72 patients (37.11%) had a PA/AA-ratio < 0.80 in the performed ECG Fig. 2 AUROC analyses of PA/AA-ratio for prediction of sPAP ≥ 40, 45 and 50 mmHg with concerning cut-off values, Youden Index, sensitivity and specificity Fig. 3 AUROC analyses of PA/AA-ratio for prediction of TRVmax ≥ 2.9 and ≥ 3.4 m/s with concerning cut-off values, Youden Index, sensitivity and specificity 1 3 triggered CTA, which corresponded to the absence of PH in this study.In contrast, 122 patients (62.89%) had a PA/AAratio ≥ 0.80 and were listed as subjects with potential PH.
A flow chart regarding study inclusion and relevant exclusion criteria is shown in Fig. 4.

Baseline characteristics
Table 1 demonstrates the collected baseline characteristics of the overall cohort and the patients classified into 2 groups according to those with a PA/AA-ratio < 0.80 and to those with a PA/AA-ratio ≥ 0.80.52.6% of the total cohort were male, with an overall mean age of 82.8 ± 4.9 years.Significant differences between patients with a PA/AA-ratio < 0.80 vs. ≥ 0.80 were related to gender (female gender with significantly higher probability of PA/AA-ratio ≥ 0.80; p = 0.006), height (p = 0.038) and weight (p = 0.033) as well as STSScore (p = 0.025) and sPAP measurements (p = 0.018) with otherwise mostly counterbalanced further characteristics.

Correlation
To investigate relationships between PA/AA-ratio or PA/ AA-ratio ≥ 0.80 and other patients' characteristics, correlation analysis using Pearson or Spearman correlation coefficient was performed (Table 2).
Overall, both the PA/AA ratio and the PA/AA ratio ≥ 0.80 generally showed no pronounced correlations with the

Binary logistic regression
In order to verify a relevant statistical relationship between the potential presence of PH via PA/AA-ratio ≥ 0.80 and other factors (especially gender, weight, height etc.), a univariate as well as a multivariate binary logistic regression was performed (Table 3).
In the univariate analysis, gender, weight, height, STSScore, sPAP, hemoglobin, hematocrit, BNP, cTnI and GDF-15 showed a relevant association (p < 0.100), so multivariate analysis was performed with these variables.None of the clinical characteristics finally showed a p value ≤ 0.050.
Patients with a PA/AA-ratio < 0.80 showed a significant lower mortality during the first year after TAVR then the corresponding group with a PA/AA-ratio ≥ 0.80 (logrank test: p = 0.034).In the group of patients with a PA/ AA-ratio < 0.80, 15/72 (20.83%) died within one year.In contrast, 44/122 (36.07%) from the group with a PA/AAratio ≥ 0.80 passed away.

Cox hazard regression
To investigate influencing factors concerning 1-year mortality after TAVR, a univariate and multivariate Cox proportional hazard regression was figured out (Table 4).The result of univariate analyses showed agreement (p < 0.100) with atrial fibrillation, mitral valve insufficiency and PA/ AA-ratio ≥ 0.80.After inclusion of these data in a multivariate analysis, atrial fibrillation (p = 0.045) and PA/AAratio ≥ 0.80 (p = 0.042) remained independent factors for estimation of mortality after 1 year.

Biomarker concentrations in dependence of new PA/ AA-ratio cut-off value
Figure 8 summarizes the corresponding plasma concentrations of the determined cardiovascular biomarkers depending on the PA/AA-ratio obtained (≥ 0.80 vs. < 0.80).

AUROC results: PA/AA-ratio and singular cardiovascular biomarker
To analyze potential biomarkers for prediction of a PA/ AA-ratio ≥ 0.80 in patients with severe AS before TAVR, AUROC-curves regarding plasma level concentration of BNP, cTnI, sST2, GDF-15, H-FABP, IGF-BP2 and suPAR were figured out.Therefore AUC, cut-off values with YI as well as sensitivity and specificity were extracted (Fig. 9; Table 5).

AUROC results: PA/AA-ratio and multiple combinations of cardiovascular biomarkers
For a better overview and also considering clinical practicability, 2 (Fig. 10; Table 6) or 3 (Fig. 11; Table 7) biomarkers were examined in combination and AUROC analyses were performed.

PA/AA-ratio: prognostic factor regarding 1-year survival
In the current clinical setting, diagnosis of PH as a sequelae of severe AS is mainly performed by TTE by determination  of TRVmax or sPAP.However, not only echocardiography offers a noninvasive way of PH detection, but also computed tomography.In the context of procedure-planning CTA   before TAVR, the main pulmonary artery diameter with a cut-off value of 29 mm and the PA/AA-ratio with a cut-off value of 1.00 (2015) and 0.90 (2022), respectively, found their way into the ESC guidelines so far [12,13].Recently, it could be shown in a publication of our group that in case of an echocardiographically obtained sPAP ≥ 40 mmHg, the cut-off value of the main pulmonary artery diameter is in accordance with the ESC guidelines.However, the main pulmonary artery parameter was not very conclusive in terms of mortality rates and also in terms of agreement with the expression of cardiovascular biomarkers [11].The PA/ AA-ratio had a very similar AUC-value (AUC 0.673; 95% CI 0.590-0.797;p < 0.001) as the main pulmonary artery diameter (AUC 0.676; 95% CI 0.580-0.771;p = 0.001) with respect to prediction of PH when sPAP ≥ 40 mmHg.Almost identical AUROC analyses were previously shown by Eberhard et al. [22], who calculated AUC-values of 0.63 for both main pulmonary artery diameter and PA/AA-ratio in a cohort of 257 TAVR patients using right heart catheterization data and a mean pulmonary artery pressure (mPAP) ≥ 25 mmHg as criteria for PH.However, in contrast to the study by Eberhardt et al., the cut-off value for PA/ AA-ratio calculated in this study was shown to be an independent prognostic factor for long-term survival after TAVR and should possibly be included in clinical considerations regarding an eventually, conservative approach.Different sPAP cut-off values (40-45-50 mmHg) were clearly inferior  PA/AA-ratio ≥ 0.80: potential for a new threshold?
The current ESC guidelines of August 2022 [13] state a PA/ AA-ratio of 0.90 as a potential threshold for the presence of PH.This is already contrasted by a paper by O'Sullivan et al. [23] with a study of 139 TAVR patients, using right heart catheterization data and multi-detector computed tomography derived pulmonary vessel measurements, where an optimal cut-off value for the presence of PH was set at a PA/AA-ratio of 0.80.The AUC-value was 0.74 (95% CI 0.65-0.83;p < 0.001) with a sensitivity of 56% and a specificity of 88%

PA/AA-ratio: to what extent can biomarkers support the diagnosis?
Due to only 51% specificity of PA/AA-ratio for PH at sPAP ≥ 40 mmHg or to 53% at TRVmax ≥ 3.4 m/s, the aim One of few papers was published by the working group of Gumauskiene et al. [24], who demonstrated that TAVR patients with an elevated sPAP (defined here ≥ 45 mmHg) had significantly higher BNP as well as GDF-15 values than TAVR patients with an sPAP < 45 mmHg.This result could be applied to our study results, because an sPAP ≥ 40 mmHg is associated with a PA/AA-ratio of ≥ 0.80, and in a group smaller or larger than the PA/ AA-ratio cut-off value, respectively, equally significantly different expressions for these two cardiovascular biomarkers could be revealed.
In contrast to PA/AA-ratio, the main pulmonary artery diameter could not contribute to an early detection of pulmonary hypertension in a recently published study of our own working group [11], even in combination with cardiovascular biomarkers.By combined use of sPAP ≥ 40 mmHg, PA/AA-ratio ≥ 0.80, a cTnI ≥ 17.50 pg/ml, GDF-15 ≥ 1118.41 pg/ml, and IGF-BP2 ≥ 106,416.89pg/ml, however, the sensitivity for the presence of PH could be increased from 78 to 81% and the specificity from 51 to 67% (Fig. 12).
Finally, the noninvasive, radiological determination of PA/AA-ratio ≥ 0.80 provides a diagnostic tool that can not only provide valuable information regarding 1-year mortality after TAVR but can also further delineate the risk for pulmonary hypertension with common cardiovascular biomarkers such as cTnI or GDF-15.
A summary of the study design and the results obtained is presented in a graphical abstract in Fig. 13.

Limitations
The present, retrospective study design is based on data from a small cohort over a circumscribed time period (2016-2018).Biomarker levels were only measured at baseline without statement regarding expression after TAVR procedure.Technical pitfalls in echocardiographic and radiological measurements which lead to misclassifications should always be conceded, even if examinations were performed by experienced clinical investigators.Additionally, invasive right heart catheterization, the gold standard of for accurate diagnosis regarding the genesis of PH (pre-capillary vs. post-capillary) was neither performed in Salzburg nor in Linz, because it is no longer a routine, diagnostic procedure before TAVR, since the findings are not a contraindication for TAVR and thus the mostly elderly and multi-morbid patients experience more Fig.11 Three-way biomarker combinations with corresponding AUROC analyses risks than actual benefits from the invasive procedure.This final point also addresses the fact that, despite exclusion of obvious factors for pre-capillary pulmonary hypertension (CTEPH, PAH, interstitial lung disease, or underlying rheumatologic diseases with pulmonary involvement), we did not include with absolute certainty a pure cohort of only left heart-related, post-capillary pulmonary hypertension patients and thus isolated pre-capillary, but also combined pre-capillary and post-capillary patients, may also be found in this noninvasive study.

Conclusion
With a PA/AA-ratio ≥ 0.80, an underlying, quick and easily measurable radiological parameter can provide information about mortality in patients undergoing TAVR.The excellent inter-and intra-reader agreement (ICC > 0.98) for CT-measured diameters underlines the reproducibility and robustness of this ratio.Combination of CT, TTE and cardiovascular biomarkers offers a potential way of noninvasive risk Table 7 AUROC analyses of a combination of 3 biomarkers for prediction of a PA/AA-ratio ≥ 0.80 with concerning Youden Index, sensitivity and specificity BNP brain natriuretic peptide, cTnI cardiac Troponin I, sST2 soluble suppression of tumorigenicity-2, GDF-15 growth/differentiation of factor-15, H-FABP heart-type fatty-acid binding protein, IGF-BP2 insulin like growth factor binding protein 2, suPAR soluble urokinase-type plasminogen activator receptor Fig. 12 AUROC analyses of PA/AA-ratio with cardiovascular biomarker cut-off values for prediction of sPAP ≥ 40 mmHg with concerning cut-off values, Youden Index, sensitivity and specificity stratification regarding pulmonary hypertension in patients with severe aortic valve stenosis.

Fig. 4
Fig. 4 Flow chart of study inclusion and exclusion criteria (created with BioRender.com)

Table 1
Baseline characteristics of study population

Table 2
Tabular overview of correlation analysis of PA/AA-ratio or PA/AA-ratio ≥ 0.80 with regard to various clinical characteristics R correlation coefficient, PA main pulmonary artery, AA ascending aorta, BMI body mass index, CVD cardiovascular disease, PAD peripheral artery disease, COPD chronic obstructive pulmonary disease, LVEF left ventricular ejection fraction, LVEDD left ventricular end diastolic diameter, IVSd interventricular septum diastolic, AV max maximal velocity over aortic valve, AV dpmean mean pressure gradient over aortic valve, AV dpmax maximal pressure gradient over aortic valve, TAPSE AVI aortic valve insufficiency, MVI mitral valve insufficiency, TVI tricuspid valve insufficiency, sPAP systolic pulmonary arterial pressure, Hb hemoglobin, Hkt hematocrit, BNP brain natriuretic peptide, cTnI cardiac troponin I, sST2 soluble suppres-GDF-15 growth/differentiation of factor-15, H-FABP heart-type fatty-acid binding protein, IGF-BP2 insulin like growth factor binding protein 2, suPAR soluble urokinase-type plasminogen activator receptor

Table 6
AUROC analyses of a combination of 2 biomarkers for prediction of a PA/AA-ratio ≥ 0.80 with concerning Youden Index, sensitivity and specificity BNP brain natriuretic peptide, cTnI cardiac Troponin I, sST2 soluble suppression of tumorigenicity-2, GDF-15 growth/differentiation of factor-15, H-FABP heart-type fatty-acid binding protein, IGF-BP2 insulin like growth factor binding protein 2, suPAR soluble urokinase-type plas-