How Do Cardiovascular Biomarkers Behave in Patients with Severe Aortic Valve Stenosis with and without Echocardiographically Proven Pulmonary Hypertension?—A Retrospective Study of Biomarker Trends before and after Transcatheter Aortic Valve Replacement

Background: Since right heart catheterization is rarely performed in patients with severe aortic valve stenosis (AS), echocardiography is currently the tool of choice to determine the presence or absence of pulmonary hypertension (PH). The systolic pulmonary artery pressure (sPAP) has established itself as a reliable measurement value for this purpose. The aim of our study was to evaluate the behavior of plasmalevel concentrations of novel cardiovascular biomarkers (sST2, GDF-15, H-FABP, IGF-BP2, and suPAR) in patients with severe AS and an sPAP < 40 mmHg in comparison to patients with an sPAP ≥ 40 mmHg before transcatheter aortic valve replacement (TAVR) and after TAVR (24 h, 96 h, 3 months, and 12 months). Methods: We retrospectively separated 85 patients with echocardiographic evidence of severe AS before TAVR procedure into two groups based on sPAP level. An sPAP of 40 mmHg was considered the cut-off value, with the absence of PH defined by an sPAP < 40 mmH (n = 32) and the presence of PH defined by an sPAP ≥ 40 mmHg (n = 53). Blood samples were drawn from each patient one day before TAVR and 24 h, 96 h, 3 months, and 12 months after TAVR. Plasma concentrations of the cardiovascular biomarkers sST2, GDF-15, H-FABP, IGF-BP2, and suPAR were determined and analyzed with univariate and multivariate binary logistic regression and AUROC curves. Results: Patients with severe AS and an sPAP ≥ 40 mmHg had significantly higher plasma concentrations of H-FABP (baseline: p = 0.022; 24 h: p = 0.012; 96 h: p = 0.037; 3 months: p = 0.006; 12 months: p = 0.030) and IGF-BP2 (baseline: p = 0.029; 24 h: p = 0.012; 96 h: p = 0.001; 3 months: p = 0.015; 12 months: p = 0.022) before and continuously up to 12 months after TAVR than did patients with an sPAP < 40 mmHg sST2, with the exception of the 12-month follow-up. We also consistently found significantly higher plasma concentrations in the sPAP ≥ 40 mmHg group (baseline: p = 0.007; 24 h: p = 0.006; 96 h: p = 0.014; 3 months: p ≤ 0.001; 12 months: p = 0.092), whereas suPAR had significantly elevated values at baseline and after 24 h in patients with echocardiographic evidence of PH and significantly decreased values after 3 months (baseline: p = 0.003; 24 h p = 0.041; 96 h: p = 0.127; 3 months: p = 0.006; 12 months: p = 0.477). Plasma concentrations of GDF-15 were only significantly different after 24 h (baseline: p = 0.075; 24 h: p = 0.016; 96 h: p = 0.101; 3 months: p = 0.244; 12 months: p = 0.090). In a multivariate binary logistic regression, atrial fibrillation, tricuspid annular plane systolic excursion (TAPSE), and sST2 at baseline were found to have a significant p-value < 0.050. Conclusion: In this descriptive study, sST2, H-FABP, and IGF-BP2 emerged as the cardiovascular biomarkers with the greatest potential with respect to echocardiographically PH detection in long-term follow-up after TAVR, as patients with an sPAP ≥ 40 mmHg had significantly continuously higher plasma biomarker concentrations than the corresponding cohort did, with an sPAP < 40 mmHg.


Introduction
Cardiovascular biomarkers have become proven tools used to predict developments of cardiovascular complications such as heart failure, myocardial infarction, or stroke. In particular, troponin in acute myocardial infarction and NT-proBNP in patients with acute or chronic heart failure are biomarkers that have already found their place in clinical routine [1][2][3]. In addition to these underlying cardiac diseases, several studies have already investigated the relationship between severe primary degenerative aortic valve stenosis (AS) and biomarkers [4,5].
Primary degenerative AS is the second most common valvular heart disease in the Western world after mitral valve regurgitation and is associated with a high 1-year mortality in symptomatic patients [6]. In patients <75 years of age and with few comorbidities, surgical valve replacement is still favored, whereas patients of advanced age and with a high frailty score are referred to the interventional technique by means of transcatheter aortic valve replacement (TAVR) ( [7]; 2021 ESC/EACTS Guidelines Valvular Heart Disease).
The typical symptom triad consisting of chest pain, syncope, and dyspnea occurs simultaneously only very rarely. The leading symptom in most cases is dyspnea, which is mainly due to elevated pressures in the left ventricle and left atrium and pulmonary circulation. Consecutive remodeling of the pulmonary vasculature results in concomitant left ventricular postcapillary pulmonary hypertension (PH), which occurs in 11 to 75% of patients, depending on the literature.
PH is often a life-limiting factor in patients with severe AS despite successful TAVR replacement and is associated with worse clinical outcome. In the past, right heart catheterization was routinely practiced as the gold standard to detect postcapillary PH before TAVR. Currently, this is only performed in larger cardiological centers for special indications. Instead, the detection of PH is assessed non-invasively by echocardiography and the determination of systolic pulmonary artery pressure (sPAP) [8][9][10].
Although numerous studies have been published on cardiovascular biomarkers and primary degenerative AS, little has been reported on the association between PH in patients with severe AS and biomarker expression.
The aim of this study was to identify novel cardiovascular biomarkers, such as soluble suppression of tumorigenicity-2 (sST2), growth differentiation factor-15 (GDF-15), hearttype fatty acid binding protein (H-FABP), insulin-like growth factor binding protein 2 (IGF-BP2), and soluble urokinase-type plasminogen activator receptor (suPAR), in patients with severe AS before TAVR and over time after TAVR (24 h, 96 h, 3 months, and 12 months) to gain information on biomarker dynamics after successful intervention. The study population was retrospectively divided into two groups based on echocardiographically determined sPAP, with the presence of PH defined as an sPAP ≥ 40 mmHg. The two study groups (sPAP < 40 mmHg; sPAP ≥ 40 mmHg) were subsequently compared with respect to plasma concentrations of the aforementioned biomarkers.

Study Population
Our study included 85 patients with severe primary degenerative AS planning to undergo the TAVR procedure between 2016 and 2018. Data analyses were performed at the Paracelsus Medical University Hospital Salzburg in accordance with the principles of the Declaration of Helsinki and good clinical practice. Approval of the study protocol was done by the local ethics committees of the Paracelsus Medical University Salzburg (415-E/1969/5-2016). Written informed consent for study participation was obtained from all patients.

Transthoracic Echocardiography
Common ultrasound devices (iE33; Philips Healthcare, Hamburg, Germany) were used to perform transthoracic echocardiography. Severe AS was classified according to the current valid guidelines of European Society for Cardiology. 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 -calculated by using continuity equation-formed the definition of severe AS. Since patients with low-flow, low-gradient AS were excluded from the study, the patient population in this study included solitary individuals with high pressure gradients without low-flow AS. Simpson's method was calculated to obtain the left ventricular ejection fraction (LVEF). Spectral and color-Doppler images were generated to identify mitral, aortic, and tricuspid valve regurgitation as mild (I), moderate (II), and severe (III). The maximum tricuspid regurgitant jet velocity (TRV) was obtained by continuous-wave Doppler over the tricuspid valve and was used to calculate the pulmonary artery pressure (PAP) via the formula 4 × TRV 2 . Finally, in order to correctly estimate the sPAP, it was necessary to determine the right atrial pressure (RAP). This corresponded to the central venous pressure and was determined by the diameter of the inferior vena cava (IVC). The RAP determination was performed uniformly in Salzburg according to the following method: A RAP of 15 mmHg was assumed, with an IVC diameter ≥21 mm and a respiratory caliber fluctuation <50%. 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 the restrictions mentioned above were ascribed an intermediate value of 8 mmHg. Finally, the simplified Bernoulli equation (4 × TRV 2 ) + RAP led to an sPAP result. An sPAP ≥ 40 mmHg was used as the cut-off value to determine PH in accordance with the current literature [11][12][13][14].

TAVR Procedure
The indication for TAVR was made by a multidisciplinary team consisting of cardiologists and cardiologic surgeons. TAVR procedure was performed as previously described [15]. Therefore, all 85 patients received TAVR via transfemoral access using a CoreValve prothesis (Medtronic, Dublin, Ireland).

Biomarker Analysis
Blood samples were obtained one day before the TAVR procedure and at follow-up times 24 h, 96 h, 3 months, and 12 months after TAVR using a vacuum-containing system. The collection tubes were centrifuged, and the plasma was separated from the blood components and frozen at −80 • C to analyze all samples at similar time points under the same conditions.
The plasma levels of sST2, GDF-15, H-FABP, IGF-BP2, and suPAR were measured using enzyme-linked immunosorbent assay (ELISA) kits (sST2: Duoset DY523, GDF-15: DY957, H-FABP: DY1678, IGF-BP2: DY674, and suPAR: DY807; R&D Systems, Minneapolis, MN, USA). The instructions were followed as described by the manufacturer. The ELISA plates (Nunc MaxiSorp flat-bottom 96 well plates, VWR International GmbH, Vienna, Austria) were pretreated overnight with a diluted capture antibody. After the appropriate washing processes and blocking with a reagent diluent, the plates were ready for the final assay procedure. After putting serum samples and standard protein onto the wells of ELISA plates (Nunc MaxiSorp flat-bottom 96 well plates, VWR International GmbH, Vienna, Austria), the probes were incubated for two hours. Afterward, the plates were washed and treated with Tween 20/PBS solution (Sigma Aldrich, St. Louis, MO, USA). A biotin-labeled antibody (detection antibody) was diluted in a reagent diluent and was added for another two hours. A further washing process was performed, and the probes were treated with streptavidin-horseradish peroxidase solution. A color reaction was generated after adding tetramethylbenzidine (TMB; Sigma Aldrich, St. Louis, MO, USA). Optical density was determined at 450 nm on an ELISA plate reader (iMark Microplate Absorbance Reader, Bio-Rad Laboratories, Vienna, Austria).

Statistical Analysis
Statistical analysis was performed using SPSS (Version 25.0, SPSSS Inc., Armonk, NY, USA). Graphical representations were created using GraphPad Prism (Version 8.0.0, GraphPad Software, San Diego, CA, USA) in addition to SPSS.
The Kolmogorov-Smirnov test was applied to test variables for normal distribution. Normally distributed metric data were expressed as the mean ± standard deviation (SD) and analyzed using an unpaired Student's t-test. Abnormally distributed metric data were expressed as the median and interquartile range (IQR), and the Mann-Whitney U test was applied for statistical analysis. Frequencies/percentages were used for categorial clinical data and compared using the chi-squared test. The Friedman test with pairwise comparison was used to test dependent samples (biomarker change over time within the sPAP group < 40 mmHg and within the sPAP group ≥40 mmHg).
To determine the optimal cut-off value according to an sPAP ≥ 40 mmHg and different serum biomarker levels over time, the area under the receiver operating characteristics (AUROC)-curves and a separate analysis of the Youden index (YI) was performed.
To recognize possible influencing factors regarding the presence of PH (sPAP ≥ 40 mmHg), a univariate binary logistic regression analysis was first completed. For better comparability, a Z transform was absolved for metric data. Subsequently, multivariate binary logistic regression was performed to assess the independent factors regarding the prediction of PH. Therefore, the covariates associated with the detection of PH in the univariate analysis (p = 0.100) were entered, and a backward variable elimination was carried out.
A p-value < 0.050 was considered statistically significant.

Study Cohort
Eighty-five patients with severe AS from the University Hospital of Salzburg were enrolled in the study. Echocardiographically, 32 patients (37.6%) showed an sPAP < 40 mmHg, which was equivalent to the absence of a PH, whereas 53 patients (62.4%) showed an sPAP ≥ 40 mmHg, which was consistent with the echocardiographic criterion of PH. Table 1 demonstrates the baseline characteristics of the overall cohort as well as those of the patients with sPAP < 40 mmHg and sPAP ≥ 40 mmHg. The sPAP groups were compared for significance, and the corresponding p-values were documented.

Baseline Characteristics of the Study
The overall cohort had a mean age of 82.34 ± 5.41 with a gender distribution male:female ratio of 50.60% vs. 48.40%. Regarding cardiovascular risk factors and concomitant diseases, 24.70% presented with diabetes mellitus, 77.60% with arterial hypertension, 9.40% with pAVK, 8.20% with COPD, and 61.20% with coronary artery disease. Echocardiographic left ventricular ejection fraction (LVEF) averaged 53.42 ± 9.99%, and sPAP was 46.36 ± 17.49 mmHg.
The plasma level of H-FABP in severe AS patients with echocardiographically proven PH was already at a significantly higher baseline level (baseline: p = 0.022). Additionally, H-FABP remained at a constant, significantly higher level in contrast to the sPAP < 40 mmHg group (24 h: p = 0.012; 96 h: p = 0.037; 3 months: p = 0.006; 12 months: p = 0.030).

Binary Logistic Regression
In order to verify a relevant statistical relationship between the presence of PH and other factors (especially investigated biomarkers), a univariate and a multivariate binary logistic regression analysis were performed (Table 3).
In the univariate analysis, the STS score, presence of atrial fibrillation, low tricuspid annular plane systolic excursion (TAPSE), creatinine, brain natriuretic peptide (BNP), cardiac troponin (cTNI), hemoglobin, sST2 at baseline, H-FABP at baseline, and IGF-BP2 at baseline showed a relevant association (p < 0.100); thus, multivariate analysis was performed with these variables. Only the atrial fibrillation, TAPSE, and sST2 at baseline were found to have a significant p-value < 0.050.

Discussion
This study focused on the plasma concentration trends of novel cardiovascular biomarkers in patients with severe AS in relation to the presence or absence of PH as determined by echocardiography. In fact, there are only few comparative studies in the literature investigating biomarker trends in severe AS patients with additional PH.

sST2
sST2 is one of two isoforms of the gene suppression of tumorigenicity-2 (ST2), which is a member of the Toll-like/IL-1 receptor family [16]. The counterpart of the soluble isoform sST2 is the transmembrane form ST2L, which forms a ligand-receptor complex with interleukin (Il-)33 to avert complex cardiac remodeling and fibrosis processes. Increased shear forces in the context of mechanical stress on the heart and lungs trigger increased secretion of sST2 from alveolar epithelial cells and cardiomyocytes. sST2 binds with markedly higher affinity to Il-33 and thus inhibits the cardioprotective signaling cascade with the membrane-associated ST2L [17,18].
This knowledge of the molecular mechanism is important in understanding the results presented here. Severe AS patients with (sPAP ≥ 40 mmHg) and without (sPAP < 40 mmHg) PH showed a similar curve progression as the overall cohort of Mirna et al. [19], who observed an additional increase in plasma concentration of sST2 in a time frame of 7 days after TAVR. In our study, the highest peak was measured after 24 h and then fell again after 96 h in patients with both an sPAP < 40 mmHg and ≥40 mmHg to then settle at this plasma level. The finding that, almost universally, patients with an sPAP ≥ 40 mmHg had significantly higher plasma sST2 concentrations than the comparison cohort with an sPAP < 40 mmHg is likely of prognostic relevance. The exception here was the determined plasma concentrations after 12 months; however, even here a relevant trend emerged with a p = 0.092. Additionally, it could be shown in multivariate binary logistic regression that sST2 was a relevant independent predictor for the detection of potential PH. This has already been confirmed recently by our own working group using a collective of patients with severe AS and available right heart catheterization data, on the basis of which PH was verified by the absolute gold standard [20]. It may well be hypothesized that removal of the aortic valve stenosis decreases the corresponding left ventricular mechanical stress; however, the already fixed pulmonary hypertension leads to continued secretion of sST2 from cardiomyocytes during sustained right ventricular stress.

GDF-15
GDF-15 as a member of the transforming growth factor beta (TGF-β) superfamily plays a relevant role in inflammatory processes, tissue injury, and apoptosis in the context of cardiovascular and pulmonary diseases [21,22]. The secretion of this growth factor occurs in numerous organs and thus also in various cells such as macrophages, cardiomyocytes, pulmonary endothelial cells, and vascular smooth muscle cells [23,24].
In the first 24 h after TAVR, there was a significant increase in the plasma concentration of GDF-15 in both the sPAP < 40 mmHg and sPAP ≥ 40 mmHg groups compared with baseline. This dropped again after 96 h, similar to sST2, which explains why Mirna et al. [19] did not observe significant changes in plasma concentration in their total cohort of severe AS patients after 7 days. In contrast to Mirna et al., plasma concentrations of GDF-15 in this study, however, were significantly increased in both groups after 3 months compared with baseline levels and persistently increased over time.
In a study by Gumaskiene et al. [25], GDF-15 was significantly elevated (p = 0.030) in the group of echocardiographically proven PH patients compared with non-PH patients. In our study, a p = 0.075 showed only a trend regarding the association between increased GDF-15 plasma level and increased sPAP. This may be due to the fact that Gumaskiene et al. set the sPAP cut-off value for the presence of PH at 45 mmHg, which was slightly higher than in our cohort. Fabiani et al. [26] pointed out in their study that GDF-15 represents a relevant biomarker that is not only elevated in patients with severe AS but also correlates with a patient's frailty. Apparently, this marker does not allow a good differentiation between severe AS patients with and without PH based on our data.

H-FABP
H-FABP is a protein that is secreted solitarily by cardiomyocytes during acute stress responses of the heart and specializes in lipid metabolism and the transport of fatty acids from the cell membrane to mitochondria [27]. Due to its small size of only 15 kDa, it is released more rapidly into the bloodstream during a myocardial infarction and has recently been proposed as a promising biomarker for an even earlier detection of myocardial infarction than troponin [28]. There are also promising data for the risk stratification of pulmonary artery embolism [29,30].
In particular, in patients with PH caused by left heart diseases or lung diseases, the plasma concentrations of H-FABP were significantly increased. This statement was made by Mirna et al. [31] in a cohort of PH patients with various underlying diseases. This could be reconciled with the data presented here, as severe AS patients with an sPAP ≥ 40 mmHg showed persistent and significantly higher plasma concentrations of H-FABP both before and up to 12 months after TAVR and thus may well be handled as a valuable biomarker for non-invasive detection of PH. This result can be interpreted as a stress situation exerted on the heart before TAVR by a chronic pressure load of severe AS with the corresponding vascular alteration at the pulmonary vessels, which continues beyond successful interventional valve replacement due to the persistent remodeling of the pulmonary pathway.

IGF-BP2
Insulin-like growth factor (IGF)-1 is a relevant growth factor produced by stimulation of growth hormone (GH) that has anabolic or proliferative effects on numerous organs. In the heart, decreased plasma IGF-1 concentrations are associated with an increased risk of cardiovascular events [32,33] due to the upregulation of the renin-angiotensin-aldosterone system [34]. As a member of the insulin-like growth factor family, IGF-BP2 exhibits an inhibitory effect on IGF-1, and therefore increased IGF-BP2 plasma concentrations lead to an effective reduction of IGF-1, which in turn leads to unimpeded cardiac remodeling [35].
In this study, IGF-BP2 had a similar prognostic value as H-FABP since the sPAP ≥ 40 mmHg group showed consecutive higher IGF-BP2 plasma levels both before and 12 months after TAVR than the sPAP < 40 mmHg group did. This finding is consistent with a study by Yang et al. [36], who demonstrated elevated circulating IGF-BP2 in patients with PH, which was associated with an increased risk of disease severity and mortality. Thus, in our study, IGF-BP2 is also a biomarker with solid discriminatory power to distinguish between elevated and depressed sPAP.

suPAR
suPAR is an inflammatory protein that has been known for several years to show a certain immune dysfunction, especially in the context of cardiovascular disease [37]. Increased plasma concentrations of suPAR have been observed in patients with coronary artery disease, myocardial infarction, chronic heart failure, and severe AS [38]. Additionally, patients with PH showed significantly increased suPAR levels compared with the normal collective [31].
In our study, suPAR showed significantly increased plasma levels at baseline, at 24 h, and at 3 months in patients with echocardiographically determined PH. However, suPAR plasma concentrations were almost simultaneously at the same level after 12 months, making non-invasive conclusions about the presence of PH difficult in this case, especially long-term.

Conclusions
This intentionally descriptive study was able to demonstrate with relative simplicity that cardiac biomarkers have the potential to predict the absence or presence of PH. Specifically, sST2, H-FABP, and IGF-BP2 demonstrated themselves as the cardiovascular biomarkers with the greatest potential with respect to echocardiographical PH detection in long-term follow-up after TAVR, as patients with an sPAP ≥ 40 mmHg had significantly continuously higher plasma biomarker concentrations than the corresponding cohort with an sPAP < 40 mmHg did. Once again, sST2 should be highlighted separately, as it was able to show its potential as an independent marker for PH prediction in binary logistic regression.

Limitation
Our single-center study is based on data from a small cohort over a circumscribed period of time (2016-2018). In addition, technical pitfalls in echocardiographic and laboratory measurements which lead to misclassifications should always be given a place, even though the examinations were performed by experienced clinical and laboratory investigators.

Outlook
The goal of follow-up studies is to figure out how biomarker concentrations behave before and after TAVR in dependence on the determined sPAP course. Further questions could be whether certain biomarkers in patients with elevated sPAP values before TAVR and normalization of sPAP after TAVR decrease in their plasma-level concentrations and behave like patients with initially normal sPAP values, or whether there are plasma-level concentrations of biomarkers that make it possible to predict whether initial PH before TAVR persists after TAVR and could therefore be prognostically relevant. To answer these research questions, large prospective studies with serial laboratory chemical determinations of biomarker concentrations over time and echocardiographic follow-up with sPAP determination are necessary in the further course.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.