Summary
Aortic stenosis (AS) is associated with alterations in the concentrations of nitric oxide (NO)-related pathway molecules, including arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA). We determined the effect of transcatheter aortic valve implantation (TAVI) on the concentrations of arginine, ADMA, and SDMA and evaluated their predictive value for post-TAVI outcomes. We found that plasma concentrations of arginine, ADMA, and SDMA were not affected by TAVI and did not predict post-TAVI MACE. ADMA and SDMA negatively correlated with AS severity, which might indicate alterations in the NO/L-arginine pathway in patients with AS.
Introduction
Aortic stenosis (AS) is the most common primary valvular disease in Europe and North America, requiring interventional treatment, with increasing prevalence due to the aging of the population and expanding indications [1]. Transcatheter aortic valve implantation (TAVI) is recommended for patients above 75 years of age, at high perioperative risk as evaluated using the STS-PROM or EuroSCORE II score or otherwise disqualified from surgical aortic valve replacement (SAVR) [2]. TAVI is also expanding to intermediate- and low-risk patients with comparable 5-year outcomes as SAVR [3, 4]. Although TAVI is less invasive than SAVR, it is still associated with about 15% annual risk of major adverse cardiovascular events (MACE), including acute myocardial infarction (MI), stroke, and cardiovascular death [3]. The main risk factors of post-TAVI MACE include decreased left ventricular ejection fraction (LVEF), reduced level of hemoglobin, atrial fibrillation (AF), and frailty [5–7].
Nitric oxide (NO)-pathway related molecules have been identified as potential prognostic biomarkers in patients with AS. NO is recognized for its vasodilatory effects, playing a crucial role in the regulation of vascular tone and hemodynamics. Furthermore, NO has demonstrated antiplatelet, antiproliferative, antiadhesive, and anti-inflammatory properties [8]. It is synthesized by endothelial nitric oxide synthase (NOS) from L-arginine and oxygen. Dimethylarginines (asymmetric dimethylarginine – ADMA and symmetric dimethylarginine – SDMA), which are endogenous competitive inhibitors of NOS, hinder NO formation via diverse mechanisms. ADMA competes for conversion of catalytic substrate [9–11], whereas SDMA inhibits arginine transport [12]. ADMA is eliminated from the body by a combination of renal excretion and metabolism by dimethylarginine dimethylaminohydrolase (DDAH), whereas SDMA is eliminated entirely by renal excretion. Therefore, renal function and DDAH expression and activity are important factors determining ADMA and SDMA levels.
In a meta-analysis of 34 studies including 32,428 patients, ADMA and SDMA were independent risk predictors of cardiovascular mortality and MACE in the general population, and in patients with cardiovascular disease (CVD) and chronic kidney diseases [13, 14]. In a study including 10 patients with severe aortic AS and 9 healthy controls, ADMA and SDMA were identified among 17 molecules that differentiated AS patients from controls [13]. The authors postulated that along with other biomarkers, they could be used to identify AS patients requiring cardiac surgery [15]. There is also evidence for ADMA being a predictor of mortality in patients with heart failure and in patients undergoing percutaneous coronary interventions or coronary angiography [16, 17]. Apart from NOS inhibitors, altered concentrations of NO-related pathway molecules may also play a role in endothelial dysfunction. For example, a deficit of NO precursor L-arginine has also been implicated in CVD development [17–22].
AS is associated with turbulent blood flow and increased shear stress as blood passes through the narrowed valve orifice, leading to oxidative stress. Oxidative stress leads to oxidative inactivation of NO by excess superoxide, with a subsequent decrease in NO availability and alterations in NO-related pathway molecules. For example, previous studies showed that plasma concentrations of ADMA are elevated in patients with AS compared to healthy controls [23]. However, there are currently no data regarding the potential predictive value of NO pathway-related molecules in AS patients undergoing TAVI. Considering the potential adverse effects of ADMA and SDMA on the vascular endothelium and their predictive role for cardiovascular events in patients with various CVD, we hypothesized that the levels of ADMA and SDMA decrease following the restoration of normal hemodynamics after TAVI and that they could be used to optimize risk stratification in AS patients after TAVI.
Aim
We aimed to determine the effect of TAVI on the concentrations of arginine, ADMA, and SDMA and to evaluate their predictive value for post-TAVI MACE.
Material and methods
Study design
This was a prospective study conducted at three reference centers in Poland, performing 100–200 TAVIs annually, in collaboration with the Department of Experimental Physiology and Pathophysiology, Medical University of Warsaw, Poland. The study protocol, designed in compliance with the Declaration of Helsinki, was approved by the Ethics Committee of Medical University of Warsaw (approval number: KB/128/2018, updated as KB/4/A2021). Informed consent for inclusion in the study was obtained from all patients.
Selection of patients
Consecutive patients diagnosed with severe AS and deemed eligible for TAVI by the local Heart Teams were enrolled. Severe AS was defined as aortic valve area (AVA) < 1.0 cm2 or indexed AVA < 0.6 cm2/m2 as calculated by the continuity equation on transthoracic echocardiography (TTE). In patients with low-flow, low-gradient AS and reduced LVEF, dobutamine stress echocardiography was performed to differentiate between true severe AS and pseudo-severe AS, and in patients with low-flow, low-gradient AS, and preserved LVEF, computed tomography was performed to assess aortic valve calcium score [24]. Exclusion criteria were valve-in-valve TAVI, chronic kidney disease (estimated glomerular filtration rate < 30 ml/min/1.73 m2, according to the Cockcroft-Gault formula), autoimmune diseases, active neoplastic disease, pregnancy, and breast-feeding. All patients provided informed written consent.
Clinical data collection
Demographic and clinical data were collected during the index hospitalization. A follow-up visit in the outpatient clinic was scheduled at 12 (3) months after TAVI, when follow-up TTE was performed and data regarding MACE (all-cause death, cardiovascular death, myocardial infarction, stroke, transient ischemic attack (TIA), decompensation of heart failure, need for re-intervention, and valve thrombosis) were recorded.
Treatment
TAVI was performed by an interventional cardiologist (J.K., Z.H., B.R.) and a thoracic surgeon (R.W.) in a hybrid operating room. Pharmacotherapy after TAVI included dual antiplatelet therapy (acetylsalicylic acid (ASA) or clopidogrel) for 6 months, followed by lifelong ASA treatment in patients with no indication for oral anticoagulation (OAC), or OAC if required [24]. Other drugs were administered at the discretion of the treating physician.
Sample collection and handling
Blood samples were collected at two time points: 1 day before TAVI and 5–7 days following the procedure (at hospital discharge). Blood was collected in 7.5 ml ethylenediaminetetraacetic acid (EDTA) plastic tubes (S-Monovette, Sarstedt) via antecubital vein puncture. Within 15 min after blood collection, plasma was prepared by double centrifugation (2500 g, 15 min, 20°C). Supernatant was collected and stored at –80°C until analyzed.
Evaluation of nitric oxide pathway-related molecules
Plasma concentrations of dimethylarginines (ADMA, SMDA) and endogenous metabolite of the NO pathway (arginine) were determined by the UPLC/MS/MS method. A Waters Acquity ultra performance liquid chromatography system coupled with a Waters TQ-S triple-quadrupole mass spectrometer was used for analyses. Analytes were separated using a Waters HILIC column (1.7 µm, 2.1 mm × 50 mm). Mobile phase A was NH4OH in water (1 ml of 25% ammonium hydroxide in 1 l of water) and mobile phase B was formic acid in acetonitrile (1 ml of formic acid in 1 l of acetonitrile). The mass spectrometer was operated in multiple-reaction monitoring (MRM) mode with positive electrospray ionization (ESI+). Samples were prepared by adding 100 µl of acetone containing internal standards to 10 µl of the sample (calibrators, plasma). After vortexing and centrifuging the mixture, 7 µl was injected. The quantification limits (LOQ) were 4.05 ng/ml, 3.64 ng/ml, and 702.89 ng/ml for ADMA, SDMA, and arginine, respectively.
Endpoints
The primary end-point was the change in plasma concentration of ADMA from baseline to post-TAVI. The secondary end-points were the changes in plasma concentrations of arginine and SDMA from baseline to post-TAVI and the predictive value of ADMA, SDMA, and arginine for the occurrence of MACE during the follow-up period.
The sample size was calculated for the mean difference in primary endpoint. In the previous study, the concentration of ADMA was 2-fold higher in patients with severe AS, compared to mild AS, with a standard deviation (SD) of 0.5 [23]. The required sample size was calculated by a two-sided t-test at a significance level of 0.05 with the following assumptions: (i) mean difference between the groups = 1, (ii) SD in each group = 2.0, and (iii) nominal test power = 0.9. Based on this sample size estimation, a total of 86 patients should be enrolled in the study to observe a mean difference in ADMA concentration from baseline to post-TAVI. Taking into account that up to 30% of patients can be potentially lost to follow-up, at least 120 patients should be included in the study.
Statistical analysis
Statistical analyses were conducted using IBM SPSS Statistics, version 27.0 (IBM, New York, USA). Categorical variables were presented as number and percentage and compared using the χ2 test. The Shapiro-Wilk test was used to assess normal distribution of continuous variables. Continuous variables were presented as mean with SD or median with interquartile range (IQR). Changes in NO pathway-related molecule concentrations before and after TAVI were calculated with the Wilcoxon signed-rank test. To assess the difference in variables between patients with and without MACE, the unpaired t-test or Mann-Whitney U-test was used to compare data with and without normal distribution, respectively. The Spearman correlation coefficient was used to evaluate correlations between plasma levels of NO pathway-related molecules and echocardiographic parameters of AS severity. A two-sided p-value below 0.05 was considered significant.
Results
Between November 2018 and September 2021, 128 were enrolled in the study. The median duration of follow-up was 13.5 months (IQR 6.5–17.0 months). During the follow-up, 21 patients developed MACE (16.4%): 4 all-cause deaths, 5 cardiovascular deaths, 1 TIA, 8 decompensation of heart failure, 1 need for reintervention, and 2 valve thromboses.
Comparison of baseline characteristics between patients who experienced post-TAVI MACE and those with did not is shown in Table I. Patients who experienced MACE were older (p < 0.001), but there were no other differences regarding comorbidities, laboratory, echocardiographic, and procedural parameters, and pharmacotherapy at discharge between the groups.
Table I
Comparison of baseline characteristics between patients who experienced MACE and those with did not during a median follow-up
[i] AVA – aortic valve area, AVAi – aortic valve area index, CABG – coronary artery bypass grafting, COPD – chronic obstructive pulmonary disease, IQR – interquartile range, MACCE – major adverse cardiovascular and cerebrovascular events, MRA drugs – mineralocorticoid receptor antagonist drugs, PCI – percutaneous coronary intervention, RAAS inhibitors – renin-angiotensin-aldosterone system inhibitors, TIA – transient ischemic attack.
There were no significant differences regarding concentrations of arginine, ADMA, and SDMA plasma concentrations before and after TAVI (p ≤ 0.70 for all; Figures 1 A–C). There were also no differences between the concentrations of NO pathway-related molecules measured before and after TAVI in patients who did and did not experience MACE during the follow-up period (p ≤ 0.88 for all; Figures 2 A–F).
Figure 1
Comparison of nitric oxide pathway-related molecules before and after transcatheter aortic valve implantation (A–C)
ADMA – asymmetric dimethylarginine, SDMA – symmetric dimethylarginine, TAVI – transcatheter aortic valve implantation.
Figure 2
Comparison of nitric oxide pathway-related molecules measured before TAVI (A–C) and after TAVI (D–F) in patients who did and did not experience MACE during the follow-up period
ADMA – asymmetric dimethylarginine, SDMA – symmetric dimethylarginine, TAVI – transcatheter aortic valve implantation.
Correlations between plasma levels of NO pathway-related molecules and echocardiographic parameters of AS severity before TAVI are shown in Table II and Figure 3. There was a significant negative correlation between plasma level of ADMA and SDMA and AVAi (p < 0.01 for both).
Table II
Correlations between plasma levels of NO pathway-related molecules and echocardiographic parameters of aortic stenosis severity before and after TAVI. Significant correlations are shown in bold and indicated with an asterisk. *p < 0.05, **p < 0.01, ***p < 0.001
| Pre TAVI | LVEF | Gradient max | Gradient mean | AVAi |
|---|---|---|---|---|
| ADMA | –0.04 | –0.1 | –0.05 | –0.25** |
| SDMA | –0.04 | –0.13 | –0.18 | –0.36** |
| Arginine | 0.12 | –0.01 | –0.01 | 0.02 |
Figure 3
Correlations between plasma levels of nitric oxide pathway-related molecules and echocardiographic parameters of AS severity before TAVI
ADMA – asymmetric dimethylarginine, SDMA – symmetric dimethylarginine, TAVI – transcatheter aortic valve implantation.
A summary of the study results is presented in Figure 4.
Figure 4
Summary of study results. Plasma concentrations of NO pathway-related molecules (ADMA, SDMA, arginine) do not change after TAVI and do not predict post-TAVI MACE. ADMA and SDMA negatively correlate with AS severity, which might indicate alterations in NO/L-arginine pathway in patients with AS
Discussion
This study is the first to examine the effect of TAVI on NO-related pathway molecules (ADMA, SDMA, arginine) and the predictive value of these molecules for post-TAVI MACE. The main findings of our study are that (i) plasma concentrations of NO pathway-related molecules do not change after TAVI, compared to the baseline, (ii) the evaluated NO pathway-related molecules do not predict post-TAVI MACE during the median follow-up period of 13.5 months, (iii) the concentrations of NO pathway-related molecules correlate with echocardiographic parameters of AS severity.
Previous studies showed that AS is associated with higher concentration of ADMA [23, 25], with a positive correlation between plasma ADMA concentration and AS severity, suggesting NO/L-arginine pathway impairment in AS patients and implying that TAVI or SAVR could decrease ADMA concentration. Although our hypothesis was that TAVI leads to a decrease in plasma level of ADMA and SDMA, this study did not confirm it. The lack of differences in NO pathway-related metabolites before and after TAVI in our study might be due to the confounding factors which we did not measure in our study, such as the differences in pre- and post-TAVI renal function, differences in expression and activity of ADMA metabolizing enzyme, DDAH, or differences in oxidative stress parameters. However, other authors also found no difference between ADMA plasma concentrations before and after TAVI, which is in line with our results [26]. The effects of TAVI on SDMA and arginine concentrations have not previously been studied. We did not observe any significant changes in SDMA and arginine concentrations. However, in one study, arginine plasma concentrations gradually increased from day 1 to day 7 after TAVI, which was associated with left ventricle mass regression at 1 year [27]. The authors postulated that arginine could be used as a predictor of left ventricle mass regression after TAVI and hypothesized that the left ventricle mass regression could be due to restoration of normal hemodynamics in AS patients after TAVI, with a subsequent decrease in myocardial stress. This would lead to restoration of normal NO/L-arginine pathway function, reflected in an increase in arginine concentration [27].
The predictive value of ADMA, SDMA, and arginine after TAVI has not been studied previously. ADMA inhibits the synthesis of endothelium-derived NO and thus contributes to endothelial dysfunction, which implicates the role of ADMA in the development of various vascular diseases [21]. In has been suggested that increased ADMA levels are associated with reduced protection of aortic valve endothelium against oxidative stress, possibly leading to AS development and progression [25, 28]. In another study, ADMA levels were associated with echocardiographic parameters of AS severity, with a positive correlation between ADMA and mean and maximum aortic gradients and a negative correlation between ADMA and AVA. Therefore, we anticipated that ADMA concentration might be predictive for post-TAVI MACE, which was not confirmed in our study. SDMA, in turn, is an indirect inhibitor of NOS, making it an important factor in endothelial dysfunction, similar to ADMA. It blocks NO/L-arginine pathways by inhibiting both NOS and L-arginine cellular intake [29]. Thus, we expected that SDMA might be an important predictor of outcomes after TAVI, which again was not confirmed by our results, nor in any other prior studies. L-arginine is an NO precursor and a substrate for NO synthesis. The lack of the precursor leads to decreased NO bioavailability and increases the risk of endothelial dysfunction [30]. In one study, L-arginine was demonstrated to prevent the pro-calcific differentiation of aortic interstitial valve cells via inhibition of osteoblast-like cell differentiation within the aortic valve [31]. The osteoblast-like cells, by their properties, initiate valve deformation followed by thickening of the cusps and finally lead to valve degeneration and AS development [32]. We hypothesized that L-arginine levels would increase after the TAVI procedure and predict post-TAVI MACE. This hypothesis was not supported by our findings.
Echocardiographic parameters of AS severity – such as transvalvular mean and maximal flow gradient and AVA/AVAi – are used as a diagnostic tool to determine patient eligibility for interventional treatment and reflect hemodynamic severity of LV overload. We found negative correlations between plasma levels of ADMA and SDMA and AVAi. Before TAVI, patients with lower AVAi had higher plasma levels of ADMA and SDMA, suggesting that greater AS severity is accompanied by higher release of NOS inhibitors, with a subsequent NO deficit. The NO deficit might be one of the mechanisms underlying higher risk of adverse cardiovascular events in patients with severe AS, including those not directly related to valve dysfunction, such as acute myocardial infarction and stroke [33, 34]. Moreover, AS progression is accelerated following acute myocardial infarction, which underlines the reciprocal relationship between AS and increased cardiovascular morbidity and mortality [35]. However, these correlations no not prove any relationship between NO pathway-related molecules and post-TAVI outcomes. Currently there is no evidence that NO/L-arginine pathway function is altered before and after TAVI and/or has predictive value for MACE [36–38].
Our study has several limitations. First, plasma levels of NO pathway-related molecules were measured only twice – before and after TAVI – which does not allow us to draw any conclusions regarding the long-term changes in the concentrations of these molecules during the follow-up period, nor their association with MACE. Second, the observed correlations between NO pathway-related molecules and echocardiographic parameters of AS severity do not prove any causal relationship between the roles of ADMA, SDMA, and arginine and adverse effects of AS on the cardiovascular system or positive post-TAVI outcomes. Third, plasma levels of ADMA and SDMA are affected by renal function and DDAH expression and activity. Hence, the difference in renal function before and after TAVI is an important parameter to consider when interpreting our results. Since we did not collect the laboratory data after TAVI, we could not take this parameter into account. Nevertheless, baseline renal function, reflected by the percentage of patients with pre-existing chronic kidney disease and by the baseline creatinine level, was comparable in patients with and without MACE. In addition, we did not measure the DDAH expression and activity, which could have affected the results. Since the activity of DDAH is reduced by oxidative stress, which is associated with cardiovascular disease, leading to elevated ADMA levels, the lack of oxidative stress parameters in our cohort is another limitation. Finally, all TAVI procedures were conducted by experienced high-volume teams, which eliminated the bias due to various expertise levels, but might limit the general applicability of the results.
Conclusions
Plasma concentrations of NO pathway-related molecules (ADMA, SDMA, arginine) do not change after TAVI and they do not predict post-TAVI MACE during the median follow-up period of 13.5 months. ADMA and SDMA negatively correlate with AS severity, which might indicate alterations in the NO/L-arginine pathway in patients with AS.
