Detrimental Effect of Plasma From Patients With Severe Aortic Stenosis on Valvular Endothelial Cells: Role of Proinflammatory Cytokines and Factor Xa

Antonin Trimaille; Sandy Hmadeh; Shinnosuke Kikuchi; Ali Mroueh; Adrien Carmona; Benjamin Marchandot; Francis Beras; Dinh Phi Truong; Manh Cuong Vu; Amandine Granier; Laurence Jesel; Patrick Ohlmann; Laurent Sattler; Valérie Schini‐Kerth; Olivier Morel

doi:10.1161/JAHA.125.041701

. 2025 Nov 26;15(6):e041701. doi: 10.1161/JAHA.125.041701

Detrimental Effect of Plasma From Patients With Severe Aortic Stenosis on Valvular Endothelial Cells: Role of Proinflammatory Cytokines and Factor Xa

Antonin Trimaille ^1,², Sandy Hmadeh ², Shinnosuke Kikuchi ^1,², Ali Mroueh ², Adrien Carmona ¹, Benjamin Marchandot ¹, Francis Beras ², Dinh Phi Truong ³, Manh Cuong Vu ¹, Amandine Granier ¹, Laurence Jesel ^1,², Patrick Ohlmann ^1,², Laurent Sattler ⁴, Valérie Schini‐Kerth ², Olivier Morel ^1,^2,^3,^✉

PMCID: PMC13055749 PMID: 41294140

Abstract

Background

The role of plasma and its components in the progression of aortic stenosis (AS) remains insufficiently investigated. This study aimed to assess whether plasma from patients with AS induces oxidative stress and contributes to valvular endothelial cells (VECs) dysfunction.

Methods

Human plasma samples were obtained from patients with severe AS (AS+, n=110), patients with cardiovascular risk factors without AS (n=30), and healthy individuals (n=15). Plasma levels of proinflammatory cytokines (IL [interleukin]‐1β, IL‐6, TNF [tumor necrosis factor]‐α, and factor Xa [FXa]) were measured. Porcine aortic VECs were then incubated with plasma (10%, 24 hours). Oxidative stress levels were assessed using dihydroethidium staining, NO formation with 4‐amino‐5‐methylamino‐2′,7′‐difluororescein diacetate, mRNA expression by quantitative reverse transcription‐polymerase chain reaction, and protein expression levels by Western blot analyses. Platelets and monocytes adhesion as well as thrombin generation were determined. Different pharmacological inhibitors were used to explore the underlying pathways.

Results

Plasma from patients with AS+ exhibited elevated levels of IL‐1β, IL‐6, TNF‐α, and FXa activity. Incubation of VECs with AS+ plasma induced a pro‐oxidant response mediated by proinflammatory cytokines, the angiotensin system‐SGLT2 (sodium‐glucose transport protein 2) pathway, and FXa. AS+ plasma also triggered VEC dysfunction, inflammation, monocytes and platelets adhesion, and thrombin generation. These detrimental effects were mitigated by empagliflozin, losartan, neutralizing antibodies targeting proinflammatory cytokines, and dabigatran and rivaroxaban.

Conclusions

Patients with AS displayed subclinical systemic inflammation and increased FXa activity. These processes contributed to aortic VEC dysfunction, inflammation, monocytes and platelets adhesion, and thrombin generation upon exposure to AS+ plasma.

Keywords: aortic stenosis, endothelial dysfunction, inflammation, SGLT2 inhibitors, thrombosis

Subject Categories: Valvular Heart Disease, Translational Studies, Oxidant Stress, Inflammation, Endothelium/Vascular Type/Nitric Oxide

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Nonstandard Abbreviations and Acronyms

AS: aortic stenosis
AS+: patients with aortic stenosis
AS−: patients without aortic stenosis with cardiovascular risk factors
HV: healthy volunteer
SGLT: sodium‐glucose transport protein
TF: tissue factor
VEC: valvular endothelial cell

Clinical Perspective.

What Is New?

Plasma from patients with severe aortic stenosis displays a specific phenotype characterized by elevated levels of proinflammatory cytokines and factor Xa activity, and plasma from patients with severe aortic stenosis strongly promotes oxidative stress in valvular endothelial cells, as compared with plasma from healthy individuals and from patients with cardiovascular risk factors without aortic stenosis.
This deleterious effect further leads to valvular endothelial dysfunction and the promotion of a prothrombotic, proinflammatory, and proadhesive phenotype.

What Are the Clinical Implications?

Further studies are needed to assess the potential therapeutic benefits of anti‐inflammatory and antithrombotic strategies in aortic stenosis and to determine the optimal timing for their introduction in the disease progression.

Aortic stenosis (AS) is the most prevalent valvular heart disease in developed countries. ¹ Despite its widespread occurrence, no medical treatment has been validated to prevent the thickening of the aortic valve and the reduction in its opening area. The only available therapeutic option to date remains surgical or transcatheter aortic valve replacement once the severity criteria are met. ² , ³ A comprehensive understanding of AS pathophysiology is therefore essential for identifying new therapeutic targets.

Initially thought to be a passive degenerative process, AS is now recognized as an active, multifaceted condition involving numerous cellular and molecular contributors. ¹ , ⁴ The progression of AS follows a chronological sequence, beginning with the damage and dysfunction of valvular endothelial cells (VECs) due to biomechanical forces acting on the aortic valve. This damage promotes intravalvular inflammation and neoangiogenesis, followed by myofibroblastic and osteoblastic differentiation of valvular interstitial cells. Additionally, several lines of evidence suggest a bidirectional interaction between the pathomechanisms of AS and various components of the hemostatic system, including platelets, tissue factor, thrombin, von Willebrand factor, and extracellular vesicles. ⁴ , ⁵ , ⁶

Given the pivotal role of VECs in maintaining valvular homeostasis under physiological conditions, and their early involvement in AS pathogenesis, they are considered key actors in the disease process. Although biomechanical factors have been identified as primary triggers of VEC dysfunction in the early stage of AS, ⁴ the molecular mechanisms underlying the interaction between plasma from patients with AS and aortic VECs remain unclear. Because plasma contains various biological effectors potentially contributing to endothelial cell dysfunction, including proinflammatory cytokines and hemostatic factors, ⁴ , ⁷ this study aimed to investigate whether plasma from patients with AS induces oxidative stress and contributes to VEC dysfunction.

METHODS

Study Design and Population

This prospective study enrolled 110 patients with severe AS (AS+) admitted to Strasbourg University Hospital, France, from May 2023 to January 2024, for scheduled transcatheter aortic valve replacement (TAVR). Severe AS was defined according to current guidelines. ² , ³ All cases were discussed within the heart team to confirm diagnosis and therapeutic decision. Arterial blood sampling was performed at the beginning of the TAVR procedure, just after sheath insertion and before any injection of heparin or other anticoagulant. Additionally, 15 healthy volunteers (HVs) and 30 patients without AS (AS−), referred to the cardiac catheterization laboratory for coronary angiography with at least 2 cardiovascular risk factors including active smoking, hypertension, dyslipidemia, diabetes, obesity, or familial history of cardiovascular disease, were enrolled for comparison.

Ethical approval for this study was obtained from the institutional review board (number FC 2016‐4), and written informed consent was obtained from all participants according to the principles of the Declaration of Helsinki. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Plasma Characterization, Cell Culture, and Biological Responses

VECs were isolated from the aortic valve of freshly collected male pig hearts from the local slaughterhouse using collagenase (type I) and cultured in MCDB 131 (Microcarrier Cell Development Buffer) with fungizone (2.5 μg/mL), penicillin (100 U/mL), streptomycin (100 μg/mL), L‐glutamine (2 mM), and 15% fetal calf serum, as described previously. ⁶ All experiments were performed with VECs at passage 1 exposed to a serum‐free culture medium for 2 hours before the addition of a treatment. In some experiments, VECs were exposed to a pharmacologic modulator for at least 30 minutes before the addition of plasma. Pharmacological modulators concentrations were selected based on prior studies on cultured endothelial cells. ⁶ , ⁷ , ⁸ All procedures on the use of animal tissues conformed to the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, and local approval was obtained.

The detailed methods of biological responses studies are extensively described in Data S1. Briefly, plasma levels of proinflammatory markers (IL [interleukin]‐1β, IL‐6, and TNF [tumor necrosis factor]‐α) were measured using commercial ELISA kits. FXa (factor Xa) activity was quantified using a fluorometer‐based method. Oxidative stress levels in VECs were assessed by fluorescence microscopy using the redox‐sensitive probe dihydroethidium, NO levels by fluorescence microscopy using the NO probe 4‐amino‐5‐methylamino‐2′,7′‐difluororescein diacetate, mRNA expression levels by quantitative reverse transcriptase–polymerase chain reaction, protein expression by Western blot analyses, thrombin generation on the surface of VECs using the chromogenic substrate of thrombin (ß‐Ala‐Gly‐Arg p‐nitroanilide diacetate), monocytes adhesion using fluorescent labeled THP‐1 cells, and platelet adhesion using fluorescent‐labeled washed human platelets.

Oxidative stress levels in VECs using dihydroethidium was used as the main surrogate marker of endothelial dysfunction following plasma stimulation, and was thus assessed for all plasma samples. Cytokines and FXa measurements were performed in the overall cohort in the limit of available plasma. For the other experiments, plasma samples with the higher activity (reactive oxygen species [ROS] formation in the highest tertile of the cohort) were used to achieve 3 to 6 different samples for each condition. Each replicate corresponded to plasma from a different patient and a separate VEC culture, ensuring biological variability in our experimental conditions.

An exploratory analysis was performed to assess oxidative stress levels in human coronary artery endothelial cells following stimulation with plasma samples from 20 patients who were AS+, 10 patients who were AS−, and 5 HVs. For this experiment, plasma samples exhibiting the highest activity on VECs were selected.

Statistical Analysis

For patients and plasma characteristics, normally continuous variables are presented as mean±SD, whereas nonnormally continuous variables are presented as median and interquartile range. The distribution normality was assessed with graphical methods for normality. Categorical variables are expressed as frequency with percentage. Continuous variables were compared with the Student t test or the Mann‐Whitney/Wilcoxon test (for 2 groups). To reduce imbalance in baseline characteristics and to minimize the potential effects of age and sex in the comparison of patients who were AS+ and AS−, the plasma characteristics were assessed using a 2:1 propensity score–matched population (patients who were AS+ versus AS−). A multivariable logistic regression model was built to estimate a propensity score for patient group, using group as the dependent variable and age and sex as covariates. A nearest‐neighbor algorithm was used to match patients who were AS+ and AS− in a 2:1 ratio, with a caliper width equal to 0.2.

For in vitro experiments, comparisons between or within 2 groups used the Student unpaired or paired t test, respectively. Comparisons of normally distributed variables among >2 groups used an ANOVA followed by Tukey post hoc test for multiple pairwise comparisons. When data were not normally distributed, a nonparametric Kruskal‐Wallis test followed by Dunn multiple comparison was used. Linear regression was used to compare 2 continuous variables with Pearson correlation presented.

A 2‐tailed P<0.05 was considered statistically significant. All statistical analysis was performed using R software (version 4.3.1; R Project for Statistical Computing) and GraphPad Prism (version 10 for Mac; GraphPad Software).

RESULTS

Patients’ Characteristics

The baseline characteristics of the study cohort are presented in Table 1. The mean age of the overall study population was 76 years, with HVs averaging 47 years of age, patients who were AS− averaging 71 years of age, and patients who were AS+ averaging 82 years of age. Hypertension, diabetes, and dyslipidemia were common in both patients who were AS− and AS+, without significant difference between these 2 groups. Smoking and family history of cardiovascular diseases were more frequently observed in patients who were AS− than AS+ (P<0.05 for both). No difference between patients who were AS− and patients who were AS+ was observed for medical history and comorbidities. No difference was observed for medications, including antithrombotic treatments, except for diuretics, which were used more in patients who were AS+ compared with patients who were AS− (P<0.001). Patients who were AS+ exhibited the typical echocardiographic features associated with AS, including an elevated mean transaortic gradient (42±12 mm Hg), increased maximal transaortic velocity (400±68 cm/s), and heightened systolic pulmonary artery pressure (38±13 mm Hg). The mean left ventricle ejection fraction was 55±14% in patients who were AS− and 58±15% in patients who were AS+ (P=0.3).

Table 1.

Baseline Characteristics of the Study Cohort (N=155)

Variables	Overall population, N=155	Healthy volunteers, N=15	Patients who were AS−, N=30	Patients who were AS+, N=110
Demographic characteristics
Age, y	76±14	47±15	71±9	82±7
Male sex	85 (54.8)	3 (20.0)	25 (83.3)	57 (51.8)
BMI, kg/m²	26.5±5.1	22.6±1.9	26.0±4.1	26.8±5.4
Cardiovascular risk factors
Hypertension	110 (71.0)	…	22 (73.3)	88 (80.0)
Diabetes	42 (27.1)	…	8 (26.7)	34 (30.9)
Dyslipidemia	91 (58.7)	…	24 (80.0)	67 (60.9)
Smoking	8 (5.2)	…	7 (23.3)	1 (0.9)
Family history of CVD	12 (7.7)	…	6 (20.0)	6 (5.5)
Sleep apnea	9 (5.8)	…	3 (10.0)	6 (5.5)
Medical history
CAD	62 (40.0)	…	15 (50.0)	47 (42.7)
Heart failure	28 (18.1)	…	6 (20.0)	22 (20.0)
Atrial fibrillation	45 (29.0)	…	7 (23.3)	38 (34.5)
Stroke	15 (9.68)	…	1 (3.3)	14 (12.7)
CKD^*	32 (20.6)	…	4 (13.3)	28 (25.5)
COPD	10 (6.45)	…	2 (6.7)	8 (7.3)
Cancer	28 (18.1)	…	4 (13.3)	24 (21.8)
Autoimmune disease	12 (7.7)	…	3 (10.0)	9 (8.2)
Medications
SAPT	63 (40.6)	…	16 (53.3)	47 (42.7)
DAPT	19 (12.3)	…	2 (6.67)	17 (15.5)
Aspirin	70 (45.2)	…	17 (56.7)	53 (48.2)
Clopidogrel	28 (18.1)	…	3 (10.0)	25 (22.7)
OAC	54 (34.8)	…	7 (23.3)	47 (42.7)
VKA	4 (2.6)	…	1 (3.3)	3 (2.7)
DOAC	48 (31.0)	…	6 (20.0)	42 (38.2)
ACEi	35 (22.6)	…	9 (30.0)	26 (23.6)
ARB	33 (21.3)	…	5 (16.7)	28 (25.5)
β‐Blocker	63 (40.6)	…	13 (43.3)	50 (45.5)
Mineralocorticoid receptor blocker	13 (8.4)	…	1 (3.3)	12 (10.9)
Statins	83 (53.5)	…	17 (56.7)	66 (60.0)
Diuretics	74 (47.7)	…	5 (16.7)	69 (62.7)
Metformin	25 (16.1)	…	4 (13.3)	21 (19.1)
SGLT2 inhibitors	17 (11.0)	…	1 (3.3)	16 (14.5)
Echocardiographic parameters
LVEF, %	57±15	…	55±14	58±15
Bicuspid aortic valve	6 (3.9)	…	0 (0.0)	6 (5.5)
Mean transaortic gradient, mm Hg	34±18	…	7±4	42±12
Maximal transaortic velocity, cm/s	347±116	…	168±32	400±68
Significant aortic regurgitation	21 (15.2)	…	5 (16.7)	16 (14.8)
Significant mitral regurgitation	9 (6.5)	…	1 (3.3)	8 (7.4)
Systolic PAP, mm Hg	37±13	…	30±11	38±13

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Values are mean±SD or n (percent). ACEi indicates angiotensin‐converting enzyme inhibitor; ARB, angiotensin receptor blocker; AS, aortic stenosis; AS+, patients with AS; AS−, patients with cardiovascular risk factors without AS; BMI, body mass index; CAD, coronary artery disease; CKD, chronic kidney disease; COPD, chronic obstructive pulmonary disease; CVD, cardiovascular disease; DAPT, dual antiplatelet therapy; DOAC, direct oral anticoagulant; LVEF, left ventricular ejection fraction; PAP, pulmonary artery pressure; OAC, oral anticoagulant; SAPT, single antiplatelet therapy; SGLT2, sodium‐glucose transport protein 2; and VKA, vitamin K antagonists.

^{^*}

CKD is defined as an estimated glomerular filtration rate <60 mL/min per 1.73 m².

Characterization of Proinflammatory Cytokines and FXa Activity Within Plasma

Higher levels of IL‐1β, IL‐6, and TNF‐α were observed in patients who were AS+ compared with values measured in both HVs and patients who were AS− (Figure 1 and Table 2). Additionally, FXa activity was also significantly higher in patients who were AS+ in comparison with HVs and patients who were AS− (Figure 1). In the AS+ cohort, no significant correlation was observed between aortic valve calcium score assessed by pre‐TAVR cardiac computed tomography and plasma cytokines levels (P>0.05 for all correlations tested).

All comparisons were performed by repeated measures analysis of variance followed by Tukey post hoc analyses (n=147 for IL‐1β, n=135 for IL‐6, n=131 for TNF‐α, n=131 for FXa).*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. AS indicates aortic stenosis; AS+, patients with AS; AS−, patients with cardiovascular risk factors without AS; FXa, factor Xa; HV, healthy volunteers; IL, interleukin; and TNF, tumor necrosis factor.

Table 2.

Circulating Cytokines, Factor Xa Activity, and Dihydroethidium Staining in Healthy Volunteers and Patients Who Were AS− and AS+

Variables	HVs, N=15	Patients who were AS−, N=30	Patients who were AS+, N=110	P value, AS+ vs HVs	P value, AS+ vs AS−
Overall population
IL‐1β, pg/mL	1.9±1.5	8.3±7.3	16.1±13.1	<0.001	0.004
IL‐6, pg/mL	11.7±7.9	37.8±25.2	192.3±153.9	<0.001	<0.001
TNF‐α, pg/mL	12.8±11.3	36.9±20.0	66.5±61.7	<0.001	0.013
FXa activity, ng/mL	25.0±20.5	79.8±33.5	128.6±75.5	<0.001	0.009
Dihydroethidium staining, AU	895±183	1197±150	1451±360	<0.001	<0.001
Propensity score–matched population
IL‐1β, pg/mL	…	8.9±7.5	15.8±13.5	…	0.016
IL‐6, pg/mL	…	38.1±26.9	181.0±120.0	…	<0.001
TNF‐α, pg/mL	…	42.4±20.1	59.8±46.5	…	0.06
FXa activity, ng/mL	…	81.9±30.2	114.0±61.9	…	0.026
Dihydroethidium staining, AU	…	1187±163	1384±324	…	0.004

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AS indicates aortic stenosis; AS+, patients with AS; AS−, patients with cardiovascular risk factors without AS; AU, arbitrary units; DHE, dihydroethidium; FXa, factor X activated; HVs, healthy volunteers; IL, interleukin; and TNF, tumor necrosis factor.

To assess the proper effect of AS on higher plasma cytokine levels and FXa activity, and to minimize the potential effects of age and sex, a sensitivity analysis was performed in a propensity score–matched population comparing 19 patients who were AS− and 38 patients who were AS+ (Table 2). The baseline characteristics of the propensity score–matched population are shown in Table S1. In this population, levels of IL‐1β, IL‐6, and FXa activity were significantly higher in patients who were AS+ than patients who were AS− (P<0.05 for all), whereas levels of TNF‐α remained increased in patients who were AS+ compared with patients who were AS−, without reaching statistical significance (P=0.06).

Pro‐oxidant State of AS Plasma‐Treated VECs

Because proinflammatory cytokines promoted oxidative stress in endothelial cells, ⁷ , ⁹ we used an in vitro approach to study the ability of AS+ plasma (n=110) to induce oxidative stress in VECs, as compared with AS− (n=30) and HV plasma (n=15). Cultured porcine VECs were exposed to either plasma at 10% for 24 hours. The exposure of VECs to AS+ plasma induced a significant increase in pro‐oxidant response in VECs with higher dihydroethidium values compared with plasma from patients who were AS− and from HVs in both the overall population (P<0.001) and in the propensity score–matched population (P=0.004) (Figure 2A and Table 2). The pro‐oxidant response of VECs induced by plasma samples was significantly correlated to the levels of IL‐1β (r=0.29; P<0.001), IL‐6 (r=0.47; P<0.001), TNF‐α (r=0.45; P<0.001), and FXa (r=0.12; P<0.001) (Figure 2B). No significant clinical differences, including sex or medications, were observed when patients were stratified by dihydroethidium value tertiles, except for the rate of atrial fibrillation, which was higher in the first tertile (P=0.03) (Table S2).

A, AS+ plasma (10%, 24 hours) induced a significant increase in pro‐oxidant response in VECs compared with HV and AS− plasma (n=155). B, Pro‐oxidant response of VECs to AS+ plasma was correlated to the levels of proinflammatory cytokines (IL‐1β, IL‐6, and TNF‐α) and FXa activity in plasma samples. C, Pro‐oxidant responses of VECs to AS+ plasma were inhibited by empagliflozin, perindoprilat, losartan, rivaroxaban, dabigatran, a single neutralizing antibody directed against either IL‐1β, IL‐6, or TNF‐α, and the PAR‐1, −2, and −4 inhibitors. Data are presented as mean±SD and compared by 1‐way ANOVA followed by Tukey multiple comparisons (A and C) or as correlation graphs with Spearman correlation test (B). Scale bars on the micrographs represent 100 μm. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Ab indicates antibody; AS, aortic stenosis; AS+, patients with AS; AS−, patients with cardiovascular risk factors without AS; AU, arbitrary units; Dabi, dabigatran; Empa, empagliflozin; FXa, factor Xa; HV, healthy volunteer; IL, interleukin; Los, losartan; PAR, protease‐activated receptor; Per, perindoprilat; Riva, rivaroxaban; SGLT2, sodium‐glucose transport protein 2; TNF, tumor necrosis factor; VAS, VAS‐2870; and VECs, valvular endothelial cells.

For subsequent experiments aiming to investigate extensive endothelial effects of plasma from patients with AS, only AS+ plasma samples that promoted dihydroethidium values within the highest tertile were used to obtain 3 to 6 samples for each condition. The baseline characteristics of these populations are described in Table 1 and Table S2. Notably, no differences of age, sex, or cardiovascular risk factors were observed among these tertiles (P>0.05).

The pro‐oxidant response induced by AS+ plasma in VECs was significantly decreased by a single neutralizing antibody directed against either IL‐1β, IL‐6, or TNF‐α (Figure 2C). This process indicated a determinant role of these cytokines in triggering the pro‐oxidant signal. Additionally, empagliflozin prevented ROS formation, suggesting the role of SGLT2 (sodium‐glucose transport protein 2) in the pro‐oxidant response in VECs following AS+ plasma exposure (Figure 2C). The pro‐oxidant response of AS+ plasma was also reduced by the inhibition of the angiotensin system by either perindoprilat (angiotensin‐converting enzyme [ACE1] inhibitor) and losartan (angiotensin‐II type 1 receptor [AT1R] antagonist, Figure 2C). An inhibitory effect was observed with the nicotinamide adenine dinucleotide phosphate (NADPH) oxidases inhibitor VAS‐2870 (Figure 2C). Finally, ROS formation was inhibited by the thrombin inhibitor dabigatran and FXa inhibitor rivaroxaban, indicating the role of these coagulation factors in the pro‐oxidant response in VECs induced by AS+ plasma (Figure 2C). This effect seems mediated, at least in part, by the protease‐activated receptors (PAR) , because the PAR‐1, −2, and −4 inhibitors were able to partially prevent the ROS formation in VECs exposed to AS+ plasma (Figure 2C).

Proinflammatory and Prothrombotic Genes and Proteins Expression Profile of AS Plasma‐Treated Endothelial Cells

Because AS+ plasma induced a pro‐oxidant signal in VECs, we explored its downstream effects on the expression profile of redox‐sensitive genes involved in endothelial cells activation (Figure 3A). Exposure of VECs to AS plasma resulted in a downregulation of the NOS3 (eNOS) and TFPI mRNA level (TFPI), and an upregulation of the mRNA levels of senescence marker TP53, CDKN1A, and CDKN2A (p53, p21, and p16), cytoadhesins ICAM1, VCAM1, SELE, and SELP (ICAM‐1, VCAM‐1, E‐selectin, and P‐selectin), modulators of thrombotic responses F3, and SERPINE (TF [tissue factor] and PAI‐1), components of the angiotensin II pathway ACE1 and AGTR1 (ACE1 and AT1R), proinflammatory cytokines IL1B, IL6, IL8, and TNFA (IL‐1β, IL‐6, IL‐8 and TNF‐α), and SLC5A1 (SGLT1) and SLC5A2 (SGLT2). The stimulatory effect of the AS+ plasma on gene expression was prevented by empagliflozin, the combination of neutralizing antibodies directed against IL‐1β, IL‐6, and TNF‐α, and dabigatran and rivaroxaban, except CDKN2A, IL6, TNFA, and TFPI (Figure S1).

A, Exposure of VECs to AS+ plasma for 24 hours was associated with a downregulation of the expression levels of *NOS3* and *TFPI* mRNA, and an upregulation of mRNA levels of senescence markers (*TP53*, *CDKN1A*, and *CDKN2A*), cytoadhesins (*VCAM1*, *ICAM1*, **SELE** , and *SELP*), modulators of thrombotic responses (F3 and *SERPINE1*), the angiotensin pathway (*ACE1* and *AGTR1*), proinflammatory cytokines (*IL1B*, *IL6*, *IL8*, and *TNFA*), and *SCL5A1* and *SLC5A2*. The stimulatory effect of the AS+ plasma on gene expression was prevented by empagliflozin, the combination of neutralizing Abs directed against IL‐1β, IL‐6, and TNF‐α, and dabigatran and rivaroxaban, except *CDKN2A*, *IL6*, *TNFA*, and *TFPI*. B, AS+ plasma promotes in VECs increased protein levels of SGLT2, VCAM‐1, and TF, and downregulation of eNOS protein level as assessed by Western blot analysis. Data are presented as mean±SD (n=4 to 7). All comparisons were performed using 1‐way ANOVA followed by Tukey post hoc analyses. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Abs indicates antibodies; AS, aortic stenosis; AS+, patients with AS; AS−, patients with cardiovascular risk factors without AS; Dabi, dabigatran; Empa, empagliflozin; eNOS, endothelial nitric oxide synthase; HV, healthy volunteers; IL, interleukin; Los, losartan; Riva, rivaroxaban; SGLT2, sodium‐glucose transport protein 2; TF, tissue factor; TNF, tumor necrosis factor; VCAM‐1, vascular cell adhesion molecule‐1; and VECs, valvular endothelial cells.

To confirm that AS+ plasma stimulates SGLT2 expression and causes VEC activation, we analyzed protein expression levels in VECs using Western blot analysis. AS+ plasma increased the protein expression levels of SGLT2, VCAM‐1, and TF, and downregulated that of eNOS (Figure 3B).

Impact of AS Plasma on NO Formation in VECs

Because AS plasma promoted oxidative stress associated with eNOS downregulation in VECs, the impact on the formation of NO, a major vasoprotective factor, was evaluated. AS plasma reduced both basal and bradykinin‐stimulated formation of NO (Figure 4A). In addition, both basal and bradykinin‐stimulated NO formation were lower after exposure of VECs with AS+ plasma compared with HV and AS− plasma (Figure 4A). The effect of AS+ plasma on NO formation was blunted by empagliflozin, losartan, the combination of 3 neutralizing antibodies directed against IL‐1β, IL‐6, and TNF‐α, and dabigatran and rivaroxaban (Figure 4B).

A, VECs studied at passage 1 demonstrated downregulation of bradykinin‐stimulated NO formation as assessed by DAF‐FM in response to AS+ plasma (10%, 24 hours), as compared with HV and AS− plasma (n=3–6). B, Empagliflozin, losartan, the combination of neutralizing antibodies directed against either IL‐1β, IL‐6, and TNF‐α, and rivaroxaban and dabigatran preserved bradykinin‐stimulated NO formation (n=4). Scale bars on micrographs represent 100 μm. Data are presented as mean±SD. All comparisons were performed using 1‐way ANOVA followed by Tukey post hoc analyses. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Abs indicates antibodies; AS, aortic stenosis; AS+ indicates patients with AS; AS−, patients with cardiovascular risk factors without AS; AU, arbitry units; BK, bradykinin; Dabi, dabigatran; DAF‐FM, 4‐amino‐5‐methylamino‐2′,7′‐difluororescein diacetate; Empa, empagliflozin; HV, healthy volunteer; IL, interleukin; Los, losartan; Riva, rivaroxaban; TNF, tumor necrosis factor; and VECs, valvular endothelial cells.

AS Plasma Promotes Inflammatory Cells and Platelets Recruitment, and Thrombin Generation on VECs

To emphasize the link between VEC dysfunction, recruitment of inflammatory cells, adherence of platelets, and thrombogenicity, we explored the impact of AS+ plasma on monocytes and platelets adhesion on VECs and procoagulant activity. Exposure of VECs to AS+ plasma caused an increased adhesion of THP1 cells (a monocytic human cell line), as compared with HV and AS− plasma (Figure 5A). The AS plasma‐induced enhanced adhesion of THP1 cells was abolished by empagliflozin, losartan, the combination of 3 neutralizing antibodies directed against IL‐1β, IL‐6, and TNF‐α, and dabigatran and rivaroxaban (Figure 5A).

A, VECs exposed to AS+ plasma (10%, 24 hours) demonstrated increased THP‐1 adherent cells count, as compared with HV and AS− plasma. Empagliflozin, losartan, the combination of neutralizing antibodies directed against either IL‐1β, IL‐6, and TNF‐α, and rivaroxaban and dabigatran exhibited decreased AS+ plasma‐mediated THP‐1 cells adherence. Data are presented as mean±SD (n=4–9). All comparisons were performed using 1‐way ANOVA followed by Tukey post hoc analyses. B, AS+ plasma increased platelets adhesion to VECs surface, as compared with HV and AS− plasma. This effect was prevented by empagliflozin, losartan, the combination of neutralizing antibodies directed against either IL‐1β, IL‐6, and TNF‐α, and rivaroxaban and dabigatran. Data are presented as mean±SD (n=4). All comparisons were performed using 1‐way ANOVA followed by Tukey post hoc analyses. C, Thrombin generation was measured in VECs upon the exposure to HV, AS−, and AS+ plasma (10%, 24 hours). AS+ plasma‐mediated thrombin generation in VECs was measured after the exposure to empagliflozin, losartan, the combination of neutralizing antibodies directed against either IL‐1β, IL‐6, and TNF‐α, and rivaroxaban and dabigatran. Scale bars on micrographs represent 100 μm. Data are presented as mean±SD (n=5). All comparisons were performed using 1‐way ANOVA followed by Tukey post hoc analyses and multiple Student paired t tests. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. Abs indicates antibodies; AngII, angiotensin II; AS, aortic stenosis; AS+ indicates patients with AS; AS−, patients with cardiovascular risk factors without AS; BK, bradykinin; CTRL, control; Dabi, dabigatran; Empa, empagliflozin; HV, healthy volunteers; IL, interleukin; Los, losartan; Riva, rivaroxaban; TNF, tumor necrosis factor; and VECs, valvular endothelial cells.

Furthermore, AS+ plasma increased platelet adhesion to the VECs surface, in comparison with HV and AS− plasma (Figure 5B). The AS+ plasma‐induced enhanced adhesion of platelets was significantly prevented by empagliflozin, losartan, the combination of the 3 neutralizing antibodies directed against IL‐1β, IL‐6, and TNF‐α, and dabigatran and rivaroxaban (Figure 5B).

AS+ plasma induced on VECs a strong procoagulant response with an enhanced thrombin generation compared with HV and AS− plasma (Figure 5C). The characterization of the AS+ plasma‐induced surface procoagulant activity on VECs indicated an inhibition by the direct thrombin inhibitor dabigatran and the direct FXa inhibitor rivaroxaban, indicating that it is mediated in part by thrombin and FXa (Figure 5C). Significant inhibition by losartan and empagliflozin, and a strong inhibition by the combination of 3 neutralizing antibodies directed against IL‐1β, IL‐6, and TNF‐α was also observed (Figure 5C).

Effect of Plasma From Patients With AS on Coronary Artery Endothelial Cells

To further explore the systemic effects of plasma from patients who were AS+, an exploratory analysis was conducted to assess its impact on human coronary artery endothelial cells. Exposure to AS+ plasma led to a significant increase in the pro‐oxidant response, as evidenced by elevated dihydroethidium fluorescence levels compared with plasma from patients who were AS− and HVs (P <0.05 for both; Figure S2).

DISCUSSION

The major findings of this study indicate that plasma from patients with severe AS displays a specific phenotype characterized by elevated levels of proinflammatory cytokines and FXa activity. This plasma exerts deleterious effects on aortic VECs, leading to ROS formation, endothelial dysfunction, and the promotion of prothrombotic, proinflammatory, and proadhesive phenotypes. These results highlight the active role of plasma components in the progression of AS and may suggest novel therapeutic targets for the treatment of this condition.

Systemic Inflammation in AS

Evidence supports the central role of inflammation in the development of AS. Intravalvular inflammation is a key factor in the natural history of AS, characterized by the infiltration of various inflammatory cells into the aortic valve, which in turn promotes myofibroblastic and osteoblastic differentiation of valvular interstitial cells. ¹⁰ , ¹¹ The expression of inflammatory cytokines, such as IL‐1, IL‐6, and TNF‐α, in the aortic valve has been associated with valvular fibrotic remodeling and AS progression. ¹² , ¹³ , ¹⁴ , ¹⁵ Accordingly, we have recently established within calcified human aortic stenotic valves a colocalization between oxidative stress, proliferation marker (proliferating cell nuclear antigen [PCNA]), and monocytic infiltration, whereas that of eNOS, a surrogate marker of endothelial health, was decreased. ⁶

However, the relationship between AS and systemic inflammation remains controversial. A cross‐sectional study demonstrated elevated CRP (C‐reactive protein) levels in patients with AS compared with matched controls. ¹⁶ Recently, a novel mechanism of shear‐induced pentameric CRP dissociation into its proinflammatory monomers was evidenced in AS, and linked with platelets and endothelial cell activation. ¹⁷ An analysis of the Framingham Heart Study cohort revealed that several inflammatory markers, including CRP, ICAM‐1, IL‐6, and MCP‐1 (monocytes chemoattractant protein) , were significantly higher in patients with AS. ¹⁸ Other prospective studies have shown that higher levels of CRP, TNF‐α, and TGF‐β (transforming growth factor) are associated with accelerated hemodynamic progression in AS. ¹⁹ , ²⁰ , ²¹ , ²² A genome‐wide association study meta‐analysis identified the IL‐6 locus as significantly associated with AS. ²³ In contrast, a Swedish retrospective study found no difference in CRP levels between patients undergoing surgical aortic valve replacement (SAVR) for AS and matched controls. ²⁴ Similarly, the ASTRONOMER (Aortic Stenosis Progression Observation Measuring Effects of Rosuvastatin) trial reported no association between CRP levels and AS severity or progression. ²⁵ The large‐scale prospective Cardiovascular Health Study also found no association between CRP levels and prevalence or incidence of AS. ²⁶

Our findings indicate higher levels of IL‐1β, IL‐6, and TNF‐α in patients with AS compared with HVs. Although some authors have suggested that elevated inflammatory biomarkers in AS might be confounded by cardiovascular risk factors common to both AS and inflammation, the originality of our work lies in the comparison of patients with AS with individuals without AS but with similar cardiovascular risk factors. In this comparison, we observed significantly higher levels of proinflammatory cytokines in patients with AS, reinforcing the notion of systemic inflammation as a hallmark of AS progression.

The precise source of elevated plasma cytokines in AS remains uncertain. It is likely that cytokine production occurs at both valvular and systemic levels. At the valvular level, inflammation plays a critical role in AS pathogenesis, with infiltrating inflammatory cells releasing proinflammatory cytokines and oxidized lipid pathways, further stimulating local cytokine expression. At the systemic level, transaortic shear‐induced activation of circulating inflammatory cells and vascular endothelial dysfunction may contribute to systemic inflammation. Coronary artery endothelial cells may represent 1 of the targets of this systemic inflammation. In an exploratory analysis, we observed that plasma from patients who were AS+ induced elevated pro‐oxidant levels in human coronary artery endothelial cells, consistent with the effects observed in VECs. Further studies are warranted to comprehensively investigate the systemic impact of AS+ plasma.

Detrimental Effects of Plasma From Patients With AS on VECs

VECs play a pivotal role in valvular homeostasis and are central in the pathophysiology of AS. Valvular endothelial dysfunction represents an early stage of AS natural history. ⁴ , ¹⁰ Biomechanical factors have been identified as key contributors to VEC dysfunction, with alterations in shear stress leading to changes in VEC phenotype. ²⁷ Disruptions in NO signaling are also critical in the early stage of AS and have been previously linked to accelerated disease progression. ²⁸ Endothelial NO deficiency is further exacerbated by eNOS uncoupling, which promotes ROS formation, particularly in pericalcific regions of human AS valves. ²⁹ VEC dysfunction, characterized by NO depletion and increased ROS generation, drives profibrotic and procalcific pathways through Rho‐kinase (ROCK), neurogenic locus notch homolog protein (NOTCH)‐1, and insulin‐like growth factor (IGF)‐1 signaling. ³⁰ In the present study, we demonstrated that plasma from patients with severe AS exerts deleterious effects on VECs, promoting ROS generation, downregulation of eNOS expression, and reduced bradykinin‐stimulated NO formation. This endothelial dysfunction transforms VECs into a prothrombotic, proinflammatory, and proremodeling surface. The functional relevance of AS+ plasma‐mediated VEC activation was highlighted by the increased recruitment of inflammatory cells and platelets to the endothelial surface, as well as enhanced thrombin generation. In contrast, plasma from HVs had no deleterious impact on VEC physiology. Interestingly, although plasma from patients with cardiovascular risk factors had a moderate effect on VECs, it was less pronounced than the impact of AS+ plasma. This finding aligns with previous studies that identified associations between traditional risk factors, including hypertension, diabetes, and dyslipidemia, and the incidence of AS. ³¹ Because patients with AS included in this study shared some cardiovascular risk factors with the control group, part of the effects observed with AS+ plasma may be attributed to these common risk factors. However, VEC dysfunction induced by AS+ plasma was significantly more pronounced than that caused by AS− plasma, suggesting that AS itself plays a distinct role in driving VEC dysfunction.

Although sex‐specific differences in vascular endothelial cells related to proinflammatory, pro‐oxidant, and angiogenic effects have been previously observed, ³² sex differences in VECs have been understudied. Prior studies suggest that female VECs may exhibit different proliferation rates due to higher thrombospondin‐2 secreation. ³³ To ensure homogeneity and eliminate potential confounding factors, we exclusively used male VECs. Future studies should investigate sex‐related differences in VEC responses to AS plasma.

Role of FXa

Beyond its central role in the coagulation process, FXa has several pleiotropic actions, including effects on endothelial physiology and pathophysiology. ³⁴ FXa can activate PAR both directly and indirectly through thrombin formation. Under physiologic conditions, FXa may provide endothelial‐protective actions through PAR‐1 or PAR‐2 activation, similarly to low concentrations of thrombin. ³⁵ At higher concentration, FXa induces oxidative stress in endothelial cells through PAR‐2 activation, mainly mediated by NADPH oxidase (NOX1). ³⁶ , ³⁷ In addition to the role of PARs in mediating endothelial dysfunction, several conditions associated with endothelial dysfunction, including atherosclerosis or diabetes, are linked to increased expression of PAR‐1 and PAR‐4. ³⁸ Increased TF activity and thrombin generation was previously observed in patients with AS. ³⁹ Our findings align with this systemic prothrombotic state in AS, indicating enhanced levels of FXa activity in plasma from patients with AS compared with plasma from patients who were AS− and HVs. Moreover, pretreatments of VECs with either rivaroxaban, a direct inhibitor of FXa, and PAR‐1, PAR‐2, and PAR‐4 inhibitors significantly mitigated the phenotypic changes induced by AS+ plasma in VECs, including increased ROS formation, reduced NO formation, enhanced adherence of inflammatory cells and platelets, and thrombin generation.

In addition to its direct effect on endothelial cells, FXa can indirectly contribute to endothelial dysfunction through thrombin generation, a well‐established mediator of endothelial dysfunction. ⁴⁰ The beneficial effects of dabigatran, a direct thrombin inhibitor, further support the role of thrombin in mediating the deleterious impact of AS+ plasma on VECs. Among hemostatic factors, FXa was selected due to its critical role in endothelial dysfunction and its potential as a pharmacological target in AS. Although thrombin is a well‐established driver of endothelial dysfunction, our functional analysis using dabigatran, a direct thrombin inhibitor, indirectly supports thrombin's role in mediating AS‐related vascular dysfunction. Future studies should further explore the contribution of additional hemostatic markers to AS pathophysiology.

Clinical Implications

There is currently no medical treatment approved for AS, and pharmacological targets remain limited. The present study provides new insights on the role of inflammation and FXa in AS pathophysiology.

Although several randomized trials have investigated the effectiveness of immunomodulatory drugs for coronary heart disease, including canakinumab ⁴¹ and colchicine, ⁴² , ⁴³ data on their role in AS remain sparse. A recent Mendelian randomization analysis explored the causal association between inflammation and AS by examining the genetically proxied effects of tocilizumab (IL‐6 receptor inhibitor), canakinumab (IL‐1β inhibitor), and colchicine. This study found that genetically proxied tocilizumab was associated with a reduced risk of AS. ⁴⁴ Further studies are needed to assess the potential therapeutic benefits of anti‐inflammatory strategies in AS and to determine the optimal timing for their introduction in disease progression.

Our findings, associated with previous observational and experimental studies, suggest that rivaroxaban and dabigatran may attenuate aortic VEC dysfunction, potentially mitigating subsequent stages of AS progression. They may therefore represent promising therapeutic strategies to slow AS progression. In patients without formal indication for anticoagulation, determining their optimal dosage is critical to avoid a prohibitive bleeding risk. Notably, the COMPASS (Rivaroxaban for the Prevention of Major Cardiovascular Events in Coronary or Peripheral Artery Disease) trial demonstrated that a low dose of rivaroxaban (2.5 mg twice daily) combined with aspirin (100 mg once daily) significantly reduced the incidence of cardiovascular death, stroke, or myocardial infarction compared with aspirin alone in patients in secondary prevention. ⁴⁵ This trial highlights the potential for low‐dose anticoagulation therapy in reducing cardiovascular events, which could be explored further in the context of AS.

Finally, our results suggest the potential benefit of empagliflozin against VEC dysfunction. Several clinical studies have demonstrated the positive cardiovascular effects of SGLT2 inhibitors. ⁴⁶ The pathophysiological mechanisms underlying their cardiovascular protective effects have not been fully elucidated to date but include decreased oxidative stress and inflammation ⁴⁷ and regulation of immune signaling pathways. ⁴⁸ Within the human calcified aortic valve, we have recently provided new evidence of enrichment of extracellular vesicles acting as potent inducers of SGLT2 expression in VECs through sustained oxidative stress. ⁶ Ex vivo, we have established that increased SGLT2 levels induce endothelial valvular dysfunction, recruitment of inflammatory cells, and thrombogenicity through nuclear factor kappa B(NF‐κB) activation. ⁶ A recent study reported a reduced risk of AS progression in patients treated with SGLT2 inhibitors. ⁴⁹ Another recent publication demonstrated the protective effects of SGLT2 inhibition against bioprosthetic valve dysfunction following aortic valve replacement. ⁵⁰ Altogether, these findings highlight the pivotal role of the SGLT2 pathway in the pathophysiology of aortic valve disease. Accordingly, in the present study, increased SGLT2 levels have a central role in the induction of VEC dysfunction, as indicated by the protective effect of empagliflozin. This beneficial effect is probably attributable to the high effectiveness of empagliflozin in impeding the sustained pro‐oxidant activator signal.

Study Limitations

The present work has several limitations. First, this study was based on a single‐center cohort of unselected patients who underwent TAVR. The control group consisted of patients with at least 2 cardiovascular risk factors but without AS who underwent coronary angiography. Although patients who were AS− share many features with patients who were AS+, we cannot account for all potential confounders influencing the plasma‐mediated effect on VECs. Given the high prevalence of AS in individuals >80 years of age, ¹ it was challenging to include patients of this age without AS. To mitigate this, a sensitivity analysis using propensity score matching for age and sex was performed, which confirmed persistent differences in cytokines levels and ROS formations in VECs induced by plasma from who were AS+ compared with patients who were AS−. Second, we did not conduct an exhaustive characterization of plasma from patients with AS. Some relevant pathways, including lipoprotein(a) or immune regulation, were not explored and deserve future research. Third, the underlying determinants of systemic inflammation in patients with AS were beyond the scope of the present study. Because previous works have emphasized the role of epicardial adipose tissue as a mediator of the adverse effects of visceral obesity and inflammation on the heart, ⁵¹ dedicated studies are required to further explore its potential contribution to AS pathophysiology. Fourth, we acknowledge that although porcine and human VECs share similarities, differences also exist. Human VECs derived from diseased aortic valves collected during aortic valve replacement surgery may not accurately model the early stages of AS pathogenesis. Although cadaveric VECs are an option, their quality is variable compared with fresh tissue‐derived cells. ⁵² Recent studies support the use of porcine VECs as a valid alternative cellular model, ⁵³ justifying this approach largely used in the literature. ⁶ , ⁵² Finally, the possible impact of TAVR on plasma activity, decreased oxidative stress, ⁵⁴ inflammation, and endothelial dysfunction improvement was not investigated.

CONCLUSIONS

In the present study, patients with AS exhibited subclinical systemic inflammation, as well as increased plasmatic FXa activity. These processes contributed to VEC dysfunction, inflammation, inflammatory cells and platelets adhesion, and thrombin generation following exposure to AS+ plasma. These deleterious effects were prevented in vitro with empagliflozin, neutralizing antibodies directed against IL‐1β, IL‐6, and TNF‐α, and dabigatran and rivaroxaban. Our data provide a benchmark for a clinical rationale supporting the need for clinical randomized trials aiming to restore VECs function and prevent aortic valve fibrotic remodeling and calcification in patients with AS.

Sources of Funding

This work was supported by GERCA (Groupe pour l'Enseignement et la Recherche Cardiologique en Alsace).

Disclosures

None.

Supporting information

Data S1

JAH3-15-e041701-s001.pdf^{(322.8KB, pdf)}

Data S2

JAH3-15-e041701-s002.pdf^{(434.9KB, pdf)}

Acknowledgments

Author contributions: Study conception and design, A.T. and O.M. Data acquisition, all authors. Statistical analysis, A.T., S.H., and O.M. Interpretation of the data, all authors. Drafting of the article, A.T., S.H., V.S.‐K., and O.M. Critical revision of the article for important intellectual content, all authors. Final approval of the article, all authors.

This article was sent to June‐Wha Rhee, MD, Associate Editor, for review by expert referees, editorial decision, and final disposition.

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/JAHA.125.041701

For Sources of Funding and Disclosures, see page 15.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Data S1

JAH3-15-e041701-s001.pdf^{(322.8KB, pdf)}

Data S2

JAH3-15-e041701-s002.pdf^{(434.9KB, pdf)}

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PERMALINK

Detrimental Effect of Plasma From Patients With Severe Aortic Stenosis on Valvular Endothelial Cells: Role of Proinflammatory Cytokines and Factor Xa

Antonin Trimaille, MD, PhD

Sandy Hmadeh, PhD

Shinnosuke Kikuchi, MD

Ali Mroueh, PhD

Adrien Carmona, MD

Benjamin Marchandot, MD

Francis Beras, MD, MSc

Dinh Phi Truong, MD

Manh Cuong Vu, MD

Amandine Granier, MD

Laurence Jesel, MD, PhD

Patrick Ohlmann, MD, PhD

Laurent Sattler, PharmD

Valérie Schini‐Kerth, PhD

Olivier Morel, MD, PhD

Abstract

Background

Methods

Results

Conclusions

Nonstandard Abbreviations and Acronyms

Clinical Perspective.

What Is New?

What Are the Clinical Implications?

METHODS

Study Design and Population

Plasma Characterization, Cell Culture, and Biological Responses

Statistical Analysis

RESULTS

Patients’ Characteristics

Table 1.

Characterization of Proinflammatory Cytokines and FXa Activity Within Plasma

Figure 1. Plasma levels of proinflammatory cytokines (IL‐1β, IL‐6, and TNF‐α), and FXa activity in HVs, patients who were AS−, and patients who were AS+.

Table 2.

Pro‐oxidant State of AS Plasma‐Treated VECs

Figure 2. Pro‐oxidant state in VECs stimulated by AS+ plasma: role of proinflammatory cytokines, the angiotensin system, SGLT2, and FXa.

Proinflammatory and Prothrombotic Genes and Proteins Expression Profile of AS Plasma‐Treated Endothelial Cells

Figure 3. Gene and protein expression in VECs exposed to AS+ plasma.

Impact of AS Plasma on NO Formation in VECs

Figure 4. NO formation in VECs exposed to AS+ plasma.

AS Plasma Promotes Inflammatory Cells and Platelets Recruitment, and Thrombin Generation on VECs

Figure 5. Effect of AS+ plasma on THP‐1 cells and platelets adherence, and thrombin generation in VECs.

Effect of Plasma From Patients With AS on Coronary Artery Endothelial Cells

DISCUSSION

Systemic Inflammation in AS

Detrimental Effects of Plasma From Patients With AS on VECs

Role of FXa

Clinical Implications

Study Limitations

CONCLUSIONS

Sources of Funding

Disclosures

Supporting information

Acknowledgments

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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