Aortic valve stenosis induced occult hemoglobin release promotes endothelial dysfunction

Rationale The impact of aortic valve stenosis (AS) on systemic endothelial function independent of standard modifiable risk factors (SMuRFs) is unknown. Objective We hypothesized that AS induces subclinical hemoglobin release from red blood cells (RBCs) following transvalvular passage due to post-stenotic aberrant blood flow and that cell-free hemoglobin (fHb) may limit endothelial NO bioavailability, affecting vascular function. Methods and Results AS induces swirling blood flow in the ascending aorta which impairs RBC integrity with consecutive release of fHb. Indeed, swirl flow magnitude assessed by 4D flow cardiac magnetic resonance correlates with fHb levels. Elevated systemic fHb reduces NO bioavailability and thus impairs endothelial cell function as evidenced by impaired flow mediated dilation (FMD). In addition, we here demonstrate impaired FMD in an experimental model of AS utilising C57BL/6 mice with preserved left ventricular function and without cardiovascular risk factors. In this model, endothelial dysfunction is accompanied by significantly increased fHb, exaggerated NO consumption and increased plasma levels of nitroso species and the final NO oxidation product, nitrate. Scavenging of fHb by infusion of haptoglobin reversed these deleterious effects. There observations were verified by transfer experiments with human plasma (sampled from patients with AS sheduled for TAVR) using a murine aortic ring bioassay system where the plasma from AS patients induced endothelial dysfunction when compared to plasma from control individuals without AS. Importantly, these deleterious effects were reversed by successful aortic valve replacement via TAVR independent of SMuRFs. Conclusions In aortic valve stenosis, increases in post-valvular swirl blood flow in the ascending aorta induces subclinical hemolysis that impairs NO bioavailability. Thus, AS itself promotes systemic endothelial dysfunction independent of other established risk factors. Transcatheter aortic valve replacement limits NO scavenging by realigning of postvalvular blood flow to normal physiological patterns. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) TRR259, Grant No. 397484323 to C.Q. and S.Z. (project S01), F.B. and U.F. (project B03). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Heinrich-Heine University Duesseldorf, Germany, gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript. All data produced in the present work are contained in the manuscript. * Ach : Acetylcholine AS : aortic valve stenosis AUC : area under the curve AVA : aortic valve area AVC : aortic valve calcification BMI : body mass index CKD : chronic kidney disease CO : cardiac output DM : diabetes mellitus dP/dtmax : maximal rate of rise of left ventricular pressure in systole dP/dtmin : minimal rate of rise of left ventricular pressure in systole EF : ejection fraction fHb : cell-free hemoglobin FMD : flow mediated dilation HLP : hyperlipoproteinemia Hp : haptoglobin HR : heart rate HT : hypertension IVSd : interventricular septum in diastole LCC : left coronary cusp LV : left ventricle LVEF : left ventricular ejection fraction LVDP : left ventricular developed pressure LVIDd : left ventricular inner diameter in diastole LVOT : left ventricular outflow tract MSCT : multi-slice computed tomography NCC : non-coronary cusp NO3- : nitrate NO2- : nitrite RBCs : red blood cells RCC : right coronary cusp RDW : RBC distribution width SVR : systemic vascular resistance TAVR : transcatheter aortic valve replacement