Novel peripheral blood immune cell populations correlate with disease progression and reveal systemic interactions in severe aortic valve stenosis patients
Authors: Anousakis-Vlachochristou N , Ktenopoulos N , Paschalidis N , Giannouli C , Vlasopoulou K , Miliou A , Divani M , Alexopoulou M , Kentepozidi E , Charitos D , Cokkinos D , Tsioufis C , Toutouzas K 
Published: October 2022

Abstract

Background
Osteoblastic differentiation is the key driver phenomenon underlying degenerative aortic valve stenosis (AS). It is characterized by a complex cellular interplay and infiltration by circulating immune cells. Moreover, AS demonstrates a diverse natural course, which is understudied.

Purpose
We sought to investigate the presence of AS clinical progression subphenotypes in the context of “AthenaValve” trial (NCT04312139).

Methods
Consecutive patients with severe AS undergoing TAVI or SAVR with complete clinical data, echocardiographic history (≥3 past years, corresponding from moderate to severe AS), along with matched healthy volunteers, were included. The median transaortic Vmax increase/month (Vi/m) was calculated and cluster analysis was used to reveal AS progression subgroups. Whole blood peripheral mononuclear cells (PBMCs) were isolated with density gradient centrifugation and cryopreserved into FBS-10% DMSO. PBMC populations were investigated with mass cytometry (CyTOF, Helios, Fluidigm) using a 30-marker antibody panel (Direct Immune Profiling Assay, Fluidigm) and analyzed with Flowjo and Cytobank software.

Results
A total of 67 patients fulfilling criteria were included (mean age 77.2±10 years, 61.2% males). Two-step cluster algorithm identified 2 clusters of AS progression: C1: 80%, mean Vi/m = 0.03±0.01 vs C2: 20%, Vi/m = 0.07±0.1 m/s/month, average silhouette = 0.7. Mean time to progression to severe AS differed significantly: C1: 59.5 (48.9,70.1) vs C2: 40.2 (29.2, 51.2) months, log rank p=0.05. Among those, 20 patients (C1: 10, C2: 10) and 10 healthy volunteers were included in CyTOF. Demographics, atherosclerotic clinical factors and renal function did not differ among patients-volunteers and C1-C2 groups. B cells, B-memory, B-naïve and pDendritic cells discriminated patients from healthy volunteers: ROC curve (AUC: 0.885, p=0.001), (0.913, p&lt;0.001), (0.770, p=0.018), (0.282, p=0.056), profile (%): (5.3±3 vs 10.2±4), (0.86±0.4 vs 2.6±2), (4.4±3 vs 7.6±4), and (0.48±0.2 vs 0.3±0.1), respectively (mean live cells analyzed: 284028.8±52213.8). pDendritic cells and CD4-Th17-like cells discriminated C1 from C2 patients: ROC curve AUC: (0.804, p=0.005) and (0.268, p=0.031), profile (%): (0.6±0.2 vs 0.3±0.2) and (0.9±0.5 vs 2.6±0.9), respectively).

Conclusions
Aortic valve stenosis patients demonstrate two distinct phenotypes of disease progression from moderate to severe stage. Patients demonstrate reduced circulating B-cells and increased plasmatocytoid dendritic cells. The rapid progression subgroup of patients demonstrates reduced pDendritic and increased CD4-Th17-like cells. Further works are needed to expand and validate these results.

Funding Acknowledgement
Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Co-Funded by Hellenic Ministry of Development and European Regional Development Fund in the context of an open call of “EATRIS-GR project: Infrastructure for preclinical and early-phase clinical development of drugs, therapeutics, and biomedical devices”