A Straightforward Cytometry-Based Protocol for the Comprehensive Analysis of the Inflammatory Valve Infiltrate in Aortic Stenosis

Abstract

Aortic stenosis (AS) is a frequent cardiac disease in old individuals, characterized by valvular calcification, fibrosis, and inflammation. Recent studies suggest that AS is an active inflammatory atherosclerotic-like process. Particularly, it has been suggested that several immune cell types, present in the valve infiltrate, contribute to its degeneration and to the progression toward stenosis. Furthermore, the infiltrating T cell subpopulations mainly consist of oligoclonal expansions, probably specific for persistent antigens. Thus, the characterization of the cells implicated in the aortic valve calcification and the analysis of the antigens to which those cells respond to is of utmost importance to develop new therapies alternative to the replacement of the valve itself. However, calcified aortic valves have been only studied so far by histological and immunohistochemical methods, unable to render an in-depth phenotypical and functional cell profiling. Here we present, for the first time, a simple and efficient cytometry-based protocol that allows the identification and quantification of infiltrating inflammatory leukocytes in aortic valve explants. Our cytometry protocol saves time and facilitates the simultaneous analysis of numerous surface and intracellular cell markers and may well be also applied to the study of other cardiac diseases with an inflammatory component.

Single-cell T cell receptor sequencing of paired tissue and blood samples reveals clonal expansion of CD8+ effector T cells in patients with calcific aortic valve disease

“…Cells were thawed at 37 °C and immediately processed downstream following established protocols 16 . Briefly, cells were counted and suspended at 1x10 6 /mL in sodium-azide free DPBS, with BD Horizon™ Fixable Viability Stain (1:1000) at room temperature for 15 minutes protected from light.…”

Procoagulant effect of phosphatidylserine exposed to extracellular vesicles, blood cells and endothelial cells in patients with aortic stenosis

“…This phenomenon has been observed in diseases such as coronary atherosclerosis 13 and atrial fibrillation 14 . There is increasing evidence that erythrocytes, leukocytes and platelets are activated in AS 1, 15, 16 . In addition, other evidence showed that ECs were activated in AS 17 .…”