PB1270 - Erythrocyte Components Affect Vascular Calcification By Modulating Nitric Oxide Signaling At Sites Of Intramural Microhemorrhage

Tuesday, July 12, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Co-Author(s)

MP

MARIA C. PAVLAKI

Senior Researcher
Dpt. of Cardiology, Democritus University of Thrace, Alexandroupolis, Greece
Alexandroupolis, Evros, Greece

Presenting Author(s)

Kateryna Moiko

Molecular Biologist
Democritus University of Thrace
Alexandroupolis, Evros, Greece

Co-Author(s)

AT

Adina Thomaidis

PhD student, MD, Cardiologist
Cardiology Dpt. University of Thrace
Alexandroupolis, Evros, Greece
AK

Annie Kourkoulis

MSc in biology
Cardiology Dpt. University of Thrace
Alexandroupolis, Evros, Greece
AM

Alexandros Markakis

Graduate Biologist
Cardiology Dpt. University of Thrace
Alexandroupolis, Evros, Greece
GC

George Chalikias

PhD, MD, Associate Professor of Cardiology
Cardiology Dpt. University of Thrace
Alexandroupolis, Evros, Greece
SK

Stavros Konstantinides

PhD, MD, Professor of Cardiology
Cardiology Dpt. University of Thrace
Alexandroupolis, Evros, Greece
DT

Dimitrios Tziakas

PhD, MD, Professor of Cardiology
Cardiology Dpt. University of Thrace
Alexandroupolis, Evros, Greece

Background: We have previously shown that red blood cells (RBCs) enhance vascular smooth muscle cell (SMC) osteogenesis via mechanisms partly dependent on nitric oxide (NO) generation. Interestingly, osteoinductive effects were exerted by RBC membranes, but not by intact or lysed RBCs.

Aims: To dissect the mechanisms underlying the osteoinductive properties of RBC membranes as opposed to intact or lysed RBCs, with particular focus on arginase activity and NO scavenging by hemoglobin.

Methods: Lysed RBCs and RBC membranes (ghosts), isolated from human blood, were examined in vitro for enhancement of osteogenic differentiation and calcification of aortic SMCs in culture as assessed by Alizarin staining. Oxidative neutralization of RBC cytoplasm served to evaluate the NO-scavenging activity of oxyhemoglobin. Arginase activity and content were assessed in the calcification assay and by sample immunoblotting followed by band densitometry analysis, respectively.

Results: The arginase content of the RBC cytoplasm exceeded that of RBC ghosts by approximately one order of magnitude. Using the calcification assay of aortic SMCs, we could show that the osteoinductive effect of RBC ghosts was enhanced by physical contact with the aortic SMCs and varied among different donors. Lysed RBC showed almost no effect when added alone to SMCs, and inhibited RBC ghost-mediated calcification. However, the inhibitory effect of the RBC cytosol could be reversed following oxidation of oxyhemoglobin and after inhibition of arginase by L-norvaline.

Conclusion(s): Incomplete clearance of lysed RBC at sites of intraplaque microhemorrhage may promote NO dependent vascular calcification. Our results suggest that, in addition to RBC membrane effects, the RBC cytosol may also exert NO dependent osteoinductive activity under oxidative conditions in association with neutralization of hemoglobin’s NO scavenging acitivity. Such an oxidative milieu has been reported to exist in human atherosclerotic lesions. Interindividual variability in the balance between NO synthase and arginase activity may determine the presence and extent of calcification.