Background: Erythrocytes (red blood cells, RBCs) participate in the control of vascular nitric oxide (NO) bioavailability. We have shown that erythrocyte membrane deposition promotes human atherosclerotic lesion calcification via mechanisms involving NO. Arginase-1 (ARG1) is expressed in rodent and human red blood cells and functional in terms of controlling NO generation from erythrocytes. Cardiovascular risk factors are associated with elevated levels of ARG1 and its metabolites in erythrocytes. The purpose of this study was to determine whether and how genetic deletion of erythrocyte ARG1 affects vascular smooth muscle cell (VSMC) NO signalling, osteoblastic differentiation and atherosclerotic lesion calcification.

Methods: Atherosclerosis-prone mice with conditional, erythrocyte-restricted deletion of ARG1 (apoE-/- RBC.ARG1 knockout, KO) were generated and vascular calcification studied using molecular imaging of the osteogenic activity agent OsteoSense™, Alizarin staining or immunohistochemistry of cell-specific antigens, qPCR of osteogenic markers and ex vivo assays.

Results: Deletion of ARG1 increased the erythrocyte NO bioactivity without affecting their numbers, morphology or removal in the spleen. Atherosclerotic lesion size at the aortic root did not differ, but calcification was significantly more pronounced in apoE-/- mice lacking erythrocyte ARG1, as shown by molecular imaging of osteogenic activity agent OsteoSense™ and verified by Alizarin staining. Incubation of murine and human VSMCs with lysed erythrocyte membranes from apoE-/- RBC.ARG1-KO mice accelerated their osteogenic differentiation, and mRNA transcripts of osteogenic markers decreased following addition of the NO scavenger PTIO or the soluble guanylal cyclase inhibitor ODQ. Elevated mRNA levels of enzymes metabolizing the arginase products L-ornithine and L-proline in SMCs also were observed, parallel to increased proliferation, myofibroblast marker and collagen type 1 expression. In addition to NO signaling via sGC, overexpression of GSNOR (S-nitrosoglutathione reductase), enhanced degradation of S-nitrosoglutathione to glutathione and reduced protein S-nitrosation of heat shock protein (HSP)-70 were identified as potential mechanisms of VSMC calcification in mice lacking ARG1 in erythrocytes, and calcium phosphate deposition was enhanced by heat shock and prevented by GSNOR inhibition. Messenger RNA levels of enzymes metabolizing the arginase products L-ornithine and L-proline also were elevated in VSMCs, paralleled by increased proliferation, myofibroblast marker and collagen type 1 expression.

Conclusion: Our findings support an important role of erythrocyte ARG1 for NO bioavailability and L-arginine metabolism in VSMCs which controls atherosclerotic lesion composition and calcification.