Deletion of arginase-1 in murine erythrocytes promotes smooth muscle lipid uptake, foam cell formation and atherosclerosis progression

Background and Aim: Experimental and clinical evidence suggests that erythrocytes have roles in cardiovascular function beyond the transport of oxygen and carbon dioxide, and that erythrocyte dysfunction contributes to cardiovascular disease processes. The endothelial isoform of nitric oxide (NO) synthase (eNOS) is also expressed and active in the erythrocyte membrane. The production of NO from erythrocytes in counterbalanced by arginase-1 (ARG1), which competes with eNOS for their common substrate L-arginine. We recently showed that genetic deletion of ARG1 in murine red blood cells (RBCs) promotes atherosclerotic lesion calcification by enhancing NO signaling and S-nitrosylation of smooth muscle cell (SMC) proteins. The aim of this study is to determine whether and how erythrocyte ARG1 deletion alters SMC transdifferentiation and atherosclerosis progression.

Methods: Primary smooth muscle cells (pSMCs) were isolated from the thoracic aorta of RBC.ARG1 knockout (KO) and RBC.ARG1 wildtype (WT) mice and cultured in DMEM (Dulbecco's Modified Eagle's medium), alone or in the presence of DIA (dexamethasone, insulin and ascorbic acid) medium to allow foam cell formation. Atherosclerosis-prone mice with conditional, erythrocyte-specific deletion of ARG1 (apoE-/- RBC.ARG1-KO) were fed western type diet for 12 and 20 weeks followed by evaluation of atherosclerotic plaque formation at the aortic root and on en face preparations.

Results: Following cultivation in DIA medium for ten days, primary aortic SMCs isolated from RBC.ARG1-KO mice exhibited a highly significantly increased accumulation of oxidized lipids, as shown by Oil Red O, BODIPY (493/503) and C11-BODIPY (581/591) lipid staining. Quantitative real-time PCR of lipid scavenger receptors showed significantly increased expression levels of CD36 (also known as fatty acid translocase or scavenger receptor class B member 3) involved in the uptake of oxidized LDL cholesterol and of ABCG1 controlling cholesterol efflux, whereas mRNA levels of macrophage scavenger receptor 1, involved in oxidized LDL clearance, and smooth muscle cell marker genes were significantly reduced. Changes in CD36 and smooth muscle cell marker expression were confirmed on the protein level, also showing higher levels of phosphorylated VASP as a marker of increased NO signaling in SMCs. A significant increase in foam cell formation was also observed in wild type pSMCs treated with supraphysiological doses (above 2 µM) of the NO donor DETA-NO, the NO-independent sGC activator BAY58-2667 or the sGC stimulator BAY41-2272, or with MY-5445, a specific inhibitor of cyclic GMP phosphodiesterase type 5, thus phenocopying the increased foam cell formation observed in RBC.ARG1-KO SMCs. On the other hand, inhibition of NO signaling using ODQ (to inhibit soluble guanylyl cyclase) or KT5823 (to inhibit protein kinase G) significantly reduced foam cell formation in vitro. Significantly increased lipid accumulation and a larger necrotic core area was also observed in atherosclerotic plaques of apoE-/- RBC.ARG1-KO mice. Endothelial dysfunction resulting in vascular leakiness in apoE-/- RBC.ARG1-KO mice was suggested by intravenous Evans Blue injection.

Conclusion: The findings of this ongoing study suggest an important role of erythrocyte ARG1 in controlling SMC differentiation and the phenotypic switch to foam cell formation thus promoting vascular lipid accumulation and atherosclerotic lesion progression in vivo.