Red blood cell-derived extracellular vesicles from type 2 diabetes patients induce endothelial dysfunction through a mechanism involving arginase 1

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Foundation for Geriatric Diseases Karolinska Institutet (2023-01860). Swedish Heart and Lung Foundation (20220210) Background The mechanisms driving the development of cardiovascular injury in type 2 diabetes (T2D) remain incompletely understood. We have recently demonstrated that red blood cells (RBCs) from patients with T2D (T2D-RBCs) act as mediators of endothelial dysfunction through the upregulation of arginase 1 and attenuation of nitric oxide bioavailability. However, the underlying mechanisms of this interaction remain unknown. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by practically all cell types, including RBCs, and represent a novel mechanism of intercellular communication. However, the involvement of RBC-derived EVs in the development of endothelial dysfunction in T2D remains to be elucidated. Purpose To test the hypothesis that EVs are secreted by RBCs and transfer signalling to induce endothelial dysfunction in T2D through arginase 1. Methods EVs released from T2D-RBCs (T2D-RBCs EVs) and RBCs from age-matched healthy controls (H-RBCs EVs) were isolated using sequential ultracentrifugation or a membrane affinity column, co-incubated with mouse aortae to evaluate endothelium-dependent relaxation (EDR), and with human carotid artery endothelial cells (HCtAEC) to study the EV uptake and alteration in gene expression. EVs were characterized based on morphology, size and particle number, uptake in endothelial cells, and arginase 1 content. Functional involvement of EV uptake and arginase were investigated using pharmacological interventions. Arginase 1 was measured in EVs, HCtAEC, and mouse aortae after co-incubation with H-RBCs EVs and T2D-RBCs EVs. Results The uptake of T2D-RBCs EVs by endothelial cells was greater than that of EVs from H-RBCs (Fig. 1A, B) despite the reduced formation of EVs by T2D-RBCs (Fig. 1C). T2D-RBCs EVs significantly impaired EDR (Fig. 1D), and this impairment was prevented by inhibiting uptake of EVs with heparin (Fig. 1E). Arginase 1 was detected in RBC-derived EVs (Fig. 2A), and endothelial function was rescued by blocking arginase activity in EVs by the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH; Fig. 2B). Immunohistochemical staining revealed upregulation of arginase 1 in the vasculature following incubation with T2D-RBCs EVs (Fig. 2C, D). Additionally, co-incubation of HCtAEC and EVs derived from T2D-RBCs significantly increased endothelial cell arginase 1 (Fig. 2E, F). Administration of ABH to the aortae following the co-incubation also attenuated the impairment of EDR induced by T2D-RBCs EVs, suggesting the involvement of vascular arginase 1 (Fig. 2G). Conclusion T2D-RBCs EVs induce endothelial dysfunction. In addition to increased uptake of EVs in endothelial cells, the signalling behind this effect of EVs is mediated by arginase 1 to induce endothelial dysfunction. These results shed new important light on the mechanism underlying vascular injury mediated by RBCs in T2D.