We recently showed that red blood cells (RBCs) from patients with type 2 diabetes mellitus (T2DM-RBCs) induce endothelial dysfunction through a mechanism involving arginase I and reactive oxygen species. Peroxynitrite is known to activate arginase in endothelial cells. Whether peroxynitrite regulates arginase activity in RBCs, and whether it is involved in the cross-talk between RBCs and the vasculature in T2DM, is unclear and elusive. The present study was designed to test the hypothesis that endothelial dysfunction induced by T2DM-RBCs is driven by peroxynitrite and upregulation of arginase. RBCs were isolated from patients with T2DM and healthy age matched controls. RBCs were co-incubated with aortae isolated from wild type rats for 18 h in the absence and presence of peroxynitrite scavenger FeTTPS. Evaluation of endothelial function in organ chambers by cumulative addition of acetylcholine as well as measurement of RBC and vessel arginase activity was performed. In another set of experiments, RBCs isolated from healthy subjects (Healthy RBCs) were incubated with the peroxynitrite donor SIN-1 with subsequent evaluation of endothelial function and arginase activity. T2DM-RBCs, but not Healthy RBCs, induced impairment in endothelial function, which was fully reversed by scavenging of RBC but not vascular peroxynitrite with FeTPPS. Arginase activity was up-regulated by the peroxynitrite donor SIN-1 in Healthy RBCs, an effect that was inhibited by FeTTPS. Healthy RBCs co-incubated with aortae in the presence of SIN-1 caused impairment of endothelial function, which was inhibited by FeTTPS or the arginase inhibitor ABH. T2DM-RBCs induced up-regulation of vascular arginase, an effect that was fully inhibited by FeTTPS. Collectively, our data indicate that RBCs impair endothelial function in T2DM via an effect that is driven by a peroxynitrite-mediated increase in arginase activity. This mechanism may be targeted in patients with T2DM for improvement in endothelial function.

Background: Patients with familial hypercholesterolemia (FH) display high levels of low‐density lipoprotein cholesterol (LDL‐c), endothelial dysfunction, and increased risk of premature atherosclerosis. We have previously shown that red blood cells (RBCs) from patients with type 2 diabetes induce endothelial dysfunction through increased arginase 1 and reactive oxygen species (ROS).Objective: To test the hypothesis that RBCs from patients with FH (FH‐RBCs) and elevated LDL‐c induce endothelial dysfunction.Methods and results: FH‐RBCs and LDL‐c >5.0 mM induced endothelial dysfunction following 18‐h incubation with isolated aortic rings from healthy rats compared to FH‐RBCs and LDL‐c 5.0 mM. Furthermore, arginase 1 but not arginase 2 was elevated in the vasculature of aortic segments after incubation with FH‐RBCs and LDL‐c >5.0 mM. A superoxide scavenger, present throughout the 18‐h incubation, attenuated the degree of endothelial dysfunction induced by FH‐RBCs and LDL‐c >5.0 mM. ROS production was elevated in these RBCs in comparison with H‐RBCs. Scavenging of vascular ROS through various antioxidants also attenuated the degree of endothelial dysfunction induced by FH‐RBCs and LDL‐c >5.0 mM. This was corroborated by an increase in the lipid peroxidation product 4‐hydroxynonenal. Lipidomic analysis of RBC lysates did not reveal any significant changes across the groups.Conclusion: FH‐RBCs induce endothelial dysfunction dependent on LDL‐c levels via arginase 1 and ROS‐dependent mechanisms.

Red blood cells (RBCs) induce endothelial dysfunction in type 2 diabetes (T2D), but the mechanism by which RBCs communicate with the endothelium is unknown. This study tested the hypothesis that extracellular vesicles (EVs) secreted by RBCs act as mediators of endothelial dysfunction in T2D. Despite a lower production of EVs derived from RBCs of T2D patients (T2D RBC-EVs), their uptake by endothelial cells was greater than that of EVs derived from RBCs of healthy individuals (H RBC-EVs). T2D RBC-EVs impaired endothelium-dependent relaxation, and this effect was attenuated following inhibition of arginase in EVs. Inhibition of vascular arginase or oxidative stress also attenuated endothelial dysfunction induced by T2D RBC-EVs. Arginase-1 was detected in RBC-derived EVs, and arginase-1 and oxidative stress were increased in endothelial cells following coincubation with T2D RBC-EVs. T2D RBC-EVs also increased arginase-1 protein in endothelial cells following mRNA silencing and in the endothelium of aortas from endothelial cell arginase-1–knockout mice. It is concluded that T2D-RBCs induce endothelial dysfunction through increased uptake of EVs that transfer arginase-1 from RBCs to the endothelium to induce oxidative stress and endothelial dysfunction. These results shed important light on the mechanism underlying endothelial dysfunction mediated by RBCs in T2D.

Red blood cells (RBCs) mediate cardioprotection via nitric oxide–like bioactivity, but the signaling and the identity of any mediator released by the RBCs remains unknown. We investigated whether RBCs exposed to hypoxia release a cardioprotective mediator and explored the nature of this mediator. Perfusion of isolated hearts subjected to ischemia-reperfusion with extracellular supernatant from mouse RBCs exposed to hypoxia resulted in improved postischemic cardiac function and reduced infarct size. Hypoxia increased extracellular export of cyclic guanosine monophosphate (cGMP) from mouse RBCs, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on RBC-soluble guanylate cyclase and cGMP transport and was sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice to increase nitric oxide bioactivity further enhanced the cardioprotective effect of hypoxic RBCs. In a placebo-controlled clinical trial, a clear cardioprotective, soluble guanylate cyclase–dependent effect was induced by RBCs collected from patients randomized to 5 weeks nitrate-rich diet. It is concluded that RBCs generate and export cGMP as a response to hypoxia, mediating cardioprotection via a paracrine effect. This effect can be further augmented by a simple dietary intervention, suggesting preventive and therapeutic opportunities in ischemic heart disease.

Background: The mechanisms underlying rupture of a coronary atherosclerotic plaque and development of myocardial ischemia‐reperfusion injury in ST‐elevation myocardial infarction (STEMI) remain unresolved. Increased arginase 1 activity leads to reduced nitric oxide (NO) production and increased formation of reactive oxygen species due to uncoupling of the NO‐producing enzyme endothelial NO synthase (eNOS). This contributes to endothelial dysfunction, plaque instability and increased susceptibility to ischemia‐reperfusion injury in acute myocardial infarction.Objective: The purpose of this study was to test the hypothesis that arginase gene and protein expression are upregulated in patients with STEMI.Methods: Two cohorts of patients with STEMI were included. In the first cohort (n = 51), expression of arginase and NO‐synthases as well as arginase 1 protein levels were determined and compared to a healthy control group (n = 45). In a second cohort (n = 68), plasma arginase 1 levels and infarct size were determined using cardiac magnetic resonance imaging.Results: Expression of the gene encoding arginase 1 was significantly elevated at admission and 24–48 h after STEMI but not 3 months post STEMI, in comparison with the control group. Expression of the genes encoding arginase 2 and endothelial NO synthase (NOS3) were unaltered. Arginase 1 protein levels were elevated at admission, 24 h post STEMI and remained elevated for up to 6 months. No significant correlation between plasma arginase 1 protein levels and infarct size was observed.Conclusion: The markedly increased gene and protein expression of arginase 1 already at admission indicates a role of arginase 1 in the development of STEMI.

Red blood cells from patients with STEMI and systemic inflammation induce endothelial dysfunction ex vivo. The RBC-induced endothelial dysfunction is mediated through increased arginase 1 and a shift in the redox balance toward oxidative stress. Inhibition of arginase or free radicals attenuates the impairment of endothelial function. The study suggests that red blood cells deserve attention as a key player in systemic inflammation and STEMI.