Red blood cells from individuals with type 2 diabetes (T2D RBC) induce endothelial dysfunction due to reduced RBC microRNA-210 levels, whereas T1D RBCs do not. We hypothesize that microR-210 plays a protective role explaining this difference. Both male and female adults with T1D and T2D matched for glycated hemoglobin, alongside age- and sex-matched healthy controls, were studied. microR-210 levels were measured by qPCR. Endothelium-dependent relaxation (EDR) in isolated rat aortas and nitric oxide (NO) production in endothelial cells following incubation with RBCs were determined using wire myograph and DAF-FM fluorescence. Protein levels of microR-210 target PTP1B and the oxidative stress marker 4-HNE were measured by immunohistochemistry. T1D RBC produced EDR and endothelial NO comparable to healthy controls, whereas T2D RBC impaired both. microR-210 levels were similar in T1D RBC and healthy controls, but reduced in T2D RBC. microR-210 inhibition in T1D RBC impaired EDR and increased vascular PTP1B and 4-HNE, while PTP1B inhibition or mitoTEMPO treatment in aortas improved EDR. RBC microR-210 regulates endothelial function differently between T1D and T2D by affecting vascular PTP1B and mitochondrial oxidative stress, highlighting a potential therapeutic target to improve vascular health.

Type 2 diabetes increases cardiovascular risk, with endothelial dysfunction playing a key role. Prolonged disease duration exacerbates cardiovascular risk, but the underlying mechanisms remain unclear. We previously demonstrated that red blood cells (RBCs) from individuals with type 2 diabetes impair endothelial function via reduced microRNA (miR)-210-3p. We investigated whether disease duration influences RBC-induced endothelial dysfunction and its link to miR-210-3p. RBCs were isolated from diabetic db/db mice of various ages and from humans with newly diagnosed (<1 year) or long-lasting type 2 diabetes (>7 years). Endothelial-dependent relaxation (EDR), miR-210-3p levels, its target protein glycerol-3-phosphate dehydrogenase 2 (GPD2), and oxidative stress marker 4-hydroxynonenal (4-HNE) were assessed. RBCs from 14- and 22-week, but not 7-week-old db/db mice impaired EDR. These RBCs showed similarly reduced miR-210-3p levels and increased vascular GPD2 and 4-HNE expression. RBCs from individuals with long-lasting, but not newly diagnosed group impaired EDR. After ≥7-years, RBCs from initially newly diagnosed individuals impaired EDR, which was rescued by miR-210-3p mimic transfection. In contrast, RBCs from healthy subjects did not impair EDR after follow-up. These findings underscore the pivotal role of disease duration for RBC-mediated vascular dysfunction, linked to miR-210-3p downregulation. RBC miR-210-3p may serve as a biomarker for diabetes-related vascular disease.

BACKGROUND:Arginase influences cardiac tolerance to ischemia-reperfusion by modulating nitric oxide (NO) signaling. In type 2 diabetes (T2D), elevated arginase activity may worsen ischemic injury through red blood cells (RBCs), but the specific roles of arginase isoforms are unclear. METHODS:C57BL/6 and db/db mice were pretreated with ARG1 or ARG2 antisense oligonucleotides (ASO) for six weeks. Conditional ARG1 knockout (ARG1fl/fl/Tie2Cretg/-) and wild-type littermates were also studied. Mice underwent coronary artery ligation and reperfusion in vivo for infarct size assessment. In ex vivo experiments, buffer-perfused hearts were subjected to global ischemia-reperfusion with or without RBCs to evaluate recovery of left ventricular developed pressure (LVDP). RESULTS:ARG1 knockdown, but not ARG2, improved post-ischemic recovery of LVDP in isolated hearts. RBCs from ARG1 ASO-treated mice enhanced recovery in wild-type hearts, while ARG1 knockout reduced infarct size compared with controls. Cardioprotection was abolished by NO synthase inhibition. RBCs from male and female ARG1 knockout mice improved LVDP recovery compared with RBCs from wild-type mice. In T2D mice, impaired recovery was restored by ARG1 ASO or RBCs from ARG1 ASO-treated T2D mice. CONCLUSIONS:Arginase 1, but not arginase 2, limits cardiac tolerance to ischemia-reperfusion and contributes to increased vulnerability in T2D.

Rationale The entero-salivary circulation of inorganic nitrate (NO3-) involves the absorption of dietary nitrate in the gut and active uptake from blood by the salivary glands, leading to a 20-25-fold concentration in saliva. This recycling process is crucial for the nitrate-nitrite-nitric oxide (NO) pathway, which helps maintaining NO signaling in mammals. While the exact uptake mechanisms are unclear, sialin (encoded by the SLC17A5 gene) has been suggested to play a key role. Interestingly, studies from the 1950s indicate that nitrate transport in the salivary glands competes with iodide (I-). This prompted us to explore the role of the sodium/iodide symporter (NIS) in salivary nitrate uptake. Method and Results Our database analysis revealed that the SLC5A5 gene (encoding NIS) and its protein are expressed at higher levels than SLC17A5 in the human salivary gland. Next, we expressed SLC5A5 in Xenopus Laevis oocytes and its functionality using electrophysiology. We could detect ion influx induced by nitrate, indicating nitrate transport. We also observed increased levels of nitrate in SLC5A5-injected oocytes and in human salivary gland cells overexpressing SLC5A5, after incubation with nitrate. Finally, to test the competition between nitrate and iodide in vivo, saliva samples were collected from patients receiving high doses of intravenous iodine (I2) contrast medium, a procedure known to generate considerable levels of circulating I-. We observed a marked decrease in salivary nitrate following the administration of contrast medium, indicating competition for salivary transport. Conclusion Our findings suggest that NIS is mediating salivary gland uptake and concentration of nitrate in saliva.

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.

Background: Recently, we have demonstrated that red blood cells (RBCs) from individuals with type 2 diabetes (T2D-RBCs) induce endothelial dysfunction via upregulation of arginase-1 in RBCs. However, the mechanism by which RBCs communicate with the vessel is unknown. 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 EVs from RBCs in the development of endothelial dysfunction remains to be elucidated. Purpose: This study was designed to test the hypothesis that EVs transfer arginase-1 protein to the vascular endothelium to induce endothelial dysfunction in T2D. Methods: RBCs from T2D patients and age-matched healthy controls (H-RBCs) were incubated for 18h for EV release. The content of arginase-1 in RBC-derived EVs was determined. The EVs were co-incubated with mouse aortas from endothelial cell arginase-1 KO mice (Arg-1 fl/fl /Tie2Cre tg/– ) and their littermates (Arg-1 fl/fl /Tie2Cre –/– ) to evaluate endothelium-dependent relaxation (EDR) and arginase-1 expression by immunohistochemistry. The functional involvement of arginase was investigated using pharmacological interventions and expression analyses. Results: Arginase-1 was detected in RBC-derived EVs ( Fig. 1A ). T2D RBC-EVs impaired EDR in vessels isolated from endothelial cell arginase-1 KO mice compared to those incubated with H RBC-EVs ( Fig. 1B ). Interestingly, incubation of aortas from endothelial cell arginase-1 KO mice with T2D RBC-EVs led to increased expression of arginase-1 in the vessel wall ( Fig. 1C-D ). Arginase-1 was co-expressed with CD31 in the aortas of Arg-1 fl/fl /Tie2Cre tg/– mice following incubation with T2D RBC-EVs ( Fig. 1E ), suggesting co-localization in the endothelium. Moreover, the impaired EDR induced by T2D RBC-EVs in aortas from endothelial cell arginase-1 KO mice was attenuated by the arginase inhibitor ABH in the vessel ( Fig. 1F ). Conclusion: EVs derived from RBCs of individuals with T2D carry arginase-1 protein to induce endothelial dysfunctionthrough the delivery of arginase-1. EVs derived from T2D-RBCs transfer arginase-1 protein to endothelial cells, leading to endothelial dysfunction. Potential therapeutic strategies that interfere with the uptake of the cargo of the EVs, or the transfer of signaling molecules by RBC-derived EVs have the potential to prevent vascular injury in T2D.

The incidence of cardiovascular disease (CVD) in young individuals is increasing. This alarming trend underscores the need to identify at-risk groups for preventive measures. Emerging evidence suggests that maternal diabetes increases the risk for metabolic diseases and early-onset CVDs in their offspring. However, the evidence is largely observational, limited by confounding factors, and lacks crucial mechanistic insight. Here, we combine experimental, epidemiological, and clinical approaches to disentangle the effects of maternal diabetes on offspring metabolism and endothelial function. In mice, we find that maternal hyperglycemia induces early-onset endothelial dysfunction specifically in male offspring, independent of metabolic disease. In humans, a case-control study and an epidemiological study confirm elevated risk of early-onset endothelial dysfunction and related CVDs in metabolically healthy sons of mothers with type 1 diabetes. Our findings identify an underrecognized risk group for early-onset CVDs and emphasize the importance of maternal conditions in shaping the cardiovascular health of future generations.

microRNAs (miRNAs) have been intensively studied as valuable biomarkers in cardiometabolic disease. Typically, miRNAs are detected in plasma or serum, but the use of samples collected in heparinized tubes is problematic for miRNA studies using quantitative PCR (qPCR). Heparin and its derivatives interfere with qPCR-based analysis, leading to a substantial reduction or even complete loss of detectable miRNA levels. Given that red blood cells (RBCs) express abundant miRNAs, whose expression is altered in cardiometabolic disease, RBCs could serve as an attractive alternative in biomarker studies. Here, we aim to explore the stability of miRNAs in RBCs collected from whole blood with different anticoagulants and thereby the potential of RBCs as alternative materials for miRNA biomarker studies. miRNA profiling was performed in human RBCs via RNA sequencing, followed by qPCR validation of selected miRNAs in RBCs and plasma in both heparinized and EDTA tubes. RNA sequencing revealed abundant miRNA presence in RBCs isolated from blood collected in EDTA tubes. miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were detected at comparable levels in RBCs isolated from both heparinized and EDTA tubes but not in plasma from heparinized tubes. Of note, miR-210-3p levels were consistently lower in RBCs from individuals with type 2 diabetes compared with healthy controls, regardless of anticoagulant type, supporting their potential as biomarker materials. In conclusion, RBCs offer a promising alternative for miRNA biomarker studies, overcoming heparin-related challenges. NEW & NOTEWORTHY microRNAs are valuable biomarkers in cardiometabolic disease, but heparinized tubes hinder their detection because of qPCR interference. RBCs, which express abundant microRNAs like miR-210-3p, may serve as an alternative. microRNAs, including miR-210-3p, are consistently detectable in RBCs at comparable levels between heparinized and EDTA tubes. miR-210-3p levels in RBCs are similarly reduced in heparinized tubes of patients with type 2 diabetes. Thus, RBCs offer a promising solution for miRNA biomarker studies, overcoming heparin-related challenges.

Background and PurposeMicroRNA (miR)-210 function in endothelial cells and its role in diabetes-associated endothelial dysfunction are not fully understood. We aimed to characterize the miR-210 function in endothelial cells and study its therapeutic potential in diabetes.Experimental ApproachTwo different diabetic mouse models (db/db and Western diet-induced), miR-210 knockout and transgenic mice, isolated vessels and human endothelial cells were used.Key ResultsmiR-210 levels were lower in aortas isolated from db/db than in control mice. Endothelium-dependent relaxation (EDR) was impaired in aortas from miR-210 knockout mice, and this was restored by inhibiting miR-210 downstream protein tyrosine phosphatase 1B (PTP1B), mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2), and mitochondrial oxidative stress. Inhibition of these pathways also improved EDR in both diabetic mouse models. High glucose reduced miR-210 levels in endothelial cells and impaired EDR in mouse aortas, effects that were reversed by overexpressing miR-210. However, plasma miR-210 levels were not affected in individuals with type 2 diabetes (T2D) following improved glycaemic status. Of note, genetic overexpression using miR-210 transgenic mice and pharmacological overexpression using miR-210 mimic in vivo ameliorated endothelial dysfunction in both diabetic mouse models by decreasing PTP1B, GPD2 and oxidative stress. Genetic overexpression of miR-210 altered the aortic transcriptome, decreasing genes in pathways involved in oxidative stress. miR-210 mimic restored decreased nitric oxide production by high glucose in endothelial cells.Conclusion and ImplicationsThis study unravels the mechanisms by which down-regulated miR-210 by high glucose induces endothelial dysfunction in T2D and demonstrates that miR-210 serves as a novel therapeutic target. image

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.

Abstract Background Histone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium. Methods We studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2−) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice. Results Incubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2− generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice. Conclusions EZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes. Graphical Abstract

Red blood cells (RBCs) regulate cardiovascular function via a mechanism involving nitric oxide-like bioactivity, but the signaling and the identity of any mediator released by RBCs have remained unknown. We have investigated whether RBCs exposed to hypoxia mediates cardioprotection during ischemia-reperfusion and explored the signaling involved. Administration of hypoxic RBCs or the extracellular supernatant from mouse RBCs exposed to hypoxia to isolated hearts subjected to ischemia-reperfusion improved post-ischemic cardiac function and reduced infarct size. This cardioprotective effect was abolished by blocking sGC in the RBCs or when exposing RBCs from sGC knockout mice to hypoxia, suggesting that RBC sGC is required for the protective effect. Exposure of RBCs to hypoxia resulted in increased extracellular levels of cGMP, and exogenous cGMP mimicked the cardioprotection induced by the supernatant. The protection induced by hypoxic RBCs was dependent on cGMP transport, sensitive to phosphodiesterase 5 and activated cardiomyocyte protein kinase G. Oral administration of nitrate to mice and humans to increase nitric oxide bioactivity further enhanced the cardioprotective effect of RBCs. Pharmacological stimulation of RBC sGC mimicked the cardioprotective effect of hypoxia and reversed the negative effect of RBCs from patients with type 2 diabetes on post-ischemic cardiac function via a mechanism involving release of cGMP and activation of cardiac protein kinase G. 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 pharmacological stimulation of RBC sGC suggesting a therapeutic target in ischemic heart disease.

To study the relationship between myocardial infarction size (IS), myocardial edema, and diastolic dysfunction after acute myocardial infarction (MI) both in the acute phase, and in the development of diastolic dysfunction in the follow-up setting. A further purpose is to study diastolic function using a mechanistic model as well as conventional parameters. Patients underwent cardiovascular magnetic resonance (CMR) imaging and echocardiography including mechanistic analysis using the parameterized diastolic filling method within 4–7 days (acute) and 6 months after a first acute anterior MI (n = 74). Linear regression modeling of echocardiographic diastolic parameters using CMR IS with and without inclusion of the myocardium at risk (MAR) and model comparisons with likelihood ratio tests were performed. Diastolic parameters at 6 months follow-up were modelled using final IS. For most parameters there was no association with acute IS, except for deceleration time (R2 = 0.24, p < 0.001), left atrial volume index (R2 = 0.13, p = 0.01) and the mechanistic stiffness parameter (R2 = 0.21, p < 0.001). Adding MAR improved only the e′ model (adjusted R2 increase: 0.08, p = 0.02). At 6 months follow-up, final IS was only associated with viscoelastic energy loss (R2 = 0.22, p = 0.001). In acute MI, both IS and MAR are related to diastolic function but only to a limited extent. At 6 months after infarction, increasing IS is related to less viscoelastic energy loss, albeit also to a limited extent. The relationship between IS and diastolic dysfunction seems to be mediated by mechanisms beyond simply the spatial extent of ischemia or infarction.

Abstract Background Recently, we have demonstrated that red blood cells (RBCs) from individuals with type 2 diabetes (T2D-RBCs) induce endothelial dysfunction. However, the mechanism by which RBCs communicate with the vessel is unknown. 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 EVs from RBC in the development of endothelial dysfunction remains to be elucidated. Purpose This study was designed to test the hypothesis that EVs are key players in the communication and the transfer of signalling between RBCs and the vascular endothelium to induce endothelial dysfunction in T2D. Methods RBCs from T2D patients and age-matched healthy controls (H-RBCs) were incubated for 18h with Krebs-Henseleit buffer (20% haematocrit) for EV release. RBC-derived EVs in the conditioned medium were isolated using a membrane affinity column. The EVs were co-incubated with mouse aortae to evaluate endothelium-dependent relaxation and with endothelial cells for expression analysis. The uptake of the EVs by endothelial cells and their content of arginase-1 were determined. The functional involvement of arginase was investigated using pharmacological interventions and expression analyses. All animal experiments were performed according to the principles of laboratory animal care (NIH Publication no. 85-23 revised 1985) and human procedures according to the declaration of Helsinki with approval by the Swedish Ethical Review Authority. Results The uptake of EVs derived from T2D-RBCs by endothelial cells was 2-fold greater than that of EVs from H-RBCs (Fig. 1A-B). Inhibiting the uptake of EVs derived from T2D-RBCs by the addition of heparin during the co-incubation rescued the endothelial function (Fig. 1C). Arginase-1 was detected in RBC-derived EVs (Fig. 2A). Arginase-1 mRNA and protein levels were increased in endothelial cells following co-incubation with EVs derived from T2D-RBCs (Fig. 2B-D). Additionally, the increase in arginase-1 protein induced by EVs derived from T2D-RBCs in endothelial cells was observed also following mRNA silencing for arginase-1 (Fig. 2E-F). Finally, mouse aortae co-incubated with EVs derived from T2D-RBCs in the presence or absence of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid significantly attenuated the impairment in endothelial function induced by EVs derived from T2D-RBCs (Fig. 2G). Conclusion Increased uptake of RBC-derived EVs by the endothelial cells is an important feature of the endothelial dysfunction induced by these EVs in T2D. In addition, these EVs carry arginase-1 protein to induce endothelial dysfunction. The mechanism underlying the increased uptake of EVs in target cells is of importance to identify in future studies, as it could lead to new treatment strategies.

Background: Red blood cells (RBCs) from patients with T2D (T2D-RBCs) induce endothelial dysfunction, but the mechanisms remain unclear. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by RBCs and represent a novel mechanism of intercellular communication. However, the functional role of T2D-RBCs EVs in communication between RBCs and the cardiovascular system remains unknown. Purpose: This study aims to investigate whether EVs induce endothelial dysfunction in T2D and, if this is mediated via increased vascular oxidative stress. Material and Methods: EVs derived from T2D-RBCs and healthy RBCs (H-RBCs) were isolated using a membrane affinity column and co-incubated with wild-type mouse aortas for evaluation of the endothelium-dependent relaxation (EDR) using the wire myograph in the presence or absence of non-selective reactive oxygen species scavenger N-acetyl cysteine (NAC; 100 μM) applied to the aortas following the 18h EV incubation. The levels of the oxidative stress marker 4-hydroxynonenal (4-HNE) were quantified by immunohistochemistry in mouse aortas incubated with EVs. Additionally, T2D-RBCs EVs and H-RBCs EVs were co-incubated with human carotid artery endothelial cells (HCtAEC) for 8h and 24h to study the effects of EVs on vascular mRNA expression levels of eNOS, NOX1, and NOX4. Results: T2D-RBCs EVs but not H-RBCs EVs impaired EDR in aortas isolated from mice ( Fig. 1A ). This impairment was reversed by inhibiting oxidative stress in the vessel by NAC ( Fig. 1B ). Immunohistochemistry showed a significant increase in 4-HNE levels in aortas incubated with T2D-RBCs EVs ( Fig. 1C, D ). The T2D-RBCs EVs also induced a significant increase in NOX4 mRNA levels in HCtAEC after 24h but not 8h ( Fig. 1E, F ). However, T2D-RBCs EVs did not affect eNOS or NOX1 in endothelial cells. Conclusion: EVs derived from T2D-RBCs induce endothelial dysfunction through increased vascular oxidative stress.

The mechanisms underlying endothelial dysfunction in Type 1 and Type 2 diabetes (T1DM and T2DM) are unresolved. The red blood cells (RBCs) with increased arginase activity induce endothelial dysfunction in T2DM, but the implications of RBCs and the role of arginase inhibition in T1DM are unexplored. We aimed to investigate the differences in endothelial function in patients with T1DM and T2DM, with focus on RBCs and arginase. Thirteen patients with T1DM and twenty-six patients with T2DM, matched for HbA1c and sex were included. In vivo endothelium-dependent and -independent vasodilation (EDV and EIDV) were assessed by venous occlusion plethysmography before and after administration of an arginase inhibitor. RBCs were co-incubated with rat aortic segments for 18h followed by evaluation of endothelium-dependent (EDR) and -independent relaxation (EIDR) in isolated organ chambers. In vivo EDV, but not EIDV, was significantly impaired in patients with T2DM compared with patients with T1DM. Arginase inhibition resulted in improved EDV only in T2DM. RBCs from patients with T2DM induced impaired EDR but not EIDR in isolated aortic segments, whereas RBCs from patients with T1DM did not affect EDR nor EIDR. The present study demonstrates markedly impaired EDV in patients with T2DM in comparison with T1DM. In addition, it highlights the divergent roles of RBCs and arginase in mediating endothelial dysfunction in T1DM and T2DM. While endothelial dysfunction is mediated via RBCs and arginase in T2DM, these phenomena are not prominent in T1DM thereby indicating distinct differences in underlying mechanisms.