The red blood cell as a mediator of endothelial dysfunction in patients with familial hypercholesterolemia and dyslipidemia.

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 <2.5 mM or RBCs from healthy subjects (H-RBCs). Inhibition of vascular but not RBC arginase attenuated the degree of endothelial dysfunction induced by 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.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling.

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia-reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia-reperfusion injury through the P2Y13 receptor and the NO-sGC-PKG pathway.

Therapeutic Potential of Sunitinib in Ameliorating Endothelial Dysfunction in Type 2 Diabetic Rats.

INTRODUCTION: Sunitinib, a multi-targeted tyrosine kinase receptor inhibitor used to treat renal-cell carcinoma and gastrointestinal stromal tumor, was recently shown to have a beneficial effect on metabolism in type 2 diabetes (T2D). Endothelial dysfunction is a key factor behind macro- and microvascular complications in T2D. The effect of sunitinib on endothelial function in T2D remains, however, unclear. We therefore tested the hypothesis that sunitinib ameliorates endothelial dysfunction in T2D. METHODS: Sunitinib (2 mg/kg/day, by gavage) was administered to T2D Goto-Kakizaki (GK) rats for 6 weeks, while water was given to GK and Wistar rats as controls. Hemodynamic, inflammatory, and metabolic parameters as well as endothelial function were measured. RESULTS: Systolic, mean arterial blood pressures, plasma tumor necrosis factor α levels, kidney weight to body weight (BW) ratio, and glucose levels were higher, while BW was lower in GK rats than in Wistar rats. Six-week treatment with sunitinib in GK rats did not affect these parameters but suppressed the increase in glucose levels. Endothelium-dependent relaxations were reduced in both aortas and mesenteric arteries isolated from GK as compared to Wistar rats, which was markedly reversed in both types of arteries from GK rats treated with sunitinib. CONCLUSIONS: This study demonstrates that sunitinib has a glucose-lowering effect and ameliorates endothelial dysfunction in both conduit and resistance arteries of GK rats.

Hemoglobin ß93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.

Altered Purinergic Receptor Sensitivity in Type 2 Diabetes-Associated Endothelial Dysfunction and Up4A-Mediated Vascular Contraction.

Purinergic signaling may be altered in diabetes accounting for endothelial dysfunction. Uridine adenosine tetraphosphate (Up4A), a novel dinucleotide substance, regulates vascular function via both purinergic P1 and P2 receptors (PR). Up4A enhances vascular contraction in isolated arteries of diabetic rats likely through P2R. However, the precise involvement of PRs in endothelial dysfunction and the vasoconstrictor response to Up4A in diabetes has not been fully elucidated. We tested whether inhibition of PRs improved endothelial function and attenuated Up4A-mediated vascular contraction using both aortas and mesenteric arteries of type 2 diabetic (T2D) Goto Kakizaki (GK) rats vs. control Wistar (WT) rats. Endothelium-dependent (EDR) but not endothelium-independent relaxation was significantly impaired in both aortas and mesenteric arteries from GK vs. WT rats. Non-selective inhibition of P1R or P2R significantly improved EDR in aortas but not mesenteric arteries from GK rats. Inhibition of A1R, P2X7R, or P2Y6R significantly improved EDR in aortas. Vasoconstrictor response to Up4A was enhanced in aortas but not mesenteric arteries of GK vs. WT rats via involvement of A1R and P2X7R but not P2Y6R. Depletion of major endothelial component nitric oxide enhanced Up4A-induced aortic contraction to a similar extent between WT and GK rats. No significant differences in protein levels of A1R, P2X7R, and P2Y6R in aortas from GK and WT rats were observed. These data suggest that altered PR sensitivity accounts for endothelial dysfunction in aortas in diabetes. Modulating PRs may represent a potential therapy for improving endothelial function.

Dietary nitrate improves cardiac contractility via enhanced cellular Ca²âº signaling.

The inorganic anion nitrate (NO3 (-)), which is naturally enriched in certain vegetables (e.g., spinach and beetroot), has emerged as a dietary component that can regulate diverse bodily functions, including blood pressure, mitochondrial efficiency, and skeletal muscle force. It is not known if dietary nitrate improves cardiac contractility. To test this, mice were supplemented for 1-2 weeks with sodium nitrate in the drinking water at a dose similar to a green diet. The hearts from nitrate-treated mice showed increased left ventricular pressure and peak rate of pressure development as measured with the Langendorff heart technique. Cardiomyocytes from hearts of nitrate-treated and control animals were incubated with the fluorescent indicator Fluo-3 to measure cytoplasmic free [Ca(2+)] and fractional shortening. Cardiomyocytes from nitrate-treated mice displayed increased fractional shortening, which was linked to larger Ca(2+) transients. Moreover, nitrate hearts displayed increased protein expression of the L-type Ca(2+) channel/dihydropyridine receptor and peak L-type Ca(2+) channel currents. The nitrate-treated hearts displayed increased concentration of cAMP but unchanged levels of cGMP compared with controls. These findings provide the first evidence that dietary nitrate can affect the expression of important Ca(2+) handling proteins in the heart, resulting in increased cardiomyocyte Ca(2+) signaling and improved left ventricular contractile function. Our observation shows that dietary nitrate impacts cardiac function and adds understanding to inorganic nitrate as a physiological modulator.

Arginase regulates red blood cell nitric oxide synthase and export of cardioprotective nitric oxide bioactivity.

The theory that red blood cells (RBCs) generate and release nitric oxide (NO)-like bioactivity has gained considerable interest. However, it remains unclear whether it can be produced by endothelial NO synthase (eNOS), which is present in RBCs, and whether NO can escape scavenging by hemoglobin. The aim of this study was to test the hypothesis that arginase reciprocally controls NO formation in RBCs by competition with eNOS for their common substrate arginine and that RBC-derived NO is functionally active following arginase blockade. We show that rodent and human RBCs contain functional arginase 1 and that pharmacological inhibition of arginase increases export of eNOS-derived nitrogen oxides from RBCs under basal conditions. The functional importance was tested in an ex vivo model of myocardial ischemia-reperfusion injury. Inhibitors of arginase significantly improved postischemic functional recovery in rat hearts if administered in whole blood or with RBCs in plasma. By contrast, arginase inhibition did not improve postischemic recovery when administered with buffer solution or plasma alone. The protective effect of arginase inhibition was lost in the presence of a NOS inhibitor. Moreover, hearts from eNOS(-/-) mice were protected when the arginase inhibitor was given with blood from wild-type donors. In contrast, when hearts from wild-type mice were given blood from eNOS(-/-) mice, the arginase inhibitor failed to protect against ischemia-reperfusion. These results strongly support the notion that RBCs contain functional eNOS and release NO-like bioactivity. This process is under tight control by arginase 1 and is of functional importance during ischemia-reperfusion.

Stimulation of Erythrocyte Soluble Guanylyl Cyclase Induces cGMP Export and Cardioprotection in Type 2 Diabetes.

Reduced nitric oxide (NO) bioactivity in red blood cells (RBCs) is critical for augmented myocardial ischemia-reperfusion injury in type 2 diabetes. This study identified the nature of "NO bioactivity" by stimulating the intracellular NO receptor soluble guanylyl cyclase (sGC) in RBCs. sGC stimulation in RBCs from patients with type 2 diabetes increased export of cyclic guanosine monophosphate from RBCs and activated cardiac protein kinase G, thereby attenuating ischemia-reperfusion injury. These results provide novel insight into RBC signaling by identifying cyclic guanosine monophosphate from RBC as a mediator of protection against cardiac ischemia-reperfusion injury induced by sGC stimulation in RBCs.

Hypoxic erythrocytes mediate cardioprotection through activation of soluble guanylate cyclase and release of cyclic GMP.

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.

Erythrocytes Induce Vascular Dysfunction in COVID-19.

Current knowledge regarding mechanisms underlying cardiovascular complications in patients with COVID-19 is limited and urgently needed. We shed light on a previously unrecognized mechanism and unravel a key role of red blood cells, driving vascular dysfunction in patients with COVID-19 infection. We establish the presence of profound and persistent endothelial dysfunction in vivo in patients with COVID-19. Mechanistically, we show that targeting reactive oxygen species or arginase 1 improves vascular dysfunction mediated by red blood cells. These translational observations hold promise that restoring the redox balance in red blood cells might alleviate the clinical complications of COVID-19-associated vascular dysfunction.

Downregulation of Erythrocyte miR-210 Induces Endothelial Dysfunction in Type 2 Diabetes.

Red blood cells (RBC) act as mediators of vascular injury in type 2 diabetes mellitus (T2DM). miR-210 plays a protective role in cardiovascular homeostasis and is decreased in whole blood of T2DM mice. We hypothesized that downregulation of RBC miR-210 induces endothelial dysfunction in T2DM. RBC were coincubated with arteries and endothelial cells ex vivo and transfused in vivo to identify the role of miR-210 and its target protein tyrosine phosphatase 1B (PTP1B) in endothelial dysfunction. RBC from patients with T2DM and diabetic rodents induced endothelial dysfunction ex vivo and in vivo. miR-210 levels were lower in human RBC from patients with T2DM (T2DM RBC) than in RBC from healthy subjects. Transfection of miR-210 in human T2DM RBC rescued endothelial function, whereas miR-210 inhibition in healthy subjects RBC or RBC from miR-210 knockout mice impaired endothelial function. Human T2DM RBC decreased miR-210 expression in endothelial cells. miR-210 expression in carotid artery plaques was lower in T2DM patients than in patients without diabetes. Endothelial dysfunction induced by downregulated RBC miR-210 involved PTP1B and reactive oxygen species. miR-210 mimic attenuated endothelial dysfunction induced by RBC via downregulating vascular PTP1B and oxidative stress in diabetic mice in vivo. These data reveal that the downregulation of RBC miR-210 is a novel mechanism driving the development of endothelial dysfunction in T2DM.

Human Cytomegalovirus Reduces Endothelin-1 Expression in Both Endothelial and Vascular Smooth Muscle Cells.

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

Dehydro-Tocotrienol-ß Counteracts Oxidative-Stress-Induced Diabetes Complications in db/db Mice.

Hyperglycemia, hyperlipidemia, and adiposity are the main factors that cause inflammation in type 2 diabetes due to excessive ROS production, leading to late complications. To counteract the effects of increased free radical production, we searched for a compound with effective antioxidant properties that can induce coenzyme Q biosynthesis without affecting normal cellular functions. Tocotrienols are members of the vitamin E family, well-known as efficient antioxidants that are more effective than tocopherols. Deh-T3ß is a modified form of the naturally occurring tocotrienol-ß. The synthesis of this compound involves the sequential modification of geranylgeraniol. In this study, we investigated the effects of this compound in different experimental models of diabetes complications. Deh-T3ß was found to possess multifaceted capacities. In addition to enhanced wound healing, deh-T3ß improved kidney and liver functions, reduced liver steatosis, and improved heart recovery after ischemia and insulin sensitivity in adipose tissue in a mice model of type 2 diabetes. Deh-T3ß exerts these positive effects in several organs of the diabetic mice without reducing the non-fasting blood glucose levels, suggesting that both its antioxidant properties and improvement in mitochondrial function are involved, which are central to reducing diabetes complications.

Erythrocytes Induce Endothelial Injury in Type 2 Diabetes Through Alteration of Vascular Purinergic Signaling.

It is well established that altered purinergic signaling contributes to vascular dysfunction in type 2 diabetes (T2D). Red blood cells (RBCs) serve as an important pool for circulating ATP and the release of ATP from RBCs in response to physiological stimuli is impaired in T2D. We recently demonstrated that RBCs from patients with T2D (T2D RBC) serve as key mediators of endothelial dysfunction. However, it remains unknown whether altered vascular purinergic signaling is involved in the endothelial dysfunction induced by dysfunctional RBCs in T2D. Here, we evaluated acetylcholine-induced endothelium-dependent relaxation (EDR) of isolated rat aortas after 18 h ex vivo co-incubation with human RBCs, and aortas of healthy recipient rats 4 h after in vivo transfusion with RBCs from T2D Goto-Kakizaki (GK) rats. Purinergic receptor (PR) antagonists were applied in isolated aortas to study the involvement of PRs. EDR was impaired in aortas incubated with T2D RBC but not with RBCs from healthy subjects ex vivo, and in aortas of healthy rats after transfusion with GK RBCs in vivo. The impairment in EDR by T2D RBC was attenuated by non-selective P1R and P2R antagonism, and specific A1R, P2X7R but not P2Y6R antagonism. Transfusion with GK RBCs in vivo impaired EDR in aortas of recipient rats, an effect that was attenuated by A1R, P2X7R but not P2Y6R antagonism. In conclusion, RBCs induce endothelial dysfunction in T2D via vascular A1R and P2X7R but not P2Y6R. Targeting vascular purinergic singling may serve as a potential therapy to prevent endothelial dysfunction induced by RBCs in T2D.

Endothelin-1 increases expression and activity of arginase 2 via ETB receptors and is co-expressed with arginase 2 in human atherosclerotic plaques.

BACKGROUND AND AIMS: Endothelin-1 (ET-1) and arginase are both suggested to be involved in the inflammatory processes and development of endothelial dysfunction in atherosclerosis. However, information regarding the roles of ET-1 and arginase, as well as the interactions between the two in human atherosclerosis, is scarce. We investigated the expression of ET-1 and its receptors, ETA and ETB, as well as arginase in human carotid atherosclerotic plaques and determined the functional interactions between ET-1 and arginase in endothelial cells and THP-1-derived macrophages. METHODS: Carotid plaques and blood samples were retreived from patients undergoing surgery for symptomatic or asymptomatic carotid stenosis. Plaque gene and protein expression was determined and related to clinical characteristics. Functional interactions between ET-1 and arginase were investigated in endothelial cells and THP-1 cells. RESULTS: Expression of ET-1 and ETB receptors was increased in plaques from patients with symptomatic carotid artery disease. ET-1 was co-localized with arginase 1 and arginase 2 in the necrotic core, together with macrophage markers CD163 and CD68. Arginase 2, ET-1 and ETB receptors were expressed in endothelial cells as well as in smooth muscle cells in the fibrous cap. ET-1 increased arginase 2 mRNA expression and arginase activity in endothelial cells and arginase activity in macrophages. Moreover, ET-1 stimulated formation of reactive oxygen species (ROS) in THP-1-derived macrophages via an arginase-dependent mechanism. CONCLUSIONS: This is the first study that demonstrates co-localization of ET-1 and arginase 2 in human atherosclerotic plaques. ET-1 stimulated arginase 2 expression and activity in endothelial cells, as well as arginase activity and ROS formation in macrophages via an arginase-dependent mechanism. These results indicate an important interaction between the ET pathway and arginase in human atherosclerotic plaques.

Mice heterozygous for both A1 and A(2A) adenosine receptor genes show similarities to mice given long-term caffeine.

Caffeine is believed to exert its stimulant effects by blocking A(2A) and A(1) adenosine receptors (A(2A)R and A(1)R). Although a genetic knockout of A(2A)R eliminates effects of caffeine, the phenotype of the knockout animal does not resemble that of caffeine treatment. In this study we explored the possibility that a mere reduction of the number of A(1)Rs and A(2A)Rs, achieved by deleting one of the two copies of the A(1)R and A(2A)R genes, would mimic some aspects of long-term caffeine ingestion. The A(1)R and A(2A)R double heterozygous (A(1)R-A(2A)R dHz) mice indeed had approximately one-half the number of A(1)R and A(2A)R, and there were little compensatory changes in A(2B) or A(3) adenosine receptor (A(2B)R or A(3)R) expression. The ability of a stable adenosine analog to activate receptors was shifted to the right by caffeine and in A(1)R-A(2A)R dHz tissue. Caffeine (0.3 g/l in drinking water for 7-10 days) and A(1)R-A(2A)R dHz genotype increased locomotor activity (LA) and decreased heart rate without significantly influencing body temperature. The acute stimulatory effect of a single injection of caffeine was reduced in A(1)R-A(2A)R dHz mice and in mice treated long term with oral caffeine. Thus at least some aspects of long-term caffeine use can be mimicked by genetic manipulation of the A(1)R and A(2A)R.

Red blood cell dysfunction: a new player in cardiovascular disease.

The primary role of red blood cells (RBCs) is to transport oxygen to the tissues and carbon dioxide to the lungs. However, emerging evidence suggests an important role of the RBC beyond being just a passive carrier of the respiratory gases. The RBCs are of importance for redox balance and are actively involved in the regulation of vascular tone, especially during hypoxic and ischaemic conditions by the release of nitric oxide (NO) bioactivity and adenosine triphosphate. The role of the RBC has gained further interest after recent discoveries demonstrating a markedly altered function of the cell in several pathological conditions. Such alterations include increased adhesion capability, increased formation of reactive oxygen species as well as altered protein content and enzymatic activities. Beyond signalling increased oxidative stress, the altered function of RBCs is characterized by reduced export of NO bioactivity regulated by increased arginase activity. Of further importance, the altered function of RBCs has important implications for several cardiovascular disease conditions. RBCs have been shown to induce endothelial dysfunction and to increase cardiac injury during ischaemia-reperfusion in diabetes mellitus. Finally, this new knowledge has led to novel therapeutic possibilities to intervene against cardiovascular disease by targeting signalling in the RBC. These novel data open up an entirely new view on the underlying pathophysiological mechanisms behind the cardiovascular disease processes in diabetes mellitus mediated by the RBC. This review highlights the current knowledge regarding the role of RBCs in cardiovascular regulation with focus on their importance for cardiovascular dysfunction in pathological conditions and therapeutic possibilities for targeting RBCs in cardiovascular disease.

The Effect of Glycemic Control on Endothelial and Cardiac Dysfunction Induced by Red Blood Cells in Type 2 Diabetes.

Red blood cells (RBCs) from patients with type 2 diabetes mellitus (T2DM) induce endothelial dysfunction and impair cardiac function following ischemia via increase in RBC arginase and oxidative stress. Here, we aimed to elucidate whether the effect of RBC-mediated cardiac impairment following ischemia and endothelial dysfunction in T2DM is dependent on glycemic control. Patients with T2DM at poor glycemic control (T2DM PGC) and at improvement in glycemic control (T2DM IGC) and healthy subjects were recruited. Isolated RBCs from subjects were incubated with aortic rings from healthy wild-type rats with subsequent evaluation of endothelium-dependent relaxation (EDR) using wire myograph. Moreover, RBCs were administered to isolated wild-type rat hearts with subsequent evaluation of left ventricular developed pressure (LVDP) during reperfusion using Langendorff setup. In separate experiments, RBCs were preincubated with an arginase inhibitor before perfusion. Blood glucose and glycated hemoglobin were 33 and 26%, respectively, lower in T2DM IGC compared with those in T2DM PGC. RBCs from T2DM PGC and T2DM IGC impaired EDR to a similar magnitude compared with RBCs from healthy subjects. LVDP was significantly impaired in hearts given RBCs from T2DM PGC as compared with those from healthy subjects. The impairment of LVDP induced by T2DM PGC was attenuated by RBCs from T2DM IGC. Arginase inhibition improved LVDP to a similar extent between T2DM PGC and IGC groups. These observations indicate that glycemic control abrogate the impairment in postischemic recovery but not endothelial dysfunction induced by RBCs from T2DM. Moreover, inhibition of RBC arginase improves cardiac function irrespective of glycemic control.

Adenosine A1 receptors regulate lipolysis and lipogenesis in mouse adipose tissue-interactions with insulin.

Adenosine acting at adenosine A1 receptors is considered to be one major regulator of adipose tissue physiology. We have examined the role of adenosine and its interactions with insulin in adipose tissue by using A1R knock out (-/-) mice. Removal of endogenous adenosine with adenosine deaminase caused lipolysis in A1R (+/+), but not A1R (-/-) adipocytes. The adenosine analogue, 2-chloroadenosine, inhibited noradrenaline-stimulated lipolysis and cAMP accumulation in A1R (+/+), but not in A1R (-/-) adipocytes. Insulin reduces lipolysis and cAMP via another mechanism than adenosine and acted additively, but not synergistically, with adenosine. Plasma levels of free fatty acids, glycerol and triglycerides were significantly lower in A1R (+/+) than in A1R (-/-) mice after administration of an adenosine analogue. 2-chloroadenosine induced lipogenesis in presence of insulin in A1R (+/+), but not in A1R (-/-) adipocytes. There were no changes in mRNA levels for several genes involved in fat synthesis in adipose tissue between genotypes. Body weight was similar in young A1R (+/+) and A1R (-/-) mice, but old male A1R (-/-) mice were heavier than wild type controls. In conclusion, adenosine inhibits lipolysis via the adenosine A1 receptor and other adenosine receptors play no significant role. Adenosine and insulin mediate additive but not synergistic antilipolytic effects and 2-chloroadenosine stimulates lipogenesis via adenosine A1 receptors. Thus deletion of adenosine A1 receptors should increase lipolysis and decrease lipogenesis, but in fact an increased fat mass was observed, indicating that other actions of adenosine A1 receptors, possibly outside adipose tissue, are also important.

Decreased behavioral activation following caffeine, amphetamine and darkness in A3 adenosine receptor knock-out mice.

We have examined behavioral consequences of genetic deletion of the adenosine A3 receptors in mice. The open field behavior of A3 adenosine receptor knock-out (A3R KO) mice was investigated both under basal conditions and after stimulation with psychostimulants. Adolescent (21 day-old) and adult A3R KO males showed an increase in overall motor activity compared to wild type (WT) males, but the type of activity differed. The motor activity, especially rearing, was also higher in A3R KO compared to WT adult females. A3 receptors have a low affinity for caffeine and it was therefore surprising to find a decreased response to stimulation with either caffeine or amphetamine in A3R KO as compared to WT mice in males as well as females. Telemetry recordings also showed a significantly smaller increase in activity upon darkness in A3R KO. There were no compensatory changes in the mRNA expression of any other adenosine receptor subtypes (A1, A2A and A2B) or any changes in dopamine D1 and D2 receptor binding in A3R KO brains. Challenge with the developmental toxicant methylmercury (1 microM in drinking water) during pregnancy and lactation did not cause any behavioral alterations in adolescent and adult WT female offspring. In contrast, the A3R KO female offspring displayed changes in locomotion indicating an interaction between perinatal methylmercury and adenosine A3 receptors. In conclusion, despite low expression of A3 receptors in wild type mouse brain we observed several behavioral consequences of genetic elimination of the adenosine A3 receptors. The possibility that this is due to a role of A3 receptors in development is discussed.