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.

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.

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.

Arginase 1 is upregulated at admission in patients with ST-elevation myocardial infarction.

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.

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.

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.

Red Blood Cells in Type 2 Diabetes Impair Cardiac Post-Ischemic Recovery Through an Arginase-Dependent Modulation of Nitric Oxide Synthase and Reactive Oxygen Species.

This study tested the hypothesis that red blood cell (RBC) arginase represents a potential therapeutic target in ischemia-reperfusion in type 2 diabetes. Post-ischemic cardiac recovery was impaired in hearts from db/db mice compared with wild-type hearts. RBCs from mice and patients with type 2 diabetes attenuated post-ischemic cardiac recovery of nondiabetic hearts. This impaired cardiac recovery was reversed by inhibition of RBCs arginase or nitric oxide synthase. The results suggest that RBCs from type 2 diabetics impair cardiac tolerance to ischemia-reperfusion via a pathway involving arginase activity and nitric oxide synthase-dependent oxidative stress.

Erythrocytes From Patients With Type 2 Diabetes Induce Endothelial Dysfunction Via Arginase I.

BACKGROUND: Cardiovascular complications are major clinical problems in type 2 diabetes mellitus (T2DM). The authors previously demonstrated a crucial role of red blood cells (RBCs) in control of cardiac function through arginase-dependent regulation of nitric oxide export from RBCs. There is alteration of RBC function, as well as an increase in arginase activity, in T2DM. OBJECTIVES: The authors hypothesized that RBCs from patients with T2DM induce endothelial dysfunction by up-regulation of arginase. METHODS: RBCs were isolated from patients with T2DM and age-matched healthy subjects and were incubated with rat aortas or human internal mammary arteries from nondiabetic patients for vascular reactivity and biochemical studies. RESULTS: Arginase activity and arginase I protein expression were elevated in RBCs from patients with T2DM (T2DM RBCs) through an effect induced by reactive oxygen species (ROS). Co-incubation of arterial segments with T2DM RBCs, but not RBCs from age-matched healthy subjects, significantly impaired endothelial function but not smooth muscle cell function in both healthy rat aortas and human internal mammary arteries. Endothelial dysfunction induced by T2DM RBCs was prevented by inhibition of arginase and ROS both at the RBC and vascular levels. T2DM RBCs induced increased vascular arginase I expression and activity through an ROS-dependent mechanism. CONCLUSIONS: This study demonstrates a novel mechanism behind endothelial dysfunction in T2DM that is induced by RBC arginase I and ROS. Targeting arginase I in RBCs may serve as a novel therapeutic tool for the treatment of endothelial dysfunction in T2DM.

Inhibition of Rho kinase protects from ischaemia-reperfusion injury via regulation of arginase activity and nitric oxide synthase in type 1 diabetes.

AIM: RhoA/Rho-associated kinase and arginase are implicated in vascular complications in diabetes. This study investigated whether RhoA/Rho-associated kinase and arginase inhibition protect from myocardial ischaemia-reperfusion injury in type 1 diabetes and the mechanisms behind these effects. METHODS: Rats with streptozotocin-induced type 1 diabetes and non-diabetic rats were subjected to 30 min myocardial ischaemia and 2 h reperfusion after being randomized to treatment with (1) saline, (2) RhoA/Rho-associated kinase inhibitor hydroxyfasudil, (3) nitric oxide synthase inhibitor NG-monomethyl-l-arginine monoacetate followed by hydroxyfasudil, (4) arginase inhibitor N-omega-hydroxy-nor-l-arginine, (5) NG-monomethyl-l-arginine monoacetate followed by N-omega-hydroxy-nor-l-arginine or (6) NG-monomethyl-l-arginine monoacetate given intravenous before ischaemia. RESULTS: Myocardial arginase activity, arginase 2 expression and RhoA/Rho-associated kinase activity were increased in type 1 diabetes ( p < 0.05). RhoA/Rho-associated kinase inhibition and arginase inhibition significantly reduced infarct size in diabetic and non-diabetic rats ( p < 0.001). The cardioprotective effects of hydroxyfasudil and N-omega-hydroxy-nor-l-arginine in diabetes were abolished by nitric oxide synthase inhibition. RhoA/Rho-associated kinase inhibition attenuated myocardial arginase activity in diabetic rats via a nitric oxide synthase-dependent mechanism. CONCLUSION: Inhibition of either RhoA/Rho-associated kinase or arginase protects from ischaemia-reperfusion injury in rats with type 1 diabetes via a nitric oxide synthase-dependent pathway. These results suggest that inhibition of RhoA/Rho-associated kinase and arginase constitutes a potential therapeutic strategy to protect the diabetic heart against ischaemia-reperfusion injury.

The role of arginase and rho kinase in cardioprotection from remote ischemic perconditioning in non-diabetic and diabetic rat in vivo.

BACKGROUND: Pharmacological inhibition of arginase and remote ischemic perconditioning (RIPerc) are known to protect the heart against ischemia/reperfusion (IR) injury. PURPOSE: The objective of this study was to investigate whether (1) peroxynitrite-mediated RhoA/Rho associated kinase (ROCK) signaling pathway contributes to arginase upregulation following myocardial IR; (2) the inhibition of this pathway is involved as a cardioprotective mechanism of remote ischemic perconditioning and (3) the influence of diabetes on these mechanisms. METHODS: Anesthetized rats were subjected to 30 min left coronary artery ligation followed by 2 h reperfusion and included in two protocols. In protocol 1 rats were randomized to 1) control IR, 2) RIPerc induced by bilateral femoral artery occlusion for 15 min during myocardial ischemia, 3) RIPerc and administration of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), 4) administration of the ROCK inhibitor hydroxyfasudil or 5) the peroxynitrite decomposition catalyst FeTPPS. In protocol 2 non-diabetic and type 1 diabetic rats were randomosed to IR or RIPerc as described above. RESULTS: Infarct size was significantly reduced in rats treated with FeTPPS, hydroxyfasudil and RIPerc compared to controls (P<0.001). FeTPPS attenuated both ROCK and arginase activity (P<0.001 vs. control). Similarly, RIPerc reduced arginase and ROCK activity, peroxynitrite formation and enhanced phospho-eNOS expression (P<0.05 vs. control). The cardioprotective effect of RIPerc was abolished by L-NMMA. The protective effect of RIPerc and its associated changes in arginase and ROCK activity were abolished in diabetes. CONCLUSION: Arginase is activated by peroxynitrite/ROCK signaling cascade in myocardial IR. RIPerc protects against IR injury via a mechanism involving inhibition of this pathway and enhanced eNOS activation. The beneficial effect and associated molecular changes of RIPerc is abolished in type 1 diabetes.

Arginase as a target for treatment of myocardial ischemia-reperfusion injury.

Two distinct enzymes of arginase (1 and 2) are critically regulating nitric oxide (NO) bioavailability by competing with NO synthase for their common substrate l-arginine. Increased expression and activity of arginase is observed in atherosclerosis and myocardial ischemia-reperfusion (I/R). Several studies have demonstrated a key pathophysiological role of increased activity of arginase during I/R. Pharmacological inhibition of arginase results in restoration of NO availability and salvage of myocardium during I/R. Arginase inhibition might be a promising therapeutic strategy for the limitation of myocardial injury in acute myocardial infarction. Current understanding of the role of arginase and efficacy of arginase inhibition during myocardial I/R is reviewed in the present article.

Myocardial protection by co-administration of L-arginine and tetrahydrobiopterin during ischemia and reperfusion.

BACKGROUND: Reduced bioavailability of nitric oxide (NO) is a key factor contributing to myocardial ischemia and reperfusion injury. The mechanism behind the reduction of NO is related to deficiency of the NO synthase (NOS) substrate L-arginine and cofactor tetrahydrobiopterin (BH4) resulting in NOS uncoupling. The aim of the study was to investigate if the combination of L-arginine and BH4 given iv or intracoronary before reperfusion protects from reperfusion injury. METHODS: Sprague-Dawley rats and pigs were subjected to myocardial ischemia and reperfusion. Rats received vehicle, L-arginine, BH4, L-arginine+BH4 with or without the NOS-inhibitor L-NMMA iv 5 min before reperfusion. Pigs received infusion of vehicle, L-arginine, BH4 or L-arginine+BH4 into the left main coronary artery for 30 min starting 10 min before reperfusion. RESULTS: Infarct size was significantly smaller in the rats (50 ± 2%) and pigs (54 ± 5%) given L-arginine+BH4 in comparison with the vehicle groups (rats 65 ± 3% and pigs 86 ± 5%, P<0.05). Neither L-arginine nor BH4 alone significantly reduced infarct size. Administration of L-NMMA abrogated the cardioprotective effect of L-arginine+BH4. Myocardial BH4 levels were 3.5- to 5-fold higher in pigs given L-arginine+BH4 and BH4 alone. The generation of superoxide in the ischemic-reperfused myocardium was reduced in pigs treated with intracoronary L-arginine+BH4 versus the vehicle group (P<0.05). CONCLUSION: Administration of L-arginine+BH4 before reperfusion protects the heart from ischemia-reperfusion injury. The cardioprotective effect is mediated via NOS-dependent pathway resulting in diminished superoxide generation.

Arginase inhibition reduces infarct size via nitric oxide, protein kinase C epsilon and mitochondrial ATP-dependent K+ channels.

Reduced bioavailability of nitric oxide (NO) contributes to the development of myocardial ischemia-reperfusion (I/R) injury. Increased activity of arginase is a potential factor that reduces NO bioavailability by competing for the substrate L-arginine. The aim of the study was to test the hypothesis that inhibition of arginase after coronary artery occlusion protects from I/R injury and to explore possible mechanisms behind this effect. Male Sprague-Dawley rats subjected to 30 min of coronary artery ligation and 2h reperfusion were given i.v. before the reperfusion: 1) saline; 2) the arginase inhibitor N-omega-hydroxy-nor-L-arginine (nor-NOHA); 3) nor-NOHA with the NO synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA); 4) nor-NOHA with the mitochondrial ATP-dependent K(+) (mitoKATP) channel blocker 5-hydroxydecanoic acid (5-HD); 5) nor-NOHA with the protein kinase C epsilon (PKCε) inhibitor ε-V1-2 or 6) ε-V1-2 alone. Infarct size in the control groups was 61±3% and it was reduced to 47±3% (P<0.01) by nor-NOHA. The cardioprotective effect was blocked by the NOS inhibitor L-NMMA. PKCε expression was reduced by I/R and this reduction was attenuated by nor-NOHA. Furthermore, the PKCε inhibitor ε-V1-2 abolished the protective effect of nor-NOHA (infarct size 69±6%). In addition, the cardioprotective effect of nor-NOHA was also abolished following blockade of the mitoKATP channel (infarct size 62±1%). Inhibition of arginase before reperfusion protects the heart from I/R injury via a NOS-dependent pathway, increased expression of PKCε and activation of mitoKATP channels.