The importance of nitric oxide availability in myocardial ischemia-reperfusion injury : regulatory roles of arginase, L-arginine and tetrahydrobiopterin

Background. Maintenance of nitric oxide (NO) availability is crucial for cardiovascular homeostasis and protection against myocardial ischemia-reperfusion (IR) injury. The mechanisms underlying reduced NO availability are multifactorial and involve de ciency of the NO synthase (NOS) substrate L-arginine and co-factor tetrahydrobiopterin (BH4) as well as increased NO inactivation by reactive oxygen species. Increased activity of arginase has emerged as a key factor behind reduced NO availability by competing with NOS for L-arginine as substrate. The aims of the studies were to investigate: 1) the potential of arginase and RhoA/Rho associated kinase (ROCK) inhibition, supplementation of L-arginine with BH4 and remote ischemic perconditioning (RIPerc) to induce cardioprotection, 2) the mechanisms behind their effects and 3) the in uence of diabetes on the cardioprotective effects. Study I. Arginase inhibition by N-omega-hydroxy-nor-L-arginine (nor-NOHA) prior to reperfusion in rats subjected to myocardial IR reduced infarct size (IS). This effect was abolished by inhibition of NOS, protein kinase C epsilon (PKCε) and blocking of the mitochondrial ATP- dependent potassium channel. The effect of nor-NOHA was associated with enhanced myocardial expression of PKCε. Study II. Combined administration of L-arginine and BH4 before and during reperfusion reduced IS in rat and pig models of myocardial IR. At the same time individual administration of L-arginine or BH4 failed to evoke cardioprotection in both species. NOS inhibition abrogated the cardioprotective effect of L-arginine and BH4. Myocardial BH4 levels were higher in pigs given BH4 with or without L-arginine. The generation of superoxide in the ischemic-reperfused myocardium was reduced only in pigs treated with the combination of L-arginine and BH4. Studies III-IV. Pharmacological enhancement of peroxynitrite decomposition and inhibition of ROCK protected from IR injury and attenuated myocardial ROCK and arginase activity in rats subjected to IR. Rats with type 1 diabetes had increased myocardial arginase activity, arginase 2 expression and ROCK activity. In addition, ROCK and arginase inhibition protected against myocardial IR in rats with type 1 diabetes. RIPerc induced by bilateral femoral artery occlusion resulted in reduction of IS, myocardial peroxynitrite formation, ROCK and arginase activity and upregulated myocardial endothelial NOS. The cardioprotective effect of RIPerc and associated changes in arginase and ROCK activity were absent in rats with type 1 diabetes. The cardioprotective effects of RIPerc, ROCK and arginase inhibition were abolished by inhibition of NOS. Conclusions. Inhibition of arginase before the onset of reperfusion reduced IS via a mechanism dependent on NOS activity, PKCε expression and activation of mitochondrial ATP-dependent potassium channels. Supplementation of L-arginine and BH4 during late ischemia and reperfusion reduced IS via a NOS-dependent pathway and reduced the generation of superoxide. Peroxynitrite and ROCK signaling pathways are involved in the upregulation of arginase activity during myocardial IR. Inhibition of either ROCK or arginase protected against myocardial IR injury via a NOS–dependent mechanism both in the presence and absence of type 1 diabetes. This suggests that reduction in arginase activity, as a result of reduced formation of peroxynitrite and ROCK activity, is of importance for the NOS-dependent cardioprotective effect of RIPerc. The cardioprotective effect of RIPerc and associated signaling effects on arginase, ROCK and NOS are abolished in type 1 diabetes. Arginase inhibition, supplementation of BH4 and L-arginine, inhibition of ROCK and RIPerc are potential tools in the treatment of acute myocardial infarction

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

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

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

BackgroundPharmacological inhibition of arginase and remote ischemic perconditioning (RIPerc) are known to protect the heart against ischemia/reperfusion (IR) injury.PurposeThe 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.MethodsAnesthetized 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.ResultsInfarct size was significantly reduced in rats treated with FeTPPS, hydroxyfasudil and RIPerc compared to controls (PConclusionArginase 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

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

Use of Robotics to Improve Upper Extremity Function in Adults with Neurological Conditions

Objectives of Presentation: 1. Describe the impact of robotics on upper extremity function among various neurological populations. 2. Differentiate which patients are appropriate for robotics interventions for upper extremity rehabiliation. 3. Defend the feasibility of robotic interventions for upper extremity rehabilitation of neurological populations. PICO: To what extend does robotic-assisted therapy improve UE function in adults with neurological conditions? Note: Handout with references available at bottom of page. Presentation: 37:3

Hemodynamic changes in Protocol 2.

<p>Values are mean ± SEM; n = 6–7. Abbreviations: MABP (mm Hg), mean arterial blood pressure; HR (beats/min), heart rate; ND-CIR, non-diabetic control ischemia/reperfusion; ND-RIPerc, non-diabeic remote ischemic perconditioning; DM-CIR: diabetes mellitus control ischemia/reperfusion; DM-RIPerc, diabetes mellitus remote perconditioning.</p><p>*<i>P</i><0.05 vs. ND-CIR.</p

ROCK activity expressed as a phosphorylation of ezrin following ischemia/reperfusion.

<p>(A) Effect of the peroxynitrite decomposition catalyst FeTPPS, the ROCK inhibitor hydroxyfasudil (H.fasudil), remote ischemic preconditioning (RIPerc) and RIPerc+the NOS inhibitor L-NMMA in protocol 1. (B and C) Effect of RIPerC in non-diabetic and diabetic rats of protocol 2. Values are means ± SEM; n = 5–7. ***<i>P</i><0.001 vs. CIR; ^###<i>P</i><0.001 vs. RIPerc and ^††<i>P</i><0.01 vs. ND-CIR.</p

The effect of RIPerc on eNOS expression and peroxynitrite formation.

<p>(A and C) Effect of remote ischemic perconditioning (RIPerc) on phosphorylated of eNOS at Ser1177 (p-eNOS), total eNOS and nitrotyrosine (3NT) in protocol 1. (B) Effect of RIPerc on NOS expression in non-diabetic and diabetic rats of protocol 2. Values are means ± SEM; n = 5–8. *<i>P</i><0.05 vs. CIR.</p