Arginase inhibition improves endothelial function in patients with type 2 diabetes mellitus despite intensive glucose-lowering therapy.

BACKGROUND: Arginase is implicated in the pathogenesis behind endothelial dysfunction in type 2 diabetes mellitus (T2DM) by its inhibition of nitric oxide formation. Strict glycaemic control is not sufficient to improve endothelial function or cardiovascular outcomes in patients with T2DM, thus other treatment strategies are needed. We hypothesized that arginase inhibition improves endothelial function beyond glucose-lowering therapy following glucose optimization in patients with poorly controlled T2DM. METHODS AND RESULTS: Endothelial function was evaluated in 16 patients with poorly controlled T2DM (visit 1) and 16 age-matched controls using venous occlusion plethysmography. T2DM patients were re-evaluated (visit 2) after intensive glucose-lowering regimen. Endothelium-dependent (EDV) and -independent (EIDV) vasodilatations were evaluated before and after 120 min intra-arterial infusion of the arginase inhibitor N(ω)-hydroxy-nor-L-arginine (nor-NOHA). HbA1c was reduced from 87 ± 17 (visit 1) to 65 ± 11 mmol mol-1 (visit 2, P < 0.001). Basal EDV, but not EIDV, was significantly lower in patients with T2DM than in healthy subjects (P < 0.05). EDV and EIDV were unaffected by glucose-lowering regimen in patients with T2DM. Arginase inhibition enhanced EDV in T2DM patients both at visit 1 and visit 2 (P < 0.01). There was no difference in improvement in EDV between the two occasions. EIDV was unaltered by nor-NOHA in T2DM at visit 1, but was slightly improved at visit 2. CONCLUSIONS: Arginase inhibition improves endothelial function in patients with poorly controlled T2DM, which is maintained following glucose optimization. Thus, arginase inhibition is a promising therapeutic target beyond glucose lowering for improving endothelial function in T2DM patients.

Long-Term Effects of Oxygen Therapy on Death or Hospitalization for Heart Failure in Patients With Suspected Acute Myocardial Infarction

Background: In the DETO2X-AMI trial (Determination of the Role of Oxygen in Suspected Acute Myocardial Infarction), we compared supplemental oxygen with ambient air in normoxemic patients presenting with suspected myocardial infarction and found no significant survival benefit at 1 year. However, important secondary end points were not yet available. We now report the prespecified secondary end points cardiovascular death and the composite of all-cause death and hospitalization for heart failure. Methods: In this pragmatic, registry-based randomized clinical trial, we used a nationwide quality registry for coronary care for trial procedures and evaluated end points through the Swedish population registry (mortality), the Swedish inpatient registry (heart failure), and cause of death registry (cardiovascular death). Patients with suspected acute myocardial infarction and oxygen saturation of ≥90% were randomly assigned to receive either supplemental oxygen at 6 L/min for 6 to 12 hours delivered by open face mask or ambient air. Results: A total of 6629 patients were enrolled. Acute heart failure treatment, left ventricular systolic function assessed by echocardiography, and infarct size measured by high-sensitive cardiac troponin T were similar in the 2 groups during the hospitalization period. All-cause death or hospitalization for heart failure within 1 year after randomization occurred in 8.0% of patients assigned to oxygen and in 7.9% of patients assigned to ambient air (hazard ratio, 0.99; 95% CI, 0.84­1.18; P=0.92). During long-term follow-up (median [range], 2.1 [1.0­3.7] years), the composite end point occurred in 11.2% of patients assigned to oxygen and in 10.8% of patients assigned to ambient air (hazard ratio, 1.02; 95% CI, 0.88­1.17; P=0.84), and cardiovascular death occurred in 5.2% of patients assigned to oxygen and in 4.8% assigned to ambient air (hazard ratio, 1.07; 95% CI, 0.87­1.33; P=0.52). The results were consistent across all predefined subgroups. Conclusions: Routine use of supplemental oxygen in normoxemic patients with suspected myocardial infarction was not found to reduce the composite of all-cause mortality and hospitalization for heart failure, or cardiovascular death within 1 year or during long-term follow-up. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01787110.

Vagal nerve stimulation reduces infarct size via a mechanism involving the alpha-7 nicotinic acetylcholine receptor and downregulation of cardiac and vascular arginase.

AIMS: Vagal nerve stimulation (VNS) protects from myocardial and vascular injury following myocardial ischaemia and reperfusion (IR) via a mechanism involving activation of alpha-7 nicotinic acetylcholine receptor (α7 nAChR) and reduced inflammation. Arginase is involved in development of myocardial IR injury driven by inflammatory mediators. The aim of the study was to clarify whether VNS downregulates myocardial and vascular arginase via a mechanism involving activation of α7 nAChR following myocardial IR. METHODS: Anaesthetized rats were randomized to (i) sham-operated, (ii) control IR (30-min ischaemia and 2-h reperfusion, (iii) VNS throughout IR, (iv) the arginase inhibitor nor-NOHA+IR, (v) nor-NOHA+VNS+IR, (vi) selective α7 nAChR blockade by methyllycaconitine (MLA) followed by VNS throughout IR and (vii) MLA+IR. RESULTS: Infarct size was reduced by VNS compared to control IR (41 ± 3% vs. 67 ± 2% of the myocardium at risk, P < 0.001). Myocardial IR increased myocardial and aortic arginase activity 1.7- and 3.1-fold respectively (P < 0.05). VNS attenuated the increase in arginase activity compared to control IR both in the myocardium and aorta (P < 0.05). MLA partially abolished the cardioprotective effect of VNS and completely abrogated the effect of VNS on arginase activity. Arginase inhibition combined with VNS did not further reduce infarct size. CONCLUSION: Vagal nerve stimulation reduced infarct size and reversed the upregulation of arginase induced by IR both in the myocardium and aorta via a mechanism depending on α7 nAChR activation. The data suggest that the cardioprotective effect of VNS is mediated via reduction in arginase activity.

Tetraspanin CD151 and integrin α6ß1 mediate platelet-enhanced endothelial colony forming cell angiogenesis.

UNLABELLED: ESSENTIALS: Platelet releasates (PRs) enhance endothelial colony forming cell (ECFC) angiogenesis. The impact of platelet membrane components on ECFC angiogenesis was studied by a tube formation assay. Platelets enhanced ECFC angiogenesis more potently than PR, via tetraspanin CD151 and integrin α6ß1. Optimal enhancement of ECFC angiogenesis by platelets requires both membrane proteins and PR. BACKGROUND: Platelets promote angiogenesis of endothelial colony forming cells (ECFCs), with the underlying mechanisms not being fully understood. OBJECTIVE: To investigate if platelets regulate the angiogenic property of ECFCs via mechanisms beyond platelet-released angiogenic regulators. METHODS AND RESULTS: Endothelial colony forming cells were generated by ECFC-directed cell culture of peripheral blood mononuclear cells. Capillary-like tube formation of ECFCs was assessed using a Matrigel assay. Platelets promoted ECFC tube formation in both basic and complete ECFC medium. Importantly, the ECFC angiogenic responses induced by platelets were stronger than those induced by platelet releasates. Thus, the branching points of ECFC tube formation (30.5 ± 9.0/field, ECFC alone) were increased by platelet releasates (58.2 ± 8.3/field) and even more profoundly by platelets (95.5 ± 17.6/field), indicating that platelet membrane components also promoted ECFC tube formation. The latter was further supported by evidence that fixed platelets did enhance ECFC tube formation. Subsequent experiments revealed that the promotion was dependent on platelet-surface glycoproteins, as removal of sialic acid from platelet glycoproteins by neuraminidase abolished the enhancement. Furthermore, platelet-expressed, but not ECFC-expressed, CD151 was important for the enhancement, as pretreatment of platelets, but not ECFCs, with a CD151-blocking antibody attenuated the effect. Integrin α6ß1 on both ECFCs and platelets also participated in platelet-promoted tube formation, as integrin α6 or ß1 blockade of either cell type markedly or totally inhibited the phenomenon. Moreover, platelets exerted the enhancement via the Src-PI3K signaling pathway of ECFCs. CONCLUSION: Platelet-enhanced ECFC angiogenesis requires platelet tetraspanin CD151 and α6ß1 integrin, as well as ECFC α6ß1 integrin and Src-PI3K signaling.

Arginase inhibition improves endothelial function in patients with familial hypercholesterolaemia irrespective of their cholesterol levels.

BACKGROUND: Elevated LDL cholesterol is an important risk factor for atherosclerosis. Endothelial dysfunction, an early event in the development of atherosclerosis, is characterized by a reduction in nitric oxide (NO) bioavailability. Arginase has emerged as a key regulator of endothelial function through competition with NO synthase for the common substrate l-arginine. Arginase in endothelial cells is activated by oxidized LDL. The study aim was to investigate the importance of arginase for endothelial dysfunction in patients with familial hypercholesterolaemia (FH). METHODS AND RESULTS: Endothelial function was evaluated in 12 patients with heterozygous FH and 12 age-matched healthy normocholesterolaemic subjects using forearm venous occlusion plethysmography. The evaluations in FH patients occurred when they were on lipid-lowering therapy and 4 weeks after withdrawal of treatment. Endothelium-dependent vasodilatation (EDV) was assessed by intrabrachial artery infusion of serotonin, and endothelium-independent dilatation was assessed by infusion of nitroprusside before and after 120 min administration of the arginase inhibitor N (ω) -hydroxy-nor-l-arginine (nor-NOHA; 0.1 mg min(-1)). In FH patients LDL cholesterol increased from 4.3 ± 0.9 mmol L(-1) at baseline to 7.6 ± 1.9 mmol L(-1) at follow-up (P < 0.001). Arginase inhibition enhanced EDV in FH patients by a similar degree independent of lipid-lowering therapy. The improvement in EDV by arginase inhibition was significantly greater in FH patients than in the control group. CONCLUSION: Arginase inhibition results in greater improvement in endothelial function in patients with FH compared to healthy controls irrespective of their cholesterol levels. Arginase may be a promising therapeutic target for improving endothelial function in patients with hypercholesterolaemia.

Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus.

BACKGROUND AND PURPOSE: Emerging evidence suggests a selective up-regulation of arginase I in diabetes causing coronary artery disease; however, the mechanisms behind this up-regulation are still unknown. Activated p38 MAPK has been reported to increase arginase II in various cardiovascular diseases. We therefore tested the role of p38 MAPK in the regulation of arginase I and II expression and its effect on endothelial dysfunction in diabetes mellitus. EXPERIMENTAL APPROACH: Endothelial function was determined in septal coronary (SCA), left anterior descending coronary (LAD) and mesenteric (MA) arteries from healthy and streptozotocin-induced diabetic Wistar rats by wire myographs. Arginase activity and protein levels of arginase I, II, phospho-p38 MAPK and phospho-endothelial NOS (eNOS) (Ser(1177) ) were determined in these arteries from diabetic and healthy rats treated with a p38 MAPK inhibitor in vivo. KEY RESULTS: Diabetic SCA and MA displayed impaired endothelium-dependent relaxation, which was prevented by arginase and p38 MAPK inhibition while LAD relaxation was not affected. Arginase I, phospho-p38 MAPK and eNOS protein expression was increased in diabetic coronary arteries. In diabetic MA, however, increased expression of arginase II and phospho-p38 MAPK, increased arginase activity and decreased expression of eNOS were observed. All these effects were reversed by p38 MAPK inhibition. CONCLUSIONS AND IMPLICATIONS: Diabetes-induced activation of p38 MAPK causes endothelial dysfunction via selective up-regulation of arginase I expression in coronary arteries and arginase II expression in MA. Therefore, regional differences appear to exist in the arginase isoforms contributing to endothelial dysfunction in type 1 diabetes mellitus.

PAR1-stimulated platelet releasate promotes angiogenic activities of endothelial progenitor cells more potently than PAR4-stimulated platelet releasate.

BACKGROUND: Endothelial progenitor cells (EPCs) are important for endothelial regeneration and angiogenesis. Thrombin protease-activated receptor 1 (PAR1) PAR1 and PAR4 stimulation induces selective release of platelet proangiogenic and antiangiogenic regulators. OBJECTIVE: To investigate if PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR regulate angiogenic properties of EPCs in different manners. METHODS AND RESULTS: EPCs were generated from peripheral mononuclear cell culture. Washed platelets (2 × 10(9) mL(-1)) were stimulated by PAR1-activating peptide (PAR1-AP; 10 µmol L(-1)) or PAR4-AP (100 µmol L(-1)) to prepare PAR1-PR and PAR4-PR, respectively. PAR1-PR or PAR4-PR had little influence on EPC proliferation. EPC migration experiments using a modified Boyden chamber showed that both platelet releasates facilitated EPC migration. As for in vitro tube formation on Matrigel, PAR1-PR and PAR4-PR similarly enhanced capillary-like network formation of EPCs in the complete EPC medium containing 10% FBS and a cocktail of growth factors, while PAR1-PR more profoundly increased EPC tube formation in basal culture medium supplemented with only 0.5% FBS than did PAR4-PR. The latter was confirmed in the murine angiogenesis model of subcutaneous Matrigel implantation. Moreover, blockade of vascular endothelial growth factor, stromal cell-derived factor 1α, or matrix metalloproteinases attenuated EPC migration and tube formation, suggesting a cooperation of these factors in the enhancements. CONCLUSIONS: PAR1-PR enhances vasculogenesis more potently than PAR4-PR, and the enhancements require a cooperation of multiple platelet-derived angiogenic regulators.

Arginase inhibition improves coronary microvascular function and reduces infarct size following ischaemia-reperfusion in a rat model.

AIM: Ischaemia-reperfusion injury is associated with reduced bioavailability of nitric oxide (NO) and microvascular dysfunction. One emerging mechanism behind reduced NO bioavailability is upregulation of arginase, which metabolizes the NO synthase substrate l-arginine. This study investigated the effects of arginase inhibition on coronary flow velocity and infarct size during reperfusion. METHODS: Anaesthetized rats, subjected to 30-min coronary artery ligation and reperfusion up to 8 days, were treated with vehicle or the arginase inhibitor N(ω) -hydroxy-nor-l-arginine (nor-NOHA; 100 mg kg(-1) ) intravenously 15 min before ischaemia. Coronary flow velocity was determined repeatedly during reperfusion. RESULTS: Arginase activity in the ischaemic-reperfused myocardium was increased already at 20 min of reperfusion and maintained at 8 days. Infarct size was reduced by arginase inhibition at 2 h (39 ± 3% of the area at risk (AAR) vs. 51 ± 2% in the vehicle group, P < 0.01) and at 8 days of reperfusion (13 ± 2% of the left ventricle (LV) vs. 22 ± 2%, P < 0.05). Basal coronary flow velocity was higher during reperfusion in the group given nor-NOHA, and it correlated inversely to infarct size (P < 0.01, r = -0.60). Hyperaemic coronary flow velocity was also increased in the nor-NOHA-treated group compared to vehicle at 24 h and at day 8 (P < 0.05). CONCLUSION: It is concluded that arginase activity is increased already during early reperfusion. Arginase inhibition increases coronary flow velocity and reduces infarct size that is sustained 8 days after reperfusion. Inhibition of arginase may thus be a promising therapeutic target to prevent the development of microvascular dysfunction and myocardial injury following ischaemia-reperfusion.

Endothelin-1 increases superoxide production in human coronary artery bypass grafts.

AIMS: Endothelin-1 (ET-1) has been shown to increase endothelial superoxide (O(2)(-)) production in experimental animal models. It is unclear whether ET-1 increases O(2)(-) production in humans. We sought to elucidate whether ET-1 increases O(2)(-) production in human vessels and to identify the mechanism behind this effect. MAIN METHODS: Segments of internal mammary artery (IMA) and human saphenous vein (HSV) were harvested from 90 patients undergoing elective coronary artery bypass graft surgery. Paired vessel rings were incubated in the presence and absence of ET-1 (10(-10)M), the ET(A) receptor antagonist BQ123 alone, or in combination with the ET(B) receptor antagonist BQ788 (dual BQ) and known inhibitors of sources of O(2)(-) and further analysed for O(2)(-) production using lucigenin-enhanced chemiluminescence and DHE fluorescence. KEY FINDINGS: ET-1 increased O(2)(-) production in both IMA (2.6 ± 1.5 vs. 1.4 ± 0.8 relative light units/s/mg tissue (RLU); n=33; p < 0.0001) and HSV (1.4 ± 0.8 vs. 1.1 ± 0.6 RLU; n=24; p<0.05). The increase in O(2)(-)production induced by ET-1 in IMA was inhibited by co-incubation with dual BQ (p < 0.05; n=15) and BQ123 (p<0.05; n = 17). Of known O(2)(-) inhibitors, only incubation with Tiron and diphenyleneiodonium resulted in a significant reduction in ET-mediated O(2)(-) production. SIGNIFICANCE: ET-1 increases O(2)(-) production especially in human arteries and less so in veins from patients with coronary artery disease via a receptor-dependent pathway involving a flavin dependent enzyme which is likely to be NADPH oxidase. Production of O(2)(-) may be an important factor underlying the negative effects of ET-1 on vascular function such as impairment of endothelium-dependent vasodilatation and pro-inflammatory effects.

The endothelin receptor antagonist bosentan improves peripheral endothelial function in patients with type 2 diabetes mellitus and microalbuminuria: a randomised trial.

AIMS/HYPOTHESIS: Endothelial dysfunction is important in the development of vascular complications in diabetes. Patients with type 2 diabetes have increased production of the vasoconstrictor and pro-inflammatory peptide, endothelin-1. Short-term intra-arterial administration of endothelin antagonists improves endothelium-dependent vasodilatation in patients with type 2 diabetes. We tested the hypothesis that oral administration of the dual endothelin receptor antagonist, bosentan, improves peripheral endothelial function in patients with type 2 diabetes and microalbuminuria. METHODS: This placebo-controlled and double-blind study was performed on 46 patients with type 2 diabetes and microalbuminuria (urine albumin/creatinine ratio >3 mg/mmol) at a medical university department. Patients were randomised to bosentan, 125 mg two times per day (n = 28), or placebo (n = 28) for 4 weeks. The computer-generated randomisation code was kept in sealed envelopes. Patients and people doing examinations or assessing outcomes were blinded. The primary endpoint was change in microvascular endothelium-dependent vasodilatation, based on change in digital reactive hyperaemia index. The secondary endpoint was change in brachial artery flow-mediated vasodilatation. RESULTS: Reactive hyperaemia index increased from 1.73 ± 0.43 (mean ± SD) at baseline to 2.08 ± 0.59 at follow-up (p < 0.05) in the bosentan group (n = 22), but did not change in the placebo group (1.84 ± 0.49 to 1.87 ± 0.47; n = 24). The change in reactive hyperaemia index from baseline was greater in the bosentan group than in the placebo group (p < 0.05). Nitroglycerine-induced digital hyperaemia was not affected. Brachial artery flow-mediated vasodilatation and blood pressure did not change during treatment. CONCLUSIONS/INTERPRETATION: Oral treatment of 4 weeks duration with the dual endothelin receptor antagonist, bosentan, improves peripheral endothelial function in patients with type 2 diabetes and microalbuminuria.

The combination of L-arginine and ischaemic post-conditioning at the onset of reperfusion limits myocardial injury in the pig.

AIM: To investigate whether ischaemic post-conditioning (IPoC) combined with i.v. infusion of the nitric oxide (NO) substrate L-arginine at the onset of reperfusion exerts cardioprotective effect that is superior to either treatment given separately. METHODS: Twenty-six anesthetized pigs were subjected to coronary artery (left anterior descending artery, LAD) ligation for 40 min followed by 4 h reperfusion. The pigs were randomized into five different groups receiving either i.v. vehicle, i.v. L-arginine, IPoC 4 × 60 s together with i.v. vehicle or IPoC together with i.v. L-arginine and a group with IPoC 8 × 30 s. All infusions were started 10 min before reperfusion. RESULTS: The infarct size of the vehicle group was 82 ± 4% of the area at risk. L-Arginine alone (79 ± 8%), IPoC 4 × 60 s vehicle (86 ± 3%) or IPoC 8 × 30 s vehicle (94 ± 7%) did not affect infarct size. l-Arginine together with IPoC significantly reduced infarct size to 59 ± 4% (P < 0.01). Except for higher LAD flow during early reperfusion in the IPoC L-arginine group, haemodynamic parameters did not differ between the four main groups. Heart rate and rate pressure product were lower during ischaemia and reperfusion in the IPoC 8 × 30 s vehicle group. In comparison with the vehicle group, there were no changes in the expression of Akt, phosphorylated Akt Ser(473) , inducible NO synthase, endothelial NO synthase (eNOS) or phosphorylated eNOS Ser(1177) in the ischaemic/reperfused myocardium. CONCLUSION: L-Arginine given systemically at the onset of reperfusion protects the pig heart against ischaemia and reperfusion injury only when combined with IPoC. These results indicate that the combination of the two treatment strategies exerts cardioprotection.

Endothelin attenuates endothelium-dependent platelet inhibition in man.

AIM: The vascular endothelium produces several substances, including nitric oxide (NO) and endothelin-1 (ET-1), which participate in the regulation of vascular tone in humans. Both these substances may exert other actions of importance for cardiovascular disease, e.g. effects on vascular smooth muscle cell proliferation and inflammation, and NO inhibits platelet function. Experiments were designed to investigate the effect of ET-1 on endothelium-dependent vasodilatation and attenuation of platelet activation. METHODS: In 25 healthy male subjects (25 +/- 1 years), forearm blood flow was measured by venous occlusion plethysmography, and platelet activity was assessed by whole blood flow cytometry (platelet fibrinogen binding and P-selectin expression) in unstimulated and adenosine diphosphate (ADP)-stimulated samples during administration of ET-1, the endothelium-dependent vasodilator acetylcholine and the NO synthase inhibitor l-NMMA. RESULTS: Acetylcholine increased forearm blood flow and significantly inhibited platelet activation in both unstimulated and ADP-stimulated samples. In samples stimulated with 0.3 microm ADP, fibrinogen binding decreased from 41 +/- 4% to 31 +/- 3% (P < 0.01, n = 11) after acetylcholine administration. The vasodilator response to acetylcholine was significantly impaired during infusions of ET-1 and l-NMMA. ET-1 did not affect platelet activity per se, whereas l-NMMA increased platelet P-selectin expression. Both ET-1 and l-NMMA attenuated the acetylcholine-induced inhibition of platelet activity. CONCLUSIONS: Our study indicates that, further to inhibiting endothelium-dependent vasodilatation, ET-1 may also attenuate endothelium-dependent inhibition of platelet activation induced by acetylcholine. An enhanced ET-1 activity, as suggested in endothelial dysfunction, may affect endothelium-dependent platelet modulation and thereby have pathophysiological implications.

Lipid lowering versus pleiotropic effects of statins on skin microvascular function in patients with dysglycaemia and coronary artery disease.

OBJECTIVES: To investigate the impact of lipid lowering therapy by different means on skin microvascular function in patients with dysglycaemia and coronary artery disease (CAD). DESIGN AND SETTING: Thirty-six patients were randomized to simvastatin 80 mg daily (S80, n = 19) or ezetimibe 10 mg and simvastatin 10 mg daily (E10/S10, n = 17) for 6 weeks. Skin microvascular function was assessed by laser Doppler fluxmetry (LDF) at rest, following arterial occlusion (peak postocclusive LDF) and following local heating on the forearm (heat arm LDF) and foot (heat foot LDF). LDF parameters and serum lipids were evaluated at baseline and follow-up. RESULTS: At follow-up, LDL cholesterol had decreased from 3.1 (2.7-3.5) to 1.6 (1.5-1.8) (mmol L(-1)) and 3.0 (2.4-3.9) to 1.3 (1.1-1.8) (mmol L(-1)) in the E10/S10 and S80 groups respectively. In the entire study group (n = 32), LDF parameters increased significantly; postocclusive LDF from 22 (17-27) to 26 (21-32) perfusion units (PU) (P < 0.001), heat foot LDF from 61 (44-82) to 66 (45-83) PU (P < 0.001) and heat arm LDF from 60 (48-121) to 75 (54-125) PU (P < 0.01). The changes in LDF parameters did not differ between the E10/S10 and S80 groups. CONCLUSIONS: Lipid lowering improves microvascular function in patients with dysglycaemia and CAD. The data suggest that lipid lowering per se is more important than pleiotropic effects of statins for this effect.

L-arginine and tetrahydrobiopterin protects against ischemia/reperfusion-induced endothelial dysfunction in patients with type 2 diabetes mellitus and coronary artery disease.

Diminished levels of L-arginine and endothelial nitric oxide synthase (eNOS) uncoupling through deficiency of tetrahydrobiopterin (BH(4)) may contribute to endothelial dysfunction. We investigated the effect of L-arginine and BH(4) administration on ischemia-reperfusion (I/R)-induced endothelial dysfunction in patients with type 2 diabetes and coronary artery disease (CAD). Forearm blood flow was measured by venous occlusion plethysmography in 12 patients with type 2 diabetes or impaired glucose tolerance and CAD. Forearm ischemia was induced for 20 min, followed by 60 min of reperfusion. The patients received a 15 min intra-brachial infusion of L-arginine (20 mg/min) and BH(4) (500 microg/min) or 0.9% saline starting at 15 min of ischemia on two separate study occasions. Compared with pre-ischemia the endothelium-dependent vasodilatation (EDV) induced by acetylcholine was significantly reduced at 15 and 30 min of reperfusion when saline was infused (P<0.001), but not following L-arginine and BH(4) infusion. EDV was also significantly less reduced at 15 and 30 min of reperfusion following L-arginine and BH(4) infusion, compared to saline infusion (P<0.02). Endothelium-independent vasodilatation (EIDV) induced by nitroprusside was unaffected by I/R. Venous total biopterin levels in the infused arm increased from 37+/-7 at baseline to 6644+/-1240 nmol/l during infusion of L-arginine and BH(4) (P<0.0001), whereas there was no difference in biopterin levels during saline infusion. In conclusion L-arginine and BH(4) supplementation reduces I/R-induced endothelial dysfunction, a finding which may represent a novel treatment strategy to limit I/R injury in patients with type 2 diabetes and CAD.

Protection against myocardial ischaemia/reperfusion injury by PPAR-alpha activation is related to production of nitric oxide and endothelin-1.

BACKGROUND: Ligands of peroxisome proliferator-activated receptor alpha (PPAR-alpha) have been shown to reduce ischaemia/reperfusion injury. The mechanisms behind this effect are not well known. We hypothesized that activation of PPAR-alpha exerts cardioprotection via a mechanism related to nitric oxide (NO) and endothelin-1 (ET-1). METHODS: Five groups of anaesthetized open-chest Sprague-Dawley rats were given the PPAR-alpha agonist WY 14643 1 mg/kg (WY; n = 7), dimethyl sulfoxide (DMSO, vehicle for WY; n = 6), the combination of WY and the NO synthase inhibitor N-nitro-L-arginine (L-NNA, 2 mg/kg) (n = 7), L-NNA only (n = 8) or 0.9% sodium chloride (NaCl, vehicle for DMSO and L-NNA; n = 8) i.v. before a 30 min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), eNOS and iNOS protein and ET-1 mRNA expression were determined. RESULTS: There were no haemodynamic differences between the groups during the experiment. The IS was 78 +/- 3% of the area at risk in the DMSO group and 77 +/- 2% in the NaCl group (P = NS). WY reduced IS to 56 +/- 3% (P < 0.001 vs. DMSO group). When WY was administered in combination with L-NNA the cardioprotective effect was abolished (IS 73 +/- 3%, P < 0.01 vs. WY 14643). L-NNA did not affect IS per se (78 +/- 2%, P = NS). The expression of eNOS but not iNOS protein in ischaemic myocardium from rats was increased in the group given WY (P < 0.05). ET-1 mRNA levels were lower in the ischaemic myocardium following WY administration. CONCLUSION: The results suggest that the PPAR-alpha activation protects the rat myocardium against ischaemia/ reperfusion injury via a mechanism related to production of NO, and possibly ET-1.

Endothelin receptor blockade improves endothelial function in atherosclerotic patients on angiotensin converting enzyme inhibition.

OBJECTIVES: Endothelin-1 (ET-1) and angiotensin II may contribute to endothelial dysfunction, which is associated with increased risk of events in patients with coronary artery disease. The objective was to test whether dual ETA/ETB receptor antagonism improves endothelium-dependent vasodilatation (EDV) in atherosclerotic patients, also on treatment with angiotensin converting enzyme (ACE) inhibitor. DESIGN AND SETTING: EDV and endothelium-independent vasodilatation were determined in 37 patients with atherosclerosis during measurement of forearm blood flow (FBF) with venous occlusion plethysmography. The patients were then randomized to treatment with ramipril 10 mg o.d. (n=21) or placebo (n=16) for 3 months in a double-blind fashion. RESULTS: Intra-arterial infusion of the ETA receptor antagonist BQ123 and the ETB receptor antagonist BQ788 (both 10 nmol min(-1)) increased basal FBF by 42 +/- 4% (P <0.001) and enhanced EDV (P <0.001). Following 3 months ramipril treatment, ET receptor blockade still enhanced EDV. Acetylcholine 10 and 30 mg min(-1) increased FBF by 68 +/- 12 and 64 +/- 12 mL min(-1)/1000 mL before vs. 101 +/- 17 and 101 +/- 16 mL min(-1)/1000 mL following ET receptor blockade in the ramipril group (P <0.001). CONCLUSIONS: Dual ETA/ETB receptor blockade improves endothelial function and exerts direct vasodilator effects in patients with atherosclerosis, also on treatment with ramipril suggesting that ET receptor blockade may have important therapeutic effects when added to ACE inhibition in these patients.

Oral pre-treatment with rosuvastatin protects porcine myocardium from ischaemia/reperfusion injury via a mechanism related to nitric oxide but not to serum cholesterol level.

AIMS: The aim of this study was to test whether oral pre-treatment with rosuvastatin at a dosage giving clinically relevant plasma concentrations protects the myocardium against ischaemia/reperfusion injury and to investigate the involvement of nitric oxide (NO) and neutrophil infiltration. METHODS: Pigs were given placebo (n = 7), rosuvastatin (80 mg day(-1), n =7), rosuvastatin (160 mg day(-1), n = 7) or pravastatin (160 mg day(-1), n = 7) orally for 5 days before being subjected to coronary artery ligation and reperfusion. An additional group was given rosuvastatin 160 mg day(-1) and a nitric oxide synthase (NOS) inhibitor. RESULTS: Rosuvastatin 80 and 160 mg day(-1) resulted in plasma concentrations of 2.6 +/- 0.7 and 5.6 +/- 1.0 ng mL(-1), respectively. Serum cholesterol was not affected. Rosuvastatin 160 mg day(-1) and pravastatin limited the infarct size from 82 +/- 3% of the area at risk in the placebo group to 61 +/- 3% (P < 0.05), and to 61 +/- 2% (P < 0.05) respectively. Rosuvastatin 80 mg day(-1) limited the infarct size to 69 +/- 2%, however, this effect was not statistically significant. Rosuvastatin 160 mg day(-1) attenuated neutrophil infiltration in the ischaemic/reperfused myocardium. The protective effect of rosuvastatin 160 mg day(-1) was abolished by NOS inhibition. The expression of NOS2 and NOS3 in the myocardium did not differ between the groups. CONCLUSIONS: Oral pre-treatment with rosuvastatin limited infarct size following ischaemia/reperfusion without affecting cholesterol levels. The cardioprotective effect is suggested to be dependent on maintained bioactivity of NO, without influencing NOS expression.

The effect of a neuropeptide Y Y1 receptor antagonist in patients with angina pectoris.

AIMS: Neuropeptide Y (NPY) is a potent vasoconstrictor released during sympathetic activation that may be involved in myocardial ischaemia. We examined the effect of a Y1 receptor antagonist on haemodynamic and ischaemic responses to exercise in patients with coronary artery disease. METHODS AND RESULTS: Eighty-two evaluable male patients were included in a randomized, double blind, two-way crossover study with a low dose (6.7 microg/kg/min; n=59)and a high dose (13.3 microg/kg/min; n=23) of the Y1 receptor antagonist AR-H040922 given as infusions for 2h or placebo. Myocardial ischaemia during a symptom-limited exercise test was monitored by conventional ST-segment analysis and heart rate (HR)-adjusted ST changes including the ST/HR slope and ST/HR recovery. Administration of the high dose AR-H040922 attenuated systolic blood pressure by 6-11 mmHg (p<0.05) during and after exercise without affecting HR. None of the two doses of AR-H040922 influenced any of the ischaemic parameters or duration of exercise, however. The maximal increase in NPY was higher during AR-H040922 (p<0.05) compared with placebo. CONCLUSIONS: Selective NPY Y1 receptor blockade attenuates the increase in blood pressure during exercise indicating a role for endogenous NPY in blood pressure regulation. Despite this effect, the Y1 receptor antagonist did not influence exercise-induced ischaemic parameters in patients with coronary artery disease.

A triglyceride-rich fat emulsion and free fatty acids but not very low density lipoproteins impair endothelium-dependent vasorelaxation.

OBJECTIVE: To investigate the effects of triglycerides and free fatty acids on endothelium-dependent and endothelium-independent vasorelaxation. METHODS: Femoral arterial rings from rats were studied in organ baths. The vascular segments were constricted with phenylephrine after 20 min of preincubation with the triglyceride-rich fat emulsion Intralipid, free fatty acids (16:0, 18:1, 18:3) bound to bovine serum albumin, or very low density lipoproteins. Endothelium-dependent and endothelium-independent relaxations were determined after administration of acetylcholine and nitric oxide donors, respectively. RESULTS: Preincubation with Intralipid caused a concentration-dependent impairment of endothelium-dependent but not endothelium-independent relaxation. Very low density lipoproteins did not affect vascular function. All free fatty acids impaired endothelium-dependent relaxation, whereas endothelium-independent relaxation was unaffected. Administration of the antioxidant vitamin C partly reversed the impairment of the endothelium-dependent relaxation induced by Intralipid and free fatty acids. CONCLUSIONS: The present study demonstrates that the triglyceride-rich fat emulsion Intralipid and individual FFAs impair endothelium-dependent relaxation of arterial rings from rat, whereas triglycerides in the form of VLDL do not affect endothelial function. The finding that the antioxidant vitamin C partly reverses this impairment indicates the involvement of oxidative mechanisms.