Effect of endothelin-1 and endothelin receptor blockade on the release of microparticles.

BACKGROUND: Increased levels of endothelial cell microparticles (EMP) are known to reflect endothelial dysfunction (ED). In diabetes mellitus type 2 (T2DM), the expression of endothelin (ET)-1 is increased. As treatment with an ET-1 antagonist significantly inhibited atherosclerosis in animal models, we sought to investigate whether treatment with ET-1 antagonists affects EMP levels in vitro and in vivo in patients with T2DM. MATERIALS AND METHODS: In vitro study: Human umbilical vein endothelial cells (HUVEC) were stimulated with ET-1 alone and ET-1 in combination with a dual ET-A and ET-B endothelin receptor blocker. In vivo study: Patients with T2DM were randomized to treatment with the ET receptor antagonist bosentan or placebo. After 4 weeks, the patients were re-examined and blood samples were obtained. EMP counts in supernatants and plasma samples were determined using flow cytometry. RESULTS: In vitro study: In supernatants of ET-1-stimulated HUVECs, the increased release of EMP was reduced significantly by co-incubation with an ET-1 receptor antagonist (e.g. CD31+/CD42b-EMP decreased from 37·1% ± 2·8 to 31·5% ± 2·8 SEM, P = 0·0078). In vivo study: No changes in EMP levels in blood samples of patients with T2DM were found after 4 weeks of bosentan treatment (n = 36, P = ns). CONCLUSIONS: Our in vitro results suggest that ET-1 stimulates the release of EMP from HUVECs via a receptor-dependent mechanism. Co-incubation with an endothelin receptor blocker abolished ET-1-dependent EMP release. However, treatment with bosentan for 4 weeks failed to alter EMP levels in patients with T2DM. Other factors seem to have influenced EMP release in patients with T2DM independent of ET-1 receptor-mediated mechanisms.

Arginase inhibition improves endothelial function in patients with coronary artery disease and type 2 diabetes mellitus.

BACKGROUND: Endothelial dysfunction plays an important role in the early development of atherosclerosis and vascular complications in type 2 diabetes mellitus. Increased expression and activity of arginase, metabolizing the nitric oxide substrate l-arginine, may result in reduced production of nitric oxide and thereby endothelial dysfunction. We hypothesized that inhibition of arginase activity improves endothelial function in patients with coronary artery disease (CAD) and type 2 diabetes mellitus. METHODS AND RESULTS: Three groups of subjects were included: 16 patients with CAD, 16 patients with CAD and type 2 diabetes mellitus (CAD+Diabetes), and 16 age-matched healthy control subjects. Forearm endothelium-dependent and endothelium-independent vasodilatation were assessed with venous occlusion plethysmography before and during intra-arterial infusion of the arginase inhibitor N(ω)-hydroxy-nor-l-arginine (nor-NOHA; 0.1 mg/min). Nor-NOHA was also coinfused with the nitric oxide synthase inhibitor (N(G)-monomethyl L-arginine). The expression of arginase was determined in the internal mammary artery of patients undergoing bypass surgery. Nor-NOHA markedly increased endothelium-dependent vasodilatation (up to 2-fold) in patients with CAD+Diabetes and CAD (P<0.001) but not in the control group. N(G)-monomethyl L-arginine completely inhibited the increase in endothelium-dependent vasodilatation induced by nor-NOHA. Endothelium-independent vasodilatation was slightly improved by nor-NOHA in the CAD+Diabetes group. Arginase I was expressed in vascular smooth muscle cells and endothelial cells, and arginase II was expressed in endothelial cells of patients with and without diabetes mellitus. CONCLUSIONS: Arginase inhibition markedly improves endothelial function in patients with CAD and type 2 diabetes mellitus suggesting that increased arginase activity is a key factor in the development of endothelial dysfunction.

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.

The effects of adrenocorticotrophic hormone and an equivalent dose of cortisol on the serum concentrations of lipids, lipoproteins, and apolipoproteins.

Previous studies have shown a strong lipid-lowering effect of adrenocorticotrophic hormone (ACTH) in healthy individuals and in patients with different kinds of dyslipoproteinemia. The mechanism behind this effect has not been established and its direct ACTH-specific nature has been questioned. Therefore, the present study was performed. Thirty healthy young males were randomized into 3 groups of equal size: one group received ACTH1-24 1 mg IM, daily for 4 days, another group was treated with cortisol 150 mg ID (50 mg tid) daily for 4 days, whereas a control group was observed for 4 days. Fasting blood samples were collected before and after treatment or observation. The serum concentrations of cholesterol (12%, P < .05), low-density lipoprotein cholesterol (24%, P < .01), and apolipoprotein (apo) B (31%, P < .01) decreased significantly in the ACTH group but not in the cortisol and control groups. The statistical workup confirmed that only ACTH had a lowering effect on the apo B-containing lipoproteins. In contrast, the results indicated conformity between the treatment groups with respect to increases in the serum apo E concentrations. There were inconsistent changes in the serum concentrations of the triglycerides, high-density lipoprotein cholesterol, apo A, and lipoprotein(a). The main results were clear: the lowering effect of ACTH on the serum concentration of apo B-containing lipoproteins could not be ascribed to cortisol. These, in combination with previous in vitro results, indicated an ACTH-specific effect.

[Transport and treatment of patients with STEMI in rural Iceland--Only a few patients receive PPCI within 120 minutes]. / Flutningstími og gæði meðferðar hjá sjúklingum með ST-hækkunar-hjartadrep á landsbyggðinni--fair ná í kransæðavíkkun innan 120 mínútna.

INTRODUCTION: ST-segment Elevation Myocardial Infarction (STEMI) is a life-threatening disease and good outcome depends on early restoration of coronary blood flow. Primary percutaneous coronary intervention (PPCI) is the treatment of choice if performed within 120 minutes of first medical contact (FMC) but in case of anticipated long transport or delays, pre-hospital fibrinolysis is indicated. The aim was to study transport times and adherence to clinical guidelines in patients with STEMI transported from outside of the Reykjavik area to Landspitali University Hospital in Iceland. MATERIALS AND METHODS: Retrospective chart review was conducted of all patients diagnosed with STEMI outside of the Reykjavik area and transported to Landspitali University Hospital in Reykjavik in 2011-2012. Descriptive statistical analysis and hypothesis testing was applied. RESULTS: Eighty-six patients had signs of STEMI on electrocardiogram (ECG) at FMC. In southern Iceland nine patients (21%) underwent PPCI within 120 minutes (median 157 minutes) and no patient received fibrinolysis. In northern Iceland and The Vestman Islands, where long transport times are expected, 96% of patients eligible for fibrinolysis (n=31) received appropriate therapy in a median time of 57 minutes. Significantly fewer patients received appropriate anticoagulation treatment with clopidogrel and enoxaparin in southern Iceland compared to the northern part. Mortality rate was 7% and median length of stay in hospital was 6 days. CONCLUSIONS: Time from FMC to PPCI is longer than 120 minutes in the majority of cases. Pre-hospital fibrinolysis should be considered as first line treatment in all parts of Iceland outside of the Reykjavik area. Directly electronically transmitted ECGs and contact with cardiologist could hasten diagnosis and decrease risk of unnecessary interhospital transfer. A STEMI database should be established in Iceland to facilitate quality control.

Effects of different doses of adrenocorticotrophic hormone on the serum lipoprotein profile in healthy subjects.

Adrenocorticotrophic hormone (ACTH) at pharmacological dosage has marked lipid-lowering effects that may have therapeutic implications. The rationale behind the present investigation was the possible use of ACTH as a lipid-lowering replacement for steroids. Thirty-two healthy individuals were randomly divided into four groups of 8 each. Three ACTH groups received different doses of ACTH(1-24) intramuscularly (0.1 mg, 0.5 mg and 1.0 mg daily for four days) and the control group received NaCl 0.9% 1 ml intramuscularly daily for four days. Moreover, 8 healthy subjects were given ACTH(1-24) 1.0 mg intramuscularly five times at an interval of four days. Blood and urine samples were collected at regular intervals. ACTH treatment at the dose of 1.0 mg daily lowered the serum concentrations of low density lipoprotein (LDL) cholesterol and apolipoprotein B by 28% and 22%, respectively, which is similar to previous observations. ACTH treatment at the doses of 0.5 mg and 0.1 mg gave smaller reductions (17% and 12%, and 9% and 8%, respectively) resulting in near linear dose-response relationships. There were no changes in the control group. Only the ACTH dose of 1.0 mg resulted in significant changes when compared with the control group. During the ACTH administration at four-days intervals, the serum concentrations of LDL cholesterol and apolipoprotein B reached the lowest values at 48 hr after an injection, remained there at 72 hr but were rising again at 96 hr. For effective lipid reduction, an ACTH dose of about 1 mg is needed and it should be given more often than every fourth day, probably every second or third day. With regard to the cortisol exposure, the results should be viewed in the light of calculations, presented in the paper, that 1 mg of ACTH is equivalent to 90 mg of cortisol administered parenterally.

Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

Background. Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation. Methods. Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays. Results. Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs. Conclusions. Human CMV may contribute to CVD via ETBR induction.

Selective endothelin ETA and dual ET(A)/ET(B) receptor blockade improve endothelium-dependent vasodilatation in patients with type 2 diabetes and coronary artery disease.

AIMS: Endothelin-1 contributes to endothelial dysfunction in patients with atherosclerosis and type 2 diabetes. In healthy arteries the ETA receptor mediates the main part of the vasoconstriction induced by endothelin-1 whilst the ETB receptor mediates vasodilatation. The ETB receptor expression is upregulated on vascular smooth muscle cells in atherosclerosis and may contribute to the increased vasoconstrictor tone and endothelial dysfunction observed in this condition. Due to these opposing effects of the ETB receptor it remains unclear whether ETB blockade together with ETA blockade may be detrimental or beneficial. The aim was therefore to compare the effects of selective ETA and dual ETA/ETB blockade on endothelial function in patients with type 2 diabetes and coronary artery disease. MAIN METHODS: Forearm endothelium-dependent and endothelium-independent vasodilatation was assessed by venous occlusion plethysmography in 12 patients before and after selective ETA or dual ETA/ETB receptor blockade. KEY FINDINGS: Dual ETA/ETB receptor blockade increased baseline forearm blood flow by 30±14% (P<0.01) whereas selective ETA blockade did not (14±8%). Both selective ETA blockade and dual ETA/ETB blockade significantly improved endothelium-dependent vasodilatation. The improvement did not differ between the two treatments. There was also an increase in endothelium-independent vasodilatation with both treatments. Dual ETA/ETB blockade did not significantly increase microvascular flow but improved transcutaneous pO2. SIGNIFICANCE: Both selective ETA and dual ETA/ETB improve endothelium-dependent vasodilatation in patients with type 2 diabetes and coronary artery disease. ETB blockade increases basal blood flow but does not additionally improve endothelium-dependent vasodilatation.

Urinary leukotriene E4 is associated with renal function but not with endothelial function in type 2 diabetes.

Leukotrienes are inflammatory and vasoactive mediators implicated in endothelium-dependent relaxations and atherosclerosis. Urinary leukotriene E4 (U-LTE4) is a validated disease marker of asthma and increases also in diabetes and acute coronary syndromes. The aim of the present study was to evaluate the association of U-LTE4 and CRP with endothelial function. Urine samples were obtained from 30 subjects (80% males; median age 65) with type 2 diabetes of at least two years duration and a median glomerular filtration rate (eGFR) of 71 (14-129) mL/min. Reactive hyperemia index (RHI) was used as a measure of microvascular endothelial function, whereas macrovascular endothelial function was determined be means of flow-mediated dilatation of the brachial artery (FMD). Decreased renal function was associated with lower concentrations of U-LTE4. In addition, U-LTE4 was correlated with serum creatinine (R = -0.572; P = 0.001) and eGFR (R = 0.517; P = 0.0036). A stepwise multiple linear regression analysis identified eGFR as an independent predictor of U-LTE4 concentrations. In conclusion, the present results did not establish an association of U-LTE4 with endothelial dysfunction. However, eGFR was an independent predictor of U-LTE4, but not CRP, in this cohort, suggesting that GFR should be considered in biomarker studies of U-LTE4.

Endothelial progenitor cells in relation to endothelin-1 and endothelin receptor blockade: a randomized, controlled trial.

AIMS: Endothelial progenitor cells (EPC) represent an endogenous repair mechanism involving rendothelialization and neoangiogenesis. Patients with both diabetes and vascular disease have low numbers of circulating EPC. The endothelium-derived peptide, endothelin-1 (ET-1), is increased in patients with type 2 diabetes and vascular complications and has been suggested to contribute to endothelial dysfunction. Therefore, we investigated the relation between EPC and plasma ET-1 and the effect of dual ET-1 receptor antagonist treatment. METHODS: In this double blind study patients with type 2 diabetes mellitus and microalbuminuria were randomized to treatment with the dual ETA/ETB receptor antagonist bosentan treatment (125mg bid; n=17) or placebo (n=19) for four weeks. Different EPC subpopulations were enumerated by flow cytometry using triple staining (CD34, CD133, KDR) at baseline at the end of treatment. Viability was assessed by 7AAD and Annexin-V-staining. RESULTS: Baseline ET-1 levels correlated significantly with C-reactive protein levels. Patients with ET-1 levels above the median value had higher levels of CD34(+)CD133(+) and CD34(+)KDR(+) EPC. There was no difference in CD34(+) and CD34(+)CD133(+)KDR(+) cells, markers of EPC apoptosis or circulating markers of endothelial damage between patients with ET-1 levels below or above the median. Four week treatment with bosentan did not change EPC levels. CONCLUSION: Among patients with type 2 diabetes and vascular disease, high plasma levels of ET-1 are associated with higher number of EPC. The recruitment of EPC does not seem to be regulated via ET-1 receptor activation since treatment with a dual ET-1 receptor blocker did not affect circulating EPC numbers.

Different subpopulations of endothelial progenitor cells and circulating apoptotic progenitor cells in patients with vascular disease and diabetes.

AIMS: Endothelial progenitor cells (EPCs) represent a repair mechanism involving reendothelialization and neoangiogenesis. Patients with both diabetes and known vascular disease have low numbers of circulating EPCs. To assess the role of diabetes in vascular disease we investigated the number and viability of circulating EPCs and related this to endothelial function. METHODS: Different EPC subpopulations were enumerated by flow cytometry using triple staining (CD34, CD133, KDR). Viability was assessed by 7AAD and Annexin-V-staining. Endothelial function was evaluated by Endo-PAT2000 in 19 patients with known vascular disease without diabetes and 20 patients with vascular disease and diabetes mellitus. RESULTS: CD34+, CD133+, CD34+/CD133+, CD34+/KDR+, CD34+/CD133+/KDR+ cell counts did not differ between patients with and without diabetes. CD34-/CD133+/KDR+ cells were lower, whereas staining for apoptosis in progenitor cells was higher in patients with diabetes. Progenitor cell counts did not correlate to peripheral arterial function test. DISCUSSION: Patients having diabetes and vascular disease have lower CD34-/CD133+/KDR+ EPC counts, a recently discovered subpopulation of EPCs, and larger proportion of apoptotic EPCs. Higher apoptotic rates of progenitor cells in diabetes could represent an important mechanism explaining lower functional capability of these cells. These observations may be of importance for the complications associated with diabetes mellitus.

The effects of adrenocorticotrophic hormone and cortisol on homocysteine and vitamin B concentrations.

BACKGROUND: Homocysteine metabolism is mainly governed by serum concentrations of folate and vitamin B(12), renal function and genetic factors. It is also well documented that endocrinological disturbances influence homocysteine metabolism. However, studies on the hormones of the hypothalamic-pituitary-adrenal axis have given conflicting results. METHODS: A total of 30 healthy young males were randomised into three groups of equal size; one group received adrenocorticotrophic hormone (ACTH)(1-24) 1 mg i.m. daily for 4 days, another group was treated with cortisol 50 mg i.m. t.i.d. for 4 days, while a control group was observed for 4 days. Fasting blood samples were collected before and after treatment or observation. RESULTS: The pattern of changes was the same for the ACTH and cortisol groups; there were significant decreases in serum concentrations of folate (23% and 24%) and cobalamines (13% and 19%) and decreases in plasma total homocysteine concentrations that did not reach significance. There were no changes in the control group. CONCLUSIONS: The virtually identical pattern of changes in both treatment groups suggests that the effects were mediated by cortisol. The reductions in serum concentrations of folate and cobalamines call for further research.