Type 2 diabetes: increased expression and contribution of IKCa channels to vasodilation in small mesenteric arteries of ZDF rats.

Impaired endothelial function, which is dysregulated in diabetes, also precedes hypertension. We hypothesized that in Type 2 diabetes, the impaired endothelium-dependent relaxation is due to a loss of endothelium-derived hyperpolarization (EDH) that is regulated by impaired ion channel function. Zucker diabetic fatty (ZDF), Zucker heterozygote, and homozygote lean control rats were used as the experimental models in our study. Third-order mesenteric arteries were dissected and mounted on a pressure myograph; mRNA was quantified by RT-PCR and channel proteins by Western blotting. Under nitric oxide (NO) synthase and cyclooxygenase inhibition, endothelial stimulation with ACh fully relaxes control but not diabetic arteries. In contrast, when small-conductance calcium-activated potassium (KCa) channels and intermediate- and large-conductance KCa (I/BKCa) are inhibited with apamin and charybdotoxin, NO is able to compensate for ACh-induced relaxation in control but not in diabetic vessels. After replacement of charybdotoxin with 1-[(2-chlorophenyl)diphenylmethyl]-(1)H-pyrazole (TRAM-34; IKCa inhibitor), ACh-induced relaxation in diabetic animals is attenuated. Specific inhibition with TRAM-34 or charybdotoxin attenuates ACh relaxation in diabetes. Stimulation with 1-ethyl-2-benzimidazolinone (IKCa activator) shows a reduced relaxation in diabetes. Activation of BKCa with 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-(2)H-benzimidazol-2-one NS619 leads to similar relaxations of control and diabetic arteries. RT-PCR and Western blot analysis demonstrate elevated mRNA and protein expression levels of IKCa in diabetes. Our results suggest that the compensatory effect of NO and EDH-associated, endothelium-dependent relaxation is reduced in ZDF rats. Specific blockade of IKCa with TRAM-34 reduces NO and EDH-type relaxation in diabetic rats, indicating an elevated contribution of IKCa in diabetic small mesenteric artery relaxation. This finding correlates with increased IKCa mRNA and protein expression in this vessel.

Endothelial dysfunction and altered mechanical and structural properties of resistance arteries in a murine model of graft-versus-host disease.

A putative involvement of the vasculature seems to play a critical role in the pathophysiology of graft-versus-host disease (GVHD). We aimed to characterize alterations of mesenteric resistance arteries in GVHD in a fully MHC-mismatched model of BALB/c mice conditioned with total body irradiation that underwent transplantation with bone marrow cells and splenocytes from syngeneic (BALB/c) or allogeneic (C57BL/6) donors. After 4 weeks, animals were sacrificed and mesenteric resistance arteries were studied in a pressurized myograph. The expression of endothelial (eNOS) and inducible nitric oxide (NO)-synthase (iNOS) was quantified and vessel wall ultrastructure was investigated with electron microscopy. The myograph study revealed an endothelial dysfunction in allogeneic-transplant recipients, whereas endothelium-independent vasodilation was similar to syngeneic-transplant recipients or untreated controls. The expression of eNOS was decreased and iNOS increased, possibly contributing to endothelial dysfunction. Additionally, arteries of allogeneic transplant recipients exhibited a geometry-independent increase in vessels strain. For both findings, electron microscopy provided a structural correlate by showing severe damage of the whole vessel wall in allogeneic-transplant recipient animals. Our study provides further data to prove, and is the first to characterize, functional and structural vascular alterations in the early course after allogeneic transplantation directly in an ex vivo setting and, therefore, strongly supports the hypothesis of a vascular form of GVHD.

Antidiabetic treatment restores adiponectin serum levels and APPL1 expression, but does not improve adiponectin-induced vasodilation and endothelial dysfunction in Zucker diabetic fatty rats.

BACKGROUND: Adiponectin is able to induce NO-dependent vasodilation in Zucker lean (ZL) rats, but this effect is clearly alleviated in their diabetic littermates, the Zucker diabetic fatty (ZDF) rats. ZDF rats also exhibit hypoadiponectinemia and a suppressed expression of APPL1, an adaptor protein of the adiponectin receptors, in mesenteric resistance arteries. Whether an antidiabetic treatment can restore the vasodilatory effect of adiponectin and improve endothelial function in diabetes mellitus type 2 is not known. METHODS: During our animal experiment from week 11 to 22 in each case seven ZDF rats received an antidiabetic treatment with either insulin (ZDF+I) or metformin (ZDF+M). Six normoglycemic ZL and six untreated ZDF rats served as controls. Blood glucose was measured at least weekly and serum adiponectin levels were quantified via ELISA in week 11 and 22. The direct vasodilatory response of their isolated mesenteric resistance arteries to adiponectin as well as the endothelium-dependent and -independent function was evaluated in a small vessel myograph. Additionally, the expression of different components of the adiponectin signaling pathway in the resistance arteries was quantified by real-time RT-PCR. RESULTS: In ZDF rats a sufficient blood glucose control could only be reached by treatment with insulin, but both treatments restored the serum levels of adiponectin and the expression of APPL1 in small resistance arteries. Nevertheless, both therapies were not able to improve the vasodilatory response to adiponectin as well as endothelial function in ZDF rats. Concurrently, a downregulation of the adiponectin receptors 1 and 2 as well as endothelial NO-synthase expression was detected in insulin-treated ZDF rats. Metformin-treated ZDF rats showed a reduced expression of adiponectin receptor 2. CONCLUSIONS: An antidiabetic treatment with either insulin or metformin in ZDF rats inhibits the development of hypoadiponectinemia and downregulation of APPL1 in mesenteric resistance arteries, but is not able to improve adiponectin induced vasodilation and endothelial dysfunction. This is possibly due to alterations in the expression of adiponectin receptors and eNOS.

Expression of fourteen novel obesity-related genes in Zucker diabetic fatty rats.

BACKGROUND: Genome-wide association studies (GWAS) are useful to reveal an association between single nucleotide polymorphisms and different measures of obesity. A multitude of new loci has recently been reported, but the exact function of most of the according genes is not known. The aim of our study was to start elucidating the function of some of these genes. METHODS: We performed an expression analysis of fourteen genes, namely BDNF, ETV5, FAIM2, FTO, GNPDA2, KCTD15, LYPLAL1, MCR4, MTCH2, NEGR1, NRXN3, TMEM18, SEC16B and TFAP2B, via real-time RT-PCR in adipose tissue of the kidney capsule, the mesenterium and subcutaneum as well as the hypothalamus of obese Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats at an age of 22 weeks. RESULTS: All of our target genes except for SEC16B showed the highest expression in the hypothalamus. This suggests a critical role of these obesity-related genes in the central regulation of energy balance. Interestingly, the expression pattern in the hypothalamus showed no differences between obese ZDF and lean ZL rats. However, LYPLAL1, TFAP2B, SEC16B and FAIM2 were significantly lower expressed in the kidney fat of ZDF than ZL rats. NEGR1 was even lower expressed in subcutaneous and mesenterial fat, while MTCH2 was higher expressed in the subcutaneous and mesenterial fat of ZDF rats. CONCLUSION: The expression pattern of the investigated obesity genes implies for most of them a role in the central regulation of energy balance, but for some also a role in the adipose tissue itself. For the development of the ZDF phenotype peripheral rather than central mechanisms of the investigated genes seem to be relevant.

High-sensitive Troponin I in acute cardiac conditions: implications of baseline and sequential measurements for diagnosis of myocardial infarction.

BACKGROUND: High-sensitive Troponin I (hsTnI) facilitates the early diagnosis of myocardial infarction (MI). However, since hsTnI has not been well characterized in non-ischemic cardiac conditions, the predictive value of hsTnI for MI remains unclear. METHODS: hsTnI (ADVIA Centaur, Siemens) on admission was analyzed in 929 patients with acute cardiac condition and invasive ascertainment of coronary status by catheterization. RESULTS: Hs-TnI upon presentation was higher in patients with STEMI (median 1.27 ng/mL, IQR 0.13-14.5 ng/mL) as compared to patients with Non-STEMI (0.66 ng/mL, IQR 0.10-4.0 ng/mL, p<0.001) whereas it did not differ from STEMI in Tako-Tsubo cardiomyopathy (2.57 ng/mL, IQR 0.17-8.4 ng/mL) and myocarditis (9.76 ng/mL, IQR 2.0-27.0 ng/mL). In patients with resuscitation of non-ischemic cause (0.31 ng/mL, IQR 0.06-1.3 ng/mL), acute heart failure (0.088 ng/mL, IQR 0.035-0.30 ng/mL) and hypertensive emergency (0.066 ng/mL, IQR 0.032-0.34 ng/mL), hs-TnI was elevated above the recommended threshold of 0.04 ng/mL. At this cutpoint of 0.04 ng/mL, hsTnI indicated acute MI (STEMI or Non-STEMI) with a sensitivity of 88% and a specificity of 45% (ROC-AUC 0.748). When patients with STEMI were excluded, hsTnI indicated Non-STEMI with a sensitivity of 87% and a specificity of 45% (ROC-AUC 0.725). When sequential measurements were taken into account in a restricted cohort, a maximum hsTnI of ≥0.40 ng/mL provided a sensitivity of 89% and a specificity of 85% (ROC-AUC 0.909) for Non-STEMI. CONCLUSIONS: HsTnI is a sensitive, albeit unspecific marker of MI. In patients with mildly elevated hsTnI and without evidence for STEMI, we suggest serial assessment of hsTnI and a 10-fold higher cutpoint of 0.40 ng/mL before Non-STEMI is assumed.

Globular and full-length adiponectin induce NO-dependent vasodilation in resistance arteries of Zucker lean but not Zucker diabetic fatty rats.

BACKGROUND: Adiponectin increases nitric oxide (NO) production in endothelial cell cultures and is reduced in the circulation of obese and diabetic patients, but its functional effect on resistance arteries is not yet studied in detail. METHODS: We assessed the direct vasodilatory response of isolated mesenteric resistance arteries of Zucker diabetic fatty (ZDF) rats and Zucker lean (ZL) rats to globular adiponectin (gAd) and full-length adiponectin (fAd) and tested the effect of additional reactive oxygen species (ROS) inhibitors in vitro. Serum adiponectin and insulin levels were measured by ELISA. The mRNA expressions of the adiponectin receptors and the downstream signaling molecules adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1), adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2), and endothelial NO synthase (eNOS) in mesenteric resistance arteries were quantified by real-time reverse transcriptase PCR. RESULTS: Both gAd and fAd induced a relevant dose-dependent vasodilation in ZL, but not in hypoadiponectinemic ZDF rats. This effect was totally blunted by L-nitroarginine-methyl-ester indicating NO dependency. The addition of ROS inhibitors could not improve the vasodilatory effect of adiponectin. Vasodilatory response to acetylcholine was reduced in ZDF rats, which could not be enhanced by low-dose adiponectin. Adiponectin receptor 1 (AdipoR1) was higher expressed than adiponectin receptor 2 (AdipoR2) with no significant differences between both animal groups, but APPL1 was significantly decreased in ZDF rats. The eNOS expression was not significantly different between ZL and ZDF rats. CONCLUSIONS: Adiponectin exerts a NO-dependent vasodilation in resistance arteries of normoglycemic ZL rats, but not diabetic ZDF rats. This may contribute to endothelial dysfunction in ZDF rats. Alterations in the expression of APPL1 may be involved in the observed insensitivity to adiponectin in ZDF rats.

Autoregulation of ventilation with neurally adjusted ventilatory assist on extracorporeal lung support.

PURPOSE: Extracorporeal membrane oxygenation (ECMO) can support oxygenation and carbon dioxide elimination in severe lung failure. Usually it is accompanied by controlled mechanical ventilation. Neurally adjusted ventilatory assist (NAVA) is a new mode of ventilation triggered by the diaphragmatic electrical activity and controlled by the patient's respiratory centre, which may allow a close interaction between ventilation and extracorporeal perfusion. This pilot study intended to measure the physiologic ventilatory response in patients with severe lung failure treated with ECMO and NAVA. We hypothesized that the combination of both methods could automatically provide a protective ventilation with optimized blood gases. METHODS: We report a case series of six patients treated with ECMO for severe lung failure. In the recovery phase of the disease, patients were ventilated with NAVA and ventilatory response and gas exchange parameters were measured under different sweep gas flows and temporarily inactivated ECMO. RESULTS: Tidal volumes on ECMO ranged between 2 and 5 ml/kg predicted body weight and increased up to 8 ml/kg with inactivated ECMO. Peak inspiratory pressure reached 19-29 cmH(2)O with active, and 21-45 cmH(2)O with inactivated ECMO. Ventilatory response to decreased sweep gas flow was rapid, and patients immediately regulated PaCO(2) tightly towards a physiological pH value. Increase in minute ventilation was a result of increased breathing frequency and tidal volumes, and protective ventilation was only abandoned if pH control was not achieved. CONCLUSIONS: With NAVA ventilatory response to decreased ECMO sweep gas flow was rapid, and patients immediately regulated PaCO(2) tightly towards a physiological pH value. Therefore, combination of NAVA and ECMO may permit a closed-loop ventilation with automated protective ventilation.

Endothelial cell seeding fails to prevent intimal hyperplasia following arterial injury in the rat carotid model.

PURPOSE: Aim of the study was to evaluate the therapeutic potential of endothelial cell seeding to inhibit the neointimal response after balloon injury of the rat carotid artery. METHODS AND RESULTS: Endothelial cells were isolated from peripheral blood after cytokine-stimulation (PB-EC), the bone marrow compartment (BM-EC), and the thoracic aorta (AO-EC) of male rats. The EC-populations were characterized by microscopy, cytofluorometry, and PCR-analysis, and displayed distinct patterns of RNA-expression of the EC-markers VEGF-receptor 1 and 2, and TIE2. 5 x 10(5) ECs were incubated in the freshly balloon-denuded arterial bed for 30 min achieving about 70% coverage with all 3 EC-populations. However, the neointimal response 2 weeks after the procedure was significantly aggravated in the EC-seeded carotids (I/M-ratio: non-seeded Control 1.00 +/- 0.10, PB-EC 1.53 +/- 0.07, BM-EC 1.64 +/- 0.12, and AO-EC 1.55 +/- 0.14; P < 0.05 vs. Control for all). We found evidence for an accelerated cell-turnover (TUNEL-assay, total cell-density, infiltration with CD45(pos) haematopoietic cells) especially in the adventitia of the treated vessels. CONCLUSION: Endothelial cell seeding fails to prevent intimal hyperplasia following arterial injury in the rat carotid model.

Electrocardiographic diagnosis of left ventricular hypertrophy in aortic valve disease: evaluation of ECG criteria by cardiovascular magnetic resonance.

BACKGROUND: Left ventricular hypertrophy (LVH) is a hallmark of chronic pressure or volume overload of the left ventricle and is associated with risk of cardiovascular morbidity and mortality. The purpose was to evaluate different electrocardiographic criteria for LVH as determined by cardiovascular magnetic resonance (CMR). Additionally, the effects of concentric and eccentric LVH on depolarization and repolarization were assessed. METHODS: 120 patients with aortic valve disease and 30 healthy volunteers were analysed. As ECG criteria for LVH, we assessed the Sokolow-Lyon voltage/product, Gubner-Ungerleider voltage, Cornell voltage/product, Perugia-score and Romhilt-Estes score. RESULTS: All ECG criteria demonstrated a significant correlation with LV mass and chamber size. The highest predictive values were achieved by the Romhilt-Estes score 4 points with a sensitivity of 86% and specificity of 81%. There was no difference in all ECG criteria between concentric and eccentric LVH. However, the intrinsicoid deflection (V6 37 +/- 1.0 ms vs. 43 +/- 1.6 ms, p < 0.05) was shorter in concentric LVH than in eccentric LVH and amplitudes of ST-segment (V5 -0.06 +/- 0.01 vs. -0.02 +/- 0.01) and T-wave (V5 -0.03 +/- 0.04 vs. 0.18 +/- 0.05) in the anterolateral leads (p < 0.05) were deeper. CONCLUSION: By calibration with CMR, a wide range of predictive values was found for the various ECG criteria for LVH with the most favourable results for the Romhilt-Estes score. As electrocardiographic correlate for concentric LVH as compared with eccentric LVH, a shorter intrinsicoid deflection and a significant ST-segment and T-wave depression in the anterolateral leads was noted.

A systemic combination therapy with granulocyte-colony stimulating factor plus erythropoietin aggravates the healing process of balloon-injured rat carotid arteries.

OBJECTIVE: Percutaneous vascular interventions lead inevitably to a destruction of the endothelial lining at the site of injury. There are conflicting data on the therapeutic benefit of hematopoietic growth factors aiming at mobilisation of circulating endothelial cells to accelerate the reendothelialisation process. Aim of our study was to evaluate the impact of a maximised 7 day-combination therapy with G-CSF plus EPO on the healing process following balloon injury of the rat carotid artery. METHODS AND RESULTS: Osmotic pumps to systemically deliver G-CSF and EPO at saturating doses during the first seven days post injury were implanted into the peritoneal cavity of splenectomised male Sprague-Dawley rats. Cytokine treatment resulted in significantly elevated numbers of white blood cells, hematocrit levels, circulating hematopoietic stem cells, and-temporarily-circulating endothelial precursors. Functional reendothelialisation as assessed by Evan's blue staining on day 14 post injury was not affected by the cytokine treatment. The neointimal response was analysed on day 7 and 28 post injury, and was significantly higher at the day 7 timepoint (Cytokines: I/M-ratio 1.10+/-0.09 vs. Saline: 0.36+/-0.06). Cytokine treated rats also displayed higher rates of thrombotic occlusion (Cytokines: 25-33% vs. Saline: 0%). Serum levels of PAI-1, TGFbeta1, and PDGF-BB were not elevated in the cytokine treated rats. The proliferative rates both in the injured vessel wall and the surrounding adventitia as assessed by BrdU incorporation were significantly higher in the cytokine treated animals. The number of CD45(pos) hematopoietic cells was significantly higher in the adventitia of the cytokine treated rats. Vasa vasorum were not found to be significantly different. The increased neointimal response was not due to expression of G-CSF- or EPO-receptors on VSMCs within the vessel wall or myofibroblasts in the adventitia. CONCLUSION: The systemic administration of G-CSF plus EPO during the first week after balloon-injury impairs the vascular healing process by increasing the neointimal response and the risk for thrombotic occlusion.

Evidence for a possible inhibitory interaction between the HO-1/CO- and Akt/NO-pathways in human endothelial cells.

OBJECTIVE: The protective properties of heme oxygenase 1 (HO-1) give reason to study this mechanism as a potential therapeutic target for inflammatory and cardiovascular diseases. Recent evidence suggests a possible interaction between the HO-1/CO- and the protein kinase Akt/NO-pathway. This study was designed to examine the effects of continuous HO-1 overexpression in endothelial cells. METHODS: Oncoretroviral vectors were constructed to achieve constitutive overexpression of HO-1, Akt, and green fluorescence protein in human umbilical vein endothelial cells. [(3)H]thymidine-incorporation and lipid-peroxidation were measured following exposure to heme and H(2)O(2). Expression of HO-1, Akt and its downstream-target endothelial NO-synthase were quantified by Western blot analysis. NO-synthase-activity was measured using the citrulline-conversion-assay. RESULTS: HO-1-overexpression reduced proliferative rates and DNA-synthesis of HUVEC, but provided potent protection from oxidative stress induced by heme and H(2)O(2). Phosphorylated-Akt and eNOS was downregulated in HO-1-HUVEC. eNOS-activity was reduced in HO-1-HUVEC. Co-infection with the Akt-retrovirus restored proliferative rates and eNOS-expression and -activity. CONCLUSION: Continuously elevated HO-1-activity protects EC from oxidative stress but inhibits Akt-mediated proliferation and eNOS-expression. This inhibitory feedback mechanism could be a limitation of HO-1 as a target for the treatment of vascular disease.

A locus on chromosome 10 influences C-reactive protein levels in two independent populations.

High sensitivity C-reactive protein (hsCRP) is an independent risk factor for cardiovascular disease, such as stroke or coronary artery disease. Genetic factors influence significantly the inter-individual variability of hsCRP. The aim of this study was to identify genomic regions influencing hsCRP levels. A genome scan was performed in two independent studies of Caucasian populations, namely 513 Western-European families ascertained for myocardial infarction (n = 1,406) and 120 French-Canadian families diagnosed with hypertension (n = 758). In the myocardial infarction families, 31% of the inter-individual variation of hsCRP levels was explained by genetic factors (P = 0.0000015) and loci influencing hsCRP were identified on chromosomes 10 (at 141 cM) and 5 (at 150 cM) with multipoint LOD scores of 3.15 and 2.23, respectively. An additional suggestive signal was detected on chromosome 2 in subset analyses. A similar degree of heritability has been observed in a second independent population of French-Canadian hypertensive families for hsCRP (30%) and linkage results for chromosome 10 were confirmed with maximum LOD score of 2.7. We identified a chromosomal region in two independent populations which influences hsCRP in addition to several unique regions. This provides targets for the identification of genes involved in the regulation of hsCRP and the development and progression of vascular disease, including stroke.

Association of the metabolic syndrome with early coronary disease in families with frequent myocardial infarction.

This study examined the extent to which the metabolic syndrome (MS) augments the risk for major cardiovascular events in healthy patients with a strong genetic background for coronary artery disease (CAD). In a prospective cohort study, we examined 1,316 patients without previously diagnosed CAD or diabetes mellitus. Patients were participants of the Regensburg Myocardial Infarction Family Study, in which > or = 2 family members had severe CAD and 1 had myocardial infarction (MI) at < 60 years of age. During a 2-year follow-up, the incidence of first cardiovascular events (MI, revascularization, and cardiac death) was compared between those with and without the MS at baseline. In all previously unaffected family members, the presence of MS increased the hazard ratio for first manifestation of CAD by a factor of 1.9 (p = 0.030), which resulted in an event rate of 7.1% during follow-up. Specifically in young patients (< or = 50 years old, n = 422), we identified the MS as a major event predictor that conferred a 5.8-fold increased relative risk for first cardiovascular events compared with patients without the MS (95% confidence interval 1.4 to 23.8, p = 0.015, event rate 6.2%). Remarkably, of the individual MS components, obesity was strongly associated with incident MI (relative risk 4.4, 95% confidence interval 1.5 to 13.0, p = 0.007). Thus, the MS strongly predicts cardiac morbidity and mortality in healthy patients with a family background of CAD.

No association of interleukin-6 gene polymorphism (-174 G/C) with myocardial infarction or traditional cardiovascular risk factors.

BACKGROUND: Recently, a polymorphism at position -174 (G>C) of the interleukin-6 (IL-6) promoter was found to be associated with an increased prevalence of myocardial infarction (MI). The aim of the present study was to further investigate the association of the IL-6 -174 G/C allele status with specific end organ damage, i.e. myocardial infarction in large population-based samples. METHODS: Individuals from two Bavarian samples of MI patients (total n=1322) and the population-based Augsburg MONICA survey (1023 unselected controls) were studied by questionnaire, physical examination, echocardiographical assessment and biochemical analyses. The -174 G/C polymorphism was genotyped using a newly established PCR-RFLP. IL-6 levels were measured in a subset of 574 MI patients. RESULTS: In the population-based sample, the IL-6 genotype was neither associated with traditional cardiovascular risk factors (systolic and diastolic blood pressure, total cholesterol, HDL and LDL cholesterol, body mass index, diabetes mellitus) nor with cardiac structural or functional parameters (left ventricular mass index, ejection fraction, diastolic inflow pattern). Moreover, the genotype distribution of the -174 G/C polymorphism was not different in MI patients (GG: 34.1%; GC: 47.4%; CC: 18.5%) and population-based controls (GG: 32.4%; GC: 48.8%; CC: 18.9%) (p=0.67). IL-6 levels were neither related to the -174 G/C polymorphism (p=0.29) nor to ACE-inhibitor treatment (2.16 with vs. 2.09 pg/ml without ACE-inhibitor, p=0.27). However, patients receiving statins displayed significantly lower IL-6 levels (1.83 vs. 2.32 pg/ml in the group without statins, p<0.0001). CONCLUSIONS: This extensive investigation failed to obtain evidence that the IL-6 -174 G/C promoter polymorphism affects traditional cardiovascular risk factors or the prevalence of myocardial infarction in a Caucasian sample.