Endothelial dysfunction in patients with peripheral arterial disease and chronic hyperhomocysteinemia: potential role of ADMA

Hyperhomocysteinemia is associated with an enhanced risk for cardiovascular disease. Patients with peripheral arterial disease (PAD) show an increased prevalence of hyperhomocysteinemia. A decreased biological activity of nitric oxide (NO) may contribute to homocysteine-associated endothelial dysfunction. This study was designed to investigate whether elevated levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) are involved in endothelial dysfunction in patients with chronic hyperhomocysteinemia and PAD. A total of 76 patients (58 males and 18 females; mean age 65.2 +/- 2.0 years) with PAD were included in the analysis and characterized according to demographic variables and cardiovascular risk factors. Flow-dependent vasodilation (FDD) was determined by high-resolution ultrasound in the radial artery. Total plasma homocysteine (plasma tHcy) and ADMA levels were measured by HPLC. Urinary nitrate was quantified using gas chromatography-mass spectrometry. Patients with plasma tHcy in the highest tertile (n = 27; i.e. > 10.6 micromol/l) had a mean plasma level of 14.4 +/- 1.21 mol/l compared with 9.9 +/- 0.1 micromol/l in those patients in the middle tertile (n = 22; p < 0.05) and 9.4 +/- 0.1 micromol/l in those in the lowest tertile (n = 27; i.e. <9.6 micromol/l; p < 0.05). The hyperhomocysteinemic individuals (highest tertile) had a significantly decreased FDD compared with healthy age-matched controls (n = 15) (7.6 +/- 1.0 vs 13.0 +/- 0.4%; p < 0.05), higher plasma ADMA concentrations (4.0 +/- 0.3 vs 2.6 +/- 0.3 micromol/l; p < 0.05), and a lower urinary nitrate excretion rate (89.5 +/- 13.4 vs 131.3 +/- 17.9 micromol/mmol creatinine; p < 0.05) compared with patients with plasma tHcy in the lowest tertile. Multivariate regression analysis including plasma tHcy, ADMA, total cholesterol, diabetes mellitus, smoking, and systolic blood pressure revealed ADMA as the only significant factor determining FDD (p < 0.05). In conclusion, we demonstrated a stronger relationship between impaired endothelial function and elevated ADMA levels in comparison with plasma tHcy concentrations in patients with PAD and chronic hyperhomocysteinemia. This may raise the question of whether different therapeutical options that interact indirectly with plasma tHcy, i.e. treatment with ACE inhibitors and AT1-receptor blockers to reduce ADMA plasma concentrations or L-arginine, could be a beneficial tool for treating patients with hyperhomocysteinemia.     

Abdominal infections in patients with acute leukaemia: a prospective study applying ultrasonography and microbiology

A prospective study of 62 chemotherapy-induced neutropenic episodes in patients with acute leukaemia was conducted to determine the incidence and causes of abdominal infections, and to assess the diagnostic value of the combined use of ultrasonography (US) and microbiology. Each patient underwent US of liver, gallbladder and complete bowel before chemotherapy, on days 2-4 after the end of chemotherapy and in cases of fever, diarrhoea or abdominal pain. US was combined with a standardized clinical examination and a broad spectrum of microbiological investigations. From January to August 2001, 243 US examinations were performed. The overall incidence of abdominal infectious diseases was 17.7% (11 out of 62, 95% confidence interval (CI): 9-29%). Four patients (6.5%) developed neutropenic enterocolitis; two of them died, two survived. Bowel wall thickening (BWT) > 4 mm in these four patients ranged from 5.8 to 23.6 mm and was detected only in one patient with mucositis. In three other patients (4.8%) Clostridium difficile, and in one patient (1.6%) Campylobacter jejuni, caused enterocolitis without BWT. Cholecystitis was diagnosed in three patients (4.8%) and hepatic candidiasis was strongly suspected in one patient. Abdominal infections caused by gastroenteritis viruses, cytomegalovirus (CMV) or Cryptosporidium were not observed. We conclude that in neutropenic patients with acute leukaemia receiving chemotherapy: (i) BWT is not a feature of chemotherapy-induced mucositis and should therefore be considered as sign of infectious enterocolitis; (ii) viruses, classic bacterial enteric pathogens (Salmonella, Shigella, Yersinia, Campylobacter, Aeromonas, Vibrio subsp., enterohaemorrhagic Escherichia coli) and Cryptosporidium have a very low incidence; and (iii) abdominal infections may be underestimated when US is not used in every patient with abdominal pain.     

The distribution of HBV, HCV and HGV among livers with fulminant hepatic failure of different aetiology

Background/Aims: The aim of the study was to assess the impact factor of HCV and HGV in fulminant hepatic failure.Methods: The 5′-untranslated regions of HCV RNA and HGV RNA and a segment of the core antigen sequence of HBV were amplified after extracting the nucleic acids from snap-frozen tissue aliquots from explanted livers of 26 consecutive patients undergoing orthotopic liver transplantation for fulminant hepatic failure preoperatively diagnosed as either autoimmune (n=2), HAV/HBV (n=8), toxic (n=4) or aetiologically unknown (n=12).Results: HCV RNA was detected in five of 26 (19.2%) livers with fulminant hepatic failure. All five HCV RNA-positive livers belonged to the group of non-toxic, non-autoimmune liver failure (n=20), three of them were found in the group of liver failure with unknown aetiology (n=12) and two in the group of HBV-associated liver failure (n=7), making an HCV incidence of 25%, 25%, and 28.6%, in the different groups, respectively. HGV RNA was detected in 10 of 17 (58.8%) explants and in all four groups of fulminant hepatic failure as defined preoperatively. HBV DNA was identified in six livers of 26 patients (23.1%) with fulminant hepatic failure. Neither HCV RNA nor HBV DNA was detected in the livers of patients with toxic or autoimmune fulminant hepatic failure.Conclusions: These results indicate that HBV and HCV, but not HGV, play an aetiologic role in fulminant hepatic failure. HCV-positive cases were concentrated either in the group of otherwise unexplained fulminant hepatic failure or in the group of HBV fulminant hepatic failure. HGV-positive cases, on the other hand, were found within all four preoperatively defined groups, indicating a role as cofactor rather than as single aetiologic agent.     

Hepatocyte cytoskeleton during ischemia and reperfusion--influence of ANP-mediated p38 MAPK activation

AIM: To determine functional consequences of this activation, whereby we focused on a potential regulation of the hepatocyte cytoskeleton during ischemia and reperfusion. METHODS: For in vivo experiments, animals received ANP (5 μg/kg) intravenously. In a different experimental setting, isolated rat livers were perfused with KH-buffer ±ANP (200 nmol/L)±SB203580 (2 μmol/L). Livers were then kept under ischemic conditions for 24 h, and either transplanted or reperfused. Actin, Hsp27, and phosphorylated Hap27 were determined by Western blotting, p38 MAPK activity by in vitro phosphorylation assay. F-actin distribution was determined by confocal microscopy. RESULTS: We first confirmed that ANP preconditioning leads to an activation of p38 MAPK and observed alterations of the cytoskeleton in hepatocytes of ANP-preconditioned organs. ANP induced an increase of hepatic F-actin after ischemia, which could be prevented by the p38 MAPK inhibitor SB203580 but had no effect on bile flow. After ischemia untreated livers showed a translocation of Hsp27 towards the cytoskeleton and an increase in total Hsp27, whereas ANP preconditioning prohibited translocation but caused an augmentation of Hsp27 phosphorylation. This effect is also mediated via p38 MAPK, since it was abrogated by the p38 MAPK inhibitor SB203580. CONCLUSION: This study reveals that ANP-mediated p38 MAPK activation leads to changes in hepatocyte cytoskeleton involving an elevation of phosphorylated Hsp27 and thereby for the first time shows functional consequences of ANP-induced hepatic p38 MAPK activation.     

Activation of Epac stimulates integrin-dependent homing of progenitor cells

Cell therapy is a novel promising option for treatment of ischemic diseases. Administered endothelial progenitor cells (EPCs) are recruited to ischemic regions and improve neovascularization. However, the number of cells that home to ischemic tissues is restricted. The GTPase Rap1 plays an important role in the regulation of adhesion and chemotaxis. We investigated whether pharmacologic activation of Epac1, a nucleotide exchange protein for Rap1, which is directly activated by cAMP, can improve the adhesive and migratory capacity of distinct progenitor cell populations. Stimulation of Epac by a cAMP-analog increased Rap1 activity and stimulated the adhesion of human EPCs, CD34(+) hematopoietic progenitor cells, and mesenchymal stem cells (MSCs). Specifically, short-term stimulation with a specific Epac activator increased the beta2-integrin-dependent adhesion of EPCs to endothelial cell monolayers, and of EPC and CD34(+) cells to ICAM-1. Furthermore, the Epac activator enhanced the beta1-integrin-dependent adhesion of EPCs and MSCs to the matrix protein fibronectin. In addition, Epac1 activation induced the beta1- and beta2-integrin-dependent migration of EPCs on fibronectin and fibrinogen. Interestingly, activation of Epac rapidly increased lateral mobility of beta1- and beta2-integrins, thereby inducing integrin polarization, and stimulated beta1-integrin affinity, whereas the beta2-integrin affinity was not increased. Furthermore, prestimulation of EPCs with the Epac activator increased homing to ischemic muscles and neovascularization-promoting capacity of intravenously injected EPCs in the model of hind limb ischemia. These data demonstrate that activation of Epac1 increases integrin activity and integrin-dependent homing functions of progenitor cells and enhances their in vivo therapeutic potential. These results may provide a platform for the development of novel therapeutic approaches to improve progenitor cell homing.     

Observation of own exploration movements impairs haptic spatial perception

The present study was designed to assess whether the visibility of ones’ own exploratory movements impairs or enhances perceptual speed and precision of haptic stimuli with varying complexity. Previous studies have shown that noninformative vision of steady surroundings improves haptic spatial perception. However, due to the serial nature of haptic processing and limited capacity of working memory resources, we hypothesized that noninformative vision of limb movements may impair haptic perception. The study sample consisted of ninety-eight healthy adults who were randomized into two groups, matched for sex and age. Participants were required to explore two-dimensional haptic stimuli with varying complexity and to recognize them visually. The difference between the two experimental groups was a screen that would prevent the participants from viewing their hands during exploration in the nonobservation condition (NonOb). The other half of participants were able to see their hands in the manual movement observation condition (MovOb) thanks to the special design of the stimuli. As hypothesized, the persons in the MovOb condition made significantly more errors. The difference in error frequency between participants of the MovOb and NonOb condition was greater for complex stimuli than for simple ones. These results suggest that incoming visual information about own manual exploration movements increases competitive pressure for limited working memory resources, and therefore, more recognition errors are made. Covering the hands during exploration may constitute a helpful simplification of the task’s demands by supporting the maintenance of information in working memory. Additionally, the relation of haptic complexity and stimulus characteristics was analyzed.     

Surface expression and function of Cav3.2 T-type calcium channels are controlled by asparagine-linked glycosylation

Low-voltage-activated T-type calcium channels play important roles in neuronal physiology where they control cellular excitability and synaptic transmission. Alteration in T-type channel expression has been linked to various pathophysiological conditions such as pain arising from diabetic neuropathy. In the present study, we looked at the role of asparagine (N)-linked glycosylation on human Ca_v3.2 T-type channel expression and function. Manipulation of N-glycans on cells expressing a recombinant Ca_v3.2 channel revealed that N-linked glycosylation is critical for proper functional expression of the channel. Using site-directed mutagenesis to disrupt the canonical N-linked glycosylation sites of Ca_v3.2 channel, we show that glycosylation at asparagine N192 is critical for channel expression at the surface, whereas glycosylation at asparagine N1466 controls channel activity. Moreover, we demonstrate that N-linked glycosylation of Ca_v3.2 not only controls surface expression and activity of the channel but also underlies glucose-dependent potentiation of T-type Ca²⁺ current. Our data suggest that N-linked glycosylation of T-type channels may play an important role in aberrant upregulation of T-type channel activity in response to glucose elevations.