Angiopoietin-1 causes reversible degradation of the portal microcirculation in mice: implications for treatment of liver disease

In many different liver diseases, such as cirrhosis, degradation of the microcirculation, including obliteration of small portal or hepatic veins contributes to disease-associated portal hypertension. The present study demonstrates the importance of angiogenesis in the establishment of arteriovenous shunts and the accompanying changes to the venous bed. One aspect of angiogenesis involves the branching of new vessels from pre-existing ones, and the molecular mechanisms controlling it are complex and involve a coordinated effort between specific endothelial growth factors and their receptors, including the angiopoietins. We modulated the hepatic vasculature in mice by conditionally expressing angiopoietin-1 in hepatocytes. In mice exposed to angiopoietin-1 during development, arterial sprouting, enlarged arteries, marked loss of portal vein radicles, hepatic vein dilation, and suggestion of arteriovenous shunting were observed. Most importantly, these phenotypic changes were completely reversed within 14 days of turning off transgene expression. Expression of excess angiopoietin-1 beginning in adulthood did not fully recapitulate the phenotype, but did result in enlarged vessels. Our findings suggest that controlling excessive angiogenesis during liver disease may promote the restoration of the portal vein circuit and aid in the resolution of disease-associated portal hypertension.     

Why do cells release vesicles?

Prokaryotic and eukaryotic cells release vesicles into their environment. To answer the question why eukaryotic cells release vesicles, we may learn from prokaryotes. Bacteria release outer membrane vesicles, resembling microparticles, which act as “multi-purpose carriers”. They contain signalling molecules for other bacteria, deliver toxins to host cells and exchange DNA encoding virulence genes between bacteria. Similarly, cell-derived microparticles and exosomes from eukaryotic cells are multi-purpose carriers containing e.g. signalling molecules, cellular waste and functional genetic information. To illustrate our rapidly increasing knowledge on the multiple roles that cellular microparticles and exosomes play in disease progression, we focus on cancer, which is one of the best studied diseases in this aspect. The clinical applications of microparticles and exosomes, including diagnosis, prognosis and therapy, in cancer are discussed.     

Protein C activity and antigen levels in childhood

Hereditary protein C deficiency is an important risk factor for thrombosis. To enable its diagnosis shortly after birth, we determined reference values of protein C antigen and activity levels for the first 3 months of life. To establish an age-related range of protein C levels we also determined median values for individuals up to 18 years of age. A good correlation between the two levels was seen from the 3rd/4th month of life onwards, whereas in the first 2 months the activity levels were significantly lower than the antigen levels. This was not due to interference by the increased plasma citrate concentration at high haematocrit values, and may suggest a dysfunctional protein C molecule in the neonatal period. We found a rapid rise in protein C activity and antigen levels until the age of 7–9 months, followed by a slower progression toward adolescence. In contrast to previous reports, our results indicate that adult values are probably not achieved until sometime during the 2nd decade of life.     

Enhanced coagulation activation in preeclampsia: the role of APC resistance,microparticles and other plasma constituents

In the Second Northwick Park Heart Study, the activation peptides of factor IX (FIXpep) and factor X (FXpep) were measured in 1261 middle-aged men by double-antibody radioimmunoassay. During follow-up 147 men who had a first coronary heart disease (CHD) event were found to have had an increased FIXpep (p = 0.003) and a reduced FXpep (p = 0.05) at baseline compared with those remaining CHD-free (controls). Plasma FIXpep and FXpep were positively associated, but the rate of rise in FIXpep with increasing FXpep was higher in cases than controls (p for interaction = 0.01). In a sample of 87 controls, FIXpep was positively and independently related to the concentrations of a polymorphonuclear-specific fibrinogen degradation product (p = 0.036) and FXpep (p = 0.004), but in larger samples no statistically significant associations were found either with C-reactive protein or with fibrinogen concentration. The findings suggested that the increased FIXpep in men at high CHD-risk may have been partly due to the generation of factor IX inactivation peptides by inflammatory proteolysis and their recognition together with true FIXpep in the radioimmunoassay. Direct evidence for this hypothesis requires development of assays for human elastase-specific factor IX inactivation peptides.     

Hyperlipoproteinemia affects cytokine production in whole blood samples ex vivo. The influence of lipid-lowering therapy

A specific and robust immunoassay for the lipoprotein-associated phospholipase A₂ (Lp-PLA₂), platelet-activating factor acetylhydrolase, is described for the first time. The immunoassay was used to evaluate possible links between plasma Lp-PLA₂ levels and atherosclerosis risk amongst susceptible individuals. Such an investigation was important because Lp-PLA₂ participates in the oxidative modification of low density lipoprotein by cleaving oxidised phosphatidylcholines, generating lysophosphatidylcholine and oxidised free fatty acids. The majority of Lp-PLA₂ was found associated with LDL (approximately 80%) and, as expected, enzyme levels were significantly positively correlated to LDL cholesterol. Plasma Lp-PLA₂ levels were significantly elevated in patients with angiographically proven coronary artery disease (CAD) when compared with age-matched controls, even though LDL cholesterol levels did not differ significantly. Indeed, when included in a general linear model with LDL cholesterol and other risk factors, Lp-PLA₂ appeared to be an independent predictor of disease status. We propose, therefore, that plasma Lp-PLA₂ mass should be viewed as a potential novel risk factor for CAD that provides information related to but additional to traditional lipoprotein measurements.     

Chromogenic endotoxin determination in blood — clinical relevance

Summary In a pilot study in 400 patients LPS-quantification in blood using chromogenic substrates, bacterial cultures and bacterial quantification were performed. Decreased plasma levels of antithrombin III and plasminogen were early predictors of gram-negative septicemia, which already were apparant 3 days prior to the first positive LPS-test. It is concluded that daily determinations of LPS may reduce the delay in proper antibiotic therapy.     

Generation of platelet-derived microparticles in patients undergoing cardiac surgery is not affected by complement activation

OBJECTIVE: The mechanisms causing the presence of platelet-derived microparticles in the circulation are unknown. In vitro platelets release platelet-derived microparticles in response to complement activation. This study evaluates the relationship between complement activation and levels of circulating platelet-derived microparticles in patients undergoing cardiac surgery. METHODS: Prospectively, 71 patients were included who underwent elective coronary artery bypass grafting with cardiopulmonary bypass. The patients were randomly allocated to one of the 3 groups: uncoated oxygenator, UnModified Surface (n = 25) or oxygenator coated with either BioPassive Surface (n = 25) or BioActive Surface (n = 21). Platelet-derived microparticles and terminal complement complexes were determined before bypass and after induction of anesthesia, 15 minutes after the start of cardiopulmonary bypass, at the end of cardiopulmonary bypass, and 30 minutes after administration of protamine sulfate. RESULTS: Demographic and cardiopulmonary bypass data were similar for the 3 groups. At the end of cardiopulmonary bypass, platelet-derived microparticle numbers were decreased in all 3 groups. No significant differences were observed among the groups at any sampling point. At the end of cardiopulmonary bypass, terminal complement complex concentrations were increased in all groups (P <.001), and significant differences among the groups were present (P =.002). CONCLUSIONS: Despite significant complement activation, no increase in numbers of circulating platelet-derived microparticles was found in the systemic blood of patients undergoing cardiac surgery with cardiopulmonary bypass. Thus complement activation in vivo does not necessarily affect generation of platelet-derived microparticles.     

Diagnostic value of the mean corpuscular volume in the detection of vitamin B12 deficiency

In clinical practice, the finding of an elevated mean corpuscular volume (MCV), macrocytic anaemia or specific neurological symptoms is often the reason to test for vitamin B12 (B12) deficiency. Use of the MCV as a test for the detection or exclusion of B12 deficiency is only justified if the diagnostic accuracy is sufficiently high. However, the sensitivity and specificity are not well known. We performed a systematic review of the diagnostic value of an elevated MCV for B12 deficiency in both anaemic and non-anaemic patients. Of approximately 3500 titles and/or abstracts that were screened, 37 original papers contained usable data. The population under study proved to be the characteristic of major influence on the study outcome. Pooling of data from different studies was performed in subsets of the data corresponding to the different populations studied. The cut-off levels of both MCV and serum B12 had a significant influence on the study outcomes. The data, however, were pooled without taking these cut-off levels into account. The pooled estimates should be interpreted with this limitation in mind. The reference standards were (1) a low serum B12 concentration and (2) a B12 deficiency confirmed by low serum B12 combined with additional diagnostic investigations. In the population that was randomly screened for low serum B12, the sensitivity of the MCV for B12 deficiency was 17%, whereas the sensitivity was 30% for B12 deficiency in patients with anaemia. When measurement of serum B12 was ordered to exclude B12 deficiency as part of the patients' treatment, the sensitivity was 30% for low serum B12 concentration, 58% for B12 deficiency and 75% for B12 deficiency in patients with anaemia. In the population with pernicious anaemia, the sensitivity was far from perfect (77%). In the five studies that reported data on the positive predictive value of the MCV for B12 deficiency, this ranged from 0% (0/6) to 55% (11/20). This systematic review shows that a considerable number of B12-deficient patients will remain unnoticed when the MCV is used to rule in patients for further evaluation. Depending on the population studied, up to 84% of cases will than be missed. The MCV can be used to make the diagnosis of B12 deficiency more--or less--probable. An elevated MCV justifies the measurement of serum B12. The MCV should not be used as the only parameter ruling out the diagnosis of B12 deficiency.     

Cell-derived microparticles contain caspase 3 in vitro and in vivo.

BACKGROUND: Microparticles (MP) from endothelial cells (endothelial microparticles; EMP) circulate in disease states, but the processes such as apoptosis or cell activation underlying their release are unclear. OBJECTIVES: We investigated whether adherent (viable) or detached (apoptotic) endothelial cells are the possible source of EMP in vitro, i.e. under control and interleukin (IL)-1alpha activation conditions, and in vivo. METHODS: Adherent and detached endothelial cells, and EMP, were isolated from human umbilical vein endothelial cell cultures (n = 6), treated without or with IL-1alpha (5 ng mL(-1); 24 h). Cell fractions were analyzed by flow cytometry for annexin V binding, propidium iodide (PI) and caspase 3 staining (n = 3). Caspase 3 in EMP was studied using Western blot (n = 6) and flow cytometry (n = 6). Plasma from healthy subjects and systemic lupus erythematosus patients (both n = 3) were analyzed for caspase 3-containing (E)MP. RESULTS: Detached but not adherent cells double-stained for annexin V and PI, confirming the apoptotic conditions of the detached cells and the viable nature of the adherent cells. Caspase 3 was solely present in the detached cells and procaspase 3 in the adherent cells. Caspase 3 was present in EMP from both control and IL-1alpha-treated cultures. Counts of EMP and detached cells, but not adherent cells, highly correlated (r = 0.959, P < 0.0001). In vivo circulating MP from nucleated (endothelial cells, monocytes) and anucleated cells (platelets, erythrocytes) contained caspase 3. CONCLUSIONS: EMP contain caspase 3 and may be mainly derived from detached (apoptotic) endothelial cells in vitro. The presence of caspase 3 in MP from anucleated cell types, however, suggests that its presence may not necessarily be related to apoptosis in vivo but may be associated with caspase 3 activation unrelated to apoptosis.