Secondary prevention of atherosclerosis, especially before the onset of symptoms, appears desirable and could be possible with a serum marker detecting atherosclerosis. Circulating, shedded forms of adhesion molecules may serve as such because their expression is upregulated in atherosclerotic plaques. In 52 patients with peripheral arterial vascular disease (Fontaine class IIa, 7 patients; class IIb, 29 patients; and class III, 16 patients), the extent of atherosclerosis was evaluated on the basis of angiograms of a large portion of the arterial system. The area diseased by atherosclerosis was determined by the percentage of vessel wall irregularities of the following calculated segments: aorta (distal from the kidney arteries), common iliac artery, external iliac artery, common femoral artery, lateral circumflex femoral artery, and popliteal artery. The maximal surface area that could exhibit atherosclerotic changes was 250 cm2. The serum concentration of circulating vascular cell adhesion molecule-1 (VCAM-1) correlated with the extent of atherosclerosis (r = .8, P < .001). In contrast, circulating intercellular adhesion molecule-1, E-selectin, P-selectin, and thrombomodulin (as markers for endothelial cell damage) did not correlate with the extent of atherosclerosis. Furthermore, circulating VCAM-1 could be used to indicate stages of atherosclerosis with a high degree of statistical significance. The potential bias of factors such as age, diabetes mellitus, hypercholesterolemia, arterial hypertension, renal failure, and history of myocardial infarction on the correlation of circulating VCAM-1 with the extent of atherosclerosis could be excluded by multivariate analysis. These findings suggest an important role of VCAM-1 in atherosclerosis and may serve as the basis for further evaluation of circulating VCAM-1 as a potential serum marker for atherosclerosis.

SUMMARYThrombomodulin is a transmembranous glycoprotein of endothelial cells. In vitro it is a marker of endothelial cell injury. In vivo the levels of serum thrombomodulin are regarded as a parameter of activity in vasculitides. The latter are pathophysiologically determined by neutrophil-derived inflammation and endothelial cell injury caused by secretion of proteases and hydrogen peroxide. It was the objective of this study to determine whether thrombomodulin is only a late marker of advanced endothelial cell injury or whether it indicates also earlier stages of cell alterations. Over 24 hr endothelial cell cultures were incubated with hydrogen peroxide or the neutrophil proteases proteinase-3, elastase and cathepsin G. The time-dependent increase of thrombomodulin in the supernatant was determined by enzymelinked immunosorbent assay and immunoblot. In addition the viability (eosin, tetrazolium dye assay), detachment (crystal-violet assay), and apoptosis (4k,6-diamine-2k-phenylindoledihydrochloride assay) of the respective endothelial cells were determined for adherent and non-adherent cells. A rapid thrombomodulin increase was found under all experimental conditions. The additional immunoblotting analysis showed the pattern of proteolytic cleavage caused by the protease reactivity. In case of hydrogen peroxide the thrombomodulin increase was closely correlated with the loss of cell viability and lysis. The incubation of endothelial cells with the different proteases resulted in a time-dependent detachment of primarily viable cells. In addition to cell necrosis apoptotic cell death was found in the subgroup of detached endothelial cells after prolonged incubation over 24 hr with proteinase-3 (23%), elastase (31%), and cathepsin G (19%). In contrast, still adhering cells did not show any signs of necrosis or apoptosis. In summary these studies confirm in vitro that soluble thrombomodulin is not only a parameter of advanced endothelial cell destruction itself but also in addition an early marker of initial endothelial cell membrane changes induced by neutrophil derived proteases and oxygen radicals.

PTX3 levels are markedly elevated in HD patients. The increase in PTX3 production in whole blood after HD indicates that the HD procedure itself contributes to elevated PTX3 levels in HD patients. The association between PTX3 and cardiovascular morbidity suggests a possible connection of PTX3 with atherosclerosis and cardiovascular disease in HD patients.

At present most quantitative proteomics investigations are focused on the analysis of protein expression differences between two or more sample specimens. With each analysis a static snapshot of a cellular state is captured with regard to protein expression. However, any information on protein turnover cannot be obtained using classic methodologies. Protein turnover, the result of protein synthesis and degradation, represents a dynamic process, which is of equal importance to understanding physiological processes. Methods employing isotopic tracers have been developed to measure protein turnover. However, applying these methods to live animals is often complicated by the fact that an assessment of precursor pool relative isotope abundance is required. Also, data analysis becomes difficult in case of low label incorporation, which results in a complex convolution of labeled and unlabeled peptide mass spectrometry signals. Here we present a protein turnover analysis method that circumvents this problem using a (15)N-labeled diet as an isotopic tracer. Mice were fed with the labeled diet for limited time periods and the resulting partially labeled proteins digested and subjected to tandem mass spectrometry. For the interpretation of the mass spectrometry data, we have developed the ProTurnyzer software that allows the determination of protein fractional synthesis rates without the need of precursor relative isotope abundance information. We present results validating ProTurnyzer with Escherichia coli protein data and apply the method to mouse brain and plasma proteomes for automated turnover studies.

Leptin is a pleiotropic hormone believed to regulate body weight. Its function in wasting during inflammatory disease in humans is unknown. We studied the effect of repeated tumor necrosis factor (TNF) infusion on serum leptin levels in six patients with solid tumors. TNF infusion on day 1 resulted in an increase in serum leptin levels from 3.1 (SEM +/- 0.28) ng/mL to 5.2 (SEM +/- 0.6) ng/mL after 12 h (P < 0.001). The serum levels returned to baseline within 24 h. Similar results were obtained when TNF was infused on subsequent days. The study shows that leptin serum levels are under control of TNF.