Werner Jaross

Secretory group II phospholipase A2 in human atherosclerotic plaques

Atherosclerotic plaques exhibit a series of features that are similar to those of chronic inflammation. Based on the fact that during inflammation several cell types synthesize and secrete a group II phospholipase A2 (PLA2), an immunohistochemical study was undertaken to explore whether this enzyme can be identified in human atherosclerotic lesions. Tissue specimens obtained from 13 patients who had undergone arteriectomy and three specimens with advanced atherosclerotic plaques obtained at autopsy were analyzed and compared to arteries free of atherosclerosis. The results showed that in all areas with atherosclerotic lesions, a staining with monoclonal antibodies raised against group II PLA2 was evident. In normal arteries without thickened intima, this immunostaining was completely negative. With the use of specific monoclonal antibodies against macrophages (anti-KP-1) and smooth muscle cells (anti-alpha-actin), PLA2-positive cells were identified as foam cells mainly derived from macrophages. In addition to these cells, other regions of the thickened intima gave a partially positive reaction with anti-PLA2 antibodies, but could not be stained with either anti-KP-1 or anti-alpha-actin. Some of these regions were localized on edges of calcification and cell necrosis. Other PLA2-positive regions seem to be associated with extracellular matrix structures. In summary, the findings of this study may be regarded as further evidence to support the link between atherosclerosis and chronic inflammatory processes. In view of the fact that the in vitro modification of lipoproteins by PLA2-treatment induces lipid deposition in macrophages, the results of this study suggest that group II PLA2 may actively be involved in the formation of foam cells in vivo.

Interleukin-1, interleukin-6 and myocardial enzyme response after coronary artery bypass grafting – a prospective randomized comparison of the conventional and three minimally invasive surgical techniques

OBJECTIVE In order to evaluate the traumatic effects of median sternotomy and cardiopulmonary bypass (CPB) in conventional and minimally invasive coronary artery bypass grafting, inflammatory response was studied in a prospective randomized trial in patients referred to single-vessel coronary artery bypass grafting. METHODS Four surgical techniques were compared: group 1, median sternotomy with CPB in ten patients (eight male, two female; aged 59.6+/-11.0 years (mean+/-SD)); group 2, median sternotomy and off-pump in ten patients (seven male, three female; aged 65.1+/-10.0 years); group 3, minithoracotomy with CPB in ten patients (seven male, three female, aged 61.2+/-10.4 years); group 4, minithoracotomy and off-pump in ten patients (nine male, one female, aged 62.9+/-9.8 years). All patients received a left internal mammary artery graft to the left anterior descending artery (LAD). Clinical data, perioperative values of cytokines and cardiac enzymes were monitored. RESULTS There were no major complications. Troponin-T and creatine kinase isoenzyme MB (CK-MB) levels were significantly higher in CPB procedures (P<0.0056; multivariate general linear model). Interleukin-6 (IL-6) levels were significantly higher in minithoracotomy procedures. Interleukin-1 (IL-1) was significantly increased in all patients compared with the preoperative values. CONCLUSIONS The use of CPB is combined with higher levels of troponin-T and CK-MB as signs of myocardial damage. Surgical access was identified as a trigger of inflammatory response, as minithoracotomy is related to higher levels of IL-6. IL-1 increased in all procedures and this occurred independently of the surgical access or the use of CPB, which points out a potential relationship between inflammatory response and anesthesia. Neither CPB nor surgical access influenced the clinical outcome in the treatment of coronary artery single-vessel bypass grafting.

Determination of cholesterol in atherosclerotic plaques using near infrared diffuse reflection spectroscopy

The aim of this investigation was to examine whether near infrared diffuse reflection spectroscopy is an acceptable tool for the determination of cholesterol content in atherosclerotic plaques. Using an FT-spectrophotometer (lambda=1000-2500 nm) and fiberoptic systems (d=4 mm), the cholesterol content could be determined in mixtures of the primary compounds of the aortic wall with acceptable precision. Considering the inhomogeneous distribution of cholesterol and cholesterol esters in atherosclerotic plaques the determination of total cholesterol using this method is of acceptable efficacy, even though the calibration procedure did not reflect the composition correctly. Using an energy dose of less than 100 mW/cm(2) to avoid damage to endothelial cells, arterial tissue of about 170-200 microm thickness attenuates the reflected NIRS signal by up to 50%. Cholesterol levels could be determined accurately in atherosclerotic lesions in human aortic specimens obtained by autopsy. The correlation coefficient between the NIRS results and those of HPLC analysis calculated in the investigation of 82 different areas of 18 human aortic specimens was 0.926 (y=0.869x+0. 771, external validation). Acceptable results were also achieved by means of a coronary-catheterlike fiberoptic strand (d=l mm), despite the worsened signal/noise ratio. The results show that the development of a coronary catheter using NIRS appears to be possible in principle.

Even moderate cigarette smoking influences the pattern of circulating monocytes and the concentration of sICAM-1

The pattern of circulating monocyte subtypes and the concentration of the soluble intercellular adhesion molecule-1 (ICAM-1) were compared in middle-aged female moderate smokers and lifetime non-smokers. Total leukocyte and monocyte counts were higher in smokers. The pattern of circulating monocytes of smokers was changed toward lower absolute counts of activated (CD16+/CD64+) monocytes and (CD16+/CD14+) monocyte-macrophages and higher counts of nonactivated monocytes. The serum concentration of soluble ICAM-1 was significantly higher in smokers than in non-smokers. It is supposed that even moderate cigarette smoking leads to an activation of the circulating monocytes and their increased adhesion to the endothelium.

Biological effects of secretory phospholipase A2 group IIA on lipoproteins and in atherogenesis

Secretory phospholipase A2 group IIA(sPLA2 IIA) can be produced and secreted by various cell types either constitutionally or as an acute‐phase reactant upon stimulation by proinflammatory cytokines. The enzyme prefers phosphatidylethanolamine and phosphatidylserine as substrates. One important biological function may be the hydrolytic destruction of bacterial membranes. It has been demonstrated, however, that sPLA2 can also hydrolyse the phospholipid monolayers of high density lipoprotein (HDL) and low density lipoprotein (LDL) in vitro. Secretory phospholipase A2‐modified LDL show increased affinity to glycosaminoglycans and proteoglycans, a tendency to aggregate, and an enhanced ability to deliver cholesterol to cells. Incubation of cultured macrophages with PLA2‐treated LDL and HDL is associated with increased intracellular lipid accumulation, resulting in the formation of foam cells. Elevated sPLA2(IIA) activity in blood serum leads to an increased clearance of serum cholesterol. Secretory phospholipase A2(IIA) can also be detected in the intima, adventitia and media of the atherosclerotic wall not only in developed lesions but also in very early stages of atherosclerosis. The presence of DNA of Chlamydia pneumoniae, herpes simplex virus, and cytomegalovirus was found to be associated with sPLA2(IIA) expression and other signs of local inflammation. Thus, sPLA2(IIA) appears to be one important link between the lipid and the inflammation hypothesis of atherosclerosis.

Analysis of secretory group II phospholipase A2 expression in human aortic tissue in dependence on the degree of atherosclerosis

Secretory non-pancreatic (group II) phospholipase A2 (sPLA2) releases precursors of important mediators of inflammation from phospholipids. Based on the inflammatory character of atherosclerosis we previously described the identification of sPLA2 in human atherosclerotic plaques. In vitro studies on lipoproteins have shown that sPLA2 is able to favour the formation of foam-like cells representing a typical feature of early atherosclerotic lesions. In the present study the expression of sPLA2 in relation to the degree of atherosclerosis was investigated. Aortic tissue samples of 25 autopsy cases ranging in age from 1 to 77 years were taken from 2 cm above the heart and 3 cm below the renal arteries. The material was classified regarding the degree of atherosclerotic changes based on staining with haemalaun and eosine as well as on staining according to Goldner. Furthermore, immunohistochemical procedures detecting sPLA2, macrophages and smooth muscle cells were performed. The study has shown that in the abdominal aorta the enzyme was present in all advanced atherosclerotic lesions, but only in some preatheromas and precursors of atherosclerosis. However, this correlation did not occur in the thoracic aorta, where sPLA2-positive results showed a similar frequency in all degrees of atherosclerotic lesion. The enzyme was found in all three layers of the vessel wall without significant differences. Round cells, scarcely smooth muscle cells and endothelial cells were identified as sPLA2-positive. However, these data do not allow a conclusion as to which type of cell is responsible for the secretion of sPLA2. In summary, the correlation between the expression of this enzyme and the degree of atherosclerosis underlines the possible importance of sPLA2 in atherogenesis.

Minimal oxidation and storage of low density lipoproteins result in an increased susceptibility to phospholipid hydrolysis by phospholipase A2

In vitro-studies have shown that phospholipid hydrolysis of low density lipoproteins (LDL) by bee venom or porcine pancreatic phospholipase A2 (PLA2) leads to an increased uptake of these lipoproteins by macrophages transforming them into foam cells. Recently, a secretory phospholipase A2, group II, was detected in human atherosclerotic plaques. In order to investigate the role of this enzyme in the pathogenesis of atherosclerosis, a structurally identical human secretory PLA2 was purified from the medium of HepG2 cells stimulated with interleukin-6 and tumor necrosis factor-alpha. The activity of the purified enzyme towards the phospholipids of native and modified low density lipoproteins was compared with the activity towards Escherichia coli-membranes and other phospholipid substrates. Compared to E. coli-membranes, native LDL proved to be a poor substrate for group II PLA2. After mild oxidation induced by copper ions or by 2,2-azobis(2-amidinopropane) (AAPH), the susceptibility of LDL to phospholipid hydrolysis was found to be increased by 25 and 23%, respectively, whereas extensive copper-mediated oxidation caused a decreased hydrolysis. Aging of LDL at 6 degrees C for weeks or at 37 degrees C for hours resulted in an increase in PLA2-catalyzed phospholipid hydrolysis of up to 26-fold. LDL protected from oxidation by probucol during aging showed a lesser increase in susceptibility to phospholipid hydrolysis. Our results suggest that PLA2, group II, can increase the atherogenicity of LDL by its ability to hydrolyze the phospholipids of these lipoproteins, especially after modifications that are likely to occur in vivo.

Determination of the cholesterol–collagen ratio of arterial atherosclerotic plaques using near infrared spectroscopy as a possible measure of plaque stability

Particular danger associated with an arteriosclerotic plaque consists in the possible rupture of its cap, dependent on the thickness of the cap covering the lipid core, its composition and different inflammatory changes. The purpose of this study was to compare the total cholesterol and collagen contents of arterial walls, both measured by near infrared spectroscopy (NIRS), and to test whether the ratios of cholesterol to collagen correlate with histochemical parameters possibly being indicators for plaque stability. NIR spectra of 118 sections from 36 human aortas were measured at 1000-2500 nm. Evaluation was performed by the partial least squares method (PLS), the chemical reference analysis by HPLC. Acceptable results were achieved for calibrations. With these calibrations 38 further aortic sections taken at autopsy were NIR-spectroscopically analysed and ordered in relation to histological findings of fatty deposits, cap thickness over the lipid core, and the ratio of fatty deposits to cap thickness. Correlations were found to exist between the spectroscopically determined total cholesterol concentrations and the histologically estimated fatty deposits (r=0.887), between the spectroscopically determined collagen concentrations and the cap thickness over the lipid core (r=0.441), and between the ratios total cholesterol to collagen and the ratios fatty deposits to cap thickness (r=0.575).

Alterations in the physicochemical characteristics of low and high density lipoproteins after lipolysis with phospholipase A2. A spin-label study

Abstract Human low and high density lipoproteins (LDL and HDL, respectively) were treated with porcine pancreatic phospholipase A 2 (PLA 2 ) in the presence of albumin resulting in hydrolysis of 40–84% of the lipoproteins phospholipids. The resulting PLA 2 -treated LDL and HDL and concurrent control lipoproteins incubated without PLA 2 were reisolated by ultracentrifugation and labelled with 5-doxyl-and 16-doxyl-stearic acid, and with a spin-labelled analogue of maleimide. Analysis of ESR spectra showed that phospholipid hydrolysis both of LDL and HDL resulted in an increase in order, micro-viscosity and polarity of lipid regions in the surface monolayer of the particles. In the temperature range from 3°C to 50–60°C, Arrhenius plots of a spectral parameter of LDL and HDL labelled with 5-doxyl-stearate exhibited alterations which suggest an increase in free cholesterol content near the surface of the lipoproteins after PLA 2 -treatment. ESR spectra of the maleimide analogue bound covalently to the protein moiety of the lipoproteins have demonstrated that, following phospholipid hydrolysis, the conformation of the apoproteins became more condensed, with more masked domains. The possible implications of the revealed alterations for enhanced delivery of LDL and HDL cholesterol to cells after phospholipolysis of the lipoproteins are discussed.

Impact of concentrations of glycated hemoglobin, α-tocopherol, copper, and manganese on oxidation of low-density lipoproteins in patients with type I diabetes, type II diabetes and control subjects

The late organ complications in diabetic patients are associated with enhanced oxidation of low-density lipoproteins (LDL). The role of vitamin and trace metal concentrations in this process is not clear. Therefore, we compared the oxidative susceptibility and alpha-tocopherol concentration of LDL with the levels of glycated hemoglobin (HbA1c), copper and manganese. Sixty-three diabetic patients (23 female and 40 male; 53 of type II, 10 of type I) and 35 control subjects (17 female and 18 male) were investigated. The in vitro-formation of conjugated dienes in purified LDL preparations in the presence of copper was followed as absorbance at 234 nm. LDL exhibited a shorter lagtime (44.5 +/- 10.1 vs. 67.8 +/- 16.0 and 50.1 +/- 14.3 vs. 68.8 +/- 14.6 min) for type I and type II diabetic patients vs. sex and age-matched controls, P < 0.001. For all subjects together the lagtime was inversely correlated to HbA1c (r = -0.230, P = 0.023) and positively correlated to LDL alpha-tocopherol/LDL (mol/mol). This ratio was lower in diabetic patients (P < 0.01 for type II) than in control subjects. The copper and manganese plasma levels were not different between diabetic and nondiabetic groups. However, parameters of LDL oxidizability (amount and rate of oxidation) were positively correlated with both copper and manganese concentrations. We conclude that in diabetes the resistance of LDL against oxidation is diminished in relation to the quality of glucose control.