Mario Menschikowski

Omega-3 fatty acids improve liver and pancreas function in postoperative cancer patients.

Epidemiologic studies have indicated that high intake of saturated fat and/or animal fat increases the risk of colon and breast cancer. Omega‐3 PUFAs in fish oil (FO) can inhibit the growth of human cancer cells in vitro and in vivo. These effects are related to the uptake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into the cellular substrate pool and their competitive metabolism with arachidonic acid (AA) at the cyclooxygenase and 5‐lipoxygenase levels. The metabolites of EPA and DHA have less inflammatory and immunosuppressant potency than the substances derived from AA. Based on previous experimental data, we hypothesized that FO supplementation after major abdominal cancer surgery would improve hepatic and pancreatic function. Ours was a prospective, randomized, double‐blinded clinical trial on 44 patients undergoing elective major abdominal surgery, randomly assigned to receive total parenteral nutrition (TPN) supplemented with either soybean oil (SO 1.0 g/kg body weight daily, n = 20) for 5 days or a combination of FO and SO (FO 0.2 + SO 0.8 g/kg body weight daily, n = 24). Compared to pure SO supplementation in the postoperative period, FO significantly reduced ASAT [0.8 ± 0.1 vs. 0.5 ± 0.1 mmol/(l · sec)], ALAT [0.9 ± 0.1 vs. 0.6 ± 0.1 mmol/(l · sec)], bilirubin (16.1 ± 5.3 vs. 6.9 ± 0.6 mmol/l), LDH (7.7 ± 0.4 vs. 6.7 ± 0.4 mmol/(l · sec) and lipase (0.6 ± 0.1 vs. 0.4 ± 0.1 μmol/(l · sec) in the postoperative course. Moreover, patients with increased risk of sepsis (IL‐6/IL‐10 ratio >8) showed a tendency to shorter ICU stay (18 hr) under omega‐3 PUFA treatment. Weight loss as encountered after the SO emulsion of 1.1 ± 2.2 kg was absent in the FO group. After major abdominal tumor surgery, FO supplementation improved liver and pancreas function, which might have contributed to the faster recovery of patients.

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.

Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways.

The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not interferon-gamma and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1beta and TNF-alpha correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-l-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1beta and TNF-alpha, and downstream by MAP kinase signaling pathways and metalloproteinases.

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.

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.

Opposing effects of HIF1α and HIF2α on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC-associated factor X

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine‐producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC‐associated factor X (MAX). To explore whether phenotypic differences among PPGLs reflect a MAX‐mediated mechanism and opposing influences of hypoxia‐inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene‐manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max‐mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re‐expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX‐related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids.

Expression of Secretory Group IIA Phospholipase A2 in Relation to the Presence of Microbial Agents, Macrophage Infiltrates, and Transcripts of Proinflammatory Cytokines in Human Aortic Tissues

Recent seroepidemiological and immunohistochemical studies have demonstrated an association between microbial infections and atherosclerosis. However, the mechanisms underlying this association are widely unknown. In the present study, arterial specimens obtained at autopsy after sudden death were analyzed concerning (1) the presence of Chlamydia pneumoniae, cytomegalovirus, herpes simplex virus, and Helicobacter pylori; (2) the expression of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) and of proinflammatory cytokines; and (3) the stage of atherosclerosis. Genomic DNA of microbial pathogens was determined by the polymerase chain reaction technique. The expression of sPLA(2)-IIA was studied immunohistochemically by using monoclonal antibodies against human sPLA(2)-IIA. Transcripts specific for sPLA(2)-IIA, interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were identified by reverse transcription-polymerase chain reaction. In 18 of 102 analyzed specimens, DNA of microbial pathogens was found. Thirteen sections were positive for C pneumoniae, whereas 2 specimens were positive either for cytomegalovirus or for herpes simplex virus. One section contained genomic DNA of all 3 pathogens simultaneously. None of the analyzed tissues exhibited nucleic acids specific for H pylori. In addition to macrophage infiltrates, the presence of microbial DNA was closely associated with the occurrence of transcripts specific for proinflammatory cytokines and sPLA(2)-IIA. Pathogens as well as sPLA(2)-IIA and cytokines were found to be present not only in advanced but also in early stages of atherosclerosis. In tissues negative for sPLA(2)-IIA and cytokine expression, none of the pathogens could be identified. Because macrophages exposed to phospholipase A(2)-treated lipoproteins are transformed into foam cells in vitro, the results of this study suggest an alternative mechanism by which microbial infections may act in a proatherogenic fashion in vessel walls.