Peter Schratzberger

Association of endotoxemia with carotid atherosclerosis and cardiovascular disease: prospective results from the Bruneck Study.

OBJECTIVES Focus of the current study was on the significance of bacterial endotoxin, which shows a variety of pro-atherogenic properties and may occur at high concentration in the circulation of infected subjects. BACKGROUND The possibility of an infectious risk factor in atherogenesis and cardiovascular disease has stimulated research interest, but the nature of such process remains obscure. METHODS We measured plasma endotoxin levels (LAL assay) in a random population of 516 men and women 50 to 79 years old at the 1990 baseline evaluation (Bruneck Study). End points of this prospective survey were incident (early) atherosclerosis in the carotid arteries as assessed with high-resolution Duplex ultrasound (five-year follow-up rate, 98%) and incident cardiovascular disease (follow-up rate, 100%). RESULTS Median endotoxin concentration amounted to 14.3 pg/ml (range, 6.0 to 209.2 pg/ml). Subjects with levels beyond 50 pg/ml (90th percentile) faced a threefold risk of incident atherosclerosis (odds ratio [95% confidence interval] 2.9 [1.4-6.3]; p < 0.01). The risk associated with high endotoxin was most pronounced in subjects with chronic infections and in current and ex-smokers. Notably, smokers with low endotoxin levels and nonsmokers did not differ in their atherosclerosis risk, whereas smokers with high levels almost invariably developed new lesions. All findings emerged as independent of vascular risk factors. Similar results were obtained for incident cardiovascular disease. CONCLUSIONS The current study yields first epidemiologic evidence that endotoxemia constitutes a strong risk factor of early atherogenesis in subjects with chronic or recurrent bacterial infections and a link in the association between cigarette smoking and atherosclerotic disease.

Mevalonate-Dependent Inhibition of Transendothelial Migration and Chemotaxis of Human Peripheral Blood Neutrophils by Pravastatin

Pravastatin, a hydrophilic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, has been reported to beneficially affect atherogenesis, plaque stability, and transient myocardial ischemia in significant coronary artery disease by influencing lipid metabolism and by intracellular signaling via mevalonate pathway products other than cholesterol. Leukocytes are implicated to play a pathophysiological role in these events. We were interested in finding out whether pravastatin could affect transendothelial migration (TEM), chemotaxis, and respiratory burst activity of the neutrophil ex vivo. In addition, effects on monocyte and T-lymphocyte chemotaxis were tested. For TEM assays, monolayers of human umbilical vein endothelial cells (HUVECs) were grown to confluence on polycarbonate filters bearing 5-microns pores in Transwell (Costar) culture plate inserts. Chemotaxis experiments were performed using modified Boyden chambers with cellulose nitrate micropore filters. Respiratory burst activity was measured fluorometrically. Treatment of neutrophils and monocytes with pravastatin at 2 to 200 mumol/L and 10 to 1000 mumol/L, respectively, significantly decreased chemotaxis triggered by fMet-Leu-Phe. This effect was abolished in the presence of mevalonic acid (500 mumol/L); no effect of pravastatin was seen on T-lymphocyte chemotaxis triggered by interleukin-8. Preincubation of neutrophils with pravastatin (200 mumol/L) also resulted in a significant reduction in the number of neutrophils that transmigrated a tumor necrosis factor-stimulated or lipopolysaccharide-stimulated HUVEC monolayer. At none of the concentrations tested (2 pmol/L to 200 mumol/L) did pravastatin affect neutrophil respiratory burst activity. We conclude that pravastatin may alter monocyte chemotaxis and neutrophil-endothelial interactions in migratory responses at concentrations obtained in vivo with cholesterol-lowering doses.

Induction of endothelial cell differentiation into capillary-like structures by substance P

Angiogenesis is an important process in inflammatory diseases and wound healing. We observed that the proinflammatory neuropeptide, substance P, stimulated angiogenesis in an in vitro model using human umbilical cord vein endothelial cells cultured on a basement membrane (Matrigel) substrate. Substance P stimulated endothelial cell differentiation into capillary-like structures in a dose-dependent manner. Stimulation of endothelial cell differentiation is a newly recognized biological function of substance P. The increased levels of substance P found in chronic inflammatory conditions may play an important role in tissue repair by promoting the development of new vessels and thus achieving compensation for ischemia.

LST1: A Gene with Extensive Alternative Splicing and Immunomodulatory Function

The gene of the leukocyte-specific transcript (LST1) is encoded within the TNF region of the human MHC. The LST1 gene is constitutively expressed in leukocytes and dendritic cells, and it is characterized by extensive alternative splicing. We identified 7 different LST1 splice variants in PBMC; thus, 14 LST1 splice variants (LST1/A-LST1/N) have been detected in various cell types. These isoforms code for transmembrane as well as soluble LST1 proteins characterized by two alternative open reading frames at their 3′ end. We demonstrate the presence of the transmembrane variant LST1/C on the cell surface of the monocytic cell lines U937 and THP1. Recombinant expression of LST1/C permitted its profound inhibitory effect on lymphocyte proliferation to be observed. In contrast, the alternative transmembrane variant LST1/A, the extracellular domain of which shows no amino acid sequence homology to LST1/C exerted a weaker but similar inhibitory effect on PBMC. These data demonstrate the protein expression of LST1 on the cell surface of mononuclear cells, and they show an inhibitory effect on lymphocyte proliferation of two LST1 proteins although they have only a very short amino acid homology.

Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used – THD, LPS, TNF, and IL-1 – inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils.

Response of Vascular Smooth Muscle Cells to the Neuropeptide Secretoneurin: A Functional Role for Migration and Proliferation in Vitro

Mesenchymal cell migration and replication are central biologic events involved in atherosclerosis and lung and hepatic fibrosis. Tissue repair and fibrosis are thought to be regulated by growth regulatory molecules, comprising both stimulators and inhibitors of mesenchymal cell functions, including platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), fibroblast growth factors, and several neuropeptides such as substance P. Secretoneurin (SN), a novel 33-amino acid neuropeptide derived from secretogranin II (chromogranin C), is widely distributed in the central and peripheral nervous and neuroendocrine systems, including afferent C-fibers, and can be released in the periphery by capsaicin. Recently, we reported that SN triggers the selective migration of human monocytes and fibroblasts in vitro, implicating its involvement in inflammatory responses. We report herein that SN stimulates specific migration (maximal response at 10(-10) M) of cultured arterial smooth muscle cells (SMCs), originating from rat thoracic aorta, and initiates DNA synthesis and SMC growth (BrdU incorporation, MTT test) with a maximum at 10(-8) M SN to a similar extent as observed by PDGF. Both functional activities of SN were inhibited by specific anti-SN immunoglobulins (dilution, 1:1000), and furthermore, a trypsinized SN peptide (10(-8) M) was unable to provoke biologic effects. Our studies suggest that SN functions as a regulatory peptide to modulate SMC migration and proliferation, which in conjunction with other factors could serve to aggravate and accelerate the development of atherosclerotic or restenotic lesions at sites of vascular injury.

Stimulation of human skin fibroblast migration by the neuropeptide secretoneurin

Fibroblasts, besides other cells, are called upon when tissue sustains an immunological, mechanical or chemical injury. Fibroblasts migrate into the site of inflammation, proliferate and synthesize and remodel a new matrix. These cellular responses are mediated locally by the release of neuropeptides from sensory nerve endings. Secretoneurin is a newly discovered 33-amino acid neuropeptide derived from secretogranin II (chromogranin C), which is found in sensory afferent C-fibers. We show here that secretoneurin triggers the selective migration of human skin fibroblasts in vitro, but does not stimulate their proliferation. The attraction of human skin fibroblasts toward secretoneurin could be blocked by specific anti-secretoneurin antibodies and is mediated by the C-terminal fragment of the peptide. The observed activity of this sensory neuropeptide is the first description of a specific effect on human skin fibroblasts and suggests a role for secretoneurin in inflammation and wound healing.

Plasma Levels of Troponin T After Electrical Cardioversion of Atrial Fibrillation and Flutter

To establish possible myocardial damage by direct-current countershock, we measured plasma levels of troponin T after electrical cardioversion in 33 nonselected patients with atrial fibrillation or flutter. Unchanged normal levels of troponin T indicate that significant myocardial cell injury by shocks in the usual dosage is unlikely to occur.

Release of chemoattractants for human monocytes from endothelial cells by interaction with neutrophils

OBJECTIVE The release of monocyte chemoattractant protein-1 (MCP-1) in the vessel wall may lead to accumulation of monocytes in the subendothelial space. The role of neutrophils (PMNL) in the initiation of this process is unknown. We tested whether PMNL are able to induce the production and release of MCP-1 in endothelial cells. METHODS PMNL were allowed to interact with human umbilical vein endothelial cell (HUVEC) monolayers in culture. Culture media were collected and assessed for chemotactic activity on mononuclear leukocytes (MNC) or purified monocytes in a modified Boyden chamber assay. Additionally, MCP-1 levels in supernatants were quantified by ELISA. RESULTS Media from unstimulated HUVEC culture supernatants induced a slight increase (1.2-fold) of MNC and purified monocyte chemotaxis, which was significantly augmented by addition of PMNL for 1 h (1.4-fold; P < 0.05). The increase in chemotaxis was time- and dose-dependent and could be blocked by an anti-MCP-1 monoclonal antibody. Media obtained after coculture of PMNL and HUVEC for 1-5 h contained increased amounts of MCP-1 as measured by ELISA; addition of cycloheximide abolished this response. CONCLUSIONS Interaction of PMNL with endothelium induces the release of functionally active MCP-1 suggesting that in the vascular wall, PMNL may play a role in the recruitment of MNC.

Secretoneurin-induced in vitro chemotaxis of human monocytes is inhibited by pertussis toxin and an inhibitor of protein kinase C

The sensory neuropeptide secretoneurin (SN) triggers chemotactic migration of monocytes. We have investigated the possibility that SN, like other chemoattractants such as formyl-Met-Leu-Phe and chemokines, might stimulate migration of monocytes by G protein and protein kinase C (PKC) activation and induce Ca2+i release. We report that preincubation of monocytes with pertussis toxin inhibited SN chemotaxis. Staurosporine, an inhibitor of PKC, significantly decreased SN-induced chemotaxis of monocytes, suggesting that PKC may be involved in the signaling. Tyrphostin-23, which inhibits tyrosin kinase, did not affect SN-induced chemotaxis of monocytes. This suggests that SN uses a signaling mechanism that is coupled to pertussis toxin-sensitive G proteins. Involvement of phospholipase C beta as a result of PKC activation is suggested by a SN-induced increase of intracellular Ca2+ concentration in monocytes.