Wolfgang Völker

Copper-induced inflammatory reactions of rat carotid arteries mimic restenosis/arteriosclerosis-like neointima formation

The pathogenesis of arteriosclerosis and of restenosis after angioplasty is linked with an inflammatory and fibroproliferative response of the arterial tissue. We have induced a non-infectious inflammation by implanting a silicon-copper cuff around rat carotid arteries. The copper ions released from the oxidized copper initiate and mimic all morphological features of post-angioplasty restenotic and arteriosclerotic lesions. The copper-induced lesions were analyzed by electron and light microscopy, immunohistochemical methods and quantified by morphometry. During the first phase of copper-induced tissue reaction (3 days), macrophages and polymorphonuclear leucocytes invaded through the endothelium, accumulated in the subendothelial space and triggered the proliferation of smooth muscle cells which then migrated from the tunica media through the lamina elastica interna into the intima. Within 3 weeks, the accumulated smooth muscle cells, macrophages, leucocytes and newly synthesized extracellular matrix formed a circular mostly eccentric fibrotic thickening that narrows the vessel lumen by 30-40%. The accompanying structural disorganization of the medial layer led to focal rupture and aneurysm-like dilatation of the vessel wall in 3 of 11 animals between day 20 and 43. The neointima progressively increased in thickness over time leading to corresponding reduction of the vessel lumen. The carotid arteries of control animals and animals treated with copper-free silicon cuffs showed no abnormal pathological appearance. Our results show that inflammation-inducing agents can contribute to and simulate restenosis- and arteriosclerosis-like lesions and that the copper-cuff model may be useful in the exploration of new approaches to intervention.

Compartmentation and characterization of different proteoglycans in bovine arterial wall.

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.

The antiatherogenic and antiinflammatory effect of HDL-associated lysosphingolipids operates via Akt → NF-kappaB signalling pathways in human vascular endothelial cells

Adhesion of mononuclear cells to the vascular endothelium and their subsequent transmigration into the arterial wall represent key events in the pathogenesis of arteriosclerosis. In previous studies we have shown that high density lipoproteins (HDL) and the HDL–associated sphingosylphosphorylcholine (SPC) have the ability to suppress the TNF–alpha–induced expression of endothelial cell E–selectin. However, the current understanding of the mechanism by which HDL reduces the expression of E–selectin is still incomplete. In the present study we show that interaction of the HDL–associated sphingosylphosphorylcholine and sphingosylgalactosyl–3–sulfate (lysosulfatide, LSF) with the G–protein–coupled EDG receptor initiates a signalling cascade that activates the protein kinase Akt and reduces the E–selectin, ICAM–1 and VCAM–1 expression on protein and mRNA level. This signalling cascade is consistently associated with a reduced translocation of TNF–alpha–activated NF–kappaB into the cell nucleus. The suppressor effect of SPC and LSF is completely reverted by inhibition of the phosphatidylinositol– 3–kinase/Akt pathway. We conclude that the antiatherogenic/antiinflammatory effect of lysosphingolipids depends on a competitive interaction of EDG receptor–induced inhibition and TNF–alpha–initiated stimulation of NF–kappaB translocation into the cell nucleus thereby preventing or stimulating inflammatory events in atherogenesis.

Differentiation of coronary smooth muscle cells to a cell cycle-arrested hypertrophic growth status by a synthetic non-toxic heparin-mimicking compound

Studies on the mode of action of basic fibroblast growth factor (bFGF) identified an essential role of heparan sulfate and heparin-like molecules in the formation of distinct bFGF-heparan sulfate-bFGF-receptor complexes that are required for bFGF-induced signal transduction. In coronary smooth muscle cells that express 6-8 ng bFGF mg(-1) cell protein, the heparan sulfate chains of membrane-associated proteoheparan sulfate are implicated in bFGF signaling and thus are involved in the regulation of proliferation and differentiation of vascular smooth muscle cells. We studied the mode of action of a synthetic non-sulfated heparin-mimicking compound termed RG-13,577 (poly-4-hydroxyphenoxy acetic acid, Mr approximately 5 kD) and found a dose-dependent antiproliferative effect that was characterized by a block of G(1)/S-phase transition indicated by a marked (80%) reduction of [3H]thymidine incorporation at a concentration of 5 microg ml(-1) RG-13,577. Cell cycle analysis showed a block of cell division in the G(1)-phase. In response to RG-13,577 the cells were converted into a hypertrophic growth status within 72 h as judged from a doubling of the cellular protein content and measurement of cell and nucleus size. The increased cell protein content resulted from a de novo synthesis and was also associated with an increase in the incorporation of [35S]sulfate into cell-associated proteoglycans, including the proteoheparan sulfate coreceptor of bFGF. In contrast, the compound-induced G(1)-phase arrest was associated with an extensive downregulation of the cellular and pericellular bFGF level. The reduced bFGF content was accompanied by downregulation of the bFGF signaling-involved protein kinase C-alpha and MAP kinase, abrogation of MAP kinase phosphorylation and overexpression of protein kinase C-gamma. RG-13,577 failed to elicit apoptotic reactions at a concentration range of 0.5-10 microg ml(-1) and its effect was reversible upon removal of the compound. It appears that RG-13,577 induces a phenotype transformation of coronary SMC into a metabolically active hypertrophic status that could promote repair processes after balloon angioplasty (PTCA) without stimulating cell proliferation. Development of non-toxic polyanionic compounds may provide an effective strategy to inhibit cell proliferation associated with restenosis following balloon angioplasty and coronary artery bypass surgery.

Cytochemical changes in a human arterial proteoglycan related to atherosclerosis

The cuprolinic blue (CB) staining method has been used to visualize and characterize proteoglycans (PG) in the extracellular matrix (ECM) of normal and atherosclerotic human arteries. Arterial tissues of 13 individuals (1-83 years of age) were obtained by autopsy. For electron microscopic visualization of PGs staining with CB was performed in the presence of a critical electrolyte concentration of 0.3 M MgCl2. Under these conditions CB selectively interacts with the polysulfated glycosaminoglycan (GAG) side chains of the molecules. Removal of PG side chains by GAG-degrading enzymes prior to CB staining selectively prevented the formation of chondroitin sulfate (CS)-rich and dermatan sulfate (DS)-rich PG-CB precipitates. The DS-rich type of PG is mainly associated with collagen fibrils, the CS-rich type of PG is preferentially localized in nonfibrous areas of the ECM (soluble matrix). When normal arterial tissues are compared with those affected by atherosclerosis quantitative and qualitative changes of PG-CB precipitates are detected. In fibrous plaques a strong accumulation of a large CS-rich type of precipitate close by smooth muscle cells (SMC) and foam cells is observed. In addition, these precipitates are significantly longer in fibrous plaques than in adjacent normal media (116 nm vs. 100 nm; P less than 0.001). This alteration is independent of the age of the donor. Small DS-rich PG-CB precipitates associated with collagen fibrils show strong variations in their length, but not a significant tendency towards elongated precipitates in atherosclerosis. The present results demonstrate that ultracytochemical and morphometric analysis are useful in providing information on the diverse types, locations, interactions, and possibly of molecular changes of PGs in normal and atherosclerotic human arteries.

Exogenous nitric oxide regulates activity and synthesis of vascular endothelial nitric oxide synthase

Backgroundu2003 Nitric oxide (NO) – a major signalling molecule of the vascular system – is constitutively produced in endothelial cells (EC) by the endothelial NO synthase (eNOS). Since a reduced NO synthesis is an early sign of endothelial dysfunction and NO delivering drugs are used to substitute the impaired endothelial NO production, we addressed the effect of exogenous NO on eNOS in human umbilical venous endothelial cell cultures.

Differential aggregation properties of alpha-synuclein isoforms

Pathologic aggregation of α-synuclein is a central process in the pathogenesis of Parkinsons disease. The α-synuclein gene (SNCA) encodes at least 4 different α-synuclein isoforms through alternative splicing (SNCA140, SNCA126, SNCA112, SNCA98). Differential expression of α-synuclein isoforms has been shown in Lewy body diseases. In contrast to the canonical α-synuclein isoform of 140 amino acid residues (SNCA140), which has been investigated in detail, little is known about the properties of the 3 alternative isoforms. We have investigated the aggregation properties of all 4 isoforms in cultured cells and analyzed fibril-formation of 3 isoforms (SNCA140, SNCA126, and SNCA98) inxa0vitro by electron microscopy. Each of the 3 alternative isoforms aggregates significantly less than the canonical isoform SNCA140. Electron microscopy showed that SNCA140 formed the well-known relatively straight fibrils while SNCA126 formed shorter fibrils, which were arranged in parallel fibril bundles and SNCA98 formed annular structures. Expression analysis of α-synuclein isoforms in different human brain regions demonstrated low expression levels of the alternative isoforms in comparison to the canonical SNCA140 isoform. Thesexa0findings demonstrate that α-synuclein isoforms differ qualitatively and quantitatively in their aggregation properties. The biological consequences of these findings remain to be explored inxa0vitro and inxa0vivo.

Binding and Endocytosis of Thrombospondin and Thrombospondin Fragments in Endothelial Cell Cultures Analyzed by Cuprolinic Blue Staining, Colloidal Gold Labeling, and Silver Enhancement Techniques'

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.

Sphingosine 1-phosphate (S1P) induces expression of E-selectin and adhesion of monocytes via intracellular signalling pathways in vascular endothelial cells.

Sphingosine 1-phosphate (S1P) - a constitutive component of human plasma - is implicated as a signalling molecule in the regulation of cell adhesion molecules (CAM) in vascular endothelial cells (EC), but the degree of the S1P-induced expression of CAM and the involvement of the S1P(1) receptor are still ambiguous. Here, we report that S1P, when added to vascular EC in the absence of other stimuli, induced a strictly proportional and concentration-dependent expression of E-selectin mRNA, of E-selectin protein and of the number of adhering THP-1 monocytes to EC. Experiments with exogenous [(3)H]S1P showed a multi-exponential influx kinetic of intracellular uptake of [(3)H]S1P up to a steady state level over 2h. This process could be inhibited or enhanced by various synthetic modulators targeting both, S1P(1) receptor-dependent (Akt, ERK1/2) as well as independent DMS-sensitive pathways. The S1P(1) receptor signalling was shown to drive the sphingosine kinase - the rate limiting enzyme for the formation of S1P - to a higher or lower activity. Furthermore, S1P as an intracellular messenger induced the phosphorylation and nuclear translocation of the p65 subunit of NF-kappaB and in turn the expression of E-selectin and monocyte adhesion. Taken together, these results suggest that the physiologically controlled variation in intracellular S1P concentrations may represent a novel not yet known mechanism of fine-tuning the expression of proinflammatory and atherogenic E-selectin cell adhesion molecule by vascular endothelial cells.

Induction of a hypertrophic growth status of coronary smooth muscle cells is associated with an overexpression of TGF-β

Hypertrophy of vascular smooth muscle cells occurs during hypertension-induced remodelling of arteries and during development of arteriosclerosis and restenosis following angioplasty but the pathogenesis of the hypertrophic status is not yet fully understood. In a previous study we demonstrated that the synthetic non-sulfated, non-toxic heparin-mimicking compound RG-13577 is capable of inducing a cell cycle-arrested hypertrophic phenotype of coronary smooth muscle cells. In this study we clarify the mode of action of RG-13577 and demonstrate that the RG-13577-induced hypertrophy is associated with an increased expression of TGF-beta1 as indicated by an increase in TGF-beta1-specific protein and mRNA level. Furthermore we show that RG-13577-treated hypertrophic smooth muscle cells maintain full metabolic activity as indicated by a continuous de novo synthesis of protein and proteoglycans and that the RG-13577-induced growth arrest is caused not only by a higher expression of TGF-beta, but also by a reduced response of RG-treated cells to the mitogenic activity of bFGF, PDGF and EGF. The growth inhibitory activity of RG-13577 is reduced in the presence of neutralizing antibodies against TGF-beta. TGF-beta itself has anti-proliferative activity in serum-depleted medium. The RG-13577 effect is reversible since incubation of hypertrophic cells in RG-13577-free medium restores cell volume and [3H]thymidine incorporation to the values of untreated control cells within 4 days. We conclude, that the active metabolic status of RG-13577-treated cells in association with the overexpression of TGF-beta could promote repair processes of injured arteries after angioplasty without stimulating cell proliferation.