Differential aggregation properties of alpha-synuclein isoforms.

Pathologic aggregation of α-synuclein is a central process in the pathogenesis of Parkinson's 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) in vitro 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. These findings demonstrate that α-synuclein isoforms differ qualitatively and quantitatively in their aggregation properties. The biological consequences of these findings remain to be explored in vitro and in vivo.

Extracellular matrix in deoxycholic acid decellularized aortic heart valves.

BACKGROUND: Only limited information is available regarding the influence of decellularization on the extracellular matrix in heart valves. Within the extracellular matrix proteoglycans (PG) play a central role in the structural organization and physical functioning of valves and in their capability of settling with endothelial and interstitial cells partially myofibroblasts. We have therefore estimated the effects of decellularization using deoxycholic acid on the structure of the extracellular matrix and PG´s in porcine aortic valves. MATERIAL/METHODS: Cupromeronic blue was used, alone or in combination with OsO4/thio-carbo-hydrazide/OsO4 for electron microscopic visualization. For PG and glycosaminoglycan (GAG) investigation a papain digestion was employed in combination with photometric determination using dimethylmethylene blue. RESULTS: The results indicate that deoxycholic acid affects the compartmentation of the PG-associated interstitial network not significantly. Compared to controls the PG-rich network was preserved even after deoxycholic acid treatment for 48 h. In parallel to electron microscopy immune assays (ELISA) showed smooth muscle cell -actin to be reduced to 0.96% ± 0.71 and total soluble protein to 6.68% ± 2.0 (n=3) of untreated controls. Protein loss corresponded well with the observations in electron micrographs of rupture and efflux of cell content. Further signs of lysis were irregular cell contours and loss of the basement membrane. CONCLUSIONS: Efficient cell-lysis without disintegration or loss of integrity of the interstitial PG network can be achieved by treatment of aortic valves with deoxycholic acid for 48h. This protocol might also be suitable for clinical use to optimize conditions for growth and autologous remodelling of valves.

Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection.

Staphylococcus aureus is a frequent cause for serious, chronic and therapy-refractive infections in spite of susceptibility to antibiotics in vitro. In chronic infections, altered bacterial phenotypes, such as small colony variants (SCVs), have been found. Yet, it is largely unclear whether the ability to interconvert from the wild-type to the SCV phenotype is only a rare clinical and/or just laboratory phenomenon or is essential to sustain an infection. Here, we performed different long-term in vitro and in vivo infection models with S. aureus and we show that viable bacteria can persist within host cells and/or tissues for several weeks. Persistence induced bacterial phenotypic diversity, including SCV phenotypes, accompanied by changes in virulence factor expression and auxotrophism. However, the recovered SCV phenotypes were highly dynamic and rapidly reverted to the fully virulent wild-type form when leaving the intracellular location and infecting new cells. Our findings demonstrate that bacterial phenotype switching is an integral part of the infection process that enables the bacteria to hide inside host cells, which can be a reservoir for chronic and therapy-refractive infections.

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.

The ARF-like GTPase ARFRP1 is essential for lipid droplet growth and is involved in the regulation of lipolysis.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a GTPase regulating protein trafficking between intracellular organelles. Here we show that mice lacking Arfrp1 in adipocytes (Arfrp1(ad-/-)) are lipodystrophic due to a defective lipid droplet formation in adipose cells. Ratios of mono-, di-, and triacylglycerol, as well as the fatty acid composition of triglycerides, were unaltered. Lipid droplets of brown adipocytes of Arfrp1(ad-/-) mice were considerably smaller and exhibited ultrastructural alterations, such as a disturbed interaction of small lipid-loaded particles with the larger droplets, suggesting that ARFRP1 mediates the transfer of newly formed small lipid particles to the large storage droplets. SNAP23 (synaptosomal-associated protein of 23 kDa) associated with small lipid droplets of control adipocytes but was located predominantly in the cytosol of Arfrp1(ad-/-) adipocytes, suggesting that lipid droplet growth is defective in Arfrp1(ad-/-) mice. In addition, levels of phosphorylated hormone-sensitive lipase (HSL) were elevated, and association of adipocyte triglyceride lipase (ATGL) with lipid droplets was enhanced in brown adipose tissue from Arfrp1(ad-/-) mice. Accordingly, basal lipolysis was increased after knockdown of Arfrp1 in 3T3-L1 adipocytes. The data indicate that disruption of ARFRP1 prevents the normal enlargement of lipid droplets and produces an activation of lipolysis.

Granulocyte macrophage colony-stimulating factor deficiency affects vascular elastin production and integrity of elastic lamellae.

BACKGROUND: Granulocyte macrophage colony-stimulating factor (GM-CSF) deficiency affects the production and fiber assembly/organization of the vascular collagenous matrix; structural alterations to the elastic system were observed. The present study elaborates the effect of GM-CSF deficiency on the vascular elastin system. METHODS AND RESULTS: Histological examination of the aorta of GM-CSF-deficient mice revealed structurally altered elastic fibers. The elastic fiber area was significantly enhanced, whereas the remaining medial area was not affected. Aortic size was significantly increased. Reverse transcription polymerase chain reaction demonstrated decreased expression levels of tropoelastin, lysyl oxidase and bone morphogenetic protein 1 (BMP-1). Cell culture studies on vascular smooth muscle cells showed that after clearance of GM-CSF with GM-CSF antibodies, the tropoelastin mRNA expression was markedly reduced. Concomitantly, lysyl oxidase and BMP-1 mRNA levels were decreased. Treatment with GM-CSF stimulated the expression of these mRNAs. CONCLUSIONS: Our studies demonstrate that disorganization of elastic lamellae as induced by GM-CSF deficiency is associated with adaptive vascular remodeling. The decreased tropoelastin expression observed is associated with elastic fiber hypertrophy. This paradox effect may be explained by decreased expression levels of lysyl oxidase and BMP-1, both mediating cross-linkage and thus assembly and organization of elastic fibers. From our data, we conclude that GM-CSF is a prerequisite for the maintenance of structural integrity of the vessel wall.

One-year results of the SCORPIUS study: a German multicenter investigation on the effectiveness of sirolimus-eluting stents in diabetic patients.

OBJECTIVES: This study sought to analyze the effectiveness of drug-eluting stents in a high-risk group of diabetic patients. Previously, this had been analyzed only in substudies of larger trials or in clinical investigations enrolling a small number of patients. BACKGROUND: Drug-eluting stents are highly effective in reducing the rate of in-stent restenosis. METHODS: Two hundred patients with diabetes and de novo coronary artery lesions were enrolled in 16 centers: 98 were randomly assigned to sirolimus-eluting stents (SES) and 102 received bare-metal stents (BMS). The primary end point was in-segment late luminal loss. Major adverse cardiac events (MACE) rate was analyzed at 30 days and 8 and 12 months. RESULTS: The extent of in-segment late luminal loss in the SES group was 0.18 mm compared with 0.74 mm in the BMS group. In-segment restenosis was identified on follow-up angiography in 8.8% of the patients in SES and in 42.1% in BMS (p < 0.0001). Target lesion revascularization was performed in 5.3% of the patients in SES and in 21.1% of the patients in BMS (p = 0.002). The SES was effective in the treatment group with oral diabetic medication as well as in the insulin-dependent treatment group (3.6% SES vs. 38.8% BMS). There was no subacute stent thrombosis in the SES group up to 1 year. The MACE rate was not significantly different at 30 days. At 12 months, MACE rate was 14.7% in SES versus 35.8% in BMS. CONCLUSIONS: The SES is safe and highly effective in patients with diabetes mellitus and coronary artery disease and associated with a significant decrease in the extent of late luminal loss.

Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells.

High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I), exert potentially anti-atherogenic properties within the arterial wall. However, it is unknown how they are transported from the blood stream into the vascular wall. Here we investigated the interaction of apoA-I with endothelial cells. At 4 degrees C endothelial cells bound 125I-apoA-I with high affinity, Kd = 2.1 microg/ml and in a saturable manner (Bmax of 35 ng/mg cell protein). At 37 degrees C, the cell association of apoA-I revealed similar affinity as at 4 degrees C (Kd = 2.2 microg/ml) but the maximum specific cell association was much enhanced (Bmax = 360 ng/mg cell protein). Binding and cell association was competed by excess unlabeled apoA-I and HDL but not by albumin. Biotinylation experiments and electron microscopy studies showed that endothelial cells internalize labeled apoA-I. Only minor amounts of the internalized apoA-I were degraded. Cultivated in a Transwell system, the cells transported a fraction of 125I-apoA-I from the apical to the basolateral compartment in a competable and temperature-sensitive manner. Furthermore, after specific transport the originally prebeta-mobile and lipid-free apoA-I was recovered as particles which have electrophoretic alpha-mobility. We conclude that endothelial cells transcytose and lipidate lipid-free apoA-I.

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.

Vascular collagens: spotlight on the role of type VIII collagen in atherogenesis.

Collagens play a central role in maintaining the integrity and stability of the undiseased as well as of the atherosclerotic vessel wall. An imbalanced metabolism may lead to uncontrolled collagen accumulation reducing vessel wall velocity, frequently resulting in arterial occlusion or thrombosis. A reduced production of collagen and its uncontrolled degradation may affect the stability of the vessel wall and especially of the atherosclerotic plaques by making them prone to rupture and aneurysm. This review presents an overview on the four groups of vascular collagens and on their role in atherogenesis. The major focus was to highlight the extraordinary role and importance of the short chain network forming type VIII collagen in the extracellular matrix of undiseased arteries and of atherosclerotic plaques. The molecular structure of type VIII collagen, its cellular origin, its implication in atherogenesis, its temporal and spatial expression patterns in human and experimental models of atherogenesis, the factors modulating its expression, and--not at least--its potential function is discussed.

Tissue engineering of heart valves: formation of a three-dimensional tissue using porcine heart valve cells.

Tissue engineering is a promising approach to obtaining lifetime durability of heart valves. The goal of this study was to develop a heart valve-like tissue and to compare the ultrastructure with normal valves. Myofibroblasts and endothelial cells were seeded on a type I collagen scaffold. The histologic organization and extracellular matrix were compared in light and electron micrographs. Radiolabeled proteoglycans were characterized by enzymatic degradation experiments. In tissue engineered specimens, cross sectional evaluation revealed that the scaffold (300 microm) was consistently infiltrated with myofibroblasts. Both sides were covered with a multicellular layer of myofibroblasts and overlaid by endothelial cells (50 microm). A newly formed extracellular matrix containing collagen fibrils and proteoglycans was found in the interstitial space. Collagen fibrils with a 60 nm banding pattern were found in both specimens. Small sized proteoglycans (65 nm) were associated and aligned at intervals of 60 nm with collagen fibrils. Large sized proteoglycans (180 nm) were located outside the collagen bundles in amorphous compartments of the extracellular matrix. The majority of glycosaminoglycans were chondroitin/dermatan sulfate, and a minority were heparan sulfate. The morphology and topography of cells and the organization of extracellular matrix in artificial tissues strongly resembles those of native valve tissues.

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

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.

Ultrastructure of proteoglycans in tissue-engineered cardiovascular structures.

Proteoglycans such as versican, decorin, and perlecan are important components of the extracellular matrix in various tissues. They play an important role in water homeostasis, tissue elasticity, prevention of calcification, and thrombogenicity. The aim of our study was to detect such proteoglycans in engineered tissue and compare them with the proteoglycans of native porcine heart valves. Myofibroblasts were seeded on a type I collagen scaffold. Thereafter, endothelial cells were seeded onto the presettled myofibroblasts. The newly formed tissue was histologically and immunohistochemically examined. Cupromeronic blue was used for ultracytochemical staining of proteoglycans. Radiolabeled proteoglycans were isolated by ion-exchange chromatography and characterized by enzymatic degradation. Three differently sized proteoglycan precipitates were found. The large-sized proteoglycan (154 nm) was located outside the collagen bundles in a rarely structured extracellular matrix compound. The small-sized proteoglycan (46 nm) was aligned along the collagen bundles at intervals of 60 nm. The intermediate-sized proteoglycan (56 nm) was detected on the cell surface of myofibroblasts. The glycosaminoglycans included 80% chondroitin and dermatan sulfate and 20% heparan sulfate. We conclude that proteoglycans play an important role in the functional integrity of cardiovascular tissues. This study shows the successful production of a heart valve-like tissue with proteoglycans resembling, in terms of type, production, and distribution, proteoglycans of native heart valves.