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

BACKGROUND: 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. MATERIALS AND METHODS: The synthetic NO donor DETA/NO (trade name, but in the following we refer to detNO), that releases NO in a strictly first order reaction with a half life of 20 h, was used in our experiments. RESULTS: Short-term (20-30 min) detNO treatment of EC increases the Ser(1177) phosphorylation of the constitutively expressed endothelial NOS and the production of endogenous NO generated by eNOS from [(3)H]arginine. The phosphorylation of eNOS is Akt-dependent and completely reverted by the phosphatidylinositol-3 kinase (PI-3K) inhibitor LY294002. A prolonged continuous exposure of EC to detNO 150 micromol L(-1) over a period of 24-48 h causes a reversible cell cycle arrest at G(1)-phase associated with a larger cell volume and increased cell protein content (hypertrophic phenotype of EC). The eNOS protein and mRNA of the hypertrophic cells and the generation of endogenous NO are reduced but eNOS phosphorylation could still be elevated by stimulation with vascular endothelial growth factor. CONCLUSIONS: Our data explain clinical studies describing a short-term but not a long-term benefit of NO treatment for patients with cardiovascular risk factors. The results could be a rational approach to develop a generation of NO donors accomplishing a retarded release from NO donors that mimic the low continuous pulsatile stress-induced release of endogenous NO.

The role of thrombospondin-1 in apoptosis.

The thrombospondins are a family of extracellular proteins that participate in cell-to-cell and cell-to-matrix communication. They regulate cellular phenotype during tissue genesis and repair. Five family members, each representing a separate gene product, probably exist in most vertebrate species. Like most extracellular proteins, the thrombospondins are composed of several structural domains that are responsible for the numerous biological functions that have been described for this protein family. Considerable progress has been made towards understanding the function of thrombospondins. The role of thrombospondin in the process of apoptosis or programmed cell death has recently come into focus. In this review we will concentrate on the role of thrombospondin-1 in the broad field of apoptotis research.

In vitro modelling of tissue using isolated vascular cells on a synthetic collagen matrix as a substitute for heart valves.

BACKGROUND: Tissue engineering is a promising approach for obtaining lifetime durability in biological heart valves. Basic questions with respect to the selection of suitable cell populations as well as scaffolds remain unsolved. The purpose of this study was to develop a tissue-like substitute in vitro for replacement of diseased valves in vivo. METHODS: Smooth-muscle cells (SMCs) were isolated from human and porcine aortic tissue using the 'explant technique' and endothelial cells from collagenase digestion. Seeding and cultivation of isolated cells was performed on a type-I collagen matrix. The scaffold-cell specimen was investigated using light and electron microscopy. Cupromeronic blue and immunoprecipitation were used for ultracytochemical staining. RESULTS: SMCs were allowed to grow to multilayers and migrate into the collagen network. We found a tissue-like morphology in these samples characterised by several layers of cells, spaces between the cell layers filled with newly formed extracellular matrix components, compartmentalisation of proteoglycans and their association with fibrilar matrix and the cell surface. Endothelium cells covered the SMCs of the scaffold with a histological topography similar to heart valves. CONCLUSIONS: This is an approach for in vitro modelling of tissue-like substitutes and preparing plane multicellular tissues as substitutes for heart valves. This model may also be used for cell biological investigations of cell-matrix interactions.

Role of the subchondral vascular system in endochondral ossification: endothelial cell-derived proteinases derepress late cartilage differentiation in vitro.

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. The subchondral vascular system is essential for this process and late chondrocyte differentiation is subject to negative control at several checkpoints. Endothelial cells of subchondral blood vessels not only are the source of vascular invasion accompanying the transition of hypertrophic cartilage to bone but also produce factors overruling autocrine barriers against late chondrocyte differentiation. Here, we have determined that the action of proteases secreted by endothelial cells were sufficient to derepress the production of the hypertrophy-markers collagen X and alkaline phosphatase in arrested populations of chicken chondrocytes. Signalling by thyroid hormones was also necessary but endothelial factors other than proteinases were not. Negative signalling by PTH/PTHrP- or TGF-beta-receptors remained unaffected by the endothelial proteases whereas signalling by FGF-2 did not suppress, but rather activated late chondrocyte differentiation under these conditions. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.

[Perimetry findings and driving performance. "How much visual field" does a motorist need?]. / Perimetriebefund und Fahrtauglichkeit. "Wieviel Gesichtsfeld" braucht ein Autofahrer?

OBJECTIVE: On 01-01-99 the new driving licence regulations came into force. Are the visual field criteria mentioned there sufficient for an adequate evaluation of driving fitness? METHODS: Typical (binocular) visual field defects were digitally superimposed onto photographs of traffic scenes to show the influence of such scotomata on visual perception. RESULTS: Exemplary cases clearly show that evaluation of visual field borders alone is not sufficient for evaluating driving fitness. They also prove that binocular scotomata affecting the (para-)central region of the visual field are of special importance to driving performance. The actual guidelines given by the traffic commission of the German Ophthalmological Society (DOG) and the legally defined limits will be critically discussed. CONCLUSION: From the ophthalmological point of view the actual visual field criteria of the new driving licence regulations do not seem to be sufficient for the evaluation of driving fitness: An additional consideration of (para-)central scotoma is mandatory. This publication provides recommendations for execution and interpretation of perimetric examinations for traffic ophthalmological purposes.

Integrin-associated protein and thrombospondin-1 as endothelial mechanosensitive death mediators.

Recently, it was reported that the offset of hemodynamic forces induces an unusual pattern of apoptosis in vascular endothelium (1). Although the apoptotic trigger covers all cells and is maintained for a longer time period, only few cells become apoptotic. So, in contrast to common apoptosis inducers, the lack of hemodynamic forces initiates only a low basal level of apoptosis, however steadily increases with time, this way preventing the complete vessel destruction upon an only transient offset of blood flow. The molecular means by which the mechanical stimulus and apoptosis are smoothly coupled have now been identified as an autocrine loop of thrombospondin-1 (TSP-1) and the alpha(v)beta(3) integrin/integrin-associated protein (IAP) complex as its receptor. Vascular EC (EC) secrete TSP-1 only in postconfluent static monolayers and not under flow. This also holds true for the IAP whereas the alpha(v)beta(3) integrin is present under static conditions, as well as under flow, assigning the IAP an essential and new switch function in the receptor complex.

Interaction of thrombospondin-1 and heparan sulfate from endothelial cells. Structural requirements of heparan sulfate.

Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.

Paracrine or virus-mediated induction of decorin expression by endothelial cells contributes to tube formation and prevention of apoptosis in collagen lattices.

Resting endothelial cells express the small proteoglycan biglycan, whereas sprouting endothelial cells also synthesize decorin, a related proteoglycan. Here we show that decorin is expressed in endothelial cells in human granulomatous tissue. For in vitro investigations, the human endothelium-derived cell line, EA.hy 926, was cultured for 6 or more days in the presence of 1% fetal calf serum on top of or within floating collagen lattices which were also populated by a small number of rat fibroblasts. Endothelial cells aligned in cord-like structures and developed cavities that were surrounded by human decorin. About 14% and 20% of endothelial cells became apoptotic after 6 and 12 days of co-culture, respectively. In the absence of fibroblasts, however, the extent of apoptosis was about 60% after 12 days, and cord-like structures were not formed nor could decorin production be induced. This was also the case when lattices populated by EA.hy 926 cells were maintained under one of the following conditions: 1) 10% fetal calf serum; 2) fibroblast-conditioned media; 3) exogenous decorin; or 4) treatment with individual growth factors known to be involved in angiogenesis. The mechanism(s) by which fibroblasts induce an angiogenic phenotype in EA.hy 926 cells is (are) not known, but a causal relationship between decorin expression and endothelial cell phenotype was suggested by transducing human decorin cDNA into EA.hy 926 cells using a replication-deficient adenovirus. When the transduced cells were cultured in collagen lattices, there was no requirement of fibroblasts for the formation of capillary-like structures and apoptosis was reduced. Thus, decorin expression seems to be of special importance for the survival of EA.hy 926 cells as well as for cord and tube formation in this angiogenesis model.

Role of the subchondral vascular system in endochondral ossification: endothelial cells specifically derepress late differentiation in resting chondrocytes in vitro.

Endochondral ossification in growth plates proceeds through several consecutive steps of late cartilage differentiation leading to chondrocyte hypertrophy, vascular invasion, and, eventually, to replacement of the tissue by bone. It is well established that the subchondral vascular system is pivotal in the regulation of this process. Cells of subchondral blood vessels act as a source of vascular invasion and, in addition, release factors influencing growth and differentiation of chondrocytes in the avascular growth plate. To elucidate the paracrine contribution of endothelial cells we studied the hypertrophic development of resting chondrocytes from the caudal third of chick embryo sterna in co-culture with endothelial cells. The design of the experiments prevented cell-to-cell contact but allowed paracrine communication between endothelial cells and chondrocytes. Under these conditions, chondrocytes rapidly became hypertrophied in vitro and expressed the stage-specific markers collagen X and alkaline phosphatase. This development also required signaling by thyroid hormone in synergy. Conditioned media could replace the endothelial cells, indicating that diffusible factors mediated this process. By contrast, smooth muscle cells, fibroblasts, or hypertrophic chondrocytes did not secrete this activity, suggesting that the factors were specific for endothelial cells. We conclude that endochondral ossification is under the control of a mutual communication between chondrocytes and endothelial cells. A finely tuned balance between chondrocyte-derived signals repressing cartilage maturation and endothelial signals promoting late differentiation of chondrocytes is essential for normal endochondral ossification during development, growth, and repair of bone. A dysregulation of this balance in permanent joint cartilage also may be responsible for the initiation of pathological cartilage degeneration in joint diseases.

Perlecan is responsible for thrombospondin 1 binding on the cell surface of cultured porcine endothelial cells.

Thrombospondin 1 (TSP1), a high molecular weight glycoprotein of the extracellular matrix, interacts with glycosaminoglycan at the cell surface of porcine endothelial cells (Schön et al., Eur.J. Cell Biol. 59, 329-339 (1992)). In this study we identified and characterized the heparan sulfate proteoglycan (HSPG) responsible for TSP1 binding and uptake in endothelial cells and investigated some properties of the TSP1-proteoglycan interaction. Porcine endothelial cells synthesize proteoglycans containing heparan sulfate (HS) or chondroitin/dermatan sulfate (CS/DS). CS/DS-containing compounds are present predominantly in the culture medium. On Sepharose CL-4B the cellular proteoglycan fraction yielded two HS-containing compounds with a Kav = 0.18 and Kav = 0.55. Only the larger HS-containing component was sensitive to alkaline treatment and was also found in the medium fraction. Trypsin treatment of endothelial cells revealed that the large HS-containing component represents a cell surface-associated proteoglycan, whereas the smaller fraction represents a pool of intracellular HS-chains. The cellular HSPG is partially localized at the apical cell surface but also incorporated and tightly bound to the subendothelial matrix. Deglycosylation of the high molecular weight HSPG resulted in the identification of a core protein of about 400 kDa. Using specific antibodies, in ELISA assays and in immunoblot analysis we observed that the large HSPG is identical to the extracellular matrix proteoglycan, perlecan. Immunohistochemical studies confirmed the location of perlecan on the apical cell surface and additionally as a dense fibrillar network surrounding the cells. Purified perlecan bound to TSP1 in a dose-dependent manner and the binding was mediated by its glycosaminoglycan side chains. In competition assays using various sulfated polysaccharides, heparin potently inhibited binding of perlecan to TSP1 immobilized on nitrocellulose. Dermatan sulfate was a less effective inhibitor. Calcium bound to TSP1 was found to influence its capacity for binding perlecan. The present data provide evidence that perlecan is required for binding and concentrating TSP1 at the apical surface of vascular endothelial cells during receptor-mediated endocytosis.

Binding and processing of multimeric vitronectin by vascular endothelial cells.

The multifunctional adhesive glycoprotein vitronectin (VN) undergoes a unique conformational transition from the plasma form into a multimeric form that represents the reactive heparin-binding form. In this study we investigated the interaction of multimeric vitronectin (VNmult) or VN-gold conjugates (which are equivalent in biochemical properties) with confluent and subconfluent monolayers of porcine endothelial cells. Time-dependent direct binding of radiolabeled VNmult to the luminal face of endothelial cells at 37 degrees C was observed which was competed by heparin, whereas plasma VN showed hardly any binding. At 4 degrees C binding of VNmult remained cell-associated, whereas after 6 hr at 37 degrees C a major portion of the ligand was translocated through cells and was associated with the subcellular matrix. Cytochemical studies with VN-gold conjugates were performed to demonstrate uptake of VNmult. At 4 degrees C only surface decoration of cells with gold label was seen, which was totally reversible in the presence of heparin. Subsequent incubation for various time intervals at 37 degrees C revealed disappearance of gold label from the surface and accumulation of conjugates in a perinuclear distribution inside the cells as judged both by electron microscopy and after silver enhancement by light microscopy. Cross-sections of endothelial cells demonstrated the inclusion of VN-gold conjugates in coated pits, endosomes, and in lysosomal compartments close to the nucleus. Within 2-6 hr a portion of VN-gold conjugates had accumulated with proteoglycans at the matrix face. These data provide strong evidence for specific routing of a portion of VNmult from the circulation into extravascular spaces, where the protein is believed to fulfill major adhesive and regulatory functions particularly as co-factor in plasminogen activation and immune defense.

Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells.

Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short-term culture. To determine the terminal carbohydrate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein-labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N-galactosamine specific lectin Wistaria floribunda agglutinin labelled only endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.

Norepinephrine and neuropeptide Y increase intracellular Ca2+ in cultured porcine aortic smooth muscle cells.

We used the fluorescent Ca2+ indicator Fura-2 in cultured porcine aortic smooth muscle cells (PASMC) to study effects of the sympathetic neurotransmitters norepinephrine (NE) and neuropeptide Y (NPY) on free intracellular Ca2+ (Cai). Both transmitters transiently increased intracellular Ca2+ in a concentration-dependent manner. Selective agonists and antagonists demonstrated that the NE-stimulated Cai increase is predominantly (if not exclusively) mediated by alpha 2-adrenoceptors, whereas the NPY response appears to be mediated by the peptide YY-insensitive Y3-like receptor subtype. Pretreatment of cells with pertussis toxin abolished NPY and alpha-adrenoceptor agonist-stimulated intracellular Ca2+ elevations (but not those stimulated by angiotensin II) suggesting involvement of a Gi-like G-protein. alpha 2-Adrenoceptor-stimulated Ca2+ increases resulted from mobilization from intracellular stores, whereas Y3-like NPY receptors mobilized Ca2+ from intracellular stores and also promoted Ca2+ influx.

Cell-associated proteoheparan sulfate mediates binding and uptake of thrombospondin in cultured porcine vascular endothelial cells.

[125I]Thrombospondin (TSP) binds to porcine endothelial cells in a specific, saturable and time-dependent fashion and is endocytosed by a receptor-mediated process. The N-terminal heparin-binding domain is necessary for the interaction with the cell surface. Binding and uptake is inhibited by heparin and to a much smaller extent by other vascular glycosaminoglycans. Chemical modification of lysine and arginine residues of TSP, but not treatment of the molecule with neuraminidase, resulted in a pronounced loss of binding at the cell surface. Treatment of cells with heparitinase but not with chondroitin ABC lyase caused inhibition of binding and uptake of TSP. Inhibition of sulfation of proteoglycans on the cell surface by chlorate leads to a dose and time-dependent inhibition of binding and degradation of TSP. In the presence of chlorate, newly synthesized TSP is not incorporated into the cell matrix but mainly released into the culture medium, whereas localization and incorporation of newly synthesized fibronectin is not altered. A cell surface proteoheparan sulfate was identified as TSP binding macromolecule by affinity chromatography. The data emphasize the role of heparan sulfate proteoglycan as a receptor-like molecule for the specific interaction with thrombospondin.

Interactions between thrombospondin and the small proteoglycan decorin: interference with cell attachment.

Decorin, a ubiquitous small interstitial dermatan sulfate proteoglycan, interacts with several extracellular matrix components, e.g., with type I collagen and fibronectin. Using a solid phase assay it is shown that the intact proteoglycan as well as its glycosaminoglycan-free core protein exhibits with KD values of about 5 nM and 2 nM, respectively, high affinity binding also to thrombospondin. However, the polysaccharide chain was required for an interaction with Sepharose-bound thrombospondin and served itself as ligand. In light of the results of binding studies with an N-terminal heparin-binding fragment of thrombospondin it is concluded that several structural features of thrombospondin and of decorin contribute to the mutual interaction of the two macromolecules. Thrombospondin substrata allowed attachment but prevented spreading of human skin fibroblasts. The addition of decorin or of its glycosaminoglycan-free core protein led to a considerable delay of cell attachment on a thrombospondin substrate. The strength of cell attachment appeared to be reduced. These data support the antiadhesive role of decorin regardless of whether subsequent cell spreading is supported or not.

Different action of heparin and fucoidan on arterial smooth muscle cell proliferation and thrombospondin and fibronectin metabolism.

Fucoidan, a sulfated fucopolysaccharide of marine algae is able to inhibit the proliferation of arterial smooth muscle cells half maximally at a concentration of 80 to 100 micrograms/ml culture medium. In comparable concentrations heparin was significantly less active than the fucopolysaccharide. Sulfation of fucoidan was found to be essential for expression of antiproliferative activity. The inhibitory effect of fucoidan is a time-dependent event with highest effectiveness during the first 6 h. Fucoidan does not influence the overall rate of synthesis of cell proteins and glycoconjugates, but led to substantial alterations in the synthesis and secretion of fibronectin and thrombospondin. Immunoprecipitation and quantitation revealed that the incorporation of [35S]methionine into fibronectin is reduced whereas thrombospondin synthesis was increased. The effect on fibronectin was not shared by heparin. Desulfation of the fucopolysaccharide abolished the observed modulation. Binding experiments with [125I]fucoidan indicate a saturable binding and a maximum of 2.8 x 10(6) bound molecules per cell. Fucoidan binding sites can be only partly displaced by heparin. The results suggest that both heparin and the structurally unrelated sulfated fucopolysaccharide act as an antiproliferative agent but differ in their modulation of cell metabolism.

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.