Increased monocyte adhesion by endothelial expression of VCAM-1 missense variation in vitro.

Objective: In whole genome and single gene analyses, genetic variation at the vascular cell adhesion molecule-1 (VCAM-1) locus has been associated with inflammatory disease and stroke in sickle cell anaemia. In the current work, we investigated the functional impact of VCAM-1 missense variants and their effect on cell-cell adhesion.

Methods And Results: To determine the functional in vitro relevance of five missense VCAM-1 variants (S318F; T384A; G413A; L555V; I716L), we generated wild type and single variant VCAM-1 forms [318F, 384A, 413A, 555V, 716L] in EA.

Molecular investigation of the functional relevance of missense variants of ICAM-1.

In genome-wide studies, the intercellular adhesion molecule-1 (ICAM-1) locus has been associated with cardiovascular and inflammatory bowel diseases. To determine the functional relevance of five missense ICAM-1 variants (G241R; I316V; P352L; K469E; R478W), we generated wild-type and variant proteins [M2(241R); M3(469E); M4(352L); M5(478W); M6(316V); M7(352L/469E)] and transiently transfected CV1 cells. Reverse transcription PCR, western blot, and ELISA did not reveal any differences in mRNA and protein expression levels for any construct.

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.

SAH gene variants are associated with obesity-related hypertension in Caucasians: the PEGASE Study.

Objective: The SAH gene locus has recently been proposed to be involved in obesity-related hypertension in Japanese individuals.

Methods: To replicate independently the initial findings in another ethnic group, we scanned the entire SAH gene in 190 Caucasian chromosomes. A total of 651 patients with essential hypertension and 776 controls (PEGASE Study) were genotyped for all identified variants using allele-specific oligonucleotides, and single nucleotide polymorphism as well as haplotype analyses were carried out.

Expression of transcription factors and matrix genes in response to serum stimulus in vascular smooth muscle cells.

During atherogenesis vascular smooth muscle cells are converted from a contractile into a synthetic phenotype characterized by enhanced matrix production. The transcription factors Gax and GATA-6 are considered negative, and Oct-1 positive regulators of the synthetic phenotype. Since the phenotype transition can be induced by culturing the cells with serum, we followed the expression of Gax, GATA-6 and Oct-1, integrins and matrix genes in quiescent porcine vascular smooth muscle cells after serum application.

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.

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.

Adhesion of osteosarcoma cells to the 70-kDa core region of thrombospondin-1 is mediated by the alpha 4 beta 1 integrin.

Thrombospondin-1 (TSP-1) is an extracellular glycoprotein that is involved in a variety of physiological processes such as tumor cell adhesion, invasion, and metastasis. It has been hypothesized that TSP-1 provides an adhesive matrix for osteosarcoma cells. Here we present data showing that TSP-1 can promote cell substrate adhesion to U2OS and SAOS cells through the alpha 4 beta 1 integrin.

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.

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.

[Perimetry findings and driving performance. "How much visual field" does a motorist need?].

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.