Endothelium-dependent relaxation of rat aorta and main pulmonary artery by the phytoestrogens genistein and daidzein.

OBJECTIVE: The dietary phytoestrogens genistein and daidzein have been shown to relax agonist-preconstricted arteries in vitro; the mechanisms of relaxation remain incompletely understood. This study aimed to determine whether the relaxation of phenylephrine (PE)-constricted rat aorta and main pulmonary artery by genistein and daidzein was endothelium-dependent. METHODS: Effects of endothelial-denudation, and pretreatment with with 100 microM L-N(G)-nitroarginine methyl ester (L-NAME) and/or 10 microM indomethacin on relaxation of PE (1 microM)-preconstricted contractures by genistein (1-100 microM) and daidzein (3-100 microM) were assessed by measuring isometric force development by rat arterial rings. The effect of L-NAME on relaxation to 17beta-estradiol (10 microM) was also measured in aorta. RESULTS AND CONCLUSIONS: Genistein and daidzein caused concentration-dependent relaxation of aorta rings preconstricted with PE (1 microM). The IC50 values were 5.7 microM (n=8, 95% confidence limits 4.3-7.7 microM) and 36.7 microM (n=12, 95% confidence limits 25.7-44.1 microM), respectively. Removal of the endothelium and pretreatment with L-NAME (100 microM) significantly inhibited relaxation at 3, 10 and 30 microM genistein and 10 and 30 microM daidzein. The contracture evoked in rat aorta by depolarization with 75 mM K+ solution was similarly relaxed by genistein in a partially endothelium-dependent manner. 17Beta-estradiol (10 microM) caused a 48.7+/-5.0% (n=11) relaxation of the PE contracture, which was significantly reduced to 25.1+/-5.3% (n=7) by L-NAME. Relaxations brought about by 17beta-estradiol, genistein, and daidzein were not significantly affected by the genomic estrogen receptor antagonist ICI 182,780 (10 microM). Similar endothelium-dependent effects of genistein were observed in the main pulmonary artery. The results show that the relaxation of these rat arteries by concentrations of genistein and daidzein which overlap those present in human plasma after ingestion of soybean-containing meals is largely endothelium dependent.

Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells.

In addition to their role as regulators of leukocyte migration and activation, chemokines and their receptors also function in angiogenesis, growth regulation, and HIV-1 pathogenesis--effects that involve the action of chemokines on nonhematopoietic cells. To determine whether chemokine receptors are expressed in human colonic epithelium, HT-29 cells were examined by RT-PCR for the expression of the chemokine receptors for lymphotactin, fractalkine, CCR1-10, and CXCR1-5. The only receptor consistently detected was CXCR4 (fusin/LESTR), although HT-29 cells did not express mRNA for its ligand, stromal cell-derived factor (SDF-1alpha). Flow cytometric analysis with anti-CXCR4 antibody indicated that the CXCR4 protein was expressed on the surface of roughly half of HT-29 cells. CXCR4 was also expressed in colonic epithelial cells in vivo as shown by immunohistochemistry on biopsies from normal and inflamed human colonic mucosa. The mRNA for SDF-1alpha and other CC and CXC chemokines was present in normal colonic biopsies. The CXCR4 receptor in HT-29 cells was functionally coupled, as demonstrated by the elevation in [Ca2+]i, which occurred in response to 25 nM SDF-1alpha and by the SDF-1alpha-induced upregulation of ICAM-1 mRNA. Sodium butyrate downregulated CXCR4 expression and induced differentiation of HT-29 cells, suggesting a role for CXCR4 in maintenance and renewal of the colonic epithelium. This receptor, which also serves as a coreceptor for HIV, may mediate viral infection of colonic epithelial cells.

Tumour necrosis factor alpha stimulated rheumatoid synovial microvascular endothelial cells exhibit increased shear rate dependent leucocyte adhesion in vitro.

OBJECTIVE: To investigate endothelial cell adhesion molecule expression and leucocyte adhesion to endothelial cells isolated from the microvasculature of rheumatoid arthritic synovial tissue (SMEC) in comparison with similar cells isolated from healthy subcutaneous adipose tissue (ADMEC) or from umbilical veins (HUVEC). METHODS: Cultured endothelial cells were treated with tumour necrosis factor alpha (TNFalpha) for 2-24 hours before the assessment of cell surface E-selectin, vascular (VCAM-1) or intercellular cell adhesion molecule-I (ICAM-1) expression. Neutrophil and T lymphocyte adhesion to TNFalpha treated endothelial cells was assessed using static and shear dependent assay systems. RESULTS: VCAM-1 expression by SMEC was significantly less sensitive to TNFalpha stimulation than HUVEC or ADMEC. E-selectin expression by SMEC appeared to be more sensitive to TNFalpha stimulation and maximal expression was about 30% greater in comparison with HUVEC or ADMEC. Sensitivity to TNFalpha induction and maximal ICAM-1 expression was similar in all three endothelial cell types. Static neutrophil adhesion to TNFalpha stimulated SMEC was significantly increased in comparison with HUVEC, however this phenomenon was dependent on the presence of neutralising antibodies to ICAM-1. At shear rates in excess of 2.4 dynes/cm(2) significantly more neutrophils and, predominantly CD45RO+, T lymphocytes adhered to TNFalpha stimulated SMEC than HUVEC. CONCLUSION: Rheumatoid synovial endothelial cells differentially regulate E-selectin and VCAM-1. The increased ability of TNFalpha stimulated synovial endothelial cells to support leucocyte adhesion may help to explain the leucocyte, in particular CD45RO+ T-lymphocyte, recruitment observed in the rheumatoid synovium.

Modulation of arachidonic acid release and membrane fluidity by albumin in vascular smooth muscle and endothelial cells.

Albumin is the major plasma protein circulating in blood. Albumin potently decreases capillary permeability, although the mechanisms are not understood completely. Albumin also effectively binds arachidonic acid (AA), which increases capillary permeability. To investigate the interactions of BSA and AA with the cell membrane, the effect of these substances on [3H]AA release and membrane fluidity was studied in vascular myocytes and endothelial cells. BSA (0.2 and 1 mg . mL-1) stimulated a significant release of [3H]AA from both intact rat aorta and cultured smooth muscle cells. This effect was not mimicked by gamma-globulin or myoglobin (both 1 mg . mL-1) in intact tissue. BSA, but not gamma-globulin and myoglobin, decreased the membrane fluidity (assessed as changes in the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3, 5-hexatriene) in a concentration-dependent manner with a half-maximum concentration between 0.007 and 0.4 mg . mL-1 in both freshly isolated and cultured rat aortic myocytes and human umbilical vein endothelial cells. AA (1 to 200 micromol/L) caused the opposite effect, increasing membrane fluidity and antagonizing the effect of BSA. BSA modified at its arginine residues, which are thought to be important in AA binding, did not stimulate [3H]AA release and was significantly less potent than native BSA in altering the membrane fluidity. The effect of BSA can be explained by a high-affinity binding of AA to the protein and extraction of AA from the cell membrane. The interaction between BSA and AA could play a role in the regulation of vascular permeability.

Modified low density lipoprotein and cytokines mediate monocyte adhesion to smooth muscle cells.

Monocyte adhesion to the arterial wall is a key event in the atherosclerotic process. We studied the interactions between human coronary arterial intimal smooth muscle cells (SMCs) and monocytes by examining (i) whether SMCs mediate monocyte adhesion when stimulated by oxidatively modified low density lipoprotein (LDL) or by the cytokines TNF alpha and IL-1, and (ii) the role of the adhesion molecules VCAM-1 and ICAM-1 (vascular cell and intercellular adhesion molecule, respectively) in this process. Preincubation of SMCs with both TNF alpha and IL-1 caused a significant 2-fold increase in VCAM-1 and ICAM-1 expression and a more than 9-fold increase in monocyte adhesion. The latter was significantly inhibited (by 1/3) by neutralising antibodies to VCAM-1 and ICAM-1. Modified LDL also induced a significant 3-fold increase in monocyte adhesion to SMCs, but did not induce VCAM-1 or ICAM-1 expression, nor was this adhesion inhibited by neutralising antibodies to VCAM-1 or ICAM-1. Oxidatively modified LDL, like the proinflammatory cytokines TNF alpha and IL-1, has the ability to enhance monocyte adhesion to human SMCs in vitro. LDL-induced monocyte adhesion to SMCs is distinct from that induced by TNF alpha and IL-1 in its lack of dependence on the classical adhesion pathways involving smooth muscle VCAM-1 and ICAM-1. SMCs are identified as a new cell population which may play an active role in recruiting monocytes to the arterial intima and atherosclerotic plaque.

Extent of oxidative modification of low density lipoprotein determines the degree of cytotoxicity to human coronary artery cells.

OBJECTIVE: To assess whether the extent of LDL oxidation influences its cytotoxic effects, thus contributing to its atherogenic potential. DESIGN AND SETTING: The effects of native and modified LDL on cultured human coronary artery smooth muscle cells (SMC) and endothelial cells (ECs) were investigated. MAIN OUTCOME MEASURES: Four indices of cytotoxicity were studied: (i) chromium-51 release; (ii) 5-bromo-2'-deoxyuridine (BrDUrd) uptake; (iii) morphological appearance; and (iv) EC migration. RESULTS: (i) Minimally modified (mm) LDL (400 micrograms/ml) causes significant 51Cr release; the cytotoxic effect was significantly greater for copper oxidised (ox) LDL (400 micrograms/ml). Native LDL had no effect. (ii) BrDUrd uptake studies showed significant inhibition of cell proliferation by 100 micrograms/ml of oxLDL and to a lesser extent by mmLDL; native LDL had no effect. (iii) Morphological appearance was not altered by native LDL. Changes in cell morphology were induced by mmLDL (400 micrograms/ml), and were more pronounced with oxLDL in concentrations of > or = 200 micrograms/ml. (iv) EC migration was significantly inhibited by oxLDL (100 micrograms/ml), but not by native or mmLDL. CONCLUSION: The extent of oxidation of LDL determined its cytotoxicity to coronary artery cells. Native LDL had no cytotoxic effect. In contrast, oxLDL and to a lesser extent mmLDL caused cytotoxicity at concentrations to which cells in vivo might be exposed. This may contribute to the atherogenicity of modified LDL by enhancing cellular injury and inflammation, and by inhibiting re-endothelialisation of areas of coronary artery damaged during the atherogenic process.

Isolation and culture of synovial microvascular endothelial cells. Characterization and assessment of adhesion molecule expression.

OBJECTIVE: In vitro studies investigating the role of the synovial endothelium in the pathogenesis of rheumatoid arthritis (RA) have, until recently, been performed using cultured endothelial cells of nonsynovial macrovascular origin. In an attempt to more correctly model in vivo conditions, a method for the isolation and culture of synovial microvascular endothelial cells (SMEC) has been developed. METHODS: SMEC were isolated, primarily, by the use of lectin-coated (Ulex europaeus agglutinin type I), magnetizable polystyrene beads. RESULTS: Isolated cells exhibit classic endothelial "cobblestone" morphology, express von Willebrand factor, metabolize acetylated low-density lipoprotein, and exhibit a cytokine (interleukin-1)-mediated expression of endothelial leukocyte adhesion molecule type 1 (ELAM-1) and intercellular adhesion molecule type 1 (ICAM-1). ELAM-1 levels were significantly elevated in SMEC, compared with human umbilical vein endothelial cells, over a range of interleukin-1 concentrations. CONCLUSION: This increased expression of ELAM-1 by SMEC may be a potentiating step in the pathogenesis of RA.

Application of methionine as a detector molecule for the assessment of oxygen radical generation by human neutrophils and endothelial cells.

Diverse cell types can generate reactive oxygen species (ROS) which are implicated in many disease processes and are ascribed both beneficial and deleterious roles. In vitro studies of this phenomenon indicate that properties of the microenvironment in culture influence the cells' behaviour with regard to ROS generation in vivo. To date, however, the assessment of cellular ROS generation has been limited to techniques which are invasive of the culture environment, or require cells to be in suspension. This study describes the application of NMR spectroscopy to the detection of ROS generation, a technique which is non-invasive of the cell culturing environment.