Binding characteristics of S fimbriated Escherichia coli to isolated brain microvascular endothelial cells

To assess the role of S fimbriae in the pathogenesis of Escherichia coli meningitis, transformants of E. coli strains with or without S fimbriae plasmid were compared for their binding to microvessel endothelial cells isolated from bovine brain cortices (BMEC). The BMEC's displayed a cobblestone appearance, were positive for factor VIII, carbonic anhydrase IV, took up fluorescent-labeled acetylated low density lipoprotein, and exhibited gamma glutamyl transpeptidase activity. Binding of S fimbriated E. coli to BMEC was approximately threefold greater than nonfimbriated E. coli Similarly S fimbriated E. coli bound to human brain endothelial cells approximately threefold greater than nonfimbriated E. coli. Binding was reduced approximately 60% by isolated S fimbriae and about 80% by anti-S adhesin antibody. Mutating the S adhesin gene resulted in a complete loss of the binding, whereas mutagenesis of the major S fimbriae subunit gene sfaA did not significantly affect binding. Pretreatment of BMEC with neuraminidase or prior incubation of S fimbriated E. coli with NeuAc alpha 2,3-sialyl lactose completely abolished binding. These findings indicate that S fimbriated E. coli bind to NeuAc alpha 2,3-galactose containing glycoproteins on brain endothelial cells via a lectin-like activity of SfaS adhesin. This might be an important early step in the penetration of bacteria across the blood-brain barrier in the development of E. coli meningitis.     

Glycosyltransferase activities in cultured endothelial cells of bovine brain microvascular origin

We report two cases of salmonella osteomyelitis isolated to the pelvis in white adolescents aged 12 and 16 years. No underlying medical condition predisposed these children to salmonella osteomyelitis, and the clinical course was prolonged before definitive diagnosis. The key to diagnosis and the localization of the site of the pathologic condition was made from radionuclide studies performed 2-3 weeks from the onset of symptoms. Clinicians should be aware of isolated salmonella osteomyelitis of the pelvis in normal children, especially when imaging studies are normal at initial presentation. Technetium-labeled bone scans may be normal < or = 2 weeks from the onset of symptoms. Definitive diagnostic testing should include a gallium scan and computed tomography scan when technetium bone scans are negative. Treatment with antibiotics alone is successful.     

Role of microvascular endothelial cells in inflammation

Endothelial cells are critical elements in the evolution of all types of cutaneous inflammation. They participate through the synthesis and secretion of pro-inflammatory cytokines, including interleukin 1 (IL-1), IL-6, and IL-8, as well as M-CSF, G-CSF, GM-CSF, gro alpha, and MCP. They also express a series of cell-surface proteins and glycoproteins known as cell adhesion molecules that allow circulating leukocytes to bind to endothelial cells and allow endothelial cells to bind to matrix proteins. The regulated expression of these molecules, including those in the integrin, immunoglobulin gene, and selection families, allows for the precise trafficking of circulating leukocytes to sites of inflammation, injury, or immunologic stimulation in the skin. Furthermore, emerging evidence clearly indicates that selected differences exist between endothelial cells of the microvasculature and those that line large blood vessels. These include differences in secreted products, differences in the expression of cell adhesion molecules, and differences in cytokine-induced regulation of commonly expressed cell adhesion molecules, among others. Thus, a precise delineation of the biology of cutaneous microvascular endothelial cells is important to our understanding of cutaneous inflammation.     

Role of adenosine in the hypoxic induction of vascular endothelial growth factor in porcine brain derived microvascular endothelial cells

The distribution of neuropeptide Y (NPY)-immunoreactive (IR) nerves, as well as the functional effects of NPY and the Y1- and Y2-receptor agonists, [Leu31,Pro34]NPY and NPY(13-36), respectively, have been investigated in vitro in both visceral and arterial smooth muscle of the horse intravesical ureter. NPY-IR nerve fibres were widely distributed along the entire length of the ureter, although the intravesical part was the most richly innervated region, and the only one where NPY-IR ganglion cells were found. NPY (10(-7) M) did not affect either basal tone or spontaneous rhythmic contractions of the isolated intravesical ureter, but significantly enhanced the increases in both tone and frequency of phasic activity elicited by noradrenaline (10(-6) and 10(-5) M). The Y1-receptor agonist, [Leu31,Pro34]NPY (10(-7) and 10(-6) M) did not significantly alter either ureteral basal tone or the contractile activity induced by noradrenaline, whereas the Y2-receptor agonist, NPY(13-36) (10(-7) M), mimicked the potentiating effect of NPY on noradrenaline responses. In ureteral resistance arteries (effective lumen diameters of 130-300 microm), NPY (10(-10) to 10(-7) M) elicited concentration-dependent contractions, which were inversely correlated with the arterial lumen diameter. Submaximal concentrations of NPY (10(-8) M) significantly increased the sensitivity of ureteral arteries to noradrenaline. [Leu31,Pro34]NPY (10(-10) to 10(-7) M), but not NPY(13-36), induced a contractile effect of similar magnitude and potency as those of NPY, and also potentiated noradrenaline responses. The present results demonstrate a rich NPY-innervation in the intravesical ureter and reveal functional effects of the peptide enhancing motor activity in both ureteral and arterial smooth muscles, although the receptors mediating such effects seem to be different. Thus, NPY potentiates the phasic contractions and tone elicited by noradrenaline through Y2-receptors, whereas it both contracts and potentiates noradrenaline vasoconstriction in ureteral arteries via Y1-receptors.     

Uptake of suramin by human microvascular endothelial cells

BACKGROUND: There are many causes of ulcer of the lower limb. In the elderly, venous ulcers and arteriosclerosis often coexist; for this reason pressure bandages might be contraindicated for the risk of precipitating a potentially critical arterial flow. In this work, the conditions which allow a safely treatment with pressure bandage in the elderly are evaluated. MATERIAL AND METHOD: Eleven self-sufficient elderly, with venous ulcerations to one leg only, and ankle pressure/omeral pressure between 0.92 and 0.86 were treated with elastic bandaging of the leg. RESULTS: All patients completed the treatment, with healing of the ulcer obtained in 3-8 months time. So far none of them relapsed. CONCLUSIONS: In the elderly, in selected cases, when Pc/Po > 0.86, pressure bandages can be safely applied to heal the ulcer, without running the risk of endangering arterial circulation.     

Differential reactivity of brain microvascular endothelial cells to TNF reflects the genetic susceptibility to cerebral malaria

Upon infection with Plasmodium berghei ANKA (PbA), various inbred strains of mice exhibit different susceptibility to the development of cerebral malaria (CM). Tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN-gamma) have been shown to be crucial mediators in the pathogenesis of this neurovascular complication. Brain microvascular endothelial cells (MVEC) represent an important target of both cytokines. In the present study, we show that brain MVEC purified from CM-susceptible (CM-S) CBA/J mice and CM-resistant (CM-R) BALB/c mice exhibit a different sensitivity to TNF. CBA/J brain MVEC displayed a higher capacity to produce IL-6 and to up-regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in response to TNF than BALB/c brain MVEC. In contrast, no difference was found in the induction of E-selectin after TNF challenge. CM-S brain MVEC were also significantly more sensitive to TNF-induced lysis. This differential reactivity to TNF was further substantiated by comparing TNF receptor expression on CM-S and CM-R brain MVEC. Although the constitutive expression of TNF receptors was comparable on cells from the two origins, TNF induced an up-regulation of both p55 and p75 TNF receptors in CM-S, but not in CM-R brain MVEC. A similar regulation was found at the level of TNF receptor mRNA, but not for receptor shedding. Although a protein kinase C inhibitor blocked the response to TNF in both the brain MVEC, an inhibitor of protein kinase A selectively abolished the response to TNF in CM-R, but not CM-S brain MVEC, suggesting a differential protein kinase involvement in TNF-induced activation of CM-S and CM-R brain MVEC. These results indicate that brain MVEC purified from CM-S and CM-R mice exhibit distinctive sensitivity to TNF This difference may be partly due to a differential regulation of TNF receptors and via distinct protein kinase pathways.     

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.     

Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases

Matrix metalloproteinase (MMP) secretion by microvascular endothelial cells is an essential first step in the formation of new blood vessels (angiogenesis). Since angiogenesis does not occur in large blood vessels, we investigated whether the secretion of MMPs and tissue inhibitor of MMP (TIMP1) differs between micro- and macro-vascular endothelial cells. We compared the secretion of MMPs and TIMP1 by human endothelial cells derived from neonatal foreskin (FSE) and umbilical vein (HUVE) sources. The cells were incubated for 24 hr in the presence or absence of the angiogenic agents, phorbol myristate acetate (PMA, 100 ng/ml) or tumour necrosis factor-alpha (TNF, 100 ng/ml). The cell supernatants were removed and assayed for MMPs and TIMP1 using a spectrophotometric assay for MMP1, zymography, Western blotting and Northern analysis. When endothelial cells were incubated in basal medium for 24 hr they secreted MMP1, MMP2 and TIMP1 but not MMP9. HUVE secreted substantially higher levels of these proteins compared to FSE. In addition, HUVE secreted two low molecular mass bands representing activated forms of MMP2. These activated forms were not present in supernatants derived from FSE. In response to PMA, both FSE and HUVE increased secretion of MMP1 and TIMP1. However, there was a dramatic difference in level of response by the two cell types with FSE secreting substantially more TIMP1 and MMP9 compared to HUVE. These data clearly show that cultured endothelial cells derived from microvascular vs macrovascular tissues exhibit different MMP and TIMP secretory profiles.     

Polyunsaturated fatty acid metabolism in retinal and cerebral microvascular endothelial cells

Docosahexaenoic acid (22:6n-3), an n-3 essential fatty acid derived from elongation and desaturation of linolenic acid (18:3n-3), is found in abundant proportion in the brain and the retina. It is generally assumed that the liver is the major source of 22:6n-3 for these organs, although some retinal and cerebral cells, such as retinal pigment epithelium (Wang and Anderson, 1993. Biochemistry. 32:13703-13709) and brain astrocytes (Moore et al. 1991. J. Neurochem. 56:518-524) have the ability to produce 22:6n-3. The aim of the present study was to determine whether retinal and cerebral microvascular endothelium could synthesize 22:6n-3. After incubation of both cultured bovine retinal and rat cerebral endothelial cells with [3-14C] 22:5n-3 in presence of serum, radioactivity was primarily recovered in 20:5n-3, indicating active retroconversion reactions in both tissues. However, 22:6n-3, 24:5n-3, and 24:6n-3 were also labeled. All of these metabolites were released in the medium as free fatty acids. Retinal endothelial cells preferentially released labeled 24-carbon metabolites, whereas cerebral endothelial cells released relatively more 20:5n-3 and 22:6n-3. With heat-inactivated serum or no serum, both endothelial cell preparations showed relatively higher retroconversion levels. However, in serum-deprived cells, the elongation/desaturation pattern was affected in retinal cells only, with an accumulation of 24:5n-3 relative to a decrease of 24:6n-3 and 22:6n-3. Fatty acid composition analyses revealed a decrease in long-chain polyunsaturated n-6 and n-3 fatty acids in retinal cells maintained in inactivated serum compared to normal serum, while no change was found in cerebral cells. Taken together, these results suggest that 1) the synthesis of 22:6n-3 by both retinal and cerebral endothelial cells is independent of a delta4-desaturase; 2) retinal and cerebral endothelia could be a source of 22:6n-3 for the retina and the brain, respectively; and 3) retinal endothelial delta6-desaturase, which converts 24:5n-3 to 24:6n-3, could be stimulated by serum components.     

Sera from patients with falciparum malaria induce substance P gene expression in cultured human brain microvascular endothelial cells

Substance P is a pluripotent neuropeptide capable of inducing neurogenic inflammation, immunoregulation, and vasodilatation. In an effort to contribute to the understanding of the pathophysiology of cerebral malaria, we have evaluated the effects of sera obtained from patients suffering from severe or mild malaria and from a healthy donor with no previous history of exposure to malaria on the expression of the substance P gene by cultured human brain microvascular endothelial cells (HBMEC) and human umbilical-vein endothelial cells. PCR, Southern blotting, hybridization with an internal probe, and densitometry demonstrated that treatment of HBMEC with sera from patients with severe malaria caused remarkably increased expression of the substance P gene. In HBMEC, substance P was not significantly influenced by serum from a healthy donor. Substance P was expressed at almost undetectable levels in untreated HBMEC. Treatment of cultured human umbilical-vein endothelial cells with the same sera produced no signal. The influence of different sera on the expression of substance P by HBMEC suggests that substance P expression may be involved in events leading to the development of severe malaria.     

Differential regulation of eosinophil adhesion and transmigration by pulmonary microvascular endothelial cells

In bronchial asthma, eosinophils (EOS) adhere to, and migrate across, the lung microvasculature to exert their effector functions in the airways. This study was conducted to determine the effect of cytokines on adhesion molecule expression on human pulmonary microvascular endothelial cells (HPMEC) and the influence of these molecules on EOS adhesion and transmigration in vitro. Unlike ICAM-1 expression (>80% positive cytokine-treated HPMEC by flow cytometry), VCAM-1 expression varied with the cytokine(s) pretreatment; the order of potency was: TNF-alpha + IL-4 (82.2 +/- 4.2% positive cells) > TNF-alpha (41.8 +/- 5.1%) > IL-1beta (20.8 +/- 4.7%). IL-4 alone had no effect on either ICAM-1 or VCAM-1 expression. EOS adhesion to cytokine-treated HPMEC followed the same order as that observed for VCAM-1 expression. Interestingly, EOS migration across cytokine-treated HPMEC varied inversely with VCAM-1 expression on, and EOS adhesion to, HPMEC; IL-1beta (21.2 +/- 1.4% migration) > TNF-alpha (12.6 +/- 2.6%) > TNF-alpha + IL-4 (9.1 +/- 2.0%). EOS adhesion was greatest with TNF-alpha + IL-4-treated HPMEC, was dependent on VCAM-1, and inhibited with anti-alpha4 integrin mAb (67.7 +/- 7.5% inhibition, p < 0.0005). In contrast, the highest EOS migration occurred across IL-1beta-treated HPMEC and was inhibited by anti-beta2 integrin mAb (40.4 +/- 2.5% inhibition, p < 0.005). Viable HPMEC were required for EOS migration but not adhesion. Our results suggest that EOS adhesion and transmigration are differentially regulated by VCAM-1 and ICAM-1 expression and the interaction of these adhesion proteins with their respective counterligands, i.e., alpha4 and beta2 integrins on EOS.     

Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells

PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.     

Environmental growth conditions influence the ability of Escherichia coli K1 to invade brain microvascular endothelial cells and confer serum resistance

A major limitation to advances in prevention and therapy of neonatal meningitis is our incomplete understanding of the pathogenesis of this disease. In an effort to understand the pathogenesis of meningitis due to Escherichia coli K1, we examined whether environmental growth conditions similar to those that the bacteria might be exposed to in the blood could influence the ability of E. coli K1 to invade brain microvascular endothelial cells (BMEC) in vitro and to cross the blood-brain barrier in vivo. We found that the following bacterial growth conditions enhanced E. coli K1 invasion of BMEC 3- to 10-fold: microaerophilic growth, media buffered at pH 6.5, and media supplemented with 50% newborn bovine serum (NBS), magnesium, or iron. Growth conditions that significantly repressed invasion (i.e., 2- to 250-fold) included iron chelation, a pH of 8.5, and high osmolarity. More importantly, E. coli K1 traversal of the blood-brain barrier was significantly greater for the growth condition enhancing BMEC invasion (50% NBS) than for the condition repressing invasion (osmolarity) in newborn rats with experimental hematogenous meningitis. Of interest, bacterial growth conditions that enhanced or repressed invasion also elicited similar serum resistance phenotype patterns. This is the first demonstration that bacterial ability to enter the central nervous system can be affected by environmental growth conditions.     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.     

Induction of apoptosis in human dermal microvascular endothelial cells and infantile hemangiomas by interferon-alpha

Post-transfusion graft-versus-host disease (PT-GVHD) is a fatal adverse effect of blood transfusion. In spite of its severity, there is no effective treatment at present for PT-GVHD. Previously, we reported that chloroquine (CH) inhibited the cytotoxicity of cytotoxic T-cell (CTL) clones and tumour necrosis factor beta (TNF beta) production by TNF beta-producing clones in vitro, both the clones being derived from peripheral blood lymphocytes (PBMCs) of PT-GVHD patients. To explore the possibility of utilizing CH for the treatment of PT-GVHD, we extended our investigation of the immunosuppressive effects of CH in vitro to PBMCs derived from healthy donors. Our results show that CH inhibits the mixed lymphocyte reaction (MLR) between allogeneic PBMCs, production of inflammatory cytokines such as TNF alpha, interleukin-1 beta (IL-1 beta) and interferon gamma (IFN gamma) in mixed lymphocyte culture and natural killer cell activity, and, further, reduces the number of alloreactive CTL precursors.     

Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells

Upon inflammation, stimulated, but not resting T lymphocytes cross the blood-brain barrier and migrate into the central nervous system. This study shows that direct contact between stimulated T lymphocytes and human brain microvascular endothelial cells (HB-MVEC) induces phenotypic and functional changes on the latter cells. Plasma membranes isolated from stimulated T lymphocytes (S-PM) up-regulated the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on isolated HB-MVEC. In addition, HB-MVEC activated by S-PM secreted interleukin (IL)-6 and IL-8. The levels of ICAM-1, E-selectin, IL-6, and IL-8 expressed in S-PM-activated HB-MVEC were similar to those observed with 1000 U/ml tumor necrosis factor (TNF). In contrast, VCAM-1 expression was 15% of that induced by TNF. Inhibitors of TNF diminished (< or = 45%), but did not abolish the expression of cell adhesion molecules and IL-6 induced by S-PM, IL-8 production being insignificantly affected (< or = 10%). This suggests that membrane-associated TNF was partially involved in HB-MVEC activation. The present study demonstrates that stimulated T lymphocytes are able to activate HB-MVEC upon direct cell contact. This novel mechanism of inducing the expression of cell adhesion molecules may prompt the initial adhesion of stimulated T lymphocytes to brain endothelium.