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Biomedical subjects

J S Alexander

Publications and source records attributed to J S Alexander.

At least 19 recordsLinked to original sources
PubMed2001

TNF-alpha induced endothelial MAdCAM-1 expression is regulated by exogenous, not endogenous nitric oxide.

BACKGROUND: MAdCAM-1 is an adhesion molecule expressed in Peyer's patches and lymphoid tissues which is mobilized by cytokines like TNF-alpha and is a major determinant of lymphocyte trafficking to the gut in human inflammatory bowel disease (IBD). It has been suggested that both reactive oxygen and nitrogen metabolites participate in regulating adhesion molecule expression in response to TNF-alpha. METHODS: To examine how exogenous and endogenous sources of NO modulate MAdCAM-1 induction by TNF-alpha, we pre-treated mouse lymphatic endothelial cells with either long or short acting NO donors prior to TNF-alpha-stimulation, and measured MAdCAM-1 induction at 24 h. RESULTS AND DISCUSSION: DETA-NO, a long-acting NO donor, and SperNO, a rapid releasing NO donor both inhibited TNF-alpha-stimulated MAdCAM-1 expression in a concentration dependent manner. Both NO donors also reduced a4b7-dependent lymphocyte endothelial adhesion. Inhibition of endogenous NO production by either L-NAME, a non-selective NOS inhibitor, or by 1400 w, a selective iNOS inhibitor failed to induce, or potentiate TNF-alpha regulated MAdCAM-1 expression. CONCLUSIONS: Exogenous NO donors may be beneficial in the treatment of IBD, while endogenous nitric oxide synthases may be less effective in controlling adhesion molecule expression in response to cytokines.

Amidines↗
PubMed2001

Interferon-gamma and interleukin-10 reciprocally regulate endothelial junction integrity and barrier function.

Inflammatory bowel disease (IBD) is associated with Th1/Th2 cytokine dysregulation, leukocyte extravasation, and tissue edema, but the mechanisms for cytokine-mediated vascular dysfunction are not understood. To investigate how cytokines might control edema in IBD, we determined vascular permeability and IFN-gamma expression in two models of murine colitis: SCID mice reconstituted with CD45RB(high T-lymphocytes (CD45RB(high)/SCID mice), and interleukin-10 gene deficient (IL-10(-/-)) mice. We also investigated the in vitro effects of IFN-gamma and IL-10 on human endothelial solute barrier and junction protein expression. Vascular permeability in CD45RB(high)/SCID and IL-10(-/-) mice was quantified using tissue (131)I-IgG accumulation. The IFN-gamma message was quantified using the ribonuclease protection assay. Endothelial barrier integrity in vitro was measured by transmonolayer electrical resistance, and junctional proteins were examined by immunoblotting and fluorescence microscopy. Both CD45RB(high)/SCID and IL-10(-/-) mice exhibit enhanced colonic microvascular leakage and IFN-gamma message levels compared to their respective controls. In vitro, IFN-gamma also reduced endothelial barrier (monolayer electrical resistance, increased albumin permeability) and reduced tight junction (occludin) expression and staining. These effects were reversed by pretreatment of monolayers with IL-10. Therefore, in vivo IFN-gamma and IL-10 may modulate microvascular leakage in IBD partly by controlling the expression of intestinal endothelial tight junctional proteins.

Animals↗
PubMed2001

Poly ADP ribose-polymerase inhibitors prevent the upregulation of ICAM-1 and E-selectin in response to Th1 cytokine stimulation.

We studied the role of poly-ADP-ribose polymerase (PARP) in the mobilization of ICAM-1, VCAM-1, and E-selectin by TNF-alpha and IL-1beta in cultured human endothelial cells. Enzyme linked immunosorbent analysis (ELISA) was used to assess if ICAM-1, VCAM-1, and E-selectin were expressed at the cell surface, and if PARP inhibition (using the selective PARP inhibitor GPI 6150) blocked the induced expression. Endothelial cell adhesion molecule expression was evaluated at 4 and at 24 h after cytokine stimulation. At 4 h ICAM-1 and E-selectin, but not VACM-1, were stimulated by both IL-1beta and TNF-alpha. Blocking PARP via GPI 6150 only affected TNF-alpha induced E-selectin expression at 4 hours. ICAM-1, VCAM-1, and E-selectin expression were all stimulated by both IL-1beta and TNF-alpha in the 24 h assays. PARP inhibition with GPI 6150 blocked the IL-1beta mediated stimulation of both ICAM-1 and E-selectin expression, and blocked TNF-alpha stimulation of ICAM-1 expression at 24 h. These experiments suggest that specific PARP inhibition may provide a novel method of controlling leukocyte dependent inflammation through the reduction of ICAM-1 and E-selectin expression in endothelial cells in response to cytokines.

Benzopyrans↗
PubMed2001

Roles of leukocyte and immune cell junctional proteins.

The restricted expression of E-cadherin on dendritic cells and intraepithelial lymphocytes has been described as a structural/adhesive system that helps to retain and integrate these cells within mucosal and dermal tissues. The activation of these cells downregulates expression of cadherins, and contributes to cell redistribution and tissue homing. It has recently been reported that lymphocytes and other leukocytes express cadherins, as well as occludin, a tight junctional component, in response to several types of stimuli. This suggests that mobilization of adherens and tight junction proteins in leukocytes may facilitate interactions of leukocytes with epithelial, endothelial, and interstitial cells that express these proteins and support homophilic adhesion. The conditions and patterns of synthesis of these adhesion molecules, in antigen-presenting cells and leukocytes, indicate that the expression of junction proteins may play roles in normal and pathological leukocyte traffic.

Animals↗
PubMed2001

Glucocorticoids and IL-10, but not 6-MP, 5-ASA or sulfasalazine block endothelial expression of MAdCAM-1: implications for inflammatory bowel disease therapy.

BACKGROUND: Enhanced MAdCAM-1 (mucosal addressin cell adhesion molecule-1) expression is associated with the aetiology of inflammatory bowel disease, but little is known about MAdCAM-1: regulation, or how inflammatory bowel disease therapies modulate MAdCAM-1. AIM: To examine how agents currently used to treat inflammatory bowel disease affect MAdCAM-1: induced by tnf-alpha in an in vitro model of inflammatory bowel disease. METHODS: Endothelial monolayers were pretreated with dexamethasone (DEX): 5-aminosalicylic acid (5-ASA), 6-mercaptopurine (6-MP), sulfasalazine or interleukin-10: (IL-10: prior to TNF-alpha (20 ng/mL), and MAdCAM-1: measured by Western blotting, RT-PCR, EMSA and lymphocyte adhesion assays. RESULTS: MAdCAM-1: was induced dose- and time-dependently by TNF-alpha on endothelial cells. Either dexamethasone or IL-10: reduced TNF-alpha-induced MAdCAM-1: protein, mRNA and lymphocyte adhesion. However, neither 5-ASA, sulfasalazine nor 6-MP blocked MAdCAM-1 induction. CONCLUSIONS: Our data indicate that dexamethasone or IL-10 can exert therapeutic activity in inflammatory bowel disease through MAdCAM-1 inhibition. 5-ASA, sulfasalazine and 6-MP, while beneficial in inflammatory bowel disease, do not directly control MAdCAM-1, and are beneficial through inhibition of other inflammatory processes.

Animals↗
PubMed2001

Regulation and distribution of MAdCAM-1 in endothelial cells in vitro.

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that regulates lymphocyte trafficking to Peyer's patches and lymph nodes. Although it is widely agreed that MAdCAM-1 induction is involved in chronic gut inflammation, few studies have investigated regulation of MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)-alpha using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-alpha induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-kappa B/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1 is regulated via MAPKs, we examined mitogen/extracellular signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that MEK-1/2 is activated by TNF-alpha within minutes and is dependent on TK and p42/44 MAPKs. Similarly, TNF-alpha activated NF-kappa B through TK, p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also shown to be frequently distributed to endothelial junctions both in vitro and in vivo. Cytokines like TNF-alpha stimulate MAdCAM-1 in high endothelium via TK, p38, p42/22 MAPKs, and NF-kappa B/PARP. MAdCAM-1 expression requires NF-kappa B translocation through both direct p42/44 and indirect p38 MAPK pathways in high endothelial cells.

Alkaloids↗
PubMed2001

H(2)O(2)-mediated permeability II: importance of tyrosine phosphatase and kinase activity.

We previously reported that exposure of endothelial cells to H(2)O(2) results in a loss of cell-cell apposition and increased endothelial solute permeability. The purpose of this study was to determine how tyrosine phosphorylation and tyrosine phosphatases contribute to oxidant-mediated disorganization of endothelial cell junctions. We found that H(2)O(2) caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues. H(2)O(2) exposure also results in increased endothelial monolayer permeability, which was attenuated by pp60, an inhibitor of src kinase. Inhibition of protein tyrosine phosphatase activity by phenylarsine oxide (PAO) demonstrated a similar permeability profile compared with H(2)O(2), suggesting that tyrosine phosphatase activity is important in maintaining a normal endothelial solute barrier. Immunofluorescence shows that H(2)O(2) exposure caused a loss of pan-reactive cadherin and beta-catenin from cell junctions that was not blocked by the src kinase inhibitor PP1. H(2)O(2) also caused beta-catenin to dissociate from the endothelial cytoskeleton, which was not prevented by PP1. Finally, we determined that PP1 did not prevent cadherin internalization. These data suggest that oxidants like H(2)O(2) produce biological effects through protein phosphotyrosine modifications by decreasing total cellular phosphatase activity combined with increased src kinase activity, resulting in increased endothelial solute permeability.

Animals↗
PubMed2001

Redox regulation of endothelial barrier integrity.

Intestinal ischemia-reperfusion is associated with the generation of reactive oxygen metabolites as well as remote, oxidant-mediated lung injury. Oxidants elicit endothelial redox imbalance and loss of vascular integrity by disorganizing several junctional proteins that contribute to the maintenance and regulation of the endothelial barrier. To determine the specific effect of redox imbalance on pulmonary vascular barrier integrity, microvascular permeability was determined in lungs of animals subjected to chemically induced redox imbalance. The effect of redox imbalance on microvascular permeability and endothelial junctional integrity in cultured lung microvascular cells was also determined. Whole lung and cultured pulmonary endothelial cell permeability both increased significantly in response to chemical redox imbalance. Thiol depletion also resulted in decreased endothelial cadherin content and disruption of the endothelial barrier. These deleterious effects of intracellular redox imbalance were blocked by pretreatment with exogenous glutathione. The results of this study suggest that redox imbalance contributes to pulmonary microvascular dysfunction by altering the content and/or spatial distribution of endothelial junctional proteins.

Animals↗
PubMed2001

The role of p38 MAP kinase in hydrogen peroxide mediated endothelial solute permeability.

OBJECTIVE: The purpose of this study was to determine the contribution of p38 MAP kinase activity during hydrogen peroxide mediated increased endothelial solute permeability. We also sought to identify the role of p38 MAP kinase-mediated changes in endothelial cell architecture due to hydrogen peroxide challenge. METHODS: Hydrogen peroxide mediated permeability of HUVEC was determined with and without inhibition of p38 MAP kinase by SB202190. Hydrogen peroxide mediated rearrangement of the endothelial actin cytoskeleton and junctional proteins occludin and ZO-1 were observed by immunofluorescence microscopy. RESULTS: Hydrogen peroxide treatment of endothelial monolayers caused a significant increase in solute permeability over a ninety-minute time period. Oxidant-mediated permeability and phosphorylation of p38 MAP kinase was significantly attenuated by SB 202190. Immunofluorescent staining for the tight junctional proteins occludin and ZO-1 demonstrated that oxidant challenge caused a loss of endothelial tight junction organization. Rhodamine phalloidin staining of the actin cytoskeleton showed that hydrogen peroxide stimulated increased stress fiber formation with concomitant gap formation between adjacent endothelial cells. Inhibition of p38 MAP kinase during oxidant challenge significantly attenuated actin stress fiber formation and prevented gap formation. CONCLUSIONS: These data demonstrate that p38 MAP kinase activity is involved in hydrogen peroxide mediated permeability, stress fiber formation, and intracellular gap formation.

Actin Cytoskeleton↗
PubMed2000

HIV-1 Tat increases endothelial solute permeability through tyrosine kinase and mitogen-activated protein kinase-dependent pathways.

OBJECTIVE: HIV-1 infection is associated with alterations of several vascular endothelial functions including adhesion molecule expression, growth, and vascular permeability. The bases of these errors are not known, but might involve secretion of the HIV-1 derived transcription factor 'Tat-1'. This study investigated Tat-1 mediated endothelial barrier changes and second message regulation of this phenomenon. METHODS: We exposed human umbilical vein endothelial cell monolayers to Tat-1 (0-150 ng/ml) for up to 48 h and measured resulting changes in monolayer permeability. We also investigated the role of tyrosine and mitogen activated protein (MAP) kinases, and protein kinase G using the pharmacological blockers genistein, PD98059 and KT5823 respectively. RESULTS: Tat-1 significantly reduced monolayer barrier and increased albumin permeability within 24 h. Tat-1 also stimulated tyrosine phosphorylation of multiple endothelial proteins, disorganized junctional phosphotyrosine staining and increased the number of these immunostaining structures. The increased permeability produced by Tat-1 was blocked by genistein and PD98059, but not by KT5823. Genistein and PD98059 pretreatment also prevented the changes in phosphotyrosine immunostaining produced by Tat-1 and blocked phosphorylation of several proteins including MAP kinase. CONCLUSION: These results suggest that HIV may dysregulate endothelial barrier through the effects of Tat-1. These blocker experiments suggest that the effects of Tat are transcription/translation-dependent. These data demonstrate that Tat increases endothelial albumin permeability in vitro through tyrosine kinase and MAP kinase, but not protein kinase G pathways.

Blotting, Western↗
PubMed2000

Inflammatory mediators induce sequestration of VE-cadherin in cultured human endothelial cells.

The mechanisms through which inflammatory mediators modify endothelial junctional structure are not well understood. Endothelial cells exposed to 1 mM H2O2, 0.1 mM histamine or 4 mM EDTA displayed decreased amounts of VE-cadherin on the cell surface in a time-dependent manner. H2O2 and EDTA-treated cells showed a sustained reduction in surface VE-cadherin, but histamine (0.1 mM) decreased cell surface VE-cadherin only at 5 and 15 min, not at 30 and 60 min. Sequestering of VE-cadherin could also be visualized as a decrease in immunofluorescent labeling of endothelial junctions in fixed, non-extracted monolayers. However, junctional staining was observed in these cells after membrane extraction. This decreased surface expression of VE-cadherin was actin-filament, but not PKC/MAP kinase dependent. VE-cadherin binding to the cytoskeleton was decreased by EDTA, but was not diminished by histamine or H2O2. Therefore, by promoting sequestration of junctional cadherins, inflammatory mediators may decrease adhesive bonds between apposed endothelial cells and increase solute permeability.

Antigens, CD↗
PubMed2000

H(2)O(2)-mediated permeability: role of MAPK and occludin.

H(2)O(2)-mediated elevation in endothelial solute permeability is associated with pathological events such as ischemia-reperfusion and inflammation. To understand how H(2)O(2) mediates increased permeability, we investigated the effects of H(2)O(2) administration on vascular endothelial barrier properties and tight junction organization and function. We report that H(2)O(2) exposure caused an increase in endothelial solute permeability in a time-dependent manner through extracellularly regulated kinase 1 and 2 (ERK1/ERK2) signal pathways. H(2)O(2) exposure caused the tight junctional protein occludin to be rearranged from endothelial cell-cell junctions. Occludin rearrangement involved redistribution of occludin on the cell surface and dissociation of occludin from ZO-1. Occludin also was heavily phosphorylated on serine residues upon H(2)O(2) administration. H(2)O(2) mediates changes in ERK1/ERK2 phosphorylation, increases endothelial solute permeability, and alters occludin localization and phosphorylation were all blocked by PD-98059, a specific mitogen-activated protein (MAP) or ERK kinase 1 inhibitor. These data strongly suggest that H(2)O(2)-mediated increased endothelial solute permeability involves the loss of endothelial tight junction integrity through increased ERK1/ERK2 activation.

Cell Membrane↗
PubMed2000

Regulation of E-selectin expression in postischemic intestinal microvasculature.

Monolayers of cultured endothelial cells exposed to hypoxia-reoxygenation exhibit a transcription-dependent increase in E-selectin expression and E-selectin-dependent neutrophil-endothelial cell adhesion. The overall objectives of this study were 1) to determine whether ischemia-reperfusion (I/R) promotes upregulation of E-selectin in vivo; 2) if so, to define the mediators of this response; and 3) to assess the contribution of E-selectin to I/R-induced neutrophil recruitment. The dual-radiolabeled monoclonal antibody (MAb) technique was used to measure E-selectin expression in the intestinal vasculature. Ischemia was induced by complete occlusion (30-60 min) of the superior mesenteric artery followed by 3-24 h of reperfusion. Increasing durations of ischemia elicited progressively increasing (2- to 5-fold) levels of E-selectin expression, with the peak response noted after 45 min of ischemia and 5 h of reperfusion. Subsequent experiments revealed that I/R-induced increase in E-selectin expression (at 5 h) is significantly blunted in transgenic mice that overexpress Cu,Zn-superoxide dismutase or by treatment of wild-type mice with either a blocking antibody against tumor necrosis factor (TNF)-alpha or an inhibitor of nuclear factor-kappaB (NF-kappaB) activation (PS341). Administration of an E-selectin-specific MAb dramatically reduced I/R-induced recruitment of neutrophils in the intestine. These findings suggest that superoxide and TNF-alpha mediate gut I/R-induced E-selectin expression via an NF-kappaB-dependent mechanism; this upregulation of E-selectin contributes significantly to I/R-induced neutrophil recruitment.

Animals↗
PubMed2000

Characterization of JOK-1, a human gastric epithelial cell line.

Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line "JOK-l." These cells express conventional epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherins, beta-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epithelial-like barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line "MDCK" also expresses several epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherin, beta-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.

Antigens, Polyomavirus Transforming↗
PubMed1999

Targeting expression with light using caged DNA.

In this report, we describe the inactivation and site-specific light induction of plasmid expression using a photosensitive caging compound. Plasmids coding for luciferase were caged with 1-(4, 5-dimethoxy-2-nitrophenyl)diazoethane (DMNPE) and transfected into approximately 1-cm diameter sites of the skin of rats with particle bombardment. Skin sites transfected with caged plasmids did not express luciferase. However, subsequent exposure of transfected skin sites to 355-nm laser light induced luciferase expression in proportion to the amount of light. Liposome transfection of HeLa cells with DMNPE-caged green fluorescent protein (GFP) plasmids showed similar results. Caging DNA with DMNPE blocks expression at the level of transcription, since in vitro production of mRNA from linearized GFP plasmid was also blocked by caging and subsequently restored by exposure to light. Under the reaction conditions of these experiments, our absorbance data indicate that each DMNPE-caged GFP plasmid contains approximately 270 caging groups. In addition to inhibition and subsequent restoration of plasmid bioactivity, the presence and photocleavage of this relatively small number of cage groups also alters electrophoretic mobility of plasmids and optical absorption characteristics. This light-induced expression strategy provides a new means to target the expression of genetic material with spatial and temporal specificity.

Animals↗