Endotoxin enhances arachidonic acid metabolism by cultured rabbit microvascular endothelial cells

1992 ◽  
Vol 263 (4) ◽  
pp. H1213-H1221 ◽  
Author(s):  
P. M. Renzi ◽  
J. T. Flynn
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Time Course ◽  
Lipid A ◽  
Primary Cultures ◽  
Microvascular Endothelial Cells ◽  
Prostaglandin Endoperoxide ◽  
Eicosanoid Production ◽  
Prostaglandin Endoperoxide Synthase ◽  
Microvascular Endothelial

This study demonstrates that bacterial lipopolysaccharide and lipid A exert a significant effect on eicosanoid formation by primary cultures of microvascular endothelial cells (MECs). Qualitative studies using [14C]-arachidonic acid demonstrated that prostaglandin E2 was the primary eicosanoid formed by MECs after 20 h of treatment with either vehicle or lipopolysaccharide. Significant, dose-dependent productions of PGE2 and prostacyclin, beginning at an endotoxin dose of 0.01 ng/ml, were quantified by radioimmunoassay in supernatants of cells treated for 20 h with lipopolysaccharide or lipid A. This eicosanoid production was inhibited by meclofenamate and cycloheximide and occurred without cellular injury. The time course and kinetics of eicosanoid production in response to endotoxin demonstrate a significant, time-related enhancement. Endotoxin-treated MECs responded to exogenous substrate with augmented PGE2 production, suggesting enhanced prostaglandin endoperoxide synthase activity. These results demonstrate a significant interaction of endotoxin with endothelial cells of microvascular origin that results in an enhanced potential for eicosanoid metabolism. This effect may be mediated in part through induction of prostaglandin endoperoxide synthase.

CULTURED HUMAN MICROVASCULAR ENDOTHELIAL CELLS SYNTHESIZE CYCLOOXYGENASE METABOLITES FROM ARACHIDONIC ACID IN RESPONSE TO LEUKOTRIENES C4 AND D4

1987 ◽  
Author(s):  
L O Carreras ◽  
J Maclouf ◽  
G Tobelem ◽  
J P Caen
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Time Course ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Microvascular Endothelial Cells ◽  
Ionophore A23187 ◽  
Total Production ◽  
Dependent Manner ◽  
Microvascular Endothelial

Several investigators have demonstrated that endothelial cells have heterogeneous intrinsic properties depending on their vascular origin. In this respect, very limited knowledge exists concerning the production of eicosanoids by human microvascular endothelial cells (HMEC). The aim of this study was to determine: 1) the pattern of the production of cyclooxygenase metabolites by cultured HMEC from omental adipose tissue as compared to the classical study of human umbilical vein endothelial cells (HUVEC); 2) the modification of this metabolism upon leukotrienes (LTs) stimulation. Cultured HMEC produced prostaglandin (PG) E2, PGF2 , 6-keto-PGF1 , and PGD2 (measured by enzymoimmunoassay). In basal conditions, PGD2 was the main product released in the supernatant. Upon stimulation with thrombin, arachidonic acid and calcium ionophore A23187, a marked increase in the production of PGE2, PGF2 , and 6-keto-PGFj , was observed; these results were quite different from HUVEC. In contrast, PGD2 remained unchanged under our experimental conditions and thromboxane B2 was always undetectable. In all cases, the release of PGE2 and PGF2 , was higher than that of 6-keto-PGFj . A considerable amount of the metabolites produced remained cell-associated. The total production (release + cell bound) of cyclooxygenase products was stimulated by LTC4 and LTD4 in a dose-dependent manner (10-9 to 10-6 M). The production of PGD2 was unchanged. LTC4 and LTD4 were almost equally potent, but LTB4 was unable to stimulate PG synthesis (n=4). The production of metabolites induced by 1 uM LTC4 or LTD4 was even higher than that obtained in the presence of high concentrations of thrombin (5 U/ml). This contrasted with the more pronounced stimulation of thrombin on HUVEC as compared to LTs. In the kinetic studies (n=2) we have observed a slow time-course of release of PGE2 and 6-keto-PGF1 into the supernatant of LTs-stimulated HMEC (half-maximal formation at 14-15 min). The stimulatory activity of LTC4 and LTD4 on the production of vasoactive cyclooxygenase metabolites by HMEC could be relevant in inflammatory processes.

Agonist-Stimulated Calcium Entry in Primary Cultures of Human Cerebral Microvascular Endothelial Cells

1999 ◽  
Vol 57 (3) ◽  
pp. 211-226 ◽  
Author(s):  
Li Li ◽  
Brian Bressler ◽  
Rukmini Prameya ◽  
Katerina Dorovini-Zis ◽  
C. Van Breemen
Keyword(s):  
Endothelial Cells ◽  
Primary Cultures ◽  
Calcium Entry ◽  
Microvascular Endothelial Cells ◽  
Microvascular Endothelial

scholarly journals L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

2002 ◽  
Vol 283 (6) ◽  
pp. C1687-C1695 ◽  
Author(s):  
Momoh A. Yakubu ◽  
Charles W. Leffler
Keyword(s):  
Endothelial Cells ◽  
Primary Cultures ◽  
Chelating Agent ◽  
Microvascular Endothelial Cells ◽  
Vasoactive Agents ◽  
Cerebral Microvessels ◽  
Thromboxane Receptor ◽  
High K ◽  
Voltage Dependent ◽  
Microvascular Endothelial

We investigated the role of intracellular calcium concentration ([Ca2+]i) in endothelin-1 (ET-1) production, the effects of potential vasospastic agents on [Ca2+]i, and the presence of L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells. Primary cultures of endothelial cells isolated from piglet cerebral microvessels were used. Confluent cells were exposed to either the thromboxane receptor agonist U-46619 (1 μM), 5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 μM) alone or after pretreatment with the Ca2+-chelating agent EDTA (100 mM), the L-type Ca2+ channel blocker verapamil (10 μM), or the antagonist of receptor-operated Ca2+ channel SKF-96365 HCl (10 μM) for 15 min. ET-1 production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT), or 3.9 (LPA) fmol/μg protein, respectively. Such elevated ET-1 biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. To investigate the presence of L-type voltage-dependent Ca2+channels in endothelial cells, the [Ca2+]isignal was determined fluorometrically by using fura 2-AM. Superfusion of confluent endothelial cells with U-46619, 5-HT, or LPA significantly increased [Ca2+]i. Pretreatment of endothelial cells with high K+ (60 mM) or nifedipine (4 μM) diminished increases in [Ca2+]i induced by the vasoactive agents. These results indicate that 1) elevated [Ca2+]i signals are involved in ET-1 biosynthesis induced by specific spasmogenic agents, 2) the increases in [Ca2+]i induced by the vasoactive agents tested involve receptor as well as L-type voltage-dependent Ca2+ channels, and 3) primary cultures of cerebral microvascular endothelial cells express L-type voltage-dependent Ca2+ channels.

scholarly journals Infectomic Analysis of Gene Expression Profiles of Human Brain Microvascular Endothelial Cells Infected withCryptococcus neoformans

2008 ◽  
Vol 2008 ◽  
pp. 1-7 ◽  
Author(s):  
Ambrose Jong ◽  
Chun-Hua Wu ◽  
Wensheng Zhou ◽  
Han-Min Chen ◽  
Sheng-He Huang
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Human Brain ◽  
Time Course ◽  
Expression Profiles ◽  
Host Cells ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
Microvascular Endothelial ◽  
Time Point

In order to dissect the pathogenesis ofCryptococcus neoformansmeningoencephalitis, a genomic survey of the changes in gene expression of human brain microvascular endothelial cells infected byC.neoformanswas carried out in a time-course study. Principal component analysis (PCA) revealed sigificant fluctuations in the expression levels of different groups of genes during the pathogen-host interaction. Self-organizing map (SOM) analysis revealed that most genes were up- or downregulated 2 folds or more at least at one time point during the pathogen-host engagement. The microarray data were validated by Western blot analysis of a group of genes, includingβ-actin, Bcl-x, CD47, Bax, Bad, and Bcl-2. Hierarchical cluster profile showed that 61 out of 66 listed interferon genes were changed at least at one time point. Similarly, the active responses in expression of MHC genes were detected at all stages of the interaction. Taken together, our infectomic approaches suggest that the host cells significantly change the gene profiles and also actively participate in immunoregulations of the central nervous system (CNS) duringC.neoformansinfection.

scholarly journals Peroxisome-proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus

2007 ◽  
Vol 406 (1) ◽  
pp. 175-183 ◽  
Author(s):  
E. Linda R. Sheldrick ◽  
Kamila Derecka ◽  
Elaine Marshall ◽  
Evonne C. Chin ◽  
Louise Hodges ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Stromal Cells ◽  
Time Course ◽  
Nuclear Factor Κb ◽  
Peroxisome Proliferator ◽  
Endometrial Stromal Cells ◽  
Prostaglandin Endoperoxide ◽  
Prostaglandin Endoperoxide Synthase ◽  
Peroxisome Proliferator Activated Receptors ◽  
Ppar Ligands

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARα and PPARδ (also known as PPARβ) (but not PPARγ). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARα (but not PPARδ or PPARγ) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4β-PMA and PGF2α, and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4β-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4β-PMA in the absence of a PPAR ligand was decreased by the NF-κB (nuclear factor κB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-κB in addition to PPAR phosphorylation. Use of NF-κB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARα to increase PTGS2 levels in bovine endometrial stromal cells.

Mechanisms of MAdCAM-1 gene expression in human intestinal microvascular endothelial cells

2005 ◽  
Vol 288 (2) ◽  
pp. C272-C281 ◽  
Author(s):  
Hitoshi Ogawa ◽  
David G. Binion ◽  
Jan Heidemann ◽  
Monica Theriot ◽  
Pamela J. Fisher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Microvascular Endothelial Cells ◽  
Cellular Density ◽  
Tnf Α ◽  
Gene And Protein Expression ◽  
Microvascular Endothelial

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a homing receptor preferentially expressed on gut-associated endothelial cells that plays a central role in leukocyte traffic into the mucosal immune compartment. Although the molecular mechanisms underlying endothelial ICAM-1 or E-selectin expression have been intensively investigated, the mechanisms that regulate human MAdCAM-1 expression have not been defined. We report MAdCAM-1 gene and protein expression in primary cultures of human intestinal microvascular endothelial cells (HIMEC) that was not demonstrated in human umbilical vein endothelial cells. Similar to ICAM-1 and E-selectin expression, MAdCAM-1 gene expression in HIMEC was inducible with TNF-α, IL-1β, or LPS activation. However, in striking contrast to ICAM-1 and E-selectin expression, MAdCAM-1 mRNA and protein expression in HIMEC was heavily dependent on culture duration and/or cellular density, suggesting a prominent role for cell-cell interaction among these endothelial cells in the expression of the mucosal addressin. MAdCAM-1 expression was inhibited by both SN-50 (NF-κB inhibitor) and LY-294002 [phosphatidylinositol 3-kinase (PI3-K) inhibitor], whereas ICAM-1 and E-selectin expression was inhibited by SN-50 but not by LY-294002. The Akt phosphorylation by TNF-α or LPS was greater at higher cell density, demonstrating a pattern similar to that of MAdCAM-1 expression. NF-κB activation was not affected by cellular density in HIMEC. MAdCAM-1 expression in human gut endothelial cells is regulated by distinct signaling mechanisms involving both NF-κB and PI3-K/Akt. These data also suggest that PI3-K/Akt is involved in the gut-specific differentiation of HIMEC, which results in expression of the mucosal addressin MAdCAM-1.

scholarly journals Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells

2003 ◽  
Vol 284 (1) ◽  
pp. H215-H224 ◽  
Author(s):  
Meetha Medhora ◽  
John Daniels ◽  
Kavita Mundey ◽  
Beate Fisslthaler ◽  
Rudi Busse ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Primary Cultures ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Arachidonic Acid Metabolite ◽  
Epoxyeicosatrienoic Acid ◽  
Adult Rats ◽  
Microvascular Endothelial ◽  
Cloned Cdna

Angiogenesis is one of the most recent physiological functions attributed to products of cytochrome P-450 (CYP450) enymes. To test this at a molecular level in human cells, we used a cloned cDNA for the human endothelial enzyme CYP450 2C9 (CYP2C9) to study growth as well as differentiation of human microvascular endothelial cells from the lung (HMVEC-L). Using adenoviral vectors overexpressing mRNA for CYP2C9, we show that the presence of CYP2C9 doubles thymidine incorporation and stimulates proliferation of primary cultures of endothelial cells compared with Ad5-GFP (control) in 24 h. In addition, there is a significant increase of tube formation in Matrigel after infection of HMVEC-L with Ad5-2C9 than with Ad5-GFP. More interestingly, Ad5-2C9 expressing the antisense product of CYP2C9 (2C9AS) inhibited tube formation compared with both Ad5-GFP as well as the Ad5-2C9 constructs. Finally, we tested the most abundant arachidonic acid metabolite of CYP2C9, 14,15-epoxyeicosatrienoic acid, which induced angiogenesis in vivo when embedded in Matrigel plugs and implanted in adult rats. These data support an important role for CYP2C9 in promoting angiogenesis.

scholarly journals iNOS expression in human intestinal microvascular endothelial cells inhibits leukocyte adhesion

1998 ◽  
Vol 275 (3) ◽  
pp. G592-G603 ◽  
Author(s):  
David G. Binion ◽  
Sidong Fu ◽  
Kalathur S. Ramanujam ◽  
Yuh Cherng Chai ◽  
Raed A. Dweik ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Intestinal Inflammation ◽  
Leukocyte Adhesion ◽  
Primary Cultures ◽  
Inos Expression ◽  
Microvascular Endothelial Cells ◽  
No Production ◽  
Nos Inhibitors ◽  
Microvascular Endothelial

Increased nitric oxide (NO) production by inducible nitric oxide synthase (iNOS) has been associated with intestinal inflammation, including human inflammatory bowel disease. However, NO can downregulate endothelial activation and leukocyte adhesion, critical steps in the inflammatory response. Using primary cultures of human intestinal microvascular endothelial cells (HIMEC), we determined the role of NO in the regulation of HIMEC activation and interaction with leukocytes. Both nonselective ( N G-monomethyl-l-arginine) and specific ( N-iminoethyl-l-lysine) competitive inhibitors of iNOS significantly increased binding of leukocytes by HIMEC activated with cytokines and lipopolysaccharide. Increased adhesion was reversible with the NOS substratel-arginine and was not observed in human umbilical vein endothelial cells (HUVEC). Activation of HIMEC significantly upregulated HIMEC iNOS expression and NO production. NOS inhibitors did not augment cell adhesion molecule levels in activated HIMEC but did result in sustained increases in intracellular reactive oxygen species. In addition, antioxidant compounds reversed the effect of NOS inhibitors on HIMEC-leukocyte interaction. Taken together, these data suggest that after HIMEC activation, iNOS-derived NO is an endogenous antioxidant, downregulating leukocyte binding and potentially downregulating intestinal inflammation.

scholarly journals Mucosal angiogenesis regulation by CXCR4 and its ligand CXCL12 expressed by human intestinal microvascular endothelial cells

2004 ◽  
Vol 286 (6) ◽  
pp. G1059-G1068 ◽  
Author(s):  
Jan Heidemann ◽  
Hitoshi Ogawa ◽  
Parvaneh Rafiee ◽  
Norbert Lügering ◽  
Christian Maaser ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Primary Cultures ◽  
Tube Formation ◽  
Microvascular Endothelial Cells ◽  
Endothelial Tube Formation ◽  
Microvascular Endothelial ◽  
Stromal Cell Derived Factor ◽  
Phosphoinositide 3 Kinase

Mice genetically deficient in the chemokine receptor CXCR4 or its ligand stromal cell-derived factor (SDF)-1/CXCL12 die perinatally with marked defects in vascularization of the gastrointestinal tract. The aim of this study was to define the expression and angiogenic functions of microvascular CXCR4 and SDF-1/CXCL12 in the human intestinal tract. Studies of human colonic mucosa in vivo and primary cultures of human intestinal microvascular endothelial cells (HIMEC) in vitro showed that the intestinal microvasculature expresses CXCR4 and its cognate ligand SDF-1/CXCL12. Moreover, SDF-1/CXCL12 stimulation of HIMEC triggers CXCR4-linked G proteins, phosphorylates ERK1/2, and activates proliferative and chemotactic responses. Pharmacological studies indicate SDF-1/CXCL12 evokes HIMEC chemotaxis via activation of ERK1/2 and phosphoinositide 3-kinase signaling pathways. Consistent with chemotaxis and proliferation, endothelial tube formation was inhibited by neutralizing CXCR4 or SDF-1/CXCL12 antibodies, as well as the ERK1/2 inhibitor PD-98059. Taken together, these data demonstrate an important mechanistic role for CXCR4 and SDF-1/CXCL12 in regulating angiogenesis within the human intestinal mucosa.

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