Cytochrome P450 Isoform Expression in Human Vascular Endothelial Cells

Hypertension ◽  
2000 ◽  
Vol 36 (suppl_1) ◽  
pp. 698-698
Author(s):  
Eduardo Barbosa-Sicard ◽  
Eva Kaergel ◽  
Dominik N Muller ◽  
Horst Honeck ◽  
Friedrich C Luft ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Vascular Endothelial Cells ◽  
Arachidonic Acid Metabolism ◽  
Vascular Endothelial ◽  
Rt Pcr ◽  
Pcr Screening ◽  
Mediators Of Inflammation ◽  
Derivatives Of

P29 Epoxy derivatives of arachidonic acid may act as important autocrine and paracrine mediators of endothelial function including regulation of vascular tone and control of inflammation. To identify potential candidates for catalyzing the synthesis of these and further arachidonic acid metabolites, we studied human vascular endothelial cells for the expression of individual cytochrome P450 isoforms belonging to the CYP families 1, 2, 3 and 4. An RT-PCR screening performed with subfamily- and isoform-specific primer pairs revealed mRNAs for the P450 forms 1A1, 1B1, 2C8, 2E1, 2J2, 3A7, 4A11 and 4F2. The identity of the RT-PCR products was confirmed by DNA sequencing. In addition, P450 1A2 mRNA was detected after induction with β-naphthoflavone which also enhanced the expresion of P450s 1A1 and 1B1. P450s 2B6 and 3A4 were not detectable. Similar P450 isoform patterns were obtained analyzing primary human endothelial cells originating from aorta, coronary arteries, dermal microvessels and umbilical veins, as well as an immortalized human endothelial cell line (HMEC-1). Further studies with HMEC-1 cells showed the expression of all human members of the P450 2C subfamily (2C8, 2C9, 2C18 and 2C19). We next used gaschromatography-mass spectrometry to identify the regioisomeric epoxeicosatrienoic acids produced by HMEC-1 cells. Among the P450 forms detected by the RT-PCR screening, P450 2C8 and 2J2 are the leading candidates for producing vasoactive epoxyeicosatrienoic acids. Using recombinant human P450 1A1, we then found that this P450 form catalyzes the formation of various regioisomeric hydroxy derivatives of arachidonic acid. We conclude that P450 1A1 known primarily for its role in polycyclic aromatic hydrocarbon metabolism, may interfere with endothelial arachidonic acid metabolism, particularly after its induction by drugs and xenobiotics. Furthermore, P450s 4A11 and 4F2 probably contribute to the degradation of lipid mediators of inflammation.

Measurement Of 1-14C Arachidonic Acid Metabolism By Cultured Human Vascular Endothelial Cells And Platelets

1981 ◽  
Author(s):  
Ch Willems ◽  
P J Laanen ◽  
G A Pool ◽  
Ph G de Groot ◽  
J A van Mourik ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Acid Metabolism ◽  
Vascular Endothelial Cells ◽  
Lipid Fraction ◽  
Relative Sensitivity ◽  
Arachidonic Acid Metabolism ◽  
Vascular Endothelial ◽  
Response Curves ◽  
Stimulation Of

To investigate the relative sensitivity of endothelial cells and platelets towards aspirin (ASA), a method was designed which allows the measurement of prostaglandin (PG) metabolism using [l-14C] arachidonic acid (AA) as precursor. When HEC were incubated with 20 μM AA for 24h at 37°C in the presence of serum, about 15-20% of the label was incorporated into phospholipids whereas 3-4% was present in the neutral lipid fraction. No PG formation could be measured, even after stimulation of the phospholipase system by thrombin or ionophore A 23187. When HEC were incubated for 20 min with AA in the absence of serum, about 8-10% of the label was incorporated into PGs (6-keto PGF1α, 2%; PGE2, 1%; PGF2α, 4%). Platelets were prelabeled with AA, in buffer and the phospholipase system could be stimulated by thrombin (measured as the formation of TXB2, HHT and HETE). Using the above methods, the IC50 of ASA (30 min, 37°C) was 4.7±2.4μM for HEC and 10.1±2.4μM for platelets (p>0.1). Preincubation with 5μM ASA resulted in 50% inhibition of PG synthesis after 30±8 min with HEC and after 40±5.6 min with platelets (p#x003E;0.1). Moreoever when HEC and platelets were combined, incubated with ASA and subsequently tested separately no difference in dose-response curves could be demonstrated. These results indicate that the sensitivity of HEC and platelets to ASA is similar.

scholarly journals Arachidonic Acid Diols Produced by Cytochrome P-450 Monooxygenases Are Incorporated into Phospholipids of Vascular Endothelial Cells

1996 ◽  
Vol 271 (24) ◽  
pp. 14001-14009 ◽  
Author(s):  
Mike VanRollins ◽  
Terry L. Kaduce ◽  
Xiang Fang ◽  
Howard R. Knapp ◽  
Arthur A. Spector
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Cytochrome P 450

Prostacyclin: Production by Vascular Endothelium and Effects on Platelet Aggregation

1979 ◽  
Author(s):  
S. Moncada ◽  
S. Bunting
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Vascular Endothelium ◽  
Vascular Endothelial Cells ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
Vascular Endothelial ◽  
Prostacyclin Production

The inhibitory effect of vascular endothelial cells on platelet aggregation is due to their ability to release prostacyclin. The existence of an ADPase has been confirmed in endothelial cells but this enzymes does not seem to be related to the anti-aggregating properties of vascular endothelium. In vitro, the release of prostacyclin by humand and rabbit endothelial cells persists after several subcultures. The production of PGI2 can be demonstrated by its inhibition by aspirin-like drugs or 15-hydroperoxy arachidonic acid (a specific inhibitor of PGI2 synthesis). Moreover, the antiaggregating activity is antagonised by an antibody to 5,6 dihydro prostacyclin which cross reacts and neutralises prostacyclin.

Influence of some phospholipase A2 and cytochrome P450 inhibitors on rat arterial smooth muscle K+ currents

1999 ◽  
Vol 77 (7) ◽  
pp. 481-489 ◽  
Author(s):  
Bert Vanheel ◽  
Patrick Calders ◽  
Isabelle Van den Bossche ◽  
Johan Van de Voorde
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Smooth Muscle ◽  
Vascular Endothelial Cells ◽  
Outward Current ◽  
Arachidonic Acid Metabolism ◽  
Phospholipase A ◽  
Muscle Membrane ◽  
Arterial Smooth Muscle ◽  
P450 Inhibitors

The hyperpolarizing factor that is liberated by vascular endothelial cells in response to various agonists, and known to induce relaxation by opening of smooth muscle K+ channels, has been suggested to be a product of cytochrome P450 dependent arachidonic acid metabolism. In this study, the direct influence of two phospholipase A2 inhibitors and of five structurally and mechanistically different cytochrome P450 inhibitors on K+ currents in freshly isolated vascular smooth muscle cells from the rat aorta was investigated. On stepping the cell membrane potential from -70 mV to a series of depolarized test potentials, a noisy outward current developed at test potentials > +10 mV, which showed no appreciable inactivation during the voltage pulse. It was largely abolished by 3 mM external tetraethylammonium chloride (TEA), suggesting that it predominantly consisted of current through large-conductance Ca2+-activated K+ channels. The phospholipase A2 inhibitor quinacrine considerably inhibited this TEA-sensitive current, while 4-bromophenacylbromide exerted no effect. The cytochrome P450 inhibitors proadifen and miconazole reversibly decreased the amplitude of IK, while clotrimazole and 1-aminobenzotriazole had no effect. Conversely, 17-octadecynoic acid increased whole-cell IK. These results show that some phospholipase A2 and cytochrome P450 inhibitors may interfere with K+ channel activation in the rat arterial smooth muscle cell. The relevance of these findings to studies on the involvement of cytochrome P450 dependent metabolism in the generation of the endothelium-derived hyperpolarizing factor in intact arteries is discussed.Key words: endothelial factors, smooth muscle, membrane currents, vasodilation, endothelium-derived hyperpolarizing factor (EDHF), arachidonic acid.

scholarly journals Effects of dietary polyphenols on gene expression in human vascular endothelial cells

2008 ◽  
Vol 67 (1) ◽  
pp. 42-47 ◽  
Author(s):  
Sonja K. Nicholson ◽  
Gregory A. Tucker ◽  
John M. Brameld
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Ferulic Acid ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Endothelial Cell ◽  
Vascular Endothelial ◽  
Rt Pcr ◽  
Physiological Concentration ◽  
Dietary Polyphenols

Previous studies have shown that consumption of fruit and vegetables plays a role in preventing the onset of CVD. These beneficial effects have been linked to the presence of polyphenolic compounds in plant-derived foods and their antioxidant capacity. It has been hypothesised that polyphenols may also have a direct effect on vascular endothelial cell growth and the expression of genes involved in angiogenesis and other roles of the endothelium. Previous studies in this area have tended to use concentrations of polyphenols that are supraphysiological (1–100 μm). The effects of more physiological concentrations (0·1 μm) of various individual polyphenols on gene expression were therefore investigated in cultured human umbilical vein endothelial cells (HUVEC) using both microarray and quantitative RT–PCR methodologies. Treatment of HUVEC with ferulic acid, quercetin or resveratrol (0·1 μm) resulted in changes to gene expression that for the three treatments amounted to significant (>2-fold) down-regulation of the expression of 363 genes and significant (>2-fold) up-regulation of 233 genes of the 10 000 genes present on the microarray. The majority of these genes were affected by resveratrol. Quantitative RT–PCR studies indicated that resveratrol (0·1 μm) significantly increased the expression of the gene encoding endothelial NO synthase (eNOS), which synthesises the vasodilator molecule NO, and both resveratrol and quercetin decreased expression of the potent vasoconstrictor, endothelin-1 (ET-1), while ferulic acid had no effect. The effects of resveratrol (0·1 μm) were also investigated when HUVEC were under oxidative stress following treatment with H2O2 (0–50 μm), which dose-dependently increased expression of eNOS and ET-1. Resveratrol stimulated eNOS mRNA in the absence of H2O2 and still allowed the increase with H2O2, but the effects were not additive. In contrast, resveratrol blocked the stimulatory effect of H2O2 on ET-1 expression. Hence, resveratrol has potent effects at a physiological concentration (0·1 μm) that would be expected to result in vasodilation and therefore help reduce blood pressure and the risk of CVD.

scholarly journals Arachidonic acid metabolism and the adhesion of human polymorphonuclear leukocytes to cultured vascular endothelial cells

Blood ◽  
1983 ◽  
Vol 62 (4) ◽  
pp. 889-895 ◽  
Author(s):  
MR Buchanan ◽  
MJ Vazquez ◽  
MA Jr Gimbrone
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Endothelial Cell ◽  
Polymorphonuclear Leukocytes ◽  
Acid Metabolism ◽  
Arachidonic Acid Metabolism ◽  
Vascular Endothelial ◽  
Endothelial Monolayers

Abstract Polymorphonuclear leukocytes (PMN) adhere to the vascular endothelial lining in vivo and to the surfaces of cultured endothelial cells in vitro, but the mechanisms of these cellular interactions remain unclear. Arachidonic acid metabolites, both cyclooxygenase- and lipoxygenase-derived, have been shown to influence PMN locomotion, secretion, and adhesion to artificial surfaces. To determine whether such mediators also are involved in regulating PMN-endothelial cell interactions, we have examined the effects of prostacyclin and various inhibitors of arachidonic acid metabolism on the adherence of radiolabeled PMN to cultured bovine aortic endothelial cells. Confluent endothelial monolayers were incubated with washed suspensions of radiolabeled human PMN (which contained less than 1% platelet contamination) at 37 degrees C for 30 min, then subjected to a standardized wash procedure and the number of adherent leukocytes determined radiometrically. Under basal conditions, i.e., in the absence of exogenous activating stimuli, 4,163 +/- 545 PMN adhered per square millimeter of endothelial surface (mean +/- SEM, n = 12). This basal adhesion (which corresponds to approximately 4–5 leukocytes per endothelial cell) was unaffected when the leukocytes and endothelial monolayers were pretreated with cyclooxygenase inhibitors (100 microM aspirin or 1–5 microM indomethacin) or PGI2 (10(-9)-10(6) M). Thus, basal PMN-endothelial adhesion in this in vitro model system does not appear to be dependent on endogenous cyclooxygenase derivatives of arachidonate or to be sensitive to inhibition by exogenous prostacyclin. In contrast, leukocyte adhesion was significantly reduced by pretreatment with 5,8,11,14- or 4,7,10,13-eicosatetraynoic acid, 0.5- 5 mM sodium salicylate, or 10–1,000 microM indomethacin, antiinflammatory agents that can interfere with the metabolism of arachidonic acid via non-cyclooxygenase-dependent mechanisms. These observations may be relevant to the interactions of circulating PMN with vascular endothelium under both physiologic and pathophysiologic conditions in vivo.

Mercury Activates Phospholipase A2and Induces Formation of Arachidonic Acid Metabolites in Vascular Endothelial Cells

2007 ◽  
Vol 17 (9) ◽  
pp. 541-557 ◽  
Author(s):  
Jessica N. Mazerik ◽  
Himabindu Mikkilineni ◽  
Vivek A. Kuppusamy ◽  
Emily Steinhour ◽  
Alon Peltz ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Arachidonic Acid Metabolites ◽  
Acid Metabolites

Biosynthesis and Apical Secretion of ADAMTS13 Metalloprotease in Vascular Endothelial Cells and in Madin-Darby Canine Kidney Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2652-2652 ◽  
Author(s):  
Dezhi Shang ◽  
X. Wu Zheng ◽  
Jihui Ai ◽  
X. Long Zheng
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Mdck Cells ◽  
Vascular Endothelial ◽  
Rt Pcr ◽  
Madin Darby Canine Kidney ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Darby Canine Kidney ◽  
Canine Kidney

Abstract ADAMTS13, a reprolysin-like metalloprotease, limits platelet aggregation by cleaving von Willebrand factor. Deficiency of plasma ADAMTS13 activity can lead to a life-threatening complication, thrombotic thrombocytopenic purpura (TTP). It has been shown that ADAMTS13 is synthesized in human and mouse hepatic stellate cells. But detection of ADAMTS13 mRNA by a reverse transcriptase polymerase chain reaction (RT-PCR) in many other tissues (brain, heart, lungs, kidneys, pancreas, adrenal gland, prostate, uterus and placenta) suggests that the vascular endothelial cells may also produce low level of ADAMTS13 protease. We showed that full-length ADAMTS13 mRNA and protein were detected in human umbilical cord vein endothelial cells (HUVEC), human aortic endothelial cells (HAEC), and microvascular endothelial cells (ECV304) by RT-PCR, SDS-polyacrylamide gel electrophoresis and Western blot. The ADAMTS13 in the cell lysates and the serum-free conditioned media of HUVEC, HAEC and ECV304 cleaved a peptidyl substrate, FRETS-VWF73, specifically, and such a proteolytic cleavage could be completely abolished by 10 mM EDTA. Immunohistochemistry showed that both endogenous and recombinant ADAMTS13 in HUVEC and HAEC appeared to be co-localized with von Willebrand factor in the endoplasmic reticulum and/or Golgi apparatus, but not in the Weibel-Parade bodies, suggesting that ADAMTS13 is readily secreted rather than stored inside the cells. To determine whether ADAMTS13 is secreted into the lumen of the vessels, we assessed the polarity of ADAMTS13 secretion in ECV304 and Madin-Darby canine kidney (MDCK) cells, two well-characterized polarized endothelial and epithelial cell lines for studying protein sorting. Both endogenous ADAMTS13 and recombinant ADAMTS13-V5-His expressed in ECV304 was predominantly secreted into the apical domain of the cells. Similarly, the recombinant ADAMTS13-V5-His in MDCK cells was also secreted predominantly into the apical domain. Apical secretion of ADAMTS13-V5-His appeared to depend on at least a transient association of ADAMTS13-V5-His with Triton X-100 insoluble glycosphingolipid and cholesterol-enriched microdomains, namely rafts. Deletion of the distal carboxyl terminal CUB (Complement C1r/C1s, Urinary EGF, and Bone morphogenic protein) domains or removal of membrane cholesterol by treatment of MDCK cells expressing recombinant ADAMTS13-V5-His with lovastatin and methyl-β-cyclodextrin completely abolished the raft association and apical secretion of ADAMTS13-V5-His in MDCK cells. Moreover, an ADAMTS13 mutation (4143insA), which naturally occurs in a patient with TTP that deletes the second CUB domain not only significantly impaired the amount of total ADAMTS13 protein secreted into the conditioned medium, but also reversed the polarity of ADAMTS13 secretion in MDCK cells. Our data demonstrate that: 1) active full-length ADAMTS13 protease is produced in human vascular endothelial cells; 2) the CUB domains are critical for apical secretion via their interaction with lipid rafts; 3) mutations in ADAMTS13 gene that disrupt sorting signals may result in congenital ADAMTS13 deficiency, leading to TTP in some cases. Considering a large surface area of the vascular endothelial cells, apically secreted ADAMTS13 from these cells may contribute significantly to plasma pool of ADAMTS13 protease.

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