Rate of Vasoconstrictor Prostanoids Released by Endothelial Cells Depends on Cyclooxygenase-2 Expression and Prostaglandin I Synthase Activity

From the Laboratory of Inflammation Mediators, Institute of Research of Santa Creu i Sant Pau Hospital, Barcelona, Spain.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—This study was undertaken to investigate the enzymatic regulation of the biosynthesis of vasoconstrictor prostanoids by resting and interleukin (IL)-1ß–stimulated human umbilical vein endothelial cells (HUVECs). Biosynthesis of eicosanoids in response to IL-1ß, exogenous labeled arachidonic acid (AA), or histamine, as well as their spontaneous release, was evaluated by means of HPLC and RIA. HUVECs exposed to IL-1ß produced prostaglandin (PG) I₂ for no longer than 30 seconds after the substrate was added irrespective of the cyclooxygenase (COX) activity, whereas the time course of PGE₂ and PGD₂ formation was parallel to the COX activity. The ratio of PGE₂ to PGD₂ produced by HUVECs was similar to that obtained by purified COX-1 and COX-2. Production of PGF₂ from exogenous AA was limited and similar in both resting and IL-1ß–treated cells. PGF₂ was the main prostanoid released into the medium during exposure to IL-1ß, whereas when HUVECs treated with IL-1ß were stimulated with histamine or exogenous AA, PGE₂ was released in a higher quantity than PGF₂. PGF₂ released into the medium during treatment with IL-1ß and the biosynthesis of PGE₂ and PGD₂ in response to exogenous AA or histamine increased with COX-2 expression, whereas this did not occur in the case of PGI₂. We observed that PGI synthase (PGIS) mRNA levels were not modified by the exposure to IL-1ß, but the enzyme was partially inactivated. When SnCl₂ was added to the incubation medium, the transformation of exogenous AA-derived PGH₂ into PGE₂ and PGD₂ was totally diverted toward PGF₂. Overall, these results support the conclusions that PGE₂ and PGD₂ (and also probably PGF₂) were nonenzymatically derived from PGH₂ in HUVECs. The concept that a high ratio of PGH₂ was released by the IL-1ß–treated HUVECs and isomerized outside the cell into PGE₂ and PGD₂ was supported by the biosynthesis of thromboxane B₂ by COX-inactivated platelets, indicating the uptake by platelets of HUVEC-derived PGH₂. The IL-1ß–induced increase in the release of PGH₂ by HUVECs was suppressed by the COX-2–selective inhibitor SC-58125 and correlated with both COX-2 expression and PGIS inactivation. An approach to the mechanism of inactivation of PGIS by the exposure to IL-1ß was performed by using labeled endoperoxides as substrate. The involvement of HO· in the PGIS inactivation was supported by the fact that deferoxamine, pyrrolidinedithiocarbamate, DMSO, mannitol, and captopril antagonized the effect of IL-1ß on PGIS to different degrees. The NO synthase inhibitor N^G-monomethyl-L-arginine also antagonized the PGIS inhibitory effect of IL-1ß, indicating that NO· was also involved. NO· reacts with O₂^-· to form peroxynitrite, which has been reported to inactivate PGIS. Homolytic fission of the O-O bond of peroxynitrite yields NO₂· and HO·. The fact that 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which reacts with NO· to form NO₂·, dramatically potentiated the IL-1ß effect suggests that NO₂· could be a species implicated in the inactivation of PGIS. Cooperation of HO· was supported by the fact that DMSO partially antagonized the effect of carboxy-PTIO. Although our results on the exact mechanism of the inactivation of PGIS caused by IL-1ß were not conclusive, they strongly suggest that both NO· and HO· were involved.

Key Words: cyclooxygenase • prostanoid • endothelial cell

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endothelium modulates the response of vascular smooth muscle to hormones, neurotransmitters, and platelet products by releasing relaxing factors, such as NO and prostaglandin (PG) I₂ (also termed prostacyclin), and contracting factors, such as endothelin-1, angiotensin II, and vasoconstrictor metabolites of arachidonic acid (AA).^{1 2 3 4 5 6 7}

PGI₂, PGF₂, and PGE₂ are the main eicosanoids detected in vitro in the incubates of endothelial cells under different experimental conditions, which include exogenously added AA and several agonists. PGD₂, 12-hydroxyheptadecatrienoic acid (HHT), 15-HETE, and 11-HETE have also been detected in minor amounts.^{8 9 10 11} All these eicosanoids are derived from cyclooxygenase (COX) activity in human umbilical vein endothelial cells (HUVECs).¹²

PGH₂ is the common precursor of the prostanoids whose formation from AA is catalyzed by 2 COX isoenzymes encoded by different genes: COX-1 and COX-2. COX-1 is expressed in a constitutive manner, and COX-2 is the isoenzyme inducible by mitogens and is overexpressed in inflammatory processes.^{13 14} Interleukin (IL)-1, together with tumor necrosis factor, plays a pivotal role in the inflammatory response and orchestrates a pleiad of secondary events that orient endothelial cells toward a proinflammatory and prothrombotic function by inducing adhesion molecules, endothelin-1, and coagulation factors.¹⁵ A characteristic activity of IL-1 is to induce COX-2 in several cell types, including endothelial cells.^{16 17 18 19}

PGI synthase (PGIS) transforms PGH₂ into what is the most characteristic prostanoid formed by resting endothelial cells, PGI₂.^{8 20} PGI₂ is the most potent platelet antiaggregatory agent, and it exhibits antiadhesive and smooth muscle–relaxing properties.^{2 21} PGH₂ also undergoes spontaneous or enzymatic transformation toward PGF₂, PGE₂, and PGD₂.^{22 23} A growing amount of data support the concept that untransformed PGH₂ is released in vivo by the vascular endothelium under different circumstances.^{24 25 26 27 28 29 30} The predominance of relaxing prostanoids, such as PGI₂, or contracting prostanoids, such as PGH₂ and PGF₂, released by endothelial cells will primarily depend on COX activity but also on the secondary pathways that yield the different prostanoids. PGI₂ release as a consequence of the exposure of endothelial cells to IL-1 has been widely reported.^{16 19 31 32 33 34 35 36 37} Nevertheless, when COX activity is overexpressed, PGIS activity could be the limiting step in the biosynthesis of PGI₂, and other prostanoids could be more stimulated by IL-1.^{33 36 37}

Although release of PGH₂ by endothelium both in vivo and ex vivo has been suggested, its regulation has not been systematically studied, and whether transformation of PGH₂ into PGF₂, PGE₂, and PGD₂ in endothelial cells is spontaneous or enzymatically catalyzed remains unclear. In fact, PGIS is the only enzyme implicated in prostanoid biosynthesis that has been found in endothelial cells in addition to COX. The objective of the present work was to evaluate the hypothesis that the release of vasoconstricting prostanoids is regulated by 2 enzyme activities, COX and PGIS, and that these prostanoids are released predominantly under inflammatory conditions, such as after exposure to IL-1.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture and Treatment
Endothelial cells were isolated from human umbilical veins and cultured as previously described.³⁵ When HUVECs cultured with 20% of FBS reached confluence, they were seeded into 6-well plates and maintained without heparin and endothelial cell growth factor for 48 hours before the addition (or not) of 10 U/mL human recombinant IL-1ß (50000 U/µg; purity, >98%; Boehringer Mannheim S.A.) in medium 199 containing 4% FBS and maintained for the indicated period of time until incubation with [¹⁴ C]AA or histamine.

Human dermal fibroblasts were isolated and cultured as described previously.³⁸ Cells cultured in 6-well plates, in passages 4 to 6, were maintained with 1% FBS for 48 hours before the addition of 10 nmol/L phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich Química S.A.). After 6 hours, COX-2 inhibition experiments were performed as described below.

The permanent endothelial cell line HUV-EC-C was cultured and treated with 10 nmol/L PMA for 6 hours as described previously³⁹ before COX-2 inhibition experiments. Experiments were performed with the HUV-EC-C line at passages 21 to 27.

Cells of the human erythroleukemia cell (HEL) line were obtained from the American Type Culture Collection (CRL 1730) and cultured in RPMI 1640 medium supplemented with 10% FBS, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂ in air and subcultured every 3 to 4 days. To study COX-1 activity, cells were used without further treatment.

Preparation of Washed Platelet Suspensions
Peripheral venous blood was drawn from healthy donors who had received no medication in the 2 weeks before extraction. Washed platelet suspensions were prepared as previously described.⁴⁰ Platelet density was 2x10⁹ platelets/mL for the PGH₂ trapping experiments and 1.7x10⁸ platelets/mL for the study of COX-1 inhibition. COX-inactivated platelet suspensions were obtained by incubating platelet-rich plasma with 200 µmol/L acetylsalicylic acid (ASA) dissolved in ethanol (final concentration of ethanol, 0.5% [vol/vol]) for 10 minutes at room temperature before the washing procedure.

Release of Prostanoids From Endogenous AA in Resting and IL-1ß–Treated HUVECs
Cells cultured in 6-well plates were incubated at 37°C in 2.5 mL medium 199 containing 4% FBS and 10 U/mL IL-1ß. At the indicated periods of time, prostanoids released into the medium were evaluated after 250-µL aliquots of media were collected and placed in tubes containing indomethacin (final concentration, 10 µmol/L). Samples were kept at -80°C until analysis of PGF₂, PGE₂, and 6-keto-PGF₁ by RIA (PGF₂ was from Amersham Ibérica; PGE₂ and 6-keto-PGF₁ were from Advanced Magnetics Inc).

Formation of Eicosanoids From Exogenous [¹⁴C]AA and Determination of COX Activity
After the indicated period of time of exposure to 10 U/mL IL-1ß, cells were incubated at 37°C in 0.5 mL medium 199 containing 10 mmol/L HEPES, and 25 µmol/L [¹⁴C]AA (55 to 58 mCi/mmol [1-¹⁴C]AA, Amersham Ibérica) was added in 5 µL of ethanol. At the indicated periods of time, the reactions were stopped by adding 1N HCl to yield pH 3 followed by 1 vol of cold methanol. COX activity was evaluated as the sum of all the eicosanoids formed through the COX pathway.

PGH₂ is an unstable prostaglandin that is converted into PGF₂ by mild reducing agents, such as SnCl₂.⁴¹ Therefore, to estimate the PGH₂ released, we calculated the difference between the PGF₂ peak of the samples from cells incubated as previously described and cells incubated in the presence of 200 µg/mL SnCl₂. Samples were kept at -80°C until analysis. HPLC analysis of eicosanoids was performed as previously described.⁴²

Release of Prostanoids From Endogenous AA in IL-1ß–Treated HUVECs in Response to Histamine
Cells untreated and treated with 10 U/mL IL-1ß for the indicated periods of time were incubated at 37°C in the presence of 0.5 mL medium 199 containing 10 mmol/L HEPES and 50 µmol/L histamine. After 10 minutes, supernatants were removed and placed in liquid N₂. Samples were then stored at -80°C until analysis of PGF₂, PGE₂, and 6-keto-PGF₁ by RIA.

COX-1–and COX-2–Specific mRNA Analysis
COX-1–and COX-2–specific mRNA levels were determined as previously described.^{38 39}

PGIS mRNA Analysis
Total RNA was isolated by phenol chloroform extraction according to the protocol described by Chomczynski and Sacchi⁴³ and quantified spectrophotometrically by absorption at 260 and 280 nm. The specific levels of PGIS mRNA were determined by means of a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Briefly, 1 µg of total RNA was reverse-transcribed into cDNA in the presence of 50 U reverse transcriptase of murine leukemia virus in a reaction buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 5 mmol/L MgCl₂, 2.5 µmol/L random hexamers, 20 U of RNAsin (Geneamp RNA PCR kit, Perkin-Elmer), and 1 mmol/L deoxynucleotide triphosphates (Epicentre Technologies) in a final volume of 20 µL. The reaction was stopped by heating for 5 minutes at 99°C and then 5 minutes at 5°C.

The primers used for the amplification of the PGIS-specific cDNA were purchased from Oxford Biomedical Research, Inc. The cDNA for GAPDH was amplified and used as the in- ternal control, and the sense and antisense primers for GAPDH were 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGGTCAGGTCCACC-3', respectively.¹⁶ The PCR was carried out in a DNA Thermal Cycler 480 (Perkin-Elmer) with a reaction mixture (100 µL) containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl₂, 0.2 mmol/L deoxynucleotide triphosphates, 0.25 µmol/L for PGIS and 1 µmol/L for GAPDH sense or anti-sense primers, 2.5 U Taq polymerase (Perkin-Elmer), and 4 µCi of [³H]deoxycytidine triphosphate (48 to 71 Ci/mmol, Amersham Ibérica). Aliquots were removed after 27, 30, and 35 cycles in order to test the linearity of the amplification. The profile of the amplification cycles was 94°C for 1 minute, 55°C for 2 minutes, and 72°C for 90 seconds. Amplification products were separated in a 1.5% low-melting-point agarose gel (GIBCO BRL) containing ethidium bromide (0.5 µg/mL, Perkin-Elmer). The bands were visualized under UV light and cut with a circular template. Radioactivity associated to the specific band was determined as previously described^{38 39} and normalized with respect to GAPDH.

Western Blotting of COX-1 and COX-2
Lysates of control cells and HUVECs treated with IL-1ß for the indicated period of time were prepared as previously described.¹⁹ Total protein equivalents of each sample were submitted to SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). After blocking nonspecific binding of antibody, membranes were incubated with a specific polyclonal antibody for COX-1 and COX-2 (Oxford Biomedical Research). Positive controls were performed by using isolated COX-1 from ram seminal vesicles (Oxford Biomedical Research) or COX-2 purified from sheep placenta (Cayman Chemical). Detection was performed by using the ECL System (Amersham) according to the manufacturer's instructions. To estimate the relative levels of each COX protein, the films were scanned in order to obtain digital images. The digital images of the bands were quantified, with the use of PC-Image analysis software (Foster Findlay Associates Ltd), by determining the sum of the gray density value of all pixels inside a rectangle drawn to encompass each band (the same-sized rectangle for all bands in a particular gel), and a ratio of gray density to rectangular area was obtained for each band. Results were then expressed as arbitrary units or normalized to the darkest band.

Trapping of HUVEC-Derived PGH₂ by Platelets
HUVECs in 6-well plates, untreated and treated with 10 U/mL IL-1ß for 24 hours, were incubated at 37°C in 0.450 mL medium 199 containing 10 mmol/L HEPES and 25 µmol/L [¹⁴C]AA. After 0.5 minutes, 10⁸ COX-inactivated platelets in 50 µL were added to the wells, and the reactions were stopped after another 9.5 minutes, as described previously. Inactivation of COX in the platelet suspensions was monitored previously by incubating 0.5-mL aliquots of the washed platelet suspensions with 25 µmol/L [¹⁴C]AA at 37°C for 5 minutes.

Trapping of PGH₂ from endogenous AA derived from resting and IL-1ß–treated HUVECs by platelets was achieved by adding 50 µL of a platelet suspension containing 10⁸ COX-inactivated platelets to HUVECs 0.5 minutes after cell challenge with 50 µmol/L histamine. After 10 minutes of the addition of histamine, supernatants were removed and placed in liquid N₂. Samples were then kept at -80°C until the analysis of 6-keto-PGF₁ and thromboxane (Tx) B₂ by RIA (TxB₂, Advanced Magnetics Inc).

Incubation With Isolated COX-1 and COX-2 and Obtaining of ¹⁴C-Labeled Endoperoxides
COX-1 (5 U/0.5 mL) isolated from ram seminal vesicles (Oxford Biomedical Research) or COX-2 purified from sheep placenta (Cayman Chemical) was incubated in 100 mmol/L Tris-HCl (pH 8.5) containing 2 mmol/L phenol and 5 µmol/L [¹⁴C]AA. Samples were incubated at 37°C for 5 minutes, after which the reaction was stopped by adding 1N HCl to yield pH 3 followed by 1 vol cold methanol. The products were subjected to HPLC analysis.

For the experiments concerning PGIS inactivation, an incubation of 180 U/mL of isolated COX-1 with 25 µmol/L [¹⁴C]AA at 30°C for 1 minute was used as a source of ¹⁴C-labeled endoperoxides. The reaction was stopped by adding an excess of indomethacin (final concentration, 10 µmol/L). In these conditions, the remaining untransformed [¹⁴C]AA was <10%, and the percentage of nondegraded endoperoxides, evaluated by the difference of PGF₂ after addition of 1 vol methanol or 1 vol methanolic solution of 400 µg/mL SnCl₂, was always >80%. Hence, the incubation mixture was immediately used as a source of labeled endoperoxides without further manipulation.

Effect of COX-2–Selective Inhibition on the Release of PGH₂
IL-1ß–treated HUVECs expressed both COX-1 and COX-2 in all experimental conditions. Therefore, in order to determine the concentration of the selective COX-2 inhibitor SC-58125^{39 44} (Laboratorios Almirall-Prodesfarma) to be used with HUVECs, cultures of PMA-treated human dermal fibroblasts and the HUV-EC-C line were used for COX-2 tests, and cultures of the HEL line and fresh human platelets were used for COX-1 tests. Fibroblasts and HUV-EC-C attached in 6-well plates, a 0.5-mL aliquot of HEL suspension (5x10⁶ cells/mL), or a 0.15-mL platelet suspension were incubated at 37°C in the presence of the indicated concentration of SC-58125 for 5 minutes. [¹⁴C]AA (25 µmol/L) was later added, and cells were incubated for another 5 minutes. Reactions were stopped, and COX-derived eicosanoids were analyzed as described above. COX activity was evaluated as the sum of all the chromatographic peaks corresponding to COX-derived eicosanoids.

As a result of these experiments, SC-58125 concentrations of 1 and 10 µmol/L were chosen to study the effect of selective inhibition of COX-2 on the release of PGH₂ by HUVECs. Resting HUVECs and HUVECs treated with 10 U/mL IL-1ß overnight were incubated with 1 and 10 µmol/L of SC-58125 for 5 minutes. HUVECs were then incubated in the presence of 25 µmol/L [¹⁴C]AA alone or in the presence of 200 µg/mL SnCl₂ for another 5 minutes as previously mentioned. The untransformed PGH₂ was then estimated as described above.

Effect of IL-1ß on the Inactivation of PGIS
To determine the PGIS activity after treatment of HUVECs with IL-1ß, cells cultured in 6-well plates were treated with 10 U/mL IL-1ß for 6 hours, without any additive or in the presence of final concentrations of the following: 1 mmol/L deferoxamine (Sigma), 1% (vol/vol) DMSO (Sigma), 1 mmol/L captopril (Laboratorios CINFA), 200 µmol/L pyrrolidinedithiocarbamate (PDTC, Sigma), 1 mmol/L 1,2,3-oxadiazolium, 5-amino-3-(4-morpholyn)-chloride (SIN-1, Cayman), 100 µmol/L 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO, Cayman), 50 U/mL superoxide dismutase (Sigma), 500 U/mL catalase (Sigma), 1 mmol/L N^G-monomethyl-L-arginine (L-NMMA, Cayman), 5 mmol/L mannitol (Merck), or 100 µmol/L tyrosine (Sigma) (all dissolved in culture medium) or 0.5 mmol/L phenylbutazone, 10 µmol/L indomethacin, or 50 µg/mL vitamin E (all from Sigma) dissolved in ethanol (final ethanol concentration, 0.25% [vol/vol]). Parallel incubations of HUVECs with the scavengers and inhibitors in the absence of IL-1ß were performed as controls. The culture medium was then removed, and HUVECs were treated with 100 µmol/L ASA in medium 199 for 10 minutes at 37°C. The ASA-containing medium was removed, and 200 µL of the solution containing the ¹⁴C-labeled endoperoxides was added. HUVECs were allowed to stand at 37°C for another 5 minutes. The final concentration of ¹⁴C-labeled endoperoxides was 18 µmol/L. ¹⁴C-labeled 6-keto-PGF₁ was evaluated by HPLC as described. Resting (control) and IL-1ß–treated HUVECs for each condition were always incubated with the same bach solution of ¹⁴C-labeled endoperoxides. Negative controls were performed using cells boiled for 15 minutes.

Synthesis of NO· in untreated and in IL-ß–treated cells was determined by assay of the culture supernatants for NO₂^- (nitrite) using the Griess reagent, which contains 1 vol of 10 g/L sulfanilamine in 50 mL/L H₃PO₄, and 1 vol of 1 g/L N-(1-naphthyl)ethylenediamine dihydrochloride in water (all from Sigma). An aliquot of 500 µL of the culture medium was reacted with an equal volume of the Griess reagent for 10 minutes at room temperature in the dark. The optical densities of the assay samples were measured at 550 nm. Concentrations of NO₂^- were determined from a standard curve prepared using NaNO₂ (Sigma) diluted in culture medium.

Statistics
Sigma-Stat software was used for statistical analysis. Statistical significance between groups was assessed using the Student t test for 2 groups. One-way ANOVA and the Student-Newman-Keus test were used to compare >2 groups. Correlations were evaluated by linear regression. A value of P<0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
AA Metabolism in HUVECs
As expected, resting and IL-1ß–treated HUVECs incubated with exogenous [¹⁴C]AA produced 6-keto-PGF₁, PGE₂, and PGF₂ as major eicosanoids, and PGD₂, HHT, 15-HETE, and 11-HETE were minor compounds.

COX-1 and COX-2 Expression
The time course of mRNA and protein expression of COX-1 and COX-2 was measured by incubating HUVECs with or without 10 U/mL of IL-1ß during various time intervals. Specific mRNAs encoding for both COX-1 and COX-2 were induced by IL-1ß in a time-dependent manner (Figure 1), although only the increase of COX-2 was statistically significant (P<0.05). The levels of COX-1 mRNA were slightly enhanced, whereas for COX-2 they were enhanced 12.5-fold by IL-1ß. The maximum levels of COX-2 mRNA were observed between 3 and 6 hours of exposure to IL-1ß.

View larger version (26K): [in this window] [in a new window]   Figure 1. Top, Levels of specific mRNA for COX-1 and COX-2 normalized with respect to GAPDH in HUVECs as a function of time of treatment with 10 U/mL IL-1ß. Values are mean+SD (n=4). *P<0.05 compared with time 0. Bottom, Representative photographs of electrophoresis of RT-PCR samples corresponding to the indicated periods of time of exposure to IL-1ß. Std indicates standard.

The Western blot analysis of the COX isoforms present in HUVECs is shown in Figure 2. In the absence of IL-1ß, the antibody against COX-1 recognized a band in HUVEC samples of 70 kDa corresponding to the migration of purified COX-1 from ram seminal vesicles. The antibody against COX-2 did not detect the purified COX-1 from ram seminal vesicles and had a slight reaction with a protein band of the unstimulated cells. In contrast, after 1 hour of IL-1ß treatment, the antibody against COX-2 peptide recognized a protein band in HUVEC samples of 70 kDa corresponding to the migration of purified COX-2 from sheep placenta. The densitometric analysis of the bands showed that after 3 hours, IL-1ß significantly increased the expression of COX-2 in a time-dependent manner, whereas modifications in the protein band corresponding to COX-1 over time were minimal. COX-2 expression reached a maximum level between 6 and 9 hours of IL-1ß exposure, followed by a slight decrease at 24 hours. Figure 2 shows that the total COX activity also increased in a time-dependent manner with the IL-1ß treatment, although it was only statistically significant after 6 hours of exposure to the cytokine. It was notable that whereas COX-2 protein levels decreased after 24 hours of exposure to IL-1ß, the total activity continuously increased until 24 hours.

View larger version (40K): [in this window] [in a new window]   Figure 2. Top, COX activity and protein expression as a function of time of exposure to IL-1ß. HUVECs were incubated with 10 U/mL IL-1ß for the indicated periods of time and were then incubated with 25 µmol/L [14C]AA for 10 minutes. The COX activity was evaluated as the sum of all HPLC peaks corresponding to eicosanoids formed through the COX pathway. Values are mean±SD (n=3). Cell lysates were subjected to Western blotting analysis as described in Material and Methods. The relative optical density (OD) of the bands, normalized for the darkest band on each gel, is shown to facilitate comparisons (mean±SD, n=4). *P<0.05 compared with time 0. Bottom, Representative photographs of Western blots. Isolated COX-1 (from ram seminal vesicles) and COX-2 (from sheep placenta) were used as positive controls.

Evaluation of Release of Prostanoids by HUVECs in Response to IL-1ß
Release of prostanoids by resting and 10 U/mL IL-1ß–treated cells was determined by monitoring the most significant prostanoids over 24 hours: PGE₂, PGF₂, and 6-keto-PGF₁. Results in Figure 3 show that after 24 hours, treatment with IL-1ß caused a higher increment in the quantity of PGF₂ (20-fold) and PGE₂ (16.5-fold) than of 6-keto-PGF₁ (3.5-fold) accumulated in the medium compared with no treatment. Levels of 6-keto-PGF₁ in the medium were abruptly enhanced 1 hour after exposure of cells to IL-1ß, whereas PGE₂ and PGF₂ were released into the medium more slowly, following the pattern of COX-2 expression. We should point out that 6-keto-PGF₁ was the only compound whose accumulated quantity in the culture medium was significantly enhanced (7-fold) 1 hour after exposure to IL-1ß, when COX-2 protein and activity was not enhanced (Figure 2). Release of PGE₂, PGF₂, and 6-keto-PGF₁ by control cells was quantitatively slight, and the 3 progression curves had similar patterns.

View larger version (17K): [in this window] [in a new window]   Figure 3. Time course of IL-1ß–stimulated release of prostanoids. HUVECs were incubated with (IL-1ß) and without (control) 10 U/mL IL-1ß for the indicated periods of time. Prostanoids in the medium were analyzed by RIA (n=3, mean±SD). *P<0.05 for IL-1ß–treated cells compared with control cells.

Biosynthesis of Eicosanoids From Exogenous AA
To observe the differences concerning synthesis of the different eicosanoids between those cells that overexpress COX-2 and resting cells, progression curves after the addition of labeled substrate were evaluated, and results are shown in Figure 4. The maximum levels of 6-keto-PGF₁ and PGF₂ were similar in both resting and IL-1ß–stimulated HUVECs (10 U/mL IL-1ß for 24 hours). Nevertheless, the amount of PGI₂ increased to a maximum 10 minutes after substrate addition in resting HUVECs, whereas the synthesis of PGI₂ practically ceased 1 minute after addition in the cells treated with IL-1ß. Furthermore, the amount of 6-keto-PGF₁ produced after 10 minutes by IL-1ß–treated cells was significantly lower than that produced by control cells. HHT was produced in a greater amount in IL-1ß–treated than in resting HUVECs. However, production stopped 1 minute after incubation, and the progression curve showed a pattern similar to that of 6-keto-PGF₁. In contrast, an increasingly higher production of PGE₂, PGD₂, and HETEs was observed in IL-1ß–treated cells compared with control cells (Figure 4).

View larger version (25K): [in this window] [in a new window]   Figure 4. Time course of prostanoid formation by HUVECs. Control cells and cells treated with 10 U/mL IL-1ß for 24 hours were incubated at 37°C for the indicated periods of time with 25 µmol/L [14C]AA, and eicosanoids were then analyzed by HPLC (n=3, mean±SD). *P<0.05 for IL-1ß–treated cells compared with control cells.

Effect of IL-1ß on the Expression of PGIS
Results presented in Figure 4 suggest that IL-1ß does not exert any effect on PGIS expression. To confirm this, PGIS mRNA levels were determined by RT-PCR, and results are shown in Figure 5. Results from 4 experiments indicated that PGIS mRNA was not increased in HUVECS exposed overnight to IL-1ß compared with control cells.

View larger version (30K): [in this window] [in a new window]   Figure 5. Levels of specific mRNA for PGIS expression normalized with respect to GAPDH in HUVECs that were untreated (control) and treated with 10 U/mL IL-1ß overnight. Data in the graph (bottom panel) are the mean of 4 separate experiments, and representative photographs (top panel) of electrophoresis of PCR samples corresponding to the indicated number of cycles are also shown. St indicates standard.

Characterization of the Biosynthetic Pathway of PGE₂ and PGD₂ in HUVECs
The slow time course of PGE₂, PGD₂, and HETE formation and the fact that they were the only eicosanoids that increased as a consequence of the induction of the COX-2 expression when an excess of exogenous substrate was supplied suggested that all of these eicosanoids could be formed in HUVECs by the exclusive intervention of COX. To verify that transformation of PGH₂ into PGE₂ and PGD₂ was not enzymatic, 2 different experimental approaches were carried out.

Figure 6 shows representative chromatograms corresponding to samples of HUVECs and isolated COX incubated with exogenous [¹⁴C]AA. Isolated COX-1 and COX-2 yield a ratio of PGE₂ to PGD₂ of 4.2±0.8 and 4.4±1.2 (mean±SD, n=5), respectively, similar to the ratio obtained in samples from HUVECs (3.9±0.99) (n=10). No 6-keto-PGF₁ was detected in the isolated COX incubations. In addition, the presence of SnCl₂ in the incubations of HUVECs with [¹⁴C]AA caused a complete diversion of the transformation of HUVEC-derived PGH₂ toward PGF₂ instead of toward PGE₂ plus PGD₂ (Figure 6).

View larger version (21K): [in this window] [in a new window]   Figure 6. Representative chromatograms of samples from IL-1ß–treated HUVECs (24 hours with 10 U/mL) incubated with 25 µmol/L of [14C]AA for 10 minutes in the absence and in the presence of SnCl2 and from 5 U of COX isolated from ram seminal vesicles incubated with 5 µmol/L [14C]AA for 5 minutes.

Comparison Between the Metabolism of Exogenous and Endogenous AA as a Function of Time of Incubation With IL-1ß
After treatment of HUVECs with 10 U/mL IL-1ß for different periods of time, cells were incubated in the presence of 25 µmol/L [¹⁴C]AA or 50 µmol/L histamine. Histamine-induced release of endogenous AA and the formation of the major PGs were evaluated by RIA. Results in Figure 7 show some differences between the effect of IL-1ß on the metabolism of exogenous and endogenous AA. When substrate was supplied exogenously, we observed a slight reduction in the ability of HUVECs to form PGI₂ (evaluated as 6-keto-PGF₁) and PGF₂ as a function of time of incubation with IL-1ß. In contrast, the ability to form PGE₂ and PGD₂ increased with the time of exposure to the cytokine and correlated with COX-2 expression. Whereas formation of PGI₂ and PGF₂ from endogenous substrate increased suddenly after 1 hour of incubation with IL-1ß and a sustained ability to form these PGs was then observed, statistically significant correlations were observed only between the increment in COX-2 protein expression and the increment in PGE₂ and PGD₂ formation from exogenous substrate and between the increment in COX-2 protein expression and the increment in PGE₂ formation in response to histamine (formation of PGD₂ from endogenous substrate was not determined; Figure 7).

View larger version (26K): [in this window] [in a new window]   Figure 7. Production of the major prostanoids formed by HUVECs from exogenous and endogenous substrate as a function of time of exposure to IL-1ß (top panels). Cells were treated with 10 U/mL IL-1ß for the indicated periods of time. They were later incubated with 25 µmol/L [14C]AA or with 50 µmol/L histamine for 10 minutes. Eicosanoids were analyzed by HPLC or RIA. Values are mean±SD (n=3). Correlations between the IL-1ß–increased prostanoids and the increment in COX-2 are also shown (bottom panels). OD indicates optical density. COX-2 protein expression after cell treatment with the cytokine for the indicated periods of time was evaluated by Western blotting in independent experiments (n=4).

Effect of IL-1ß on the Release of Untransformed PGH₂ by HUVECs From Exogenous and Endogenous Substrate
The amount of untransformed PGH₂ released by HUVECs from exogenous [¹⁴C]AA was estimated as the difference in the labeled PGF₂ between the samples incubated in the presence and absence of SnCl₂. After any period of time of exposure to IL-1ß, the formation of 6-keto-PGF₁ and HETEs from exogenous substrate was only minimally modified by the presence of SnCl₂ in the medium. In contrast, PGE₂ and PGD₂ completely disappeared in the samples containing SnCl₂, and the levels of PGF₂ increased concomitantly. HHT levels were also substantially reduced in the presence of SnCl₂ but were not totally suppressed (not shown). Figure 8 shows the estimated release of PGH₂ and PGI₂ (evaluated as 6-keto-PGF₁) relative to the resting cells as a function of time of incubation with IL-1ß. The increment in the release of PGH₂ into the medium was statistically correlated with the increase in COX-2 protein and with the decrement in PGI₂ formation (Figure 8). The ratio of PGH₂ to PGI₂ increased 4-fold as a consequence of treatment with IL-1ß.

View larger version (14K): [in this window] [in a new window]   Figure 8. Increment of untransformed PGH2 and PGI2 released by HUVECs as a function of time of exposure to IL-1ß relative to resting HUVECs (top panel). The PGH2 released by HUVECs was estimated as the difference between the PGF2 produced by HUVECs from exogenous AA in the presence and in the absence of SnCl2. PGI2 was evaluated as 6-keto-PGF1. Values are mean±SD (n=3). Correlations between the increment in PGH2 released and in COX-2 protein and between the increment in PGH2 released and the decrement in PGI2 are also shown (middle and bottom panels). OD indicates optical density. COX-2 protein after cell treatment with the cytokine for the indicated periods of time was evaluated by Western blotting in independent experiments (n=4).

PGE₂, PGF₂ and 6-keto-PGF₁ released from endogenous sources in HUVECs stimulated with histamine were also evaluated, after several periods of time of exposure to IL-1ß, both in the absence and presence of SnCl₂. Formation of 6-keto-PGF₁ was lower in the presence of SnCl₂ than in its absence (Figure 9), in contrast with results from the experiments adding exogenous [¹⁴C]AA. As expected, PGE₂ levels were substantially lower in the presence of SnCl₂ but were not totally suppressed. In addition, the production of PGF₂ was similar both in the absence and in the presence of SnCl₂ in particular when the activity of COX was maximum. Data from exogenous substrate experiments indicated that SnCl₂ did not modify COX and PGIS activities. Hence, the lower levels of prostanoids observed in the presence of SnCl₂ should be due to a less effective release of AA. This led to a decreased formation of PGH₂ and therefore a decreased production of PGI₂. The less effective mobilization of endogenous AA would also explain why PGF₂ did not increase as much as expected as a consequence of the diversion of the PGH₂ toward formation of PGF₂ instead of PGE₂ and PGD₂. Hence, although the results indicated that untransformed PGH₂ was also released from endogenous AA, the estimation of the amount of PGH₂ released was not possible when this indirect method was used.

View larger version (22K): [in this window] [in a new window]   Figure 9. Production of the major prostanoids formed by HUVECs from endogenous AA in response to histamine as a function of time of exposure to IL-1ß in the presence and in the absence of SnCl2 in the medium. Cells were treated with 10 U/mL IL-1ß for the indicated period of time and were later challenged with 50 µmol/L histamine for 10 minutes in the presence and in the absence of SnCl2. Values are mean±SD (n=3). *P<0.05 compared with time 0.

To evaluate the untransformed PGH₂ released by HUVECs from endogenous sources, PGH₂ was trapped by ASA-treated platelets, and its transformation by Tx synthase was evaluated. First, platelets treated with 200 µmol/L ASA were added to the wells of HUVECs and incubated in the presence of [¹⁴C]AA. ASA-treated platelets did not produce any eicosanoid derived from COX activity. Preliminary experiments served to choose the appropriate time to add the platelet suspensions to the HUVECs. COX-inactivated platelets were added to the HUVECs treated with IL-1ß at increasing periods of time after the addition of 25 µmol/L [¹⁴C]AA to the HUVECs. The maximum conversion of labeled PGH₂ into labeled TxB₂ and HHT was observed when 10⁸ platelets were added to HUVECs 0.5 minutes later than the addition of [¹⁴C]AA (not shown). Therefore, a delay of 0.5 minutes between the addition of [¹⁴C]AA and platelet suspensions was chosen for the following experiments.

Results in the Table show that the presence of platelets in the incubation mixture did not modify the amount of PGI₂ produced by HUVECs from endogenous or exogenous AA. ASA-treated platelets trapped HUVEC-derived PGH₂ from both exogenous and endogenous AA. HHT in platelets is formed by the action of Tx synthase.²⁰ The ratio of HHT to TxB₂ formed in our conditions was evaluated by incubating suspensions of platelets untreated with ASA with 25 µmol/L [¹⁴C]AA and determining both compounds by HPLC. This ratio was 1.47±0.18 (mean±SD, n=5), which allowed us to estimate the amount of PGH₂ transformed by ASA-treated platelets into TxB₂ plus HHT in the experiments using histamine. The estimated amounts of PGH₂ trapped by platelets from HUVECs incubated with [¹⁴C]AA and those incubated with 50 µmol/L histamine were comparable, indicating a substantial release of PGH₂ from endogenous AA.

View this table: [in this window] [in a new window]   Table 1. Production of PGI2 (Evaluated as 6-Keto-PGF1) and Amount of PGH2 Produced by IL-1ß–Stimulated HUVECs That Was Trapped by ASA-Treated Platelets

Effect of the Selective Inhibition of COX-2 on the Release of PGH₂
To select the most suitable concentration of the selective inhibitor in our experimental system, concentration-dependent inhibition curves on both COX-1 and COX-2 whole-cell systems were obtained by using human platelets and HEL line suspensions for COX-1 and the HUV-EC-C line and human dermal fibroblasts stimulated with PMA for COX-2. The [¹⁴C]AA concentration used was the same as that in HUVEC experiments. The COX activity in these systems was measured as the sum of all COX-derived compounds. As a result of these experiments, we chose 1 and 10 µmol/L SC-58125, since at these concentrations, inhibition of COX-2 was high and COX-1 was not substantially inhibited (see Figure 10). As expected, the inhibition of the release of PGH₂ evaluated using the SnCl₂ indirect method was consistent with a major COX-2 origin.

View larger version (29K): [in this window] [in a new window]   Figure 10. Effect of the selective COX-2 inhibitor SC-58125 on the release of PGH2 (bars). HUVECs treated with 10 U/mL IL-1ß overnight were incubated with and without the indicated concentrations of SC-58125 for 5 minutes before the addition of 25 µmol/L [14C]AA. The release of PGH2 was then estimated by the indirect method of the SnCl2. Error bars represent individual values (n=2). Inhibition curves of SC-58125 of COX-2 and COX-1 in whole-cell systems are also shown. The HUV-EC-C line and human dermal fibroblasts treated with PMA were used as COX-2 systems. In the experimental conditions used, COX-1 was slightly expressed in both fibroblasts and the HUV-EC-C line (not shown). The HEL line and human platelets were used as the COX-1 system. COX activity was evaluated as a sum of all COX-derived eicosanoids formed after incubation of cells with [14C]AA. Values are mean±SD (n=4).

Inactivation of PGIS During Incubation of HUVECs With IL-1ß
After treatment of HUVECs with 10 U/mL IL-1ß for 6 hours, cells were incubated with ¹⁴C-labeled endoperoxides, and the formation of PGI₂ as 6-keto-PGF₁ was measured. A significant decrease in the formation of PGI₂ was observed in cells after IL-1ß treatment compared with resting cells. This decrease was similar to that observed when cells were incubated with [¹⁴C]AA (Figure 11). These results clearly indicate that PGIS was partially inactivated during the treatment with IL-1ß.

View larger version (31K): [in this window] [in a new window]   Figure 11. Effect of several radical scavengers and inhibitors on the inactivation of PGIS during the exposure of HUVECs to IL-1ß. Cells were treated with 10 U/mL IL-1ß for 6 hours in the absence of any drug or in the presence of final concentration of 1 mmol/L captopril, 500 U/mL catalase, 100 µmol/L carboxy-PTIO (C-PTIO), 1 mmol/L deferoxamine, 1% (vol/vol) DMSO, 10 µmol/L indomethacin, 5 mmol/L mannitol, 1 mmol/L L-NMMA, 0.5 mmol/L phenylbutazone, 200 µmol/L PDTC, 50 U/mL superoxide dismutase (SOD), 100 µmol/L tyrosine (L-Tyr), 50 µg/mL vitamin E, or the indicated combinations of substances. Parallel incubations in the absence of IL-1ß were performed as controls. a-Tocopherol indicates -tocopherol. Cells were then incubated with [14C]endoperoxides as described in Materials and Methods. Results are expressed as percentage of increment with respect to their own controls. Values are mean±SD; the numbers in parentheses indicate the number of experiments. *P<0.05 compared with their own controls; #P<0.05 compared with the cells without scavengers or inhibitors (none); and @P<0.05 compared with C-PTIO alone.

To approach the mechanism of inactivation of PGIS in response to IL-1ß, cells were incubated with several radical scavengers and inhibitors during exposure to IL-1ß. Vitamin E and extracellular addition of catalase and superoxide dismutase did not exert any effect. In contrast, the Fe³⁺-chelating agent deferoxamine and the free radical scavengers PDTC and DMSO totally reverted the effect of IL-1ß. Mannitol and captopril also significantly prevented the effect of the cytokine. Indomethacin and phenylbutazone partially avoided the effect of IL-1ß, but no statistical significance was achieved. The effect of IL-1ß was totally suppressed by the NO synthase L-NMMA but not by extracellular addition of L-tyrosine. This was consistent with a significant 1.5-fold increase of the NO₂^- levels in the culture medium of IL-1ß–treated cells compared with control cells (492.3±117 and 320.5±63.3 pmol/10⁶ cells for 6 hours, respectively; mean±SD, n=6, P<0.05). In contrast, the presence the NO· scavenger carboxy-PTIO dramatically increased the PGIS inhibitory effect of IL-1ß. The effect of carboxy-PTIO was significantly prevented by DMSO but not by L-tyrosine. The inhibitors or scavengers referred in Figure 11 did not exert any statistically significant effect on the PGIS activity in IL-1ß–untreated cells. Additional experiments were performed exposing the cells to 1 mmol/L of the NO· and ·O₂^- (peroxynitrite) donor SIN-1 in the absence or presence of 10 U/mL IL-1ß for 6 hours, and a significant inhibition of PGIS was achieved in resting and IL-1ß–treated cells (51.51±16.14% and 41.2±19.9%, respectively; mean±SD, n=3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In 1978, Marcus et al⁸ suggested that PGE₂ and PGD₂ could be formed nonenzymatically in HUVECs. Their suggestion was based on the observation that PGH₂ incubated in the absence of cells yielded a mixture of all the prostanoids except PGI₂. Nevertheless, COX-2 had not yet been discovered at that time. We have now extended this research in order to verify the hypothesis that endothelial cells express only 2 enzymes that are involved in prostanoid biosynthesis, COX and PGIS, and that when COX-2 is overexpressed, accumulated PGH₂ is effectively released outside the cell.

Our results indicate that HUVECs do not transform PGH₂ into PGE₂ and PGD₂ enzymatically. This concept is supported by the following: (1) Progression curves indicate that the formation of PGE₂ and PGD₂ is substantially slower than that of the other prostanoids and overlap those of 15-HETE and 11-HETE, whose formation does not require later enzymatic steps.⁴⁵ (2) The ratio of PGE₂ to PGD₂ formed by HUVECs from exogenous substrate was almost identical to that yielded by isolated COX. (3) The presence of SnCl₂ totally diverted exogenous AA-derived PGH₂ toward PGF₂ instead of PGE₂ and PGD₂.

In agreement with other reports,^{16 33 34 36 46} we observed that release of PGI₂ by HUVECs was increased in cells exposed to IL-1ß compared with resting cells. However, the release of PGF₂ and PGE₂ was substantially more induced by IL-1ß than was the release of PGI₂. The mobilization of AA from cell lipids induced by IL-1ß^{46 47} could account for the fact that the cytokine-induced release of PGI₂ occurred (Figure 3) before an appreciable increase in the COX expression and activity (Figure 2). In contrast, a slow PGF₂ and PGE₂ accumulation in the medium was observed as COX-2 increased. This indicated that a COX-2–dependent excess of PGH₂, which was not transformed into PGI₂, was formed during the incubation with IL-1ß. On the other hand, the ability of cells to form PGE₂ and PGD₂ from exogenous AA was greatly enhanced with the expression of COX-2 (Figure 7). The same phenomenon was observed when mobilization of endogenous AA was induced by histamine; the ability to synthesize PGE₂ (in this case PGD₂ was not measured) also correlated with COX-2 expression (Figure 7). This was consistent with the data reported by Bull et al,³⁶ who found that PGE₂ was the most enhanced eicosanoid in response to histamine after pretreatment of dermal microvascular endothelial cells with IL-1ß. The fact that the increased ability to form PGI₂ in response to histamine was already observed after 1 hour of IL-1ß-exposure, when COX activity was not appreciably induced, suggests that IL-1ß exerted priming in the histamine-induced AA mobilization.

Since biosynthesis of PGE₂ and PGD₂ are nonenzymatic in HUVECs, PGH₂ could be transformed into a mixture of these position isomers outside the cell. The ratio of untransformed [¹⁴C]AA-derived PGH₂ to PGI₂ released by HUVECs as a function of time of exposure to IL-1ß overlapped with the increase in the COX activity and COX-2 expression. This indicates that the presence of COX-2 causes a dramatic increase in the ratio of PGH₂ to PGI₂ released by HUVECs when exogenous substrate is supplied. The fact that the COX-2–selective inhibitor SC-58125^{39 44} inhibited, in a concentration-dependent manner, the release of PGH₂ at concentrations that did not appreciably inhibit COX-1 also supports this conclusion (Figure 10). A substantial release of PGH₂ formed from endogenous AA could be demonstrated by trapping with ASA-treated platelets. In contrast to that which occurred in the exogenous substrate experiments, SnCl₂ was unable to completely suppress PGE₂ formation when IL-1ß–treated cells were stimulated with histamine. This may be due to the endogenous and exogenous AA having access to the COX located in different intracellular compartments. PGH₂ generated from endogenous sources could be partially converted nonenzymatically into PGE₂ inside the cell.

The formation of PGF₂ by 2-electron reduction of PGH₂ could occur nonenzymatically inside or partially outside the cells. Our results are not conclusive with respect to these points. Nevertheless, unlike 6-keto-PGF₁, formation of PGF₂ showed a slow progression curve consistent with a nonenzymatic reduction of PGH₂. In contrast with that which occurred with PGE₂ and PGD₂, PGF₂ did not increase with the COX activity when an excess of exogenous substrate was supplied. This was consistent with an enzymatic and/or a electron donor–dependent synthesis. These facts taken together strongly suggest that formation of PGF₂ in endothelial cells is limited by the presence of some cell-derived electron donor rather than by an enzymatic activity posterior to COX. The cellular nature of the electron donor is suggested by the fact that the relative quantity of PGF₂ formed in the incubations with isolated COX was negligible. We can speculate that a slow turnover of such an electron donor(s) could account for the predominant formation of PGF₂ when the slow release of prostanoids caused by the presence of IL-1ß was measured (Figure 3). In contrast, when an excess of exogenous AA was supplied, or a high quantity of AA was suddenly released by the action of histamine, a great amount of PGH₂ was rapidly formed. As a consequence, this factor(s) could be rapidly depleted and finally exhausted, thus limiting the transformation of PGH₂ into PGF₂. Under such conditions, PGE₂ and PGD₂ would be the predominant nonenzymatic transformations of PGH₂ (Figures 4 and 7).

These considerations lead to the idea that COX and PGIS are the only enzymes involved in the biosynthesis of prostanoids in endothelial cells. Moreover, progression curves indicate that PGE₂, PGD₂, and PGF₂ were formed in detectable amounts, mainly when PGIS became inactive, which occurred faster in the IL-1ß–treated cells than in the resting HUVECs (Figure 4). The faster inactivation of PGIS under COX-stimulated conditions yielding an excess of PGH₂ is consistent with the "suicide" behavior of PGIS.⁴⁸ The fact that the levels of mRNA for PGIS were not induced by IL-1ß indicates that the only enzyme activity regulated at the transcriptional level was COX, particularly COX-2. This was consistent with the fact that the PGI₂ that formed when an excess of exogenous substrate was supplied did not increase according to the increment of COX-2 protein and activity. Moreover, PGI₂ formation ability was reduced as a function of time of treatment with IL-1ß, reaching a minimum 6 to 9 hours after the addition of IL-1ß (Figure 8), when the expression of COX was maximum (Figure 2).

The partial inactivation of PGIS as a consequence of exposure of HUVECs to IL-1ß was further confirmed by supplying ¹⁴C-labeled endoperoxides and analyzing the formation of labeled 6-keto-PGF₁. The inhibition of PGIS observed after exposure to IL-1ß using labeled endoperoxides as substrate was comparable to that obtained the addition of [¹⁴C]AA (Figure 10). The partial inactivation of PGIS also contributed to the accumulation of an excess of PGH₂, and the increase of PGH₂ released by HUVECs correlated not only with COX-2 expression but also with PGIS inactivation (Figure 8).

Evidence that the HO· radical was involved in the inactivation of PGIS was provided by the following: (1) The use of DMSO, PDTC, mannitol, and captopril, powerful HO· scavengers,^{49 50 51 52} partially or totally antagonized the effect of IL-1ß. (2) Iron-dependent HO· formation from ·O₂^--generating systems such as Fenton or Haber-Weiss reactions was inhibited by deferoxamine, a powerful Fe³⁺ chelator,⁴⁹ which also blocked the inactivation of PGIS induced by IL-1ß. (3) -Tocopherol did not antagonize the IL-1ß–induced inactivation of PGIS, since -tocopherol did not react efficiently with HO·.⁵³ Catalase and superoxide dismutase added to the extracellular phase did not exert any effect on the inactivation of PGIS, probably because of the fast intracellular generation of HO· from H₂O₂ generated by dismutation of ·O₂^- or from homolytic fission of ONOOH, with the extracellular presence of these enzymes being not effective.

Peroxidase activity as a source of free radicals has been reported to be involved in the inactivation of PGIS.⁵⁴ Deferoxamine and PDTC are also inhibitors of the peroxidase activity of COX.⁵⁵ Since most of the COX inhibitors do not exert any effect on peroxidase activity,⁵⁵ the fact that indomethacin and phenylbutazone partially (but without statistical significance) inhibited the inactivation of PGIS by IL-1ß would indicate that PGG₂ contributed to the production of the inactivating oxygen-derived free radicals.

Another explanation has been given by Zou and Ullrich,⁵⁶ who found that peroxynitrite irreversibly inhibits PGIS. Our results from L-NMMA experiments, which inhibited inactivation of PGIS by IL-1ß, are consistent with those of these authors, who suggested that NO· formation is the limiting step in the peroxynitrite production in endothelial cells and may be induced by the inflammatory cytokines. Actually, exposition to IL-1ß caused a significant 1.5-fold increase in the NO· released by HUVECs in terms of NO₂^-, although we did not evaluate the expression of the inducible NO synthase. The fact that the NO· scavenger carboxy-PTIO⁵⁷ significantly increased the inhibitory effect of IL-1ß in the present study was in apparent contradiction with this. Nevertheless, carboxy-PTIO equimolarly reacts with NO· to yield 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl plus NO₂·.⁵⁷ Homolytic fission of peroxynitrous acid, formed by protonation of peroxynitrite, yields NO₂· and HO·.^{58 59} Zou et al⁶⁰ recently reported that nitration of an active site–related tyrosine could account for the PGIS inactivation produced by peroxynitrite. These authors proposed an O-O homolytic cleavage in the peroxynitrite molecule caused by the heme iron of the PGIS. This could finally yield NO₂⁺-nitrating species, which may attack tyrosine or tyrosinate.⁶⁰ Nevertheless, nitration of tyrosyl residues by NO₂· is also possible through the formation of tyrosyl radicals,^{61 62} which could be favored by HO·. Our results from carboxy-PTIO experiments are consistent with an inactivation of the PGIS mediated by NO₂·. The fact that the presence of DMSO in addition of carboxy-PTIO decreased the ratio of inactivated PGIS, when compared with carboxy-PTIO alone, suggests that HO· was also involved. Consistently, another mechanism by which deferoxamine could prevent the effect of IL-1ß on PGIS inactivation may be the direct reaction with peroxynitrite.⁵⁹ We also observed that SIN-1, which releases both NO· and ·O₂^- as a source of peroxynitrite, inactivated PGIS in both resting and IL-1ß–treated HUVECs. In summary, our results about the exact mechanism of the partial inactivation of PGIS caused by the exposure of HUVECs to IL-1ß are not conclusive, but they strongly suggest that both NO· (which could generate peroxynitrite that undergoes homolytic cleavage of the O-O bond to yield NO₂·) and HO· were involved.

Present results provide a biochemical explanation for the production and release of the constrictive prostanoids, PGH₂ and PGF₂, by endothelial cells. Predominance of relaxing or constricting prostanoids will depend on both COX and PGIS activities. An impairment of endothelial cell function occurs under inflammatory conditions, and COX activity is increased by means of the induction of COX-2 and by partial inactivation of PGIS. Under such conditions, endothelial cells could produce an excess of PGH₂ that could exert proinflammatory and prothrombotic activities before its conversion to other prostaglandins inside and outside the endothelium. PGH₂ from endothelial cells enriched in COX-2 could also be converted into TxA₂ by platelets and macrophages. Microvessel expression of COX-2 could contribute to the platelet activation observed in the disseminated intravascular coagulation, usually associated with systemic inflammatory responses. By use of an antagonist of the PGH₂/TxA₂ receptor, it has been shown that endogenous PGH₂ decreases the thrombolytic activity of tissue plasminogen activator.⁶¹ PGH₂ and TxA₂ stimulate proliferation of vascular smooth muscle cells in vitro.^{62 63 64 65 66} Expression of COX-2 could also contribute to the myointimal hyperplasia observed in atherosclerosis by promoting the formation of PGH₂ (and transcellular TxA₂) and hydroxy linoleic acids,¹⁹ which elicit smooth muscle cell mitogenesis.⁶⁷

In summary, our results show that resting HUVECs and HUVECs exposed to IL-1ß produce only PGH₂ and PGI₂ enzymatically. Production of PGF₂ could apparently be linked to the presence of a limiting intracellular electron donor. Overexpression of COX-2 turns the endothelial cell toward a phenotype characterized by an increased production of PGH₂, which cannot be totally transformed into PGI₂, partly because of its inactivation. Despite the fact that caution and more research is required to assess the biological relevance of our findings in vivo, the present data increase our knowledge about the metabolism of AA by endothelial cells and about the possible biological role of the induction of COX-2 in the vascular endothelium.

	Acknowledgments

 
This study was supported by grants from Institut de Recerca of the HSCSP, FIS 94/1559, and SAF96-0138. The authors wish to thank Dr de Castellarnau for her suggestions and Cristina Gerbolés, Esther Gerbolés, and Aurora Sierra for their technical assistance.

	Footnotes

 
Reprint requests to Dr Luis Vila, H.S. Creu i S. Pau (Casa de Convalecencia), S. Antonio M^a Claret 167, 08025-Barcelona, Spain.

Preliminary data were presented as a poster at the 9th International Conference on Prostaglandins and Related Compounds, Florence, Italy, June 6–10, 1994.

Received March 13, 1998; accepted May 18, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Vane JR, Änggard EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med. 1990;323:27–36.[Medline] [Order article via Infotrieve]

2. Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane A₂, and prostacyclin. Pharmacol Rev. 1979;30:293–331.[Medline] [Order article via Infotrieve]

3. Gryglewski RJ, Botting RM, Vane JR. Mediators produced by endothelial cell. Hypertension. 1988;12:530–548.[Abstract/Free Full Text]

4. Rees DD, Palmer RMJ, Hodson HF, Moncada S. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol. 1989;96:418–424.[Medline] [Order article via Infotrieve]

5. Richard V, Tanner FC, Tschudi MR, Lüscher TF. Differential activation of the endothelial L-arginine pathway by bradykinin, serotonin and clonidine in porcine coronary arteries. Am J Physiol. 1990;259:H1433–H1439.[Abstract/Free Full Text]

6. Tschudi M, Richard V, Bühler FR, Lüscher TF. Importance of endothelium-derived nitric oxide in intramyocardial porcine coronary arteries. Am J Physiol. 1990;160:H13–H20.

7. Lüscher TF. The endothelium in hypertension: bystander, target or mediator? J Hypertens. 1994;12(suppl 10):S105–S116.

8. Marcus AJ, Weksler BB, Jaffe EA. Enzymatic conversion of prostaglandin endoperoxide H₂ and arachidonic acid to prostacyclin by cultured human endothelial cells. J Biol Chem. 1978;253:7138–7141.[Free Full Text]

9. Alhenc-Gelas F, Tsai SJ, Callahan KS, Campbell WB, Johnson AR. Stimulation of prostaglandin formation by vasoactive mediators in cultured human endothelial cells. Prostaglandins. 1982;24:723–742.[Medline] [Order article via Infotrieve]

10. Kühn H, Pönicke K, Halle W, Wiesner R, Schewe T, Förster W. Metabolism of [1-¹⁴C]-arachidonic acid by cultured calf aortic endothelial cells: evidence for the presence of a lipoxygenase pathway. Prostaglandins Leukot Med. 1985;17:291–303.[Medline] [Order article via Infotrieve]

11. Hopkins NK, Oglesby TD, Bundy GL, Gorman RR. Biosynthesis and metabolism of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid by human umbilical vein endothelial cells. J Biol Chem. 1984;259:14048–14053.[Abstract/Free Full Text]

12. López S, Vila L, Breviario F, de Castellarnau C. Interleukin-1 increases 15-hydroxyeicosatetraenoic acid formation in cultured human endothelial cells. Biochim Biophys Acta. 1993;1170:17–24.[Medline] [Order article via Infotrieve]

13. Smith WL. Prostanoid biosynthesis and mechanisms of action. Am J Physiol. 1992;263:F181–F191.[Abstract/Free Full Text]

14. Hla T, Ristimäki A, Appleby S, Barriocanal JG. Cyclooxygenase gene expression in inflammation and angiogenesis. Ann N Y Acad Sci. 1993;696:197–204.[Medline] [Order article via Infotrieve]

15. Dinarello CA. Interleukin-1 and interleukin-1 antagonism. Blood. 1991;77:1627–1652.[Abstract/Free Full Text]

16. Maier JAM, Hla T, Maciag T. Cyclooxygenase is an immediate-early gene induced by interleukin-1 in human endothelial cells. J Biol Chem. 1990;265:10805–10808.[Abstract/Free Full Text]

17. Hla T, Neilson K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci U S A.. 1992;89:7384–7388.[Abstract/Free Full Text]

18. Jones DA, Carlton D, McIntyre MT, Zimmerman GA, Prescott SM. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J Biol Chem. 1993;268:9049–9054.[Abstract/Free Full Text]

19. Camacho M, Godessart N, Antón R, García M, Vila L. Interleukin-1 enhances the ability of cultured umbilical vein endothelial cells to oxidize linoleic acid. J Biol Chem. 1995;270:17279–17286.[Abstract/Free Full Text]

20. Hecker M, Ullrich V. On the mechanism of prostacyclin and thromboxane A₂ biosynthesis. J Biol Chem. 1989;264:141–150.[Abstract/Free Full Text]

21. Honn KV, Cicone B, Skoff A. Prostacyclin: a potent antimetastatic agent. Science. 1981;212:1270–1272.[Abstract/Free Full Text]

22. Nugteren DH, Christ-Hazelhof E. Chemical and enzymatic conversions of prostaglandin endoperoxide PGH₂. Adv Prostaglandin Thromboxane Res. 1980;6:129–137.[Medline] [Order article via Infotrieve]

23. Smith WL. Prostanoid biosynthesis and mechanisms of action. Am J Physiol. 1992;263:F181–F191.

24. Koga T, Takayata Y, Kobayashi K, Takishita S, Yamashita Y, Fujishima M. Age and hypertension promote endothelium-dependent contractions to acetylcholine in the aorta of the rat. Hypertension. 1989;14:542–548.[Abstract/Free Full Text]

25. Pagano PJ, Lin L, Sessa WC, Nasjletti A. Arachidonic acid elicits endothelium-dependent release from the rabbit aorta of a constrictor prostanoid resembling prostaglandin endoperoxides. Circ Res. 1991;69:396–405.[Abstract/Free Full Text]

26. Shimizu K, Muramatsu M, Kakegawa Y, Asano H, Toki Y, Miyazaki Y, Okumura K, Hashimoto H, Ito T. Role of prostaglandin H₂ as an endothelium-derived contracting factor in diabetic state. Diabetes. 1993;42:1246–1252.[Abstract]

27. Williams SP, Campbell AK, Roscell N, Myatt L, Leikauf GD, Rapoport RM. Modulation of phorbol ester-induced contraction by endogenously released cyclooxygenase products in rat aorta. Am J Physiol. 1994;267:H1654–H1662.[Abstract/Free Full Text]

28. Fu-Xiang D, Skopec J, Diederich A, Diederich D. Prostaglandin H₂ and thromboxane A₂ are contractile factors in intrarenal arteries of spontaneously hypertensive rats. Hypertension. 1992;19:795–798.[Abstract/Free Full Text]

29. Lin L, Balazy M, Pagano PJ, Nasjletti A. Expression of prostaglandin H₂–mediated mechanism of vascular contraction in hypertensive rats: relation to lipoxygenase and prostacyclin synthase activities. Circ Res. 1994;74:197–205.[Abstract/Free Full Text]

30. Asano H, Shimizu K, Muramatsu M, Iwama Y, Toki Y, Miyazaki Y, Okumura K, Hashimoto H, Ito T. Prostaglandin H₂ as an endothelium-derived contracting factor modulates endothelin-1-induced contraction. J Hypertens. 1994;12:383–390.[Medline] [Order article via Infotrieve]

31. Dejana E, Breviario F, Balconi G, Rossi V, Remuzzi G, de Gaetano G, Mantovani A. Stimulation of prostacyclin synthesis in vascular cells by mononuclear cell products. Blood. 1984;64:1280–1283.[Abstract/Free Full Text]

32. Rossi V, Breviario F, Ghezzi P, Dejana E, Mantovani A. Prostacyclin synthesis induced in vascular cells by interleukin-1. Science. 1985;229:174–176.[Abstract/Free Full Text]

33. Albrightson CR, Baenziger NL, Needleman P. Exaggerated human vascular cell prostaglandin biosynthesis mediated by monocytes: role of monokines and interleukin 1. J Immunol. 1985;135:1872–1877.[Abstract]

34. Rustin MHA, Bull HA, Dowd PM. Effect of human recombinant interleukin-1 on release of prostacyclin from human endothelial cells. Br J Dermatol. 1989;120:153–159.[Medline] [Order article via Infotrieve]

35. Bull HA, Dowd PM. Interleukin-1 potentiates histamine-induced release of prostacyclin from human endothelial cells. Br J Pharmacol. 1990;101:703–709.[Medline] [Order article via Infotrieve]

36. Bull HA, Cohen J, Dowd PM. Responses of human dermal microvascular endothelial cells to histamine and their modulation by interleukin 1 and substance P. J Invest Dermatol. 1991;97:787–792.[Medline] [Order article via Infotrieve]

37. Ristimäki A, Garfinkel S, Wessendorf J, Maciag T, Hla T. Induction of cyclooxygenase-2 by interleukin-1 evidence for post-transcriptional regulation. J Biol Chem. 1994;269:11769–11775.[Abstract/Free Full Text]

38. Godessart N, Camacho M, López-Belmonte J, Antón R, García M, de Moragas J-M, Vila L. Prostaglandin H-synthase-2 is the main enzyme involved in the biosynthesis of octadecanoids from linoleic acid in human dermal fibroblasts stimulated with IL-1ß. J Invest Dermatol. 1996;107:726–732.[Medline] [Order article via Infotrieve]

39. Miralpeix M, Camacho M, López-Belmonte J, Canalías F, Beleta J, Palacios JM, Vila L. Selective induction of cyclooxygenase-2 activity in the permanent human endothelial cell line HUV-EC-C: biochemical and pharmacological characterization. Br J Pharmacol. 1997;121:171–180.[Medline] [Order article via Infotrieve]

40. Vila L, Cullaré C, Solá J, Puig L, de Castellarnau C, de Moragas JM. Cyclooxygenase activity is increased in platelets from psoriatic patients. J Invest Dermatol. 1991;97:922–926.[Medline] [Order article via Infotrieve]

41. Hamberg M, Svensson J, Wakabayashi T, Samuelsson B. Isolation and structure of two prostaglandin endoperoxides that cause platelet aggregation. Proc Natl Acad Sci U S A.. 1974;71:345–349.[Abstract/Free Full Text]

42. Solá J, Godessart N, Vila L, Puig L, de Moragas JM. Epidermal cell-polymorphonuclear leukocyte cooperation in the formation of leukotriene B₄ by transcellular biosynthesis. J Invest Dermatol. 1992;98:333–339.[Medline] [Order article via Infotrieve]

43. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]

44. Penning TD, Talley JJ, Bertenshaw SR, Carter JS, Collins PW, Docter S, Graneto MJ, Lee LF, Malecha JW, Miyashiro JM, Rogers RS, Rogier DJ, Yu SS, Anderson GD, Burton EG, Cogburn JN, Gregory SA, Koboldt CM, Perkins WE, Seibert K, Veenhuizen AW, Zhang YY, Isakson PC. Synthesis and biological evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methyl-phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-58635, Celecoxib). J Med Chem. 1997;40:1347–1365.[Medline] [Order article via Infotrieve]

45. Hecker M, Ullrich V, Fischer C, Meese CO. Identification of novel arachidonic acid metabolites formed by prostaglandin H synthase. Eur J Biochem. 1987;169:113–123.[Medline] [Order article via Infotrieve]

46. Breviario F, Proserpio P, Bertocchi F, Lampugnani MG, Mantovani A, Dejana E. Interleukin-1 stimulates prostacyclin production by cultured human endothelial cells by increasing arachidonic acid mobilization and conversion. Arteriosclerosis. 1990;10:129–134.[Abstract/Free Full Text]

47. García JGN, Stasek JE, Bahler C, Natarajan V. Interleukin 1-stimulated prostacyclin synthesis in endothelium: lack of phospholipase C, phospholipase D, or protein kinase C involvement in early signal. J Lab Clin Med. 1992;120:929–940.[Medline] [Order article via Infotrieve]

48. Wade ML, Voelkel NF, Fitzpatrick FA. "Suicide" inactivation of prostaglandin I₂ synthase: characterization of mechanism-based inactivation with isolated enzyme and endothelial cells. Arch Biochem Biophys. 1995;321:453–458.[Medline] [Order article via Infotrieve]

49. Halliwell B, Gutteridge JMC. Protection against oxidants in biological systems: the superoxide theory of oxygen toxicity. In: Halliwell B, Gutteridge JMC, eds. Free Radicals in Biology and Medicine. 2nd ed. Oxford, UK: Oxford University Press; 1989:86–187.

50. Tsai J-C, Jain M, Hsieh C-M, Lee W-S, Yoshizumi M, Patterson C, Perrella MA, Cooke C, Wang H, Haber E, Schlegel R, Lee M-E. Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells. J Biol Chem. 1996;271:3667–3670.[Abstract/Free Full Text]

51. Abello PA, Fidler SA, Bulkley GB, Buchman TG. Antioxidants modulate induction of programmed endothelial cell death (apoptosis) by endotoxin. Arch Surg. 1994;129:134–141.[Abstract/Free Full Text]

52. Bagchi D, Prasad R, Das DK. Direct scavenging of free radicals by captopril, and angiotensin converting enzyme inhibitor. Biochem Biophys Res Commun. 1989;158:52–57.[Medline] [Order article via Infotrieve]

53. Liebler DC. The role of metabolism in the antioxidant function of vitamin E. Crit Rev Toxicol. 1993;23:147–169.[Medline] [Order article via Infotrieve]

54. Ham EA, Egan RW, Soderman DD, Gale PH, Kuehl FA Jr. Peroxidase-dependent deactivation of prostacyclin synthetase. J Biol Chem. 1979;254:2191–2194.[Abstract/Free Full Text]

55. Tam SSC, Lee DHS, Wang EY, Munroe DG, Lau CY. Tepoxalin, a novel dual inhibitor of the prostaglandin-H synthase cyclooxygenase and peroxidase activities. J Biol Chem. 1995;270:13948–13955.[Abstract/Free Full Text]

56. Zou M-H, Ullrich V. Peroxynitrite formed by simultaneous generation of nitric oxide and superoxide selectively inhibits bovine aortic prostacyclin synthase. FEBS Lett. 1996;382:101–104.[Medline] [Order article via Infotrieve]

57. Akaike T, Yoshida M, Miyamoto Y, Sato K, Kohno M, Sasamoto K, Miyazaki K, Ueda S, Maeda H. Antagonistic action of imidazolineoxyl N-oxides against endothelium-derived relaxing factor/·NO through a radical reaction. Biochemistry. 1993;32:827–832.[Medline] [Order article via Infotrieve]

58. Hogg N, Darley-Usmar VM, Wilson MT, Moncada S. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem J. 1992;281:419–424.

59. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A.. 1990;87:1620–1624.[Abstract/Free Full Text]

60. Zou MH, Martin C, Ullrich V. Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite. Biol Chem. 1997;378:707–713.

61. Prütz WA, Mönig H, Butler J, Land EJ. Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins. Arch Biochem Biophys. 1985;243:125–134.[Medline] [Order article via Infotrieve]

62. Van der Vliet A, Eiserich JP, O'Neill CA, Halliwell B, Cross CE. Tyrosine modification by reactive nitrogen species: a closer look. Arch Biochem Biophys. 1995;319:341–349.[Medline] [Order article via Infotrieve]

63. Golino P, Rosolowsky M, Yao S-K, McNatt J, De Clerck F, Buja LM, Willerson JT. Endogenous prostaglandin endoperoxides and prostacyclin modulate the thrombolytic activity of tissue plasminogen activator: effects of simultaneous inhibition of thromboxane A₂ synthase and blockade of thromboxane A₂/prostaglandin H₂ receptors in a canine model of coronary thrombosis. J Clin Invest. 1990;86:1095–1102.

64. Morinelli TA, Zhang L-M, Newman WH, Meier KE. Thromboxane A₂/prostaglandin H₂-stimulated mitogenesis of coronary artery smooth muscle cells involves activation of mitogen-activated protein kinase and S6 kinase. J Biol Chem. 1994;25:5693–5698.

65. Sachinidis A, Flesch M, Ko Y, Schrör K, Böhm M, Düsing R, Vetter H. Thromboxane A₂ and vascular smooth muscle cell proliferation. Hypertension. 1995;26:771–780.[Abstract/Free Full Text]

66. Jones DA, Benjamin CW, Linseman DA. Activation of thromboxane and prostacyclin receptors elicits apposing effects on vascular smooth muscle cell growth and mitogen-activated protein kinase signaling cascades. Mol Pharmacol. 1995;48:890–896.[Abstract]

67. Rao GN, Alexander RW, Runge MS. Linoleic acid and its metabolites, hydroperoxy-octadecadienoic acids, stimulate c-fos, c-jun, and c-myc mRNA expression, mitogen activated protein kinase activation, and growth in rat aortic smooth muscle cells. J Clin Invest. 1995;96:842–846.

This article has been cited by other articles:

				C. Bolego, C. Buccellati, A. Prada, R. M. Gaion, G. Folco, and A. Sala Critical role of COX-1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone FASEB J, February 1, 2009; 23(2): 605 - 612. [Abstract] [Full Text] [PDF]

				N. Foudi, L. Louedec, T. Cachina, C. Brink, and X. Norel Selective cyclooxygenase-2 inhibition directly increases human vascular reactivity to norepinephrine during acute inflammation Cardiovasc Res, February 1, 2009; 81(2): 269 - 277. [Abstract] [Full Text] [PDF]

				R. Schuligoi, M. Sedej, M. Waldhoer, A. Vukoja, E. M. Sturm, I. T. Lippe, B. A. Peskar, and A. Heinemann Prostaglandin H2 induces the migration of human eosinophils through the chemoattractant receptor homologous molecule of Th2 cells, CRTH2 J. Leukoc. Biol., January 1, 2009; 85(1): 136 - 145. [Abstract] [Full Text] [PDF]

				P. M. Vanhoutte and E. H. C. Tang Endothelium-dependent contractions: when a good guy turns bad! J. Physiol., November 15, 2008; 586(22): 5295 - 5304. [Abstract] [Full Text] [PDF]

				M. Camacho, C. Rodriguez, J. Salazar, J. Martinez-Gonzalez, J. Ribalta, J.-R. Escudero, L. Masana, and L. Vila Retinoic acid induces PGI synthase expression in human endothelial cells J. Lipid Res., August 1, 2008; 49(8): 1707 - 1714. [Abstract] [Full Text] [PDF]

				A. Hirao, K. Kondo, K. Takeuchi, N. Inui, K. Umemura, K. Ohashi, and H. Watanabe Cyclooxygenase-dependent vasoconstricting factor(s) in remodelled rat femoral arteries Cardiovasc Res, July 1, 2008; 79(1): 161 - 168. [Abstract] [Full Text] [PDF]

				T.-T. Hong, J. Huang, T. D. Barrett, and B. R. Lucchesi Effects of cyclooxygenase inhibition on canine coronary artery blood flow and thrombosis Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H145 - H155. [Abstract] [Full Text] [PDF]

				X. Tan, S. Essengue, J. Talreja, J. Reese, D. J. Stechschulte, and K. N. Dileepan Histamine Directly and Synergistically with Lipopolysaccharide Stimulates Cyclooxygenase-2 Expression and Prostaglandin I2 and E2 Production in Human Coronary Artery Endothelial Cells J. Immunol., December 1, 2007; 179(11): 7899 - 7906. [Abstract] [Full Text] [PDF]

				P. Gluais, J. Paysant, C. Badier-Commander, T. Verbeuren, P. M. Vanhoutte, and M. Feletou In SHR aorta, calcium ionophore A-23187 releases prostacyclin and thromboxane A2 as endothelium-derived contracting factors Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2255 - H2264. [Abstract] [Full Text] [PDF]

				Z. Qi, H. Cai, J. D. Morrow, and M. D. Breyer Differentiation of Cyclooxygenase 1- and 2-Derived Prostanoids in Mouse Kidney and Aorta Hypertension, August 1, 2006; 48(2): 323 - 328. [Abstract] [Full Text] [PDF]

				A. Razmara, D. N. Krause, and S. P. Duckles Testosterone augments endotoxin-mediated cerebrovascular inflammation in male rats Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1843 - H1850. [Abstract] [Full Text] [PDF]

				L. H. Smith, M. S. Petrie, J. D. Morrow, J. A. Oates, and D. E. Vaughan The sterol response element binding protein regulates cyclooxygenase-2 gene expression in endothelial cells J. Lipid Res., May 1, 2005; 46(5): 862 - 871. [Abstract] [Full Text] [PDF]

				A. Virdis, R. Colucci, M. Fornai, C. Blandizzi, E. Duranti, S. Pinto, N. Bernardini, C. Segnani, L. Antonioli, S. Taddei, et al. Cyclooxygenase-2 Inhibition Improves Vascular Endothelial Dysfunction in a Rat Model of Endotoxic Shock: Role of Inducible Nitric-Oxide Synthase and Oxidative Stress J. Pharmacol. Exp. Ther., March 1, 2005; 312(3): 945 - 953. [Abstract] [Full Text] [PDF]

				F. Miceli, A. Tropea, F. Minici, M. Orlando, G. Lamanna, M. F. Gangale, B. Panetta, F. Tiberi, S. Vaccari, R. Canipari, et al. Effects of Insulin-Like Growth Factor I and II on Prostaglandin Synthesis and Plasminogen Activator Activity in Human Umbilical Vein Endothelial Cells J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 372 - 378. [Abstract] [Full Text] [PDF]

				I. Eskurza, K. D. Monahan, J. A. Robinson, and D. R. Seals Effect of acute and chronic ascorbic acid on flow-mediated dilatation with sedentary and physically active human ageing J. Physiol., April 1, 2004; 556(1): 315 - 324. [Abstract] [Full Text] [PDF]

				J. Quilley and Y.-J. Chen Role of COX-2 in the Enhanced Vasoconstrictor Effect of Arachidonic Acid in the Diabetic Rat Kidney Hypertension, October 1, 2003; 42(4): 837 - 843. [Abstract] [Full Text] [PDF]

				M. E. Widlansky, D. T. Price, N. Gokce, R. T. Eberhardt, S. J. Duffy, M. Holbrook, C. Maxwell, J. Palmisano, J. F. Keaney Jr, J. D. Morrow, et al. Short- and Long-Term COX-2 Inhibition Reverses Endothelial Dysfunction in Patients With Hypertension Hypertension, September 1, 2003; 42(3): 310 - 315. [Abstract] [Full Text] [PDF]

				N. K. Chanani, D. B. Cowan, K. Takeuchi, D. N. Poutias, L. M. Garcia, P. J. del Nido, and F. X. McGowan Jr Differential Effects of Amrinone and Milrinone Upon Myocardial Inflammatory Signaling Circulation, September 24, 2002; 106(12_suppl_1): I-284 - I-289. [Abstract] [Full Text] [PDF]

				R. Maas, E. Schwedhelm, J. Albsmeier, and R. H Boger The pathophysiology of erectile dysfunction related to endothelial dysfunction and mediators of vascular function Vascular Medicine, August 1, 2002; 7(3): 213 - 225. [Abstract] [PDF]

				L. H. Smith, O. Boutaud, M. Breyer, J. D. Morrow, J. A. Oates, and D. E. Vaughan Cyclooxygenase-2-Dependent Prostacyclin Formation Is Regulated by Low Density Lipoprotein Cholesterol In Vitro Arterioscler. Thromb. Vasc. Biol., June 1, 2002; 22(6): 983 - 988. [Abstract] [Full Text] [PDF]

				I. M. Robbins, R. J. Barst, L. J. Rubin, S. P. Gaine, P. V. Price, J. D. Morrow, and B. W. Christman Increased Levels of Prostaglandin D2 Suggest Macrophage Activation in Patients With Primary Pulmonary Hypertension Chest, November 1, 2001; 120(5): 1639 - 1644. [Abstract] [Full Text] [PDF]

				S. T. Davidge Prostaglandin H Synthase and Vascular Function Circ. Res., October 12, 2001; 89(8): 650 - 660. [Abstract] [Full Text] [PDF]

				R. A. Houliston, J. D. Pearson, and C. P. D. Wheeler-Jones Agonist-specific cross talk between ERKs and p38mapk regulates PGI2 synthesis in endothelium Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1266 - C1276. [Abstract] [Full Text] [PDF]

				G. E. Caughey, L. G. Cleland, P. S. Penglis, J. R. Gamble, and M. J. James Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-2 J. Immunol., September 1, 2001; 167(5): 2831 - 2838. [Abstract] [Full Text] [PDF]

				M. Soler, M. Camacho, J.-R. Escudero, M. A. Iniguez, and L. Vila Human Vascular Smooth Muscle Cells but Not Endothelial Cells Express Prostaglandin E Synthase Circ. Res., September 15, 2000; 87(6): 504 - 507. [Abstract] [Full Text] [PDF]

				T. O. Daniel, H. Liu, J. D. Morrow, B. C. Crews, and L. J. Marnett Thromboxane A2 Is a Mediator of Cyclooxygenase-2-dependent Endothelial Migration and Angiogenesis Cancer Res., September 1, 1999; 59(18): 4574 - 4577. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Camacho, M. Articles by Vila, L. Search for Related Content PubMed PubMed Citation Articles by Camacho, M. Articles by Vila, L. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB HISTAMINE