[Frontiers in Bioscience E4, 2654-2669, June 1, 2012]
Imidazolineoxyl N-oxide induces COX-2 in endothelial cells: role of free radicals
Mercedes Camacho1, Jose Martinez-Gonzalez2, Cristina Rodriguez2, Laura Siguero1, Cristina Seriola1, Jose-Maria Romero1,3, Luis Vila1
1
TABLE OF CONTENTS
1. ABSTRACT
cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) exerts beneficial actions on systemic inflammatory response. Besides its nitric oxide (NO) scavenging properties cPTIO could exert beneficial effects through modulation of arachidonic acid metabolism. We studied the effect of cPTIO on the biosynthesis of vasoactive prostaglandins (PG) by endothelial cells. Human cord umbilical vein endothelial cells (HUVEC) were treated with cPTIO, and expression of cycloxygenase (COX) isoenzymes in terms of mRNA and protein was determined by real-time-PCR and immunoblotting. Release of PGE2 (as index of untransformed PGH2 release) and 6-oxo-PGF1alpha (PGI2 stable metabolite) was determined by enzyme-immunoassay. cPTIO significantly increases the release of untransformed PGH2 associated to the induction of COX-2 expression. Experiments with NO-synthase inhibitors and radical scavengers showed that induction of COX-2 by cPTIO was mediated by free radical species, likely caused by the mobilization of NO from cellular stores. Finally, using specific signal-transduction inhibitors we show the involvement of Src/PI3-K/PKC pathway. Additional effects other than a direct NO scavenging activity may confer therapeutic advantages to cPTIO as compared with NO-synthase inhibitors for the treatment of systemic inflammation-associated vascular hyporeactivity.
3. MATERIALS AND METHODS
3.1. Synthesis of cPTIO and cPTI
cPTIO and cPTI potassium salt were synthesized as previously described (6).
3.2. Cell culture and treatment
Endothelial cells were isolated from human cord umbilical veins (HUVEC) and cultured as previously described (19). Cells in the first passage were seeded in 6 well plates and cultured in M199 medium containing 20% fetal bovine serum (FBS) without heparin and endothelial cell growth factor for 48 hours prior to the addition of cPTIO (0-100 microM) in M199 containing 1 % FBS. cPTIO stimulation was maintained for the indicated (in the results section) period of time until thrombin stimulation or enzyme expression (protein or mRNA) studies were performed.
3.3. Determination of arachidonic acid metabolism from endogenous substrate mobilized with thrombin
After pretreatment of HUVEC with or without cPTIO, medium was replaced and cells were incubated at 37oC with 1 U/mL thrombin for 10 minutes. Next, supernatants were removed, frozen in liquid N2 and stored at -80oC until PGE2 and 6-oxo-PGF1alpha were analyzed by specific enzyme immunoassay (EIA, GE Healthcare, Buckighamshire, UK), following the manufacturer's instructions.
3.4. COX-1 and COX-2 protein levels
After the appropriate treatments, HUVEC lysates were prepared and proteins were analyzed by immunoblotting as previously described (19,20). The effect of cPTI on COX-2 expression was analyzed incubating HUVEC with or without 100 microM of cPTI for 24 hours.
3.5. COX-2 mRNA levels
Total RNA was extracted by chloroform isopropanol precipitation using UltraspecTM (Biotecx Laboratories, Inc, Houston, Texas, USA) according to the manufacturer's instructions. Reverse transcription was performed with 1 microg of RNA per 20 microL reaction mixture and COX-2 mRNA levels were studied by real-time PCR as previously described (20). Gene expression data were normalized to b
-actin as endogenous control and RNA of untreated cells was used as a calibrator sample.
3.6. NO-dependence of the induction of COX-2 expression by cPTIO
HUVEC were incubated with or without 100 microM cPTIO for 24 hours, in the absence or presence of 1 mM of NO-synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), 1 mM N5-(imino(nitroamino)methyl)-L-ornithine, methyl ester, (L-NAME), 120 microM of the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP, Cayman Chemical, Ann Arbor, MI), or 10 m
M oxyhaemoglobin (oxyHb). Thereafter, COX-2 protein expression was evaluated as aforementioned 3.7. Effect of radical scavengers on the induction of COX-2 expression by cPTIO
HUVEC were incubated with or without 100 microM cPTIO for 24 hours, in the absence or presence of 10 microL/mL DMSO, 100 microM N-acetylcysteine (NAC), 120 microM vitamin C or 100 microM vitamin E. Thereafter, COX-2 protein expression was evaluated as described above. The effect of O2- on the ability of cPTIO to induce COX-2 expression in HUVEC was evaluated by incubating cells with 100 microM cPTIO or 10 microM oxyHb for 24 hours, in the presence or absence of the indicated combinations of 500 microM xanthine sodium salt, 100 mU/mL microbial xanthine oxidase and 100 U/mL bovine superoxide dismutase-polyethylene glycol (SOD) (all compounds were purchased from Sigma). Involvement of NADPH oxidase in the action of cPTIO was investigated by incubating HUVEC with or without 100 microM cPTIO for 24 hours in the presence or absence of 30 microM diphenyleneiodonium chloride (DPI) or 1 mM of 4-(-2-aminoethyl)-benzenesulfonyl fluoride (AEBSF).
3.8. Signalling pathways involved in cPTIO-induced expression of COX-2
HUVEC were incubated with 100 microM cPTIO for 24 hours, To analyse activation of MAPK family, HUVEC were exposed to 100 microM cPTIO for the indicated period of time (in the Results section). Afterwards, cells were lysed and the phosphorylated MAPKs were analysed by immunoblotting, as described above for COX isoenzymes, by using polyclonal antibodies against phosphorylated p38-MAPK
3.9. Statistics
SigmaPlot 11 software was used for statistical analysis. Statistical significance between more than two groups was assessed using one way ANOVA test, when normality test failed, one-way ANOVA on ranks was used. Student t test was used to compare the two groups. A "p" value below 0.05 was considered significant.
4. RESULTS AND DISCUSSION
4.1. Prostanoid release by HUVEC exposed to cPTIO
In Figure 1 we show the effect of cPTIO on the accumulation of prostanoids in the culture medium. cPTIO significantly increased levels of both PGI2 (analysed in terms of 6-oxo-PGF1alpha, stable hydrolysis product of PGI2) and PGE2. These results indicate that cPTIO was able to mobilize AAc from the membrane phosphoglycerides to be oxidized towards prostanoids. We and others have reported that PGE2 is formed by non-enzymatic isomerization of PGH2 outside of endothelial cells (13,16-18), since these cells do not express mPGES-1, the main isoform of PGES involved in PGE2 biosynthesis (17,18). Therefore, PGE2 may be representative of untransformed PGH2 released by the cells.
4.2. Effect of cPTIO on the expression of COX-isoenzymes
An excess of untransformed PGH2 seems to be a consequence of COX-2 over-expression as we and others previously observed (13,16,18), thus, next we explored the effect of cPTIO on the expression of COX-2. cPTIO increased COX-2 expression in a concentration- and time-dependent manner in terms of protein (Figure 2 A and B). There was no effect of cPTIO on the expression of COX-1 (data not shown). The effect of cPTIO on COX-2 expression was at a transcriptional level since mRNA was also strongly induced in a time-dependent manner (Figure 2C), and the transcription inhibitor actinomycin-D totally suppressed the effect of cPTIO (Figure 2D). As expected, the translation inhibitor cycloheximide also inhibited cPTIO-induced expression of COX-2. To explore the effect of cPTIO on prostanoid biosynthesis in response to a phospholipase stimulus, HUVEC were incubated in the presence of several concentrations of cPTIO for 24 hours. Medium was then replaced, and HUVEC were stimulated with thrombin for 10 min to induce endogenous AAc release. Next PGE2 and 6-oxo-PGF1alpha were measured in the cell incubation media. In these conditions cPTIO significantly increased the ability of HUVEC to produce PGE2 but not 6-oxo-PGF1alpha (Figure 3). These findings were consistent with the up-regulation of COX-2 expression by cPTIO. The ratio PGE2 to 6-oxo-PGF1alpha following thrombin challenge, as representative of the ratio untransformed PGH2 to PGI2, increased about 17 times after 48 hours of cell exposure to 100 microM cPTIO.
Our results show that exposure of endothelial cells to cPTIO increased the ability of endothelial cells to release an excess of the vaso-constricting prostanoid PGH2 when mobilization of AAc is stimulated. Release of untransformed PGH2 by endothelial cells is regulated by COX-2/PGIS activity ratio (13,16). In a previous work we showed that in the presence of high amounts of NO, as a consequence of strong inflammatory conditions, cPTIO inactivates PGIS probably by the generation of NO2 (6,13,14). Here, we explore the action of cPTIO on endothelial cells without any other treatment. The COX-2/PGIS activity ratio in endothelial cells is primarily increased by over-expression of the inducible COX-2.
4.3. Role of NO in the up-regulation of COX-2 expression induced by cPTIO
Commonly, cPTIO is merely viewed as a NO-scavenger and its biological effects are usually attributed to this property. The reaction of cPTIO with NO yields cPTI and NO2 (11). We therefore investigated whether NO2 or other NO derivatives mediated the cPTIO-induced COX-2 expression. Results in Figure 4A show that two NO-synthase inhibitors (L-NMMA and L-NAME), a NO-donor (SNAP) and cPTI (the reaction product of cPTIO with NO) did not exert any effect on COX-2 expression. The fact that cPTI was inactive in inducing COX-2 indicates that the oxygen of the N-oxide in position 3 of the imidazoline ring, which is transferred to NO to form NO2 during NO-scavenging, is essential for the induction of COX-2 expression. OxyHb oxidizes NO to nitrate thereby acting as a NO-scavenger (22). We found that oxyHb also increased COX-2 expression, but in a lesser extend than cPTIO. Data in Figure 4B show that the presence of L-NMMA modified neither the effect of cPTIO nor the effect of oxyHb on COX-2 expression. Similarly, the presence of oxyHb did not modify the effect of cPTIO. The NO-donor SNAP used at low concentration attenuated the effect of cPTIO by a 47.6+/-3.7 % (mean+/-SEM, determined by densitometry, n=3) probably by transforming cPTIO to cPTI outside the cells. These results indicate that the effect of both cPTIO and oxyHb on COX-2 induction was not due to its interaction with the NO formed during the incubation period since NO synthesized de novo was inhibited in the presence of the NO-synthase inhibitors. The chemistry associated with the interaction of oxyHb and NO and NO-related compounds is complex. Indeed, oxyHb could reduce nitrite to generate NO, and it may also generate superoxide radical by autoxidation (23,24,25). Nevertheless, as both cPTIO and oxyHb induced COX-2 expression, this effect could be the result of the interaction of the NO-scavengers with NO from sources other than de novo-synthesized NO such as nitroso-derivatives. The concept that NO used by cPTIO could come from cellular stores, is consistent with our previous report (14). Indeed, we observed that the hyporesponsiveness to phenylephrine of rat aortic rings exposed to IL-1β was mainly due to NO stores formed during the incubation with the cytokine rather than those formed in the organ bath during the assay (14).
4.4. Redox states of cPTIO in HUVEC
Figure 5 shows the UV-visible spectrophotometry analysis of culture medium containing cPTIO and cPTIO plus L-NMMA after 24 hours of incubation with or without cells compared with cPTI alone. No changes in the cPTI spectra were observed by the incubation with cells, either in the presence or in the absence of L-NMMA (data not shown). Data in Figure 5A show that cPTIO incubated without cells exhibited two maximum at 347 nm and 558 nm, whereas cPTI had only a maximum at 329 nm. No differences were observed between cPTIO alone and cPTIO plus L-NMMA in the absence of cells (data not shown). When cPTIO or cPTIO plus L-NMMA were incubated in the presence of HUVEC, the spectrum in the range of 300-400 nm was different than that of cPTIO without cells, and quite similar to that of cPTI. In the visible zone, spectra of cPTIO and cPTIO plus L-NMMA irrespectively of the presence of cells during the incubation, were qualitatively similar to that of cPTIO alone with a maximum at 558 nm, but different than that of cPTI. When HUVEC were present during the incubation, differential spectra, subtracting cPTI spectrum, in the zone of 300-400 were similar independently of the presence of L-NMMA, but different to that of cPTIO without cells (Figure 5B, left panel). Differential spectra analysis subtracting cPTIO shows that cPTI had a minimum at 558, whereas this valley disappeared in that of cPTIO plus L-NMMA incubated with cells (Figure 5B, right panel). The differential spectrum of cPTIO incubated with HUVEC in the absence of L-NMMA exhibited an intermediated profile between cPTI and cPTIO plus L-NMMA (Figure 5B, right panel). These results from the UV-visible spectrometric analysis were consistent with those reported by others (26). cPTIO could suffer reversible oxidation. Data regarding 500-600 nm indicated that cPTI was present in very small amount in cells incubated in the absence of L-NMMA, while it was apparently absent when L-NMMA was present. Our results regarding 300-400 nm, compared with spectra reported by Goldstein et al. (26) suggest that after incubation with HUVEC, the hydroxylamine form (c-PTIO-H) of cPTIO was present. Janssen et al. (27) also found that cPTIO-H was present in pulmonary cells incubated with cPTIO. Data reported by Goldstein et al. (26) show that cPTI, cPTIO-H and the oxoammonium cation (cPTIO+) exhibit a maximum at about 330 nm, whereas cPTIO have a maximum at about 360 nm. In addition, cPTIO+ has another maximum at about 450 nm, which was not observed in our conditions.
4.5. Role of reactive oxygen species in the up-regulation of COX-2 by cPTIO
NO reacts rapidly with superoxide anion (O2-) to yield the strong oxidant species peroxynitrite (ONOO-) (28). Endothelial cells are a relevant source of O2- formed from molecular oxygen by the action of NADPH oxidases (29,30). The interaction between NO and O2- regulates the availability of NO, O2- and ONOO- , NO being normally produced in excess by the endothelium (31). Depletion of endogenous NO may disrupt the balance between NO and O2- and may promote O2- depending responses. We then explored the effect of several radical scavengers on cPTIO-induced expression of COX-2. DMSO, NAC, vitamin C and vitamin E (Figure 6A) per se did not modify the expression of COX-2, whereas they significantly reduced the effect of cPTIO on COX-2 expression (Figure 6B). These data suggested that free radicals were involved in the effect of cPTIO. We then tested the effect SOD on the COX-2 induction caused by cPTIO and oxyHb (Figure 7A). SOD slightly decreased oxyHb-induced expression of COX-2, although after densitometric evaluation, difference between oxyHb with and without SOD was not statistically significant. In contrast, SOD significantly increased the effect of cPTIO on COX-2 expression by a 40.8+/-11.3 % (mean+/-SEM, determined by densitometry, n=3, p<0.05). We then tested the effect of an O2- generating system on COX-2 expression. Results in Figure 7A shows that incubation with xanthine/xanthine oxidase induced COX-2 in HUVEC. Next, we explore the effect of two NADPH oxidase inhibitors on cPTIO- and oxyHb-induced expression of COX-2. Results in the Figure 7B show that both DPI and AEBSF reduced the effect of the NO-scavengers on COX-2 expression. Collectively, these results indicated that oxygen derived free radicals were involved in the action of cPTIO and oxyHb, and that SOD could have an additional activity in the case of cPTIO. Indeed, SOD can also liberate NO from small-molecular-weight SNO (32). These results are consistent with the fact that reactive oxygen intermediates induce COX-2 expression (33). In fact, cPTIO is itself a relatively stable oxygen-free radical that could react with free radicals other than NO, such as O2-. Superoxide reduced cPTIO to the corresponding hydroxylamine (cPTIO-H) which does not react with NO (26,34). It has been reported that nitroxides have SOD mimetic activity (35-37). Reaction of nitroxides with O2- to mimic SOD activity involves an oxoammonium/nitroxide redox couple (36,37). Oxoammonium cation (cPTIO+, in the case of cPTIO), could react with NADH or NADPH to yield NAD+ or NADP+ plus the corresponding hydroxylamine (cPTIO-H, in the case of cPTIO) (36,37). As indicated above the spectophotometric analysis of cPTIO incubated with cells indicated that part of cPTIO was in the cPTIO-H form.
4.6. PGE2 released in response to cPTIO is synthesized by COX
To dismiss the possibility that PGE2 and its isomers, which would be detectable by EIA, could be generated from the oxidation of AAc by free radical lipid peroxidation during the incubation with cPTIO, PGE2 release was determined incubating cells with cPTIO in the presence of 10 microM indomethacin. Indomethacin did not modify cPTIO-induced COX-2 expression and totally suppressed PGE2 levels in the culture medium under any condition (data not shown). These data indicate that COX-derived PGH2 was the origin of PGE2 present in the extracellular medium.
4.7. Generation of nitrosylating species by cPTIO
cPTIO yields NO2 when reacts with NO (11), which is a nitrating species forming nitrotyrosine residues in proteins, but also can react with NO to yield the powerful thiol-nitrosating species N2O3 (38). To explore the possibility that cPTIO could act by generating nitrosating species, we incubated cells with cPTIO in the presence of GHS, a known quencher of reactive nitrogen intermediates particularly N2O3. Results in Figure 7C show that GSH effectively reduced cPTIO-induced expression of COX-2. Moreover, treatment of HUVEC with 1 mM of nitroso glutathione (GSNO), a nitrosating agent (39), also induced COX-2 expression. Altogether, our results suggest that cPTIO could alter the redox state of cells. cPTIO may modify nitrosylation-denitrosylation cycle of sulfhydryls, and thereby modify cell signalling (38). A point that reinforces this concept is that cPTIO-mediated COX-2 induction was not observed in cells with a basal rate of NO biosynthesis lower than endothelial cells, such as cultured fibroblasts and vascular smooth muscle cells (data not shown). Assuming that NO could come from nitrosated proteins and/or nitrite, cPTIO could favour nitrosation and nitrosylation processes involving the mediation of NO2 (40). This, not necessarily must result in cPTI formation, since cPTIO can be regenerated if it interacts with NO in the oxidized oxoammonium form (26).
4.8. Signalling pathways involved in the cPTIO-induced expression of COX-2
To characterize signalling pathways involved in the cPTIO-induced COX-2 expression, cells were treated with several inhibitors during cPTIO stimulation. As a first approximation, we analyzed COX-2 expression in cells stimulated with cPTIO in the presence of a constant concentration of inhibitor, as follows: Ro31-3220 (10 m M), GF109203X (5 m M), Gö6976 (1 m M), rapamycin (1 microM), SB203580 (10 m M), U0126 (10 m M), PD98059 (50 m M), LY294002 (50 microM), Akt-inhibitor (25 microM) and PP-1 and PP-2 (10 microM). Densitometric evaluation of the immunoblottings showed that the PI3-K inhibitor LY294002 significantly inhibited the effect of cPTIO by a percentage of 99.2+/-6.8 (n=3, p<0.05 when compared with cPTIO sample without inhibitor), whereas the Akt-inhibitor did not modify COX-2 expression at all. The PKC general inhibitors Ro31-3220 and GF109203X significantly inhibited the effect of cPTIO by a percentage of 100+/-0.0 (mean+/-SEM) (n=2, p<0.05) and 45.4+/-2.0 (n=3, p<0.01), respectively. The Ca++-dependent PKC inhibitor Gö6976 also significantly inhibited the cPTIO effect by a percentage of 61.0+/-2.6 (n=3, p=0.01). The Src family protein tyrosine kinase inhibitors PP-1 and PP-2 at concentration of 10 microM partially abrogated cPTIO-induced COX-2 expression by a 39.5+/-6.8 and 41.8+/-4.3 % respectively (mean+/-SEM, n=3, p<0.05). The MEK1/2 inhibitors U0126 and PD98059, the p38 MAPK inhibitor SB203580 and the mTOR inhibitor rapamycin, did not exert a significant inhibition of COX-2 expression induced by cPTIO.
Since the above results suggested that PI3-K was involved in the cPTIO-induced COX-2, further experiments were carried out to observe the concentration-dependent effect of two PI3K inhibitors (LY294002 and wortmannin) and therefore rule-out the possibility that the effects observed in the preliminary experiments were unspecific. Figure 8 depict representative western-blots showing that both inhibitors suppressed the effect of cPTIO on the COX-2 protein expression in a concentration-dependent manner. Since Akt is a canonical downstream effector of PI3-K, we also tested the effect of Akt-inhibitor on cPTIO-induced COX-2 expression. However, results in Figure 8 clearly show that 50 m M of Akt-inhibitor per se neither modify the expression of COX-2, nor the induction of COX-2 by cPTIO. Our results are consistent with the fact that PI3-K has been described as a critical link in signalling of reactive oxygen species (41). These results also rule out the involvement of PI3-K/Akt pathway in the induction of COX-2 by cPTIO.
PKC isoforms have been classified into three groups according to their structure and cofactor requirements: conventional PKCs calcium- and DAG-dependent, novel PKCs DAG-dependent and calcium independent and atypical PKCs calcium independent, which are not activated by phorbol esters. PI3-K could also regulate conventional PKC (42). Consistently we confirmed that the general PKC inhibitor Ro31-3220 and the calcium-dependent PKC inhibitor Gö6976 reduced cPTIO-induced expression of COX-2 in a concentration-dependent manner (Figure 8). The inhibition of cPTIO-induced expression of COX-2 by a calcium-dependent PKC inhibitor suggests the particular implication of this class of PKCs in the action of cPTIO. Taken together, these results suggest that PI3-K/PKC is involved in cPTIO-induced COX-2 expression. Direct PKC activation by oxidant species has also been described (43), which should be consistent with the involvement of oxygen-derived reactive species such as O2- on cPTIO-induced COX-2 expression.
The Src family protein tyrosine kinase inhibitor PP-1 also concentration-dependently suppressed the effect of cPTIO on the COX-2 protein expression (Figure 8). Src protein tyrosine kinases are non receptor tyrosine kinases that initiate a sequential phosphorylation through MAPK pathway that includes Ras, Raf/MEK and ERK1/2 and can also activate Ras/PI3-K pathway. Src and Ras can be activated either by nitrosylation or by O2- (44-47).
Three subfamilies of MAPKs, ERK, JNK and p38-MAPK, play pivotal roles in a variety of cellular functions and they are involved in the regulation of many genes. To further evaluate the involvement of the MAPK family in the induction of COX-2 by cPTIO we examined phosphorylation of p38-MAPK, JNK and ERK1/2 after treatment of HUVEC with cPTIO for several periods of time. Anti-phospho-MAPKs antibodies were used to perform immunoblotting analysis. No effect was observed in response to cPTIO regarding either JNK or p38-MAPK phosphorylation in the incubation period ranging from 10 min to 48 hours (data not shown). In contrast, cPTIO triggered ERK1/2 phosphorylation in a time dependent manner, although this was observed 6 hours after the addition of cPTIO to the cells (Figure 9). ERK1/2 phosphorylation usually occurs within minutes after cell challenge, but cPTIO induced a late phosphorylation. One possible explanation for this finding could be that the phosphorylation of ERK1/2 was secondary to the cPTIO-mediated induction of other effector/s. The fact that neither actinomycin D, (a transcription inhibitor), nor cycloheximide (a translation inhibitor) suppressed ERK1/2 phosphorylation (data not shown), rules out the possibility that this putative secondary effector was a protein transcriptionally regulated by cPTIO. In order to establish if phosphorylation of ERK1/2 mediated cPTIO-induced expression of COX-2, we tested whether the transduction signal inhibitors related with the pathways involved in the cPTIO-induced expression of COX-2, Ro31-3220, Gö6976, LY294002 and Akt-inhibitor, inhibited ERK1/2 phosphorylation. We observed that not only none of them attenuated ERK1/2 phosphorylation but that they even increased this, indicating that this MAPK was not involved in the up-regulation of COX-2 expression by cPTIO in HUVEC (not shown). Phosphorylation of ERK1/2 is nevertheless consistent with the activation of Src/Ras pathway caused by cPTIO (48).
We previously reported that cPTIO could act indirectly on the metabolism of AAc inhibiting PGIS activity, likely throughout the generation of NO2 (6,13,14). Herein, we show that cPTIO induces COX-2 expression. Together these actions result in the release of untransformed PGH2 by endothelial cells, suggesting it could contribute to the positive vasopressor activity of cPTIO (2,4,7) irrespectively of its action as an NO scavenger. This could be particularly relevant for systemic inflammatory responses characterized by a vascular hyporeactivity such as endotoxic shock (1-6), since cPTIO could add a therapeutic value compared with NO-synthase inhibitors. Additionally, our results show that caution must to be taken into account when cPTIO is used to ascertain the role of NO in a particular physiopathologic process, since cPTIO could exert diverse effects. Indeed, cPTIO was able to trigger signalling pathways that could potentially influence gene expression, cell apoptosis or cell cycle progression. This wide range of biological effects of cPTIO could account for a number of non-expected observations in experimental approaches using this drug (9,10,49).
In our previous work (6) we show that cPTIO improved blood pressure and mortality in an experimental shock model by inhibition of PGI-synthase in addition to NO scavenging. This leads to a reduced formation of the potent vasodilator PGI2. We also have shown that the presence of cPTIO in the organ bath, added after the treatment of aortic rings with IL-1β, improved contractility more than L-NAME. The hyporeactivity of rat aorta exposed to IL-1b was mainly due to pre-formed NO-stores rather to a NO formed during the stimulation in the organ bath (14). Our current results indicate that cPTIO could induce COX-2 expression in the vascular endothelium resulting in the release of the potent vaso-constrictor prostaglandin PGH2. All these additional effects may confer therapeutic advantages to cPTIO as compared with NO-synthase inhibitors for the treatment of systemic inflammation-associated vascular hyporeactivity.
In conclusion, in this study we found that in HUVEC cPTIO significantly increases the release of untransformed PGH2 (measured as PGE2) associated to a time- and concentration-dependent induction of COX-2 expression. The induction of COX-2 by cPTIO was mediated by free radical-species. Although we have not identified the molecular species and the exact mechanism involved in cPTIO induced expression of COX-2 expression, taken together, our results strongly suggest that this effect was dependent of the reaction with NO coming from intracellular pools probably nitroso-derivatives. NO quenching by cPTIO produces as outcome an increased availability of superoxide and generation of NO2, which could result in the generation of the nitrosating agent N2O3. These facts could lead to an activation of Src/PI3-K/PKC pathway through nitrosylation and/or directly mediated by superoxide (this is schematized in Figure 10). More research is needed to establish the most relevant way leading COX-2 expression.
5. ACKNOWLEDGMENTS
This study was partially supported by grants from the Ministerio de Ciencia e Innovación (SAF2008-01777, SAF2010-21392 and SAF2009-11949) and Red Temática de Investigación Cardiovascular (RECAVA) (RD06/0014/1005 and RD06/0014/0027) from the Instituto de Salud Carlos III. The authors thank Sonia Alcolea for their technical assistance and Dr Jordi Mancebo for his critical review of this manuscript.
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Abreviations: AAc: arachidonic acid, COX: cycloxygenase, AEBSF: 4-(-2-aminoethyl)-benzenesulfonyl fluoride, cPGES: cytosolic PGE-synthase, cPTI: 2-(4-carboxyphenyl)-4:4:5:5-tetramethylimidazoline-1-oxyl, cPTIO (2-(4-carboxyphenyl)-4:4:5:5-tetramethylimidazoline-1-oxyl-3-oxide, DPI: Diphenyleneiodonium chloride, FBS: fetal bovine serum, HUVEC: umbilical vein endothelial cells, GSH: reduced glutathione, GSNO: nitroso-glutathione, JNK: Jun N-terminal kinase, L-NAME: N5-(imino(nitroamino)methyl)-L-ornithine: methyl ester, L-NMMA: NG-monomethyl-L-arginine, mPGES-1: microsomal PGE-synthase-1, MEK1/2: mitogen-activated protein kinase kinase-1 and 2, mTOR, mammalian target of rapamycin, NAC: N-acetylcysteine, NO: nitric oxide, oxyHb: oxyhaemoglobin: p38-MAPK: p38 mitogen activated protein kinase, PG:prostaglandin, PGES: PGE-synthase, PGIS: PGI-synthase, PI3-K: phosphoinoside 3-kinase, PKC: protein kinase C, PP1: 4-Amino-5-(methylphenyl)-7-(t-butyl)pyrazolo-(3:4-d)pyrimidine, PP2: 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo(3:4-d)pyrimidine, SNAP: S-Nitroso-N-acetyl-D:L-penicillamine, TxA2: thromboxane A2
Key Words: cPTIO, COX-2, Endothelial cell, Nitric oxide, PI3K, PKC, Src
Send correspondence to: Luis Vila, Laboratory of Angiology, Vascular Biology and Inflammation, Hospital de la Santa Creu i Sant Pau, c/ Antoni Ma Claret 167, 08025 Barcelona, Spain, Tel: 34-93-2919105, Fax: 34-93-4552331, E-mail:lvila@santpau.cat