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CARDIOVASCULAR
Department of Internal Medicine (A.V., E.D., S.P., S.T., A.S.), Division of Pharmacology and Chemotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine (R.C., M.F., C.B., L.A., M.D.T.), and Department of Human Morphology and Applied Biology, Section of Histology and General Embryology (N.B., C.S.), University of Pisa, Pisa, Italy
Received September 10, 2004; accepted November 15, 2004.
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). Endothelial dysfunction, characterized by reduced NO availability and increased production of reactive oxygen species (ROS), is regarded as a crucial and early mechanism whereby vascular inflammation leads to atherosclerosis (Ross, 1999). A major involvement of endothelial dysfunction has been found also in severe forms of acute inflammation, such that occurring in endotoxemia elicited by Gram-negative bacterial lipopolysaccharide (LPS). Indeed, in vitro and in vivo evidence indicates an impairment of endothelium-dependent relaxation during endotoxemia by LPS (Szabo et al., 1997; Brandes et al., 1999; Piepot et al., 2000; Hernanz et al., 2004b). When considering the underlying mechanisms, increased levels of inflammatory cytokines have been reported to interfere with endothelial function during endotoxemia (Greenberg et al., 1993). Moreover, such cytokines promote the expression of several enzymes, including inducible nitric-oxide synthase (iNOS) and cyclooxygenase (COX)-2, which in turn lead to an increased generation of NO as well as vasodilator and vasoconstrictor prostanoids (Moncada and Higgs, 1993; Camacho et al., 1998). This picture is further complicated by the evidence that COX-2 may act as a source of ROS (Katusic, 1996; O'Banion, 1999), which are responsible for NO breakdown (Cai and Harrison, 2000). However, the contribution of iNOS and COX-2, and particularly their putative synergistic or antagonistic roles in determining endothelial dysfunction, have not been clarified. The present study was designed to examine endothelium-dependent relaxations of mesenteric small arteries in a rat model of endotoxic shock to evaluate whether a reduction in NO availability and changes in COX-2 expression play a role in the pathogenesis of endothelial dysfunction secondary to endotoxemia. The participation of iNOS, thromboxane A2 (TXA2), and ROS in endotoxin-induced endothelial dysfunction was also investigated.
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; Yamagata et al., 2001).
Preparation of Small Mesenteric Arteries for Reactivity Experiments. Superior mesenteric arteries were taken from the part of the mesenteric vascular bed that feeds the jejunum 8 to 10 cm distal to the pylorus and placed in cold PSS of the following composition: 120 mmol/l NaCl, 25 mmol/l NaHCO3, 4.7 mmol/l KCl, 1.18 mmol/l KH2PO4, 1.18 mmol/l MgSO4, 2.5 mmol/l CaCl2, 0.026 mmol/l EDTA, and 5.5 mmol/l glucose. A third order branch of the mesenteric arterial tree (
2 mm in length) was carefully dissected 1 mm from the intestine and cleaned of all adherent connective tissue under a dissecting microscope. The arterial segments were mounted in a pressure myograph chamber and slipped into two glass micro-cannulae. Both ends of the arterial segment were secured to the microcannulae with nylon ties. Intraluminal pressure was set to 45 mm Hg with a servocontrolled pump. Vessels were then equilibrated for 1 h with PSS that was bubbled with 95% air and 5% CO2 to give a pH of 7.4 and heated to 37°C (Virdis et al., 2002). Lumen dimensions were determined by light microscopy and, via a video camera, displayed on a monitor and automatically measured by software (Myoview; DMT, Aarhus, Denmark). Arterial segments were considered viable and used for experiments if they constricted by more than 50% of their resting lumen diameter in response to an extraluminal application of high-potassium (125 mM KCl) PSS containing 10 µM noradrenaline (NA).
Experimental Design. Endothelium-dependent and -independent relaxations were assessed by measuring the responses of mesenteric arteries retrieved from control or LPS-treated rats (n = 8 each group) to cumulative concentrations of acetylcholine (ACh; 1 nM–100 µM) and sodium nitroprusside (0.01–100 µM), respectively. All experiments were performed in vessels precontracted with NA (10 µM). Since exposure of mesenteric vessels to LPS was previously shown to induce hyporeactivity to NA (Briones et al., 2000; Hernanz et al., 2004a), in preliminary experiments, we performed a concentration-response analysis of NA effects (from 1 nM–100 µM) to establish at which level this compound was able to elicit similar contractions in vessels from control and LPS-treated rats. After the titration study, the dose of 10 µM NA, which induced similar contracting responses in both groups, was selected.
Influence of COX-1 and COX-2 Activity on NO Availability and Endothelial Function. The participation of COX-1 and COX-2 isoenzymes in modulation of endothelial function was assessed by construction of concentration-response curves to ACh following 30-min preincubation of mesenteric vessels with SC-560 [5-(4-clorophenyl)-1-(4-metoxyphenyl)-3-trifluoromethylpirazole; 1 µM; selective COX-1 inhibitor] (Smith et al., 1998) or 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)-phenyl-2(5H)-furanone (DFU; 1 µM; selective COX-2 inhibitor). The latter drug was used because of its documented high selectivity for COX-2. For instance, in hamster ovarian cells expressing human COX-1 and COX-2, DFU was more selective for COX-2 (COX-1/COX-2 IC50 > 1000) as compared with NS-398 (COX-1/COX-2 IC50 300). In addition, DFU was less potent than NS-398 in blocking COX-1-derived prostaglandin E2 production (Riendeau et al., 1997).
Concentrations of SC-560 and DFU were chosen according to preliminary experiments (n = 4 mesenteric vessels from LPS-treated rats), where a concentration-ranging analysis was performed for SC-560 and DFU (0.1, 0.3, 1, and 10 µM) to identify their maximal effects on relaxant responses to ACh. Increasing concentrations of SC-560 failed to modify the relaxation to ACh. By contrast, DFU concentration-dependently increased the response to ACh, with a maximal effect observed at 1 µM (data not shown).
To verify the possibility that the effects of DFU were specific for cholinergic relaxations, vessels isolated from control and LPS-treated rats (n = 4 each) were used to construct concentration-response curves to bradykinin (BDK; from 0.1 nM to 1 µM), another endothelial agonist acting through cholinergic-independent receptor pathways (Flavahan et al., 1991), either in the absence or in the presence of DFU. Moreover, to assess whether the effect of DFU was selective for endothelium-dependent relaxation, this COX-2 inhibitor was tested against concentration-response curves to sodium nitroprusside.
To evaluate NO availability, concentration-response curves to ACh were constructed after 30-min preincubation with the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 µM). Then, to examine whether COX-2 could influence NO availability, ACh was tested in an additional group (n = 6) of LPS-treated rats under simultaneous presence of L-NMMA and DFU.
Finally, to ascertain the possible contribution of COX-2-derived TXA2 on endothelial dysfunction elicited by endotoxemia, mesenteric vessels were isolated from LPS-treated animals (n = 4), and concentration-response curves to ACh were constructed after 30-min incubation with the TxA2 receptor antagonist SQ-29548 (7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-[1S(1alpha,2alpha(Z),3alpha,4alpha)]-5-heptenoic acid) (1 µM).
Interaction between iNOS and COX-2. To examine the possible role of iNOS in the modulation of COX-2 expression and function, both control and LPS-treated rats (n = 6 each) were administered with the selective iNOS inhibitor S-methylisothiourea (SMT; 25 mg/kg i.p., 30 min before LPS or PSS injection) (Muntane et al., 2000). Six hours after LPS or PSS treatment, mesenteric vessels were isolated, and concentration-response curves to ACh were constructed either in the absence or in the presence of DFU.
Influence of ROS on Endothelial Function. In this further set of experiments (n = 6 for each group), concentration-response curves to ACh were determined after 30 min preincubation with the antioxidant compounds ascorbic acid (10 mM) or superoxide dismutase (SOD; 100 U/ml). In addition, to evaluate whether impaired NO availability in endotoxemic rats was attributable to increased ROS production, ACh was infused during simultaneous incubation with L-NMMA and ascorbic acid or SOD. Subsequently, to investigate whether COX-2 could be implicated in the generation of ROS, additional mesenteric vessels from LPS rats (n = 6 for each group) were assayed with ACh after simultaneous incubation with DFU and ascorbic acid or SOD.
RNA Extraction and Reverse Transcription-Polymerase Chain Reaction. Expression of mRNA for COX and NOS isoforms was assessed by reverse transcription (RT)-polymerase chain reaction (PCR). Specimens of mesenteric vessels were disrupted with cold glass pestles, and total RNA was isolated by Trizol (Invitrogen, Carlsbad, CA) and chloroform. Total RNA (1 µg) served as template for cDNA synthesis in a reaction using 2 µl of random hexamers (0.5 µg/µl) with 200 U of Moloney murine leukemia virus-reverse transcriptase in a buffer containing 500 µM deoxynucleotide triphosphate mixture and 10 mM dithiothreitol. cDNA samples were subjected to PCR in the presence of specific primers based on the nucleotide sequences of cloned rat COX and NOS isoforms (Ferraz et al., 1997; Tanaka et al., 2002). PCR, consisting of 5 µl of RT products, 2.5 U of Taq polymerase, 100 µM deoxynucleotide triphosphate mixture, and 0.5 µM primers, was carried out by a PCR-Express thermocycler (Hybaid; Ashford, Middlesex, UK). For COX isoforms, amplification conditions were: 1 min at 94°C, 2 min at 60°C, and 1 min at 72°C for 35 cycles. For NOS isoforms, after 3 min at 94°C, the cycle condition was 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C for 30 cycles, followed by 7 min at 72°C. Aliquots of RNA not subjected to RT were included in PCR reactions to verify the absence of genomic DNA. The efficiency of RNA extraction, RT, and PCR was evaluated by primers for rat 
-actin. PCR products were separated by 1.8% agarose gel electrophoresis in a 40 mM Tris buffer containing 2 mM EDTA and 20 mM acetic acid (pH 8) and stained with ethidium bromide. PCR products were then visualized by UV light and subjected to densitometric analysis by NIH image software (Scion Corporation, Frederick, MD). The relative expression of target mRNA was normalized to that of -actin.
Immunostaining of COX-2. After collection, mesenteric specimens were immediately fixed in cold 4% paraformaldehyde diluted in phosphate-buffered saline (PBS). Samples were dehydrated with ethanol, treated with xylene, and embedded in paraffin at 56°C. Sections were sequentially exposed to the following solutions as previously described (Bernardini et al., 1999): 1% hydrogen peroxide in methanol for 5 min; proteinase K (0.05 mg/ml; Roche Diagnostics, Mannheim, Germany) in PBS for 10 min; normal swine serum (1:20; Sigma-Aldrich, St. Louis, MO); and primary antibody solutions, 1:100 diluted in 0.1% bovine serum albumin (BSA) and 0.1% sodium azide in PBS (overnight at 4°C). Goat anti-COX-2 polyclonal antibody (no. sc-1746; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was employed (1:100 diluted in 0.1% BSA and 0.1% sodium azide-PBS). Detection of immunoprecipitates was performed by an indirect streptavidine-peroxidase method, based on biotinylated immunoglobulins, peroxidase-labeled streptavidin complex, and finally to 3,3'-diaminobenzidine tetra-hydrochloride (DAB; Dakopatts, Glostrup, Denmark), as previously reported (Bernardini et al., 1996). All reactions were performed at room temperature in a chamber humidified with PBS unless otherwise specified; washing between each step was performed with PBS. Negative controls were obtained by substituting the primary antibodies with a nonimmune goat serum or with PBS plus 0.1% BSA. Endogenous peroxidases and avidin-binding activity were assayed by incubation of slides with DAB alone or with streptavidin-horseradish peroxidase complex/DAB, respectively.
Cytokine Assays. At the time of sacrifice, serum and plasma from trunk blood were collected for the determination of tumor necrosis factor-
(TNF-), interleukin (IL)-1, and IL-6 by means of enzyme-linked immunosorbent assay commercial kits (R&D Systems, Minneapolis, MN).
Drugs and Solutions. Salmonella-derived LPS, SMT, SC-560, L-NMMA, ACh, BDK, NA, sodium nitroprusside, ascorbic acid, and SOD were purchased from Sigma-Aldrich, and SQ-29548 was purchased from Cayman Chemical (Ann Arbor, MI). DFU was kindly provided by Merck Research Labs (West Point, PA). Drug solutions were made in distilled water except for SMT (dissolved in PSS) and SC-560, DFU, and SQ-29548 (dimethylsulphoxide). Further dilutions were prepared with distilled water.
Data Analysis. Results are presented as mean ± S.E.M. and analyzed by repeated measures ANOVA, followed by a Student-Newman-Keuls test or by unpaired Student's t test where appropriate. P < 0.05 was considered statistically significant. Maximal NA-, ACh-, BDK-, and sodium nitroprusside-induced responses (Emax) were calculated as maximal percentage decrements or increments of lumen diameter. n indicates the number of experiments.
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, IL-1, and IL-6 compared with controls. Lumen diameters of vessels from control and LPS-treated rats did not significantly differ (Table 1).
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Contractile Reactivity to NA. NA concentration-dependently induced contractions in mesenteric vessels from both control and LPS-treated animals. Vessels from LPS-treated rats showed hyporeactivity to the first four concentrations of NA compared with controls, a difference that no longer occurred when higher concentrations were applied (Fig. 1). Indeed, NA reached its maximum contracting effect at the concentration of 10 µM, with similar values in both groups (Emax 60.6 ± 1.3% and 55.6 ± 2.6% in controls and LPS-treated rats, respectively). No further significant increments were detected at the concentration of 100 µM (Fig. 1). Based on these findings, 10 µM NA was used to precontract vessels in subsequent experiments.
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Effects of SC-560, DFU, and L-NMMA on Endothelium-Dependent Relaxations. In control rats, relaxation of resistance arteries evoked by ACh (Emax 97.8 ± 0.9%) was not modified by exposure to SC-560 (98.5 ± 0.6%) or DFU (97.8 ± 1.0%) (Fig. 2A), whereas it was significantly blunted by L-NMMA (Emax 64.1 ± 1.6%; inhibition –33.7 ± 1.0%) (Fig. 2A; Fig. 6).
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In LPS-injected rats, relaxation to ACh was attenuated (P < 0.001) in comparison with controls (Emax 69.9 ± 3.6%) and significantly improved, but not normalized, by DFU (84.6 ± 2.9%, P < 0.05 versus control) (Fig. 2B). Relaxation to ACh was unaffected by SC-560 (70.2 ± 3.9%) (Fig. 2B).
Similarly to results obtained with ACh, relaxation to BDK was significantly (P < 0.001) blunted in vessels from LPS-treated rats (Emax 73.3 ± 2.1%) compared with controls (Emax 95.2 ± 1.9%). Moreover, DFU, although not modifying the response to BDK in controls (Emax 94.7 ± 2.1%), significantly (P < 0.01) improved, but did not normalize, relaxation to BDK in vessels from LPS-treated rats (Emax 82.1 ± 1.3%, P < 0.05 versus controls).
In vessels from LPS-treated rats, the inhibitory effect exerted by L-NMMA on relaxation evoked by ACh (62.4 ± 2.5%; inhibition –7.5 ± 1.6%) was significantly lower compared with controls (Fig. 6). Under the same conditions, DFU application not only improved the relaxant responses to ACh (ACh alone, 71.1 ± 0.3%; ACh plus DFU, 84.2 ± 1.1%) but also enhanced the inhibitory effect of L-NMMA on endothelial-mediated relaxation (ACh plus L-NMMA, 62.1 ± 1.8%; inhibition, –9.0 ± 1.4%; ACh plus L-NMMA and DFU, 61.4 ± 1.9%; inhibition, –22.8 ± 1.4%) (Fig. 2C; Fig. 6). Upon incubation with DFU, the blunting effect of L-NMMA on maximal response to ACh, although improved, was still significantly lower compared with controls (Fig. 6).
In vessels from LPS-treated rats, the relaxant response to ACh (Emax 70.3 ± 1.7%) was not affected by incubation with SQ-29548 (Emax 71.2 ± 2.6%).
Endothelium-independent relaxation by sodium nitroprusside was similar in control (Emax 96.6 ± 0.7%) and LPS-treated vessels (Emax 96.5 ± 0.8%) and was not modified by DFU (Emax 96.2 ± 1.1 and 96.9 ± 1.5 in control and LPS-treated vessel, respectively).
Interaction between iNOS and COX-2. In vessels from animals treated with SMT plus LPS, relaxation to ACh, although attenuated in comparison with controls, was significantly increased (Emax 84.1 ± 1.6%) when compared with LPS alone (Fig. 3). Interestingly, following iNOS inhibition, the relaxant response to ACh was no longer enhanced by DFU (Emax 86.1 ± 1.9%) (Fig. 3). In rats treated with SMT alone, the response to ACh (Emax 96.9 ± 2.8%) was similar to that recorded in controls and was not affected by DFU (Emax 96.5 ± 2.7%).
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Effect of Ascorbic Acid on Responses to Acetylcholine. In control rats, relaxation to ACh (98.1 ± 0.7%) was not modified by ascorbic acid (98.3 ± 0.7%). By contrast, the antioxidant drug significantly improved endothelium-dependent relaxation in endotoxemic rats (ACh, 71.0 ± 0.6%; ACh with ascorbic acid, 96.4 ± 0.9%; Fig. 4A). Moreover, ascorbic acid restored the inhibitory effect of L-NMMA on agonist-induced relaxation (ACh plus L-NMMA, 62.8 ± 0.6%; inhibition, –8.2 ± 1.1%; ACh plus L-NMMA and ascorbic acid, 61.7 ± 0.6%; inhibition, –34.7 ± 1.2%; Fig. 4A; Fig. 6). Under ascorbic acid, the relaxing effect of ACh on vessels from LPS-treated rats was no longer different from that exerted by ACh alone in control rats (Fig. 6). Similarly, upon exposure to ascorbic acid, the blunting effect of L-NMMA on maximal relaxation elicited by ACh in endotoxemic rats (–34.7 ± 1.2%) was similar to that measured in control animals (–32.9 ± 1.0%).
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When ascorbic acid and DFU were applied to vessels from LPS-treated rats, the COX-2 inhibitor did not further improve the enhancing action of ascorbic acid on the maximal relaxant response elicited by ACh (ACh, 70.5 ± 0.6%; ACh plus DFU, 84.8 ± 0.5%; ACh plus ascorbic acid, 96.6 ± 0.3%; ACh plus ascorbic acid and DFU, 95.5 ± 0.2%; Fig. 4B).
Effect of SOD on Responses to Acetylcholine. As expected, in control rats, relaxation to ACh (98.5 ± 0.6%) was not modified by SOD (98.0 ± 0.9%). By contrast, and similarly to that observed with ascorbic acid, in endotoxemic rats SOD administration normalized the vascular response to ACh and restored the inhibitory effect of L-NMMA on ACh-induced relaxation (ACh, 68.3 ± 0.8%; ACh with SOD, 97.2 ± 1.1%; ACh plus L-NMMA, 61.4 ± 0.9%; inhibition, –6.9 ± 0.9%; ACh plus L-NMMA and SOD, 64.7 ± 1.1%; inhibition, –32.5 ± 1.1%; Figs. 5A and 6). When SOD and DFU were simultaneously applied to mesenteric vessels from LPS-treated rats, no further improvement of the maximal relaxant response elicited by ACh was observed (ACh, 70.1 ± 0.8%; ACh plus DFU, 82.4 ± 0.7%; ACh plus SOD, 95.9 ± 0.4%; ACh plus SOD and DFU, 96.2 ± 0.3%; Fig. 5B).
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RT-PCR Analysis of COX and NOS Isoform Expression. RT-PCR analysis showed a basal expression of mRNA encoding COX-1, COX-2, and endothelial nitric-oxide synthase (eNOS), but not iNOS, in control rats. After treatment with LPS, a significant induction of iNOS and COX-2 was detected, whereas eNOS and COX-1 expressions were not affected (Fig. 7).
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As expected, treatment with SMT alone failed to modify both COX-1 and COX-2 expressions. By contrast, although not modifying COX-1, the iNOS inhibitor significantly reduced, without completely abolishing, the LPS-induced COX-2 expression in mesenteric vessels (Fig. 8).
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Immunohistochemical Analysis of COX-2. In control mesenteric arteries, a weak COX-2 immunopositivity was detected (Fig. 9A). By contrast, in vessels from LPS-injected rats, a marked enhancement in COX-2 immunostaining was observed, both in endothelial cells and within the muscle layer (Fig. 9B).
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; Brandes et al., 1999; Piepot et al., 2000).
With respect to the mechanisms responsible for endothelial dysfunction, to assess NO availability, we employed the NOS inhibitor L-NMMA. In LPS-injected rats, the inhibition by L-NMMA on cholinergic relaxations was lower than in controls. Concomitantly, we detected an enhanced expression of iNOS in mesenteric vascular walls, whereas eNOS was un-affected. These findings, although extending to the mesenteric vascular district the concept that endotoxemia is associated with up-regulated iNOS expression (Hocherl et al., 2002), indicate that LPS-induced endothelial dysfunction is characterized by a significant reduction in stimulated NO availability. The different behavior of NOS isoforms in response to LPS (up-regulation of iNOS and unmodified eNOS expression), together with the reduced NO availability, is in line with a previous report on rabbit carotid arteries exposed to inflammatory cytokines (Kessler et al., 1997), showing that iNOS up-regulation was associated with an unmodified eNOS expression and an impaired endothelial function.
The major novel finding of our study concerns the involvement of COX-2 in endothelial dysfunction elicited by endotoxic shock. In mesenteric vessels from LPS-treated rats, DFU, but not SC-560, improved endothelium-dependent relaxation, indicating that COX-2, but not COX-1, contributes to the pathogenesis of endothelial dysfunction associated with endotoxemia. The selectivity of the COX-2 inhibitor for endothelium-dependent relaxation was supported by the observation that DFU improved the relaxant response of mesenteric vessels to different endothelial agonists, such as ACh and bradykinin, without effects on the response to sodium nitroprusside. Of note, DFU improved, without normalizing, endothelium-dependent relaxation, suggesting that COX-2-independent pathways are also responsible for LPS-induced vascular effects. In keeping with our functional results, an up-regulation of COX-2 expression in the mesenteric district, secondary to endotoxemia, was detected by RT-PCR. Furthermore, immunohistochemical analysis revealed a marked up-regulation of COX-2, but not COX-1, in the endothelial layer of mesenteric arteries from LPS-treated rats. These findings provide the first demonstration of COX-2 localization in endothelial cells of rat small resistance arteries and strongly support our findings that endothelial COX-2 is involved in endotoxin-induced endothelial dysfunction. Interestingly, DFU restored partly the inhibitory effect of L-NMMA on cholinergic relaxation in LPS-injected rats, indicating that selective COX-2 blockade improves NO availability and providing the first evidence for the existence of a close correlation between endothelial COX-2 expression and reduced NO-dependent relaxation in endotoxic shock.
A cross talk between iNOS and COX-2, with a strict dependence of COX-2 up-regulation on iNOS activation, has been previously demonstrated in the heart (Shinmura et al., 2002), whereas in vitro studies in other tissues gave conflicting results (Goodwin et al., 1999). Our experiments revealed that, after in vivo iNOS inhibition, the LPS-induced endothelial dysfunction was attenuated and that COX-2 blockade by DFU was no longer able to improve the response to ACh. Concomitantly, RT-PCR analysis showed that, under iNOS inhibition, LPS-induced COX-2 up-regulation was reduced but not completely abrogated. Taken together, these findings indicate that the enhancement of COX-2 expression and activity occurs downstream of iNOS induction, thus demonstrating the existence of a hierarchical relationship between iNOS and COX-2 in mesenteric vessels. Of importance, under iNOS inhibition, the endothelium-dependent relaxation, although improved, was still attenuated compared with controls, thus supporting the concept that COX-2-independent pathways contribute to LPS-induced effects.
In line with a recent study on patients with Crohn's disease, a clinical condition characterized by severe inflammation of gut wall and mesenteric district (Tabernero et al., 2003), LPS up-regulated COX-2 expression also in the mesenteric muscle layer. However, at least in our experimental conditions, the increased muscular expression of COX-2 does not appear to interfere with relaxant responses mediated by NO pathways, as demonstrated by the unmodified relaxations to sodium nitroprusside in vessels from LPS-treated rats. Rather, it is conceivable that up-regulated muscular COX-2 interacts with other endogenous mediators involved in the control of vascular contractility. This hypothesis is in keeping with previous data (Tabernero et al., 2003), where COX-2 inhibition attenuated NA-induced vasoconstriction, suggesting an involvement of COX-2 products in the regulation of vascular responses to vasoconstrictor mediators.
The increased iNOS expression, together with the decreased NO availability secondary to endotoxemia, raises the possibility that NO might be inactivated by an enhanced production of ROS, as supported by previous evidence (Brandes et al., 1999; Macarthur et al., 2000; Hernanz et al., 2004b). To address this issue, we first used ascorbic acid, an antioxidant compound capable of scavenging superoxide anions at high concentrations (Jackson et al., 1998). In LPS vessels, the blunted endothelium-dependent relaxation was completely reversed by exposure to ascorbic acid. Furthermore, in the presence of ascorbic acid, L-NMMA increased its inhibiting effect on endothelium-dependent relaxations to values not different from those observed in control vessels. These findings suggest a major involvement of ROS in reducing NO availability in endotoxemia. However, the beneficial effect of ascorbic acid might depend on mechanisms other than scavenging superoxide, such as stimulation of NO production, reduced lipid peroxidation, or increased production of tetrahydrobiopterin (Tomasian et al., 2000). For these reasons, we also tested SOD, a specific superoxide anion scavenger. The results obtained with this scavenger were similar to those achieved with ascorbic acid. Taken in conjunction, such findings indicate that, in the presence of antioxidant compounds, the activity of NO pathway is restored, thus demonstrating that ROS represent the main mechanism accounting for NO breakdown during endotoxemia in resistance arteries. In line with this proposal, SOD abolished the LPS-induced endothelial dysfunction in isolated cerebral arteries (Hernanz et al., 2004b). Moreover, previous reports demonstrated that the enhanced NO production, secondary to iNOS up-regulation (Stoclet et al., 1999; Piepot et al., 2000), succeeded in reacting with superoxide anions to generate peroxynitrite, a substance responsible for endothelial cytotoxic effects (Katusic, 1996). Overall, it is conceivable that enhanced ROS production could play a crucial role in reducing endothelial NO availability in rats with endotoxic shock. However, tissue ROS production and nitrite/nitrate concentrations were not measured in our study; therefore, the above hypothesis could not be substantiated by direct mechanicistic evidence. Furthermore, it is worth mentioning that different durations of endotoxemia might lead to the recruitment of other pathophysiological mechanisms, with induction of different vascular responses. For instance, SOD did not restore, but further deteriorated, ACh-induced relaxation in aorta rings from animals exposed to LPS for 12 to 30 h (Brandes et al., 1999).
As far as the pathogenesis of oxidative injury is concerned, there is evidence that COX-2 may be a source of oxygen radicals (Katusic, 1996; O'Banion, 1999). Indeed, the catalytic activity of cyclooxygenase consists of a series of radical reactions that use molecular oxygen and generate intermediate ROS (Marnett, 2000). Thus, it is conceivable that an increase in COX-2 activity during endotoxemia might promote an overproduction of ROS. In our experiments the reduced endothelium-dependent relaxation was partly restored by COX-2 blockade, whereas being completely normalized by ascorbic acid or SOD. Moreover, when mesenteric vessels were simultaneously exposed to DFU plus ascorbic acid or SOD, no further potentiation of the relaxing responses to ACh was observed. Taken together, these data suggest that ROS production, occurring in the presence of endotoxic shock, can be partly ascribed to COX-2-dependent mechanisms, leading to endothelial dysfunction. Although the molecular mechanisms whereby COX-2 promotes ROS production, and ROS can then impair the endothelial function during endotoxemia, were not examined in the present study, some pathways putatively involved in these processes have been schematically outlined in Fig. 10. It is also noteworthy that COX-2 seems to account for a great part, but not for the whole production of ROS, suggesting that COX-2-independent sources of ROS might contribute to endothelial dysfunction. Although elucidation of this aspect deserves further investigation, inflammatory cytokines, with particular regard for TNF--induced activation of NADPH oxidase, are likely candidates (Frey et al., 2002).
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Besides enhancing ROS generation, COX-2 overexpression might also produce vasoconstrictor prostanoids (Cipollone et al., 2001). To address this issue, we used the TxA2 receptor antagonist SQ-29548 and found that this drug failed to modify the endothelium-dependent relaxation of mesenteric vessels from LPS-treated rats, a finding which is in keeping with previous data obtained in cerebral arteries isolated from WKY rats (Hernanz et al., 2004b). Overall, these data, together with our results showing that the response to sodium nitroprusside was similar in control and LPS-treated rats, exclude the participation of vasoconstrictor prostanoids acting on TxA2 receptor in COX-2 mediated endothelial dysfunction.
In conclusion, the present study indicates that small mesenteric vessels from rats with endotoxic shock are characterized by impaired endothelial function due to reduced NO availability and that such an alteration is partly dependent on iNOS-dependent COX-2 overexpression. It also suggests that ROS represent the main mechanism responsible for reduced NO availability and that COX-2 could contribute to their generation.
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Footnotes |
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ABBREVIATIONS: ROS, reactive oxygen species; LPS, lipopolysaccharide; iNOS, inducible nitric-oxide synthase; COX, cyclooxygenase; TXA2, thromboxane A2; PSS, physiological salt solution; SBP, systolic blood pressure; NA, noradrenaline; ACh, acetylcholine; SC-560, 5-(4-clorophenyl)-1-(4-metoxyphenyl)-3-trifluoromethylpirazole; DFU, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)-phenyl-2(5H)-furanone; NS-398, N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide; BDK, bradykinin; NOS, nitric-oxide synthase, L-NMMA, NG-monomethyl-L-arginine; SQ-29548, 7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-[1S(1alpha,2alpha(Z),3alpha,4alpha)]-5-heptenoic acid; SMT, S-methylisothiourea; SOD, superoxide dismutase; RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DAB, 3,3'-diaminobenzidine tetrahydrochloride; TNF-, tumor necrosis factor-; IL, interleukin; eNOS, endothelial nitric-oxide synthase.
Address correspondence to: Dr. Agostino Virdis, Department of Internal Medicine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy. E-mail: a_virdis{at}yahoo.com
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, P < 0.01 versus LPS + DFU.
