• View in gallery
    Figure 1—

    Cumulative concentration-response curves (mean ± SEM) of acetylcholine (A) or sodium nitroprusside (B) in phenylephrine-preconstricted intact EDVs. Responses were obtained in control EDVs (white squares [n = 6]) or LPS-treated EDVs in the absence (black squares [6]) or the presence (black circles [6]) of SOD (200 U/mL). *Significantly (P < 0.05) different control value versus value in the LPS-treated group. Significantly (P < 0.05) different value in the LPS-treated group versus value in the LPS + SOD-treated group.

  • View in gallery
    Figure 2—

    Cumulative concentration-response curves (mean ± SEM) of isoprenaline (A) or SR 58611A (B) in phenylephrine-precon-stricted intact EDVs. See Figure 1 for key.

  • View in gallery
    Figure 3—

    Cumulative concentration-response curves (mean ± SEM) of isoprenaline (A) or SR 58611A (B) in phenylephrine-precon-stricted endothelium-denuded EDVs. See Figure 1 for key.

  • View in gallery
    Figure 4—

    Cumulative concentration-response curves (mean ± SEM) of isoprenaline in phenylephrine-preconstricted intact EDVs. The responses were obtained in control EDVs (white squares [n = 6]) or LPS-treated EDVs in the absence (black squares [6]) or the presence (black circles [6]) of indomethacin (^M) or NS-398 (^M; black diamonds [6]). *Significantly (P < 0.05) different control values versus value in LPS-treated group. Significantly (P < 0.05) different value in the LPS-treated group versus value in the LPS + indomethacin-treated group. Significantly (P < 0.05) different value in the LPS-treated group versus value in the LPS + NS-398-treated group.

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Evaluation of the role of superoxide anions in endotoxin-induced impairment of β-adrenoceptor-mediated vasodilation in equine digital veins

Mohamed Y. MallemFrom ONIRIS, Unit of Animal Pathophysiology and Functional Pharmacology (UPSP 5304), Atlanpole-La C.hantrerie, BP 40706, Nantes, F-44307, France and Université Nantes Angers Le Mans, Nantes, France.

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Aurélie ThuleauFrom ONIRIS, Unit of Animal Pathophysiology and Functional Pharmacology (UPSP 5304), Atlanpole-La C.hantrerie, BP 40706, Nantes, F-44307, France and Université Nantes Angers Le Mans, Nantes, France.

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Jacques NoireaudFrom ONIRIS, Unit of Animal Pathophysiology and Functional Pharmacology (UPSP 5304), Atlanpole-La C.hantrerie, BP 40706, Nantes, F-44307, France and Université Nantes Angers Le Mans, Nantes, France.

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Jean-Claude DesfontisFrom ONIRIS, Unit of Animal Pathophysiology and Functional Pharmacology (UPSP 5304), Atlanpole-La C.hantrerie, BP 40706, Nantes, F-44307, France and Université Nantes Angers Le Mans, Nantes, France.

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Marc GognyFrom ONIRIS, Unit of Animal Pathophysiology and Functional Pharmacology (UPSP 5304), Atlanpole-La C.hantrerie, BP 40706, Nantes, F-44307, France and Université Nantes Angers Le Mans, Nantes, France.

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Abstract

Objective—To investigate the role of superoxide anions in the lipopolysaccharide (LPS)-induced impairment of β-adrenoceptor-mediated equine digital vein (EDV) vasodilation.

Sample Population—EDVs isolated from forelimbs of 24 healthy adult horses.

Procedures—Endothelium-intact or endothelium-denuded EDV rings were incubated with or without LPS (10 μg/mL) of Escherichia coli (O55:B5) for 4 hours. Cumulative concentration-relaxation curves resulting from administration of isoprenaline, a nonselective β-adrenoceptor agonist, or from administration of SR 58611A, a selective β3-adrenoceptor agonist, were recorded in phenylephrine-preconstricted EDVs in the absence or the presence of superoxide dismutase (200 U/mL). Isoprenaline-induced relaxation was also evaluated with or without the cyclooxygenase inhibitors indomethacin (10μM) and NS-398 (10μM).

Results—Isoprenaline and SR 58611A induced concentration-dependent relaxation of EDV rings, which was inhibited by LPS exposure. Superoxide dismutase abolished the inhibitory effect of LPS on the isoprenaline- and SR 58611A-mediated relaxation. Pretreatment of the LPS-treated EDVs with indomethacin or NS-398 restored the isoprenaline-mediated relaxation and abolished the LPS-induced impairment to a similar extent as superoxide dismutase.

Conclusions and Clinical Relevance—Results supported a role of superoxide anions in the LPS-induced impairment of β-adrenoceptor-mediated EDV vasodilation. The LPS-induced oxidative stress in EDVs may contribute to vascular dysfunctions associated with laminitis in horses.

Abstract

Objective—To investigate the role of superoxide anions in the lipopolysaccharide (LPS)-induced impairment of β-adrenoceptor-mediated equine digital vein (EDV) vasodilation.

Sample Population—EDVs isolated from forelimbs of 24 healthy adult horses.

Procedures—Endothelium-intact or endothelium-denuded EDV rings were incubated with or without LPS (10 μg/mL) of Escherichia coli (O55:B5) for 4 hours. Cumulative concentration-relaxation curves resulting from administration of isoprenaline, a nonselective β-adrenoceptor agonist, or from administration of SR 58611A, a selective β3-adrenoceptor agonist, were recorded in phenylephrine-preconstricted EDVs in the absence or the presence of superoxide dismutase (200 U/mL). Isoprenaline-induced relaxation was also evaluated with or without the cyclooxygenase inhibitors indomethacin (10μM) and NS-398 (10μM).

Results—Isoprenaline and SR 58611A induced concentration-dependent relaxation of EDV rings, which was inhibited by LPS exposure. Superoxide dismutase abolished the inhibitory effect of LPS on the isoprenaline- and SR 58611A-mediated relaxation. Pretreatment of the LPS-treated EDVs with indomethacin or NS-398 restored the isoprenaline-mediated relaxation and abolished the LPS-induced impairment to a similar extent as superoxide dismutase.

Conclusions and Clinical Relevance—Results supported a role of superoxide anions in the LPS-induced impairment of β-adrenoceptor-mediated EDV vasodilation. The LPS-induced oxidative stress in EDVs may contribute to vascular dysfunctions associated with laminitis in horses.

Equine laminitis is believed to be related to vascular disorders in the foot,1–4 and digital vascular reactivity has received increasing interest in the investigation of the pathophysiologic mechanisms underlying its development. Blood flow disturbance in digital microcirculation has been involved in the genesis of foot ischemia5 of the sensitive dermal lamellae, which leads to failure of the attachment between the distal phalanx and the inner hoof wall. The initial vascular dysfunctions associated with equine laminitis seem to occur preferentially at the digital vein level.6 The preferential aspect of this location is supported by the presence of a significant increase in postcapillary resistance in the laminar microcirculation7 and by the fact that laminar veins8 and digital palmar veins9 are more sensitive than digital palmar and laminar arteries to various vasoconstrictor stimuli. However, it should be mentioned that the precise pathophysiologic mechanisms involved in acute equine laminitis remain incompletely defined. The view that equine laminitis is attributed to reduced digital blood flow is now challenged. One study10 failed to find any evidence that digital hypoperfusion is linked with the onset of equine laminitis. More interestingly, some investigators have found vasodilation and increased digital blood flow10,11 rather than vasoconstriction during the development of equine laminitis. Moreover, recent investigations suggest that the lamellar separation of laminitis involves vascular-independent mechanisms such as excessive release of matrix metalloproteinases12 or decreased glucose metabolism.13

On the basis of the carbohydrate overload model, it has been hypothesized that endotoxins released from the cell walls of lysed gram-negative bacteria could be the trigger factor for acute laminitis in horses.14 However, the relationship between endotoxemia and laminitis seems to be indirect because systemic endotoxin administration fails to induce clinical signs of laminitis in horses.15,16 Although the relationship still needs to be clarified, in vivo LPS administration is reported to reduce digital blood flow.17 Moreover, in vitro studies reveal impairment of reactivity in equine digital vessels incubated with LPS18 or isolated from horses that have been administered LPS.19 However, the precise mechanism involved in the LPS-induced alteration of vascular digital reactivity has not yet been completely elucidated. The deleterious effects of LPS are well known to involve an immune and inflammatory response leading to release of different mediators including cytokines and arachidonic acid-derived products from various cell types.20–22 At the vascular level, LPS may induce endothelial dysfunction19,23 or vascular damage.24,25 Arachidonic acid products alter EDV responses after LPS exposure,18,26 although the role played by COX-2 and the mechanism underlying its action have not yet been specified. Lipopolysaccharide via the reduced form of NADPH oxidase27 or COX-228 may act as a source of ROS. Thus, increased production of superoxide anions and other ROS could be responsible for the LPS-induced vascular impairment of the contractile and relaxant responses observed in laminitic horses.

In a previous study,18 we determined in an experimental model of laminitis that β-adrenoceptor-mediated relaxation of EDVs was impaired in the presence of LPS of Escherichia coli. β-Adrenoceptors play an important role in the control of blood vessel tone and have been reported to be functionally expressed in EDVs and equine digital arteries.18,29,30 The purpose of the study reported here was to investigate a possible role of superoxide anions in the LPS-induced impairment of β-adrenoceptor-mediated EDV vasodilation.

Materials and Methods

Specimen preparation—At an abattoir (Cholet, France), distal portions of forelimbs were collected from 30 healthy adult horses immediately after euthanasia via stunning and exsanguination. Digital veins were quickly removed as close to the coronary band as possible and flushed with ice-cold Kreb solution containing 118.3mM NaCl, 4.7mM KCl, 1.2mM MgSO4, 1.2mM KH2PO4, 20mM NaHCO3, 0.016mM EDTA, 11.1mM glucose, and 2.5mM CaCl2 and oxygenated with 95% O2 and 5% CO2 (pH, 7.4). After collection, vessels were maintained in ice-cold Kreb solution for transport to the laboratory (30 to 35 minutes). In the laboratory, EDVs were dissected free from surrounding tissue and cut into rings 3 to 4 mm long.

LPS treatment of EDV rings—The EDV rings were exposed at 37°C to LPS of E colia (10 μg/mL) in 2-mL tubes containing media (Dulbecco modified Eagle mediuma) supplemented with 10% horse serum,a 25mM HEPESa buffer (pH, 7.4), penicillin (100 U/mL), and streptomycin (100 mg/mL) for 4 hours in an incubator with 95% O2 and 5% CO2. Rings exposed to media without LPS were used as a negative control.

Isometric tension recording and vasodilation studies—Following incubation, control (LPS-untreat-ed) or LPS-treated EDV rings were washed with Kreb solution and suspended between stainless steel wires in a 5-mL organ bathb containing Kreb solution (95% O2 and 5% CO2; 37°C). Isometric tension was recorded as described.18 Briefly, 1 side of each ring was attached to the base of the organ bath, and the other side was fixed to a force isometric transducer.b Then, EDV rings were stretched progressively to a resting tension of 2 g. Isometric tension changes were continuously measured by force-displacement transducersb and recorded on data-acquisition software.c After a 1-hour equilibration period with the Kreb solution changed every 15 minutes, EDV rings were contracted with 2μM phenylephrine,a an a1-adrenoreceptor agonist. The presence of functional endothelium was checked by the presence of at least 70% relaxation to 1μM acetylcholinea for intact EDVs unexposed to LPS or 50% for LPS-treated intact EDVs. This low threshold of relaxation was based on preliminary experiments that revealed that the amplitude of the relaxation evoked by acetylcholine was substantially lower in LPS-treated EDV rings than in controls. In some experiments, at the end of the incubation period, EDV rings were denuded of endothelium by gentle rubbing of the intimal surface with a pair of small, fine forceps. Endothelium removal was confirmed by the absence of acetylcholine-induced relaxation.

After a second washing period of 30 minutes, some EDV rings were incubated in Kreb solution containing 200 U of SODa/mL (a superoxide anions scavenger), 10μM indomethacina (a nonselective COX inhibitor), or 10μM NS-398a (a selective COX-2 inhibitor) for 30 minutes. Control EDV rings were not treated during that period. The superoxide anions’ scavenging effect for SOD was checked in preliminary experiments that revealed that the alteration of acetylcholine-induced relaxation by xanthine-xanthine oxidase, a superoxide-generating system (xanthine,a 500 μM; xanthine oxidase,a 0.01 U/mL), was abolished by 200 U of SOD/mL.

The EDV rings were contracted again with phenylephrine, the concentration (0.3 to 2μM) of which was adjusted to induce a similar level of tone for each experimental condition (5 ± 0.2 g). Once the contraction reached a plateau, CCRCs to acetylcholine (1nM to 3μM), an endothelium-dependent vasodilator, or SNPa (0.1nM to 30μM), an endothelium-independent vasodilator, were performed in control intact EDVs or in LPS-treated intact EDVs. The CCRCs to isoprenalinea (1nM to 3μM [a nonspecific β12-adrenoceptor agonist]) or SR 58611Ad (0.1μM to 300μM [a selective β3-adrenoceptor agonist]) were constructed in control or in LPS-treated EDVs with or without endothelium.

Statistical analysis—Relaxant responses were calculated as the percentage change in the maximal tension of vessel rings after addition of phenylephrine. Data were reported as mean ± SEM, and n refers to the number of animals examined. Comparisons of whole CCRCs were performed by use of a 2-way ANOVA with repeated measures. The EC50 value, the Emax, and the Hill slope factor were calculated by means of a computerized curve fitting technique based on the Hill equation (E/Emax = Cn/(Cn + [EC50]nH, where E = effect, Cn = concentration, and nH = Hill slope factor) when the maximal response reached a plateau.e The Emax and pD2 values (pD2 = -log EC50) calculated for the different experimental conditions were compared by use of 1-way ANOVA with a post hoc Bonferroni test. For each statistical test, a value of P < 0.05 was considered to be significant.

Results

In intact EDV rings, acetylcholine caused a concentration-dependent relaxation (pD2 = 8 ± 0.2; Emax = 83.3 ± 4.6%; n = 6). Treatment with LPS shifted the acetylcholine CCRC to the right and decreased the maximum response (pD2 = 7.4 ± 0.1 [P = 0.01]; E = 65.3 ± 5.3% [P < 0.05]; n = 6). Treatment with SOD totally restored the relaxant response of the LPS-treated EDV rings (pD2 = 7.9 ± 0.02 [P < 0.01]; Emax = 87.3 ± 2.7% [P = 0.01]; n = 6; Figure 1). In intact EDV rings, SNP induced a concentration-dependent relaxation (pD2 = 7.3 ± 0.1; Emax = 98.3 ± 2.9%; n = 6) that was unaltered after LPS exposure (pD2 = 7.4 ± 0.1; E = 102.9 ± 1%; 6).

Figure 1—
Figure 1—

Cumulative concentration-response curves (mean ± SEM) of acetylcholine (A) or sodium nitroprusside (B) in phenylephrine-preconstricted intact EDVs. Responses were obtained in control EDVs (white squares [n = 6]) or LPS-treated EDVs in the absence (black squares [6]) or the presence (black circles [6]) of SOD (200 U/mL). *Significantly (P < 0.05) different control value versus value in the LPS-treated group. Significantly (P < 0.05) different value in the LPS-treated group versus value in the LPS + SOD-treated group.

Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.773

In intact EDV rings, isoprenaline and SR 58611A induced concentration-dependent relaxations (pD2 = 7.3 ± 0.09; Emax = 93.7 ± 4.9%; n = 6 [for isoprenaline]) that were significantly inhibited by LPS exposure (pD2 = 7.2 ± 0.1; Emax = 54 ± 3.2% [P = 0.01]; 6 [for isoprenaline]). Pretreatment of the LPS-treated EDVs with SOD (Figure 2) abolished the inhibitory effect of LPS on isoprenaline and SR 58611A-mediated EDV relaxations (pD2 = 7.6 ± 0.1; Emax = 74.8 ± 6.7% [P < 0.05]; n = 6 [for isoprenaline]). In denuded EDV rings (Figure 3), CCRCs to isoprenaline (pD2 = 6.8 ± 0.09; Emax = 66.5 ± 6%; n = 6) and SR 58611A were not modified by LPS exposure in either the absence (pD2 = 6.7 ± 0.1; E = 73.5 ± 5.2%; n = 6) or the presence of SOD (pD2 = 6.7 ± 0.1; Emax = 74.8 ± 5%; 6).

Figure 2—
Figure 2—

Cumulative concentration-response curves (mean ± SEM) of isoprenaline (A) or SR 58611A (B) in phenylephrine-precon-stricted intact EDVs. See Figure 1 for key.

Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.773

Figure 3—
Figure 3—

Cumulative concentration-response curves (mean ± SEM) of isoprenaline (A) or SR 58611A (B) in phenylephrine-precon-stricted endothelium-denuded EDVs. See Figure 1 for key.

Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.773

Vasodilation in response to isoprenaline was also examined in LPS-treated intact EDVs in the absence or the presence of COX inhibitors. The relaxant response to isoprenaline (pD2 = 7.3 ± 0.07; Emax = 88 ± 6.2%; n = 6) was significantly (P = 0.01) reduced by LPS exposure (pD2 = 7.2 ± 0.1; Emax = 50.8 ± 4.1%; 6). Indomethacin or NS-398 significantly (P = 0.01) restored the isoprenaline-mediated relaxation of the LPS-treated EDVs (indomethacin pD2 = 7.1 ± 0.2; E = 94 ± 5.6%; n = 6; NS-398 pD2 = 7.3 ± 0.2; Emax = 90.6 ± 8%; 6) and abolished the LPS-induced impairment (Figure 4).

Figure 4—
Figure 4—

Cumulative concentration-response curves (mean ± SEM) of isoprenaline in phenylephrine-preconstricted intact EDVs. The responses were obtained in control EDVs (white squares [n = 6]) or LPS-treated EDVs in the absence (black squares [6]) or the presence (black circles [6]) of indomethacin (^M) or NS-398 (^M; black diamonds [6]). *Significantly (P < 0.05) different control values versus value in LPS-treated group. Significantly (P < 0.05) different value in the LPS-treated group versus value in the LPS + indomethacin-treated group. Significantly (P < 0.05) different value in the LPS-treated group versus value in the LPS + NS-398-treated group.

Citation: American Journal of Veterinary Research 71, 7; 10.2460/ajvr.71.7.773

Discussion

Results of some studies indicate that horses exposed to LPS have decreased digital blood flow17 and impairment of endothelium-dependent vasodilation.31 However, the underlying mechanism has not been fully investigated. Because vascular cells can produce superoxide anions on exposure to LPS,27 we hypothesized that LPS-induced impairment of β-adrenoceptor-mediated EDV relaxation could be related to increased ROS production. In EDV rings in the present study, treatment with LPS led to impairment of β1-, β2-, and β3-adrenoceptor-mediated vasodilation dependent on superoxide anion generation. As reported, the interaction between LPS and cells in the host is markedly enhanced by a specific cell-associated LPS receptor, termed CD14, and a plasma protein (LPS-binding protein) that facilitates transfer of LPS to CD14.32 In isolated blood vessels, LPS might be expected to be ineffective because the endothelial and the vascular smooth muscle cells do not contain the membrane-bound form of CD14.33 However, LPS signaling could occur through an LPS-binding protein and membrane CD14-independent pathway.34 In the present study, EDVs were incubated with LPS of E coli in the presence of horse serum components. The presence of a soluble form of CD14 could be a prerequisite for the LPS-induced effect in EDVs potentially lacking membrane CD14 receptor. Future experiments are required to address the involvement of

CD14 in the LPS-mediated effect in EDV. Isolated EDVs or endothelial cultures incubated with LPS and leukocytes have been used as models of equine laminitis.26 In those models, vascular alteration induced by LPS could involve ROS generation, likely through leukocyte activation. Although ROS production was not measured in the present study, restoration of relaxation by SOD treatment in EDVs incubated with LPS without activated neutrophils suggested that superoxide anion release may also occur as a result of a direct LPS-EDV interaction. This assumption was supported by preliminary results that indicated that 200 U of SOD/mL was able to quench superoxide anions produced by the X/XO generating system, and the assumption was corroborated with a direct action of LPS on the vascular cells.35 Although the involvement of endotoxin in equine laminitis is not completely elucidated, the use of isolated digital vessels under LPS exposure as a model of equine laminitis18,26 can yield useful information for the understanding of the modification of digital vascular reactivity in this disease. Nevertheless, caution should be exercised when extrapolating the results obtained to in vivo conditions because the concentrations of LPS used in that model were higher than those observed in clinical situations. In the present study, we chose 10 μg of LPS/mL because, in preliminary experiments, 1 μg/mL induced much smaller effects. The need to use such a high concentration of LPS to induce a cellular response in vitro may be ascribed either to the absence of cofactors that would facilitate the effect of LPS in vivo or to the possibility that, in an vivo situation, the effect of LPS at the vascular level could occur mainly indirectly through cytokine mediators (tumor necrosis factor and interleukin-1) released from activated leukocytes.

In the present study, treatment with LPS reduced endothelium-dependent relaxation that occurred in response to acetylcholine. These findings were consistent with previous reports that endotoxin induces impairment of endothelium-dependent relaxation in equine digital arteries and veins19,31 and in other vascular beds from nonequine species.36,37 It is not clear how endotoxin impairs endothelium-dependent relaxation. Decreased NO production, hyporesponsiveness of vascular smooth muscle to NO, or altered receptor coupling may be possible explanations. The present findings suggested that the impairment of acetylcholine-mediated EDV relaxation was caused by an increase in inactivation of NO by superoxide anions because this impairment was restored by SOD. In agreement with this suggestion, the response to the direct NO donor SNP remained unchanged, indicating that the LPS-induced alteration in response to muscarinic stimulation was not attributable to changes in the activity of guanylate cyclase or other distal elements in the signal transduction pathway.

Concerning responses to β-adrenoceptor stimulation, the present study revealed that LPS induced an impairment of β1-, β2-, and β3-adrenoceptor-mediated EDV relaxations that were entirely restored by antioxidant treatment. Impairment of β-adrenoceptor-mediated response following LPS exposure has been reported38 but without specifying the contribution of ROS. In the present study, overproduction of superoxide anions in endothelial cells could be responsible for enhanced inactivation of NO, resulting in impaired endothelial response to isoprenaline and SR 58611A. There are several lines of evidence to support this contention. First, the β1-, β2-, and β3-adrenoceptor-mediated EDV vasodilation is reported to be mediated partly through an endothelium-NO pathway.18 Second, in the present study, endothelium-independent vasodilation in response to isoprenaline and SR 58611A was unaltered after LPS treatment. Third, LPS-treated EDVs had normal responses to SNP, an NO donor, which directly activates smooth muscle guanylyl cyclase. Therefore, the LPS-induced deleterious effect was not extended to the smooth muscle cells. However, it is not known whether 10 μg/mL is sufficient to elicit vascular smooth muscle dysfunction if a prolonged incubation (> 4 hours) was allowed. The fact that LPS did not modify the potency of the isoprenaline indicated that the coupling of β-and β2-adrenoceptors was not affected by superoxide anions. In contrast, this seemed to be not the case for β3-adrenoceptors, suggesting that β1-, β2- and β3-adrenoceptors may be affected differently by LPS treatment. The β3-adrenoceptors differ from β1-adrenoceptors and β2-adrenoceptors in that they lack the phosphorylation sites for β-adrenoceptor kinases and cAMP-dependent protein kinase39 and may not be downregulated in pathophysiologic conditions associated with a sympathetic overdrive such as endotoxemia.40 Whether the different sensitivity of β3-adrenoceptors to superoxide anions could be related to those properties remains to be determined.

In endothelial cells, numerous sources of ROS have been described, including prostaglandin metabolism and cytochrome p450 electron transport.41,42 Importantly, it has been reported that COX-2 may be responsible for ROS generation.28,43 It is speculated that an increase in COX-2 activity, in the presence of LPS,44 might induce an overproduction of ROS. In the present experiments, the nonselective COX inhibitor, indomethacin, restored the relaxant responses to isoprenaline in LPS-treated EDVs. Interestingly, the COX-2 selective inhibitor, NS-398,45 induced a similar effect as indomethacin, which strongly suggested that COX-2 but not COX-1 contributes to the prostanoid-mediated alteration by LPS of β-adrenoceptor-induced relaxation. This finding is consistent with observations of upregulation only of COX-2 in response to LPS in distinct cell types including equine digital vessels.46 Because ROS can be generated by the COX-2 pathway, the beneficial effects of SOD on the isoprenaline and SR 58611A-induced vasodilation after LPS exposure could be attributable to scavenging of ROS by the COX-2 pathway. Although, from the present results, COX-2 seemed to account for the entire production of ROS, we cannot exclude that a COX-2-independent origin of ROS might contribute to β-adrenoceptor dysfunction in EDV7. Moreover, it is possible that COX-2-induced ROS formation could occur indirectly through NADPH oxidase or XO activation. Other experiments with selective NADPH oxidase or XO inhibitors such as apocinin or allopurinol are needed to address this possibility. However, it should be mentioned that ROS can stimulate prostaglandin H2 and thromboxane A2 production by vascular cells.41 Thus, the possibility is raised that the superoxide anion-mediated

LPS effect in EDV may be ascribed to these endothelial vasoconstrictor mediators. Furthermore, reaction of superoxide anions with NO is well documented27 and not only causes its inactivation, but also results in the formation of peroxynitrite (ONOO-), a highly reactive and cytotoxic molecule.47 In addition to reduced NO availability, peroxynitrite has been reported to be able to directly alter the β-adrenoceptors’ affinity.48 Thus, it can be suggested that superoxide anion-induced alteration of vasorelaxation in EDV may involve an impairment of β-adrenoceptor-linked mechanisms and a decrease in sensitivity of the β-adrenoceptors themselves.

Limitations of this study included the in vitro nature of the study, the concentration of the endotoxin used, and the potential differences in the vascular reactivity in various digital territories. Many investigations have used palmar digital vessels isolated from healthy horses or from horses with experimentally induced laminitis.9,49 The use of that peculiar vascular bed has limitations because modifications of the vascular reactivity at that level may not correspond to the responses of the blood vessels within the laminae. The use of laminar veins or laminar arteries8,50 has the advantage of studying vascular reactivity in vessels that contribute importantly to control blood flow in the laminar microvasculature. Although it has been reported that palmar digital vessels and laminar vessels can have a similar pattern of responses (ie, veins more sensitive to vasoconstrictors than arteries),8,9 we cannot reject the possibility that the modifications in venous reactivity under LPS exposure to β-adrenoceptor agonists or in venous sensitivity to ROS might not be identical in these vessels. Thus, care must be taken not to extrapolate the present results to laminar veins. Further investigations should be focused on the laminar veins to determine whether β-adrenoceptor-mediated vasodilation may undergo an oxidative action following LPS exposure.

Although the relationship between endotoxemia and equine laminitis remains controversial, the present study has provided functional evidence that the LPS-induced impairment of β-adrenoceptor-mediated vasodilation involves superoxide anion generation most likely through the COX-2 pathway. Impairment of vasodilation that might be induced by LPS during the developmental stage of laminitis could involve an impairment of β-adrenoceptor-mediated relaxation that can be attributable to ROS generation. This oxidative action may be considered as another operative mechanism that occurred additionally to reperfusion injury. Further investigations about the pathophysiologic relevance of oxidative stress in equine digital vessels and about the clinical use of antioxidant drugs for the treatment of laminitis in horses are warranted.

ABBREVIATIONS

CCRC

Cumulative concentration-relaxation curve

CD14

Cluster of differentiation 14

COX-2

Cyclooxygenase-2

EC50

Half maximal effective concentration

EDV

Equine digital vein

Emax

Maximal effect

LPS

Lipopolysaccharide

NADPH

Nicotinamide adenine dinucleotide phosphate (reduced)

ROS

Reactive oxygen species

SNP

Sodium nitroprusside

SOD

Superoxide dismutase

a.

Sigma Chemical Co, Lyon, France.

b.

EMKA Technologies, Paris, France.

c.

Acqknowledge BIOPAC system, MP100, Goleta, Calif.

d.

Provided by Sanofi Synthelabo Recherche, Labege, France.

e.

Prism, version 4, GraphPad Software Inc, San Diego, Calif.

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Contributor Notes

Address correspondence to Dr. Mallem (yassine.mallem@oniris-nantes.fr).