• View in gallery
    Figure 1—

    Time course of production (mean ± SEM values from 6 horses) of PGI2 by EDVECs exposed to LPS (1 μg/mL [black squares]) or saline (0.9% NaCl) solution (white squares). *Significant (P ≤ 0.05) difference between LPS-treated and control cells.

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    Figure 2—

    Time course of production (mean ± SEM values from 6 horses) of cGMP by EDVECs exposed to LPS (1 μg/mL [black squares]) or saline solution (white squares). See Figure 1 for key.

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    Figure 3—

    Effect of cycloheximide (100μM), ibuprofen (10μM), and L-NAME (100μM) on LPS (1μg/mL)-induced cGMP production (mean ± SEM values from 6 horses) by EDVECs. †Significant (P ≤ 0.05) difference between LPS-treated and LPS plus inhibitortreated cells. See Figure 1 for remainder of key.

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    Figure 4—

    Effect of cycloheximide (100μM), ibuprofen (10μM), and L-NAME (100μM) on LPS (1μg/mL)-induced PGI2 production (mean ± SEM values from 6 horses) by EDVECs. See Figures 1 and 3 for key.

  • View in gallery
    Figure 5—

    Time course of production (mean ± SEM values from 6 horses) of ET-1 by EDVECs exposed to LPS (1 μg/mL [black squares]) or saline solution [white squares]).

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    Figure 6—

    Effect of LPS concentration on expression of COX-2 (mean ± SEM values for cells from 7 horses [24-hour incubation time]) by EDVECs. Panel illustrates a representative western blot. *Significant (P < 0.05) difference from baseline value. †Significant (P < 0.01) difference from baseline value.

  • 1.

    Robertson TP, Peroni JF, Noschka E, et al. Prostanoids and isoprostanes as inflammatory and vasoactive conduits in the development of laminitis. Recent advances in the physiology of equine laminitis Havemeyer Foundation Workshop 2007;120.

    • Search Google Scholar
    • Export Citation
  • 2.

    Bailey SR. The pathogenesis of acute laminitis: fitting more pieces into the puzzle. Equine Vet J 2004;36:199203.

  • 3.

    Hunt JM, Edwards GB, Clarke KW. Incidence, diagnosis and treatment of postoperative complications in colic cases. Equine Vet J 1986;18:264270.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Sprouse RF, Garner HE, Green EM. Plasma endotoxin levels in horses subjected to carbohydrate induced laminitis. Equine Vet J 1987;19:2528.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Morris DD. Endotoxemia in horses. A review of cellular and humoral mediators involved in its pathogenesis. J Vet Intern Med 1991;5:167181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Peroni JF, Harrison WE, Moore JN, et al. Black walnut extractinduced laminitis in horses is associated with heterogenous dysfunction of the laminar microvasculature. Equine Vet J 2005;37:546551.

    • Search Google Scholar
    • Export Citation
  • 7.

    Hood DM, Grosenbaugh DA, Mostafa MB, et al. The role of vascular mechanisms in the development of acute equine laminitis. J Vet Intern Med 1993;7:228234.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Baxter GM. Alterations of endothelium-dependent digital vascular responses in horses given low-dose endotoxin. Vet Surg 1995;24:8796.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Zerpa H, Vega F, Vasquez J, et al. Effect of acute sublethal endotoxemia on in vitro digital vascular reactivity in horses. J Vet Med A Physiol Pathol Clin Med 2005;52:6773.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Turek JJ, Lamar CH, Fessler JF, et al. Ultrastructure of equine endothelial cells exposed to endotoxin and flunixin meglumine and equine neutrophils. Am J Vet Res 1987;48:13631366.

    • Search Google Scholar
    • Export Citation
  • 11.

    Bottoms GD, Johnson MA, Lamar CH, et al. Endotoxin-induced eicosanoid production by equine vascular endothelial cells and neutrophils. Circ Shock 1985;15:155162.

    • Search Google Scholar
    • Export Citation
  • 12.

    Rodgerson DH, Belknap JK, Moore JN, et al. Investigation of mRNA expression of tumor necrosis factor-A, interleukin1B, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin. Am J Vet Res 2001;62:19571963.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Frangos JA, McIntire LV, Eskin SG. Shear stress-induced stimulation of mammalian cell metabolism. Biotechnol Bioeng 1988;32:10531060.

  • 14.

    Busse R, Trogisch G, Bassenge E. The role of endothelium in the control of vascular tone. Basic Res Cardiol 1985;80:475490.

  • 15.

    Janssens SP, Shimouchi A, Quertermous T, et al. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem 1992;267:1451914522.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest 1991;21:361374.

  • 17.

    Akarasereenont P, Chotewuttakorn S, Techatraisak K, et al. The effects of COX-metabolites on cyclooxygenase-2 induction in LPS-treated endothelial cells. J Med Assoc Thai 2001;84(suppl 3):S696–S709.

    • Search Google Scholar
    • Export Citation
  • 18.

    Bottoms GD, Johnson M, Ward D, et al. Release of eicosanoids from white blood cells, platelets, smooth muscle cells, and endothelial cells in response to endotoxin and A23187. Circ Shock 1986;20:2534.

    • Search Google Scholar
    • Export Citation
  • 19.

    Katwa LC, Johnson PJ, Ganjam VK, et al. Expression of endothelin in equine laminitis. Equine Vet J 1999;31:243247.

  • 20.

    Fleming I, Busse R. NO: the primary EDRF. J Mol Cell Cardiol 1999;31:514.

  • 21.

    Ramaswamy CM, Eades SC, Venugopal CS, et al. Plasma concentrations of endothelin-like immunoreactivity in healthy horses and horses with naturally acquired gastrointestinal tract disorders. Am J Vet Res 2002;63:454458.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Bunting S, Moncada S, Vane JR. The prostacyclin—thromboxane A2 balance: pathophysiological and therapeutic implications. Br Med Bull 1983;39:271276.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Nawroth PP, Stern DM, Dietrich M. Thromboxane production by perturbed bovine aortic endothelial cells in culture. Blut 1985;50:711.

  • 24.

    Pfister SL, Hughes MJ, Rosolowsky M, et al. Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells. Prostaglandins Other Lipid Mediat 2002;70:3949.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Bailey SR, Elliott J. Evidence for different 5-HT1B/1D receptors mediating vasoconstriction of equine digital arteries and veins. Eur J Pharmacol 1998;355:175187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Katz LM, Marr CM, Elliott J. Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1. Am J Vet Res 2003;64:14381443.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Peroni JF, Moore JN, Noschka E, et al. Predisposition for venoconstriction in the equine laminar dermis: implications in equine laminitis. J Appl Physiol 2006;100:759763.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    King JN, Gerring EL. Detection of endotoxin in cases of equine colic. Vet Rec 1988;123:269271.

  • 29.

    Allen D Jr, Clark ES, Moore JN, et al. Evaluation of equine digital Starling forces and hemodynamics during early laminitis. Am J Vet Res 1990;51:19301934.

    • Search Google Scholar
    • Export Citation
  • 30.

    Eaton SA, Allen D, Eades SC, et al. Digital Starling forces and hemodynamics during early laminitis induced by an aqueous extract of black walnut (Juglans nigra) in horses. Am J Vet Res 1995;56:13381344.

    • Search Google Scholar
    • Export Citation
  • 31.

    Menzies-Gow NJ, Bailey SR, Katz LM, et al. Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators. Equine Vet J 2004;36:273278.

    • Search Google Scholar
    • Export Citation
  • 32.

    Kalogeris TJ, Kevil CG, Laroux FS, et al. Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells. Am J Physiol 1999;276:L9–L19.

    • Search Google Scholar
    • Export Citation
  • 33.

    Bailey SR, Wheeler-Jones CP, Elliott J. Uptakes of 5-hydroxytryptamine by equine digital vein endothelial cells: inhibition by amines found in the equine caecum. Equine Vet J 2003;35:164169.

    • Search Google Scholar
    • Export Citation
  • 34.

    Akarasereenont P, Mitchell JA, Bakhle YS, et al. Comparison of the induction of cyclooxygenase and nitric oxide synthase by endotoxin in endothelial cells and macrophages. Eur J Pharmacol 1995;273:121128.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Berhane Y, Bailey SR, Harris PA, et al. In vitro and in vivo studies of homocysteine in equine tissues: implications for the pathophysiology of laminitis. Equine Vet J 2004;36:279284.

    • Search Google Scholar
    • Export Citation
  • 36.

    Lees P, Ewins CP, Taylor JB, et al. Serum thromboxane in the horse and its inhibition by aspirin, phenylbutazone and flunixin. Br Vet J 1987;143:462476.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Katz LM, Elliott J, Marr CM. Measurement of plasma endothelin-1 from normal and laminitic horses and ponies using a quantitative sandwich enzyme immunoassay technique. J Vet Intern Med 2002;16:355.

    • Search Google Scholar
    • Export Citation
  • 38.

    Janicke H, Taylor PM, Bryant CE. Lipopolysaccharide and interferon gamma activate nuclear factor kappa B and inducde cyclooxygenase-2 in equine vascular smooth muscle cells. Res Vet Sci 2003;75:133140.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Mirza MH, Seahorn TL, Oliver JL, et al. Detection and comparison of nitric oxide in clinically healthy horses and those with naturally acquired strangulating large colon volvulus. Can J Vet Res 2005;69:106155.

    • Search Google Scholar
    • Export Citation
  • 40.

    Bishop-Bailey D, Larkin SW, Warner TD, et al. Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture. Br J Pharmacol 1997;121:125133.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Kis B, Snipes JA, Simandle SA, et al. Acetaminophen-sensitive prostaglandin production in rat cerebral endothelial cells. Am J Physiol Regul Interg Comp Physiol 2005;288:R897–R902.

    • Search Google Scholar
    • Export Citation
  • 42.

    Bailey SR, Elliott J. The role of prostanoids and nitric oxide in endotoxin-induced hyporesponsiveness of equine digital blood vessels. Equine Vet J 1999;31:212218.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Kosonen O, Kankaanranta H, Malo-Ranta U, et al. Inhibition by nitric oxide-releasing compounds of prostacyclin production in human endothelial cells. Br J Pharmacol 1998;125:247254.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Swierkosz TA, Mitchell JA, Warner TD, et al. Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids. Br J Pharmacol 1995;114:13351342.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Akarasereenont P, Bakhle YS, Thiemermann C, et al. Cytokinemediated induction of cyclo-oxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide. Br J Pharmacol 1995;115:401408.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46.

    Chen JX, Berry LC, Christman BW, et al. Glutathione mediates LPS-stimulated transient p42/44 MAPK activation. J Cell Physiol 2003;197:8693.

  • 47.

    Huang H, Rose JL, Hoyt DG. p38 Mitogen-activated protein kinase mediates synergistic induction of inducible nitric-oxide synthase by lipopolysaccharide and interferon-gamma through signal transducer and activator of transcription 1 Ser727 phosphorylation in murine aortic endothelial cells. Mol Pharmacol 2004;66:302311.

    • Search Google Scholar
    • Export Citation
  • 48.

    Radomski MW, Palmer RM, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci U S A 1990;87:1004310047.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49.

    MacNaul KL, Hutchinson NI. Differential expression of iNOS and cNOS mRNA in human vascular smooth muscle cells and endothelial cells under normal and inflammatory conditions. Biochem Biophys Res Commun 1993;196:13301334.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50.

    Marumo T, Nakaki T, Adachi H, et al. Nitric oxide synthase mRNA in endothelial cells: synergistic induction by interferongamma, tumor necrosis factor-alpha and lipopolysaccharide and inhibition by dexamethasone. Jpn J Pharmacol 1993;63:327334.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Kilbourn RG, Belloni P. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin. J Natl Cancer Inst 1990;82:772776.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52.

    Oswald IP, Eltoum I, Wynn TA, et al. Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide. Proc Natl Acad Sci U S A 1994;91:9991003.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 53.

    Greenberg SS, Jie O, Zhao X, et al. Role of PKC and tyrosine kinase in ethanol-mediated inhibition of LPS-inducible nitric oxide synthase. Alcohol 1998;16:167175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54.

    Connelly L, Madhani M, Hobbs AJ. Resistance to endotoxic shock in endothelial nitric-oxide synthase (eNOS) knock-out mice. J Biol Chem 2005;280:1004010046.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55.

    Kan WH, Yan WS, Jiang Y, et al. Role of p38 mitogen-activated protein kinase in lipopolysaccharide-induced expression of inducible nitric oxide synthase in human endothelial cells. Di Yi Jun Yi Da Xue Xue Bao 2002;22:388392.

    • Search Google Scholar
    • Export Citation
  • 56.

    Fulger LA, Eades SC, Truax RE, et al. Nitric oxide and endothelin-1 synthesis by cultured equine digital endothelial cells in response to endotoxin and cytokines, in Proceedings. 1st Equine Laminitis Research Meet Panel 2004;114115.

    • Search Google Scholar
    • Export Citation
  • 57.

    Ros J, Leivas A, Jimenez W, et al. Effect of bacterial lipopolysaccharide on endothelin-1 production in human vascular endothelial cells. J Hepatol 1997;26:8187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 58.

    MacEachern KE, Smith GL, Nolan AM. Methods for the isolation, culture and characterisation of equine pulmonary artery endothelial cells. Res Vet Sci 1997;62:147152.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 59.

    Sugiura M, Inagami T, Kon V. Endotoxin stimulates endothelinrelease in vivo and in vitro as determined by radioimmunoassay. Biochem Biophys Res Commun 1989;161:12201227.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 60.

    Malek A, Izumo S. Physiological fluid shear stress causes downregulation of endothelin-1 mRNA in bovine aortic endothelium. Am J Physiol 1992;263:C389–C396.

    • Search Google Scholar
    • Export Citation

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Evaluation of the induction of vasoactive mediators from equine digital vein endothelial cells by endotoxin

Nicola J. Menzies-GowDepartment of Veterinary Clinical Sciences, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, England.

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Simon R. BaileyDepartment of Veterinary Basic Sciences, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, England.

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Yoel BerhaneDepartment of Veterinary Basic Sciences, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, England.

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Andrew C. BrooksDepartment of Veterinary Basic Sciences, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, England.

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Jonathan ElliottDepartment of Veterinary Basic Sciences, Royal Veterinary College, North Mymms, Hertfordshire AL9 7TA, England.

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Abstract

Objective—To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs).

Sample Population—EDVECs obtained from forelimb digital veins of 7 healthy adult horses.

Procedures—EDVECs were incubated with or without LPS (1 μg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 μg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A2 were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined.

Results—LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration–dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A2 by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner.

Conclusions and Clinical Relevance—LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.

Abstract

Objective—To determine the effect of endotoxin (lipopolysaccharide [LPS]) on vasoactive mediator production by cultured equine digital vein endothelial cells (EDVECs).

Sample Population—EDVECs obtained from forelimb digital veins of 7 healthy adult horses.

Procedures—EDVECs were incubated with or without LPS (1 μg/mL) for 0, 2, 4, 6, 22, and 24 hours. The EDVECs were incubated for 18 hours with LPS (10 pg/mL to 1 μg/mL) with or without ibuprofen, cycloheximide, or L-nitroarginine methyl ester. Medium concentrations of prostacyclin, cyclic guanosine monophosphate, endothelin-1, and thromboxane A2 were determined. Changes in inducible nitric oxide synthase and cyclooxygenase-2 expression were determined.

Results—LPS stimulated mean 4.2- and 14.1-fold increases in EDVEC prostacyclin and cyclic guanosine monophosphate production, respectively, after 22 hours. These effects were LPS concentration–dependent (LPS concentrations that induced a response halfway between the maximum response and baseline of 1.50 and 1.22 ng/mL, respectively). The LPS-induced cyclic guanosine monophosphate production was significantly inhibited (to basal concentrations) by L-nitroarginine methyl ester, and prostacyclin production was inhibited by cycloheximide and ibuprofen. Production of thromboxane A2 by EDVECs was not detected. Endothelin-1 accumulated in the medium, but LPS did not enhance its production. Inducible nitric oxide synthase expression in EDVECs was not detected with the available antibodies, whereas LPS stimulated cyclooxygenase-2 expression in a time- and concentration-dependent manner.

Conclusions and Clinical Relevance—LPS stimulated vasoactive mediator production by equine endothelial cells, which may play a role in LPS-induced digital hypoperfusion.

Although the precise mechanisms underlying the pathogenesis of laminitis in horses remain unclear, the prodromal stages appear to be associated with concurrent inflammatory and microvascular perturbations.1 The role of endotoxin (ie, LPS) has long been debated2 because laminitis is a sequel to certain forms of colic3 and endotoxin can be detected in the plasma of horses given carbohydrate overload at the onset of Obel grade-3 lameness.4 Lipopolysaccharide exposure initiates multiple pathophysiologic events, including cellular activation and subsequent systemic release of inflammatory and vasoactive mediators.5 Vascular dysfunction plays a central role in the etiology of both laminitis6,7 and endotoxemia,8,9 and LPS may damage the vascular endothelium directly10 or indirectly via the effects of endogenous vasoactive or inflammatory mediator production.11

Vascular tone is influenced by circulating endogenous vasoactive substances and by mediators produced locally within the vascular wall (particularly by the endothelium), which cause smooth muscle cells to change shape or express proteins including cytokines and vasoactive mediators.12 Thus, the endothelium plays an important role in the normal physiologic control of vascular tone and in the hemodynamic changes that develop in pathologic states including endotoxemia. Despite this, the role of endothelium-derived mediators in equine digital perfusion regulation and pathologic states associated with endotoxemia and digital hypoperfusion, such as laminitis, remains poorly understood.

Endothelial cells release vasoactive mediators in response to several physical13 and chemical stimuli.14 Vasodilator mediators include prostacyclin (ie, PGI2) and NO, which are produced by the enzymes COX and NOS, respectively.15 These enzymes exist in multiple isoforms and endothelial cells express constitutively the isoforms COX-1 and eNOS, and also the inducible isoforms COX-2 and iNOS, which are expressed after cellular stimulation by agents, including LPS,16,17 resulting in increased mediator synthesis. Lipopolysaccharide (5 to 100 μg/mL) dosedependently stimulates increased equine pulmonary vascular endothelial cell PGI2 production,18 whereas LPS (20 to 160 μg/mL) increases equine digital artery smooth muscle cell COX-2 expression in a non–dosedependent manner.12

Endothelium-derived vasoconstrictors include ET-1 and Txα2. Endothelin-1 is the most potent endogenous vasoconstrictor yet to be identified19 and interacts with endothelial-derived relaxing factors such as NO to maintain normal local vascular flow.20 Plasma ET-1 concentrations are increased in certain diseases associated with vascular injury, including endotoxemia in horses.21 Thromboxane α2 is a potent vasoconstrictor22 produced by some endothelial cells. High LPS concentrations increase equine pulmonary vascular18 and bovine aortic23 endothelial cell Txα2 production, whereas human vascular endothelial cell Txα2 production has been attributed to contaminating adherent platelets.24

Determination of LPS-induced vasoactive mediator expression in equine digital vasculature may aid understanding of the pathophysiologic events that occur in conditions associated with altered vascular tone and endotoxemia, including laminitis. Although important research has been undertaken to evaluate digital vascular physiologic and pharmacologic phenomena by use of isolated rings of either the digital vessels8,9,25,26 or, more recently, the laminar microvessels,1,7,27 little research has been done on vasoactive mediator production by equine digital endothelial or vascular smooth muscle cells. The only 2 previous studies12,18 addressing these issues have used single, high LPS concentrations, relative to the circulating concentrations in endotoxemia in horses28 or carbohydrate-induced laminitis,4 and those studies did not evaluate digital endothelial cells in isolation. Because digital venoconstriction has been documented in experimentally induced laminitis29,30 and following LPS infusion,31 equine digital veins appear to be more sensitive than other peripheral veins and digital arteries to the effects of vasoconstrictor agonists,25,27 and there is some evidence to suggest that venous endothelial cells are more sensitive to the effects of LPS than arterial endothelial cells,32 the purpose of the study reported here was to evaluate the effect of clinically relevant concentrations of LPS on the production of vasoactive mediators known to be important in LPS-related diseases by equine digital venous cells, as opposed to arterial endothelial cells.

Materials and Methods

Cell culture—The EDVECs were cultured as described33 from the digits of horses euthanized at an abattoir by use of the free bullet method. Briefly, as soon as possible postmortem, the digits were flushed to remove the blood with sterile PBS solution (150 mL) by cannulating the medial and lateral digital veins 3 to 4 cm proximal to the coronary band. The medial and lateral digital arteries were ligated, the digit was infused from the venous side with type II collagenasea (20 mL [1 mg/mL], prewarmed at 37°C), and the limb was incubated in a water bath for 30 minutes at 37°C. The endothelial cells were flushed out and collected by use of sterile PBS solution before being centrifuged (300 × g for 10 minutes). The supernatant was removed and the cells resuspended in culture medium (DMEMb containing 10% fetal calf serum, 10% newborn calf serum, penicillin [100 U/mL], and streptomycin [100 mg/mL]) and transferred to a 75-cm3 flask for incubation at 37°C in 5% CO2 and 95% air. After 24 hours, erythrocyte contamination was removed with warm sterile PBS solution and fresh culture medium was added. After 90% confluency was achieved, characterized by the typical cobblestone morphology and positive results of immunostaining for von Willebrand factor, the cells were removed with trypsin and EDTA solution (1 and 0.25 mg/mL, respectively), resuspended in culture medium, transferred evenly to 24-well plates, and incubated at 37°C for 48 hours to allow the cells to adhere and become confluent.

Effect of LPS on mediator production by ED-VECs—Confluent EDVEC monolayers were made quiescent in serum-free medium for 3 hours and preincubated with the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-2,6(1H,3H)-putinedione-methyl-3-isobutylxzanthinec (IBMX [1mM]) for 30 minutes at 37°C to inhibit breakdown of cGMP. The EDVECs were incubated with DMEM containing 1% bovine serum with or without Escherichia coli LPS O55:B5c (1 μg/mL). The culture medium was sampled after 0, 2, 4, 6, 22, and 24 hours of incubation. The EDVECs were also incubated for 18 hours with LPS ranging in concentration from 10 pg/mL to 1 μg/mL with or without either ibuprofenc (10μM), cycloheximidec (100μM), or L-NAMEc (100μM).

Protein and mediator determinationA radioimmunoassay was used to measure the stable metabolite 6-keto-PGF and so indirectly determine PGI2 concentrations.34 Cyclic GMP concentrations were measured as an index of endothelial NO production by use of a commercial enzyme immunoassay systemd according to the manufacturer's instructions (protocol 2), as described.35 The Txα2 concentrations were determined indirectly by use of a radioimmunoassay, as described.36 The ET-1 concentrations were measured by use of a sandwich ELISA assay for human ET-1e that had been validated for measurement of equine ET-1 in culture medium.37 Mediator concentrations were expressed per milligram of protein in the well, measured after cell lysis by use of a protein assay kit.f

Effect of LPS on COX-2 and iNOS expression by EDVECs—Confluent EDVEC monolayers, in 6-well tissue-culture plates, were made quiescent in serumfree medium for 3 hours at 37°C. The EDVECs were then incubated with DMEM containing 1% bovine serum with or without E coli LPS O55:B53 (1 μg/mL). The EDVECs were also incubated for 24 hours with LPS ranging in concentration from 10 pg/mL to 1 μg/mL. The culture medium was removed, and the cell monolayers were lysed in Laemmli sample buffer, boiled (10 minutes at 95°C), and stored at −80°C. Western blotting was used to assess COX-2 and iNOS expression, as described.38,39

Determination of COX-2 and iNOS expression Equal amounts of protein (50 μg/lane) were separated via SDS-PAGE by use of 7.5% (iNOS) and 10% (COX-2) polyacrylamide gels,g and proteins were transferred onto a nitrocellulose membrane. Immunodetection of iNOS and COX-2 was performed with primary antibodies against iNOSh and COX-2i and isotype-matched secondary horseradish peroxidase (HRP-conjugated) antibodies.d Protein bands were detected by use of an enhanced chemiluminescence kit.d Blots were then scanned, and band density was determined with computer software.j

Statistical analysisStatistical analyses were carried out with computer software.k Values represent mean ± SEM from n = 4 to 7, referring to the number of individual animals from which cells were derived. Values at each time point were compared between the LPS-exposed and control cells by either a 2-way ANOVA followed by Bonferroni post hoc test or a 1-way ANOVA with a Dunnett post hoc test. The effects of the inhibitors cycloheximide, ibuprofen, and L-NAME on LPS-induced mediator production were assessed by use of 1-way ANOVA followed by a Bonferroni post hoc test. Concentration response curves for the effects of LPS on prostacyclin and cGMP production were fitted by a computerized nonlinear iterative regression procedure to a single process logistic equation, as follows:

article image
where E is the response, Emax is the maximum response, EC50 is the LPS concentrations that induced a response halfway between the maximum response and the baseline, n is the slope that describes the steepness of the curve, and C is the LPS concentration. The appropriateness of the monophasic equation was confirmed by comparing the sum of squares of the residuals and the scatter of the points about the line (expressed as the coefficient of variation) with a 2-site equation.

The best fit values for EC50, Emax, and n obtained from each experimental replicate were used to calculate the geometric mean and 95% confidence intervals (EC50) and the arithmetic mean ± SEM (Emax and n). The threshold LPS concentration at which a response was first detected was calculated as the LPS concentration corresponding to the upper 95% confidence interval value for the bottom of the dose response curve. Significance was accepted at P ≤ 0.05.

Results

Endothelial cell mediator production—Lipo poly-saccharide (1 μg/mL) stimulated significant mean ± SEM increases of 4.2 ± 1.4 times and 14.1 ± 5.6 times in EDVEC production of PGI2 and cGMP, respectively, compared with basal concentrations, after 22 hours (Figures 1 and 2). These stimulations were LPS concentration–dependent (Table 1). The LPS-induced cGMP production was significantly (P = 0.01) inhibited to basal concentrations by L-NAME but was unaffected by cycloheximide and ibuprofen (Figure 3). The LPS-induced PGI2 production was partially inhibited by L-NAME, although significance was not reached (P = 0.09), and was significantly inhibited to less than basal concentrations by cycloheximide and ibuprofen (Figure 4). The TxA2 concentration, at all time points and irrespective of LPS concentration, was not significantly different from that of the culture media (0.78 ng/mL). Endothelin-1 accumulated in the medium, with the medium concentration increasing by 82 ± 35 times during the 24-hour incubation period, but mediator production was not enhanced by LPS (Figure 5). There was a large amount of variation in ET-1 production after 22 and 24 hours of incubation both in the presence (313 ± 168 pg of protein/mg and 328 ± 123 pg of protein/mg, respectively) and absence (210 ± 87 pg of protein/mg and 299 ± 131 pg of protein/mg, respectively) of LPS.

Table 1—

Concentration response curve values (n = 6 horses) for PGI2 and cGMP production by EDVECs after 18 hours of exposure to LPS (10 pg/mL to 1 μg/mL).

MediatorEC50 (geometric mean ng/mL [95% CI])Emax (mean ± SEM ng/mg of protein)E0 (mean ± SEM ng/mg of protein)Threshold LPS concentration (mean ± SEM ng/mL)Hillslope (mean ± SEM)
PGI21.50 (0.75-3.04)1,682 ± 54*373 ± 440.25 ± 0.071.19 ± 0.24
cGMP1.22 (0.75-1.98)22.62 ± 0.6*4.23 ± 0.590.126 ± 0.061.09 ± 0.26

Significant (P < 0.001) difference from E0 values.

EC50 = LPS concentrations that induced a response halfway between the maximum response and the baseline. Emax = Maximum response. E0 = Basal response. CI = Confidence interval.

Figure 1—
Figure 1—

Time course of production (mean ± SEM values from 6 horses) of PGI2 by EDVECs exposed to LPS (1 μg/mL [black squares]) or saline (0.9% NaCl) solution (white squares). *Significant (P ≤ 0.05) difference between LPS-treated and control cells.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.349

Figure 2—
Figure 2—

Time course of production (mean ± SEM values from 6 horses) of cGMP by EDVECs exposed to LPS (1 μg/mL [black squares]) or saline solution (white squares). See Figure 1 for key.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.349

Figure 3—
Figure 3—

Effect of cycloheximide (100μM), ibuprofen (10μM), and L-NAME (100μM) on LPS (1μg/mL)-induced cGMP production (mean ± SEM values from 6 horses) by EDVECs. †Significant (P ≤ 0.05) difference between LPS-treated and LPS plus inhibitortreated cells. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.349

Figure 4—
Figure 4—

Effect of cycloheximide (100μM), ibuprofen (10μM), and L-NAME (100μM) on LPS (1μg/mL)-induced PGI2 production (mean ± SEM values from 6 horses) by EDVECs. See Figures 1 and 3 for key.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.349

Figure 5—
Figure 5—

Time course of production (mean ± SEM values from 6 horses) of ET-1 by EDVECs exposed to LPS (1 μg/mL [black squares]) or saline solution [white squares]).

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.349

Endothelial cell COX-2 and iNOS expression—Lipopolysaccharide (1 μg/mL) induced a significant 5-times and 23-times increase in COX-2 expression after 6 and 24 hours, respectively. This increase was concentration-dependent (Figure 6), with 1 ng/mL of LPS significantly increasing expression to 273 ± 51% of basal expression (n = 7). Inducible NOS expression could not be detected in EDVECs with the commercially available antibodies used in this study.

Figure 6—
Figure 6—

Effect of LPS concentration on expression of COX-2 (mean ± SEM values for cells from 7 horses [24-hour incubation time]) by EDVECs. Panel illustrates a representative western blot. *Significant (P < 0.05) difference from baseline value. †Significant (P < 0.01) difference from baseline value.

Citation: American Journal of Veterinary Research 69, 3; 10.2460/ajvr.69.3.349

Discussion

Endotoxin induced timeand concentration-dependent increases in PGI2 and NO production by EDVECs through increased COX-2 expression and increased eNOS activity that was evident over the range of LPS concentrations reported in clinical equine endotoxemia28 and carbohydrate overload-induced laminitis.4 Although ET-1 accumulated in the medium and this was unaltered by LPS, there was considerable variation in the results obtained. Endothelial cells did not appear to release Txα2.

To the authors' knowledge, the production of vasoactive mediators by equine digital endothelial cells has not been evaluated previously. Lipopolysaccharide (10 μg/mL) increased PGI2 production by segments of rat aorta after 24 to 48 hours of incubation40 and by rat cerebral endothelial cells after 6 hours.41 Incubation of human umbilical17 and bovine aortic34 endothelial cells with LPS (0.001 to 1 μg/mL for 24 hours) revealed a timeand concentration-dependent (EC50 not reported) increase in COX metabolite accumulation, enhanced COX activity, and COX-2 protein induction that was inhibited by cycloheximide, dexamethasone, aspirin, and indomethacin pretreatment. Lipopolysaccharide (5 μg/mL for 1 hour) significantly increased PGI2 production by equine pulmonary arterial endothelial cells and increasing LPS concentration (5 to 100 μg/mL) further increased PGI2 production.18 Lipopolysaccharide (20 μg/mL) induced COX-2 upregulation in equine digital artery smooth muscle cells.13 Lipopolysaccharide (1 μg/mL) also increased cultured equine digital blood vessel PGI2 production, an effect that was completely inhibited by cycloheximide and ibuprofen and partially inhibited by L-NAME.42 Human umbilical vein endothelial cells expressed COX-2 and produced prostacyclin in a NS-398– sensitive manner, suggesting that PGI2 production was derived principally by the COX-2 pathway.43 Results of the present study support and extend these previous findings and revealed timeand LPS concentration–dependent increases in EDVEC PGI2 production. The threshold concentration at which LPS-induced PGI2 production was detectable (250 ± 70 pg/mL) suggests that these findings are relevant to clinical endotoxemia in horses28 but are at the high end of the LPS concentrations reported in horses with carbohydrate overload–induced laminitis.4

Prostacyclin production was inhibited to less than basal concentrations by cycloheximide and ibuprofen and reduced by L-NAME. The inhibitory effects of the nonselective COX inhibitor ibuprofen and the protein synthesis inhibitor cycloheximide suggest that LPS-induced endothelial prostanoid release probably results from increased inducible COX, that is, COX-2 synthesis and activity. This was confirmed by the LPS-induced timeand concentration-dependent increase in COX-2 expression. Cross talk between the NO and COX pathways, with NO stimulating COX-2, has been detected in murine macrophages.44 The apparent partial inhibitory effect of the NOS inhibitor L-NAME on PGI2 production by EDVECs may suggest that this also occurs in equine endothelial cells. A link between NO and regulation of eicosanoid synthesis could represent an important mechanism in controlling vascular and inflammatory responses in pathophysiologic states. Induction of COX-2 by LPS in bovine aortic endothelial cells is mediated by a tyrosine kinase45 and LPS-stimulated COX-2 gene and protein expression, and PGI2 release by bovine pulmonary artery endothelial cells is mediated by activation of p38 or p42/44 mitogen-activated protein kinases.46 However, the signalling pathways involved in equine digital endothelial cell COX-2 activation remain unknown.

In some species, LPS upregulates vascular NO production. High LPS concentrations (10 μg/mL) stimulated NO production by cultured segments of rat aorta that peaked after 48 hours40 but failed to stimulate NO production by murine aortic endothelial cells.47 Slightly lower LPS concentrations (1 μg/mL) failed to stimulate nitrite production by either bovine aortic endothelial cells34 or segments of equine digital vessels.42 In all of those studies, the Griess reaction was used to quantify NO production, which is not very sensitive. One study48 found that LPS-induced endothelial cell NO production was detectable by use of the oxyglobin assay but not the Griess reaction. Evidence of iNOS induction was not detected in human49 and rat50 aortic endothelial cells exposed to LPS or cytokines by use of the chemiluminescence assay. Nitric oxide production may have been detectable in those studies if cellular cGMP production had been used as a marker of its biological activity, as used in the present study. Lipopolysaccharide, in combination with other cytokines, induced iNOS activity in murine endothelial cells,51 whereas LPS alone did not increase nitrite production52 or iNOS mRNA.50 Thus, it is also possible that LPS requires cytokines, generated in vivo from several cell types directly or indirectly, to induce iNOS. In the present study, L-NAME–sensitive NO production was significantly increased in response to LPS in a timeand LPS concentration– dependent manner in the absence of additional cytokines. Thus, it would appear that EDVECs differ from other endothelial cells in their LPS sensitivity, resulting in increased NO production and lack of requirement for cytokines for NOS upregulation. Similar to PGI2, LPS-induced cGMP production was detectable from 126 ± 60 pg of LPS/mL, suggesting that these responses may be relevant to clinical endotoxemia in horses28 but occur at the high end of the LPS concentrations reported in horses with carbohydrate overload–induced laminitis.4

Upregulation of iNOS expression has been reported to be the mechanism by which endothelial cells increase NO production in response to LPS.53 The expression of iNOS and subsequent high-output NO production is believed to underlie the systemic hypotension, inadequate tissue perfusion, and organ failure associated with endotoxemia in humans.54 In 1 study,55 LPS induced human endothelial cell iNOS expression via the p38 mitogen-activated protein kinase signalling pathway. In another study, LPS activated human umbilical vein endothelial NOS through phosphoinositide 3-kinase and Akt-protein kinase B–dependent enzyme phosphorylation with the resultant NO acting as a costimulus for the expression of iNOS.54 The absence of an effect of the protein synthesis inhibitor, cycloheximide, at a concentration that inhibited LPS-induced mediator production by segments of equine digital vessel42 on LPS-stimulated EDVEC NO production in the present study suggests that increased activity of a constitutively expressed enzyme (eNOS) is involved rather than synthesis of an inducible isoform. The means by which the activity of eNOS may be upregulated in these circumstances remains unclear. Unfortunately, the commercially available antibodies for iNOS protein tested in the present study did not recognize the equine form of the protein, so it was not possible to confirm that increased iNOS expression did not occur in response to LPS.

Lipopolysaccharide increased equine pulmonary endothelial cell thromboxane production but only at concentrations greater than 10 μg/mL,18 which is much higher than the concentrations used in the present study and the concentrations measured in clinical endotoxemia. Lower concentrations of LPS (100 ng/mL for 6 hours) stimulated Txα2 production by rat cerebral endothelial cells41 but not bovine aortic endothelial cells (1 ng/mL to 1 μg/mL for 24 hours).34 In the present study, EDVECs did not appear to produce Txα2.

Cultured equine pulmonary artery, equine digital artery and vein, and human umbilical vein endothelial cells produced ET-1 continuously over 24 hours, although the rate of release decreased after the first 4 hours of incubation.56–58 The pattern of ET-1 production by control EDVECs in the present study was similar. Lipopolysaccharide (0.1 to 10 μg/mL) promoted ET-1 release from cultured bovine aortic endothelial cells59 and from human umbilical vein endothelial cells in a concentration-dependent manner over a concentration range of 1 to 250 ng/mL, whereas concentrations between 250 and 1,000 ng/mL significantly decreased production.57 Lipopolysaccharide (100 μg/mL) decreased equine digital arterial and venous endothelial cell ET-1 production.56 Thus, the LPS concentration used in the present study was potentially inhibitory. Nonetheless, basal ET-1 production was not reduced by LPS, suggesting that EDVEC ET-1 production is unaffected by 1 μg of LPS/mL. Furthermore, these results are in agreement with the results from an equine in vivo experiment, in which LPS (30 ng/kg) infusion leading to peak plasma LPS concentrations of 13 pg/mL failed to increase plasma ET-1 concentrations.31 However, it must be acknowledged that there was a large amount of variability in EDVEC ET-1 production after 22 and 24 hours of incubation both with and without LPS, which confounded interpretation of these findings.

Endotoxin stimulated EDVEC COX-2 expression and constitutive NOS activity, resulting in increased PGI2 and cGMP production but not ET-1 or Txα2 production, which was evident over the range of LPS concentrations reported in clinical endotoxemia28 and carbohydrate overload–induced laminitis.4 Although cultured endothelial cells are clearly not exposed to the same conditions as they would be in vivo58 (in particular, shear stress that is believed to modulate endothelial cell vasoactive mediator release60), equine digital vein endothelium appeared to be capable of increased vasoactive mediator production in response to LPS. The prodromal stages of laminitis appear to be associated with selective dysfunction of the laminar veins; there is a physiologic predisposition for venoconstriction in the equine digital microvasculature,27 and there are reduced contractile responses to vasoconstrictor mediators following experimental induction of laminitis.7 The increased production of vasodilator mediators induced by LPS may in part explain this reduced response to vasoconstrictor agonists and may play an important role in the alterations in digital perfusion evident in experimental and clinical endotoxemia and laminitis. Further investigations are required to determine the intracellular signalling pathways involved.

ABBREVIATIONS

LPS

Lipopolysaccharide

PGI2

Prostaglandin I2

NO

Nitric oxide

COX

Cyclooxygenase

eNOS

Endothelial nitric oxide synthase

iNOS

Inducible nitric oxide synthase

ET-1

Endothelin-1

TxA2

Thromboxane A2

EDVEC

Equine digital vein endothelial cell

DMEM

Dulbecco modified Eagle medium

cGMP

Cyclic guanosine monophosphate

L-NAME

L-nitroarginine methyl ester

a.

Worthington Biochemical Corp, Lakewood, NJ.

b.

Life Technologies Ltd, Paisley, Scotland.

c.

Sigma-Aldrich Co Ltd, Poole, Dorset, England.

d.

Biotrak cGMP EIA Amersham Pharmacia Biotech, Amersham Biosciences, Buckinghamshire, England.

e.

QuantiGlo, R&D Systems Inc, Minneapolis, Minn.

f.

Pierce Bio Science Ltd, Tattenhall, Cheshire, England.

g.

Biorad Mini Protean II system, Biorad, Hemel Hempstead, England.

h.

Mouse monoclonal anti-human iNOS and rabbit polyclonal anti-human iNOS antibodies, BD Biosciences, Erembodegem, Belgium.

i.

Monoclonal mouse anti-human COX-2 (amino acids 580-599), Cayman Chemical Co, Ann Arbor, Mich.

j.

Image J image analysis software, Company, city, state, country. Available at: rsb.info.nih.gov/ij. Accessed Month, year.

k.

GraphPad Prism, version 3.00 for Windows, GraphPad Software, San Diego, Calif.

References

  • 1.

    Robertson TP, Peroni JF, Noschka E, et al. Prostanoids and isoprostanes as inflammatory and vasoactive conduits in the development of laminitis. Recent advances in the physiology of equine laminitis Havemeyer Foundation Workshop 2007;120.

    • Search Google Scholar
    • Export Citation
  • 2.

    Bailey SR. The pathogenesis of acute laminitis: fitting more pieces into the puzzle. Equine Vet J 2004;36:199203.

  • 3.

    Hunt JM, Edwards GB, Clarke KW. Incidence, diagnosis and treatment of postoperative complications in colic cases. Equine Vet J 1986;18:264270.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Sprouse RF, Garner HE, Green EM. Plasma endotoxin levels in horses subjected to carbohydrate induced laminitis. Equine Vet J 1987;19:2528.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Morris DD. Endotoxemia in horses. A review of cellular and humoral mediators involved in its pathogenesis. J Vet Intern Med 1991;5:167181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Peroni JF, Harrison WE, Moore JN, et al. Black walnut extractinduced laminitis in horses is associated with heterogenous dysfunction of the laminar microvasculature. Equine Vet J 2005;37:546551.

    • Search Google Scholar
    • Export Citation
  • 7.

    Hood DM, Grosenbaugh DA, Mostafa MB, et al. The role of vascular mechanisms in the development of acute equine laminitis. J Vet Intern Med 1993;7:228234.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Baxter GM. Alterations of endothelium-dependent digital vascular responses in horses given low-dose endotoxin. Vet Surg 1995;24:8796.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 9.

    Zerpa H, Vega F, Vasquez J, et al. Effect of acute sublethal endotoxemia on in vitro digital vascular reactivity in horses. J Vet Med A Physiol Pathol Clin Med 2005;52:6773.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Turek JJ, Lamar CH, Fessler JF, et al. Ultrastructure of equine endothelial cells exposed to endotoxin and flunixin meglumine and equine neutrophils. Am J Vet Res 1987;48:13631366.

    • Search Google Scholar
    • Export Citation
  • 11.

    Bottoms GD, Johnson MA, Lamar CH, et al. Endotoxin-induced eicosanoid production by equine vascular endothelial cells and neutrophils. Circ Shock 1985;15:155162.

    • Search Google Scholar
    • Export Citation
  • 12.

    Rodgerson DH, Belknap JK, Moore JN, et al. Investigation of mRNA expression of tumor necrosis factor-A, interleukin1B, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin. Am J Vet Res 2001;62:19571963.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13.

    Frangos JA, McIntire LV, Eskin SG. Shear stress-induced stimulation of mammalian cell metabolism. Biotechnol Bioeng 1988;32:10531060.

  • 14.

    Busse R, Trogisch G, Bassenge E. The role of endothelium in the control of vascular tone. Basic Res Cardiol 1985;80:475490.

  • 15.

    Janssens SP, Shimouchi A, Quertermous T, et al. Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase. J Biol Chem 1992;267:1451914522.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 16.

    Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest 1991;21:361374.

  • 17.

    Akarasereenont P, Chotewuttakorn S, Techatraisak K, et al. The effects of COX-metabolites on cyclooxygenase-2 induction in LPS-treated endothelial cells. J Med Assoc Thai 2001;84(suppl 3):S696–S709.

    • Search Google Scholar
    • Export Citation
  • 18.

    Bottoms GD, Johnson M, Ward D, et al. Release of eicosanoids from white blood cells, platelets, smooth muscle cells, and endothelial cells in response to endotoxin and A23187. Circ Shock 1986;20:2534.

    • Search Google Scholar
    • Export Citation
  • 19.

    Katwa LC, Johnson PJ, Ganjam VK, et al. Expression of endothelin in equine laminitis. Equine Vet J 1999;31:243247.

  • 20.

    Fleming I, Busse R. NO: the primary EDRF. J Mol Cell Cardiol 1999;31:514.

  • 21.

    Ramaswamy CM, Eades SC, Venugopal CS, et al. Plasma concentrations of endothelin-like immunoreactivity in healthy horses and horses with naturally acquired gastrointestinal tract disorders. Am J Vet Res 2002;63:454458.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22.

    Bunting S, Moncada S, Vane JR. The prostacyclin—thromboxane A2 balance: pathophysiological and therapeutic implications. Br Med Bull 1983;39:271276.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23.

    Nawroth PP, Stern DM, Dietrich M. Thromboxane production by perturbed bovine aortic endothelial cells in culture. Blut 1985;50:711.

  • 24.

    Pfister SL, Hughes MJ, Rosolowsky M, et al. Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells. Prostaglandins Other Lipid Mediat 2002;70:3949.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Bailey SR, Elliott J. Evidence for different 5-HT1B/1D receptors mediating vasoconstriction of equine digital arteries and veins. Eur J Pharmacol 1998;355:175187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Katz LM, Marr CM, Elliott J. Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1. Am J Vet Res 2003;64:14381443.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Peroni JF, Moore JN, Noschka E, et al. Predisposition for venoconstriction in the equine laminar dermis: implications in equine laminitis. J Appl Physiol 2006;100:759763.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    King JN, Gerring EL. Detection of endotoxin in cases of equine colic. Vet Rec 1988;123:269271.

  • 29.

    Allen D Jr, Clark ES, Moore JN, et al. Evaluation of equine digital Starling forces and hemodynamics during early laminitis. Am J Vet Res 1990;51:19301934.

    • Search Google Scholar
    • Export Citation
  • 30.

    Eaton SA, Allen D, Eades SC, et al. Digital Starling forces and hemodynamics during early laminitis induced by an aqueous extract of black walnut (Juglans nigra) in horses. Am J Vet Res 1995;56:13381344.

    • Search Google Scholar
    • Export Citation
  • 31.

    Menzies-Gow NJ, Bailey SR, Katz LM, et al. Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators. Equine Vet J 2004;36:273278.

    • Search Google Scholar
    • Export Citation
  • 32.

    Kalogeris TJ, Kevil CG, Laroux FS, et al. Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells. Am J Physiol 1999;276:L9–L19.

    • Search Google Scholar
    • Export Citation
  • 33.

    Bailey SR, Wheeler-Jones CP, Elliott J. Uptakes of 5-hydroxytryptamine by equine digital vein endothelial cells: inhibition by amines found in the equine caecum. Equine Vet J 2003;35:164169.

    • Search Google Scholar
    • Export Citation
  • 34.

    Akarasereenont P, Mitchell JA, Bakhle YS, et al. Comparison of the induction of cyclooxygenase and nitric oxide synthase by endotoxin in endothelial cells and macrophages. Eur J Pharmacol 1995;273:121128.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35.

    Berhane Y, Bailey SR, Harris PA, et al. In vitro and in vivo studies of homocysteine in equine tissues: implications for the pathophysiology of laminitis. Equine Vet J 2004;36:279284.

    • Search Google Scholar
    • Export Citation
  • 36.

    Lees P, Ewins CP, Taylor JB, et al. Serum thromboxane in the horse and its inhibition by aspirin, phenylbutazone and flunixin. Br Vet J 1987;143:462476.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37.

    Katz LM, Elliott J, Marr CM. Measurement of plasma endothelin-1 from normal and laminitic horses and ponies using a quantitative sandwich enzyme immunoassay technique. J Vet Intern Med 2002;16:355.

    • Search Google Scholar
    • Export Citation
  • 38.

    Janicke H, Taylor PM, Bryant CE. Lipopolysaccharide and interferon gamma activate nuclear factor kappa B and inducde cyclooxygenase-2 in equine vascular smooth muscle cells. Res Vet Sci 2003;75:133140.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 39.

    Mirza MH, Seahorn TL, Oliver JL, et al. Detection and comparison of nitric oxide in clinically healthy horses and those with naturally acquired strangulating large colon volvulus. Can J Vet Res 2005;69:106155.

    • Search Google Scholar
    • Export Citation
  • 40.

    Bishop-Bailey D, Larkin SW, Warner TD, et al. Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture. Br J Pharmacol 1997;121:125133.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41.

    Kis B, Snipes JA, Simandle SA, et al. Acetaminophen-sensitive prostaglandin production in rat cerebral endothelial cells. Am J Physiol Regul Interg Comp Physiol 2005;288:R897–R902.

    • Search Google Scholar
    • Export Citation
  • 42.

    Bailey SR, Elliott J. The role of prostanoids and nitric oxide in endotoxin-induced hyporesponsiveness of equine digital blood vessels. Equine Vet J 1999;31:212218.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43.

    Kosonen O, Kankaanranta H, Malo-Ranta U, et al. Inhibition by nitric oxide-releasing compounds of prostacyclin production in human endothelial cells. Br J Pharmacol 1998;125:247254.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44.

    Swierkosz TA, Mitchell JA, Warner TD, et al. Co-induction of nitric oxide synthase and cyclo-oxygenase: interactions between nitric oxide and prostanoids. Br J Pharmacol 1995;114:13351342.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45.

    Akarasereenont P, Bakhle YS, Thiemermann C, et al. Cytokinemediated induction of cyclo-oxygenase-2 by activation of tyrosine kinase in bovine endothelial cells stimulated by bacterial lipopolysaccharide. Br J Pharmacol 1995;115:401408.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46.

    Chen JX, Berry LC, Christman BW, et al. Glutathione mediates LPS-stimulated transient p42/44 MAPK activation. J Cell Physiol 2003;197:8693.

  • 47.

    Huang H, Rose JL, Hoyt DG. p38 Mitogen-activated protein kinase mediates synergistic induction of inducible nitric-oxide synthase by lipopolysaccharide and interferon-gamma through signal transducer and activator of transcription 1 Ser727 phosphorylation in murine aortic endothelial cells. Mol Pharmacol 2004;66:302311.

    • Search Google Scholar
    • Export Citation
  • 48.

    Radomski MW, Palmer RM, Moncada S. Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc Natl Acad Sci U S A 1990;87:1004310047.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49.

    MacNaul KL, Hutchinson NI. Differential expression of iNOS and cNOS mRNA in human vascular smooth muscle cells and endothelial cells under normal and inflammatory conditions. Biochem Biophys Res Commun 1993;196:13301334.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 50.

    Marumo T, Nakaki T, Adachi H, et al. Nitric oxide synthase mRNA in endothelial cells: synergistic induction by interferongamma, tumor necrosis factor-alpha and lipopolysaccharide and inhibition by dexamethasone. Jpn J Pharmacol 1993;63:327334.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51.

    Kilbourn RG, Belloni P. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumor necrosis factor, interleukin-1, or endotoxin. J Natl Cancer Inst 1990;82:772776.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 52.

    Oswald IP, Eltoum I, Wynn TA, et al. Endothelial cells are activated by cytokine treatment to kill an intravascular parasite, Schistosoma mansoni, through the production of nitric oxide. Proc Natl Acad Sci U S A 1994;91:9991003.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 53.

    Greenberg SS, Jie O, Zhao X, et al. Role of PKC and tyrosine kinase in ethanol-mediated inhibition of LPS-inducible nitric oxide synthase. Alcohol 1998;16:167175.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 54.

    Connelly L, Madhani M, Hobbs AJ. Resistance to endotoxic shock in endothelial nitric-oxide synthase (eNOS) knock-out mice. J Biol Chem 2005;280:1004010046.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 55.

    Kan WH, Yan WS, Jiang Y, et al. Role of p38 mitogen-activated protein kinase in lipopolysaccharide-induced expression of inducible nitric oxide synthase in human endothelial cells. Di Yi Jun Yi Da Xue Xue Bao 2002;22:388392.

    • Search Google Scholar
    • Export Citation
  • 56.

    Fulger LA, Eades SC, Truax RE, et al. Nitric oxide and endothelin-1 synthesis by cultured equine digital endothelial cells in response to endotoxin and cytokines, in Proceedings. 1st Equine Laminitis Research Meet Panel 2004;114115.

    • Search Google Scholar
    • Export Citation
  • 57.

    Ros J, Leivas A, Jimenez W, et al. Effect of bacterial lipopolysaccharide on endothelin-1 production in human vascular endothelial cells. J Hepatol 1997;26:8187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 58.

    MacEachern KE, Smith GL, Nolan AM. Methods for the isolation, culture and characterisation of equine pulmonary artery endothelial cells. Res Vet Sci 1997;62:147152.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 59.

    Sugiura M, Inagami T, Kon V. Endotoxin stimulates endothelinrelease in vivo and in vitro as determined by radioimmunoassay. Biochem Biophys Res Commun 1989;161:12201227.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 60.

    Malek A, Izumo S. Physiological fluid shear stress causes downregulation of endothelin-1 mRNA in bovine aortic endothelium. Am J Physiol 1992;263:C389–C396.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Supported in part by a project grant from the Horse Race Betting Levy Board.

Dr. Menzies-Gow was supported by a Home of Rest for Horses Clinical Training Scholarship.

Presented in part at 42nd British Equine Veterinary Association Annual Congress, Birmingham, England, September 2003.

Address correspondence to Dr. Menzies-Gow.