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Investigation of mRNA expression of tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2 in cultured equine digital artery smooth muscle cells after exposure to endotoxin

Dwayne H. Rodgerson DVM, MS1,2, James K. Belknap DVM, PhD3, James N. Moore DVM, PhD4, and Genevieve L. Fontaine DVM, MS5,6
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  • 1 Department of Large Animal Surgery and Medicine, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 2 Present address is the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.
  • | 3 Department of Large Animal Surgery and Medicine, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 4 Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30628.
  • | 5 Department of Large Animal Surgery and Medicine, College of Veterinary Medicine, Auburn University, Auburn, AL 36849.
  • | 6 Present address is the Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610-0136.

Abstract

Objective—To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin- (IL)-1β from cultured equine smooth muscle cells (SMC).

Sample Population—Segments of palmar digital artery harvested from 6 clinically normal adult horses.

Procedure—Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 µg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-α, and IL-1β was performed, using isolated total cellular RNA.

Results—Although no message was detected for IL-1β or TNF-α in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1β and TNF-α mRNA.

Conclusions and Clinical Relevance—Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis. (Am J Vet Res 2001;62:1957–1963)

Abstract

Objective—To determine messenger RNA expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin- (IL)-1β from cultured equine smooth muscle cells (SMC).

Sample Population—Segments of palmar digital artery harvested from 6 clinically normal adult horses.

Procedure—Explants were collected from the tunica media of arteries for primary culture of SMC. Equine mononuclear cells were used as control cells. Subcultured vascular SMC and control cells were exposed to lipopolysaccharide (20 µg/ml and 100 ng/ml, respectively). Northern blot analysis with equine-specific probes for COX-2, TNF-α, and IL-1β was performed, using isolated total cellular RNA.

Results—Although no message was detected for IL-1β or TNF-α in control or endotoxin-exposed equine vascular SMC from all horses, COX-2 underwent a distinct substantial up-regulation after endotoxin exposure. Endotoxin-exposed equine mononuclear cells had up-regulation of IL-1β and TNF-α mRNA.

Conclusions and Clinical Relevance—Increased expression of COX-2 mRNA by equine vascular SMC may be an important early pathophysiologic event in the onset of endotoxemia in horses. Potentiated local vascular production of various prostanoids after increased expression of mRNA for COX-2 may result in vasoactive events observed with laminitis. (Am J Vet Res 2001;62:1957–1963)