Cloning, partial sequencing and expression of cyclooxygenase isoforms in the common digital artery of the horse: an in vitro study.

Introduction. The cyclooxygenase (COX) enzyme represent the target of nonsteroidal anti-inflammatory drugs (NSAIDs), a class of heterogeneous compounds. Actually, two distinct forms of COX have been identified. Cyclooxygenase 1 (COX1) is considered a constitutive form of the enzyme, responsible for the synthesis of protective prostaglandins in several tissues; cyclooxygenase 2 (COX2), on the contrary, is an inducible form and it is mainly involved in inflammation (1). In the horse, COX2 might play a crucial role in musculoskeletal diseases (i.e. laminitis), possibly by inducing vascular alterations (2). In the present study, COX1 and COX2 expression was estimated in the digital artery of the horse in vitro, in basal conditions and after the exposure to a bacterial toxin (Escherichia coli lipopolysaccharide, LPS). Materials and Methods. Adult horse fore legs were collected at the slaughterhouse and transferred to the laboratory. Small rings were obtained from endothelium-denuded segments of the digital artery as reported elsewhere (3), placed into Petri dishes and incubated in a controlled atmosphere (37°C, 5% CO2) for 1, 2, 4 and 16 hours in the presence of 10, 20 and 30 μg ml-1 LPS. Total RNA was isolated and check for its purity and concentration. Primers for an housekeeping gene (gliceraldehyde 3-phosphate dehydrogenase, GAPDH), COX1 and COX2 were designed by alignment and identification of highly conserved regions of respective genes; in particular, mouse, rat and dog (COX1); mouse, rat and pig (COX2); horse, dog, cat, cattle, goat and pig (GADPH). Then, GAPDH, COX1 and COX2 were amplified by RT-PCR. Complementary DNA was cloned, cut with specific restriction enzymes and, finally, sequenced according to Cantiello et al. (2005: 4). Therefore, the effect of LPS upon both cyclooxygenase isoforms was evaluated by RT-PCR. Results. Selected primers were able to amplify fragments of 727 bp, 917 bp and 668 bp for COX1, COX2 and GAPDH, respectively. Horse COX1 and COX2 were successfully cloned, sequenced and submitted to GenBank (http://www.ncbi.nlm.nih.gov/; COX1 accession number DQ246452). As regards the effect of different LPS concentrations as well as time of incubation upon COX1 and COX2 gene expression levels, an increase of COX2 expression was observed, at all times of incubation, at 20 μg ml-1 LPS concentration. On the contrary, no differences in COX1 expression were ever noticed. Discussion and Conclusion. On a knowledge basis, numerous studies concerning the effects of NSAIDs upon the horse COX isoforms have been done (5, 6). Recent advances in biotechnology provided an array of molecular technologies which allow the single or the global gene expression analysis; in this respect, a few papers on the horse COX isoenzyme gene expression have been recently published (2, 7-9). Pharmacologically, a rapid and physiologically sound screening method is usually requested for the evaluation of different COX-inhibitors efficacies (1). The in vitro equine digital artery method has been successfully used in previous pharmacodynamic studies (3, 10-11) and we were able, by using this same methodology, to clone and partially sequence COX1 and COX2 genes, although other partial sequences of these same ones (as well as for GADPH) were yet available in GenBank; moreover, a set of primers useful to evaluate, qualitatively or semi-quantitatively, both COX isoforms gene expression levels have been designed. Finally, the best LPS concentration to be used for studies on COX2-inhibitors efficacy has been identified. Future perspectives might be represented by the validation of an useful quantitative real-time PCR protocol to be eventually used jointly with the pharmacodynamic approach in this same in vitro method or, as an alternative, in other ones which have been already took into account previously (i.e. whole blood assay: 1, 12). References. 1. Brideau C. et al. Am. J. Vet. Res. 2001; 62: 1755-1760. 2. Rodgerson D.H. et al. Am. J Vet. Res. 2001; 62: 1957-1963. 3. Barbero R. et al. Vet. Res. Comm. 2005; 29 (spl.2): 273-275. 4. Cantiello et al. Pharmacologyonline. 2005; 3: 66-76. 5. Moses A.S. and Bertone A.L. Vet. Clin. North Am. Equine Pract. 2002; 18: 21-37. 6. Less P. et al. J. Vet. Pharmacol. Therap. 2004; 27: 491-502. 7. Neil K.M. et al. Am. J. Vet. Res. 2005; 66: 1861-1869. 8. Farley J. et al. Am J. Vet. Res. 2005; 66: 1985-1911. 9. Waguespack R.W. et al. Am J. Vet. Res. (2004); 65: 1724-1729. 10. Zizzadoro C. et al. J. Vet. Pharmacol. Therap. 2003; 26 (spl.1): 174-175. 11. Belloli C. et al. J. Vet. Pharmacol. Therap. 2004; 27: 247-254. 12. Belloli C. et al. J. Vet. Pharmacol. Therap. 2003; 26 (spl.1): 175-176. Acknowledgements. This study was partly supported by a grant from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR-FIRB RBAU01JLN3/02) to G.R.