Restenosis is a major limitation of balloon angioplasty. Balloon injury activates leukocytes and neointimal growth is attenuated by inhibiting leukocyte adhesion or depleting leukocytes in the rabbit carotid artery (Miller et al., 2001). Protease-activated receptor 2 (PAR-2) belongs to a class of 7 transmembrane domain G protein coupled receptors, activated by proteolytic cleavage. Both neutrophils and T cells express PAR-2 (Hou et al., 1998). PAR-2 activation increases leukocyte rolling, adhesion and extravasation in vivo and the receptor is upregulated following balloon angioplasty (Damiano et al., 1999). Trypsin and novel synthetic peptides, 2furoyl-LIGKV-OH (2f-KV) (Ferrell et al., 2003) and 2furoyl-LIGRL-NH 2 (2f-RL) (Kawabata et al., 2004), induce receptor activation. With a view to deciphering a link between PAR-2, leukocyte activation and the progression of restenosis, the main objectives of this study were to characterise the vascular response to PAR-2 activation in carotid arteries from PAR-2 wild type ^(+/+) or deletion ^(-/-) mice and assess the effect of autologous isolated leukocytes on vessel reactivity.

Male PAR-2^(+/+) or PAR-2^(-/-) mice, 28±0.5g, were killed by CO₂ asphyxiation. Whole blood samples obtained from cardiac puncture were placed on 6% dextran for 1hour. The resultant cell suspension centrifuged (3500rpm for 10mins) and red blood cells (RBCs) lysed with distilled water. The suspension was then placed on top of a 2-step percoll gradient and after further centrifugation for 1hour the polymorphonuclear leukocytes (PMNs) were removed from between the two percoll densities (1.093 and 1.081) and added to RPMI medium. To isolate spleen leukocytes the spleen was disrupted over nitex mesh in medium (RPMI and foetal calf serum), filtered and centrifuged (1000rpm for 10mins). RBCs were lysed as above, collected by centrifugation and suspended in 1ml of medium. Cell viability was checked with trypan blue and cells were counted using a haemocytometer and the final concentration adjusted with medium to 10⁶– 5x10⁶cells/ml. Rings (2mm) from the carotid artery of mice were set up in a 10ml myograph (37 °C) containing Krebs solution and gassed with 95%O₂/5%CO₂ for measurements of isometric contraction and relaxation. Rings were preconstricted with phenylephrine (EC₅₀) and subjected to cumulative additions of leukocytes (10⁴– 10⁶cells/ml), trypsin (1-1000U/ml), 2f-KV (10^-7– 3x10 ^-4M) or 2f-RL (10^-9–10^-5M).

Trypsin, 2f-KV and 2f-RL caused concentration-dependent relaxation of phenylephrine-preconstricted arteries in the presence of endothelium. The EC₅₀ values were 224 ± 62U/ml (trypsin, n=4), 10.5 ± 5 µM (2f-KV, n=4), and 0.063 ± 0.03 µM (2f-RL, n=4). This response was absent in arteries from PAR-2^(-/-) mice and significantly abrogated following endothelium denudation in PAR-2^(+/+) mice (n=3). PMNs contracted PAR-2^(+/+) carotid arteries, causing a significant contraction of 4.5±1.2 mN at a concentration of 3x10⁵ cells/ml (n=4). Splenocytes also induced a concentration-dependent contraction of PAR-2^(+/+) carotid artery rings. Endothelial denudation and PAR-2 deletion abolished the contractile ability of the PMNs and the splenocytes.

This study demonstrates that activation of PAR-2 in the mouse carotid artery induces an endothelium-dependent relaxation. Deletion of the PAR-2 receptor eliminated the response to trypsin and the PAR-2 activating peptides. Autologous leukocytes induced a strong contraction which appears to be abrogated in arteries from PAR-2^(-/-) mice and by removal of the endothelium. Future work will determine the involvement of the PAR-2 receptor in the leukocyte-induced contraction.

Damiano et al., 1999 Thromb Haemost. 81: 808-814.
Ferrell et al.,2003 J.Clin.Invest 111 , 35-41.
Hou et al., 1998 Br.J.Haematol. 101, 1-9.
Kawabata et al., 2004 J Pharmacol Exp Ther. 309, 1098-107.
Miller et al., 2001 Cardiovas. Res. 49; 838-850.