Expression And Significance Of CCL25/CCR9 In Chronic Rejection Of Cardiac Allografts In Rats

Background In recent years, the heart donor shortage problem which restricted the development of heart transplant has been alleviated with the distribution system of hearts donated by brain death patients being established and the appearance of mechanical circulatory support. How to further improve the long term survival rate of patients with heart transplant is in the spotlight again.Cardiac allograft vasculopathy and myocardial fibrosis are the characteristic pathological changes in chronic rejection of cardiac allografts. Both of them are the main reasons for the lower long-term survival rate. Researches show that a variety of chemokines and their receptors play important roles in organ transplant rejection. So targeted blocking their combination has become a hot direction of delaying the chronic rejection. CC chemokine ligand 25(CCL25) is a newly discovered member in CC subtribe chemokine family. It is mainly expressed in the thymus and the small intestinal epithelium. It can promote the proliferation and chemotaxis of the inflammatory cells which expressed CC chemokine receptor 9(CCR9).Currently, the studies of CCL25/ CCR9 are concentrated in rheumatoid arthritis, inflammatory bowel disease, hepatitis, tumor etc,but is rare in chronic rejection of cardiac allografts at home and abroad.Objective This experiment is going to use anti-CCL25 neutralizing antibody targeted blocking the combination of CCL25 and CCR9 to investigate their expression and significance in chronic rejection of cardiac allografts in rats by making chronic rejection models.Methods Inbred Wistar rats and inbred SD rats were selected as donors and recipients. Cervical heterotopic heart transplantation was performed with "cuff technique". The recipients were divided into 3 groups randomly, with 10 rats in each group:CsA group, CsA+IgG group, and CsA+anti-CCL25 group. All recipients were received lOmg/Kg cyclosporin A (CsA) immediately after the surgery, then once every other day for 10 times. CsA+IgG group were injected with 0.lmg/Kg IgG isotype antibody immediately after the surgery, then once every other day for 10 times. CsA+anti-CCL25 group were injected with 0.1mg/Kg anti-rat CCL25 neutralizing antibody immediately after the surgery, then once every other day for 10 times. The survival conditions of cardiac allografts were judged by inspecting and touching the right cervical of rat recipients. All allografts were harvested on the 70th day postoperatively, and made into tissue slices. HE,Van Gieson and sirus red staining were used to observe the pathologic changes of the allograft myocardia. Additionally, immunohistochemistry and western blotting were adopted to detect and analyse the expression of CCL25 and CCR9 in the myocardium.Results HE and Van Gieson staining:CsA group’s and CsA+IgG group’s large flake cardiac cells were degeneration and necrosis, the coronary artery was stenotic and even occlusion, inflammatory cells infiltration was widespread and more crowded around the blood vessels, CsA+anti-CCL25 group’s pathological changes were improved more significantly than the other two groups’(P＜0.05). Sirius red staining:CsA group’s and CsA+IgG group’s myocardial collagen fibers hyperplasia were in diffuse, like hyperchromatic and high density fiber bundle, CsA+anti-CCL25 group’s collagen fibers were loose, the degree of fibrosis were improved more significantly than the other two groups’(P＜0.05). Immunohistochemistry:CCL25 and CCR9 were expressed in all cardiac allografts. Western blotting:the expression of CCL25 and CCR9 were in accord with the result of immunohistochemistry; the expression level of CCL25 and CCR9 in CsA+anti-CCL25 group were significantly lower than that in the other two groups’(P<0.05).Conclusions CCL25 and CCR9 are expressed in the chronic rejection of cardiac allografts in rats. Anti-CCL25 neutralizing antibody can significantly alleviate the pathologic changes of cardiac allografts through targeted blocking of CCL25/CCR9. Our study indicates that CCL25/CCR9 plays important roles in the chronic rejection of cardiac allografts in rats.