EOTAXIN AND CAPPING PROTEIN IN EXPERIMENTAL VASCULOPATHY

Ischemia-induced tissue activation may contribute to the pathogenesis of graft vasculopathy, but the mediators implicated have only partiall y been characterized. To gain further insight into the molecular mecha nisms involved, syngeneic rat aortic transplants with cold-storage-ind uced vasculopathy were studied for differentially expressed mRNA trans cripts. Vessel segments were exposed to either 1 or 18 hours of cold i schemia, followed by transplantation into syngeneic recipients. After 3 days or 4 weeks, the grafts were removed and total mRNA was isolated and used for differential display to identify modulation of transcrip t expression related to prolonged storage. Using 15 sets of random pri mers, 17 polymerase chain reaction products were up-regulated and 2 we re downregulated in grafts exposed to 18 hours of ischemia. Sequencing of these amplicons showed that 6 had a high degree of homology to kno wn sequences whereas 13 had no homology to any of the genes in the dat abase. Two of the differentially displayed amplicons (capping protein and eotaxin) were cloned, re-amplified, and used as probes for Norther n blot analysis to confirm their differential expression. Immunohistoc hemistry using monoclonal antibodies against capping protein-a and eot axin confirmed that both proteins are expressed in the media of normal aortas and that there was an increased expression in vessels exposed to prolonged ischemia albeit that the increase at the protein level se emed less compared with changes in transcript expression. Northern blo ts with RNA from aortic allografts exposed to prolonged ischemic stora ge also showed increased levels of capping protein and eotaxin mRNA wh ereas there was a decrease in the relative amount of these transcripts in vessels exposed to balloon denudation, suggesting that the increas e after prolonged ischemic exposure is not the result of a nonspecific response to injury. Based on the biological characteristics of cappin g protein and eotaxin it is conceivable that they play a pathogenetic role in ischemia-induced vessel wall remodeling. It remains to be esta blished whether these genes or their products serve as target molecule s for therapeutic interventions to prevent or treat cold-storage-induc ed graft vasculopathy.