445: Preferential expression of CD44 v1 isoforms by intragraft fibroblasts from cardiac allografts with chronic rejection

Abstract

Purpose: CD44 is a transmembrane glycoprotein primarily involved in cell-cell interactions and cell migration. This study investigated the genetic composition and the functional implication of CD44 species expressed by intragraft fibroblasts (IF). Methods and Materials: Intragraft fibroblasts recovered from the chronically rejecting heart allografts of rats were examined for CD44 gene expression using quantitative real time PCR. CD44 specific cDNA libraries were constructed and selected CD44 clones were analyzed in automate nucleotide sequencing. The role of CD44/hyaluronan interaction was studied using in vitro migration and proliferation assays. Results: IF displayed a 4.5-fold increase in expression of CD44 mRNA when compared with that of the fibroblasts isolated from non-rejecting heart allografts (p 0.01). The IF preferentially expressed CD44 variant isoforms containing v1 exon transcript. Automate nucleotide sequence analysis revealed that the majority (90.12%) of the CD44 v1 isoforms expressed by the rejecting graft fibroblasts were encoded by a mutated CD44 mRNA, which contained 2 point mutations and a codon deletion in the v1 coding region. Histochemistry demonstrated a massive deposition of extracellular hyaluronan in the rejecting heart allografts. Hyaluronan promoted in vitro fibroblast adhesion, migration in a CD44 dependent manner, and survival in a serum-free culture condition. Conclusions: A significant increase in CD44 expression by the rejecting graft fibroblasts was coupled with augmentation of extracellular hyaluronan synthesis in the heart allografts, indicating that CD44-HA interaction is critically involved in the pathogenesis of graft fibrosis. CD44v1 isoforms may play a role, in a way similar to other documented v exons such as v3 or v6, to provide additional signaling for enhancing or suppressing the biological activities of intragraft fibroblasts. CD44 antagonism may affect multiple cellular activities mediated by the CD44-HA pathway, leading to alleviation of allograft fibrosis and chronic inflammation.