Abstract 464: Novel Anti-Medin Antibodies Detect Medin Amyloid Deposits in the Aorta of Patients with Thoracic Aneurysms or Marfan Syndrome

Abstract

Introduction: Medin, a 50 aa cleavage fragment of MFG-E8/ lactadherin appears to self assemble into fibrils and form the amyloid deposits seen in patients with aortic aneurysms. While the pathogenic nature of these aggregates is not fully understood, it appears medin may perturb smooth muscle cell function and thereby weaken the integrity of the aorta wall. One approach to treat aortic aneurysms may be to sequester medin and thereby block aggregation or remove the amyloid deposits from the aorta using a monoclonal antibody. Methods: Mice were immunized with a c-terminal peptide or full-length human medin, hybridomas cloned and antibodies screened for activity using ELISA, Western blot, Biacore and immunocytochemistry. Results: Initial ELISA studies revealed that antibodies raised against full-length 50 aa medin (e.g. 6B3) bind both medin peptide and MFG-E8, while antibodies raised against a c-terminal peptide (e.g. 18G1) appeared to be medin specific. Subsequent studies confirmed these observations and revealed that 18G1 recognized the c-terminal end of medin, a neo epitope created when medin is cleaved from MFG-E8. Data from Western blot also showed that both 6B3 and 18G1 were capable of binding both monomeric and oligomeric forms of medin. To assess the specificity for endogenous human medin, a series of immunohistochemical studies were conducted with thoracic aorta samples from aneurysm or Marfan syndrome patients. The results demonstrated that anti-medin antibodies were able to bind endogenous medin, although the intensity and pattern of staining appeared to be antibody specific. While 6B3 stained dense aggregated (Thioflavin S+) material or amyloid deposits with high affinity in and around the patient aneurysm, 18G1 showed little to no specific staining. In contrast, 18G1 widely stained structures in the tunica media, the region of the aorta that contains the elastin fibers and smooth muscle cells. Conclusions: These data demonstrated that anti-medin antibodies can detect endogenous medin peptide present in aneurysm and Marfan patient samples and showed that the antibody epitope can play an important role in determining the size (monomer, dimer, trimer, etc.) and aggregation state (oligomer, protofibril, aggregate) of medin detected.