9 - Chlorination and oxidation of human plasma fibronectin by myeloperoxidase-derived oxidants, and its consequences for smooth muscle cell function

Abstract

Background Fibronectin (FN) occurs as both a soluble form, in plasma and at sites of tissue injury, and a cellular form in tissue extracellular matrices (ECM). FN is critical to wound repair, ECM structure and assembly, cell adhesion and proliferation. FN is reported to play a critical role in the development, progression and stability of cardiovascular atherosclerotic lesions, with high FN levels associated with a thick fibrotic cap, stable disease and a low risk of rupture. Evidence has been presented for FN modification by inflammatory oxidants, and particularly myeloperoxidase (MPO)-derived species including hypochlorous acid (HOCl). The targets and consequences of FN modification are poorly understood. Hypothesis That HOCl and MPO-derived oxidants generate specific modifications (oxidation and chlorination) on FN, and that this alters the adhesion, proliferation and gene expression in human coronary artery smooth muscle cells. Results Mass spectrometry using a novel peptide mass mapping protocol show that HOCl and an enzymatic MPO system, generate site-specific, dose-dependent, chlorination and dichlorination of Tyr residues (up to 16 of 100 residues modified), and oxidation of Trp (7 of 39 residues), Met (3 of 26) and His (1 of 55) residues. This damage occurs within selected FN domains, and particularly the heparin- and cell-binding regions. These alterations increase FN binding to heparin-containing columns. Studies using primary human coronary artery smooth muscle cells (HCASMC) show that exposure to HOCl-modified FN, results in decreased adherence, increased proliferation and altered expression of genes involved in ECM synthesis and remodelling. Conclusions These findings indicate that the presence of modified fibronectin may play a major role in the formation, development and stabilization of fibrous caps in atherosclerotic lesions and may play a key role in the switching of quiescent contractile smooth muscle cells to a migratory, synthetic and proliferative phenotype.