Thus, sequestration of MPO by perlecan, and consequent localization of oxidant production to this target, potentiates oxidative damage to the perlecan protein core

Thus, sequestration of MPO by perlecan, and consequent localization of oxidant production to this target, potentiates oxidative damage to the perlecan protein core. an Ethynylcytidine impairment of its ability to support endothelial cell adhesion, with this observed at a pathologically-achievable oxidant dose of 425 nmol oxidant/mg protein. In contrast, the heparan sulfate chains of HOCl/HOBr-modified perlecan retained their ability to bind FGF-2 and collagen V and were able to promote FGF-2-dependent cellular proliferation. Collectively, these data highlight the potential role of perlecan oxidation, and consequent deregulation of cell function, in vascular injuries by myeloperoxidase-derived HOCl and HOBr. nzymatic degradation of perlecan is a potentially important mechanism for promoting growth factor bioavailability and activity within the vessel wall (Whitelock et al., 1996). The protein core of perlecan has also been identified as an adhesive molecule for human vascular cells (Whitelock et al., 1999). Oxidation of endothelial cell-derived matrix by the MPO-H2O2-Cl? system results in the release of perlecan-derived material (Klebanoff et al., 1993) and similar release of proteoglycan-derived material occurs upon exposure of smooth muscle cell-derived matrix to HOCl, HOBr or the MPO-H2O2-Cl? system (McGowan, 1990; Rees et al., 2007; Woods and Davies, 2003). These data indicate that modification and release of perlecan from extracellular matrix may be an important process when MPO-derived HOCl and HOBr are generated within the subendothelial compartment, however the mechanisms underlying this process and its potential biological consequences are unclear. In particular, it is unknown whether damage occurs principally to its protein core or to its heparan sulfate chains. Studies with isolated heparan sulfate show that HOCl and HOBr react rapidly with the free amino groups present on its GlcNH2 residues, resulting in modification and fragmentation of its polysaccharide backbone (Rees and Davies, 2006; Rees et al., 2003; Rees et al., 2007; Rees et al., 2005). It has yet to be established whether HOCl and HOBr can degrade heparan sulfate present on intact proteoglycans. Moreover, the effects of these oxidants on the diverse biological activities of the perlecan protein core and its heparan sulfate chains, such as growth factor binding and cell adhesion, are unknown. In the light of these data, the ability of endothelial cell-derived perlecan to bind MPO, and Ethynylcytidine the structural and functional consequences of its oxidation by HOCl, HOBr and MPO-H2O2-halide systems were determined. 2. Materials and Methods 2. 1 Materials Solutions and media were prepared using water filtered through a four-stage MilliQ system. pH control was achieved using Chelex-treated 0.1 M phosphate buffer or PBS, pH 7.4. HCAECs and HCAEC Growth Medium were from Cell Applications. Medium 199 containing Earles salts were from Gibco BRL. RMPI-1640 medium was from Sigma. Tissue culture plastic ware was from BD Falcon. Sodium hypochlorite (NaOCl) was from Sigma. H2O2 and HOCl solutions were prepared immediately before use and their concentrations were determined spectrophotometrically at pH 12 (H2O2, 243.6 M?1 cm?1; HOCl, 292 350 M?1 cm?1). Stock solutions of HOBr were prepared by reaction of Rabbit polyclonal to Bcl6 20 mM NaOCl with 22.5 mM NaBr in water for 5 min at 22C and were used immediately. MPO (isolated from human polymorphonuclear leukocytes; purity index A430/A280 0.84) was from Planta Natural Products. Alkaline phosphate substrate (<0.05 taken as significant. 3. Results 3.1 Isolation and characterization of endothelial cell-derived perlecan Perlecan was immunopurified from media conditioned by HCAECs as described previously (Whitelock et al., 1999). The purified proteoglycan migrated as a single broad band on 3C8% gradient Ethynylcytidine SDS-PAGE gels, which was recognized by antibodies against the perlecan protein core (CSI-076, against domain I (Fig. 1A; Fig. 4); 7B5, against domain III; CSI-074, against domain V) and against heparan sulfate (10E4) (Fig. 1A; Fig. 4). The perlecan was recognized by Ethynylcytidine the same antibodies in ELISAs and by other antibodies against heparan sulfate (HepSS-1 and JM403) (Fig. 1B and Fig. 2). After removal of heparin sulfate by digestion with heparinase III, the perlecan was resolved as a narrow band at a molecular mass consistent with.