The susceptibility of bioprosthetic heart valve leaflets to oxidation

Abstract

The clinical use of bioprosthetic heart valves (BHV) is limited due to device failure caused by structural degeneration of BHV leaflets. In this study we investigated the hypothesis that oxidative stress contributes to this process. Fifteen clinical BHV that had been removed for device failure were analyzed for oxidized amino acids using mass spectrometry. Significantly increased levels of ortho-tyrosine, meta-tyrosine and dityrosine were present in clinical BHV explants as compared to the non-implanted BHV material glutaraldehyde treated bovine pericardium (BP). BP was exposed in vitro to oxidizing conditions (FeSO4/H2O2) to assess the effects of oxidation on structural degeneration. Exposure to oxidizing conditions resulted in significant collagen deterioration, loss of glutaraldehyde cross-links, and increased susceptibility to collagenase degradation. BP modified through covalent attachment of the oxidant scavenger 3-(4-hydroxy-3,5-di-tert-butylphenyl) propyl amine (DBP) was resistant to all of the monitored parameters of structural damage induced by oxidation. These results indicate that oxidative stress, particularly via hydroxyl radical and tyrosyl radical mediated pathways, may be involved in the structural degeneration of BHV, and that this mechanism may be attenuated through local delivery of antioxidants such as DBP.

Keywords: Antioxidant; Bioprosthesis; Calcification; Collagen; Heart valve; Oxidation.

Fig. 1. Oxidized amino acid levels are elevated in clinical BHV

(A)–(C) Quantification of oxidized amino acid levels in non-implanted control tissue (Glut BP, n = 5) and clinical BP BHV samples (n = 15). *p < 0.05 vs Glut BP.

Fig. 2. Covalent modification of BHV material bovine pericardium with the oxidant scavenger DBP

(A) Schematic of the carbodiimide-driven reaction used to modify collagen-based tissues with DBP. The carboxyl groups of the collagen fibers are activated with EDC. SuOH stabilizes the O-acylisourea intermediates to enable the amine of DBP to react and form an amide bond. (B) Quantification of Asp and Glu in pericardium by HPLC. Asp and Glu provide carboxyl groups that react with DBP. (C) Dose curve of DBP attachment to pericardium, generated with ¹⁴C-DBP.

Fig. 3. DBP scavenges oxidants after attachment to BP

Oxidation of Amplex Red to resorufin with (A) Myeloperoxidase (MPO)/H₂O₂/Cl (B) MPO/H₂O₂/NO₂.* p < 0.05 vs Glut BP.

Fig. 4. BHV oxidative damage in vitro is mitigated with DBP modification

BP exposed to 1% H₂O₂/100 μM FeSO₄ for 7 days. (A–D) Picrosirius red staining of collagen (200x magnification) (A) Glut BP no treatment (B) Glut BP oxidized (C) Glut-EDC/SuOH oxidized (D) Glut-DBP oxidized (E) Dry weight loss of oxidized BP (F) Collagenase treatment of oxidized BP (G) Stability of ³H-glutaraldehyde cross-linking in PBS and oxidizing conditions. *p < 0.05 vs Glut BP.