Inhibition of cross-links in collagen is associated with reduced stiffness of the aorta in young rats
Introduction
Collagen and elastin are the major structural proteins of the aortic wall, providing the vascular tissue with tensile strength and elasticity [1]. Collagen and elastin are stabilized by covalent cross-links and the formation of these cross-links is mediated by the enzyme lysyloxidase 2, 3, 4. In collagen, cross-links bind the collagen molecules and fibres together and are essential in providing the tensile strength and mechanical stability of the collagen fibrils and fibres 5, 6, 7, 8. The first step in the cross-linking process of collagen is the oxidative deamination of specific lysyl- or hydroxylysyl-residues by lysyloxidase in the non-helical telopeptide regions of the collagen molecules, and lysyl- and hydroxylysyl-aldehydes are generated by this reaction. The divalent cross-links, dehydro-hydroxylysinonorleucine (HLNL) and dehydro-dihydroxylysinonorleucine (DHLNL) can then spontaneously be formed by a condensation reaction between these aldehydes and the ϵ-amino group of a lysyl- or a hydroxylysyl-residue of a neighbouring collagen molecule. DHLNL may then react with a third lysyl- or hydroxylysyl-residue, forming a mature trivalent cross-link, which can be detected in two forms, the pyridinoline and deoxy-pyridinoline 9, 10, 11. During early growth and development of the aorta, HLNL and DHLNL decrease and pyridinolines and other mature collagen cross-links increase 12, 13, 14.
The characteristic cross-links of elastin are the desmosines: desmosine and isodesmosine. The cross-linking process in elastin is also initiated by an oxidative deamination of lysyl-residues to lysyl-aldehydes and the subsequent formation of the tetrafunctional desmosines probably occurs as a series of spontaneous condensation reactions of three reactive lysyl-aldehydes with a fourth lysyl-residue 15, 16. As in collagen, the cross-links in elastin are crucial for a normal function of the elastin [7].
β-aminopropionitrile (BAPN) is a lathyrogen which irreversibly inhibits the lysyloxidase, thereby preventing the formation of cross-links in collagen and elastin [17]. As only newly synthesized collagen and elastin are rendered lathyritic, the effect of BAPN is most marked in young growing animals [18].
The purpose of the present study was to investigate how a reduction in the content of cross-links in collagen and elastin would influence the stiffness and strength of the aorta. We examined the biomechanical properties, the amounts of collagen type I and III, elastin, pyridinoline and desmosines of the thoracic aorta from young rats treated with BAPN for five weeks.
Section snippets
Materials
Sixty-two female SPF Wistar rats, 6 weeks old, were obtained from Møllegård (Skensved, Denmark). Two experiments were performed, and the experimental setting was identical except for the number of rats in each group. For biomechanical examination (experiment A) 26 rats were randomly allocated by body weight into three different groups. The first group served as a start control group (n=7), these animals were killed at the beginning of the experiment. The second group (n=11) was a saline
Results
Table 1 gives the number of animals in each group, initial and final body weight. In experiment A (aortas used for the biomechanical analysis) no difference in final body weight was found between the BAPN-treated and the control group. In experiment B (aortas used for biochemical analysis), however, there was a significant reduction of 12% of the final body weight of the BAPN-treated group compared with the control group (169±4 g vs 192±4 g, 2P<0.05).
The biomechanical results are given in Fig. 1
Discussion
This study investigated the changes in biomechanical and biochemical parameters of the aorta from rats treated with BAPN, a treatment which, due to an irreversible blocking of the enzyme lysyloxidase, hampered the formation of cross-links in collagen and elastin. The extent of inhibition of the cross-linking process was estimated by measuring the actual content of pyridinoline and desmosines, mature cross-links in collagen and elastin. To our knowledge this has not been done earlier by others
Acknowledgements
This study was supported by P. Carl Petersen Foundation, The Danish Medical Research Council, Agnes and Knut Mørks Foundation and The Danish Heart Association. We thank Ellen Noer for linguistic assistance.
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