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Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation.

ABSTRACT: Mechanical property elaboration of engineered tissues is often assumed on the basis of gene and protein characterizations, rather than mechanical testing. However, we recently demonstrated that mechanical properties are not consistently correlated with matrix content and organization during embryonic tissue development. Based on this, mechanical properties should be assessed independently during natural or engineered tissue formation. Unfortunately, mechanical testing is destructive, and thus alternative means of assessing these properties are desirable. In this study, we examined lysyl oxidase (LOX)-mediated crosslinks as markers for mechanical properties during embryonic tendon formation and the potential to detect them non-destructively. We used tandem mass spectrometry (LC-MS/MS) to quantify changes in hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) crosslink density in embryonic chick tendon as a function of developmental stage. In addition, we assessed a multiphoton imaging approach that exploits the natural fluorescence of HP and LP. With both techniques, we quantified crosslink density in normal and LOX-inhibited tendons, and correlated measurements with mechanical properties. HP and LP crosslink density varied as a function of developmental stage, with HP-to-dry mass ratio correlating highly to elastic modulus, even when enzymatic crosslink formation was inhibited. Multiphoton optical imaging corroborated LC-MS/MS data, identifying significant reductions in crosslink density from LOX inhibition. Taken together, crosslink density may be useful as a marker of tissue mechanical properties that could be assessed with imaging non-destructively and perhaps non-invasively. These outcomes could have significant scientific and clinical implications, enabling continuous and long-term monitoring of mechanical properties of collagen-crosslinked tissues or engineered constructs.

SUBMITTER: Marturano JE

PROVIDER: S-EPMC4053294 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation.

Marturano Joseph E JE Xylas Joanna F JF Sridharan Gautham V GV Georgakoudi Irene I Kuo Catherine K CK

Acta biomaterialia 20131206 3

Mechanical property elaboration of engineered tissues is often assumed on the basis of gene and protein characterizations, rather than mechanical testing. However, we recently demonstrated that mechanical properties are not consistently correlated with matrix content and organization during embryonic tissue development. Based on this, mechanical properties should be assessed independently during natural or engineered tissue formation. Unfortunately, mechanical testing is destructive, and thus al ...[more]

PMID: 24316363

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Project description:The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼ 16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

Project description:BackgroundLysyl oxidases (LOX) have been extensively studied in mammals, whereas properties and functions of recently found homologues in prokaryotic genomes remain enigmatic.MethodsLOX open reading frame was cloned from Haloterrigena turkmenica in an E. coli expression vector. Recombinant Haloterrigena turkmenica lysyl oxidase (HTU-LOX) proteins were purified using metal affinity chromatography under denaturing conditions followed by refolding. Amine oxidase activity has been measured fluorometrically as hydrogen peroxide release coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting.ResultsCultured H. turkmenica has no detectable amine oxidase activity. HTU-LOX may be expressed in E. coli with a high protein yield. The full-length protein gives no catalytic activity. For this reason, we hypothesized that the hydrophobic N-terminal region may interfere with proper folding and its removal may be beneficial. Indeed, truncated His-tagged HTU-LOX lacking the N-terminal hydrophobic signal peptide purified under denaturing conditions can be successfully refolded into an active enzyme, and a larger N-terminal truncation further increases the amine oxidase activity. Refolding is optimal in the presence of Cu2+ at pH 6.2 and is not sensitive to salt. HTU-LOX is sensitive to LOX inhibitor 3-aminopropionitrile. HTU-LOX deaminates usual substrates of mammalian LOX such as lysine-containing polypeptides and polymers. The major difference between HTU-LOX and mammalian LOX is a relaxed substrate specificity of the former. HTU-LOX readily oxidizes various primary amines including such compounds as taurine and glycine, benzylamine being a poor substrate. Of note, HTU-LOX is also active towards several aminoglycoside antibiotics and polymyxin. Western blotting indicates that epitopes for the anti-HTU-LOX polyclonal antibodies coincide with a high molecular weight protein in H. turkmenica cells.ConclusionH. turkmenica contains a lysyl oxidase gene that was heterologously expressed yielding an active recombinant enzyme with important biochemical features conserved between all known LOXes, for example, the sensitivity to 3-aminopropionitrile. However, the native function in the host appears to be cryptic.SignificanceThis is the first report on some properties of a lysyl oxidase from Archaea and an interesting example of evolution of enzymatic properties after hypothetical horizontal transfers between distant taxa.

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Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation.

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Lysyl oxidase-mediated collagen crosslinks may be assessed as markers of functional properties of tendon tissue formation.

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