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An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry.

ABSTRACT: Current literature contains scant information regarding the extent of enzymatic collagen cross-linking in the keratoconus (KC) cornea. The aim of the present study was to examine levels of enzymatic lysyl oxidase-derived cross-links in stromal collagen in KC tissue, and to correlate the cross-link levels with collagen fibril stability as determined by thermal denaturation temperature (Tm).Surgical KC samples (n = 17) and Eye-Bank control (n = 11) corneas of age 18 to 68 years were analyzed. The samples were defatted, reduced (NaBH4), hydrolyzed (6N HCl at 110°C for 18 hours), and cellulose enriched before analysis by C8 high-performance liquid chromatography equipped with parallel fluorescent and mass detectors in selective ion monitoring mode (20 mM heptafluorobutyric acid/methanol 70:30 isocratic at 1 mL/min). Nine different cross-links were measured, and the cross-link density was determined relative to collagen content (determined colorimetrically). The Tm was determined by differential scanning calorimetry.Cross-links detected were dihydroxylysinonorleucine (DHLNL), hydroxylysinonorleucine, lysinonorleucine (LNL), and histidinohydroxylysinonorleucine in both control and KC samples. Higher DHLNL levels were detected in KC, whereas the dominant cross-link, LNL, was decreased in KC samples. Decreased LNL levels were observed among KC ? 40 corneas. There was no difference in total cross-link density between KC samples and the controls. Pyridinolines, desmosines, and pentosidine were not detected. There was no notable correlation between cross-link levels with fibril instability as determined by Tm.Lower levels of LNL in the KC cornea suggest that there might be a cross-linking defect either in fibrillar collagen or the microfibrillar elastic network composed of fibrillin.

SUBMITTER: Takaoka A

PROVIDER: S-EPMC4727527 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry.

Takaoka Anna A Babar Natasha N Hogan Julia J Kim MiJung M Price Marianne O MO Price Francis W FW Trokel Stephen L SL Paik David C DC

Investigative ophthalmology & visual science 20160101 1

<h4>Purpose</h4>Current literature contains scant information regarding the extent of enzymatic collagen cross-linking in the keratoconus (KC) cornea. The aim of the present study was to examine levels of enzymatic lysyl oxidase-derived cross-links in stromal collagen in KC tissue, and to correlate the cross-link levels with collagen fibril stability as determined by thermal denaturation temperature (Tm).<h4>Methods</h4>Surgical KC samples (n = 17) and Eye-Bank control (n = 11) corneas of age 18 ...[more]

PMID: 26780316

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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.

Dataset Information

An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry.

Publications

An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry.

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