Changes in matrix protein biochemistry and the expression of mRNA encoding matrix proteins and metalloproteinases in posterior tibialis tendinopathy.

OBJECTIVES: Adult-acquired flat foot secondary to a dysfunctional posterior tibialis tendon (PTT) is often treated by surgical transfer of the flexor digitorum longus tendon (FDLT). In this study, the authors compared normal PTT, stage II dysfunctional PTT and replacement FDLT, aiming to define changes in collagen modification, glycosaminoglycan (GAG) and the expression of matrix and metalloproteinase mRNA. METHODS: Normal PTTs were obtained from patients with no history of tendon problems. Samples of dysfunctional PTT and replacement FDLT tissue were obtained from patients undergoing surgical reconstruction. Tissue samples were analysed for total collagen and GAG, pentosidine and collagen cross-links. Total RNA was assayed for mRNA encoding matrix proteins and metalloproteinases, using real-time reverse transcription PCR. Differences between clinical groups were assessed using non-parametric statistics. RESULTS: Dysfunctional PTT contained higher levels of GAG and lower levels of pentosidine than normal PTT or FDLT. In contrast, collagen in FDLT contained fewer ketoimine and more aldimine cross-links than either normal or dysfunctional PTT. mRNA encoding types I and III collagens, aggrecan, biglycan, matrix metalloproteinase (MMP)-2, -13 and -23, and a disintegrin and metalloproteinase (ADAM)-12L each showed increased levels in dysfunctional PTT compared with either normal PTT or (except MMP-13) FDLT. In contrast, MMP-3 and ADAM with thrombospondin domain (ADAMTS)-5 mRNA were lower in both dysfunctional PTT and FDLT than in normal PTT, while ADAMTS-1 mRNA was lower in dysfunctional PTT than in FDLT. CONCLUSIONS: Stage II dysfunctional PTT shows biochemical and molecular changes consistent with a chronic remodelling of the extracellular matrix, rather than rupture, while the replacement FDLT resembles normal PTT in many, but not all, parameters.

Thermal stabilization of collagen in skin and decalcified bone.

The state of collagen molecules in the fibres of tail tendon, skin and demineralized bone has been investigated in situ using differential scanning calorimetry (DSC). Hydroxyproline analysis and tissue digestion with bacterial collagenase and trypsin were used to confirm that the common cause of all the DSC endotherms was collagen denaturation. This occurred within a narrow temperature range in tendons, but over a wide temperature range in demineralized bone and old skin and demonstrated that in tendon and demineralized bone at least the same type I collagen molecule exists in different thermal states. Hypothesizing that this might be caused by different degrees of confinement within the fibre lattice, experiments were performed to measure the effect of changing the lattice dimensions by extracting the collagen into dilute solution with pepsin, swelling the lattice in acetic acid, and contracting the lattice by dehydration. A theoretical analysis was undertaken to predict the effect of dehydration. Results were consistent with the hypothesis, demonstrating that collagen molecules within the natural fibres of bone and old skin are located at different intermolecular spacings, revealing differences between molecules in the magnitude of either the attractive or repulsive forces controlling their separation. One potential cause of such variation is known differences in covalent cross-linking.

Lower strength of the human posterior patellar tendon seems unrelated to mature collagen cross-linking and fibril morphology.

The human patellar tendon is frequently affected by tendinopathy, but the etiology of the condition is not established, although differential loading of the anterior and posterior tendon may be associated with the condition. We hypothesized that changes in fibril morphology and collagen cross-linking would parallel differences in material strength between the anterior and posterior tendon. Tendon fascicles were obtained from elective ACL surgery patients and tested micromechanically. Transmission electron microscopy was used to assess fibril morphology, and collagen cross-linking was determined by HPLC and calorimetry. Anterior fascicles were markedly stronger (peak stress: 54.3 +/- 21.2 vs. 39.7 +/- 21.3 MPa; P < 0.05) and stiffer (624 +/- 232 vs. 362 +/- 170 MPa; P < 0.01) than posterior fascicles. Notably, mature pyridinium type cross-links were less abundant in anterior fascicles (hydroxylysylpyridinoline: 0.859 +/- 0.197 vs. 1.416 +/- 0.250 mol/mol, P = 0.001; lysylpyridinoline: 0.023 +/- 0.006 vs. 0.035 +/- 0.006 mol/mol, P < 0.01), whereas pentosidine and pyrrole concentrations showed no regional differences. Fibril diameters tended to be larger in anterior fascicles (7.819 +/- 2.168 vs. 4.897 +/- 1.434 nm(2); P = 0.10). Material properties did not appear closely related to cross-linking or fibril morphology. These findings suggest region-specific differences in mechanical, structural, and biochemical properties of the human patellar tendon.

quantitative determination of collagen cross-links.

The primary functional role of collagen is as a supporting tissue and it is now established that the aggregated forms of the collagen monomers are stabilised to provide mechanical strength by a series of intermolecular cross-links. In order to understand the mechanical properties of collagen, it is necessary to identify and quantitatively determine the concentration of the cross-links during their changes with maturation, ageing and disease. These cross-links are formed by oxidative deamination of the epsilon-amino group of the single lysine or hydroxylysine in the amino and carboxy telopeptides of collagen by lysyl oxidase, the aldehyde formed reacting with a specific lysine or hydroxylysine in the triple helix. The divalent Schiff base and keto-amine bonds so formed link the molecules head to tail and spontaneously convert during maturation to trivalent cross-links, a histidine derivative and cyclic pyridinolines and pyrroles, respectively. These latter bonds are believed to be transverse inter-fibrillar cross-links, and are tissue rather than species specific. We describe the determination of these cross-links in detail.Elastin is also stabilised by cross-linking based on oxidative deamination of most of its lysine residues to yield tetravalent cross-links, desmosine and iso-desmosine, the determination of which is also described.A second cross-linking pathway occurs during ageing (and to a greater extent in diabetes mellitus) involving reaction with tissue glucose. The initial product glucitol-lysine can be determined as furosine and pyridosine, and determination of advanced glycation end-products believed to be cross-links, such as pentosidine, are also described.

Collagen in its fibrillar state is protected from glycation.

To assess the impact collagen structures may have on glycation, the effects of glucose upon bovine serum albumin, guinea pig skin collagen, rat tail tendon and monomeric collagen were compared under near physiological conditions. Proteins were incubated with or without 50 mM glucose for 64 d in pH 7.4 50 mM phosphate buffer, followed by reduction, acid/alkaline hydrolysis, and analysis. Yields of non-reducible fructose-lysine, in the form of the acid-degradation products furosine and pyridosine, were significantly higher from skin collagen when compared to albumin. Yields of reducible fructose-lysine, in the form of glucitol- and mannitol-lysine, were conversely much greater for albumin, while tail tendon reported intermediate values. Fructose-lysine and unmodified lysine within collagen fibres prior to incubation was therefore protected by the tight packing of the collagen helices, where milling of tail tendon to increase the surface area exposed much of it to reduction protocols. Together with an analysis of pentosidine formation and other products, these results have shown that the interior of the tightly packed skin collagen fibres is protected from both glycation and reduction, and that glycation products differ depending on the protein incubated. Amino acid analysis then showed that our glycated skin collagen was similar to human diabetic skin collagen. Significant quantities of glucose-independent unknowns form in control incubations; their composition again being protein-dependent. The four compound Ks as previously reported were found to be unique to glycated rat tail tendon and soluble collagen, while another glycation product detected in collagen but not albumin may be attributable to carboxymethyl-arginine.

Increased proteolysis of collagen in an in vitro tensile overload tendon model.

Presently, there is a lack of fundamental understanding regarding changes in collagen's molecular state due to mechanical damage. The bovine tail tendon (BTT; steers approximately 30 months) was characterized and used as an in vitro model for investigating the effect of tensile mechanical overload on collagen susceptibility to proteolysis by acetyltrypsin and alpha-chymotrypsin. Two strain rates with a 1000-fold difference (0.01 and 10 s(-1)) were used, since molecular mechanisms that determine mechanical behavior were presumed to be strain rate dependent. First, it was determined that the BTTs were normal but immature tendons. Water content and collagen content (approx. 60% of wet weight and 80% of dry weight, respectively) and mechanical properties were all within the expected range. The collagen crosslinking was dominated by the intermediate crosslink hydroxylysinonorleucine. Second, tensile overload damage significantly enhanced proteolysis by acetyltrypsin and, to a lesser degree, by alpha-chymotrypsin. Interestingly, proteolysis by acetyltrypsin was greatest for specimens ruptured at 0.01 s(-1) and seemed to occur throughout the specimen. Understanding damage is important for insight into injuries (as in sports and trauma) and for better understanding of collagen fiber stability, durability, and damage mechanisms, aiding in the development of durable tissue-based products for mechanically demanding surgical applications.

Altered collagen in tartrate-resistant acid phosphatase (TRAP)-deficient mice: a role for TRAP in bone collagen metabolism.

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein that is highly expressed by osteoclasts, macrophages, and dendritic cells. The enzyme is secreted by osteoclasts during bone resorption, and serum TRAP activity correlates with resorptive activity in disorders of bone metabolism. TRAP is essential for normal skeletal development. In knockout mice lacking TRAP, bone shape and modeling is altered with increased mineral density. Here, we report the effect of TRAP on the biochemical and biomechanical properties of collagen, the major protein constituting the bone matrix, using these mice. Femurs from TRAP-/- and wild-type mice were used in these studies. The biomechanical properties were investigated using a three-point bending technique. Collagen synthesis was determined by measuring cross-link content using high-performance liquid chromatography and amino acid analysis. Collagen degradation was determined by measuring matrix metalloproteinase-2 (MMP-2) activity. The rates of collagen synthesis and degradation were significantly greater in bones from TRAP-/- mice compared with wild type. At 8 weeks, there was an increase in the intermediate cross-links but no significant difference in animals aged 6 months. There was a significant increase in mature cross-links at both ages. A significant increase in MMP-2 production both pro and active was observed. A significant increase in ultimate stress and Young's modulus of elasticity was needed to fracture the bones from mice deficient in TRAP. We conclude that both synthesis as well as degradation of collagen are increased when TRAP is absent in mice at 8 weeks and 6 months of age, showing that TRAP has an important role in the metabolism of collagen.

Altered tryptophan metabolism in FIV-positive cats.

BACKGROUND: Feline immunodeficiency virus (FIV) is analogous to human immunodeficiency virus, the causative agent of human acquired immunodeficiency syndrome (AIDS). In AIDS patients, a progressive reduction in serum tryptophan concentration occurs because of activation of an inducible tryptophan degradation pathway mediated by elevated lamda-interferon production. HYPOTHESIS: Cats infected with FIV have increased tryptophan catabolism evidenced by reduced circulating concentrations of tryptophan and increased concentrations of the tryptophan catabolite kynurenine. ANIMALS: Convenience sample of 235 cats submitted for diagnostic FIV serology (115 FIV-negative and 120 FIV-positive cats). METHODS: Retrospective, cross-sectional study. Serum was assayed for tryptophan and kynurenine using a high performance liquid chromatography assay with fluorescence and ultraviolet detection, respectively. RESULTS: Tryptophan and kynurenine concentrations were log-normally distributed. Geometric mean concentrations were: tryptophan: FIV-positive 30.6 microM (95% CI: 26.8 34.8 microM), FIV-negative 48.9 [microM (95% CI: 43.6-54.9 microM) (P < .001); kynurenine: FIV-positive 22.7 microM (95% CI: 25.5-10.9 microM), FIV-negative 9.9 microM (95% CI: 20.3-9.03 microM) (P < .001). The ratio of kynurenine to tryptophan was: FIV-positive 4.93 (95% CI: 5.62-4.32), FIV-negative 1.34 (95% CI: 1.53 1.17) (P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: Serum tryptophan concentration was significantly lower and serum kynurenine concentration was significantly higher in FIV-positive cats. The kynurenine: tryptophan ratio was >3-fold higher in FIV-positive animals, indicating increased tryptophan catabolism in this group. Dietary or pharmacologic intervention to support serum tryptophan concentrations has been shown to be clinically useful in humans with AIDS and might be applicable to cats with FIV infection.

The increase in denaturation temperature following cross-linking of collagen is caused by dehydration of the fibres.

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.

Identification of a new cross-link and unique histidine adduct from bovine serum albumin incubated with malondialdehyde.

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1epsilon-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nepsilon-(2-carboxyl,2-aminoethane)-Nepsilon-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.