Cross-linking and fluorescence changes of collagen by glycation and oxidation

The non-enzymatic glucosylation of collagen in vivo and in vitro produces blue-fluorescent cross-links very slowly. The mechanism of their formation is unknown. We investigated the role of oxidation in glycation. When native fluorescent collagen from old-rat tail tendon and its CNBr peptides were oxidized by chemically generated singlet oxygen, cross-linking occurred immediately, and the cross-linked products showed an increased blue fluorescence. Further cross-linking and development of blue fluorescence also were accelerated by singlet oxygen when oxidizing in vitro glucosylated collagen CNBr peptides. It was noted that the blue fluorescence developed at the expense of a near-UV fluorescence. This near-UV fluorophore, which is also present in native collagen, was found to be produced by the in vitro glucosylation of collagen and during the cross-linking by glucosylation was slowly converted to the blue fluorophore. These changes indicate the autoxidation of near-UV fluorescent intermediates to blue fluorescent cross-links during glucosylation. Non-enzymatic fructosylation, which occurs in vivo in certain proteins, was more effective than glucosylation in forming fluorophores and cross-links with collagen in vitro. Fructosylated fluorophores were found different from glucosylated products in their oxidation reactivities with singlet oxygen.     

Determination of the relationship between collagen cross-links and the bone-tissue stiffness in the porcine mandibular condyle

Although bone-tissue stiffness is closely related to the degree to which bone has been mineralized, other determinants are yet to be identified. We, therefore, examined the extent to which the mineralization degree, collagen, and its cross-links are related to bone-tissue stiffness. A total of 50 cancellous and cortical bone samples were derived from the right mandibular condyles of five young and five adult female pigs. The degree of mineralization of bone (DMB) was assessed using micro-computed tomography. Using high-performance liquid chromatography, we quantified the collagen content and the number of cross-links per collagen molecule of two enzymatic cross-links: hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and one non-enzymatic cross-link: pentosidine (Pen). Nanoindentation was used to assess bone-tissue stiffness in three directions, and multiple linear regressions were used to calculate the correlation between collagen properties and bone-tissue stiffness, with the DMB as first predictor. Whereas the bone-tissue stiffness of cancellous bone did not differ between the three directions of nanoindentation, or between the two age groups, cortical bone-tissue stiffness was higher in the adult tissue. After correction for DMB, the cross-links studied did not increase the explained variance. In the young group, however, LP significantly improved the explained variance in bone-tissue stiffness. Approximately half of the variation in bone-tissue stiffness in cancellous and cortical bone was explained by the DMB and the LP cross-links and thus they cannot be considered the sole determinants of the bone-tissue stiffness.     

Enzymatic and nonenzymatic cross-linking of collagen and elastin.

Knowledge regarding the steps and mechanisms related to the intra- and interchain cross-linking of collagen and elastin has evolved steadily during the past 30 years. Recently, effort has been directed at identifying the location and types of cross-links that are found in collagen and elastin. There are two major groups of cross-links: those initiated by the enzyme lysyl oxidase and those derived from nonenzymatically glycated lysine and hydroxylysine residues. The formation of enzymatic cross-links depends on specific enzymes, amino acid sequences, and quaternary structural arrangements. The cross-links that are derived nonenzymatically occur more adventitiously and are important to pathobiological processes. Considerable progress has been made in elucidating the pathways of synthesis for several of the enzymatically mediated cross-links, as well as possible mechanisms regulating the specificity of cross-linking. Although less is known about the chemistry of cross-links arising from nonenzymatically glycated residues, recent progress has also been made in understanding possible biosynthetic pathways and control mechanisms. This review focuses on such progress and hopes to underscore the biological importance of collagen and elastin cross-linking.     

Modeling the tensile mechanical behavior of bone along the longitudinal direction

The tensile stress-strain behavior of bone along its longitudinal axis is modeled by using a simple shear-lag theory, wherein, stresses and strains in a unit cell consisting of an organic matrix reinforced by overlapped mineral platelets are computed. It is assumed that loads are transferred between overlapped mineral-platelets by shear in the organic matrix. The mechanical behavior of bone in which the matrix partially or completely debonds from the sides of the overlapped mineral platelets (after an ultimate interfacial shear stress value is exceeded) is modeled. It is shown that the tensile mechanical behavior of bone can be modeled only by assuming little or no debonding of the organic from the mineral. A physical phenomenon that explains the tensile behavior of bone is, after the interfacial shear stress has reached a constant value over the length of the mineral platelets, the collagen molecules/microfibrils (with the associated mineral platelets) move relative to one another. The tensile stress-strain curve of bovine bone is modeled using this model. The theory predicts the mechanical behavior of the tissue in the elastic, yield and post-yield region. The ultimate strain and strengths are not predicted in the present model.     

Collagen cross-linking. Isolation of cross-linked peptides from collagen of chicken bone

Cross-linked peptides were isolated from chicken bone collagen that had been digested with CNBr or with bacterial collagenase. Analyses of (3)H radioactivity in disc electrophoretic profiles of the CNBr peptides from bone collagens that had been treated with NaB(3)H indicated that a major site of intermolecular cross-linking in chicken bone collagen is located between the carboxy-terminal region of an alpha1 chain and a small CNBr peptide, probably situated near the amino-terminus of an alpha1 or alpha2 chain in an adjacent collagen molecule. A small amount of this cross-linked CNBr peptide was isolated from a CNBr digest of chicken bone collagen by column chromatography. Amino acid analysis showed that the CNBr peptide, alpha1CB6B, the carboxy-terminal peptide of the alpha1 chain, was the major CNBr peptide in the preparation, and the reduced cross-linking components were identified as hydroxylysinohydroxynorleucine (HylOHNle), with a smaller amount of hydroxylysinonorleucine (HylNle). However, the composition and the low recovery of the cross-linking amino acids suggested that the preparation was a mixture of CNBr peptides alpha1CB6B and alpha1CB6B cross-linked to a small CNBr peptide whose identity could not be determined. A small cross-linked peptide was isolated from chicken bone collagen that had been reduced with NaB(3)H(4) and digested with bacterial collagenase. This peptide was the major cross-linked peptide in the digest and contained a stoicheiometric amount of the reduced cross-linking compounds. A peptide which had the same amino acid composition, but contained the cross-linking compounds in their reducible forms, was isolated from a collagenase digest of chicken bone collagen that had not been treated with NaBH(4). The absence of the reduced cross-links from this peptide indicates that, at least for the cross-linking site from which the peptide derives, natural reduction is not a significant pathway for biosynthesis of stable cross-links. However, most of the reducible cross-linking component in the peptide appeared to stabilize in the bone collagen by rearrangement from aldimine to ketoamine form.     

T3 affects expression of collagen I and collagen cross-linking in bone cell cultures

Thyroid hormones (T3, T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links.T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.     

Conformation and sequence evidence for two-fold symmetry in left-handed beta-helix fold

The glycosaminoglycan (GAG) side-chains of small leucine-rich proteoglycans have been postulated to mechanically cross-link adjacent collagen fibrils and contribute to tendon mechanics. Enzymatic depletion of tendon GAGs (chondroitin and dermatan sulfate) has emerged as a preferred method to experimentally assess this role. However, GAG removal is typically incomplete and the possibility remains that extant GAGs may remain mechanically functional. The current study specifically investigated the potential mechanical effect of the remaining GAGs after partial enzymatic digestion.A three-dimensional finite element model of tendon was created based upon the concept of proteoglycan mediated inter-fibril load sharing. Approximately 250 interacting, discontinuous collagen fibrils were modeled as having a length of 400 μm, being composed of rod elements of length 67 nm and E-modulus 1 GPa connected in series. Spatial distribution and diameters of these idealized fibrils were derived from a representative cross-sectional electron micrograph of tendon. Rod element lengths corresponded to the collagen fibril D-Period, widely accepted to act as a binding site for decorin and biglycan, the most abundant proteoglycans in tendon. Each element node was connected to nodes of any neighboring fibrils within a radius of 100 nm, the slack length of unstretched chondroitin sulfate. These GAG cross-links were the sole mechanism for lateral load sharing among the discontinuous fibrils, and were modeled as bilinear spring elements. Simulation of tensile testing of tendon with complete cross-linking closely reproduced corresponding experiments on rat tail tendons. Random reduction of 80% of GAG cross-links (matched to a conservative estimate of enzymatic depletion efficacy) predicted a drop of 14% in tendon modulus. Corresponding mechanical properties derived from experiments on rat tail tendons treated in buffer with and without chondroitinase ABC were apparently unaffected, regardless of GAG depletion. Further tests for equivalence, conservatively based on effect size limits predicted by the model, confirmed equivalent stiffness between enzymatically depleted tendons and their native controls.Although the model predicts that relatively small quantities of GAGs acting as primary collagen cross-linking elements could provide mechanical integrity to the tendon, partial enzymatic depletion of GAGs should result in mechanical changes that are not reflected in analogous experimental testing. We thus conclude that GAG side chains of small leucine-rich proteoglycans are not a primary determinant of tensile mechanical behavior in mature rat tail tendons.     

Cross-linking connectivity in bone collagen fibrils: the COOH-terminal locus of free aldehyde.

Quantitative analyses of the chemical state of the 16c residue of the alpha 1 chain of bone collagen were performed on samples from fetal (4-6-month embryo) and mature (2-3 year old) bovine animals. All of this residue could be accounted for in terms of three chemical states, in relative amounts which depended upon the age of the animal. Most of the residue was incorporated into either bifunctional or trifunctional cross-links. Some of it, however, was present as free aldehyde, and the content increased with maturation. This was established by isolating and characterizing the aldehyde-containing peptides generated by tryptic digestion of NaB3H4-reduced mature bone collagen. We have concluded that the connectivity of COOH-terminal cross-linking in bone collagen fibrils changes with maturation in the following way: at first, each 16c residue in each of the two alpha 1 chains of the collagen molecule is incorporated into a sheet-like pattern of intermolecular iminium cross-links, which stabilizes the young, nonmineralized fibril as a whole. In time, some of these labile cross-links maturate into pyridinoline while others dissociate back to their precursor form. The latter is likely due to changes in the molecular packing brought about by the mineralization of the collagen fibrils. The resultant reduction in cross-linking connectivity may provide a mechanism for enhancing certain mechanical characteristics of the skeleton of a mature animal.     

Glycated collagen cross-linking alters cardiac mechanics in volume-overload hypertrophy

Alteration of hemodynamic loading induces remodeling that includes changes in myocardial properties and extracellular matrix structure. We investigated the hypothesis that cardiac hypertrophy due to volume overload produces changes in myocardial diastolic mechanics and stiffness that are in part due to alterations in advanced glycation end-product (AGE) collagen cross-linking. Rats developed volume overload induced by arteriovenous fistula (AVF). To assess the dependence of AGE cross-linking on mechanics, we prevented AGE formation by administering the drug aminoguanidine (AG) to one group of AVF rats (AG+AVF). Volume overload did not modify collagen concentration. Right ventricular AGE cross-links were modestly elevated in AVF hearts but were significantly reduced by AG. AVF rats exhibited significantly increased septal AGE cross-links that were inhibited in the AG+AVF group. AVF-induced increases in left ventricular longitudinal stiffness and septal circumferential stiffness were prevented in AG+AVF hearts. Volume overload appears to regionally modify AGE collagen cross-linking and stiffness, and AG treatment prevented these increases, demonstrating that AGE cross-linking plays a role in mediating diastolic compliance in volume-overload hypertrophy.     

The influence of water removal on the strength and toughness of cortical bone

Although the effects of dehydration on the mechanical behavior of cortical bone are known, the underlying mechanisms for such effects are not clear. We hypothesize that the interactions of water with the collagen and mineral phases each have a unique influence on mechanical behavior. To study this, strength, toughness, and stiffness were measured with three-point bend specimens made from the mid-diaphysis of human cadaveric femurs and divided into six test groups: control (hydrated), drying in a vacuum oven at room temperature (21 degrees C) for 30 min and at 21, 50, 70, or 110 degrees C for 4 h. The experimental data indicated that water loss significantly increased with each increase in drying condition. Bone strength increased with a 5% loss of water by weight, which was caused by drying at 21 degrees C for 4 h. With water loss exceeding 9%, caused by higher drying temperatures (> or =70 degrees C), strength actually decreased. Drying at 21 degrees C (irrespective of time in vacuum) significantly decreased bone toughness through a loss of plasticity. However, drying at 70 degrees C and above caused toughness to decrease through decreases in strength and fracture strain. Stiffness linearly increased with an increase in water loss. From an energy perspective, the water-mineral interaction is removed at higher temperatures than the water-collagen interaction. Therefore, we speculate that loss of water in the collagen phase decreases the toughness of bone, whereas loss of water associated with the mineral phase decreases both bone strength and toughness.     

Collagen cross-linking. Synthesis of collagen cross-links in vitro with highly purified lysyl oxidase.

In this paper, the synthesis of collagen cross-links in vitro was investigated in a defined system consisting of highly purified chick cartilage lysyl oxidase and chick bone collagen fibrils. Cross-link synthesis in vitro was quite similar to the biosynthesis of collagen cross-links in vivo. Enzyme-dependent synthesis of cross-link intermediates and cross-linked collagen derived from lathyritic collagen occurred. The concentration of the two principal reducible cross-links, N6:6'-dehydro-5,5'-dihydroxylysinonorleucine and N6:6'-dehydro-5-hydroxylysinonorleucine, increased to a peak value of approximately two cross-links per molecule and then decreased. Synthesis of histidinohydroxymerodesmosine and a second polyfunctional cross-link of unknown structure began after synthesis of bifunctional cross-links was largely completed and proceeded linearly afterwards. Inhibition of lysyl oxidase after the bulk of bifunctional cross-link synthesis had occurred did not alter the rate of decrease in reducible cross-link concentration but did inhibit further histidinohydroxymerodesmosine synthesis. These results indicate that lysyl oxidase and collagen fibrils are the only macromolecules required for cross-link biosynthesis in vivo. It is likely that the decrease in reducible cross-links observed during fibril maturation results from spontaneous reactions within the collagen fibril rather than additional enzymatic reactions.     

Cartilage type IX collagen is cross-linked by hydroxypyridinium residues

Type IX collagen, a recently discovered, unusual protein of cartilage, has a segmented triple-helical structure containing interchain disulfides. Its polymeric form and function are unknown. When prepared by pepsin from bovine articular cartilage, type IX collagen was found to contain a high concentration of hydroxypyridinium cross-links, similar to that in type II collagen. Fluorescence spectroscopy located the hydroxylysyl pyridinoline and lysyl pyridinoline cross-linking residues exclusively in the high-molecular-weight collagen fraction, from which they were recovered predominantly in a single CNBr-derived peptide. The results point to a structural role for type IX collagen in cartilage matrix, possibly as an adhesion material to type II collagen fibrils.     

1,25(OH)2D3 regulates collagen quality in an osteoblastic cell culture system

The active form of vitamin D, 1,25(OH)₂D₃, has a broad range of effects on bone, however, its role in the quality of bone matrix is not well understood. In this study, using an osteoblastic cell (MC3T3-E1) culture system, the effects of 1,25(OH)₂D₃ on collagen cross-linking and related enzymes, i.e., lysyl hydroxylases (LH1-3) and lysyl oxidases (LOX, LOXL1-4), were examined and compared to controls where cells were treated with cholecalciferol or ethanol. When compared to the controls, gene expressions of LH1, LH2b and LOXL2 were significantly upregulated by 1,25(OH)₂D₃ up to 72 h of culture. In addition, hydroxylysine (Hyl), Hyl aldehyde (Hyl^ald), Hyl^ald-derived cross-links and a total number of cross-links of collagen were significantly higher and the cross-link maturation was accelerated in the 1,25(OH)₂D₃ treated group. These results demonstrate that 1,25(OH)₂D₃ directly regulates collagen cross-linking in this culture system likely by upregulating gene expression of specific LH and LOX enzymes.     

p-Dimethylaminobenzaldehyde-reactive substances in tail tendon collagen of streptozotocin-diabetic rats: temporal relation to biomechanical properties and advanced glycation endproduct (AGE)-related fluorescence

In the present work, pepsin digests of tail tendons from streptozotocin-diabetic rats were found to contain material that reacted rapidly at room temperature with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to give an adduct with an absorbance spectrum characteristic of the Ehrlich chromogen of pyrrolic nature determined in ageing collagens. A significant correlation of the Ehrlich adduct with tendon mechanical strength and collagen fluorescence characteristic of advanced glycation endproducts was observed. Collagen content of the Ehrlich-positive material was found to be significantly elevated in tendons of diabetic rats compared with age-matched healthy controls. The results indicate that the p-dimethylaminobenzaldehyde-reactive pyrrole moieties may contribute to the increased cross-linking of diabetic matrix collagen. Profound inhibitory effect of aminoguanidine was observed, underlining the role of non-enzymatic mechanisms of advanced glycation in pyrrolisation and cross-linking of collagen exposed to hyperglycaemia. It is hypothesised that quantification of the p-dimethylaminobenzaldehyde-reactive material in matrix collagen may provide a tissue measure of integrated hyperglycaemia over prolonged periods of time. Further research is to assess the significance of p-dimethylaminobenzaldehyde-reactive substances in diabetic collagen tissues and to reveal their relationship to enzyme-mediated physiological pyrrolisation of ageing collagens.     

Effects of ultraviolet-A and riboflavin on the interaction of collagen and proteoglycans during corneal cross-linking

Corneal cross-linking using riboflavin and ultraviolet-A (RFUVA) is a clinical treatment targeting the stroma in progressive keratoconus. The stroma contains keratocan, lumican, mimecan, and decorin, core proteins of major proteoglycans (PGs) that bind collagen fibrils, playing important roles in stromal transparency. Here, a model reaction system using purified, non-glycosylated PG core proteins in solution in vitro has been compared with reactions inside an intact cornea, ex vivo, revealing effects of RFUVA on interactions between PGs and collagen cross-linking. Irradiation with UVA and riboflavin cross-links collagen α and β chains into larger polymers. In addition, RFUVA cross-links PG core proteins, forming higher molecular weight polymers. When collagen type I is mixed with individual purified, non-glycosylated PG core proteins in solution in vitro and subjected to RFUVA, both keratocan and lumican strongly inhibit collagen cross-linking. However, mimecan and decorin do not inhibit but instead form cross-links with collagen, forming new high molecular weight polymers. In contrast, corneal glycosaminoglycans, keratan sulfate and chondroitin sulfate, in isolation from their core proteins, are not cross-linked by RFUVA and do not form cross-links with collagen. Significantly, when RFUVA is conducted on intact corneas ex vivo, both keratocan and lumican, in their natively glycosylated form, do form cross-links with collagen. Thus, RFUVA causes cross-linking of collagen molecules among themselves and PG core proteins among themselves, together with limited linkages between collagen and keratocan, lumican, mimecan, and decorin. RFUVA as a diagnostic tool reveals that keratocan and lumican core proteins interact with collagen very differently than do mimecan and decorin.     

Bone material properties and mineral matrix contributions to fracture risk or age in women and men

The strength of bone is related to its mass and geometry, but also to the physical properties of the tissue itself. Bone tissue is composed primarily of collagen and mineral, each of which changes with age, and each of which can be affected by pharmaceutical treatments designed to prevent or reverse the loss of bone. With age, there is a decrease in collagen content, which is associated with an increased mean tissue mineralization, but there is no difference in cross-link levels compared to younger adult bone. In osteoporosis, however, there is a decrease in the reducible collagen cross-links without an alteration in collagen concentration; this would tend to increase bone fragility. In older people, the mean tissue age (MTA) increases, causing the tissue to become more highly mineralized. The increased bone turnover following menopause may reduce global MTA, and would reduce overall tissue mineralization. Bone strength and toughness are positively correlated to bone mineral content, but when bone tissue becomes too highly mineralized, it tends to become brittle. This reduces its toughness, and makes it more prone to fracture from repeated loads and accumulated microcracking. Most approved pharmaceutical treatments for osteoporosis suppress bone turnover, increasing MTA and mineralization of the tissue. This might have either or both of two effects. It could increase bone volume from refilling of the remodeling space, reducing the risk for fracture. Alternatively, the increased MTA could increase the propensity to develop microcracks, and reduce the toughness of bone, making it more likely to fracture. There may also be changes in the morphology of the mineral crystals that could affect the homogeneity of the tissue and impact mechanical properties. These changes might have large positive or negative effects on fracture incidence, and could contribute to the paradox that both large and small increases in density have about the same effect on fracture risk. Bone mineral density measured by DXA does not discriminate between density differences caused by volume changes, and those caused by changes in mineralization. As such, it does not entirely reflect material property changes in aging or osteoporotic bone that contribute to bone's risk for fracture.     

Glycation cross-linking induced mechanical–enzymatic cleavage of microscale tendon fibers

Recent molecular modeling data using collagen peptides predicted that mechanical force transmitted through intermolecular cross-links resulted in collagen triple helix unwinding. These simulations further predicted that this unwinding, referred to as triple helical microunfolding, occurred at forces well below canonical collagen damage mechanisms. Based in large part on these data, we hypothesized that mechanical loading of glycation cross-linked tendon microfibers would result in accelerated collagenolytic enzyme damage. This hypothesis is in stark contrast to reports in literature that indicated that individually mechanical loading or cross-linking each retards enzymatic degradation of collagen substrates. Using our Collagen Enzyme Mechano-Kinetic Automated Testing (CEMKAT) System we mechanically loaded collagen-rich tendon microfibers that had been chemically cross-linked with sugar and tested for degrading enzyme susceptibility. Our results indicated that cross-linked fibers were > 5 times more resistant to enzymatic degradation while unloaded but became highly susceptible to enzyme cleavage when they were stretched by an applied mechanical deformation.     

The nature of the collagen cross-links in bone in the chronic uraemic state.

The cross-links from NaB3H4-reduced bone collagen of chronically uraemic rats and pairfed controls were compared. The ratio of the reduced cross-links deltadelta'-dihydroxylysinonorleucine to delta-hydroxylysinonorleucine was significantly increased in the uraemic animals. The observed increment in the dihydroxylysinonorleucine:hydroxylysinonorleucine ratio was accentuated as the uraemic state advanced. The data indicate that osteodystrophy of chronic renal insufficiency is characterized by an alteration of the quantitative relations between cross-links and aldehydic precursors of bone collagen.     

Thermal stabilization of collagen molecules in bone tissue

Differential thermal calorimetry (DSC) analysis of partially dehydrated bovine bone, demineralized bone and bovine tendon collagen was performed up to 300 °C to determine factors influencing stability of mineralized collagen in bone tissue. Two endothermal regions were recognized. The first, attributed to denaturation of collagen triple helix, was hydration dependent and had a peak at 155–165 °C in bone, 118–137 °C in tendon and 131–136 °C in demineralized bone. The second region extended from 245 to 290 °C in bone and from 200 to 280 °C in tendon and was connected with melting and decomposition of collagen. Differences in thermodynamic parameters between cortical and trabecular bone tissue were stated. Activation energy of collagen unfolding in native bone tissue increased with dehydration of the bone. From the results of the present study we conclude that dehydrated bone collagen is thermally very stable both in native and in demineralized bone. Presence of mineral additionally stabilizes bone tissue.     

Minoxidil exerts different inhibitory effects on gene expression of lysyl hydroxylase 1, 2, and 3: implications for collagen cross-linking and treatment of fibrosis.

Collagen deposits in fibrotic lesions often display elevated levels of hydroxyallysine (pyridinoline) cross-links. The relation between the occurrence of pyridinoline cross-links and the irreversibility of fibrosis suggests that these cross-links contribute to the aberrant accumulation of collagen. Based on its inhibitory effect on lysyl hydroxylase activity minoxidil has been postulated to possess anti-fibrotic properties by limiting the hydroxylysine supply for hydroxyallysine cross-linking. However, to interfere with hydroxyallysine cross-linking specifically lysyl hydroxylation of the collagen telopeptide should be inhibited, a reaction predominantly catalysed by lysyl hydroxylase (LH) 2b. In this study, we demonstrate that minoxidil treatment of cultured fibroblasts reduces LH1>LH2b>LH3 mRNA levels dose-and time-dependently, but has essentially no effect on the total number of pyridinoline cross-links in the collagen matrix. Still the collagen produced in the presence of minoxidil displays some remarkable features: hydroxylation of triple helical lysine residues is reduced to 50% and lysylpyridinoline cross-linking is increased at the expense of hydroxylysylpyridinoline cross-linking. These observations can be explained by our finding that LH1 mRNA levels are the most sensitive to minoxidil treatment, corroborating that LH1 has a preference for triple helical lysine residues as substrate. In addition, the non-proportional increase in cross-links (20-fold) with respect to the decrease in lysyl hydroxylation state of the triple helix (2-fold) even suggests that LH1 preferentially hydroxylates triple helical lysine residues at the cross-link positions. We conclude that minoxidil is unlikely to serve as an anti-fibroticum, but confers features to the collagen matrix, which provide insight into the substrate specificity of LH1.