The composition of collagen in the aneurysm wall of men and women.

BACKGROUND: Loss of vessel wall integrity by degradation is essential for the development of abdominal aortic aneurysm (AAA) and ultimately its rupture. The observed greater rupture rate in women with AAA might be related to gender differences in the biomechanical properties of the aneurysm wall. The aim of the study was to compare the biomechanically important structure of collagen between men and women with AAA. METHODS: Biopsies of the aneurysm walls were obtained during elective open repair of men (n = 14) and women (n = 14) treated for AAA. High-performance liquid chromatography (HPLC), Western blot, messenger RNA expression, and histochemical analyses were performed to assess the cross-linking and the amount and the composition of collagen. RESULTS: There was neither a difference in the thickness of the aneurysm wall, nor in the histological evaluation of the collagen composition between the sexes. Relative collagen content in the aneurysm wall was similar in men and women, as assessed by messenger RNA expression and HPLC. Collagen cross-linking differed between the sexes; women had more lysyl pyridinoline (LP) than men (0.140 vs 0.07; P = .005), resulting in a lower hydroxyl pyridinoline (HP):LP ratio (3.28 vs 8.41; P = .003). There was no difference in messenger RNA and protein expressions of lysyl hydroxylase and lysyl oxidase to associate with the lower HP:LP ratio in women. CONCLUSIONS: The composition of collagen in the aneurysm wall of men and women are in several aspects similar, with the exception of collagen cross-linking, suggesting that the difference in rupture rate between the sexes rather depend on the composition of other vessel wall structures.

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.

Development of a convenient peptide-based assay for lysyl hydroxylase.

Hydroxylysine (Hyl) is a posttranslationally modified amino acid found mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) catalyzes the hydroxylation of the C-5 position of a Lys residue, resulting in the production of Hyl. Mechanistically, LH incorporates one oxygen atom into both Lys and 2-oxoglutarate; the latter is decarboxylated to form succinate and CO(2). To develop a convenient, RP-HPLC based LH assay, we used Fmoc solid-phase methodology to synthesize three different peptides designed as LH substrates and one peptide corresponding to an LH product. Peptides were characterized by RP-HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. Separation of peptides was examined under a variety of RP-HPLC conditions. The best results were achieved using peptide derivatization (1-anthroylnitrile for organic phase and dansyl chloride for aqueous phase) prior to RP-HPLC analysis. The products (di- and tetra-substituted Lys- and Hyl-containing peptides) were well resolved by RP-HPLC. The resolution of each peak allows for quantification of peak areas, which in turn, when examined as a function of time, can be utilized for studying the kinetics of LH catalyzed reactions. Most significantly, the RP-HPLC assay directly monitors the Hyl containing product. Prior LH assay methods are multi-step, require radio-labeled substrates, and/or measure depletion of 2-oxoglutarate or formation of CO(2). Since the LH reaction with 2-oxoglutarate is uncoupled from Lys hydroxylation, the most accurate assay of LH activity should monitor the formation of Hyl.

Biochemical and functional modulation of the cartilage collagen network by IGF1, TGFbeta2 and FGF2.

OBJECTIVE: Examine effects of insulin-like growth factor 1 (IGF1), transforming growth factor beta2 (TGFbeta2) and fibroblast growth factor 2 (FGF2) on proteoglycan and collagen network and biomechanical properties of the newly formed cartilage matrix. METHODS: Bovine articular chondrocytes were cultured in alginate beads for 3 weeks with or without FGF2, TGFbeta2 or IGF1 in the presence of 10% FCS. Proteoglycan content, collagen content, hydroxylysylpyridinoline cross-links and overall matrix metalloproteinase (MMP) activity in the culture medium were measured. Alginate disks cultured for 5 weeks were used to evaluate the effect of growth factors on mechanical properties of the construct by determining the equilibrium aggregate modulus and secant modulus. RESULTS: IGF1 increased collagen and proteoglycan deposition. FGF2 mainly decreased collagen deposition and TGFbeta2 proteoglycan deposition. A decrease in cross-links was observed in matrix produced by chondrocytes cultured in the presence of TGFbeta2. IGF1 and FGF2 had no influence on the number of cross-links per collagen molecule. Overall MMP activity was significantly higher in culture medium of cells cultured with FGF2. TGFbeta2 and IGF1 had no effect on MMP activity. After 35 days of culture, the matrix produced under influence of IGF1 had a lower permeability and a trend to increase stiffness. FGF2 showed a trend to lower both properties. TGFbeta2 had no effect on these parameters. CONCLUSION: IGF1, TGFbeta2 and FGF2 had differential effects on collagen network formation. Of the three growth factors tested, IGF1 seems to be best in promoting the formation of a functional collagen network since it increased proteoglycan and collagen deposition and improved the mechanical properties.

Differential gene expression of collagen-binding small leucine-rich proteoglycans and lysyl hydroxylases, during mineralization by MC3T3-E1 cells cultured on titanium implant material.

Titanium implants create a unique ultrastructure (composed of a collagenous zone with relatively disorganized fibril morphology) at the bone-implant interface. The objective of this study was to investigate the temporal mRNA expression patterns, using real-time polymerase chain reaction, of type I collagen (COLI) and regulators for collagen fibrillogenesis, collagen-binding small leucine-rich proteoglycans (SLRPs) and lysyl hydroxylases (LHs), during mineralization, by MC3T3-E1 cells cultured on titanium (Ti). Lysates of the cultures on Ti and on plastic wells (Pl) for 10-50 d were used for the quantification of calcium and mRNA. Although the onset of calcium accumulation in the cultures on Ti (30-40 d) was slower than that of cultures on Pl (20-30 d), the gene expression patterns during mineralization were similar in cells cultured on either material. COLI and fibromodulin were up-regulated just before the onset of mineralization and then down-regulated. Lumican and LH1 were up-regulated just before the onset of mineralization and then returned to the baseline level. Decorin and LH2 were up-regulated at the late mineralization stage. Biglycan was down-regulated once at the early mineralization stage and then returned to the original level. LH3 was maintained at a steady level throughout. This study suggests actual but distinct roles of SLRPs and LHs in the formation of a unique ultrastructure at the bone-implant interface.

Proteomic identification of collagens and related proteins in human fibroblasts.

Fibroblasts are used for diagnosis of a series of metabolic diseases and are particularly suitable for the diagnosis of collagen disorders. We aimed to generate a skin fibroblast map that would be suitable for the concomitant determination of collagen and collagen-related proteins.A human skin fibroblast cell line was cultivated, homogenised, proteins extracted and subject to two-dimensional gel electrophoresis with subsequent in-gel-digestion of protein spots and mass spectrometrical identification (MALDI-TOF). Collagen alpha1 (I) chain precursor, collagen alpha1 (III) chain precursor, collagen alpha2 (VI) precursor and collagen modifying enzymes prolyl 4-hydroxylase alpha-2-subunit precursor, procollagen-lysine 2-oxoglutarate 5-dioxygenase 1 and 2, protein disulfide isomerase ER-60 precursor and peptidyl-prolyl cis-trans isomerase were among the abundant proteins. The finding of collagen and collagen-related structures as well as the identification of other metabolic enzyme systems on one 2D gel may propose the use of this proteomic method for further characterization of collagen and collagen-related proteins or for preliminary screening of metabolic disorders.

Lysyl hydroxylase-2b directs collagen cross-linking pathways in MC3T3-E1 cells.

UNLABELLED: To elucidate the roles of LH2b in collagen cross-linking, MC3T3-E1 cell clones expressing higher (S) or lower (AS) levels of LH2b were established. Compared with controls, the collagen cross-linking pattern was shifted toward hydroxylysine-aldehyde (S clones)- or lysine-aldehyde (AS clones)-derived pathways. The data indicate that LH2b directs collagen cross-linking pathways through its action on telopeptidyl lysine residues. INTRODUCTION: Lysine (Lys) hydroxylation is a post-translational modification of collagen critical for cross-linking and glycosylation. Currently, three isoforms of lysyl hydroxylase (LH) have been identified, but their specific functions are still not well defined. Recently, we proposed that LH2 might modulate collagen cross-linking pattern through its action on Lys residues located in the telopeptide domains of collagen. MATERIALS AND METHODS: To directly test this hypothesis, several MC3T3-E1 cell-derived clones expressing higher (sense [S]) or lower (antisense [AS]) levels of LH2b, the predominant form of LH2 in this cell line, were established and cultured for 2 weeks, and collagen cross-links and precursor aldehydes in the matrices were analyzed. RESULTS: In S clones tested, the ratio of dihydroxylysinonorleucine (DHLNL) to hydroxylysinonorleucine (HLNL) was significantly higher than the average of controls (76% and 140% increase, respectively), and the level of pyridinoline (Pyr) was elevated (100% and 150% increase, respectively). In contrast, when MC3T3-E1 cells were transfected with a LH2b antisense construct (AS clones), the DHLNL/HLNL ratios were significantly lower than that of controls (56% and 73% decrease, respectively), and Pyr was not detected. Furthermore, significant amounts of an aldol-derived cross-link, dehydrohistidinohydroxymerodesmosine, were produced ( approximately 0.3 mol/mol of collagen) in AS clones. CONCLUSIONS: The data clearly show a critical role of LH2b in determining collagen cross-linking pathways, most likely through its action on telopeptidyl Lys residues.

Brittle cornea syndrome and its delineation from the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VI): report on 23 patients and review of the literature.

The brittle cornea syndrome (BCS) is a generalized connective tissue disorder characterized by corneal rupture following only minor trauma, keratoconus or keratoglobus, blue sclerae, hyperelasticity of the skin without excessive fragility, and hypermobility of the joints. It is inherited as an autosomal recessive trait but the underlying genetic defect remains undetermined. We present 23 patients (11 male) from 13 nuclear families followed at the King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia, aged 3-28 years at last follow-up. A total of 28 events of corneal rupture were noted in 17 patients (eight male), among whom nine had had bilateral ruptures, and eight had had unilateral ruptures (four of the right cornea), while two had experienced re-rupture 2 and 4 years, respectively, after surgery; six patients (aged 3-21 years) had had no ruptures. We describe the natural history of our cases and discuss them together with those others reported in the literature. Because of similarities between the BCS and the kyphoscoliotic type of the Ehlers-Danlos syndrome (EDS VI), both disorders tend to have been confounded. Here, we show that all of our BCS patients tested in this regard had biochemical findings reflective of normal activity of lysyl hydroxylase, characteristically deficient in EDS VI, such as normal urinary total pyridinoline ratios and/or normal electrophoretic migration of collagen chains produced by dermal fibroblasts. The BCS is, therefore, an entity distinct from the kyphoscoliotic type of EDS, which has a much poorer prognosis.

Developmental changes in skin collagen biosynthesis pathway in posthatch male and female chickens.

The developmental changes in skin collagen biosynthesis pathway in male and female chickens were evaluated. Concentration of collagen, levels of mRNA for collagen type I subunits and for lysyl hydroxylase, and the level of three lysyl oxidase-derived cross-links: dehydro-dihydroxylysinonorleucine (DHLNL), dehydro-hydroxylysinonorleucine (HLNL), and dehydro-histidinohydroxymerodesmosine (HHMD) were determined during 4 wk posthatching. Skin collagen content increased with age and was higher in males than in females. In both sexes, the expression of the genes coding for alpha 1 and alpha 2 of collagen type I decreased with age: alpha 1(I) gene expression decreased from Day 3 onwards, whereas the reduction in alpha 2(I) gene expression started 1 wk later. At all ages examined, the expression of both genes was higher in male than in female skin. Males and females lysyl hydroxylase gene expression remained low until Day 16, after which an increase in the enzyme gene expression was observed. An increase in skin HLNL content was observed from Day 3 in both sexes reaching a peak in males at Day 9 and in females 1 wk later. The DHLNL content, which was higher in males than in females at all ages tested, dramatically decreased in both male and female skin from 3 d of age, reaching its lowest level at Day 16, and remained at that low level thereafter. The skin content of HHMD in males and females followed an oscillatory behavior with higher peaks in the male skin. The results suggest that the higher tensile strength of male skin than female skin may be due to the elevated skin collagen content that resulted from increased expression in collagen type I genes on the one hand, and from the higher amounts of various collagen cross-links on the other.

A homozygous stop codon in the lysyl hydroxylase gene in two siblings with Ehlers-Danlos syndrome type VI.

Ehlers-Danlos syndrome (EDS) is characterized by joint hypermobility, alterations in the skin and additional signs of connective tissue involvement. EDS type VI was the first connective tissue disorder for which a specific defect in collagen metabolism was identified, namely a deficiency of lysyl hydroxylase activity. We now report a homozygous single basepair substitution converting the CGA codon (Arg319) to a TGA termination codon in two siblings with EDS type VI. The healthy parents, who are first cousins, and two of the three healthy siblings of the patients are heterozygous. The mutation leads to an almost complete absence of lysyl hydroxylase activity in extracts derived from fibroblasts of the patients.

A paradoxical effect of hydralazine on prolyl and lysyl hydroxylase activities in cultured human skin fibroblasts.

The effect of hydralazine on several parameters of collagen biosynthesis has been studied in cultured human skin fibroblasts. Cells treated with hydralazine synthesized procollagen which was severely deficient in hydroxyproline and hydroxylysine, indicating inhibition of prolyl and lysyl hydroxylase reactions in the cell. Assays of prolyl and lysyl hydroxylase activities, however, revealed markedly increased levels in hydralazine-treated cells. The stimulatory effect of hydralazine could not be simulated in cell extracts, demonstrating its requirement for intact cells. The effect occurred slowly over a period of 96 h and was dependent on hydralazine concentration between 10 and 100 microM. This phenomenon was also observed in lysyl hydroxylase-deficient mutants. In both normal and mutant cells the relative magnitude of the hydralazine effect could be modified by ascorbic acid in the culture medium. Ascorbic acid increased the response of prolyl hydroxylase to hydralazine from 1.5- to 2-fold to 3- to 7-fold, whereas it decreased the response of lysyl hydroxylase to hydralazine from 4- to 8-fold to 2- to 3-fold. Total collagen synthesis was substantially reduced in hydralazine-treated cells; the time course and the dose-response relationship were similar to those observed for the hydroxylases. alpha, alpha'-Dipyridyl, an iron chelator, mimicked these effects of hydralazine. The studies suggest the existence in cultured cells of a compensatory mechanism for overproduction of these crucial enzymes in collagen biosynthesis, a mechanism which remains functional in cells derived from patients afflicted with hydroxylysine-deficient collagen disease.

Human osteopetrosis: a histological, ultrastructural, and biochemical study.

UNLABELLED: Histological ultrastructural, and biochemical studies of the tissues of a patient with osteopetrosis were done. The bone contained an increased number of osteoclasts which were characterized ultrastructurally by the absence of ruffled borders and clear zones, hallmarks of actively resorbing osteoclasts. In contrast with normal human bone, tissue collagenase was not detected in osteopetrotic bone cultured in vitro, nor was tissue collagenase activity released when the osteopetrotic bone was incubated with parathyroid hormone. No striking abnormalities of parathyroid hormone or calcitonin were found in the blood or parathyroid and thyroid glands. Except for a slight increase in the extent of lysine hydroxylation of bone collagen, no significant biochemical abnormality of collagen was found. The histological, ultrastructural, and biochemical data support the hypothesis that the basic defect in osteopetrosis is cellular and the osteoclasts, in particular, are abnormal. They appear to be unable to resorb bone and cartilage, and they do not appear to respond to parathyroid hormone in a completely normal way. CLINICAL RELEVANCE: Bone marrow transplantation has produced improvement in both experimental animals and humans and should be actively considered in a child with osteopetrosis who is not thriving.