Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome

Cultured fibroblasts of 13 patients with the Menkes syndrome and two with a new subtype (type IX) of the Ehlers-Danlos syndrome (E-D IX patients) showed many very similar abnormalities in their copper and collagen metabolism. Both cell types had markedly increased copper concentrations and 64Cu incorporation, and this cation accumulated in metallothionein or a metallothionein-like protein, as previously established for Menkes cells. Histochemical staining indicated that copper was distributed diffusely throughout the cytoplasm in both cell types, this location being consistent with the accumulation in metallothionein. Both fibroblast types also had markedly low lysyl oxidase activity and distinctly increased extractability of newly synthesized collagen, whereas no abnormalities were present in cell viability, duplication rate, prolyl 4-hydroxylase activity, or collagen synthesis rate. A high negative correlation (P less than 0.001) was found in the pooled group of Menkes and E-D IX cells between cellular copper concentration (r = 0.804) or 64Cu incorporation (r = 0.863) and the logarithm of lysyl oxidase activity. There was also a high positive correlation (P less than 0.001) between cellular copper concentration and incorporation (r = 0.869). One of the two E-D IX patients was also shown to have similar changes in lysyl oxidase activity and collagen extractability in the skin biopsy specimen, suggesting that the abnormalities observed in cultured cells are similar to those present in vivo. The only distinct abnormality found in the cells of the parents of the E-D IX patients was an increased 64Cu incorporation in those of the mother, this finding being consistent with X-linked inheritance of the disorder.     

Response of rat connective tissues to streptozotocin-diabetes. Tissue-specific effects on collagen metabolism

We assessed the effect of streptozotocin-diabetes on in vivo collagen metabolism in skin, aorta and intestine by injecting [³H]proline into rats, 20 days after administering the diabetogen, streptozotocin. One day after [³H]proline injection, diabetic and control animals were killed, their tissues analyzed for both ³H-labeled and unlabeled hydroxyproline and results expressed per entire tissue. Thereby, the effect of diabetes on net collagen synthesis and tissue collagen mass, respectively, was evaluated.Diabetes resulted in a lower content of [³H]collagen in skin and aorta, suggesting decreased net collagen synthesis. This decrease in net synthesis was accompanied by a decrease of collagen mass in skin, whereas aortic collagen mass was unaffected. Consequently, an acceleration of collagen degradation in skin is postulated to have accompanied the expected depression of collagen synthesis; alterations of the physiochemical properties of skin from diabetic rats support this interpretation. For intestine, both net collagen synthesis and mass increased in diabetic rats, reflecting increased collagen synthesis—possibly associated with polyphagy.In conclusion, with regard to collagen metabolism, representative connective tissues respond differently to experimental diabetes, and we suggest that this insight will be useful in future studies aimed at understanding the pathophysiology of connective tissues affected by diabetes.     

Mechanism of impaired skin collagen maturity in riboflavin or pyridoxine deficiency

To elucidate the biochemical basis of impaired skin collagen maturity in pyridoxine-or riboflavin-deficient rats the following two mechanistic possibilities were tested: (i) Reduction in the activity of skin lysyl oxidase (EC 1·4·3·13) which initiates the cross-linking of collagen and (ii) putative rise in homocysteine level leading to neutralization of allysine (α-aminoadipic acid δ-5-semialdehyde)or hydroxyallysine (hydroxy α-aminoadepic acid (δ-semialdehyde) in collagen by the formation of thiazine complexes. Skin lysyl oxidase activity was not affected in pyridoxine deficiency suggesting that pyridoxal phosphate may not be its cofactor. In riboflavin deficiency, lysyl oxidase activity was not altered in the newly regenerated rat skin but a slight reduction was observed in the skin of 18-day-old rat pups. This could be related to the body weight deficit rather than deficiencyper se. Aldehyde content of purified salt soluble collagen of regenerated skin was significantly reduced in both the deficiencies. A 2 to 4-fold increase in the concentration of skin homocysteine was observed in both the deficiencies. The results suggest that increase in skin homocysteine level may be responsible for the impaired skin collagen maturity in riboflavin or pyridoxine deficiency.     

Age-Related Changes in Collagen and Glycosaminoglycans of Rat Gingiva1

Age-related changes in collagen and glycosaminoglycans (GAGs) of rat gingiva were studied biochemically and histologically. The components of isolated GAGs were identified as dermatan sulphate, hyaluronic acid, and heparan sulphate. In histological studies, hyaluronic acid was present in all regions of the gingiva, whereas dermatan sulphate and heparan sulphate were present only in gingival connective tissue. However, there was no significant difference in the relationship between aging, and the content or distribution of GAGs. On the other hand, histological findings showed that collagen fibers were markedly increased in number and the vascular composition was decreased with increasing age. In biochemical studies, the content of collagen, especially of insoluble collagen, was greatly increased with age, whereas collagen biosynthesis and collagenolytic activity were markedly decreased. In addition, lysyl oxidase activity was also significantly decreased with age. The results indicate that the rate of collagen turnover decreases and collagen fibers increase in stability in rat gingiva with increasing age. These observed age-related changes may affect the ability of gingiva to respond to local irritants.     

Collagen synthesis by regenerating rabbit ear tissue

The principal collagen types synthesized during two distinct phases of regeneration in rabbit ears have been investigated, in order to relate altered phenotypic expression in connective tissue cells to regeneration of cartilage. To do this, radioactively labeled collagens synthesized in short-term culture by selected regenerating ear tissues were analyzed by ion-exchange chromatography and SDS-gel electrophoresis of the intact collagens and of the cyanogen bromide peptides derived from them. Prior to the appearance of cartilage, rabbit ear holes are filled by an outgrowth of mesenchyme-like cells derived locally from adjacent tissues. These cells produce a mixture of collagens including type I, [α1(I)]₂α2, and the type I trimer, [α1(I)]₃, but not type II collagen. Trimer production represents about one-fourth of the collagen synthesized in either a 4-, 10-, or a 24-hr incubation. Trimer is not made by tissues from healing skin wounds nor is it present in normal, uninjured ear tissues. Type II collagen synthesis was detected in tissues taken from late regenerates containing histologically recognizable cartilage, and direct analysis of regenerated cartilage confirmed the presence of type II collagen in the matrix. Thus, regenerated cartilage in the rabbit ear system contains the normal cartilage collagen, type II, while the proliferating cell mass from which the cartilage develops synthesizes the unusual collagen, [α1(I)]₃.     

Alterations of Dermal Connective Tissue Collagen in Diabetes: Molecular Basis of Aged-Appearing Skin

Alterations of the collagen, the major structural protein in skin, contribute significantly to human skin connective tissue aging. As aged-appearing skin is more common in diabetes, here we investigated the molecular basis of aged-appearing skin in diabetes. Among all known human matrix metalloproteinases (MMPs), diabetic skin shows elevated levels of MMP-1 and MMP-2. Laser capture microdissection (LCM) coupled real-time PCR indicated that elevated MMPs in diabetic skin were primarily expressed in the dermis. Furthermore, diabetic skin shows increased lysyl oxidase (LOX) expression and higher cross-linked collagens. Atomic force microscopy (AFM) further indicated that collagen fibrils were fragmented/disorganized, and key mechanical properties of traction force and tensile strength were increased in diabetic skin, compared to intact/well-organized collagen fibrils in non-diabetic skin. In in vitro tissue culture system, multiple MMPs including MMP-1 and MM-2 were induced by high glucose (25 mM) exposure to isolated primary human skin dermal fibroblasts, the major cells responsible for collagen homeostasis in skin. The elevation of MMPs and LOX over the years is thought to result in the accumulation of fragmented and cross-linked collagen, and thus impairs dermal collagen structural integrity and mechanical properties in diabetes. Our data partially explain why old-looking skin is more common in diabetic patients.     

The role of decorin in collagen fibrillogenesis and skin homeostasis

Decorin, a prototype member of the growing family of the small leucine-rich proteoglycans (SLRP's), plays significant roles in tissue development and assembly, as well as playing both direct and indirect signaling roles. This review will concentrate on decorin's function in collagen fibrillogenesis as determined through the study of mice with a disrupted decorin gene. The fragile skin and abnormal tendon phenotypes initially observed were found to be due to fundamental alterations in collagen fibers, highlighting the crucial role of proteoglycans in general and SLRP's in particular in collagen fibrillogenesis. The altered fibril formation within tissues in turn leads to observable and quantifiable changes at the organismal level. Research into certain fibrotic processes with concomitant upregulation or reduction of decorin makes interesting comparisons with the collagen malformations seen in Dcn ^–/– mice. Overall, decorin is shown to be a vital player in maintaining skin and tendon integrity at the molecular level, among other functions. Published in 2003.     

Topical application of B-aminopropionitrile

Application of ¹⁴CB-aminopropionitrile (BAPN) to the skin of rats followed by measurement of urinary radioactivity was used for comparative studies of the absorption of ¹⁴C-BAPN fumarate and ¹⁴C-BAPN free base (F.B.). ¹⁴C-BAPN (F.B.) was absorbed more rapidly and to a greater extent than the fumarate salt. Six hours after topical administration of ¹⁴C-BAPN (F.B.) only traces of ¹⁴C were found on the skin and less than 1% of the dose within the skin section suggesting rapid drug absorption. Lysyl oxidase activity of sponge granuloma tissue was significantly inhibited by a single small dose (20 μl) of BAPN (F.B.) applied to the intact skin over the implant. Chronic application of twice daily 5 μl doses of BAPN (F.B.) significantly inhibited lysyl oxidase of the granuloma tissue underlying the area of topical application as well as that on the opposite side not receiving BAPN. Extractibility of collagen was significantly increased only in the granuloma tissue under the skin receiving BAPN.     

Liver proline oxidase activity and collagen synthesis in rats with cirrhosis induced by carbon tetrachloride

In rats treated with CCl₄ for 7 weeks, liver proline oxidase activity was drastically reduced 24 h after the initial administration of the toxic agent and remained low throughout the treatment period. This was accompanied by a larger accumulation of added proline in the incubation medium and a lesser release of ¹⁴CO₂ from [¹⁴C]proline during incubation.Collagen synthesis by liver slices of CCl₄-treated rats increased in proportion to proline concentration, a plateau being reached at 0.48 mM proline. The plateau did not occur within the range studied with liver slices of normal liver.Increased collagen synthesis in vitro was accompanied by increased deposition of collagen in vivo only during the first 3 weeks of CCl₄ treatment. No further increase in liver collagen content occurred thereafter. Discontinuance of CCl₄-administration was followed by a return to normal of proline oxidase activity and in vitro collagen synthesis within 2 weeks. Nevertheless, collagen content remained elevated.The results suggest that proline oxidase activity, together with the previously shown increased formation of proline from precursor amino acids, may control the amount of proline available for collagen biosynthesis; and that the rate of degradation of collagen, perhaps by collagenase, may determine the levels of collagen remaining after discontinuance of CCl₄-administration.     

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen or fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by η-amino-caproic acid (ηACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva. © 1996 Wiley-Liss, Inc.     

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous,Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.     

Inhibition of lysyl oxidase and prolyl hydroxylase activity in glucocorticoid treated rats

Triamcinolone diacetate produced a dose dependent decrease in lysyl oxidase activity in the skin of new born rats when administered over a three day period. Maximum inhibition by this glucorticoid resulted in less than 10% of control lysyl oxidase activity. A similar though less dramatic effect was observed on skin prolyl hydroxylase activity. These results suggest that the antianabolic effect of glucocorticoids on collagen synthesis extend to enzymes involved in the intra- and extracellular modifications of collagen.     

Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

Changes in collagen cross-linking and lysyl oxidase by estrogen.

Dermal collagen solubility and lysyl oxidase activity of bones were measured in DDD mice of advancing age. Insoluble fractions of the dermal collagen increased more rapidly in females than in males after 5 weeks of age. Activity of the lysyl oxidase extracted from bones was higher in females than in males after 4 weeks of age. After sexual maturation, such sex differences were always observed in skin as well as in bone tissue. In other experimental animals, dermal collagen solubility was markedly decreased by estrogen treatment and lysyl oxidase was remarkably activated by estrogen in both skin and bone. Thus it is clear that estrogen stimulates the enzyme activity and accelerates the maturation of collagen and elastin in extracellular space.     

The function of Ca+ in the action of mammalian collagenases.

The removal of extrinsic Ca²⁺ from human skin, rat skin, and postpartum rat uterus collagenases results in a reversible loss of enzymatic activity, which becomes irreversible with increasing length of Ca²⁺-free incubation at physiological temperature and pH. Ca²⁺ is necessary for thermostabilization both in the presence and absence of the collagen substrate. Enzymes from all three sources display linear rates of reaction at Ca²⁺ concentrations from 0.5 to 20 mm and are half-maximally activated at 0.5 mm Ca²⁺. The increase in collagenase activity with increasing Ca²⁺ concentration is associated with an increase in thermostabilization. Ba²⁺ and Sr²⁺ are effective substitutes for Ca²⁺ in human skin collagenase but not in the collagenases from rat tissues. These studies also indicate that Ca²⁺ plays no role in the binding of collagenases to their substrate.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

MULTIPLE FORMS OF MONOAMINE OXIDASE IN DEVELOPING BRAIN: TISSUE AND SUBSTRATE SPECIFICITIES

Brains, hearts and livers from newborn and adult rats were assayed for monoamine oxidase activity using gel electrophoretic techniques. The results suggest that each of the tissues possesses multiple forms (isoenzymes) of monoamine oxidase and that these forms are different for the various tissues. Further, the forms of monoamine oxidase in the neonatal tissues differ from those in the corresponding adult tissue. These different forms of monoamine oxidase have different substrate specificities. Using 5-hydroxy[¹⁴C]tryptamine as substrate, we have demonstrated that the monoamine oxidase patterns appearing on the gel do indeed possess monoamine oxidase activity.     

Glycation-induced matrix stability in the rabbit achilles tendon

Connective tissue susceptibility to nonenzymatic glycation was examined following 0, 2, 4, 6, 8, and 10 weeks of incubating the rabbit Achilles tendon in phosphate-buffered saline containing ribose (glycated). The biomechanical integrity of the glycated tendons was then compared to control tendons incubated in phosphate-buffered saline (non-glycated) at each time interval, while the biochemical stability of both groups of tendons was determined by examining collagen extractability and the formation of pentosidine at 8 weeks. Whereas there were no significant biomechanical differences between control and glycated tendons at 0- and 2-week intervals (P > 0.05), moderately significant increases in maximum load, energy to yield, and toughness of glycated tendons were observed at 4 weeks. Beyond 4 weeks of incubation, the differences between glycated and non-glycated tendons became highly significant, as glycated tendons withstood more load and tensile stress (P < 0.01 for each variable), attained significantly higher modulus of elasticity (P < 0.01), absorbed more energy (P < 0.01), and became tougher (P < 0.01) than controls. These differences in the biomechanical indices of the effects of glycation were stable between the 6th and 10th week of glycation. The maximum increases in the biomechanical measurements as a result of glycation were 29% for maximum load, 125% for stress, 19% for strain, 106% for Young's modulus of elasticity, 14% for energy to yield, and 57% for toughness. Biochemical analysis showed a 61% reduction in the extractability of neutral salt-soluble collagen, a 48% decrease in acid-soluble collagen, and a 29% decline in pepsin-soluble collagen in glycated tendons (P < 0.01). In contrast, there was a 28% increase in the amount of insoluble collagen and significantly higher amounts of pentosidine (P < 0.01) in glycated tendons. Collectively, these biomechanical and biochemical results suggest that nonenzymatic glycation may explain the altered stability of connective tissue matrix induced by the processes of diabetes and aging.