scholarly journals Effects of Copper and Cross-Linking on the Extracellular Matrix of Tissue-Engineered Arteries

2005 ◽  
Vol 14 (6) ◽  
pp. 367-374 ◽  
Author(s):  
Shannon L. M. Dahl ◽  
Robert B. Rucker ◽  
Laura E. Niklason
Keyword(s):  
Extracellular Matrix ◽  
Collagen Fibril ◽  
Lysyl Oxidase ◽  
Copper Ion ◽  
Ion Concentration ◽  
Cross Linking ◽  
Cross Link ◽  
Ion Concentrations ◽  
Mechanical Strengths ◽  
Link Formation

In many cases, the mechanical strengths of tissue-engineered arteries do not match the mechanical strengths of native arteries. Ultimate arterial strength is primarily dictated by collagen in the extracellular matrix, but collagen in engineered arteries is not as dense, as organized, or as mature as collagen in native arteries. One step in the maturation process of collagen is the formation of hydroxylysyl pyridinoline (HP) cross-links between and within collagen molecules. HP cross-link formation, which is triggered by the copper-activated enzyme lysyl oxidase, greatly increases collagen fibril stability and enhances tissue strength. Increased cross-link formation, in addition to increased collagen production, may yield a stronger engineered tissue. In this article, the effect of increasing culture medium copper ion concentration on engineered arterial tissue composition and mechanics was investigated. Engineered vessels grown in low copper ion concentrations for the first 4 weeks of culture, followed by higher copper ion concentrations for the last 3 weeks of culture, had significantly elevated levels of cross-link formation compared to those grown in low copper ion concentrations. In contrast, vessels grown in high copper ion concentrations throughout culture failed to develop higher collagen cross-link densities than those grown in low copper ion concentrations. Although the additional cross-linking of collagen in engineered vessels may provide collagen fibril stability and resistance to proteolysis, it failed to enhance global tissue strength.

scholarly journals Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta

1986 ◽  
Vol 237 (1) ◽  
pp. 17-23 ◽  
Author(s):  
D Tinker ◽  
J Geller ◽  
N Romero ◽  
C E Cross ◽  
R B Rucker
Keyword(s):  
Messenger Rna ◽  
Copper Deficiency ◽  
Degradation Products ◽  
Lysyl Oxidase ◽  
Proteolytic Processing ◽  
Cross Linking ◽  
Cross Link ◽  
Dietary Copper ◽  
Lysine Residues ◽  
Link Formation

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.

scholarly journals A matricellular protein fibulin-4 is essential for the activation of lysyl oxidase

2020 ◽  
Vol 6 (48) ◽  
pp. eabc1404
Author(s):  
Kazuo Noda ◽  
Kaori Kitagawa ◽  
Takao Miki ◽  
Masahito Horiguchi ◽  
Tomoya O. Akama ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Lysyl Oxidase ◽  
Copper Ion ◽  
Pivotal Role ◽  
Cross Linking ◽  
Matricellular Protein ◽  
Ion Transfer ◽  
Wild Type ◽  
Copper Transporter ◽  
Golgi Network

Fibulin-4 is a matricellular protein required for extracellular matrix (ECM) assembly. Mice deficient in fibulin-4 (Fbln4−/−) have disrupted collagen and elastin fibers and die shortly after birth from aortic and diaphragmatic rupture. The function of fibulin-4 in ECM assembly, however, remains elusive. Here, we show that fibulin-4 is required for the activity of lysyl oxidase (LOX), a copper-containing enzyme that catalyzes the covalent cross-linking of elastin and collagen. LOX produced by Fbln4−/− cells had lower activity than LOX produced by wild-type cells due to the absence of lysine tyrosyl quinone (LTQ), a unique cofactor required for LOX activity. Our studies showed that fibulin-4 is required for copper ion transfer from the copper transporter ATP7A to LOX in the trans-Golgi network (TGN), which is a necessary step for LTQ formation. These results uncover a pivotal role for fibulin-4 in the activation of LOX and, hence, in ECM assembly.

scholarly journals Site-specific cross-linking of proteins to DNA via a new bioorthogonal approach employing oxime ligation

2018 ◽  
Vol 54 (49) ◽  
pp. 6296-6299 ◽  
Author(s):  
Suresh S. Pujari ◽  
Yi Zhang ◽  
Shaofei Ji ◽  
Mark D. Distefano ◽  
Natalia Y. Tretyakova
Keyword(s):  
Cross Linking ◽  
Cross Link ◽  
Site Specific ◽  
Link Formation

Model site-specific DNA–protein cross-link formation by bioorthogonal oxime ligation.

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links

2019 ◽  
Vol 63 (3) ◽  
pp. 349-364 ◽  
Author(s):  
Sylvain D. Vallet ◽  
Sylvie Ricard-Blum
Keyword(s):  
Extracellular Matrix ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Lysyl Oxidase ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Copper Binding ◽  
Molecular Features ◽  
Ecm Proteins ◽  
Cross Links

Abstract The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.

scholarly journals A SEX-LINKED DEFECT IN THE CROSS-LINKING OF COLLAGEN AND ELASTIN ASSOCIATED WITH THE MOTTLED LOCUS IN MICE

1974 ◽  
Vol 139 (1) ◽  
pp. 180-192 ◽  
Author(s):  
David W. Rowe ◽  
Ermona B. McGoodwin ◽  
George R. Martin ◽  
Michael D. Sussman ◽  
Douglas Grahn ◽  
...  
Keyword(s):  
Coat Color ◽  
Cross Linking ◽  
Genetic Abnormality ◽  
Cross Link ◽  
Skin Collagen ◽  
The Cross ◽  
Tissue Abnormalities ◽  
Link Formation ◽  
Experimental Lathyrism

A genetic abnormality in collagen and elastin cross-linking resembling experimental lathyrism has been identified in mice. The defect is an X-linked trait, attributed to the mottled locus which also influences coat color. The affected mice have aneurysms of the aorta and its branches, weak skin, and bone deformities in a spectrum of severity varying with the alleles at the mottled locus. A defect in the cross-linking of collagen was demonstrated in the skin of the affected animals by a marked increase in collagen extractability and a reduced proportion of cross-linked components in the extracted collagen. A decrease in lysine-derived aldehyde levels was found in both skin collagen and aortic elastin similar to that found in lathyritic tissue. Furthermore the in vitro formation of lysine-derived aldehyde was reduced. Thus the cause of the connective tissue abnormalities in these mice appears to be a defect in cross-link formation due to an impairment in aldehyde formation.

scholarly journals The inhibition of lysyl oxidase in vivo by isoniazid and its reversal by pyridoxal. Effect on collagen cross-linking in the chick embryo

1984 ◽  
Vol 221 (3) ◽  
pp. 837-843 ◽  
Author(s):  
M J Carrington ◽  
T A Bird ◽  
C I Levene
Keyword(s):  
Pyridoxal Phosphate ◽  
Isonicotinic Acid ◽  
Lysyl Oxidase ◽  
Acid Hydrazide ◽  
Chick Embryos ◽  
Cross Link ◽  
Irreversible Inhibitor ◽  
Link Formation

Isonicotinic acid hydrazide (isoniazid) causes a large increase in the salt-solubility of collagen when injected into chick embryos; this change is accompanied by the inactivation of lysyl oxidase (EC 1.4.3.13), the enzyme responsible for initiating cross-link formation in collagen and elastin. In addition, isoniazid markedly decreases the liver content of pyridoxal phosphate. The depletion of pyridoxal phosphate takes approx. 6 h, whereas the inhibition of lysyl oxidase and the increase in collagen solubility occur more slowly. A reversal of these effects of isoniazid can be produced by the subsequent injection of a stoichiometric amount of pyridoxal, supporting the role of pyridoxal as a cofactor for lysyl oxidase. Treatment of chick embryos with beta-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, causes an inhibition of the enzyme, which begins to recover within 24 h but which is not affected by the administration of pyridoxal; with isoniazid inhibition, however, lysyl oxidase activity does not show any sign of recovery by 48 h. It is proposed that isoniazid may cause the inhibition of lysyl oxidase by competing for its obligatory cofactor, pyridoxal phosphate. The potential clinical implications in the therapeutic control of fibrosis are briefly discussed.

Mechanistic and Pharmacodynamic Studies of Adct-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1559-1559 ◽  
Author(s):  
Michael J. Flynn ◽  
Patrick H. van Berkel ◽  
Francesca Zammarchi ◽  
Peter C. Tyrer ◽  
Ayse U. Akarca ◽  
...  
Keyword(s):  
Cell Surface ◽  
Clinical Development ◽  
Cross Linking ◽  
Cross Link ◽  
Employment Equity ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Equity Ownership ◽  
Dose Dependent ◽  
Link Formation

Abstract ADCT-301, currently in Phase I clinical trial, is an ADC composed of a recombinant human IgG1, HuMax®-TAC against human IL-2R-α (CD25) conjugated through a cleavable linker to a PBD dimer warhead with a drug-antibody ratio of 2.3. In vitro and ex vivo, ADCT-301 binds human CD25 with picomolar affinity. ADCT-301 has highly potent and targeted cytotoxicity against a panel of human lymphoma cell lines. On release, PBD dimers bind in the DNA minor groove and exert their cytotoxic action via the formation of DNA interstrand cross-links. In vivo, ADCT-301 demonstrates dose-dependent antitumor activity against subcutaneous and disseminated lymphoma models. For example, in the Karpas 299 xenograft model, 10/10 tumor-free survivors are observed following a single dose of 0.5 mg/kg, whereas Adcetris® gives only a modest delay in mean tumor growth at 0.5 mg/kg, despite this tumor expressing three-fold higher target antigen levels for this drug. The current study aimed to further define the mechanism of action of ADCT-301 and validate pharmacodynamic assays for clinical development. In Karpas 299 cells, evidence for internalization of ADCT-301 was shown by a reduction of CD25 molecules on the cell surface over the first three hours post-treatment followed by a return to pre-treatment levels by 16 hours. This is consistent with the documented rapid recycling of CD25 to the membrane after exposure to IL-2 (Hemar et al Journal of Cell Biology 1995). Furthermore, ADCT-301 on the cell surface declined by >70% over four hours. Following a two-hour exposure to ADCT-301, DNA interstrand cross-linking, measured using a modification of the single cell gel electrophoresis (comet) assay, reached a peak between 4 and 8 hours after which cross-links persisted up to 36 hours. In contrast, the peak of cross-link formation for an equimolar concentration of warhead was immediately following drug exposure and a non-targeted PBD-containing ADC did not produce crosslinks in these cells. A strong correlation (r = 0.97) between loss of viability and DNA cross-link formation provides support for this DNA damage being the critical initiating mechanism of cytotoxicity of ADCT-301. We have previously shown that PBD-induced DNA interstrand cross-links elicit a robust, but delayed γ-H2AX response (Wu et al Clinical Cancer Research 2013). In Karpas 299 cells phosphorylation of H2AX was observed 24 hours after a two-hour exposure to sub-GI50 concentrations of ADCT-301. In these cells continuous exposure to ADCT-301 resulted in a dose-dependent G2/M arrest, peaking at 48 hours, later than for the naked warhead. The peak of the early apoptosis marker annexin-V on the cell surface of Karpas 299 cells was observed between 60 and 72 hours and maximal loss of viability was at 96 hours. Significant bystander killing of CD25-negative human Burkitt's lymphoma-derived Ramos cells was demonstrated for ADCT-301 both by co-culture experiments with CD25-positive Karpas 299 cells, and by media transfer from Karpas 299 cells treated with ADCT-301. This is important as many lymphomas are heterogeneous in their CD25 expression profile (Strauchen et al American Journal of Pathology 1987). In SCID mice with Karpas 299 subcutaneous tumors a single dose of ADCT-301 was administered at 0.2 or 0.6 mg/kg. 24 hours after treatment, excised tumors showed a dose proportional increase in intensity of membrane and cytoplasmic staining by an anti-PBD payload antibody. Cross-linking was determined as 23% (0.2 mg/kg) vs 49% (0.6 mg/kg) (p ≤ 0.01) reduction in Tail Moment using the comet assay and dose-dependent γ-H2AX formation measured by immunohistochemistry was observed. No cross-linking was observed in matched lymphocyte samples. These data confirm the mechanism of cell killing of ADCT-301 and provide relevant pharmacodynamic assays for use in the clinical development of PBD-based ADCs. Disclosures Flynn: Spirogen/Medimmune: Employment. van Berkel:ADC Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zammarchi:ADC Therapeutics: Employment. Tyrer:Spirogen/Medimmune: Employment. Williams:Spirogen/Medimmune: Employment. Howard:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Patents & Royalties. Hartley:ADCT Spirogen/Medimmune: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Characterization of Genipin-Modified Dentin Collagen

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Hiroko Nagaoka ◽  
Hideaki Nagaoka ◽  
Ricardo Walter ◽  
Lee W. Boushell ◽  
Patricia A. Miguez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lysyl Oxidase ◽  
Biomechanical Properties ◽  
Biochemical Properties ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cross Link ◽  
Concentration Dependent Manner ◽  
Cross Links ◽  
Dentin Collagen

Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE), on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5%) for several treatment times (0–24 h). Changes in biochemical properties of NaB3H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P< 0.05). The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB3H4.

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