Collagen Cross-link Formation Research Articles

Inhibiting Lysyl Oxidases prevents pathologic cartilage calcification

Lysyl oxidases (LOX(L)) are enzymes that catalyze the formation of cross-links in collagen and elastin fibers during physiologic calcification of bone. However, it remains unknown whether they may promote pathologic calcification of articular cartilage, an important hallmark of debilitating arthropathies. Here, we have studied the possible roles of LOX(L) in cartilage calcification, related and not related to their cross-linking activity. We first demonstrated that inhibition of LOX(L) by β-aminoproprionitrile (BAPN) significantly reduced calcification in murine and human chondrocytes, and in joint of meniscectomized mice. These BAPN’s effects on calcification were accounted for by different LOX(L) roles. Firstly, reduced LOX(L)-mediated extracellular matrix cross-links downregulated Anx5, Pit1 and Pit2 calcification genes. Secondly, BAPN reduced collagen fibrotic markers Col1 and Col3. Additionally, LOX(L) inhibition blocked chondrocytes hypertrophic differentiation (Runx2 and COL10), pro-inflammatory IL-6 release and reactive oxygen species (ROS) production, all triggers of chondrocyte calcification. Through unbiased transcriptomic analysis we confirmed a positive correlation between LOX(L) genes and genes for calcification, hypertrophy and extracellular matrix catabolism. This association was conserved throughout species (mouse, human) and tissues that can undergo pathologic calcification (kidney, arteries, skin). Overall, LOX(L) play a critical role in the process of chondrocyte calcification and may be therapeutic targets to treat cartilage calcification in arthropathies.

The effects of collagen cross-link deficiency on osseointegration process of pure titanium implants.

This study aimed to investigate the effect of collagen cross-link deficiency on collagen fiber formation around an implant and its effect on the osseointegration process. Wistar rats were fed 0.1% beta-aminopropionitrile (BAPN) dissolved in water to induce collagen cross-link deficiency. Custom-made mini-implants with machined surfaces were placed proximal to the tibia. At 1, 2, and 4 weeks postoperatively, the bone area around the implant, bone-implant contact ratio, osteoclast/osteocyte activity, and osseointegration strength were evaluated using histological and immunohistochemical analyses and biomechanical tests. Long-term disturbance of collagen cross-link formation in the BAPN group resulted in faster collagen fiber maturation than that in controls, with a defective collagen structure, low bone formation quantity, and low bone-implant contact values. Deficiency of collagen cross-links resulted in increased bone resorption and decreased osteocyte activity. Collagen cross-linking is important for the formation of the collagen matrix, and their deficiency may impair bone activity around implants, affecting the osseointegration process.

Collagen cross-link profiles and mineral are different between the mandible and femur with site specific response to perturbed collagen

Compromises to collagen and mineral lead to a decrease in whole bone quantity and quality in a variety of systemic diseases, yet, clinically, disease manifestations differ between craniofacial and long bones. Collagen alterations can occur through post-translational modification via lysyl oxidase (LOX), which catalyzes enzymatic collagen cross-link formation, as well as through non-enzymatic advanced glycation end products (AGEs) such as pentosidine and carboxymethyl-lysine (CML). Characterization of the cross-links and AGEs, and comparison of the mineral and collagen modifications in craniofacial and long bones represent a critical gap in knowledge. However, alterations to either the mineral or collagen in bone may contribute to disease progression and, subsequently, the anatomical site dependence of a variety of diseases. Therefore, we hypothesized that collagen cross-links and AGEs differ between craniofacial and long bones and that altered collagen cross-linking reduces mineral quality in an anatomic location dependent. To study the effects of cross-link inhibition on mineralization between anatomical sites, beta-aminoproprionitrile (BAPN) was administered to rapidly growing, 5–8 week-old male mice. BAPN is a dose-dependent inhibitor of LOX that pharmacologically alters enzymatic cross-link formation. Long bones (femora) and craniofacial bones (mandibles) were compared for mineral quantity and quality, collagen cross-link and AGE profiles, and tissue level mechanics, as well as the response to altered cross-links via BAPN. A highly sensitive liquid chromatography/mass spectrometry (LC-MS) method was developed which allowed for quantification of site-dependent accumulation of the advanced glycation end-product, carboxymethyl-lysine (CML). CML was ∼8.3× higher in the mandible than the femur. The mandible had significantly higher collagen maturation, mineral crystallinity, and Young's modulus, but lower carbonation, than the femur. BAPN also had anatomic specific effects, leading to significant decreases in mature cross-links in the mandible, and an increase in mineral carbonation in the femur. This differential response of both the mineral and collagen composition to BAPN between the mandible and femur highlights the need to further understand how inherent compositional differences in collagen and mineral contribute to anatomic-site specific manifestations of disease in both craniofacial and long bones.

Novel Assessment of Collagen and Its Crosslink Content in the Humerus from Primiparous Dairy Cows with Spontaneous Humeral Fractures Due to Osteoporosis from New Zealand.

Simple SummaryCollagen and collagen crosslinking are important determinants of bone strength. For the first time, collagen and its crosslink content were measured in the humerus from primiparous dairy cows with spontaneous humeral fracture due to osteoporosis from New Zealand. The results were compared with the humerus from unfractured dairy cows. Cows with a humeral fracture have a lower total collagen content but higher total collagen crosslink content compared with unfractured cows. The results suggest that protein/calorie undernutrition might be an important factor associated with the incidence of spontaneous humeral fractures in dairy cows from New Zealand.Numerous cases of spontaneous humeral fracture in primiparous dairy cows from New Zealand have prompted the study of the condition to establish probable causes or risk factors associated with the condition. Previous studies identified inadequate protein-calorie malnutrition as an important contributory factor. Earlier case studies also reported that ~50% of cows have low liver and/or serum copper concentration at the time of humeral fracture. Because copper is so closely associated with the formation of collagen cross-links, the aim of this study was to compare collagen and collagen crosslink content in the humerus from primiparous cows with and without humeral fractures and to determine the role of copper in the occurrence of these fractures. Humeri were collected from cows with and without humeral fractures, ground, and the collagen and collagen cross-link content measured using high-performance liquid chromatography. Collagen content was significantly higher in the humeri of cows without humeral fractures, while total collagen crosslink content was significantly higher in the humerus of cows with humeral fractures. These results indicate other factor/s (e.g., protein-calorie undernutrition) might be more important than the copper status in the occurrence of humeral fractures in dairy cows in New Zealand.

Halogenation Activity of Mammalian Heme Peroxidases.

Mammalian heme peroxidases are fascinating due to their unique peculiarity of oxidizing (pseudo)halides under physiologically relevant conditions. These proteins are able either to incorporate oxidized halides into substrates adjacent to the active site or to generate different oxidized (pseudo)halogenated species, which can take part in multiple (pseudo)halogenation and oxidation reactions with cell and tissue constituents. The present article reviews basic biochemical and redox mechanisms of (pseudo)halogenation activity as well as the physiological role of heme peroxidases. Thyroid peroxidase and peroxidasin are key enzymes for thyroid hormone synthesis and the formation of functional cross-links in collagen IV during basement membrane formation. Special attention is directed to the properties, enzymatic mechanisms, and resulting (pseudo)halogenated products of the immunologically relevant proteins such as myeloperoxidase, eosinophil peroxidase, and lactoperoxidase. The potential role of the (pseudo)halogenated products (hypochlorous acid, hypobromous acid, hypothiocyanite, and cyanate) of these three heme peroxidases is further discussed.

Increased Fibrosis in a Mouse Model of Anti-Laminin 332 Mucous Membrane Pemphigoid Remains Unaltered by Inhibition of Aldehyde Dehydrogenase.

Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by autoantibodies against the basal membrane zone of skin and surface-close epithelia and predominant mucosal lesions. The oral cavity and conjunctivae are most frequently affected, albeit clinical manifestations can also occur on the skin. MMP-associated lesions outside the oral cavity typically lead to scarring. Mechanisms underlying scarring are largely unknown in MMP and effective treatment options are limited. Herein, we assessed the collagen architecture in tissue samples of an antibody-transfer mouse model of anti-laminin-332 MMP. In MMP mice, increased collagen fibril density was observed in skin and conjunctival lesions compared to mice injected with normal rabbit IgG. The extracellular matrix of MMP skin samples also showed altered post-translational collagen cross-linking with increased levels of both lysine- and hydroxylysine-derived collagen crosslinks supporting the fibrotic phenotype in experimental MMP compared to control animals. In addition, we evaluated a potential anti-fibrotic therapy in experimental anti-laminin-332 MMP using disulfiram, an inhibitor of the aldehyde dehydrogenase (ALDH), which has been implicated in immune-mediated mucosal scarring. In addition, disulfiram also acts as a copper chelator that was shown to block lysyl oxidase activity, an enzyme involved in formation of collagen crosslinks. Topical use of disulfiram (300 μM in 2% [w/v] methocel) did not improve ocular lesions in experimental MMP over the 12-day treatment period in disulfiram-treated mice compared to vehicle-treated mice (n=8/group). Furthermore, C57BL6/J mice (n=8/group) were treated prophylactically with 200 mg/kg p.o. disulfiram or the solvent once daily over a period of 12 days. Systemic treatment did not show any reduction in the severity of oral and ocular lesions in MMP mice, albeit some improvement in skin lesions was observed in disulfiram- vs. vehicle-treated mice (p=0.052). No reduction in fibrosis was seen, as assessed by immunohistochemistry. Whilst blocking of ALDH failed to significantly ameliorate disease activity, our data provide new insight into fibrotic processes highlighting changes in the collagenous matrix and cross-linking patterns in IgG-mediated MMP.

Vitamin B12 status is a risk factor for bone fractures among vegans

Several studies consistently showed vegans having a higher risk of bone fractures compared to individuals from other diet groups. While researchers have focused on several factors as possible explanation of these findings, both dietary (e.g. calcium) and non-dietary (e.g. weight/BMI status), the widespread inadequate vitamin B12 (B12) status among vegans as a risk factor for bone fractures has not received adequate scrutiny. The detrimental impact of inadequate B12 status on bone tissue is both direct, via the impairment of the insulin-like growth hormone 1 and taurine synthesis, and indirect, induced through its hyperhomocysteinemic effect, via at least the following mechanisms: 1) reducing bone mineral content and density by accumulating in the extracellular matrix, 2) reducing osteoblasts and increasing osteoclasts function, 3) reducing blood flow to bone tissue, 4) inducing apoptosis via the reactive oxygen-species-mediated mitochondrial pathway, and 5) obstructing the formation of collagen cross-links, impeding lyslyl oxidase, and hampering insolubility of fibrils. Considering the widespread B12 deficiency prevalence among vegans, its role in bone fracture risk should not be overlooked.

Intraradicular Dentin Biomodification with Natural Agents for Bonding Glass-fiber Posts.

To evaluate the effects of pretreatment with different crosslinking agents on glass-fiber-post adhesive luting. Single-rooted human teeth (n = 20) were randomly assigned to four groups: proanthocyanidins (PA) from grape-seed extract, cardol and cardanol (separated from cashew nut-shell liquid) and negative control (hydroethanolic solution). The solutions were applied on 37% phosphoric acid-etched dentin for 60 s. Glass-fiber posts were cemented using a three-step etch-and-rinse adhesive (Scotchbond Multi-Purpose, 3M Oral Care) and composite cement (RelyX ARC, 3M Oral Care). Slices for the push-out bond strength test were cut and tested after 24-h or 6-month storage in distilled water. The dentin underlying the adhesive layer was analyzed by micro-Raman spectroscopy to evaluate vibrational formation of collagen crosslinks. Three additional slices per group were also made and the adhesive in-situ degree of conversion (DC) was analyzed by micro-Raman spectroscopy. The results were analyzed using two-way ANOVA and Tukey's test (p < 0.05). No statistically significant changes in bond strength were found over time for any of the groups, except with cardol, which increased bond strength (8.4 ± 3.9 MPa at 24 h to 15.0 ± 2.9 MPa after 6 months, p < 0.001) after aging. The formation of peaks at 1117 cm-1 and 1235 cm-1 showed the presence of collagen crosslinks for all three biomodification agents. The DC outcomes showed no statistically significant differences between groups (p = 0.514). Biomodification agents did not impair adhesive polymerization. Cardol demonstrated a positive influence on intraradicular dentin bonding for glass-fiber post luting.

Abstract 303: Decreasing Microenvironment Stiffness Improves Exogenous Extracellular Matrix Induced Heart Regeneration in Neonatal Mouse

Introduction: Transplanting cardiac extracellular matrix (ECM) has been demonstrated to influence healing in post-ischemic hearts. We propose that altering mechanical properties can stimulate a regenerative response in ECM-treated hearts. In this study, we investigate the role of mechanical unloading and solubilized ECM to modulate matrix-induced heart regeneration in low-regenerative P5 neonatal mice after acute myocardial infarction and mouse ventricle explants. Methods: P5 neonatal mouse heart stiffness was lowered by inhibiting formation of new collagen crosslinks. Solubilized fetal ECM was injected immediately after myocardial infarction (MI). Heart function and histology were conducted at week 3 post-MI. Cardiac ventricle explants were also used to investigate relevant signaling pathways. Results: We observed that lowering tissue stiffness increased the regenerative influence of fetal ECM treatment on heart function, fibrosis, and cardiomyocyte proliferation. Decrease heart stiffness inhibits fibrosis and better preserves heart function in fetal ECM treated hearts (Figure 1). We further provide evidence that yes-associated protein (Yap) signaling pathway plays a role in ECM-induced cardiomyocyte proliferation possibly through cytoskeleton polymerization.The results suggest that the native microenvironment stiffness, particularly with aging or post-ischemia, affects the therapeutic efficacy of drugs for heart disease. Figure 1 . Fetal ECM treatment P5 mouse hearts showed a higher ejection fraction in comparison with the control hearts at 3-weeks post-MI. Decreasing heart stiffness in P5 mouse heart further promoted increased ejection fraction in fetal ECM treated animals. (n=5, two-way ANOVA test and Tukey’s test, *p&lt;0.05, ****p&lt;0.0001.)

Enhancement of tendon-bone interface healing and graft maturation with cylindrical titanium-web (TW) in a miniature swine anterior cruciate ligament reconstruction model: histological and collagen-based analysis

BackgroundTendon-bone interface healing and ligamentization of the graft in anterior cruciate ligament (ACL) reconstruction with autografts are important factors affecting treatment outcome. This study aimed to investigate the effectiveness of a cylindrical titanium-web (TW) in tendon-bone interface healing and graft maturation in ACL reconstruction.MethodsFourteen mature female CLAWN miniature swine underwent bilateral ACL reconstructions with patellar tendon (PT) autografts. In one limb, the TW/tendon complex was placed into the proximal side of the tibial tunnel. Only the graft was transplanted into the tunnel in the control limb. The proximal side of the graft was sutured into the stump of the native ACL and the distal end was stapled to the tibia. The animals were euthanized at 4 and 15 weeks postoperatively, for histological and biochemical analyses.ResultsMicroscopic images in TW limbs showed that ingrowth of tendon-like tissue and mineralized bone tissue into the TW connected the bone and the tendon directly. In contrast, fibrous tissue intervened between the bone and tendon in the control limbs. The total amount of collagen cross-links (which defines the strength of collagen fibers) and the maturation of collagen cross-links in TW tendons were significantly higher (p < 0.05) than those of control limbs. There was no significant difference in the ratio of dihydroxy-lysinonorleucine to hydroxy-lysinonorleucine (an indicator of tissue specific collagen maturation) between TW tendons and that of the native PT.ConclusionsTW promoted the maturation and formation of collagen cross-links in the grafted tendon while maintaining the cross-links pattern of native tendon collagen, and enabled direct binding of tendon to bone.

Tocotrienol-rich fraction from annatto ameliorates expression of lysyl oxidase in human osteoblastic MG-63 cells.

Lysyl oxidase (LOX) is required for the formation of bone collagen cross-links. Inactivation of the LOX gene in osteoblasts by DNA methylation and JAK signaling has been reported to cause loss of cross-links and an increased risk of fractures. Tocotrienols (T3s) have proven benefits on bone strength, but their potential effects on LOX remain largely unknown. Thus, the present study investigates the in vitro effects of T3s on LOX expression in human osteoblastic MG-63 cells. Results indicated that Tocotrienol-Rich Fraction (TRF), the δ-T3 rich oil extracted from Annatto was the most effective and significantly increased LOX expression. TRF treatment decreased de-novo methyltransferases (DNMTs), DNMT3A and DNMT3B levels. In addition, TRF significantly inhibited JAK2 activation and decreased expression of Fli1, a transcription factor of DNMTs. We conclude that TRF induced an increase in LOX expression via inhibition of de-novo methylation and reduction of Fli1 expression by the inactivation of JAK2.Abbreviations: CpG: cytosine-guanine dinucleotide; DNMT: DNA methyltransferase; Fli1: friend leukemia virus integration 1; JAK: janus kinase; LOX: lysyl oxidase; PCR: polymerase chain reaction; STAT: signal transducers and activators of transcription; T3s: tocotrienols; TPs: tocopherols; TRF: Tocotrienol-Rich Fraction.

Identification and Optimization of Mechanism-Based Fluoroallylamine Inhibitors of Lysyl Oxidase-like 2/3.

Lysyl oxidase-like 2 (LOXL2) is a secreted enzyme that catalyzes the formation of cross-links in extracellular matrix proteins, namely, collagen and elastin, and is indicated in fibrotic diseases. Herein, we report the identification and subsequent optimization of a series of indole-based fluoroallylamine inhibitors of LOXL2. The result of this medicinal chemistry campaign is PXS-5120A (12k), a potent, irreversible inhibitor that is >300-fold selective for LOXL2 over LOX. PXS-5120A also shows potent inhibition of LOXL3, an emerging therapeutic target for lung fibrosis. Key to the development of this compound was the utilization of a compound oxidation assay. PXS-5120A was optimized to show negligible substrate activity in vitro for related amine oxidase family members, leading to metabolic stability. PXS-5120A, in a pro-drug form (PXS-5129A, 12o), displayed anti-fibrotic activity in models of liver and lung fibrosis, thus confirming LOXL2 as an important target in diseases where collagen cross-linking is implicated.

Differential cleavage of lysyl oxidase by the metalloproteinases BMP1 and ADAMTS2/14 regulates collagen binding through a tyrosine sulfate domain

Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N- and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloid-like families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen cross-links, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solid-phase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1- and ADAMTS-processing sites contains several conserved tyrosine residues, of which some are post-translationally modified by tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1- and ADAMTS2/14-mediated cleavage of a tyrosine-sulfated domain.

Development of a High-Throughput Lysyl Hydroxylase (LH) Assay and Identification of Small-Molecule Inhibitors against LH2

Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.

Lysyl hydroxylase 3 increases collagen deposition and promotes pulmonary fibrosis by activating TGFβ1/Smad3 and Wnt/β-catenin pathways

IntroductionLysyl hydroxylase 3 (LH3) is a collagen post-translational modifying enzyme; it is abnormally activated during the formation of collagen cross-links. iCRT3 is an inhibitor of both Wnt and β-catenin responsive transcription. We hypothesized that LH3 is regulated by TGFβ1/Smad3 signaling and Wnt/β-catenin signaling pathways. Some evidence suggested that there is complicated cross-talk between the two signal pathways in the genesis of pulmonary fibrosis.Material and methodsThe normal culturing human lung cancer cell line A549 was derived from pulmonary epithelial cells. Transforming growth factor-β1 (TGF-β1) was induced A549 cells of pulmonary fibrosis. MTT assays detected cell growth stimulation by TGF-β1; collagen pyridine-crosslinking contents were detected by ELISA kits. Immunofluorescence were used to evaluate expression of key molecules in PLOD3 (LH3), Wnt/β-catenin and TGFβ1/Smad3 pathways.ResultsOur findings suggested that iCRT3 could decrease LH3 protein expression (p < 0.01), Wnt1, β-catenin and p-Smad3 protein expression (p < 0.05). Knock-down PLOD3 could decrease LH3, collagen I gene and protein expression (p < 0.05). These effects were associated with decreasing collagen pyridine-crosslinking production (p < 0.05). However, ovexpression PLOD3 could increase LH3, collagen I gene and protein expression (p < 0.05). The result showed that LH3 plays an important role in collagen post-translational modifications, and it is regulated by Wnt/β-catenin and TGFβ1/Smad3 pathways.ConclusionsThis study suggests that PLOD3 (LH3) represents a target to prevent pulmonary fibrosis.

Molecular processes of corneal collagen cross-linking in keratoconus therapy

Abstract Corneal collagen cross-linking (CXL) with riboflavin and UVA light is a therapeutic procedure to restore the mechanical stability of corneal tissue. The treatment method applies to pathological tissue changes, such as keratoconus. It induces the photochemical formation of new collagen cross-links. Although therapeutic effects are indisputable, the exact molecular process of CXL and how cross-links are formed is still unclear. In this work, Fouriertransform infrared (FT-IR) spectroscopy is used to investigate the cross-linking process. For that purpose, in-situ experiments with porcine corneas are carried out using attenuated total reflection (ATR) spectroscopy. Furthermore, IR micro-spectroscopic imaging in transmission mode is used to investigate thin tissue sections of the cornea and initial approaches for the distinction of cross-linked and untreated tissue by IR microspectroscopic imaging were performed. Multivariate methods are applied to access changes that occur as a result of CXL. It is shown that spectral changes after cross-linking are caused predominantly by an increase of methyl- and methylene groups as well as primary and secondary amines. In addition, a decrease of carbonyl groups could be observed.

Exogenous Lysyl Oxidase-Like 2 and Perfusion Culture Induce Collagen Crosslink Formation in Osteogenic Grafts.

Lysyl oxidase (LOX)-mediated collagen crosslinking can regulate osteoblastic phenotype and enhance mechanical properties of tissues, both areas of interest in bone tissue engineering. The objective of this study is to investigate the effect of lysyl oxidase-like 2 (LOXL2) on osteogenic differentiation of mesenchymal stem cells (MSCs) cultured in perfusion bioreactors, enzymatic collagen crosslink formation in the extracellular matrix (ECM), and mechanical properties of engineered bone grafts. Exogenous LOXL2 to MSCs seeded in composite scaffolds under perfusion culture for up to 28 days is administered. Constructs treated with LOXL2 appear brown in color and possess greater DNA content and osteogenic potential measured by a twofold increase in bone sialoprotein gene expression. Collagen expression of LOXL2-treated scaffolds is lower than untreated controls. Functional outputs such as calcium deposition, osteocalcin expression, and compressive modulus are unaffected by LOXL2 supplementation. Excitingly, LOXL2-treated constructs contain 1.8- and 1.4-times more pyridinoline (PYD) crosslinks per mole of collagen and per wet weight, respectively, than untreated constructs. Despite these increases, compressive moduli of LOXL2-treated constructs are similar to untreated constructs over the 28-day culture duration. This is the first report of LOXL2 application to engineered, three-dimensional bony constructs. The results suggest a potentially new strategy for engineering osteogenic grafts with a mature ECM by modulating crosslink formation.

Hydroxyhomocitrulline Is a Collagen-Specific Carbamylation Mark that Affects Cross-link Formation

Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during agingand is a risk factor for various diseases. The mostcommon product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group hasreacted with urea-derived cyanate. HCit has recently beendetected in collagen; however, given that 15%-90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and invivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys933, a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation.

Pre-steady-state Kinetics Reveal the Substrate Specificity and Mechanism of Halide Oxidation of Truncated Human Peroxidasin 1

Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV.

Assessment of collagen quality associated with non-enzymatic cross-links in human bone using Fourier-transform infrared imaging

Aging and many disease conditions, most notably diabetes, are associated with the accumulation of non-enzymatic cross-links in the bone matrix. The non-enzymatic cross-links, also known as advanced glycation end products (AGEs), occur at the collagen tissue level, where they are associated with reduced plasticity and increased fracture risk. In this study, Fourier-transform infrared (FTIR) imaging was used to detect spectroscopic changes associated with the formation of non-enzymatic cross-links in human bone collagen. Here, the non-enzymatic cross-link profile was investigated in one cohort with an in vitro ribose treatment as well as another cohort with an in vivo bisphosphonate treatment. With FTIR imaging, the two-dimensional (2D) spatial distribution of collagen quality associated with non-enzymatic cross-links was measured through the area ratio of the 1678/1692cm−1 subbands within the amide I peak, termed the non-enzymatic crosslink-ratio (NE-xLR). The NE-xLR increased by 35% in the ribation treatment group in comparison to controls (p<0.005), with interstitial bone tissue being more susceptible to the formation of non-enzymatic cross-links. Ultra high-performance liquid chromatography, fluorescence microscopy, and fluorometric assay confirm a correlation between the non-enzymatic cross-link content and the NE-xLR ratio in the control and ribated groups. High resolution FTIR imaging of the 2D bone microstructure revealed enhanced accumulation of non-enzymatic cross-links in bone regions with higher tissue age (i.e., interstitial bone). This non-enzymatic cross-link ratio (NE-xLR) enables researchers to study not only the overall content of AGEs in the bone but also its spatial distribution, which varies with skeletal aging and diabetes mellitus and provides an additional measure of bone's propensity to fracture.