Cross-linking Of Matrix Proteins Research Articles

Linking inflammation and angiogenesis with fibrogenesis: Expression of FXIIIA, MMP-9, and VEGF in oral submucous fibrosis

ObjectivesInterplay of Factor XIIIa (FXIIIa), a transglutaminase, responsible for cross-linking of matrix proteins, Matrix Metalloproteinase-9 (MMP-9), a gelatinase, and Vascular Endothelial Growth Factor (VEGF), an angiogenic inducer, were studied in relation to fibrogenesis and disease progression in oral submucous fibrosis (OSMF). Material and methodsImmunohistochemical expression of markers was studied in 60 formalin-fixed paraffin-embedded tissue blocks of OSMF and 20 normal oral mucosal tissues. FXIIIa was studied quantitatively while MMP-9 and VEGF were assessed semi-quantitatively. Expression was compared with histopathological grades of OSMF. ResultsFXIIIa expression significantly increased in OSMF (p-value 0.000). However, expression decreased and cells became quiescent with increasing grades (p-value 0.000). MMP-9 (p-value epithelium 0.011, p-value connective tissue 0.000) and VEGF expression (p-value epithelium 0.000, connective tissue 0.000) increased in OSMF. A negative correlation between FXIIIa and MMP-9 (−0.653) in early grade (p-value of 0.021) and a positive correlation between FXIIIa and VEGF (0.595) (p-value of 0.032) was found in the moderate grade OSMF. Regression analysis showed a significant association (p<0.01) of FXIIIa in OSMF and with increasing grades of OSMF. ConclusionFXIIIa may play a crucial role in initiation of fibrosis in OSMF. MMP-9 may have a diverse role to play in OSMF as a regulator of fibrosis. VEGF may show an angio-fibrotic switch and contribute to fibrosis in OSMF. These cytokines may show altered function and can contribute to fibrosis and chronicity of disease due to changes in the microenvironment. Tissue stiffness in OSMF itself creates an environment that enhances the chronicity of the disease.

Extracellular vesicles and the extracellular matrix: a new paradigm or old news?

Extracellular vesicles (EV) are implicated in a variety of functions affecting the extracellular matrix (ECM), including matrix degradation, cross-linking of matrix proteins and matrix calcification. These processes are important in many physiological contexts such as angiogenesis and wound healing, and dysregulation of ECM homeostasis contributes to a wide range of diseases including fibrosis, cancer and arthritis. Most studies of EV have focussed on their roles in cell:cell communication, but EV can exist as integral components of the ECM. By far the most well-characterised ECM-resident EV are matrix vesicles (MV) in bone, but the broader role of EV in the ECM is not well understood. This review will explore what is known of the roles of EV in the ECM and will also highlight the similarities and differences between MV and other EV.

Methylsulfonylmethane increases osteogenesis and regulates the mineralization of the matrix by transglutaminase 2 in SHED cells.

Methylsulfonylmethane (MSM) is a naturally occurring, sulfate-containing, organic compound. It has been shown to stimulate the differentiation of mesenchymal stem cells into osteoblast-like cells and bone formation. In this study, we investigated whether MSM influences the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into osteoblast-like cells and their osteogenic potential. Here, we report that MSM induced osteogenic differentiation through the expression of osteogenic markers such as osterix, osteopontin, and RUNX2, at both mRNA and protein levels in SHED cells. An increase in the activity of alkaline phosphatase and mineralization confirmed the osteogenic potential of MSM. These MSM-induced effects were observed in cells grown in basal medium but not osteogenic medium. MSM induced transglutaminase-2 (TG2), which may be responsible for the cross-linking of extracellular matrix proteins (collagen or osteopontin), and the mineralization process. Inhibition of TG2 ensued a significant decrease in the differentiation of SHED cells and cross-linking of matrix proteins. A comparison of mineralization with the use of mineralized and demineralized bone particles in the presence of MSM revealed that mineralization is higher with mineralized bone particles than with demineralized bone particles. In conclusion, these results indicated that MSM could promote differentiation and osteogenic potential of SHED cells. This osteogenic property is more in the presence of mineralized bone particles. TG2 is a likely cue in the regulation of differentiation and mineral deposition of SHED cells in response to MSM.

Elevated transglutaminase-2 expression mediates fibrosis in areca quid chewing-associated oral submucocal fibrosis via reactive oxygen species generation.

Transglutaminase-2 (TGM-2) protein is involved in the cross-linking of matrix proteins resulting in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Little is known about its role in the development of oral submucocal fibrosis (OSF). Hence, we hypothesize that TGM-2 may have a role in the pathogenesis of areca quid chewing-associated OSF and arecoline, a major areca nut alkaloid, could regulate TGM-2 via ROS generation. Forty OSF specimens from areca quid chewing-associated OSF and ten normal buccal mucosa biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The expression of TGM-2 from fibroblasts cultured from OSF and normal buccal mucosa was evaluated by Western blot. The effect of arecoline on normal buccal mucosa fibroblasts (BMFs) was used to elucidate whether TGM-2 expression could be affected by arecoline by using 2', 7'-dichlorofluorescein diacetate assay and Western blot. In addition, glutathione precursor N-acetyl-L-cysteine (NAC) and epigallocatechin-3 gallate (EGCG) were added to find the possible regulatory mechanisms. TGM-2 expression was significantly higher in OSF specimens than normal specimens (p<0.05). Fibroblasts derived from OSF were found to exhibit higher TGM-2 expression than BMFs in protein levels (p<0.05). Arecoline significantly upregulated the intracellular ROS generation in a dose-dependent manner (p<0.05). TGM-2 protein induced by arecoline was found in BMFs in a dose-dependent manner (p<0.05). Treatment with NAC and EGCG markedly inhibited TGM-2 expression induced by arecoline (p<0.05). Our results suggest that TGM-2 expression is significantly upregulated in OSF tissues from areca quid chewers. Arecoline-upregulated TGM-2 expression may be mediated by ROS generation. TGM-2 protein is upregulated in areca quid chewing-associated OSF. Using this in vitro model, antioxidants could inhibit arecoline-upregulated TGM-2 expression. NAC and EGCG may serve as a useful agent in controlling OSF.

Two functional loci in the promoter of EPAS1 gene involved in high-altitude adaptation of Tibetans.

EPAS1 involves in the hypoxic response and is suggested to be responsible for the genetic adaptation of high-altitude hypoxia in Tibetans. However, the detailed molecular mechanism remains unknown. In this study, a single nucleotide polymorphism rs56721780:G>C and an insertion/deletion (indel) polymorphism −742 indel in the promoter region showed divergence between Tibetans and non-Tibetan lowlanders. rs56721780:G>C regulated the transcription of EPAS1 by IKAROS family zinc finger 1 (IKZF1), which was identified as a new transcriptional repressor for EPAS1 gene. It demonstrated that the C allele of rs56721780:G>C decreased the binding of IKZF1, leading to the attenuated transcriptional repression of EPAS1 gene. The insertion at −742 indel provided a new binding site for Sp1 and was related to the activation of EPAS1 promoter. Further functional analysis revealed that lysyl oxidase (LOX) gene, which was reported to be responsible for extracellular matrix protein cross-linking of amnion previously, was a direct target of EPAS1. The CC genotype at rs56721780:G>C and the insertion genotype at −742 indel were found associated with higher EPAS1 and LOX expression levels in amnion, as well as higher birth weight of Tibetan newborns, suggesting that EPAS1 gene might play important roles in the development of amnion, fetus growth and high-altitude adaptation of Tibetans.

Arterial stiffness and hypertension: chicken or egg?

Hypertension is highly prevalent in the United States and worldwide. In the United States, 1 in 3 adults (≈73 million people) have hypertension. The financial and human costs of hypertension are substantial. The estimated costs attributable to hypertension in the United States alone in 2010 were $70 billion direct costs for medical treatments and $24 billion indirect costs because of lost productivity.1 Awareness and treatment of hypertension have improved considerably during the past several decades: >80% of all people with hypertension are aware of the condition and 75% are using antihypertensive medications.2,3 However, despite enormous effort and treatment-related expenditures, only 53% of those with documented hypertension have their blood pressure controlled.3 Older age (Figure 1), left ventricular hypertrophy, and obesity are important risk factors for uncontrolled or drug-resistant hypertension.4 Importantly, the foregoing are also risk factors for increased aortic stiffness.5 Figure 1. Hypertension prevalence and control rates as a function of age in the National Health and Nutrition Examination Survey 2003 to 2004. Data derived from Wong et al.44 During the past 2 decades, measures of aortic stiffness have emerged as important risk factors for progression of blood pressure and incident cardiovascular disease. Carotid femoral pulse wave velocity (CFPWV), a measure of aortic wall stiffness, increases markedly with age, particularly after midlife. For example, Framingham Heart Study investigators have shown that the prevalence of CFPWV ≥12 m/s increases from a few percentage before 50 years of age to ≥60% after 70 years of age (Figure 2A). Importantly, elevated CFPWV is associated with high risk for incident hypertension (Figure 2B) and cardiovascular disease (Figure 2C). Figure 2C should be interpreted within the context that a normal value (<95th percentile) for CFPWV in a healthy Framingham Heart Study reference sample <50 years of age was 8.1 …

Increased tissue transglutaminase activity contributes to central vascular stiffness in eNOS knockout mice

Nitric oxide (NO) can modulate arterial stiffness by regulating both functional and structural changes in the arterial wall. Tissue transglutaminase (TG2) has been shown to contribute to increased central aortic stiffness by catalyzing the cross-linking of matrix proteins. NO S-nitrosylates and constrains TG2 to the cytosolic compartment and thereby holds its cross-linking function latent. In the present study, the role of endothelial NO synthase (eNOS)-derived NO in regulating TG2 function was studied using eNOS knockout mice. Matrix-associated TG2 and TG2 cross-linking function were higher, whereas TG2 S-nitrosylation was lower in the eNOS(-/-) compared with wild-type (WT) mice. Pulse-wave velocity (PWV) and blood pressure measured noninvasively were elevated in the eNOS(-/-) compared with WT mice. Intact aortas and decellularized aortic tissue scaffolds of eNOS(-/-) mice were significantly stiffer, as determined by tensile testing. The carotid arteries of the eNOS(-/-) mice were also stiffer, as determined by pressure-dimension analysis. Invasive methods to determine the PWV-mean arterial pressure relationship showed that PWV in eNOS(-/-) and WT diverge at higher mean arterial pressure. Thus eNOS-derived NO regulates TG2 localization and function and contributes to vascular stiffness.

Copper Deficiency Induced Emphysema Is Associated with Focal Adhesion Kinase Inactivation

BackgroundCopper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK).MethodologyTo examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA).Principal FindingsCopper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.ConclusionsThese data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.

Influence of copper on early development: Prenatal and postnatal considerations

Copper (Cu) is an essential nutrient whose requirement is increased during pregnancy and lactation. These represent times of critical growth and development, and the fetus and neonate are particularly vulnerable to deficiencies of this nutrient. Genetic mutations that predispose the offspring to inadequate stores of Cu can be life threatening as is observed in children with Menkes disease. During the last decade, severe Cu deficiency, once thought to be a rare condition, has been reported in the literature at an increasing frequency. Secondary Cu deficiencies can be induced by a variety of ways such as excessive zinc or iron intake, certain drugs, and bariatric surgery. Premature and low birth weight infants can be born with low Cu stores. A number of mechanisms can contribute to the teratogenicity of Cu including decreased activity of select cuproenzymes, increased oxidative stress, decreased nitric oxide availability, altered iron metabolism, abnormal extracellular matrix protein crosslinking, decreased angiogenesis and altered cell signaling among others. The brain, heart, and vessels as well as tissues such as lung, skin and hair, and systems including the skeletal, immune, and blood systems, are negatively affected by suboptimal Cu during development. Additionally, persistent structural, biochemical, and functional adverse effects in the offspring are noted even when Cu supplementation is initiated after birth, supporting the concept that adequate Cu nutriture during pregnancy and lactation is critical for normal development. Although Cu-containing IUDs are an effective method for increasing intrauterine Cu concentrations and for reducing the risk of pregnancy, high amounts of dietary Cu are not thought to represent a direct developmental risk.

Transglutaminase 2 Deficiency Decreases Plaque Fibrosis and Increases Plaque Inflammation in Apolipoprotein-E-Deficient Mice

Aim: Transglutaminase 2 (TG2) is important for the deposition and stability of the extracellular matrix via effects on cross-linking of matrix proteins and transforming growth factor β (TGFβ) activity. The purpose of this study was to investigate the effect of TG2 deficiency on the composi- tion of atherosclerotic plaques. Methods: Apolipoprotein E (ApoE)^–/– mice were crossbred with TG2^–/– mice to obtain ApoE^–/–TG2^–/– mice. ApoE^–/– and ApoE^–/–TG2^–/– mice were fed a Western-type diet for 16 or 30 weeks to determine the effect of TG2 deficiency on early and advanced atherosclerosis, respectively. Results: Atherosclerotic plaques of ApoE^–/–TG2^–/– mice showed decreased cross-linking of matrix proteins, as well as decreased nuclear staining for phospho-Smad2/-Smad3, indicative of decreased TGFβ activity. Compared to ApoE^–/– mice, plaque area was decreased by 45 and 48% in ApoE^–/–TG2^–/– mice after 16 and 30 weeks, respectively. Sirius red staining showed a significant decrease in collagen content in early and advanced atherosclerotic plaques of ApoE^–/–TG2^–/– mice. Furthermore, there was a significant increase in macrophages in advanced atherosclerotic plaques of ApoE^–/–TG2^–/– mice. Conclusion: TG2 deficiency resulted in a decreased collagen content and increased inflammation, which are features of a more unstable plaque.

Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications

This review summarises the functions of the enzyme tissue transglutaminase (TG2) in the extracellular matrix (ECM) both as a matrix stabiliser through its protein cross-linking activity and as an important cell adhesion protein involved in cell survival. The contribution of extracellular TG2 to the pathology of important diseases such as cancer and fibrosis are discussed with a view to the potential importance of TG2 as a therapeutic target. The medical applications of TG2 are further expanded by detailing the use of transglutaminase cross-linking in the development of novel biocompatible biomaterials for use in soft and hard tissue repair.

Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures

Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and beta-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms--TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin-collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.

Antisense inhibition of transglutaminase 2 affects development of mouse embryo submandibular gland in organ culture

Tissue transglutaminase (TGase 2) has been implicated in numerous cellular functions, i.e., apoptosis, differentiation, extracellular matrix protein cross-linking and organogenesis. Earlier report of the strong transient expression of TGase 2 localized at the anchoring sites of muscle bundles of human embryo and recent findings of a similar transient expression of the TGase 2 in the salivary myoepithelial cells of mouse embryo indicated a definitive role of TGase 2 in the cytodifferentiation of myoepithelial cells. To understand functional role(s) of TGase 2 in the organogenesis of salivary gland, antisense inhibition of TGase 2 expression was performed in the organ culture of mouse embryo submandibular gland. The antisense of TGase 2 transfection tested using oral keratinocyte cell line, KB cells, elicited significant inhibition of cellular transglutaminase expression. The same antisense treatment of submandibular glands in organ culture also resulted in the suppression of cellular TGase 2 expression as indicated by weak immunoreaction against anti-TGase 2 in the myoepithelial cells of submandibular glands in contrast to strong reaction in those of the normal and sense-treated glands. Antisense to TGase 2 treatment induced retarded growth of salivary epithelium in 1 week and severe aberrant growth of salivary ducts and acini in 2 weeks and also expression of apoptotic inducer, Bax specifically localized in the myoepithelial cells, suggesting apoptotic state of myoepithelial cells. These data suggest that the antisense inhibition of TGase 2 expression affects the cytodifferentiation of ductal cells and myoepithelial cells, and resulted in severe retardation of tubuloalveolar structure formation of salivary gland.

Selective expression of lysyl oxidase (LOX) in the stromal reactions of broncho-pulmonary carcinomas.

Lysyl oxidase (LOX) is the extracellular enzyme that initiates the main pathway of collagen and elastin cross-linking. LOX has also been correlated with the ras recision gene, a putative tumour suppressor isolated from revertants of ras-transformed fibroblasts. The present study investigates the potential correlation of LOX-dependent matrix protein cross-linking in the stromal reaction of lung carcinomas, with reference to the architecture of the main stromal reactions accompanying the neoplastic breast tissues. A strong LOX expression was associated with the hypertrophic scar-like stromal reaction found at the front of tumour progression in squamous carcinomas, adenocarcinomas, large cell carcinomas, or at sites of initial extense in bronchiolo-alveolar carcinomas. In contrast, little or no LOX expression was found within the stromal reaction of invasive carcinomas, small cell carcinomas, and neuro-endocrine carcinomas. The significance of LOX expression and of the stromal reaction are discussed, in light of data that associate LOX expression with tumours displaying a rather good prognosis.

Copper, lysyl oxidase, and extracellular matrix protein cross-linking

Protein-lysine 6-oxidase (lysyl oxidase) is a cuproenzyme that is essential for stabilization of extracellular matrixes, specifically the enzymatic cross-linking of collagen and elastin. A hypothesis is proposed that links dietary copper levels to dynamic and proportional changes in lysyl oxidase activity in connective tissue. Although nutritional copper status does not influence the accumulation of lysyl oxidase as protein or lysyl oxidase steady state messenger RNA concentrations, the direct influence of dietary copper on the functional activity of lysyl oxidase is clear. The hypothesis is based on the possibility that copper efflux and lysyl oxidase secretion from cells may share a common pathway. The change in functional activity is most likely the result of posttranslational processing of lysyl oxidase. Copper is essential for organic cofactor formation in amine oxidases such as lysyl oxidase. Copper-containing amine oxidases have peptidyl 2,4,5 tri(oxo)phenylalanine (TOPA) at their active centers. TOPA is formed by copper-catalyzed oxidation of tyrosine, which takes place as part of Golgi or trans-Golgi processing. For lysyl oxidase, recent evidence (Science 1996;273:1078-84) indicates that as an additional step, a lysyl group at the active center of lysyl oxidase reacts with TOPA or its precursor to form lysyl tyrosylquinone.

Effect of cross-linking on matrix permeability. A model for AGE-modified basement membranes.

There is much evidence that basement membranes, such as in the renal glomerulus, act as macromolecular sieves, restricting the passage of proteins. Cross-linking of matrix proteins, as occurs because of advanced glycosylation end products (AGEs) in diabetes, may have an effect on the sieving properties of the basement membrane. To test this hypothesis, Matrigel, a basement membrane-like matrix, was cross-linked with glycolaldehyde and control and cross-linked matrices compared. Control matrices allowed less bovine serum albumin to pass through than did cross-linked matrices, with sieving coefficients (SCs) of 0.38 +/- 0.02 and 0.52 +/- 0.02, respectively (P < 0.0005). The control matrices also allowed less cross-linked albumin through than did the cross-linked matrices: 0.13 +/- 0.01 vs. 0.17 +/- 0.02 (P < 0.002). The SCs of a series of fluorescein isothiocyanate dextrans (four sizes, Mr 16,000- 168,000) were lower for the control matrix than for the cross-linked matrix (P < 0.03). In addition, the SC for glycated albumin (incubated with glucose-6-phosphate) was higher than that of normal albumin for both the control (P < 0.04) and cross-linked matrices (P < 0.001). These data indicate that cross-linking of the matrix increases permeability to macromolecules. Analysis of the data using fiber-matrix theory suggests that the mean fiber radius was increased in the cross-linked matrix. The data also indicate that glycated albumin filters through the matrices more easily than does normal albumin. In relation to the situation seen in vivo, it is possible that glycation of circulating proteins and AGE modification of glomerular basement membrane proteins may both contribute to the proteinuria seen in diabetes.