Extracellular Matrix Stability Research Articles

The Compact and Biologically Relevant Structure of Inter-α-inhibitor Is Maintained by the Chondroitin Sulfate Chain and Divalent Cations

Inter-α-inhibitor is a proteoglycan of unique structure. The protein consists of three subunits, heavy chain 1, heavy chain 2, and bikunin covalently joined by a chondroitin sulfate chain originating at Ser-10 of bikunin. Inter-α-inhibitor interacts with an inflammation-associated protein, tumor necrosis factor-inducible gene 6 protein, in the extracellular matrix. This interaction leads to transfer of the heavy chains from the chondroitin sulfate of inter-α-inhibitor to hyaluronan and consequently to matrix stabilization. Divalent cations and heavy chain 2 are essential co-factors in this transfer reaction. In the present study, we have investigated how divalent cations in concert with the chondroitin sulfate chain influence the structure and stability of inter-α-inhibitor. The results showed that Mg(2+) or Mn(2+), but not Ca(2+), induced a conformational change in inter-α-inhibitor as evidenced by a decrease in the Stokes radius and a bikunin chondroitin sulfate-dependent increase of the thermodynamic stability. This structure was shown to be essential for the ability of inter-α-inhibitor to participate in extracellular matrix stabilization. In addition, the data revealed that bikunin was positioned adjacent to both heavy chains and that the two heavy chains also were in close proximity. The chondroitin sulfate chain interacted with all protein components and inter-α-inhibitor dissociated when it was degraded. Conventional purification protocols result in the removal of the Mg(2+) found in plasma and because divalent cations influence the conformation and affect function it is important to consider this when characterizing the biological activity of inter-α-inhibitor.

Biochemical Characterization of Medaka (Oryzias latipes) Transglutaminases, OlTGK1 and OlTGK2, as Orthologues of Human Keratinocyte-Type Transglutaminase.

Calcium-dependent transglutaminases (TGs) are a family of enzymes that catalyze protein cross-linking and/or attachment of primary amines in a variety of organisms. Mammalian TGs are implicated in multiple biological events such as skin formation, blood coagulation, and extracellular matrix stabilization. Medaka (Oryzias latipes) has been used as a model fish to investigate the physiological functions of mammalian proteins. By analysis of the medaka genome, we found seven TGs orthologues, some of which apparently corresponded to the mammalian TG isozymes, TG1, TG2, and Factor XIII. All orthologues had preserved amino acid residues essential for enzymatic activity in their deduced primary structures. In this study, we analyzed biochemical properties of two orthologues (OlTGK1 and OlTGK2) of mammalian epithelium-specific TG (TG1) that are significantly expressed at the transcriptional level. Using purified recombinant proteins for OlTGK1 and OlTGK2, we characterized their catalytic reactions. Furthermore, immunohistochemical analyses of fish sections revealed higher expression in the pancreas (OTGK1), intervertebral disk (OlTGK2) and pharyngeal teeth (OlTGK2) as well as in the skin epidermis.

Inhibition of versican expression by siRNA facilitates tropoelastin synthesis and elastic fiber formation by human SK-LMS-1 leiomyosarcoma smooth muscle cells in vitro and in vivo

Versican is an extracellular matrix (ECM) molecule that interacts with other ECM components to influence ECM organization, stability, composition, and cell behavior. Versican is known to increase in a number of cancers, but little is known about how versican influences the amount and organization of the ECM components in the tumor microenvironment. In the present study, we modulated versican expression using siRNAs in the human leiomyosarcoma (LMS) smooth muscle cell line SK-LMS-1, and observed the formation of elastin and elastic fibers in vitro and also in vivo in a nude mouse tumor model. Constitutive siRNA-directed knockdown of versican in LMS cells resulted in increased levels of elastin, as shown by immunohistochemical staining of the cells in vitro, and by mRNA and protein analyses. Moreover, versican siRNA LMS cells, when injected into nude mice, generated smaller tumors that had significantly greater immunohistochemical and histochemical staining for elastin when compared to control tumors. Additionally, microarray analyses were used to determine the influence of versican isoform modulation on gene expression profiles, and to identify genes that influence and relate to the process of elastogenesis. cDNA microarray analysis and TaqMan low density array validation identified previously unreported genes associated with downregulation of versican and increased elastogenesis. These results highlight an important role for the proteoglycan versican in regulating the expression and assembly of elastin and the phenotype of LMS cells.

A role for repressive complexes and H3K9 di-methylation in PRDM5-associated brittle cornea syndrome.

Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 (PRDM5) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 (PRDM5 Δ exons 9-14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and Δ exons 9-14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures (COL13A1, COL15A1, NTN1, CDH5) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9-14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and Δ exons 9-14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease.

Annotated Gene and Proteome Data Support Recognition of Interconnections Between the Results of Different Experiments in Space Research

In a series of studies, human thyroid and endothelial cells exposed to real or simulated microgravity were analyzed in terms of changes in gene expression patterns or protein content. Due to the limitation of available cells in many space research experiments, comparative and control experiments had to be done in a serial manner. Therefore, detected genes or proteins were annotated with gene names and SwissProt numbers, in order to allow searches for interconnections between results obtained in different experiments by different methods. A crosscheck of several studies on the behavior of cytoskeletal genes and proteins suggested that clusters of cytoskeletal components change differently under the influence of microgravity and/or vibration in different cell types. The result that LOX and ISG15 gene expression were clearly altered during the Shenzhou-8 spaceflight mission could be estimated by comparison with the results of other experiments. The more than 100-fold down-regulation of LOX supports our hypothesis that the amount and stability of extracellular matrix have a great influence on the formation of three-dimensional aggregates under microgravity. The approximately 40-fold up-regulation of ISG15 cannot yet be explained in detail, but strongly suggests that ISGylation, an alternative form of posttranslational modification, plays a role in longterm cultures.

Low expression of ITIH5 in adenocarcinoma of the lung is associated with unfavorable patients' outcome

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression and DNA methylation, as well as its potential clinical impact in non-small-cell lung carcinoma (NSCLC). We examined ITIH5 mRNA expression in tumor and adjacent normal lung tissue specimens of NSCLC patients. In addition, methylation frequency of the ITIH5 promoter was investigated using methylation-specific PCR and pyrosequencing. Significance of our data was validated by independent data sets from The Cancer Genome Atlas and the Kaplan-Meier Plotter platform. Furthermore, ITIH5 protein expression was evaluated by immunohistochemistry utilizing a tissue microarray with 385 distinct lung tissue samples. Based on our tissue collections, ITIH5 mRNA expression was significantly decreased in NSCLC compared to normal lung tissue in line with an increased methylation frequency in lung cancer tissue. Independent TCGA data confirmed significant expression loss of ITIH5 in lung cancer concordant with ITIH5 promoter hypermethylation in NSCLC. Of interest, low ITIH5 mRNA expression was particularly found in the magnoid and squamoid ADC expression subtype, concordant with an unfavorable patients' outcome in squamoid as well as tobacco smoking ADC patients. In conclusion, ITIH5 may be a novel putative tumor suppressor gene in NSCLC with a potential molecular significance in the squamoid ADC subtype and further clinical impact for risk stratification of adenocarcinoma patients. In addition, ITIH5 may serve as a novel biomarker for prognosis of tobacco smoking ADC patients.

Abstract 380: Resident Cardiac Fibroblasts Give Rise to Periostin+ Myofibroblasts Which are the Primary Mediators of Cardiac Fibrosis

Resident cardiac fibroblasts (CFs) are potential therapeutic targets in treating or preventing heart failure since they play a critical role in cardiac remodeling and fibrosis after injury or with prolonged stress stimulation. Heterogeneity among activated fibroblasts within the heart has been noted by a number of previous studies in the literature. In addition to resident CFs, many cell types such as endothelial, perivascular and bone marrow cells have been suggested to go through a mesenchymal transition and acquire a myofibroblast-like phenotype during disease conditions. Hence, the cellular origin of the activated myofibroblast within the heart remains uncertain, in part because of a lack in reliable genetic strategies to define cellular lineage. Recent studies suggest that epicardial precursor cells expressing transcription factor 21 (Tcf21) give rise to resident CFs in the adult heart. In addition, the secreted matricellular protein periostin (Postn), appears to be expressed only within activated fibroblasts (myofibroblasts) within the heart. Here we used Tcf21-MerCreMer (Tcf21MCM) knockin mice and Postn-MerCreMer (PostnMCM) knock-in (KI) mice to lineage trace resident CFs and myofibroblasts with injury stimulation. To account for other potential cellular lineages giving rise to fibroblasts in the heart we also performed lineage tracing with the mouse genetic models including LysM-Cre (macrophage), ckit-Cre (bone marrow), Tie2CreERT2 (endothelial) and Myh11-CreERT2 (smooth muscle) in conjunction ROSA26 (R26) locus based loxP inactivated reporter alleles. Results of this study indicate that the Tcf21+ resident CFs are the predominant source for the activated periostin+ MFs which are the key mediators of extracellular matrix (ECM) production and ECM stability in heart whereas the contribution of other lineages to MFs are minimal. Additionally, we have performed single cell RNA sequencing on TCF21+ and Postn+ isolated CFs pre and post myocardial injury in order to define the fibroblast lineage itself at greater molecular depth.

Compensatory fetal membrane mechanisms between biglycan and decorin in inflammation.

Preterm premature rupture of fetal membranes (PPROM) is associated with infection, and is one of the most common causes of preterm birth. Abnormal expression of biglycan and decorin, two extracellular matrix proteoglycans, leads to preterm birth and aberrant fetal membrane morphology and signaling in the mouse. In humans and mice, decorin dysregulation is associated with inflammation in PPROM. We therefore investigated the link between biglycan and decorin and inflammation in fetal membranes using mouse models of intraperitoneal Escherichia coli injections superimposed on genetic biglycan and decorin deficiencies. We assessed outcomes in vivo as well as in vitro using quantitative PCR, Western blotting, and enzyme-linked immunosorbent assays. Our results suggest that biglycan and decorin compensate for each other in the fetal membranes, but lose the ability to do so under inflammation, leading to decreased latency to preterm birth. Furthermore, our findings suggest that biglycan and decorin play discrete roles in fetal membrane signaling pathways during inflammation, leading to changes in the abundance of MMP8 and collagen α1VI, two components of the fetal membrane extracellular matrix that influence the pathophysiology of PPROM. In summary, these findings underline the importance of biglycan and decorin as targets for the manipulation of fetal membrane extracellular matrix stability in the context of inflammation.

RvD1 Activates Resolution of Inflammation Following Myocardial Infarction Leading To Improved Ventricular Function

Unresolved inflammation is a major contributor to heart failure following myocardial infarction (MI). Pro-resolving lipid mediators, including resolvins (e.g. RvD1), are biosynthesized endogenously. The role of RvD1 in resolving post-MI inflammation has not been addressed. Here, we tested whether liposomal RvD1 (Lipo-RvD1) or Pure-RvD1 limit left ventricle (LV) and splenic remodeling by resolving post-MI inflammation. 8 to 12 week old male C57BL/6 mice were subjected to coronary artery ligation and Lipo-RvD1 or pure-RvD1 (3ug/kg/day) was injected 3 hour post-MI for day (d)1 or until d5. No-MI mice and MI mice were treated with saline as controls. RvD1 injected groups showed improved fractional shortening post-MI (p<0.05) and preserved transient changes in splenic reservoir compared to MI control. RvD1 groups showed early exit of neutrophils from LV and spleen compared to MI-controlat d5 post-MI. RvD1 groups showed reduced macrophage density compared to MI-control with reduced ccr5 and cxcl5 levels at d5 post-MI (p<0.05). Increased transcripts of mrc-1, arg-1 and Ym-1; p<0.05 suggests macrophage-mediated clearance of necrotic cells in RvD1 groups. RvD1-injected mice exhibited reduced post-MI fibrosis with reduced collagen deposition. Pro-fibrotic genes (colla1, coll2a1 and Tnc; p<0.05) reduction indicated a stabilization of extracellular matrix at d5 in RvD1 groups. RvD1-mediated pro-resolving actions were also supported by increased 5-lipoxygenase (LOX) and decreased 12-LOX levels at d5 post-MI. In summary, these results indicated that liposomal and pure-RvD1 show potential pro-resolving activity to remit uncontrolled inflammation and delay heart failure.

The expression of periostin in dental pulp cells

Background and objectiveDental pulp repair is a common process triggered by microbial and mechanical challenges. Matricellular modulators, such as periostin, are key for extracellular matrix stability and tissue healing. In the scope of the dental pulp, periostin expression has been reported during development and active dentinogenesis. However, the specific dental pulp cell population capable of expressing periostin in response to known regulators has not been clearly defined. Among the different relevant cell populations (i.e., stem cells, fibroblasts and pre-odontoblasts) potentially responsible for periostin expression in the dental pulp, this study aimed to determine which is the primary responder to periostin regulators. MethodsHuman dental pulp stem cells (DPSCs), human dental pulp fibroblasts (DPFs), and rat odontoblast-like cells (MDPC-23) were treated with different concentrations of TGF-β1 or different regimens of biomechanical stimulation to evaluate periostin expression by qRT-PCR, Western blot and ELISA. Statistical analyses were performed by Student's t-test and ANOVA with Fisher's LSD post hoc tests (p≤0.05). ResultsDPSC and MDPC-23 showed a statistically significant increase in periostin mRNA expression after exposure to TGF-β1 for 48h. TGF-β1 also up-regulated periostin protein levels in DPSC. However, periostin significantly down-regulated protein expression in DPF. Different regimens of biomechanical stimulation showed different patterns in protein and mRNA periostin expression. ConclusionsExpression of periostin was identified in each of the analysed dental pulp cell lines, which can be regulated by TGF-β1 and biomechanical stimulation. Overall, DPSCs are the most responsive cells to stimulation.

The Role of Transglutaminase 2 (TG2) in Definition of Cancer Hallmarks

Over the past three decades, TGM2, a stress-responsive gene encoding transglutaminase 2 (TG2) has been identified as one of the several genes that may be involved in carcinogenesis and cancer physiology. TG2 is a pleiotropic calcium-dependent enzyme belonging to the transglutaminase family of enzymes, which post-translationally modify glutaminyl and lysyl side chains on the surface of both in vivo and in vitro substrate proteins. Unlike other members of the transglutaminase family, TG2 has additional Ca-independent enzymatic and non-enzymatic activities, which have been directly or indirectly implicated in diverse cellular physiological events, including cell growth and differentiation, cell adhesion and morphology, extracellular matrix stabilization, wound healing, cellular development, receptor-mediated endocytosis, apoptosis, and disease pathology. TG2 has specialized biochemical, structural and functional elements, wide tissue distribution and subcellular localisation, as well as broad substrate specificity. These specialised features of TG2 account for its multiple patho-physiological functionalities. Considering the multiplicity of TG2 Mini-review Article Odii et al.; BJMMR, 6(3): 297-311, 2015; Article no.BJMMR.2015.205 298 functions and its importance in disease pathology, including cancer; we have reviewed herein, the importance of TG2 in the definition of the hallmark capabilities of cancer cells. This was done with the view to deepen our understanding of the role of TG2 in carcinogenesis and recapitulating its potential as a therapeutic target for cancer treatment.

Abstract B76: Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma

Abstract 3D in vitro skin models are valuable research tools to investigate normal keratinocyte as well as skin carcinoma cell interaction with dermal fibroblasts. Currently, the models lack either a structured vital human extracellular matrix or long-term stability and longevity. Therefore, we aimed at a new 3D in vitro skin model for normal human epidermis and tumorigenic stages of skin cancer. This model is based on dermal equivalents containing a highly structured cell-derived matrix (CDM) produced by human dermal fibroblasts. First, we show that the CDM dermal equivalent supports a high structural integrity of the skin model and maintains a vital epidermis for six months. The epidermis is well-stratifying and differentiates in a regular orthokeratotic pattern. Transmission electron microscopy reveals ultrastructural entities as basement membranes, hemidesmosomes, desmosomes, keratohyalin granulae and lamellar bodies. In line, the molecular differentiation markers are properly distributed. Second, five non-tumorigenic and tumorigenic HaCaT keratinocyte variants are tested for their proliferation, differentiation and invasion capacities. After four weeks of co-cultivation the tumorigenic HaCaT variants show hyperproliferative epithelia and lose the capability of an ordered stratification and differentiation. Furthermore, they destroy the basement membrane as well as the extracellular matrix and invade the dermal equivalent. Moreover, seven squamous cell carcinoma (SCC) cell lines were co-cultivated with the CDM dermal equivalent. All SCC cells form hyperproliferative epithelia that heavily invade the extracellular matrix. Remarkably, the epithelia are individually distinct for their invasion and differentiation capacity. Thus, the CDM-based skin cancer model is suitable for demonstrating the heterogeneity of SCC tumors and their interaction with the microenvironment. In conclusion, the CDM model for normal skin and tumorigenic keratinocytes provides a novel tool to investigate keratinocyte-fibroblast interactions and microenvironment modulations during SCC development. Therefore, this system will provide further insights into mechanistic processes and the interaction of SCCs with their tumor microenvironment. Citation Format: Manuel Berning, Petra Boukamp. Novel human 3D in vitro invasion model identifies tumor and stroma heterogeneity in squamous cell carcinoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B76. doi:10.1158/1538-7445.CHTME14-B76

Use of laser capture microdissection for the assessment of equine lamellar basal epithelial cell signalling in the early stages of laminitis.

Dysadhesion of laminar basal epithelial cells (LBECs) from the underlying dermis is the central event leading to structural failure in equine laminitis. Although many studies of sepsis-related laminitis have reported multiple events occurring throughout the lamellar tissue, there is minimal information regarding signalling events occurring specifically in LBECs. To determine signalling events in the LBECs during the early stages of carbohydrate-induced laminitis. Experimental study. Eight horses were given an overload of carbohydrate (CHO) consisting of corn starch mixture via nasogastric tube. Prior to administration of CHO, lamellar biopsies were taken from the left forefoot (control [CON]). Biopsies were taken from the left hind foot at the onset of fever (developmental [DEV]) and from the right forefoot at the onset of Obel grade 1 lameness (OG1). Laminar basal epithelial cells were isolated from cryosections using a laser capture microdissection (LCM) microscope. Next generation sequencing (RNA-seq) was used to identify transcripts expressed in the LBECs for each time point and bioinformatic analysis was performed with thresholds for between group comparisons set at a greater than 2-fold change and P value ≤0.05. Forty genes (22 increased/18 decreased) were significantly different from DEV time vs. CON and 107 genes (57 increased/50 decreased) were significantly different from OG1 time vs. CON. Significant increases in inflammatory genes were present in addition to significantly altered expression of genes related to extracellular matrix composition, stability and turnover. Signalling related to inflammatory response and extracellular matrix regulation was strongly represented at the DEV and OG1 times. These results indicate that the LBEC is not only a casualty but also an active participant in lamellar events leading to structural failure of the digital lamellae in equine laminitis.

Structural characterization of heparinase III from Bacteroides thetaiotaomicron

Glycosaminoglycans (GAGs) are linear polysaccharides comprised of disaccharide repeat units, a hexuronic acid, glucuronic acid (GlcA) or iduronic acid (IdoA), linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine (GalNAc). GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix (ECM), control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior, and differentiation. Niche adapted bacteria expresses enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. Polysaccharide lyases have been classified into 23 sequence-related families. Comparison of three-dimensional structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level and identified occurrences of convergent evolution. We have characterized structurally and enzymatically Heparinase III (HepIII) from Bacteroides thetaiotaomicron, which is classified within the PL12 family. HepIII is a 72.5KDa protein. We will present the X-Ray structures of two crystal forms of HepIII of resolution 1.8 Å and 2.6 Å. HepIII contains two domains, the N-terminal α-helical domain forming a toroid and the C-terminal β-sheet domain. Comparison with recently determined structures of two other heparinases from the same PL12 family allowed us to identify structural flexibility in the arrangement of the domains indicating open-close movement. Based on comparison with other GAG lyases we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of HepIII toward sulfate poor heparan sulcate substrate.

Epigenetic inactivation of the novel candidate tumor suppressor gene ITIH5 in colon cancer predicts unfavorable overall survival in the CpG island methylator phenotype

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression as well as its potential clinical and functional impact in colon cancer. Based on 30 tumor and 30 adjacent normal tissues we examined ITIH5 mRNA expression and promoter methylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA) platform. In addition, ITIH5 protein expression was evaluated using immunohistochemistry. ITIH5 mRNA expression loss was significantly associated (P < 0.001) with hypermethylation of the ITIH5 promoter in primary colon tumors. In addition, treatment of tumor cell lines with demethylating (DAC) and histone acetylating (TSA) agents induced ITIH5 expression. In line, independent TCGA data revealed a significant expression loss of ITIH5, particularly in the MSI-high and CIMP-positive phenotype concordant with an increased ITIH5 hypermethylation in CIMP-positive colon tumors (P < 0.001). In proximal, i.e., right-sided tumors, abundant ITIH5 expression was associated with longer overall survival (OS, P = 0.049) and the CIMP-positive (P = 0.032) subgroup. Functionally, ITIH5 re-expression mediated a reduced proliferation in HCT116 and CaCo2 cells. In conclusion, our results indicate that ITIH5 is a novel putative tumor suppressor gene in colon cancer with a potential impact in the CIMP-related pathway. ITIH5 may serve as a novel epigenetic-based diagnostic biomarker with further clinical impact for risk stratification of CIMP-positive colon cancer patients.

Distinct functions of the laminin β LN domain and collagen IV during cardiac extracellular matrix formation and stabilization of alary muscle attachments revealed by EMS mutagenesis in Drosophila.

BackgroundThe Drosophila heart (dorsal vessel) is a relatively simple tubular organ that serves as a model for several aspects of cardiogenesis. Cardiac morphogenesis, proper heart function and stability require structural components whose identity and ways of assembly are only partially understood. Structural components are also needed to connect the myocardial tube with neighboring cells such as pericardial cells and specialized muscle fibers, the so-called alary muscles.ResultsUsing an EMS mutagenesis screen for cardiac and muscular abnormalities in Drosophila embryos we obtained multiple mutants for two genetically interacting complementation groups that showed similar alary muscle and pericardial cell detachment phenotypes. The molecular lesions underlying these defects were identified as domain-specific point mutations in LamininB1 and Cg25C, encoding the extracellular matrix (ECM) components laminin β and collagen IV α1, respectively. Of particular interest within the LamininB1 group are certain hypomorphic mutants that feature prominent defects in cardiac morphogenesis and cardiac ECM layer formation, but in contrast to amorphic mutants, only mild defects in other tissues. All of these alleles carry clustered missense mutations in the laminin LN domain. The identified Cg25C mutants display weaker and largely temperature-sensitive phenotypes that result from glycine substitutions in different Gly-X-Y repeats of the triple helix-forming domain. While initial basement membrane assembly is not abolished in Cg25C mutants, incorporation of perlecan is impaired and intracellular accumulation of perlecan as well as the collagen IV α2 chain is detected during late embryogenesis.ConclusionsAssembly of the cardiac ECM depends primarily on laminin, whereas collagen IV is needed for stabilization. Our data underscore the importance of a correctly assembled ECM particularly for the development of cardiac tissues and their lateral connections. The mutational analysis suggests that the β6/β3/β8 interface of the laminin β LN domain is highly critical for formation of contiguous cardiac ECM layers. Certain mutations in the collagen IV triple helix-forming domain may exert a semi-dominant effect leading to an overall weakening of ECM structures as well as intracellular accumulation of collagen and other molecules, thus paralleling observations made in other organisms and in connection with collagen-related diseases.

Peroxidasins: novel players in tissue genesis

Stabilization of extracellular matrix by protein crosslinking is a universal and essential process in multicellular organisms. Recent findings revealed that peroxidasin, a unique heme-peroxidase, produces hypohalides to support matrix synthesis. Unexpectedly, the highly reactive and potentially damaging hypohalides mediate the formation of sulfilimine bonds between adjacent collagen IV protomers. This crosslink is a fundamental feature of basal membranes, defining peroxidasin-dependent oxidant generation and sulfilimine crosslink formation as an elemental mechanism of tissue biogenesis.

Chemical stabilization of dentin extracellular matrix detected by FEISEM and EDS

Dentin collagen degradation represents an important limit to the stability of the resin-dentin interface in conservative dentistry. In vitro application of 1-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC), an imide-based zerolength cross-linking agent, showed [1] the capability to inactivate the endogenous dentin matrix metalloproteinases and to increase the mechanical properties of the organic dentin extracellular matrix. In the present study, a correlative high resolution SEM (FEISEM) with an Energy Dispersive Spectroscopy (EDS) analysis was performed to characterize the fine structure and the chemical modifications of EDCstabilized human dentin, after compressive mechanical stress (Chewing Simulation - CS). Demineralized human dentin disks were assigned to four groups: (1) artificial saliva at 37°C for 30 days; (2) pre-treatment with 0.5M EDC for 60 s, then as in Group 1; (3) CS challenge for 30 days; (4) 0.5M EDC as in Group 2 and CS challenge as in Group 3. The FEISEM analysis revealed that the EDC-pretreatment made the collagen fibrillar network more compact, in comparison to controls and this effect was particularly evident on the surface of not stressed samples. Along with the increased compactness of the collagen complex, the EDS analysis showed a significant semi-quantitative increase of sulfur. The presence of chlorine in EDC treated samples was also detectable. The increase of sulfur, not present in EDC composition, suggests a possible implication of sulfate glycosaminoglycans containing proteoglycans during the extracellular matrix stabilization, as also suggested by the concurrent increase of the amorphous matrix. The presence of chlorine in EDC treated samples induces to conclude that the activity of the cross-linking agent is stable even after the experimental time intervals.

Biological functionalities of transglutaminase 2 and the possibility of its compensation by other members of the transglutaminase family.

Transglutaminase 2 (TG2) is the most widely distributed and most abundantly expressed member of the transglutaminase family of enzymes, a group of intracellular and extracellular proteins that catalyze the Ca2+-dependent posttranslational modification of proteins. It is a unique member of the transglutaminase family owing to its specialized biochemical, structural and functional elements, ubiquitous tissue distribution and subcellular localization, and substrate specificity. The broad substrate specificity of TG2 and its flexible interaction with numerous other gene products may account for its multiple biological functions. In addition to the classic Ca2+-dependent transamidation of proteins, which is a hallmark of transglutaminase enzymes, additional Ca2+-independent enzymatic and nonenzymatic activities of TG2 have been identified. Many such activities have been directly or indirectly implicated in diverse cellular physiological events, including cell growth and differentiation, cell adhesion and morphology, extracellular matrix stabilization, wound healing, cellular development, receptor-mediated endocytosis, apoptosis, and disease pathology. Given the wide range of activities of the transglutaminase gene family it has been suggested that, in the absence of active versions of TG2, its function could be compensated for by other members of the transglutaminase family. It is in the light of this assertion that we review, herein, TG2 activities and the possibilities and premises for compensation for its absence.

Neomycin and pentagalloyl glucose enhanced cross-linking for elastin and glycosaminoglycans preservation in bioprosthetic heart valves

Glutaraldehyde cross-linked bioprosthetic heart valves fail within 12-15 years of implantation due to limited durability. Glutaraldehyde does not adequately stabilize extracellular matrix components such as glycosaminoglycans and elastin, and loss of these components could be a major cause of degeneration of valve after implantation. We have shown earlier that neomycin-based cross-linking stabilizes glycosaminoglycans in the tissue but fails to stabilize elastin component. Here, we report a new treatment where neomycin and pentagalloyl glucose (PGG) were incorporated into glutaraldehyde cross-linking neomycin-PGG-Glutaraldehyde (NPG) to stabilize both glycosaminoglycans and elastin in porcine aortic valves. Invitro studies demonstrated a marked increase in extracellular matrix stability against enzymatic degradation after cross-linking and 10 month storage in NPG group when compared to glutaraldehyde controls. Tensile properties showed increased lower elastic modulus in both radial and circumferential directions in NPG group as compared to glutaraldehyde, probably due to increased elastin stabilization with no changes in upper elastic modulus and extensibility. The enhanced extracellular matrix stability was further maintained in NPG-treated tissues after rat subdermal implantation for three weeks. NPG group also showed reduced calcification when compared to glutaraldehyde controls. We conclude that NPG cross-linking would be an excellent alternative to glutaraldehyde cross-linking of bioprosthetic heart valves to improve its durability.