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Multiple unique aspects of liver biology make this organ an excellent paradigm for novel cell and gene therapy applications. In recent years, insights were obtained into how transplanted cells engraft and proliferate in the liver, including in the context of pre-existing disease. Also, a variety of animal models were studied to establish the basis of cell and gene therapy applications in specific disorders. Through ongoing research activity, additional mechanisms in liver repopulation have been uncovered, where manipulation of specific cell compartments and cellular processes,e.g., those aimed at extracellular matrix component receptors or soluble signals in transplanted and native cells can be exploited for enhancing cell engraftment and proliferation. Such studies demonstrate the possibility of applying biotechnology and/or bioengineering principles to organ replacement aimed at cell and gene therapy. Joining of these disciplines with research in stem cell biology, particularly in efforts concerning targeting of transplanted stem cells to given organs with achievement of lineage-specific cell differentiation and function, will be particularly important for future cell and gene therapy applications. This review offers an overview of relevant mechanisms in liver repopulation.

Recent evidence has shown that retinoic acid (RA) signalling is required for early pancreatic development in zebrafish and frog but its role in later development in mammals is less clear cut. In the present study, we determined the effects of RA on the differentiation of the mouse embryonic pancreas. Addition of all-trans retinoic acid (atRA) to embryonic pancreatic cultures induced a number of changes. Branching morphogenesis and exocrine differentiation were suppressed and there was premature formation of endocrine cell clusters (although the total area of beta cells was not different in control and atRA-treated buds). We investigated the mechanism of these changes and found that the premature formation of beta cells was associated with the early expression of high-level Pdx1 in the endocrine cell clusters. In contrast, the suppressive effect of RA on exocrine differentiation may be due to a combination of two mechanisms (i) up-regulation of the extracellular matrix component laminin and (ii) enhancement of apoptosis. We also demonstrate that addition of fibroblast growth factor (FGF)-10 is able to partially prevent apoptosis and rescue exocrine differentiation and branching morphogenesis in atRA-treated cultures but not in mice lacking the FGF receptor 2-IIIb, suggesting the effects of FGF-10 are mediated through this receptor.

A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3′,5′-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose., Author Summary Biofilms are specialized and highly differentiated three-dimensional communities of bacteria encased in an extracellular polymeric matrix (EPM), and the bacteria’s mechanisms to form biofilm are closely linked to their virulence. The EPM often consists of polysaccharides, proteins, nucleic acids, and lipids. Compared to extracellular polysaccharides, little is known about the protein components in the biofilm matrix of Vibrio vulnificus, a foodborne pathogen. In this study, we identified and characterized cabABC genes which were preferentially expressed in biofilms. CabA is a calcium-binding protein and is secreted through functional CabB and CabC. Our results indicated that CabA contributes to the development of biofilm and rugose colony morphology under elevated c-di-GMP conditions. CabA is an extracellular matrix protein crucial for the structural integrity of robust biofilm in flow cells and on oyster shells. Calcium binding induces conformational changes and multimerization of CabA that may render the protein functional to build a well-structured matrix. CabA can assemble a functional matrix extracellularly only when exopolysaccharides (EPS) coexist, indicating that both CabA and EPS are required for the scaffold of V. vulnificus biofilm matrix. This is the first report on a non-polysaccharide matrix component that is essential for the development of the V. vulnificus biofilm structure.

Despite the significant advances in the meniscus tissue engineering field, it is difficult to recreate the complex structure and organization of the collagenous matrix of the meniscus. In this work, we developed a meniscus prototype to be used as substitute or scaffold for the regeneration of the meniscal matrix, recreating the differential morphology of the meniscus by electrospinning. Synthetic biocompatible polymers were combined with the extracellular matrix component, collagen and used to replicate the meniscus. We studied the correlation between mechanical and structural properties of the polymer blend as a function of collagen concentration. Fibers were collected on a surface of a rapidly rotating precast mold, to accurately replicate each sectional morphology of the meniscus; different electro-tissues were produced. Detailed XRD analyses exhibited structural changes developed by electrospinning. We achieved to integrate all these electro-tissues to form a complete synthetic meniscus. Vascularization tests were performed to assess the potential use of our novel polymeric blend for promising meniscus regeneration.

In this issue of Science Signaling, Mills et al. show that the amino acid ʟ-arginine increases the concentration of the second messenger c-di-GMP in Salmonella enterica serovar Typhimurium through a specific diguanylate cyclase, leading to increased production of the exopolysaccharide cellulose, which is an extracellular matrix component of environmental and host-associated biofilms.

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an "outside-in" signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.

Hyaluronan (HA), a long-chain polysaccharide, is currently being evaluated as a potential therapeutic agent for pulmonary emphysema, based on previous studies from this laboratory indicating its protective effect against elastic fiber breakdown. To determine whether exogenously administered HA might replace a loss of this extracellular matrix component in this disease, we measured the content of HA in lung biopsies from both healthy individuals and alpha-1 antiprotease-deficient (AAPD) COPD patients with pulmonary emphysema.Tissue samples (9 from COPD patients, 5 from controls) were digested with papain to isolate glycosaminoglycans, and lung HA was quantified with an enzyme-linked immunoabsorbent assay.HA was significantly decreased in the AAPDCOPD population compared to normal individuals (13.5 vs 21.7 ng/mg wet lung; p 0.01). Furthermore, there was a positive correlation between HA levels and the following parameters: 1) percent predicted FEV1 (r = 0.78; p 0.001), 2) percent predicted DLCO (r = 0.74; p 0.05), and 3) serum levels of AAP (r = 0.61; p 0.05).These findings support the hypothesis that depletion of lung HA plays a role in the pathogenesis of pulmonary emphysema, and that replacement of this matrix component could slow the progression of the disease.

This review focusses on impact of a better knowledge of pathogenic mechanisms of Marfan and related disorders on their treatment strategies. It was long believed that a structural impairment formed the basis of Marfan syndrome as deficiency in the structural extracellular matrix component, fibrillin-1 is the cause of Marfan syndrome. However, the study of Marfan mouse models has revealed the strong involvement of the transforming growth factor-beta signalling pathway in the pathogenesis of Marfan. Similarly, this pathway was demonstrated to be key in the pathogenesis of Loeys-Dietz and Shprintzen-Goldberg syndrome. The elucidation of the underlying pathogenic mechanisms has led to new treatment strategies, targeting the overactive TGF-beta pathway. Various clinical trials are currently investigating the potential new treatment options. A meta-analysis will contribute to a better understanding of the various trial results. (C) 2015 Elsevier Masson SAS. All rights reserved.

Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the β1 and α6 integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the β1 subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

Laminin-2/4 is the major laminin isoform of normal muscle and nerve tissues and plays an important role in tumor invasion and metastasis. Despite the fact that laminin-2/4 has been found in the skin basement membrane, insufficient evidence is available on the effect of laminin-2/4 on the behavior of both normal and transformed skin cells. A comparison of the contribution of alpha2beta1, alpha3beta1, alpha6beta4 integrins and 67 kDa laminin receptor on the surface of the human epidermoid carcinoma cell, A-431, to interaction with laminin-2/4 was carried out. The cell interaction with extracellular matrix component is a multistage process. We employed new methods for studying different stages of the interaction of A-431 cells with laminin-2/4. We demonstrated that integrins alpha2beta1, alpha3beta1, alpha6beta4 and 67 kDa laminin receptor are involved in the interaction of A-431 cells with laminin-2/4. We found that contribution of the same receptors to different stages of the interaction with laminin can be different. alpha2beta1 integrins are involved in EGF-induced A-431 cells' migration on laminin-2/4. We demonstrated the cooperation between alpha2beta1 and alpha3beta1 integrins during adhesion and spreading of A-431 cells on laminin-2/4-coated substrate. These results provide information about laminin-2/4 receptors and their contribution to different stages of the interaction with cells.

Airway epithelium is emerging as a regulator of local inflammation and immune responses. However, the cellular and molecular mechanisms responsible for the immune modulation by these cells have yet to be fully elucidated. At the cellular level, the hallmarks of airway inflammation are mucus gland hypertrophy with excess mucus production, accumulation of inflammatory mediators, inflammation in the airway walls and lumen, and breakdown and turnover of the extracellular matrix. We demonstrate that fragments of the extracellular matrix component hyaluronan induce inflammatory chemokine production in primary airway epithelial cells grown at an air-liquid interface. Furthermore, hyaluronan fragments use two distinct molecular pathways to induce IL-8 and IFN-γ-inducible protein 10 (IP-10) chemokine expression in airway epithelial cells. Hyaluronan-induced IL-8 requires the MAP kinase pathway, whereas hyaluronan-induced IP-10 utilizes the NF-κB pathway. The induction is specific to low-molecular-weight hyaluronan fragments as other glycosaminoglycans do not induce IL-8 and IP-10 in airway epithelial cells. We hypothesize that not only is the extracellular matrix a target of destruction in airway inflammation but it plays a critical role in perpetuating inflammation through the induction of cytokines, chemokines, and modulatory enzymes in epithelial cells. Furthermore, hyaluronan, by inducing IL-8 and IP-10 by distinct pathways, provides a unique target for differential regulation of key inflammatory chemokines.

Anterior–posterior axis formation in the Drosophila oocyte requires activation of the EGF receptor (EGFR) pathway in the posterior follicle cells (PFC), where it also redirects them from the default anterior to the posterior cell fate. The relationship between EGFR activity in the PFC and oocyte polarity is unclear, because no EGFR-induced changes in the PFC have been observed that subsequently affect oocyte polarity. Here, we show that an extracellular matrix receptor, Dystroglycan, is down-regulated in the PFC by EGFR signaling, and this down-regulation is necessary for proper localization of posterior polarity determinants in the oocyte. Failure to down-regulate Dystroglycan disrupts apicobasal polarity in the PFC, which includes mislocalization of the extracellular matrix component Laminin. Our data indicate that Dystroglycan links EGFR-induced repression of the anterior follicle cell fate and anterior–posterior polarity formation in the oocyte.

Extracellular matrix abnormalities have been found in both human and animal models of polycystic kidney disease (PKD). A new mouse PKD model has been produced through insertion of a PGKneo cassette in an intron of the gene that encodes laminin alpha5 (Lama5), a major tubular and glomerular basement membrane component that is important for glomerulogenesis and ureteric bud branching. Lama5neo represents a hypomorphic allele as a result of aberrant splicing. Lama5neo/neo mice exhibit PKD, proteinuria, and death from renal failure by 4 wk of age. This contrasts with mice that totally lack Lama5, which die in utero with multiple developmental defects. At 2 d of age, Lama5neo/neo mice exhibited mild proteinuria and microscopic cystic transformation. By 2 wk, cysts were grossly apparent in cortex and medulla, involving both nephron and collecting duct segments. Tubular basement membranes seemed to form normally, and early cyst basement membranes showed normal ultrastructure but developed marked thickening as cysts enlarged. Overall, Lama5 protein levels were severely reduced as a result of mRNA frameshift caused by exon skipping. This was accompanied by aberrant accumulation of laminin-332 (alpha3beta3gamma2; formerly called laminin-5) in some cysts, as also observed in human PKD. This constitutes the first evidence that a primary defect in an extracellular matrix component can cause PKD.

p38 MAPKs (mitogen-activated protein kinases) play important roles in the regulation of cellular responses to environmental stress. Recently, this signalling pathway has also been implicated in the regulation of processes unrelated to stress, for example, in T lymphocytes and cardiomyocytes. In order to identify molecular targets responsible for the housekeeping functions of p38 MAPKs, we have analysed the differences in the transcriptomes of normally proliferating wild-type and p38alpha knockout immortalized embryonic cardiomyocytes. Interestingly, many potential components of the myocardium extracellular matrix were found to be upregulated in the absence of p38alpha. Further analysis of the microarray data identified TEF-1 (transcriptional enhancer factor-1), a known regulator of heart-specific gene expression, and C/EBPbeta (CCAAT/enhancer-binding protein beta), as the two transcription factors the binding sites of which were most enriched in the promoters of p38alpha-regulated genes. We have focused on the study of the extracellular matrix component COL1A1 (alpha1 chain of type I collagen) and found evidence for the involvement of both TEF-1 and C/EBPbeta in the p38alpha-dependent inhibition of COL1A1 transcription. Our data therefore show that p38 MAPKs regulate TEF-1 and C/EBPbeta transcriptional activity in the absence of environmental stress and suggests a role for p38alpha in the expression of extracellular matrix components that maintain organ architecture.

Human papillomaviruses (HPVs) have previously been shown to adsorb to cultured cells via membrane-associated heparan sulfate (HS) and alpha6 integrin. We demonstrate that cultured keratinocytes uniquely secrete a component into the basal extracellular matrix (ECM) which can function to adsorb HPV particles which can then be internalized by adherent cells. This uncharacterized basal ECM adsorption receptor was secreted by normal human epidermal keratinocytes (NHEK) and by each of the four keratinocyte-derived cell lines we examined, but not by non-keratinocyte cell lines. Multiple HPV types bound preferentially to this keratinocyte-specific receptor over the membrane-associated receptor, and binding to the basal ECM adsorption receptor was refractory to inhibition by heparin. Like the membrane-associated receptor, this basal ECM component was functional as an adsorption receptor in our in vitro infection model using HPV-11. Unlike particle adsorption, however, successful infection with HPV-11 virions remained sensitive to the pretreatment of virions with heparin. The secreted basal ECM receptor did not colocalize with antibodies against HS, perlecan, or alpha6 integrin, but colocalized with antibody against laminin-5, a marker of keratinocyte ECM and an abundant component of the basement membrane in mucosa and skin. These findings suggest a model for natural infections in which HPV virions, nonspecifically adsorbed to HS on suprabasal keratinocytes throughout an epithelial wound, might be transferred to mitotically active migrating keratinocytes via an intermediate association with the ECM secreted by these cells as they reestablish the basement membrane.

The effects of chemically induced hypoxia and ionizing radiation on the adhesive properties of MG-63 human osteosarcoma three-dimensional spheroids were investigated. Hypoxia was induced by addition of CoCl2 to small, nonhypoxic spheroids and verified by HIF-1alpha expression. In addition, the possible role of important cell adhesion molecules involved in tumor dissemination in inducing adhesive changes were also studied. In particular, two key integrins (i.e., the alpha chain of the fibronectin receptor, alpha5, and the alpha chain of the collagen receptor, alpha2), an important member of the immunoglobulin superfamily (CD54 or ICAM-1) and the strategic molecule CD44 (H-CAM, the principal receptor for hyaluronan) were examined. Because of the important role of fibronectin in adhesive processes, variations in this extracellular matrix component were also examined. The results seem to indicate that CoCl2-induced hypoxia greatly increases adhesion of MG-63 spheroids to both tissue culture plates and plates coated with fibronectin or collagen when compared to controls, while ionizing radiation induces a great decrease in this attachment. Furthermore, chemically induced hypoxia also partially inhibits the effects of ionizing radiation. The data also show that these adhesive changes are accompanied by concomitant variations in the expression of alpha5 and alpha2 integrins, CD44, and CD54 and fibronectin.

Background. Hyaluronan (HA) is a prominent extracellular matrix component undergoing continuous production and degradation. Increased HA levels have been described in a variety of tumors. The objective of this study was to examine the staining patterns of HA and two of its associated receptors (CD44 and HARE) in relation to the metastatic potential of mucoepidermoid carcinoma (MC). Immunohistochemical staining of preserved surgical specimens was used. Methods. Tissues from 12 patients with a histologic diagnosis of salivary MC (10 parotid, one submandibular gland, one minor salivary gland) were studied. Half (six of 12) of the patients had regional metastases. Tumor, normal salivary tissue, and regional lymph nodes were stained for HA, CD44, and HARE expression. Specimens were graded for staining intensity and a percent of the specimen stained. Results. Normal salivary tissue did not demonstrate epithelial cell surface HA expression, whereas HA was expressed on tumor cells and in regional lymph nodes containing metastases. These differences were both significant using Student's t test (p < .00002, and p < .0022, respectively). Tumors with positive nodes tended to have greater cell surface HA. Decreased expression or downregulation of HARE was also noted in involved lymph nodes. No differences in CD44 expression were seen between primary specimens and lymph nodes. The observed staining patterns for CD44 and HARE were not reflective of the metastatic potential of the primary MC. Conclusions. Increased HA expression was seen on mucoepidermoid carcinoma cells compared with adjacent normal salivary gland epithelium. This observation may assist in explaining the development of regional metastasis in these tumors. We did not identify specific HA, CD44, or HARE staining patterns in primary lesions that were predictive of regional metastases. © 2005 Wiley Periodicals, Inc. Head Neck27: XXX–XXX, 2005

Hyaluronan (HA), the major extracellular matrix component, is often anchored to CD44, a family of structurally/functionally important cell surface receptors. Recent results indicate that UV irradiation (UVR)-induced cutaneous squamous cell carcinomas (SCC) overexpress a variety of CD44 variant isoforms (CD44v), with different CD44v isoforms appear to confer malignant SCC properties. UVR also stimulates HA degradation in epidermal keratinocytes. Both large HA polymers and their UVR-induced catabolic products (small HA) selectively activate CD44-mediated cellular signaling in normal keratinocytes and SCC cells, with all of the downstream processes being mediated by RhoGTPases (e.g., Rac1 and Rho). Importantly, we found that the hormonally active form of vitamin D 1,25(OH)2D3 not only prevents the UVR-induced small HA activation of abnormal keratinocyte behavior and SCC progression, but also enhances large HA stimulation of normal keratinocyte activities and epidermal function(s). The aim of this hypothesis and theory article is to question whether matrix HA and its UVR-induced catabolic products (e.g., large and small HA) can selectively activate CD44-mediated cellular signaling such as GTPase (Rac and RhA) activation. We suggested that large HA–CD44 interaction promotes Rac-signaling and normal keratinocyte differentiation (lipid synthesis), DNA repair, and keratinocyte survival function. Conversely, small HA–CD44 interaction stimulates RhoA activation, NFκB/Stat-3 signaling, and miR-21 production, resulting in inflammation and proliferation as well as SCC progression. We also question whether vitamin D treatment displays any effect on small HA–CD44v-mediated RhoA signaling, inflammation, and SCC progression, as well as large HA–CD44-mediated differentiation, DNA repair, keratinocyte survival, and normal keratinocyte function. In addition, we discussed that the topical application of signaling perturbation agents (e.g., Y27623, a ROK inhibitor) may be used to treat certain skin diseases displaying upregulation of keratinocyte proliferation such as psoriasis and actinic keratoses in order to correct the imbalance between Rac and RhoA signaling during various UV irradiation-induced skin diseases in patients. Finally, we proposed that matrix HA/CD44-signaling strategies and matrix HA (HAS vs. HAL or HAS → HAL)-based therapeutic approaches (together with vitamin D) may be used for the treatment of patients suffering a number of UV irradiation-induced skin diseases (e.g., inflammation, skin cancer, and chronic non-healing wounds).

Oligodendrogliomas are diffuse infiltrating gliomas of adulthood. The WHO 2007 classification criteria classifies oligodendrogliomas as well-differentiated tumors (Grade 2) and anaplastic tumors (Grade 3). Osteopontin is a sialic acid-rich phosphoglycoprotein extracellular matrix component and it is an immune marker shown to be associated with parameters such as grade and recurrence in different tumors. The purpose of this study was to investigate the relationship between osteopontin and histological grades, recurrence and survival in the oligodendroglioma cases by analyzing its expression rates. Twenty-seven cases of oligodendroglioma operated at the Turkish Ministry of Health Ankara Dışkapı Education and Research Hospital, Brain Surgery Clinic between 2008 and 2011 and diagnosed at our hospitals’ pathology clinic were included in the study. Fourteen cases were diagnosed as low-grade (Grade 2, WHO 2007) oligodendroglioma and 12 as high-grade (anaplastic, Grade 3, WHO 2007), and one case was evaluated as glioblastoma with oligodendrogliomal component (Grade 4, WHO 2007). Osteopontin staining density was below the 25% in 10 cases. Nine out of those 10 cases were low-grade oligodendroglioma and one was high-grade. Immunostaining below the 25% was observed in the remaining five low-grade cases. On the basis of histological grade and expressions of osteopontin, density of staining was significantly higher in the high-grade tumors (p, ÖzetOligodendrogliom erişkin dönemin yaygın infiltratif bir gliomudur. DSÖ 2007 sınıflaması oligodendrogliomları iyi differensiye tümörler (Derece II) ve anaplastik tümörler (Derece III) olmak üzere iki derecede sınıflandırır. Osteopontin ekstrasellüler matriks ilişkili sialik asitten zengin fosfoglikoprotein yapısında integrin bağlayan bir protein olup, farklı tümörlerde derece ve rekürens gibi parametreler ile ilişkili olduğu yönünde veriler ortaya konmuş olan bir immün belirteçtir. Bu çalışmamızda Oligodendrogliomlarda Osteopontin ekspresyon oranlarını immünohistokimyasal olarak değerlendirerek histolojik derece, rekürens ve sağ kalım ile ilişkisini araştırmayı amaçladık. S.B Ankara Dışkapı Eğitim Araştıma Hastanesi Beyin Cerrahisi Kliniği tarafından 2008- 2011 yılları arasında ameliyat edilen ve hastanemiz patoloji kliniğinde tanı konulmuş olan toplam 27 oligodendrogliom olgusu çalışma kapsamına alındı. Bu değerlendirmede olguların 14 tanesi düşük dereceli (Derece 2, DSÖ 2007), 12 tanesi yüksek dereceli (Anaplastik, Derece 3, DSÖ 2007) oligodendrogliom olarak tanı alırken, 1 olgu Oligodendroglial komponentli Glioblastom (Derece 4, DSÖ 2007) olarak değerlendirildi. Olguların 10 tanesinde Osteopontin ile boyanma yaygınlığı %25’in altında izlendi. Bu 10 olgunun 9 tanesi düşük dereceli oligodendrogliom, 1 tanesi yüksek dereceli oligodendrogliomdu. Düşük dereceli olgulardan geri kalan 5 tanesinde %25’in altında immün boyanma izlendi. Histolojik derece ve Osteopontin ekspresyonları göz önüne alındığında yüksek dereceli tümörlerde boyanma yaygınlığı ve boyanma yoğunluğu düşük dereceli tümörlerle karşılaştırıldığında belirgin olarak yüksek düzeyde saptandı (p

Objective— Elastin is the major structural extracellular matrix component of the arterial wall that provides the elastic recoil properties and resilience essential for proper vascular function. Elastin-derived peptides (EDP) originating from elastin fragmentation during vascular remodeling have been shown to play an important role in cell physiology and development of cardiovascular diseases. However, their involvement in thrombosis has been unexplored to date. In this study, we investigated the effects of EDP on (1) platelet aggregation and related signaling and (2) thrombus formation. We also characterized the mechanism by which EDP regulate thrombosis. Approach and Results— We show that EDP, derived from organo-alkaline hydrolysate of bovine insoluble elastin (kappa-elastin), decrease human platelet aggregation in whole blood induced by weak and strong agonists, such as ADP, epinephrine, arachidonic acid, collagen, TRAP, and U46619. In a mouse whole blood perfusion assay over a collagen matrix, kappa-elastin and VGVAPG, the canonical peptide recognizing the elastin receptor complex, significantly decrease thrombus formation under arterial shear conditions. We confirmed these results in vivo by demonstrating that both kappa-elastin and VGVAPG significantly prolonged the time for complete arteriole occlusion in a mouse model of thrombosis and increased tail bleeding times. Finally, we demonstrate that the regulatory role of EDP on thrombosis relies on platelets that express a functional elastin receptor complex and on the ability of EDP to disrupt plasma von Willebrand factor interaction with collagen. Conclusions— These results highlight the complex nature of the mechanisms governing thrombus formation and reveal an unsuspected regulatory role for circulating EDP in thrombosis.

The interaction between epithelial tumor cells and their surrounding stroma is important in tumor progression and metastasis. This is accomplished through a number of transmembrane receptors that interact with stromal extracellular matrix molecules. One of these receptors, CD44, binds to extracellular matrix component hyaluronic acid (HA). The purpose of this study was to evaluate the significance of HA, CD44s, and CD44v6 in benign, hyperplastic, atypical, and malignant endometrial epithelia. Archival paraffin-embedded cell blocks from proliferative endometrium (n = 11), secretory endometrium (n = 12), simple hyperplasia (n = 13), complex hyperplasia without atypia (n = 9), complex hyperplasia with atypia (n = 17), and adenocarcinoma (n = 21) were stained for HA, CD44s, and CD44v6. HA was detected throughout the normal menstrual cycle but was more intense during the secretory phase. Only during the secretory phase was CD44s expressed in the stromal cells in 11 cases (92%), whereas CD44v6 was detected in glandular epithelium in 9 (75%). CD44s was expressed in the glandular epithelium in 2 (15%) cases of simple hyperplasia, 4 (44%) of complex hyperplasia without atypia, 14 (82%) of complex hyperplasia with atypia, and in 16 (76%) of adenocarcinoma. CD44v6 was expressed in the glandular epithelium in 1 (11%) case of complex hyperplasia without atypia, 17 (100%) cases of complex hyperplasia with atypia, and in 18 (86%) cases of adenocarcinoma, but in none of the cases of simple hyperplasia. The endometrial stromal cells expressed CD44v6 in 1 (8%) case of simple hyperplasia, 6 (67%) of complex hyperplasia without atypia, 8 (47%) of complex hyperplasia with atypia, and in 3 (14%) of adenocarcinoma. We concluded that in the normal menstrual cycle, the timing of peak staining of HA and CD44s in the stroma and the up-regulation of CD44v6 in secretory glands are coincident with the period in which the endometrium is most receptive to embryo implantation. HA is more abundant in the stroma adjacent to the tumor, suggesting that interactions between tumor cells and stromal HA promote tumorigenesis. With progression from hyperplasia and with increasing atypia to adenocarcinoma, levels of stromal HA, glandular CD44v6, and glandular and stromal CD44s all increase. Thus, HA and CD44 are both involved in the development and progression of endometrial cancer.

Various invasive pathogens attach to host tissues via the extracellular matrix component laminin, the major glycoprotein found within basement membranes. Previous investigations identified the laminin-binding adhesin Tp0751 within the spirochete bacteriumTreponema pallidum. In the current study, Tp0751 was shown to attach to a variety of laminin isoforms that are widely distributed throughout the host, including laminins 1, 2, 4, 8, and 10. Such universal attachment is conducive for an adhesin present within a highly invasive pathogen that encounters a variety of tissue sites during the course of infection. Additional studies systematically identified the amino acid residues within Tp0751 that contribute to laminin binding using synthetic peptides designed from the mature protein sequence. The minimum laminin-binding region of the adhesin was localized to 10 amino acids; peptides containing these residues inhibited attachment of Tp0751 andT. pallidumto laminin. Further, Tp0751-specific antibodies inhibited attachment ofT. pallidumto laminin. This study furthers our knowledge of the interaction ofT. pallidumwith laminin, an association that is proposed to facilitate bacterial traversal of basement membranes and subsequent entry into the circulation and tissue invasion. As such, these investigations will reveal new targets for possible prevention of bacterial dissemination and establishment of chronic infection.

During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

Targeting with radionuclide labelled substances that bind specifically to the epidermal growth factor receptor, EGFR, is considered for intracavitary therapy of EGFR-positive glioblastoma multiforme, GBM. Relevant literature is reviewed and examples of EGFR expression in GBM are given. The therapeutical efforts made so far using intracavitary anti-tenascin radionuclide therapy of GBM have given limited effects, probably due to low radiation doses to the migrating glioma cells in the brain. Low radiation doses might be due to limited penetration of the targeting agents or heterogeneity in the expression of the target structure. In this article we focus on the possibilities to target EGFR on the tumour cells instead of an extracellular matrix component. There seems to be a lack of knowledge on the degree of intratumoral variation of EGFR expression in GBM, although the expression seemed rather homogeneous over large areas in most of the examples (n=16) presented from our laboratory. The observed homogeneity was surprising considering the genomic instability and heterogeneity that generally characterises highly malignant tumours. However, overexpression of EGFR is, at least in primary GBMs, one of the steps in the development of malignancy, and tumour cells that lose or downregulate EGFR will probably be outgrown in an expanding tumour cell population. Thus, loss of EGFR expression might not be the critical factor for successful intracavitary radionuclide therapy. Instead, it is likely that the penetration properties of the targeting agents are critical, and detailed studies on this are urgent.

Adhesive properties of cells undergoing morphogenetic rearrangements can be regulated either at the cellular level or by altering the environment in which rearrangements occur. Here, we describe the identification of a mutation ( my F11 ) in the mouse extracellular matrix component Frem2, and provide evidence that suggests Frem2 expression creates an environment conducive to morphogenetic events. Loss of Frem2 function results in defects in developmental events associated with morphogenetic rearrangements of the vasculature and of tissues arising from all germ layers. The Frem2 transcript is restricted both spatially and temporally and appears in advance of cell rearrangement events. Thus, expression of Frem2 may dynamically alter the extracellular matrix to provide a substrate for cell migration and rearrangements during embryogenesis.

Pterygia are inflammatory, invasive, and proliferative lesions of the human ocular surface in which the matrix metalloproteinase (MMP) collagenase-1 (MMP-1) is highly expressed. Pterygia development may involve MMP-1 activity against interstitial fibrillar collagen, an abundant extracellular matrix component of the cornea, and its induction by ultraviolet light (UVB). We examined the pathways responsible for enhanced expression of MMP-1 in pterygium epithelial cells after UVB exposure and/or treatment with chemical inhibitors of mitogen-activated protein kinases or epidermal growth factor receptor. The induction of MMP-1 by UVB was comparable to that mediated by heparin-binding epidermal growth factor-like growth factor and epidermal growth factor. The epidermal growth factor receptor inhibitor PD153035 partially blocked the UVB-mediated induction of MMP-1 and totally abrogated its production after stimulation with either heparin-binding epidermal growth factor-like growth factor or epidermal growth factor. UVB exposure enhanced the phosphorylated form of ERK1/2 in a time-dependent manner whereas the ERK1/2 inhibitor PD98059 decreased this induction by at least fivefold. Transcripts for c-jun and c-fos were detected as early as 2 hours after UVB exposure and were suppressed by PD98059. The identification of a specific intracellular signaling pathway responsible for the enhanced production of a key enzyme that denatures intact fibrillar collagen has important implications for understanding the pathophysiology and future therapy for pterygia.

Interactions of tumour and stromal cells influence tumour cell proliferation and differentiation, stromal cell phenotypic transdifferentiation and secretion of extracellular matrix (ECM) components. In this study, we established a monolayer and a three-dimensional cell-to-cell interaction model between canine mammary stromal cells and human colonic carcinoma cell lines (Caco-2 and HT-29) to investigate mutual paracrine effects of tumour cells and stromal cells on (i) tumour cell differentiation, (ii) production of ECM components and (iii) phenotypic transdifferentiation of stromal cells. We showed that when Caco-2 or HT-29 cells are cultured in collagen gels, they form a few small solid cell clusters with no lumina, but when cocultured with stromal cells, the tumour cells formed glandular structures with central lumina. This fibroblast-induced organization and differentiation of Caco-2 cells (not HT-29 cells) appeared to be mediated by transforming growth factor-beta (TGF-beta). Culturing of stromal cells, Caco-2 cells or HT-29 cells alone in both monolayers and gels resulted in weak tenascin-C expression in stromal cells and HT-29 cells and no expression in the Caco-2 cells. Coculturing of stromal cells with tumour cells resulted in increased tenascin-C expression in the stromal cells and HT-29 cells and induced expression of tenascin-C in the Caco-2 cells. This induction and increased expression of tenascin-C appeared to be mediated by TGF-beta. Culturing of stromal cells, Caco-2 cells or HT-29 cells alone on monolayers and in gels resulted in a weak expression of chondroitin sulfate (CS), chondroitin-6-sulfate (C-6-S) and versican in stromal cells and no expression in Caco-2 and HT-29 cells. Coculturing of stromal cells with tumour cells on monolayers and in gels resulted in increased CS, C-6-S and versican expression in stromal cells. This tumour cell-induced expression of CS, C-6-S and versican appeared to be mediated by TGF-beta and platelet-derived growth factor (PDGF). Coculturing of Caco-2 and HT-29 and stromal cells promoted the transdifferentiation of stromal cells into myofibroblasts, and this appeared to be mediated by TGF-beta. These results suggest that TGF-beta and PDGF are part of a paracrine system involved in stromal-epithelial cell interaction important in stromal cell differentiation and ECM component production.

The construction of renal glomerular tissue has provided an important tool not only for the understanding of renal physiology and pathology in blood ultrafiltration and cell dysfunction, but also in the application of tissue engineering to glomeruli regeneration and nephritic therapy. In this study, a novel method to reconstruct glomerular tissue combining cultured cells on a collagen vitrigel scaffold is described. The method consists of two newly developed techniques, one to isolate glomerular epithelial and mesangial cells rapidly from kidney, which facilitates the prolongation of cell population doublings and allows a long-term cell culture without losing cellular features, and another to prepare a stable and thin transparent collagen gel membrane termed collagen vitrigel that can facilitate three-dimensional cultures for reconstructing an epithelial-mesenchymal model. By combining the two methods, we cocultured glomerular epithelial and mesangial cells on both surfaces of the collagen vitrigel by the manipulation of two-dimensional cultures, resulting in the successful reconstruction of a three-dimensional glomerular organoid. The coculture results showed that the collagen vitrigel maintains cell growth and cell viability for more than 1 month, and surprisingly, the epithelial layer demonstrated polarity formation, which usually appears in in vivo normal epithelial cells existing at the glomerular basement membrane, but seldom appears in epithelial cells cultured in vitro. Moreover, the coculture results showed that fibronectin, an extracellular matrix component, and integrin beta1, a receptor of fibronectin, were detected in high amounts on both cells, suggesting our collagen vitrigel can provide a suitable environment for cell-cell interactions that stabilize the cell structure and may contribute to the polarity formation of epithelial cells.

Hyaluronan, a major extracellular matrix component in the epidermis, has been shown to control keratinocyte proliferation and differentiation in vitro. We assayed hyaluronan and hyaluronan synthases (has1-3) in mouse epidermis during fetal development, postnatal life, and trauma reaction in vivo. Hyaluronan increased in the epidermis when keratinocytes started to stratify on day E15, remained high until birth, and then rapidly declined, with corresponding changes in the mRNA levels of has2 and has3. The hyaluronan in adult mouse epidermis mainly resided around the orifices of the hair follicles, and the overall concentration was about one order of magnitude lower than in adult human epidermis. In adult mice, epidermal trauma caused by tape stripping rapidly increased hyaluronan, leading to a 6-fold increase in epidermal hyaluronan on day 3 following trauma. The hyaluronan response was associated with a strong induction of has2 and has3 mRNA, slightly higher CD44 expression, and considerable epidermal hyperplasia. The data show that the pre- and postnatal fluctuations in epidermal hyaluronan content correlate with the expression levels of has2 and has3. Stimulated hyaluronan synthesis through upregulated has expression is an inherent feature of the keratinocyte activation triggered by tissue trauma, and presumably important for a proper healing response.

Bovine type I collagen (BIC), which is widely used as a fibrous extracellular matrix component in cell culture models, inhibits the progression of melanoma cell cycle via p27 up-regulation. BIC also induces nitric oxide synthase in macrophages through JunB/AP-1 and NF-κB activation. Given the previous observations, this study investigates the effect of BIC on the cell cycle progression and regulatory function of Raw264.7 macrophage cells and the responsible signaling pathways. Cell cycle analysis revealed that BIC completely suppressed proliferation of Raw264.7 cells with inhibition of the percentage of cells in the S phase and the reciprocal decrease in the G0/G1 phase. DNA synthesis was also inhibited by BIC, as evidenced by a decrease in the cellular incorporation of [3H]thymidine. The G1/S arrest induced by BIC was reversed by chemical inhibition of phosphatidylinositol 3-kinase (PI3-kinase) or overexpression of the p85 subunit of PI3-kinase. Either PD98059 or stable transfection with mitogen-activated protein kinase kinase-1 [MKK1(−)] or c-Jun N-terminal kinase 1 [JNK1(−)] also released the cell cycle arrest. Immunoblot analyses revealed that the levels of cyclins D1, A and B1 were partly or completely down-regulated by BIC, but cyclin E, p21 and p27 were minimally changed. Chemical inhibition and dominant negative mutant overexpression experiments revealed that either PI3-kinase inhibition or JNK1(−) transfection prevented the decreases in cyclin D1, A and B1 by BIC, indicating that the PI3-kinase and JNK1 pathways were associated with disruption of the cyclins. The pathway involving MKK1-extracellular signal-regulated kinase-1/2 (ERK1/2) was responsible for the suppression of cyclin A and B1, but not that of cyclin D1. The present study showed that BIC inhibited proliferation of Raw264.7 cells and that the pathways involving PI3-kinase and mitogen-activated protein kinases regulate the cell cycle arrest.

Transformation of chick embryo fibroblasts (CEFs) by the v-Jun oncoprotein correlates with a downregulation of the alpha 2 (I) collagen gene. To investigate whether this gene constitutes a direct target of v-Jun, an analysis of a large proximal fragment of the promoter, extending from position -1080 to +109, was performed. Transient transfections with -1080/+109 and deleted derivatives revealed that a short proximal fragment, -433/+11, is the target for repression by v-Jun. Extensive analysis, conducted in CEFs and in Sp1/3-deficient Drosophila SL2 cells, further showed that (i) high constitutive activity of -433/+11 requires a direct binding of the ubiquitous Sp1 and/or Sp3 transcription factors acting on two distinct motifs, that is, a proximal TCC-rich region and an upstream GC box, and that (ii) repression by v-Jun does not require any direct binding of the oncoprotein to the DNA, but an indirect binding within a v-Jun-Sp1/3-DNA chromatin-associated complex. This situation is reminiscent of a situation previously reported with the tata-less, SPARC (secreted protein, acidic, and rich in cysteine) target promoter that regulates the expression of another extracellular matrix component in the same model of cell transformation. Taken together, these data reinforce the view that, at least in CEFs, v-Jun downregulates a family of direct target genes by binding to the DNA indirectly through Sp1/3.

After the first description of the myofibroblast in granulation tissue of an open wound by means of electron microscopy, as an intermediate cell between the fibroblast and the smooth muscle cell, the myofibroblast has been identified both in normal tissues, particularly in locations where there is a necessity of mechanical force development, and in pathological tissues, in relation with hypertrophic scarring, fibromatoses and fibrocontractive diseases as well as in the stroma reaction to epithelial tumors. It is now accepted that fibroblast/myofibroblast transition begins with the appearance of the protomyofibroblast, whose stress fibers contain only beta- and gamma-cytoplasmic actins and evolves, but not necessarily always, into the appearance of the differentiated myofibroblast, the most common variant of this cell, with stress fibers containing alpha-smooth muscle actin. Myofibroblast differentiation is a complex process, regulated by at least a cytokine (the transforming growth factor-beta1), an extracellular matrix component (the ED-A splice variant of cellular fibronectin), as well as the presence of mechanical tension. The myofibroblast is a key cell for the connective tissue remodeling that takes place during wound healing and fibrosis development. On this basis, the myofibroblast may represent a new important target for improving the evolution of such diseases as hypertrophic scars, and liver, kidney or pulmonary fibrosis.

Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-beta-mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-beta activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-beta. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-beta activity and disease, suggesting new therapeutic strategies for osteoarthritis.

For some time, estrogen has been suspected to play a negative role in anterior cruciate ligament (ACL) fibroblast biosynthesis; however, reports on this issue have been controversial. In a recent study, our group demonstrated a negative combined effect of estrogen and mechanical loading on the gene expression of major extracellular matrix component molecules in ACL fibroblasts.

We investigated laminin, an important extracellular matrix component, to elucidate mechanisms of invasion and metastasis in colorectal cancer, and whether preoperative serum laminin is a predictive marker of high-risk groups.We measured preoperative serum laminin levels using a two-step sandwich enzymeimmunassay (EIA) method in 205 patients with colorectal cancer, 109 with colon cancer and 96 with rectal cancer, 52 with hepatic metastases, and 153 with no hepatic metastases.Mean serum laminin in patients with colon cancer was 606.3+/-260.2 ng/ml, significantly higher than that of 258.0+/-92.0 ng/ml in normal controls (P0.0001). The positive rate was higher at 89.3% for laminin vs. 38.0% for carcinoembryonic antigen (CEA) and 19.5% for CA19-9. Mean serum laminin in patients with hepatic metastases was 668.0+/-274.7 ng/ml, significantly higher than that of 585.2+/-252.5 ng/ml in patients without hepatic metastases (P=0.0472). Survival rates were significantly lower in the high (or = 520 ng/ml) than in the low laminin group (350 ng/ml; P=0.0451). Univariate and multivariate analysis, using Cox's proportional hazard regression model, showed serum laminin is an independent prognostic factor in colorectal cancer, along with hepatic, pulmonary and peritoneal metastases.Preoperative serum laminin levels are a useful predictive marker of high-risk groups in colorectal cancer.

We have shown previously that components of the extracellular matrix (ECM) modulate neuronal development. Here, we searched for additional ECM elements that might play roles in retinal histogenesis and identified a secreted glycoprotein that is heavily expressed in the retina. This molecule, named by others Wnt Inhibitory Factor-1 (WIF-1), is expressed during and after the period of rod photoreceptor morphogenesis in the mouse. We show that a potential WIF-1 ligand, Wnt4, as well as a potential Wnt4 receptor, fzd4, and a potential Wnt4 coreceptor, LRP6, are expressed in the region of, and at the time of, rod photoreceptor genesis. WIF-1 and Wnt4 are coexpressed during retinal development and bind to each other; therefore, they are likely to interact during rod production. WIF-1 protein inhibits rod production, and anti-WIF-1 antibodies increase rod production; in contrast, Wnt4 promotes rod production. Together, these data suggest that WIF-1 and Wnt4, both components of the ECM, regulate mammalian photoreceptor development.

Prognosis and survival of patients with hepatocellular carcinoma (HCC) is still very poor, and no therapies are currently available to inhibit tumour growth and metastases. Recently, we reported that the expression of an extracellular matrix component (ECM), namely Laminin-5 (Ln-5), is directly related to poor prognosis in HCC patients. The aim of our study is to investigate the preclinical effect of gefitinib in an in vitro HCC model. We found that the IC(50) of gefitinib in HCC cells ranged from 0.7 to 10.0 muM, whereas Ln-5 inhibited the activity of gefitinib in a dose-dependent manner. Complete inhibition of phosphorylated (p)-EGFR (epidermal growth factor receptor) was obtained within 6 h exposure to gefitinib and complete restoration of the receptor status was obtained after 24 h. A downstream effect yields a decrease in p-Akt and p-Erk 1/2. The addition of exogenous Ln-5 has no effect on p-EGFR, whereas it restores p-Erk 1/2 and p-Akt. Consistently, Ln-5 induces recovery of HCC cells from Gefitinib-induced apoptosis. In conclusion, gefitinib inhibits HCC cell growth and we report for the first time that Ln-5, but not other ECM molecules, reduces the ability of gefitinib to inhibit cell growth via Akt. As patients with HCC have different Ln-5 expression levels, these results may help to better understand which patients might benefit from gefitinib treatment.

Because the hyperpolarization-activated cation-selective current I(h) makes important contributions to neural excitability, we examined its long-term regulation by vitronectin, an extracellular matrix component commonly elevated at injury sites and detected immunochemically in activated microglia. Focusing on mouse hippocampal pyramidal neurones in organotypic slice cultures established at postnatal day 0 or 1 and examined after 3-4 days in vitro, we observed differences in the amplitude and activation rate of I(h) between neurones in naive and vitronectin-exposed slices (10 microg ml(-1) added to serum-free medium), and between neurones in slices derived from wild-type and vitronectin-deficient mice. The potassium inward rectifier I(K(ir)), activated at similar voltages to I(h), was not affected by vitronectin. In CA1, differences in I(h) amplitude primarily reflected changes in maximum conductance (G(max)): a 23.3% increase to 3.18 +/- 0.64 nS from 2.58 +/- 0.96 nS (P 0.05) or vitronectin deficiency (-83.8 +/- 3.1 mV versus -82.0 +/- 2.9 mV in wild-type neurones; P > 0.05). In CA3 neurones, changes in I(h) reflected differences in both G(max) and V(1/2): in vitronectin-exposed neurones there was a 35.4% increase in G(max) to 1.30 +/- 0.49 nS from 0.96 +/- 0.26 nS (P < 0.01), and a +3.0 mV shift in V(1/2) to -89.8 mV from -92.8 mV (P < 0.05). The time course of I(h) activation could be fitted by the sum of two exponential functions, fast and slow. In both CA1 and CA3 neurones the fast component amplitude was preferentially sensitive to vitronectin, with its relatively larger contribution to total current in vitronectin-exposed cells contributing to the acceleration of I(h) activation. Further, HCN1 immunoreactivity appeared elevated in vitronectin-exposed slices, while HCN2 levels appeared unaltered. We suggest that vitronectin-stimulated increases in I(h) may potentially affect excitability under pathological conditions.

Fibronectin (FN) is the main extracellular matrix component in glomerulosclerotic lesions. There are different FN isoforms that result from alternative splicing at the EDA and EDB regions of FN mRNA. Increased inclusion of EDA and EDB, which can be elicited by TGFbeta, may be conducive to the development of glomerulosclerosis (GS). TGFbeta and IL-4 have previously been shown to play a role in the development of GS. In this study, the mRNA splicing patterns for EDA+ and EDB+ fibronectin were investigated in vivo in various experimental sclerotic glomerulopathies, in vitro in rat mesangial cells (MC) that were stimulated by TGFbeta or transfected with IL-4, and in human kidney biopsies with GS from patients with various kidney diseases. Analysis of glomerular FN mRNA demonstrated inclusion of both ED regions in rats with anti-Thy1 nephritis or chronic serum sickness and in mice with anti-GBM glomerulonephritis. Inclusion of both the EDA and EDB regions was associated with glomerular TGFbeta expression. In contrast, in mice with Th2-mediated graft-versus-host disease, a model for lupus nephritis, the FN transcripts included neither the EDA nor the EDB region, and renal TGFbeta expression was absent. Compared to normal MCs in culture, MCs transfected with IL-4 produced lower amounts of FN and demonstrated less EDA inclusion, while MC that had been treated with TGFbeta showed increased production of FN and more EDA inclusion. Renal biopsies from patients with renal diseases, except those taken from patients with lupus nephritis, showed higher TGFbeta levels, higher FN levels, and more EDA inclusion than controls. TGFbeta may be a key player in the development of GS by inducing local FN production and alternative splicing of FN mRNA. In lupus glomerulonephritis, in which the involvement of TGFbeta in GS is less prominent, Th2 cytokines such as IL-4 probably account for increased intrarenal collagen synthesis and subsequent FN accumulation from the circulation. In conclusion, neither alternative FN splicing, nor a high transcription level of TGFbeta, appears to be a general prerequisite for the development of GS.

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide–dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, namely impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus. This developmental abnormality was accounted for by failed activation of myogenic regulatory factors that normally drive esophageal muscle transdifferentiation. Therefore, these findings identify collagen XIX as the first structural determinant of sphincteric muscle function, and as the first extrinsic factor of skeletal myogenesis in the murine esophagus.

Human pathogenic viruses are increasingly common vehicles for the development of novel therapeutic agents against malignant glioma (Gromeier, 2001; Gromeier and Wimmer, 2001; Gromeier et al., 2000a). The first reported incidences of viral oncolysis were due to nonintended exposure to naturally occurring viruses or after administration of live attenuated vaccine strains (DePace, 1912; reviewed in Sincovics and Horvath [1993]). New prospects for genetically manipulating viruses have opened possibilities for increasing the tumor specificity and lowering the toxicity of oncolytic viral agents (reviewed in Gromeier [2002]). These efforts have given rise to oncolytic adenoviruses (Bischoff et al., 1996), herpesviruses (Martuza et al., 1991), reoviruses (Coffey et al., 1998), vesicular stomatitis virus (Stojdl et al., 2000), and most recently, polioviruses (Gromeier et al., 2000b). The antineoplastic effects of oncolytic viruses are subject to multifaceted interactions with the host cell. The primary prerequisite for viral oncolysis is expression of cellular receptors mediating viral entry in malignant cell types. In preparation for clinical applications using oncolytic viruses, analysis of viral receptor expression in the target tissue is highly desirable in order to select tumor types and patients most likely to respond favorably to therapeutic intervention. The critical influence of virus tropism determinants on therapeutic efficacy has recently become evident with oncolytic adenoviruses and adenovirus-derived gene delivery vehicles directed to malignant tissues. Targeting of adenoviral vectors to malignant tissues is greatly limited by low or absent expression of a critical adenovirus attachment and entry molecule, the Coxsackie adenovirus receptor (CAR3; Asaoka et al., 2000; Bergelson et al., 1997; Douglas et al., 2001; Li, D., et al., 1999; Li, Y., et al., 1999; Tomko et al., 1997). Moreover, CAR expression levels in established cell lines frequently used for pre-clinical testing of these agents did not correspond to expression in tumor tissues. The failure of adenovirus-based therapeutic agents to reach and infect the intended target due to low CAR expression has inspired efforts to alter receptor specificity of adenoviruses in order to broaden its tropism (reviewed in Curiel [1999]). Cellular entry of poliovirus appears to be mediated by a single cell-surface molecule, the immunoglobulin superfamily (IGSF) member CD155 (Mendelsohn et al., 1989). Although a physiological function for CD155 is unknown, the protein binds specifically to the extracellular matrix component vitronectin (Lange et al., 2001). Transcriptional regulation of the CD155 gene is tightly controlled (Solecki et al., 1999, 2000). In accordance with the restricted tropism of poliovirus for anterior horn spinal cord motor neurons, activity of the CD155 promoter was restricted to the floor plate, notochord, and the anterior spinal cord (Gromeier et al., 2000a). Confirming the activity pattern of the CD155 promoter in the developing spinal cord, morphogenic factors active in the floor plate and notochord — the transcription factors sonic hedgehog (shh) and its downstream gli effectors—strongly activate the CD155 promoter and induce CD155 expression (Solecki et al., 2002). Both shh and gli transcription factors have been implicated in the oncogenesis of neuroectodermal tumors (Goodrich et al., 1997; Kinzler et al., 1987). Thus, the role of shh and gli transcription factors in CD155 gene regulation suggested that CD155 expression may occur ectopically in neuroectodermal malignancies. TagE4, a rodent member of the nectin family recently proposed to be the true rodent CD155 homolog (Baury et al., 2001), is ectopically upregulated in colon and mammary tumors in mice (Denis, 1998). CD155 has been reported to be overexpressed in human colorectal tumors (Masson et al., 2001). Evidence for CD155 expression in neuroectodermal tumors stems mainly from studies of neuroectodermal tumor cell lines that are susceptible to oncolytic poliovirus-based agents (Gromeier et al., 2000b). However, no systematic CD155 expression analysis has been performed on CNS tumor tissues. Determination of CD155 expression levels is notoriously difficult because of the exceedingly low expression levels, even in cell lines routinely used for the propagation of poliovirus and in confirmed sites of expression, such as human spinal cord motor neurons (Bernhardt et al., 1994). To address this problem, we have generated new polyclonal α-CD155 antibodies directed against a recombinant soluble segment of the antigen. Combining solid-phase antigen capture procedures with Western blot detection of CD155 using our new antibodies proved to be an efficient means of conducting comprehensive and reliable expression analyses in tissue samples and cell lines. Analysis of 6 cases indicated that CD155 overexpression is commonly associated with high-grade malignant glioma (HGL). CD155 expression levels in tumor tissues corresponded to those in primary tissue cultures derived from the tumors. Furthermore, CD155 expression in primary glioma explant cultures was equivalent to that found in established glioma cell lines used in preclinical evaluations of oncolytic poliovirus recombinants. In accordance with consistent expression of CD155, primary explant cultures of glioma tissue from all 6 cases displayed exquisite sensitivity to the oncolytic poliovirus recombinant PVS-RIPO. We have thus provided evidence for aberrant expression of a molecular target for oncolytic poliovirus recombinants in HGL. Our findings demonstrate that individual tumor characterization for viral cellular receptor expression can aid in the identification of targets most likely to respond to oncolytic virus therapy.

Over 30 genes responsible for human hereditary hearing loss have been identified during the last 10 years. The proteins encoded by these genes play roles in a diverse set of cellular functions ranging from transcriptional regulation to K+ recycling. In a few cases, the genes are novel and do not give much insight into the cellular or molecular cause for the hearing loss. Among these poorly understood deafness genes is DFNA5. How the truncation of the encoded protein DFNA5 leads to an autosomal dominant form of hearing loss is not clear. In order to understand the biological role of Dfna5, we took a reversegenetic approach in zebrafish. Here we show that morpholino antisense nucleotide knock-down of dfna5 function in zebrafish leads to disorganization of the developing semicircular canals and reduction of pharyngeal cartilage. This phenotype closely resembles previously isolated zebrafish craniofacial mutants including the mutant jekyll. jekyll encodes Ugdh [uridine 5′-diphosphate (UDP)-glucose dehydrogenase], an enzyme that is crucial for production of the extracellular matrix component hyaluronic acid (HA). In dfna5 morphants, expression of ugdh is absent in the developing ear and pharyngeal arches, and HA levels are strongly reduced in the outgrowing protrusions of the developing semicircular canals. Previous studies suggest that HA is essential for differentiating cartilage and directed outgrowth of the epithelial protrusions in the developing ear. We hypothesize that the reduction of HA production leads to uncoordinated outgrowth of the canal columns and impaired facial cartilage differentiation.

More than 1.5 million percutaneous coronary interventions are performed annually. The most important long-term complication is restenosis caused by inflammation, neointimal vascular smooth muscle cell (VSMC) hyperplasia, and excess accumulation of extracellular matrix. Delineating mechanisms that contribute to inflammation, neointimal hyperplasia, and constrictive vascular remodeling will, therefore, improve and expand the options for prophylactic and therapeutic percutaneous coronary interventions for the growing population of patients with arterial disease. See accompanying article on page e9 Hyaluronan is a pericellular and extracellular matrix component of normal vasculature located throughout vessels from the luminal surface of the endothelium, to most prominently in the adventitia. The marked upregulation of hyaluronan production and accumulation in atherosclerotic lesions and in the neointima of restenotic vessels has generated great interest as to its role in vascular disease. Endothelial pericellular hyaluronan promotes leukocyte adhesion and transmigration, whereas hyaluronan binding to CD44 on leukocytes regulates inflammatory gene expression. Platelet-mediated hyaluronan cleavage generates bioactive hyaluronan fragments that stimulate leukocyte production of cytokines and chemokines. Moreover, hyaluronan is involved in the processes underlying the tissue response after vascular injury, in particular, VSMC proliferation and migration. In most cases, the implication of hyaluronan in vascular disease has been based on the circumstantial evidence of elevated hyaluronan levels in vascular lesions or deduced from deleting or blocking hyaluronan receptors (such as CD44 and receptor for HA-mediated motility). Designing approaches to more directly define the role of hyaluronan in vascular disease has been difficult in view of the multitude of synthases (HAS1, HAS2, and HAS3) and multiple hyaluronidases involved in its metabolism. …

Hyaluronic acid (hyaluronan) is a ubiquitous extracellular matrix component, and present at high concentrations in skin, joints and cornea. In the skin, it is synthesized primarily by dermal fibroblasts and by epidermal keratinocytes. Hyaluronic acid usually exists as a high molecular mass (600,000-1,000,000) and non-sulfated glycosaminoglycan composed of a disaccharide unit of [bond]3GlcNAc beta 1[bond]4GlcA beta 1[bond]. Hyaluronic acid has been widely used not only for osteoarthritis and ophthalmology but also for cosmetics for skin care. To examine the biological activities of hyaluronic acid, we have to accurately determine the quantity and molecular masses in biological samples. We review recent development in the analysis of hyaluronic acid having various molecular sizes using electrophoretic and chromatographic techniques. Recently, interactions between hyaluronic acid oligomers and hyaluronic acid-binding proteins have attracted the interest for understanding the biological functions. We show some interesting reports on biological interactions of hyaluronic acid and its oligomers with some proteins.

The fibrous cap of a lipid-containing atherosclerotic plaque consists of collagen produced by arterial smooth muscle cells (SMCs) of synthetic phenotype. A thick cap protects the lipid-rich core, whereas a thin cap predisposes it to rupture, with ensuing acute clinical complications, such as myocardial infarction. Among the pathological mechanisms leading to plaque weakening and rupture, one possibility is loss of the matrix-synthesizing SMCs. Indeed, caps of ruptured coronary plaques contain a reduced number of SMCs. In contrast, in such lesions, the number of activated inflammatory cells, such as mast cells, is increased, suggesting that they may regulate the SMC number. We have shown that heparin proteoglycans secreted by activated mast cells can efficiently inhibit proliferation of SMCs in vitro and reduce their ability to produce collagen. Chymase, a neutral serine protease secreted by activated mast cells, can also inhibit SMC-mediated collagen synthesis by a transforming growth factor-beta-dependent and -independent mechanism, and moreover, cause degradation of the collagen matrix by activating latent interstitial collagenase (MMP-1). Furthermore, chymase can induce SMC apoptosis by degrading the extracellular matrix component fibronectin necessary for SMC adhesion, with subsequent disruption of focal adhesions and loss of outside-in survival signaling. Thus, activated mast cells may participate in the weakening and rupture of atherosclerotic plaques by secreting mediators, such as heparin proteoglycans and chymase, which affect the growth, function and death of arterial SMCs.

Glypican-1 is an extracellular matrix component found by microsequencing in a medium conditioned by cultured rat-sciatic nerves (CM). This CM was concentrated by ultrafiltration and fractionated by quaternary ammonium chromatography, followed by Hi-Trap blue affinity chromatography to obtain the active fraction B1.2. Previously, we have reported a 54 kDa neuregulin (NRG) in the same B1.2 fraction [Villegas et al., Brain Res. 852 (2001) 304]. The effect of Glypican-1 on the neuron-like differentiation of PC12 cells was investigated by immunoprecipitation, Western blot and cellular image analysis. Removal of glypican-1 by immunoprecipitation with increasing concentrations of specific antibodies revealed a gradual decrease of the differentiation activity of fraction B1.2, which paralleled the results obtained by removal of the 54 kDa NRG protein. Colorless native electrophoresis and Western blot analysis was used to identify a glypican-1-NRG protein complex, which could be afterwards separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis into its individual components. Additionally, it was demonstrated that glypican-1, in cooperation with the 54 kDa NRG, is involved in the neuronal-like differentiation of PC12 cells and could play an important role on the regeneration responses of peripheral nerves.

Elastin is synthesized and secreted by vascular smooth muscle cells and is the major extracellular matrix component deposited in the arterial wall. When last reviewed by this journal in 1994, the link between elastin and a rare occlusive vascular disease had just been established. Since that time, it has become increasingly clear that elastin is a critical autocrine factor that maintains vascular homeostasis through a combination of biomechanical support and biologic signaling. This review examines the complexity of elastin-smooth muscle cell interactions, and how new insights may impact understanding of the pathogenesis and treatment of vascular disease.