Showing papers on "Extracellular matrix component published in 2003"

Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-kappa B (RelB/p50) in myeloma cells.

Abstract: The microenvironment has been shown to influence tumor cell phenotype with respect to growth, metastasis, and response to chemotherapy. We have utilized oligonucleotide microarray analysis to identify signal transduction pathways and gene products altered by the interaction of myeloma tumor cells with the extracellular matrix component fibronectin that may contribute to the antiapoptotic phenotype conferred by the microenvironment. Genes with altered expression associated with fibronectin cell adhesion, either induced or repressed, were numerically ranked by fold change. FN adhesion repressed the expression of 469 gene products, while 53 genes with known coding sequences were induced by twofold or more. Of these 53 genes with two fold, or greater increase in expression, 11 have been reported to be regulated by the nuclear factor-kappa B (NF-kappa B) family of transcription factors. EMSA analysis demonstrated NF-kappa B binding activity significantly increased in cells adhered to fibronectin compared to cells in suspension. This DNA binding activity consisted primarily of RelB-p50 heterodimers, which was distinct from the NF-kappa B activation of TNF alpha. These data demonstrate the selectivity of signal transduction from the microenvironment that may contribute to tumor cell resistance to programmed cell death.

Extracellular Polysaccharides Associated with Thin Aggregative Fimbriae of Salmonella enterica Serovar Enteritidis

Abstract: Lipopolysaccharide (LPS) O polysaccharide was identified as the principle factor impeding intercellular formation of intact thin aggregative fimbriae (Tafi) in Salmonella enterica serovar Enteritidis. The extracellular nucleation-precipitation assembly pathway for these organelles was investigated by quantifying fimbrial formation between deltaagfA (AgfA recipient) and deltaagfB (AgfA donor) cells harboring mutations in LPS (galE::Tn10) and/or cellulose (deltabcsA) synthesis. Intercellular complementation could be detected between deltaagfA and deltaagfB strains only when both possessed the galE mutation. LPS O polysaccharide appears to be an impenetrable barrier to AgfA assembly between cells but not within individual cells. The presence of cellulose did not restrict Tafi formation between cells. Transmission electron microscopy of w+ S. enterica serovar Enteritidis 3b cells revealed diffuse Tafi networks without discernible fine structure. In the absence of cellulose, however, individual Tafi fibers were clearly visible, appeared to be occasionally branched, and showed the generally distinctive appearance described for Escherichia coli K-12 curli. A third extracellular matrix component closely associated with cellulose and Tafi was detected on Western blots by using immune serum raised to whole, purified Tafi aggregates. Cellulose was required to tightly link this material to cells. Antigenically similar material was also detected in S. enterica serovar Typhimurium and one diarrheagenic E. coli isolate. Preliminary analysis indicated that this material represented an anionic, extracellular polysaccharide that was distinct from colanic acid. Therefore, Tafi in their native state appear to exist as a complex with cellulose and at least one other component.

Mast Cell Chymase Induces Smooth Muscle Cell Apoptosis by a Mechanism Involving Fibronectin Degradation and Disruption of Focal Adhesions

Abstract: Objective— Chymase released from activated mast cells has been shown to induce apoptosis of vascular smooth muscle cells (SMCs) in vitro. The proteolytic activity of chymase is essential for the proapoptotic effect, but the mechanism of chymase-induced apoptosis has remained unknown. Methods and Results— Here we show by means of FACS analysis, immunohistochemistry, and Western blotting that mast cell–derived chymase induces SMC apoptosis by a mechanism involving degradation of an extracellular matrix component, fibronectin (FN), with subsequent disruption of focal adhesions. The FN degradation products induced SMC apoptosis of similar magnitude and with similar changes in outside-in signaling, as did chymase. Sodium orthovanadate, an inhibitor of tyrosine phosphatases, inhibited the chymase-induced SMC apoptosis. Focal adhesion kinase (FAK), one of the key mediators of integrin–extracellular matrix interactions and cell survival, was rapidly degraded in the presence of chymase or FN degradation products. Loss of phosphorylated FAK (p-FAK) resulted in a rapid dephosphorylation of the p-FAK–dependent downstream mediator Akt. Conclusions— The results suggest that chymase-secreting mast cells can mediate apoptosis of neighboring SMCs through a mechanism involving degradation of pericellular FN and disruption of the p-FAK–dependent cell-survival signaling cascade.

Laminin-5 Chains Are Expressed Differentially in Metastatic and Nonmetastatic Hepatocellular Carcinoma

Abstract: Purpose: The purpose of this work was to study the expression of the extracellular matrix protein laminin-5 (Ln-5) in hepatocellular carcinoma (HCC), which is the fifth most frequent cancer and the third most common cause of tumor-related death in the world. The occurrence of metastasis is the main problem in HCC patients. Ln-5 is an extracellular matrix component that promotes adhesion and migration; it is present at the basement membrane and has recently been associated with cancer metastasis. Although Ln-5 has been shown to promote motility and scatter of rat liver cells, it has never been found in the liver. Experimental Design: We studied the expression and localization of the α3, β3, and γ2 chains of Ln-5 in 40 HCC patients. We analyzed tissue samples collected from the HCC primary nodule and from peritumoral and metastatic tissues. The presence of Ln-5 was investigated by immunohistochemistry, reverse transcription-PCR, and Northern blot analysis. The clinical outcome of the patients was evaluated over a 4-year follow-up period. Results: This study provides the first report that Ln-5 is present in the HCC primary nodule, but not in normal or peritumoral cirrhotic tissues. In particular, the γ2 chain is strongly associated with the occurrence of metastasis (96%; P < 0.001) and with worse prognosis. In peritumoral tissues, Ln-5 has been detected along the advancing edge of the metastatic nodule. Conclusions: Ln-5 is associated with a more metastatic phenotype of HCC, and its detection could be an important finding both as an unfavorable prognostic factor and as a diagnostic marker for detecting micrometastasis in peritumoral tissues.

Age-dependent iris abnormalities in collagen XVIII/endostatin deficient mice with similarities to human pigment dispersion syndrome.

Abstract: Purpose Collagen XVIII is expressed in ocular basement membranes (BMs) and inactivating mutations cause Knobloch syndrome, with several ocular abnormalities. In this study we investigated ocular structures in collagen XVIII/endostatin (Col18a1(-/-))-deficient mice to elucidate the role of this extracellular matrix component in the eye. Methods Eyes of Col18a1(-/-) and control mice were examined by light and transmission electron microscopy, laser scanning ophthalmoscopy, and fluorescence angiography. Immunohistochemical analysis of neuronal, epithelial, and immune cells in the eye was performed with antibodies against established cell markers. Results Col18a1(-/-) mice showed a disruption of the posterior iris pigment epithelial (IPE) cell layer with release of melanin granules. The BM of the posterior IPE was attached to the lens and the nonpigmented epithelium of the ciliary body, which was flattened in mutant mice. In aged mutant mice a severe thickening of the stromal iris BM zone was found, and pigmented cells migrated out of the iris and covered the retina along the inner limiting membrane (ILM), sometimes penetrating into the retina. These cells resembled iris clump cells, and immunohistochemistry demonstrated that they were macrophage-like cells. Furthermore, morphologically abnormal retinal vasculature was seen by fluorescence angiography. Conclusions The abnormalities in the iris and ciliary body of Col18a1(-/-) mice demonstrate an important role of collagen XVIII for the function of ocular BMs. The absence of this collagen alters the properties of BMs and leads to severe defects in the iris, showing striking similarities to human pigment dispersion syndrome. In addition, loss of collagen XVIII creates changes that allow clump cells to migrate out of the iris. These cells have not been well characterized previously. In the current study we showed that they are macrophage-like cells and are able to penetrate the ILM in mutant mice. The disease mechanism of human pigment dispersion syndrome is not well understood, but Col18a1(-/-) mice may serve as a model and demonstrate the potential importance of alterations in extracellular matrix components in this disease.

Fibronectin-α4β1 Integrin-Mediated Blockade Protects Genetically Fat Zucker Rat Livers from Ischemia/Reperfusion Injury

Abstract: We tested a hypothesis that interactions between fibronectin (FN), the major extracellular matrix component, and its integrin α4β1 receptor is important in the development of ischemia/reperfusion injury of steatotic liver transplants. We examined the effect of connecting segment-1 (CS1) peptide-facilitated blockade of FN-α4β1 interaction in a well-established steatotic rat liver model of ex vivo cold ischemia followed by iso-transplantation. In this model, CS1 peptides were administered through the portal vein of steatotic Zucker rat livers before and after cold ischemic storage. Lean Zucker recipients of fatty liver transplants received an additional 3-day course of CS1 peptides after transplant. CS1 peptide therapy significantly inhibited the recruitment of T lymphocytes, neutrophil activation/infiltration, and repressed the expression of proinflammatory tumor necrosis factor-α and interferon-γ. Moreover, it resulted in selective inhibition of inducible nitric oxide synthase expression, peroxynitrite formation, and hepatic necrosis. Importantly, CS1 peptide therapy improved function/histological preservation of steatotic liver grafts, and extended their 14-day survival in lean recipients from 40% in untreated to 100% in CS1-treated OLTs. Thus, CS1 peptide-mediated blockade of FN-α4β1 interaction protects against severe ischemia/reperfusion injury experienced otherwise by steatotic OLTs. These novel findings document the potential of targeting FN-α4β1 in vivo interaction to increase the transplant donor pool through modulation of marginal steatotic livers.

Induction of myocardial biglycan in heart failure in rats—an extracellular matrix component targeted by AT1 receptor antagonism

Abstract: Objective: Cardiac remodelling associated with congestive heart failure typically involves dilatation of the ventricular cavities, cardiomyocyte hypertrophy and alterations of extracellular matrix. Biglycan is an extracellular proteoglycan with several recently appreciated functions including cell adhesion, collagen fibril assembly, and growth factor interactions. The aims of this study were to investigate the regulation of biglycan expression and to elucidate the site(s) of synthesis of biglycan in myocardial tissue in an experimental model of heart failure (HF). Methods: Myocardial tissue samples were obtained from rats with myocardial infarction (MI) subsequent to ligation of the left coronary artery. Northern blot analysis and real-time quantitative RT-PCR were employed to investigate mRNA levels. The cellular distribution of biglycan was analysed by in situ hybridisation and immunohistochemistry. Results: Myocardial biglycan mRNA levels in non-ischemic tissue of both left and right ventricles of heart failure rats were substantially elevated as compared to sham-operated rats. Although expression levels peaked 7 days after MI (13-fold increase compared to the sham group, P <0.05), substantial elevations of biglycan mRNA were observed throughout the study period. Analysis of cellular distribution revealed that biglycan expression was confined to myocardial fibroblasts and vascular endothelial cells. In cardiac fibroblasts isolated from failing hearts, biglycan mRNA levels were markedly elevated compared with fibroblasts from sham-operated rats. In addition, in rats with ischemic heart failure treatment with the AT1 receptor antagonist losartan (12.5 mg·kg−1 b.i.d. per os, for 25 days) prevented the increase of myocardial biglycan as well as TGF-β1 mRNA. Conclusion: This report demonstrates global induction of myocardial biglycan mRNA in heart failure. Myocardial biglycan expression could be targeted by AT1 receptor antagonism, an intervention well documented to halt cardiac remodelling in heart failure. Furthermore, the study provides evidence that angiotensin II is a regulator of biglycan expression in cardiac fibroblasts.

Clonogenic Culture of Normal Spermatogonia: In Vitro Regulation of Postnatal Germ Cell Proliferation

Abstract: The stem cell properties of neonatal germ cells have recently been demonstrated by in vivo transplantation. Regulation of proliferation of these cells, however, is not yet understood, and an in vitro system is needed for directly testing the action of differentiation and proliferation-related factors for germ cells. We developed an in vitro model involving micromanipulation and a single-cell clonogenic assay in which results from independent experiments on spermatogonia and gonocytes have been analyzed and compared. Neonatal germ cells can be distinguished by their large size both in vivo and in vitro in a single-cell suspension. These cells are picked up singly using a micropipette and deposited into a 96-well plate precoated with an extracellular matrix component, e.g., collagen IV. The effect of growth factors or cocultured somatic cells was assayed by counting the percentage of wells containing a colony and comparing this percentage with that of control cultures. Addition of platelet-derived growth factor significantly shifted the modal colony size for gonocytes from >16-64 to >64-128 cells/colony (P < 0.001, chi2) but had no effect on spermatogonia-derived colony size and number. For testis somatic cell underlays, there was a profound inhibition of all colony types, and immunohistochemical staining of testis cell underlays showed inhibin/activinbetaA subunit expression. This finding suggests that negative regulation of germ cell proliferation is mediated by inhibin. Addition of activin A to these cultures resulted in significant recovery (P = 0.046) of gonocyte-derived colony numbers but not spermatogonia-derived colonies, which may reflect the functional regulation by these factors observed in vivo. This proliferation assay also highlights many similarities in the regulation of gonocyte and spermatogonia proliferation in vitro, suggesting that proliferation potential is not noticeably affected by the transition of gonocytes to spermatogonia. For example, the average colony cloning efficiency was 80% for gonocytes and 76% for spermatogonia. This technology forms a basis for optimizing growth of neonatal germ cells for applications such as introduction of genetic material into the germ line to produce transgenic mice and to explore gene therapy.

Increased Fibronectin Deposition in Embryonic Hearts of Retinol-Binding Protein–Null Mice

Abstract: Precise regulation of retinoid levels is critical for normal heart development. Retinol-binding protein (RBP), an extracellular retinol transporter, is strongly secreted by cardiogenic endoderm. This study addresses whether RBP gene ablation affects heart development. Despite exhibiting an >85% decrease in serum retinol, adult RBP-null mice are viable, breed, and have normal vision when maintained on a vitamin A-sufficient diet. Comparison of RBP-null with wild-type (WT) hearts from embryos at day 9.0 (E9.0) through E12.5 revealed an RBP-null phenotype similar to that of other retinoid-deficient models. At an early stage, RBP-null hearts display retarded trabecular development, which recovers by E9.5. This is accompanied at E9.5 and E10.5 by precocious differentiation of subepicardial cardiac myocytes. Most remarkably, RBP-null hearts display augmented deposition of fibronectin protein in the cardiac jelly at E9.0 through E10.5 and in the outflow tract at E12.5. This phenomenon, which was detected by immunohistochemistry and Western blotting without increased fibronectin transcript levels, is accompanied by increased numbers of mesenchymal cells in the outflow tract but not in the atrioventricular canal. RBP-null cardiac myocytes, especially in the subepicardial layer, display increased cell proliferation. This phenotype may present a model of subclinical retinoid insufficiency characterized by alteration of an extracellular matrix component and altered cellular differentiation and proliferation, changes that may have functional consequences for adult cardiac function. This murine model may have relevance to fetal development in human populations with inadequate retinoid intake.

Prostaglandin-J(2) upregulates expression of matrix metalloproteinase-1 independently of activation of peroxisome proliferator-activated receptor-gamma.

Abstract: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-inducible nuclear receptor that functions as a transcription factor involved in lipid metabolism, inflammatory response and angiogenesis. The most potent endogenous PPARgamma activator is 15-deoxy-Delta(12,14)prostaglandin-J(2) (15d-PGJ(2)), whereas synthetic ligands include the oral antidiabetic drugs thiazolidinediones (TZDs). Activation of PPARgamma was reported to decrease the synthesis of matrix metalloproteinases (MMPs) in vascular smooth muscle cells and macrophages. We aimed to investigate the effect of PPARgamma ligands on expression of MMP-1 and urokinase plasminogen activator (uPA) in human microvascular endothelial cells (HMEC-1). We found that treatment of HMEC-1 with 15d-PGJ(2) increased the synthesis of MMP-1 protein up to 168% comparing to untreated cells. TZDs (ciglitazone and troglitazone), more potent activators of PPARgamma in HMEC-1, did not influence MMP-1 production, arguing against the involvement of PPARgamma in this process. Importantly, the stimulatory effect of 15d-PGJ(2) was reversed by the antioxidant N-acetyl-cysteine (NAC), suggesting a contribution of oxidative stress. We demonstrated also that 15d-PGJ(2) did not change the activity of MMP-1 promoter, but increased the stability of MMP-1 mRNA. In contrast, 15d-PGJ(2) very potently inhibited the synthesis of uPA. This effect was in part mimicked by ciglitazone and troglitazone implying an involvement of PPARgamma. Accordingly, NAC did not modify the inhibitory effect of 15d-PGJ(2) on uPA expression. In conclusion, we postulate that 15d-PGJ(2) may differently regulate the synthesis of proteases involved in angiogenesis: it upregulates MMP-1 expression in HMEC-1 through induction of oxidative stress, and inhibits uPA synthesis partly by activation of PPARgamma.

Investigations of RPE cells of choriodal neovascular membranes from patients with age-related macula degeneration.

Abstract: Age-related macular degeneration (AMD) is the most frequent cause for blindness in older age. The most severe complication is the development of choroidal neovascularisation (CNV). The retinal pigment epithelium (RPE) plays an important role in the induction of angiogenesis in AMD (Glaser et al., 1985; Young, 1987; Morse et al., 1989; Bird, 1991) by secreting the majority of angiogenic factors (Kliffen et al., 1997). The most important angiogenic factor is VEGF (vascular endothelial growth factor). CNV membranes contain high amounts of VEGF and it is known that overexpression of VEGF in the RPE leads to choroidal neovascularisation (Schwesinger et al., 2001). In vitro and in situ investigations revealed several extracellular stimuli inducing secretion of angiogenic factors by the RPE. A major part takes a hypoxia-comparable situation in the area of soft drusen (Eagle, 1984). In consequence to this, VEGF and factors inducing the VEGF production are secreted by the RPE and the neuronal retina (D' Amore, 1994; Shima et al., 1995; Stone et al., 1995; Frank et al., 1996; Kliffen et al., 1997; Tanihara et al., 1997). VEGF secretion is stimulated by advanced-glycation endproducts (AGE) accumulating in CNV membranes, and also by vitronectin, the major extracellular matrix component in drusen (Hammes et al., 1996; Hageman et al., 1999). In addition, insulin-like growth factor-1 (IGF-1) secreted by the neuronal retina and tumour necrosis factor-a (TNFa) secreted by macrophages which migrate towards the retina during the development of the first blood vessels are known to induce VEGF secretion by RPE cells (Punglia et al., 1997; Oh et al., 1999).

Trypanosoma cruzi Cell Invasion Mechanisms

Abstract: Mammalian cell invasion by Trypanosoma cruzi is a multi-step process that requires the interaction of various parasite and host cell molecules and the activation of signal transduction pathways in both cells. Depending on the parasite strain, developmental stage and the type of target cell, distinct sets of molecules may be engaged. The metacyclic trypomastigote, which initiates infection of the mammalian host, expresses stage-specific surface glycoprotein gp82 and the mucin-like molecule gp35/50. The binding of these proteins to the target cell during infection triggers Ca2+ responses in both cells. Cell-derived trypomastigotes trigger signaling pathways from receptors for agonists, such as bradykinin and TGFα, and from receptors for a trypomastigote Ca2+-agonist, generated from a precursor molecule by oligopeptidase B. Trypomastigote surface molecules, such as trans-sialidase, the stage-specific mucin SSp-3 and Tc85, which contains binding sites for laminin and cytokeratin 18, may also be involved in cell invasion. In host cells, Ca2+ released from internal compartments in IP3-dependent manner is required for translocation of lysosomes to the site of trypomastigote entry, a process that contributes to the formation of the vacuole membrane surrounding the parasite during penetration.

Modulation of fibrinolytic system components in mesothelial cells by hyaluronan.

Abstract: ← ObjectiveHyaluronan (HA) is an important extracellular matrix component and is involved in fluid homeostasis, tissue repair, and response to infections. Previous studies have shown that supplemen...

Manufacturing method of bracket material for applying in myocardial tissue engineering

Abstract: The invention belongs to the tissue engineering field, more specifically relates to a manufacturing method of stent material for cardiac muscle tissue enginerering. The stent simulates extracellular matrix component. Its original form is a liquid hydrogel. It mainly comprises liquid collagen(I type and III type), elastin, laminin, chicken embryo extractive, 2i‡DMEM and cow embryo serum. Blending these constituents in proper ratio to obtain natural cardiac muscle analog stent materials and blending them with cardiac cell can generate cardiac cell-bracket materials in various shape, by which a tissue engineered cardiac muscle tissue capable of beating synchronistically can be generated.

Preparation method of artificial endometrium

Abstract: The present invention belongs to the tissue engineering field and is especially a preparation process of artificial endometrium. New type biodegradable material is used in making stereo rack, endometrium cell of experiment animal is used as seed cell, and artificial endometrium is in vitro constituted via special culture process. The artificial endometrium is a 3D structure, has the similar shapeand function to the in vivo endometrium, can secrete extracellular matrix component and can emulate the configuration and function of in vivo endometrium. The present invention provides the research of embryo nidatin mechanism with ideal platform and provides treating way for endometrium damage caused by induced abortion, tuberculosis, etc.

New extracts of Cryptomeria japonica buds, are stimulants of extracellular matrix component synthesis by dermal cells and cytoprotectants for the skin, useful in cosmetic compositions

Abstract: New extracts (A) of Cryptomeria japonica (Japanese cedar) buds are obtained by (i) solid-liquid extraction, (ii) solid-liquid separation and (iii) recovery of the liquid phase.

Induction of Hepatocyte Nuclear Factor 3γ Gene Expres- sion by the Extracellular Matrix Component from Fish Skin

Abstract: The skin of the halibut (Reinhararditus hippoglossoides) has long been discarded as a waste product by the food industry in large quantities; hence, we examined the biological activity in the skin for utilization of the waste. In the present study, we determined induction of hepatocyte nuclear factor 3γ (HNF3γ), which is involved in liver function, using human hepatoma cell line HepG2 and extracellular matrix components extracted from the fish skin. Induction of the HNF3γ was detected by reporter plasmid constructed with the promoter region of the HNF3γ gene and luciferase or green fluorescent protein genes. The alkali extract containing proteoglycan showed HNF3γ induction, but the acid-extract enriched in collagen did not. Consequently, dermatan sulfate showed the strongest activity in HNF3γ induction. These observations suggested that fish skin has some significant biological activity, and indicates its possible function in the food and drug industry.

CD44 mediates polymorphonuclear leukocyte motility on hyaluronan.

Abstract: OBJECTIVES To investigate the behavior of polymorphonuclear (PMN) leukocytes on the extracellular matrix carbohydrate component, hyaluronan (HA), in the presence and absence of the chemokine, interleukin-8 (IL-8). METHODS The present study was conducted at the Department of Hematology, University of Liverpool, United Kingdom, between the period 2000 to 2001. Polymorphonuclear cells were isolated from whole venous blood using Mono-Poly-Resolving Medium. Purified PMN were added alone or with IL-8 to HA-coated plates and the behavior of these cells monitored by time-lapse video microscopy over a period of 40 minutes. For the identification of surface receptor(s) mediating PMN migration on HA, PMN were incubated with blocking and non-blocking antibodies against cluster of differentiation 44 (CD44) and Receptor for Hyaluronan Mediated Motility (RHAMM) prior to addition to HA-coated surfaces. RESULTS Approximately 55% of PMN were found to interact and migrate on HA-coated plates with a mean speed of 6.4 +/- 0.7 mm/min. Addition of IL-8 reduced both the percentage moving cells (7.5%) and the average speed of the remaining moving cells (2.0 +/- 0.3 mm/min). The inhibitory effect of IL-8 on PMN migration was associated with reorganization of the cytoplasmic fibrillar form of actin. Anti-CD44 blocking antibody substantially reduced the speed of PMN (2.5 +/-0.9 mm/min), while non-blocking anti-CD44 and anti-RHAMM antibodies had no effect. CONCLUSIONS The present study demonstrates for the first time that PMN are able to interact and migrate on the widely distributed extracellular matrix component, HA, using the cell surface receptor, CD44. Such interaction is modified by the chemokine, IL-8, in a way that optimizes the host defense against invading pathogens.