Differential expression of the extra domain-containing form of cellular fibronectin in human placentas at different stages of maturation

The distribution of the extra domain-containing form of cellular fibronectin was studied in human placentas at different stages of maturation by using the monoclonal antibody 52DH1 in indirect immunofluorescence. In early chorionic tissue (7 to 10 weeks post menstruationem) cellular fibronectin was codistributed with laminin and type IV collagen in the trophoblastic basement membranes. At weeks 11 to 12 the trophoblastic basement membranes were negative but positivity was typically revealed in distinct aggregates in the stromal tissue. In second-trimester and term placentas the immunoreactivity was confined to the vessel endothelia of villous stroma. Extravillous trophoblast cells seen in placentas at different stages did not show positivity. Double staining with the 52DH1 monoclonal antibody and polyclonal fibronectin antibodies showed that both in the early and term placentas there was much fibrillar positivity only revealed with the polyclonal antibodies. The present results show that cellular fibronectin is a prominent component of early trophoblastic basement membranes and may thus play a special role in the maturation of chorionic villi.     

Extra cellular matrix remodelling after heterotopic rat heart transplantation: gene expression profiling and involvement of ED-A+ fibronectin, alpha-smooth muscle actin and B+ tenascin-C in chronic cardiac allograft rejection

Chronic cardiac rejection is represented by cardiac allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) known to cause severe complications. These processes are accompanied by remarkable changes in the cardiac extra cellular matrix (cECM). The aim of our study was to analyse the cECM remodelling in chronic rejection and to elucidate a potential role of ED-A domain containing fibronectin (ED-A(+) Fn), alpha smooth muscle actin (ASMA) and B domain containing tenascin-C (B(+) Tn-C). A model of chronic rejection after heterotopic rat heart transplantation was used. Allografts, recipient and control hearts were subjected to histological assessment of rejection grade, to real-time PCR based analysis of 84 genes of ECM and adhesion molecules and to immunofluorescence labelling procedures, including ED-A(+) Fn, ASMA and B(+) Tn-C antibodies. Histological analysis revealed different grades of chronic rejection. By gene expression analysis, a relevant up-regulation of the majority of ECM genes in association with chronic rejection could be shown. For 8 genes, there was a relevant up-regulation in allografts as well as in the corresponding recipient hearts. Association of ASMA positive cells with the grade of chronic rejection could be proven. In CAV and also in CIF there were extensive co-depositions of ED-A(+) Fn, ASMA and B(+) Tn-C. In conclusion, chronic cardiac allograft rejection is associated with a cECM remodelling. ASMA protein deposition in CAV, and CIF is a valuable marker to detect chronic rejection. Interactions of VSMCs and Fibro-/Myofibroblasts with ED-A(+) Fn and B(+) Tn-C might functionally contribute to the development of chronic cardiac rejection.     

Citrullinated fibronectin inhibits apoptosis and promotes the secretion of pro-inflammatory cytokines in fibroblast-like synoviocytes in rheumatoid arthritis

Introduction

Rheumatoid arthritis (RA) is characterized by synovial lining hyperplasia, in which there may be an imbalance between the growth and death of fibroblast-like synoviocytes (FLSs). Antibodies against citrullinated proteins are proposed to induce RA. This study aimed to investigate the pathogenic role of citrullinated fibronectin (cFn) in RA.

Methods

The distribution of fibronectin (Fn) and cFn in synovial tissues from RA and osteoarthritis (OA) patients was examined by immunohistochemical and double immunofluorescence analysis. FLSs were isolated from RA and OA patients and treated with Fn or cFn. Apoptosis was detected by flow cytometry and TUNEL assay. The expression of survivin, caspase-3, cyclin-B1, Bcl-2 and Bax was detected by real-time PCR. The secretion of proinflammatory cytokines was measured by ELISA.

Results

Fn formed extracellular aggregates that were specifically citrullinated in synovial tissues of RA patients, but no Fn deposits were observed in those of OA patients. Fn induced the apoptosis of RA and OA FLSs while cFn inhibited the apoptosis of RA and OA FLSs. Fn significantly increased the expression of caspase-3 and decreased the expression of survivin and cyclin-B1 in FLSs from RA and OA patients. cFn significantly increased the expression of survivin in RA FLSs. Furthermore, cFn increased the secretion of TNF-α and IL-1 by FLSs.

Conclusions

cFn plays a potential pathophysiologic role in RA by inhibiting apoptosis and increasing proinflammatory cytokine secretion of FLSs.     

Comparison of the specific binding of cortisol in human amnion, amniotic fluid, and plasma

The purpose of this study was to compare the specific cortisol-binding protein found associated with human amnion with specific cortisol binding in human amniotic fluid and plasma. The electrophoretic mobility on polyacrylamide gels of the specific cortisol binding in amnion, amniotic fluid, and maternal plasma was identical. The influence of pH on cortisol binding activity was similar in all tissues and the cortisol binding was immunoprecipitable by a polyclonal antibody raised against human corticosteroid-binding globulin. The interaction of the cortisol binding protein with concanavalin A was studied in preterm amniotic fluid, term amniotic fluid, term amnion, and plasma from pregnant women at term and women under oral contraceptive treatment. Binding to concanavalin A was similar in term amnion and term amniotic fluid but was less than that found with both preterm amniotic fluid and term plasma. These results indicate that the cortisol binding protein associated with human amnion has similar characteristics to plasma corticosteroid-binding globulin, but that its state of glycosylation appears to be more like that of the cortisol binding protein in term amniotic fluid rather than in plasma.     

Cell type-specific regulation of fetal fibronectin expression in amnion: conservation of glucocorticoid responsiveness in human and nonhuman primates

The appearance of oncofetal fibronectin (FFN) in cervical and vaginal secretions is predictive of human labor. Levels of FFN in amnion increase with the onset of labor in rhesus monkeys. Since glucocorticoid (GC) levels in serum and amniotic fluid increase in association with parturition, we compared GC-mediated regulation of FFN expression in cultures of amnion epithelial cells and fibroblasts isolated from human and baboon amnions. Cells were maintained with and without dexamethasone (DEX), and levels of FFN in the conditioned media were determined by ELISA. We observed that DEX treatment suppressed FFN levels in both human and baboon amnion epithelial cells, whereas it increased FFN levels in amnion fibroblasts. DEX treatment reduced FFN levels in cytotrophoblasts from human placenta and increased FFN levels in placental fibroblasts. Northern blots revealed that DEX reduced levels of fibronectin (FN) mRNA in amnion epithelial cells and cytotrophoblasts, whereas it increased FN mRNA in amnion and placental fibroblasts. We conclude that GC differentially regulates FFN expression in epithelial and mesenchymal cells from amnion and placenta. In addition, this pattern of cell type-specific FFN regulation by GC is conserved in human and nonhuman primates and may be responsible for parturition-dependent changes in FFN expression in gestational tissues.     

Intermediate filament cytoskeleton of amnion epithelium and cultured amnion epithelial cells: expression of epidermal cytokeratins in cells of a simple epithelium

Using immunofluorescence microscopy and two-dimensional gel electrophoresis, we compared the cytoskeletal proteins expressed by human amnion epithelium in situ, obtained from pregnancies of from 10-wk to birth, with the corresponding proteins from cultured amnion epithelial cells and cultures of cells from the amniotic fluid of 16 week pregnancies. Epithelia of week 16 fetuses already display tissue-specific patterns of cytokeratin polypeptides which are similar, although not identical, to those of the corresponding adult tissues. In the case of the simple amnion epithelium, a complex and characteristic complement of cytokeratin polypeptides of Mr 58,000 (No. 5), 56,000 (No. 6), 54,000 (No. 7), 52,500 (No. 8), 50,000 (No. 14), 46,000 (No. 17), 45,000 (No. 18), and 40,000 (No. 19) is present by week 10 of pregnancy and is essentially maintained until birth, with the addition of cytokeratin No. 4 (Mr 59,000) and the disappearance of No. 7 (Mr 54,000) at week 16 of pregnancy. In full-term placentae, the amnion epithelium displays two morphologically distinct regions, i.e., a simple and a stratified epithelium, both of which express the typical amnion cytokeratin polypeptides. However, in addition the stratified epithelium also synthesizes large amounts of special epidermal cytokeratins such as No. 1 (Mr 68,000), 10 (Mr 56,500), and 11 (Mr 56,000). In culture amnion epithelial cells obtained from either 16-wk pregnancies or full-term placentae will continue to synthesize the amnion-typical cytokeratin pattern, except for a loss of detection of component No. 4. This pattern is considerably different from the cytokeratins synthesized by cultures of cells from amniotic fluids (cytokeratins No. 7, 8, 18, and 19, sometimes with trace amounts of No. 17) and from several so-called "amnion epithelial cell lines." In addition, amnion epithelial cells in situ as well as amnion epithelial cell cultures appear to be heterogeneous in that they possess some cells that co-express cytokeratins and vimentin. These observations lead to several important conclusions: In contrast to the general concept of recent literature, positively charged cytokeratins of the group No. 4-6 can be synthesized in a simple, i.e., one-layered epithelium. The change from simple to stratified amnion epithelium does not require a cessation of synthesis of cytokeratins of the simple epithelium type, but in this case keratins characteristic of the terminally differentiated epidermis (No. 1, 10, and 11) are also synthesized.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells

Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, Fl-amnion (transformed amnion), WISH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (WI-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 micrograms/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.     

Monoclonal antibodies to human cytokeratins: application to various epithelial and mesothelial cells

Immunohistological analysis of human tissue using monoclonal antibodies against cytokeratins, which are confined to cells of epithelial origin, is a valuable technique. Using human epidermal keratins as antigen, we prepared monoclonal antibodies against cytokeratins (ZK1, ZK7, ZK61 and ZK99) and against a desmosomal protein (ZK31). Immunohistochemical staining of human skin sections using these antibodies showed a specific reaction with the epidermis: ZK1 stained the entire epidermis, ZK7 only the basal layer, ZK61 and ZK99 the suprabasal layers, and ZK31 the cellular interfaces. In order to test for antibody specificity, immunoblots with human epidermal and amnion epithelial cytokeratin polypeptides, as well as immunofluorescence microscopy of simple epithelia (glandular and simple columnar epithelia) were performed. ZK1, ZK61 and ZK99 reacted preferentially with cytokeratin polypeptides of stratified squamous epithelia and ZK7 recognized cytokeratins of stratified and simple epithelia. When the ZK antibodies were tested on mesothelial cells in pleural effusions, only ZK7 reacted with these cells. Biochemical analysis of cytokeratin accumulation in cells of primary and long-term cultures indicated that the cytokeratin pattern of mesothelial cells was quite unstable, while that of amnion epithelial cells showed only minor quantitative changes. The use of these antibodies to determine the epithelial origin of cells present in pleural effusions is proposed.     

Dibutyryl cyclic AMP decreases expression of ED-A fibronectin by canine chondrocytes

The presence of Extra Domain A (ED-A) in fibronectins from cartilage and from the culture medium of chondrocytes cultured under various conditions was determined with monoclonal antibody IST-9. Less than 2% of the fibronectin extracted from cartilage was fibronectin which contained the ED-A sequence (ED-A fibronectin). Chondrocytes placed in primary culture began to synthesize increased amounts of ED-A fibronectin (6% to 36% of the total fibronectin). Expression of ED-A fibronectin was selectively reduced when the chondrocytes were cultured in the presence of 0.5 mM dibutyryl cyclic AMP.     

Immunohistochemical localization of glucose transporters and insulin receptors in human fetal membranes at term

The localization has been investigated of the isoforms GLUT1, GLUT3 and GLUT4 of glucose transporter proteins as well as of insulin receptors. Fetal membranes (n=10) were examined by immunohistochemical methods at the light and electron microscopic levels using mono- and polyclonal antibodies. In all amnion epithelial cells, GLUT1 and GLUT3 antibodies were bound to the apical membrane. Very rarely the GLUT1 antibody also immunostained the basolateral membrane and reacted weakly with the endomembrane system and membranes of the lateral cell protrusions. Fibroblasts reacted with the antibodies against GLUT1, GLUT4 and insulin receptor, whereas they were labelled only in one case with GLUT3 antibody. Cytotrophoblast cells were only stained with antibodies against GLUT1 and GLUT3. Antibodies against GLUT4 only reacted with fibroblasts in the membranes. On amnion epithelial cells, weak immunoreactivity with insulin receptor antibodies was detected only at the electron microscopic level. The data indicate: (1) GLUT1 is located on all cells of the amnion, whereas GLUT3 is present in detectable amounts only on amnion epithelial cells and cytotrophoblast; (2) GLUT1 and GLUT3 on amnion epithelial cells are predominantly located on the apical surface; (3) GLUT4 and insulin receptors are not regularly expressed. We suggest that amnion epithelial cells cover their basal glucose requirements from the amniotic fluid and not from the maternal circulation.     

Epithelial cell-derived neutrophil-activating peptide-78 is present in fetal membranes and amniotic fluid at increased concentrations with intra-amniotic infection and preterm delivery

Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased ( approximately 4-fold) with term labor in amnion (n = 15), and with preterm labor ( approximately 30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r2 = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1beta and tumor necrosis factor alpha. Production of ENA-78 by choriodecidual explants was increased modestly after 2-4 h of exposure to lipopolysaccharide (5 microg/ml). An immunoreactive doublet ( approximately 8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.     

Comparative analysis of oncofetal fibronectin and tenascin-C expression in right atrial auricular and left ventricular human cardiac tissue from patients with coronary artery disease and aortic valve stenosis

Aortic valve stenosis (AVS) and coronary artery disease (CAD) are accompanied by changes in the cardiac extra cellular matrix (cECM) including the re-expression of oncofetal fibronectin (Fn) and tenascin-C (Tn-C) variants. Human antibodies against these variants are usable for targeted therapy. Aim of the study was the comparative analysis of cECM remodelling in tissue samples from right atrial auricle (RAA) and left ventricular septum (LVS). RAA and LVS specimens from 30 patients (17?×?AVS; 13?×?AVS+CAD) were analysed with respect to histological changes and ECM remodelling using PCR based ECM gene expression profiling. Re-expression of ED-A(+) Fn and A1(+) Tn-C was investigated on the mRNA and on the protein level. For immunofluorescence, human recombinant small immunoprotein (SIP) format antibodies were used. There was a positive correlation of the grade of histological changes in RAA and corresponding LVS samples (r?=?0.695). ECM gene expression levels were higher in LVS compared to RAA. For 24 genes, a corresponding relevant (>2.5-fold) up- or down-regulation in RAA and LVS occurred. Using SIP antibodies, a positive correlation of protein deposition levels in RAA and corresponding LVS (r?=?0.818) could be shown for ED-A(+) Fn. Cardiac tissue remodelling is likely a process involving the entire heart reflected by intra-individually comparable histology and cECM changes in RAA and LVS samples. ED-A(+) Fn might be an excellent target for an antibody-mediated delivery of diagnostic or therapeutic agents. The RAA is a valuable and representative tool to evaluate cardiac remodelling and to plan individualized therapy.     

Changes in the levels of human tropomyosins IEF 52,55, and 56 do not correlate with the loss of actin cables observed in SV 40 transformed MRC-5 fibroblasts

Summary A mouse monoclonal antibody (mAb 1D122G9) raised against human tropomyosin IEF 52 (HeLa protein catalogue number, Mr=35 kd) has been characterized both in terms of specificity and patterns of immunofluorescence staining in Triton extracted cultured cells. As determined by two dimensional gel immunoblotting of HeLa cell proteins the antibody recognized IEF 52 and two other acidic proteins (IEF 55, Mr=31.8 kd; IEF 56, Mr=31 kd) previously identified as putative tropomyosin-like proteins. Immunofluorescence staining of Triton extracted cultured cells revealed the striated or interrupted pattern on the actin cables characteristic of tropomyosin staining. Quantitation of the three tropomyosins in Triton cytoskeletons from normal and SV 40 transformed human MRC-5 fibroblasts showed that the latter contained significantly less of tropomyosin IEF's 52 (52%) and 56 (72%) as compared to their normal counterparts. The ratios of these two tropomyosins to actin however was very similar for both types of cytoskeletons. This was not the case for tropomyosin IEF 55, which was present in nearly twice the amount in the cytoskeletons from the SV 40 transformed cells. The ratio of actin to total tropomyosin for whole cells was found to be unchanged on transformation. This ratio however was 31% lower in the cytoskeletons from the transformed cells. These and other results presented here suggest that changes in the levels of these three tropomyosins are not enough to account for the magnitude of the loss of actin cables observed in the transformed cells.Abbreviations IEF isoelectric focusing - mAb monoclonal antibody - NEPHGE non equilibrium pH gradient electrophoresis     

Basally located epithelial cell surface component identified by a novel monoclonal antibody technique

Tubular aggregates of glandular epithelial cells (gland fragments) were isolated from human endometrium by collagenase digestion of surrounding stroma, thus exposing the basal surfaces of the cells. Using these aggregates as immunogen, monoclonal antibodies could be derived that recognized basally located antigens. One such antibody, G71, is described, that binds to a basal epithelial cell antigen present in a variety of human epithelia. Epitope-bearing molecules in the range Mr 60 000-180 000 are present in two of the tissues studied, amnion and endometrium. The epitope is associated with areas of epithelial cell-extracellular matrix contact.     

Pemphigoid gestationis autoantigen, transmembrane collagen XVII, promotes the migration of cytotrophoblastic cells of placenta and is a structural component of fetal membranes.

In pemphigoid gestationis (PG), autoantibodies target collagen XVII, a hemidesmosomal transmembrane protein, which is an important element in cutaneous epithelial adhesion and signalling. We report that collagen XVII is expressed in the first trimester and term syncytial and cytotrophoblastic cells of normal placenta and in epithelial cells of amniotic membrane. Immunoelectron microscopy confirmed the localization of collagen XVII to the hemidesmosomes of amniotic epithelium. Examination of three PG placentas showed mild villitis, but there were no differences between collagen XVII expression levels or immunostaining signals as compared to normal placenta. Collagen XVII expression was also detected in cultured extravillous trophoblast HTR-8/SVneo cells, where collagen XVII expression was upregulated by PMA and TGF-beta1. Interestingly, the presence of Col15, the cell migration domain of collagen XVII, induced the migration of HTR-8/SVneo cells in transmigration assay. Analysis of amniotic fluid samples at different gestational weeks revealed that a large quantity of collagen XVII ectodomain was shed into amniotic fluid throughout pregnancy. Biochemical and immunoblotting analysis indicated that the ectodomain in amniotic fluid is structurally very similar to the ectodomain produced by cultured keratinocytes. Cultured cells from amniotic fluid samples also expressed collagen XVII. Our results suggest that collagen XVII may contribute to the invasion of extravillous trophoblasts during placental development and is also required for the integrity of amniotic basement membrane. Although the exact pathomechanism of PG is still largely unknown, the clinical symptoms of PG are initiated after the expression of collagen XVII in placenta during the first trimester of pregnancy.     

Differential expression of the extra domain-containing form of cellular fibronectin in human placentas at different stages of maturation

Summary The distribution of the extra domain-containing form of cellular fibronectin was studied in human placentas at different stages of maturation by using the monoclonal antibody 52DHl in indirect immunofluorescence. In early chorionic tissue (7 to 10 weeks post menstruationem) cellular fibronectin was codistributed with laminin and type IV collagen in the trophoblastic basement membranes. At weeks 11 to 12 the trophoblastic basement membranes were negative but positivity was typically revealed in distinct aggregates in the stromal tissue. In second-trimester and term placentas the immunoreactivity was confined to the vessel endothelia of villous stroma. Extravillous trophoblast cells seen in placentas at different stages did not show positivity. Double staining with the 52DHl monoclonal antibody and polyclonal fibronectin antibodies showed that both in the early and term placentas there was much fibrillar positivity only revealed with the polyclonal antibodies. The present results show that cellular fibronectin is a prominent component of early trophoblastic basement membranes and may thus play a special role in the maturation of chorionic villi.