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Static pressure regulates connective tissue growth factor expression in human mesangial cells.

Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders such as renal fibrosis and atherosclerosis. Fibrosis is a common final pathway of renal diseases of diverse etiology, including inflammation, hemodynamics, and metabolic injury. Mechanical strains such as stretch, shear stress, and static pressure are possible regulatory elements in CTGF expression. In this study, we examined the ability of static pressure to modulate CTGF gene expression in cultured human mesangial cells. Low static pressure (40-80 mm Hg) stimulated cell proliferation via a protein kinase C-dependent pathway. In contrast, high static pressure (100-180 mm Hg) induced apoptosis in human mesangial cells. This effect was reversed by treatment with CTGF antisense oligonucleotide but not with transforming growth factor beta1-neutralizing antibody or protein kinase C inhibitor. High static pressure not only up-regulated the expression of CTGF, but also the expression of extracellular matrix proteins (collagen I and IV, laminin). This up-regulation of extracellular matrix proteins was also reversed by treatment with CTGF antisense oligonucleotide. As judged by mRNA expression of a total of 1100 genes, including apoptosis-associated genes using DNA microarray techniques, recombinant CTGF protein induced apoptosis by down-regulation of a number of anti-apoptotic genes. Overexpression of CTGF in mesangial cells by transient transfection had similar effects. Taken together, these results suggest that high blood pressure up-regulates CTGF expression in mesangial cells. High levels of CTGF in turn enhance extracellular matrix production and induce apoptosis in mesangial cells, and may contribute to remodeling of mesangium and ultimately glomerulosclerosis.

Apoptosis↗

Cells of the connective tissue differentiate and migrate into pollen sacs.

In angiosperms, archesporial cells in the anther primordium undergo meiosis to form haploid pollen, the sole occupants of anther sacs. Anther sacs are held together by a matrix of parenchyma cells, the connective tissue. Cells of the connective tissue are not known to differentiate. We report the differentiation of parenchyma cells in the connective tissue of two Gordonia species into pollen-like structures (described as pseudopollen), which migrate into the anther sacs before dehiscence. Pollen and pseudopollen were distinguishable by morphology and staining. Pollen were tricolpate to spherical while pseudopollen were less rigid and transparent with a ribbed surface. Both types were different in size, shape, staining and surface architecture. The ratio of the number of pseudopollen to pollen was 1:3. During ontogeny in the connective tissue, neither cell division nor tetrad formation was observed and hence pseudopollen were presumed to be diploid. Only normal pollen germinated on a germination medium. Fixed preparations in time seemed to indicate that pseudopollen migrate from the connective tissue into the anther sac.

Asteraceae↗

Expression of human group II phospholipase A2 in transgenic mice.

Group II phospholipase A2 (PLA2) has been proposed to play an important role in inflammation and defense against bacterial infection. We investigated tissues of transgenic mice expressing the human group II PLA2 gene by immunohistochemistry using rabbit anti-human group II PLA2 antibodies, and by in situ hybridization by probing with human group II PLA2 mRNA anti-sense (test) and sense (control) riboprobes. By immunohistochemistry, human group II PLA2 was found in various mouse tissues and cell types including hepatocytes, proximal tubule cells of the kidney, epithelial cells of the renal pelvis, urinary bladder and ureter, granulosa cells of Graafian follicles, aortic intima and media, cartilage, epiphyseal bone, bronchial epithelial cells, and connective tissue cells in the dermis. By in situ hybridization, group II PLA2 mRNA was localized in hepatocytes, epidermal cells, dermal cells, connective tissue fibroblasts, epithelial and smooth muscle cells of the urinary bladder, and cells of Bowman's capsule. These results show that human group II PLA2 is expressed in large amounts in hepatocytes and many extrahepatic tissues of the transgenic mice. These animals provide a useful new tool for studies on the metabolism, in vivo effects, and physiological and pathological roles of phospholipase A2.

Animals↗

[Effect of temperature on the duration of mitosis in mammalian cells cultivated outside the body].

Nine cell strains of different origin were cultivated at 28--36 degrees with the interval of 2 degrees. During the phase of logarithmic culture growth, the duration of mitosis (Tm) was determined by means of colchicine method. A strict temperatural dependence Tm, obeyed to Arrenius' law was revealed. Temperature range within which Arreinius' law is valid in different cell strains is not alike. Cultivation of L cells and connective tissue cells from Chinese hamster to 39, 41and 42 degrees demonstrated their upper critical point Tm to be for L cells 39 degrees, for connective tissue cells from the Chinese hamster--41 degrees. Electron microscopic investigations demonstrated that cell cultivation within physiological (mitosis destroying) range of temperatures does not notably effect their ultrastructural organization.

Animals↗

Connective tissue growth factor expression in human bronchial epithelial cells.

Connective tissue growth factor (CTGF) is a cysteine-rich protein that promotes extracellular matrix deposition. CTGF is selectively induced by transforming growth factor beta and des-Arg kallidin in lung fibroblasts and increases steady-state mRNA levels of alpha type I collagen, 5alpha-integrin and fibronectin in fibroblasts. Bronchial epithelial cells have been proposed to functionally interact with lung fibroblasts. We therefore investigated if bronchial epithelial cells are able to synthesize CTGF. Human bronchial epithelial cells were grown to subconfluence in standard growth media. Proliferating cells grown in small airway growth media were harvested following starvation for up to 24 h. Expression of CTGF transcripts was measured by PCR. Immunocytochemistry was also completed using a commercially available antibody. The cells expressed readily detectable CTGF transcripts. Starvation of these cells resulted in a quantitative decline of CTGF transcripts. Direct sequencing of the PCR product identified human CTGF. Immunocytochemistry confirmed intracellular CTGF in the cells and none in negative control cells. We conclude that bronchial epithelial cells could be a novel source of CTGF. Bronchial epithelial cell-derived CTGF could thus directly influence the deposition of collagen in certain fibrotic lung diseases.

Bronchi↗

Epithelial dendritic cells and connective tissue macrophages in oral carcinogenesis and the effects of systemic Corynebacterium parvum.

Epithelial dendritic cells (EDC) and connective tissue macrophages were examined during the induction and growth of oral squamous cell carcinomas in Sprague-Dawley rats treated with the carcinogen 4-nitroquinoline N-oxide (4NQO) and the immune potentiator Corynebacterium parvum. Splenomegaly was induced in all animals receiving C. parvum. Acetone-fixed frozen sections of the palate and tongue were stained using an indirect immunoperoxidase technique and monoclonal antibodies to rat Ia (MRC OX-6) and macrophage subpopulations (ED1, ED2, ED3). EDC were predominantly Ia+, ED1-, ED2- and ED3-. The lamina propria contained Ia+, ED1+ and ED2+ cells; ED3-reactive cells were rare. ED2+ cells predominated in the interstitial connective tissue of deeper muscle. In the non-invasive tissues, the number of positive cells (Ia+EDC and connective tissue Ia+, ED1+ and ED3+ cells) increased significantly throughout the experimental period (0-9 months), were significantly more prevalent in the test tissues (4NQO, 4NQO + C.parvum, C.parvum) compared to untreated controls and, at 9 months, the carcinogen-treated rats (4NQO, 4NQO + C.parvum) had significantly more Ia+ EDC and connective tissue Ia+ cells than C.parvum controls. Irrespective of the marker under study, there were no significant differences between rats treated with 4NQO or 4NQO + C.parvum at any time during the experimental period. Similarly, intra-epithelial Ia+ and ED1+ cells increased significantly throughout the experimental period in all test groups compared to untreated controls, but no significant differences were evident between carcinogen-treated animals (4NQO, 4NQO + C.parvum) and C.parvum controls. Significant positive correlations between connective tissue Ia+ and ED1+ cells and also intra-epithelial Ia+ and ED1+ cells were present in all experimental groups; connective tissue ED2+ and ED3+ cell numbers did not correlate with any of the other phenotypes and intra-epithelial ED2+ and ED3+ cells were rare/absent. Palatal and/or lingual tumours developed in 80% of carcinogen-treated rats by 9 months and the tumour incidence was similar in rats treated with either 4NQO or 4NQO + C.parvum. There were no significant differences in the number of Ia+ EDC between the infiltrating and the non-invasive overlying epithelium of the lingual carcinomas and the non-invasive lingual epithelium treated with either 4NQO or 4NQO + C.parvum.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Nitroquinoline-1-oxide↗

Natural killer cells in connective tissue disorders.

NK cells may be important in the elimination of cells infected by virus, in the regulation of antibody production, and in tissue destruction. The significance of NK cells in rheumatic disorders is unknown, but NK cells and NK-like cells have been found in the peripheral blood and synovial tissues of patients with autoimmune disease. In particular, defects in NK cell activity have been reported in SLE, RA, PSS and SS. Of these diseases, SLE appears to be the best characterized with obvious abnormalities in NK cell numbers, impaired cytotoxicity of individual NK cells, decreased release of cytotoxic factors, deranged IFN modulation of the NK cell, and associated abnormalities in the IL-2 system. The association of these abnormalities with the underlying disease process is currently under investigation.

Arthritis, Rheumatoid↗

Increased MMP-2 expression in connective tissue growth factor over-expression vascular smooth muscle cells.

Connective tissue growth factor (CTGF) is abundantly expressed in the vascular smooth muscle cells (VSMC) of atherosclerotic lesions but not in normal vessels. CTGF is able to promote VSMC proliferation and migration and influences the composition of extracellular matrix. The mechanisms for controlling these events remain unclear. We studied the effects of CTGF on matrix metalloproteinases (MMPs) by introducing a CTGF over-expression construct into VSMC. We found that the over-expression of CTGF significantly increased the activity of MMP-2 in VSMC conditioned medium. MMP-2 activity was similarly increased by exogenous CTGF treatment, and this effect could be blocked by an anti-CTGF antibody. We also showed that the increased MMP-2 activity was due to an increase in MMP-2 mRNA levels in VSMC. We further studied the mechanisms involved in the regulation of MMP-2 mRNA levels and found that the AP-2 transcription factor is responsible for most of the CTGF-induced MMP-2 transcription. Because MMP-2 is an important factor directly involved in controlling cell movement and the turnover of extracellular matrix, our study may provide a mechanism for CTGF-promoted VSMC migration.

Animals↗

Extracellular localization of human connective tissue mast cell granule contents.

In early phases of cutaneous inflammation, connective tissue mast cell degranulation is associated with apparent secretion and externalization of immunoreactive chymotryptic serine proteinase. To determine whether this event is associated with structural evidence of granule externalization, we studied the sequential evolution of IgE-mediated hypersensitivity in vivo, as well as mast cell degranulation provoked by a variety of stimuli in cultured explants of human skin. By 1 min after intradermal antigen challenge with ragweed extract, mast cell degranulation was associated with apparent extrusion of intragranule constituents into the pericellular connective tissue. Similar features typified cultured skin explants exposed for 45 min to anti-IgE and other mast cell secretagogues (morphine sulfate, calcium ionophore A23187, compound 48/80, and substance P). Once externalized, granule constituents could be identified within the dermal matrix by their rounded contour and structural similarity to solubilized granule matrices remaining within actively secreting cells. These data indicate that externalization of connective tissue mast cell granule contents occurs early after secretagogue exposure, potentially accounting for infrequent documentation of this event in naturally occurring dermatoses. The ability to recognize externalized granule products at a morphologic level should facilitate the understanding of interactions between mast cell-derived mediators and target structures of the dermal microvasculature.

Adult↗

A neoplastic connective tissue mast cell capable of continuous growth in tissue culture.

A neoplastic connective tissue mast cell from a dog mast cell sarcoma has been grown in tissue culture for 50 passages over a period of 2 years. The cells were grown as monolayer cultures in glass bottles, using Eagle's basal medium fortified with calf serum. The cultures were contaminated with an Alkaligenes sp. for 10 months but finally were sterilized bacteriologically by treatment with specific antiserum combined with antibiotics. The cells grow in a fibroblastic pattern, and contain mitochondria, mast cell granules, and lipid granules or droplets. The mast cell granules stain basophilic with Giemsa's stain and metachromatically with azure A or toluidine blue. They also stain with Sudan black B and with periodic acid-Schiff stain. The interphase nuclei are vesicular, contain from 1 to 20 nucleoli, and frequently show bizarre outlines. Multinucleate cells are often seen, as are mitotic figures. Extracellular fibrous material occurs in all cultures and apparently originates from the cell surface. This material does not have the structure of connective tissue fibers and has not been identified. The cells develop an increased number of metachromatic granules when grown in medium containing heparin and an increased number of sudanophilic granules when grown in medium containing stearic acid. Only small amounts of histamine were present in the tumor from which this cell line was derived and in the cells grown in tissue culture.

Animals↗

Neurogenic tumors of the skin.

A classification of peripheral nerve sheath tumors in the skin is based on analysis of light-, electron microscopy, immunohistochemistry and cellular composition of the different tumor types. Not only Schwann cells, but also perineurial cells and fibroblasts may be involved in tumor formation. Schwann cell rich and connective tissue cell rich tumors are distinguished. Generally all tumors show low proliferating activity as investigated by H3-Thymidine autoradiography. Only in few connective tissue cell rich tumors higher labelling indices can be demonstrated. These findings are correlated with the clinical course and prognosis of the different tumor types. The question remains, whether connective tissue cell rich tumors are at higher risk for malignant transformation.

Humans↗

Growth of connective tissue progenitor cells on microtextured polydimethylsiloxane surfaces.

Growth of human connective tissue progenitor cells (CTPs) was characterized on smooth and microtextured polydimethylsiloxane (PDMS) surfaces. Human bone-marrow-derived cells were cultured for 9 days under conditions promoting osteoblastic differentiation on smooth PDMS surfaces and on PDMS post microtextures that were 6 microm high and 5, 10, 20, and 40 microm in diameter, respectively. Glass tissue-culture dishes were used as controls. The number of viable cells was determined, and an alkaline phosphatase stain was used as a marker for osteoblastic phenotype. CTPs attached, proliferated, and differentiated on all surfaces. Cells on the smooth PDMS and control surfaces spread and proliferated as colonies in proximity to other cells and migrated in random directions, with cell process lengths of up to 80 microm. In contrast, cells on the PDMS post microtextures grew as sparsely distributed networks of cells, with processes, occasionally up to 300 microm, that appeared to interact with the posts. Cell counts revealed that there were fewer (50%) CTPs on the smooth PDMS surface than were on the glass control surfaces. However, there were consistently more (>144%) CTPs on the PDMS post textures than on the controls. In particular, the 10-microm-in-diameter posts (268%) exhibited a significantly (p < 0.05) greater cell number than did the smooth PDMS.

Bone Marrow Cells↗

The role of p42/44 MAPK and protein kinase B in connective tissue growth factor induced extracellular matrix protein production, cell migration, and actin cytoskeletal rearrangement in human mesangial cells.

Connective tissue growth factor (CTGF) is a member of an emerging family of immediate-early gene products that coordinate complex biological processes during differentiation and tissue repair. Here we describe the role of CTGF in integrin-mediated adhesive signaling and the production of extracellular matrix components in human mesangial cells. The addition of CTGF to primary mesangial cells induced fibronectin production, cell migration, and cytoskeletal rearrangement. These functional responses were associated with recruitment of Src and phosphorylation of p42/44 MAPK and protein kinase B. The inhibition of CTGF-induced p42/44 MAPK or phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway activities abrogated the induction of fibronectin expression. In addition, anti-beta(3) integrin antibodies attenuated the activation of both the p42/44 MAPK and protein kinase B and the increase in fibronectin levels. CTGF also induced mesangial cell migration via a beta(3) integrin-dependent mechanism that was similarly sensitive to the inhibition of the p42/44 MAPK and PI3K pathways, and it promoted the adhesion of the mesangial cells to type I collagen via up-regulation of alpha(1) integrin. Transient actin cytoskeletal disassembly was observed following treatment with the ligand over the course of a 24-h period. CTGF induced the loss of focal adhesions from the mesangial cell as evidenced by the loss of punctate vinculin. However, these processes are p42/44 MAPK and PI3K pathway-independent. Our data support the hypothesis that CTGF mediates a number of its biological effects by the induction of signaling processes via beta(3) integrin. However, others such as actin cytoskeleton disassembly are modulated in a beta(3) integrin/MAPK/PI3K-independent manner, indicating that CTGF is a complex pleiotropic factor with the potential to amplify primary pathophysiological responses.

Actins↗

Connective tissue mast cells in contact with fibroblasts express IL-3 mRNA. Analysis of single cells by polymerase chain reaction.

Mast cell-fibroblast interactions have been extensively investigated in the last few years. Fibroblasts support the in vitro survival but not proliferation of mouse connective-tissue type mast cells. However, the factor(s) that allow their survival on fibroblast monolayers has not been identified. We have investigated the presence of mRNA for IL-3 and granulocyte-macrophage-CSF in single mouse mast cells, before and after co-culture with 3T3 fibroblasts, using the polymerase chain reaction technique. The system was calibrated first by using in vitro generated population of mouse bone-marrow derived mast cells (BMMC). Significant differences in the amplification of IL-3 cDNA were observed in each of the BMMC cells examined, whereas the amplification of cDNA for the alpha-subunit of the Fc epsilon RI were similar. Inasmuch as murine cultured IL-3-dependent mast cells differentiate into connective tissue-like mast cells when co-cultured with 3T3 fibroblasts without any exogenous supply of growth factors, it was of interest to determine whether these connective tissue-like mast cells produce IL-3 message. Separation of the differentiated BMMC from the fibroblast monolayer, by either trypsinization or by single cell manipulation revealed the synthesis of a detectable amount of IL-3 mRNA in these mast cells. Whether this IL-3 mRNA was induced by fibroblasts was further investigated using connective tissue mast cells freshly purified from the mouse peritoneal cavity. Only about 20% of these connective tissue mast cells produced detectable amount of granulocyte-macrophage-CSF mRNA whereas in less than 10% of the cells IL-3 mRNA was detected. However, when these connective tissue mast cells were co-cultured with 3T3 fibroblasts for 18 hours and then separated, IL-3 mRNA were detected in most of the cells whereas no such mRNA was detected in tissue mast cells incubated for 18 h with medium derived from 3T3 fibroblasts. Therefore we conclude that fibroblasts induce the accumulation of IL-3 mRNA in connective tissue mast cells. The production of IL-3 may play a role in the survival of this type of mast cells on the fibroblast monolayer.

Animals↗