Ultrastructural and experimental evidence of myocardial cell differentiation into connective tissue cells in embryonic chick heart.
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Epithelial and connective tissue cells were isolated from rat palate by sequential enzymatic digestion. Differences between the two populations were noted with respect to proline uptake and incorporation, % collagen synthesized, effects of parathyroid hormone and metabolism of D-valine. From these studies it can be concluded that the cell populations are viable and distinct with respect to the biochemical parameters examined.
In human placental villi the connective tissue is constructed by mesenchymal cells, small and large reticulum cells and fibroblasts. During early pregnancy mesenchymal cells dominate; starting with the third month of gestation the reticulum cells are in the majority within the terminal villi, the fibroblasts within the stem villi. Ultrastructurally intermediary types of cells can be differentiated. Together with reticular and collagenous fibres the reticulum cells form the basic architecture of the villous stroma during the first 2/3 of gestation: the "reticular type of stroma". This consists of a network of cells and fibres with fetal vessels fitted in between. The remaining interspaces form a fluid system of compartments in which Hofbauer cells are suspended. They are called stromal channels. During the last trimester these channels and the Hofbauer cells as well are progressively replaced either by voluminous masses of fibres ("fibrous type of stroma", mainly in the stem villi) or by sinusoidal enlargements of fetal capillaries ("sinusoidal type of stroma", mainly in the terminal villi).
The surface features and behavior of the connective tissue cell in normal areas and in healing incisions of the aponeuroses of the abdominal wall of the albino rat were studied with the scanning electron microscope (SEM). The normal cell is smooth, oval or triangular in outline and has a generally rounded surface and very short or no cell processes. The behavior of the fibroblasts and the condition of the fibers in and around the wounds were visualized. Shortly after the incision, the fibroblasts accumulated around the wound, possibly by increased cell division. At the site of the incision the cells responded by a self-protective mechanism in which they withdrew their processes and acquired a spheroid form. During the maximum period of a fibrillogenesis the fibroblasts enlarged, acquired a fusiform outline and long processes, exhibited a roughened surface and were surrounded with small rounded cell particles polymerizing into fine fibrils. The process of collagen secretion and three stages of collagen maturation were visualized. Some abnormal cells were seen; they could be evidence of merocrine or holocrine mode of secretion or possibly signs of cell senility.
The ubiquitous connective tissues contain a wide range of cells including fibroblasts, osteoblasts and chondroblasts. Recently it has been demonstrated that another principal cell of the connective tissue is the smooth muscle cell in several organ systems. These have been shown to be responsible for the synthesis of the connective tissue matrix components of the uterine myometrium and of the arterial system, including collagen, both elastic fibre proteins and glycosaminoglycan. Microtubule inhibitors such as colchicine and vinblastine, and iron chelators such as alpha,alpha -dipyridyl have been used to study their morphologic and chemical effects on collagen synthesis and secretion. Colchicine produces an increase in large Golgi-associated vacuoles, which sometimes contain material reminiscent of aggregates of collagen macromolecules. Vinblastine produces alterations in the endoplasmic reticulum cisternae similar to alterations seen in ascorbic acid deficiency, and alpha,alpha-dipyridyl increases the frequency of regions in cells, interpretable as potential sites of communication of rough endoplasmic reticulum cisternae with the cell surface. Ferritin conjugated anti-procallagen sera were used to localize procollagen in cells and demonstrated procollagen not only in the cisternae of rough endoplasmic reticulum but in all of the elements of the Golgi complex as well. The studies reported in this review have shown that in cell culture arterial smooth muscle will produce not only the microfibrillar protein of the elastic fibre but soluble and/or insoluble elastin as well. Recent studies on serum factors responsible for the proliferation of connective tissue cells have demonstrated that at least one of the principal factors responsible for fibroblast and/or smooth muscle cell proliferation in culture is derived from thrombocytes. Medium containing serum derived from cell-free plasma lacks most of this proliferative effect which can be reinstated when platelets are present during recalcification to form serum. This effect is due to the platelet release reaction as shown by combining supernatant factors derived from platelets exposed to purified thrombin to cell-free, plasma derived serum. Studies with macrophages have also suggested that phagocytic macrophages release factor(s) into a cell culture medium that may also participate in stimulating fibroblasts to proliferate in vitro. The means by which these factors stimulate fibroblast proliferation and connective tissue synthesis remains to be elucidated.
A study of the quantitative parameters of DNA in cells nuclei of epithelium and connective tissue allowed establishing certain periods which are characterized by an increase in the percentage of ploidy. The causes and importance of this phenomenon for the development of pathological processes in tissues of the prostate gland and the possibility of its use for the diagnosis are discussed.
In 60 rabbits with experimental myocardial infarction induces by ligation of the anterior branch of the left coronary artery, DNA synthesis was studied by means of H3-thymidine in muscular and connective tissue cells depending on the period of myocardial infarction. The development of myocardial infarction in the cardiac muscle was stated to be accompanied by activation of DNA synthesis in the connective tissue cells not only in the necrotic zone but in the adjacent, as well as in distant areas of the myocardium and in stromal cells of the auriculum. Indices of H3-thymidine labeled nuclei were of high value during the acute period of myocardial infarction and gradually decreased with the time elapsed since the operation. During the period of the myocardical infarction organization high activity in DNA synthesis was revealed in connective tissue elements of the epicardium and the subepicardial zone of the heart. Myocardial cells of the cardiac auriculi incorporated H3-thymidine but extremely seldom-single labels per thousands of nuclei. In the auriculi with application of prolonged sessions of the labeled precussor introduction, DNA synthesising nuclei were revealed, sometimes with paired nuclei of cardiomyocytes.
Mild treatment with trypsin causes a radical change in the heavy meromyosin (HMM) binding properties of intermediate filaments in glycerinated, myosin-extracted cultured chick embryo connective tissue cells. In non-trypsin-treated cells, HMM labeling of filaments was often indistinct and variable in its distribution. By contrast, in cells treated with trypsin (under conditions which allowed most intermediate filaments to survive), virtually all filaments, including those of intermediate size, decorated with HMM to give distinct arrowhead patterns. We suggest that most intermediate filaments in such cells contain a core of F-actin masked by trypsin-labile accessory proteins.
Human and rat decalcified bone matrix preparations were shown to be active in inducing cartilage formation by subcutaneous implantation in the rat. When young rat fibroblastic cells were grown in cultures, which also contained bone matrix preprations in particulate form, the fibroblastic cells underwent a uniform and consistent morphological alteration. These altered cells showed higher rates of synthesis of hyaluronic acid and chondroitin sulfate than the controls and exhibited very active amino-sugar-nucleotide metabolism. It is suggested that this approach to the culture of connective tissue cells will allow a more precise definition of the early steps of connective tissue differentiation.
A model has been developed that proposes a cell surface-associated protein meshwork, composed in part of fibronectin and collagen, for a connective tissue cell attached to a substratum. In support of this model are the observations that collagen and fibronectin interact and that these proteins are similarly distributed on the fibroblast cell surface. We suggest that this external meshwork interacts directly or indirectly with the internal cytoskeleton and with the extracellular matrix and thereby mediates several cellular properties, including adhesion, shape, and motility. Loss of cell surface fibronectin as a result of viral transformation, or due to treatment of normal cells with tunicamycin, an inhibitor of protein glycosylation, may contribute to the reduced adhesion and altered morphology observed in these circumstances. We therefore predict that the changes in these properties observed with virally transformed cells, mitotic cells, and cells treated with proteolytic enzymes are related to alterations in the external protein meshwork.
CTAP-I from lymphocytes and CTAP-III from platelets markedly stimulated 35SO4= incorporation into chondroitin 4/6 sulfate and dermatan sulfate synthesized by human synovial, dermal, and cartilage connective tissue cells in vitro. These agonists promoted synthesis of the GAG carbon chain as well as sulfate incorporation. Both RNA and protein synthesis were required for these mediators to be effective in stimulating synthesis of connective tissue matrix components. A major part of the capacity of normal serum to stimulate sulfate incorporation into GAG's may reside in CTAP-III.
Guinea pig lung fibroblasts "activated" in vitro by exposure to connective tissue-activating peptides I and III, and guinea pig tissue extracts showed enhanced glycolysis and accelerated glycosaminoglycan synthesis. Formation of hyaluronic acid, and to a lesser extent, chondroitin 4/6-sulfate was stimulated by these agents.
1. The effects of two diphosphonates (compounds containing a P-C-P bond), disodium dichloromethanediphosphonate and disodium 1-hydroxyethane-1,1-diphosphonate, on the metabolism of cultured rat calvaria cells, rabbit ear cartilage cells and rat skin fibroblasts were investigated. 2. The diphosphonates had no effect on the growth of cartilage cells and on the exponential growth of the calvaria cells and the fibroblasts. However, dichloromethanediphosphonate stopped the growth of the calvaria cells and the fibroblasts after the beginning of confluence, whereas the untreated cells were still growing to a certain extent. This inhibition was dose-dependent. After the drug was withdrawn, the cells recovered slowly. 1-Hydroxyethane-1,1-diphosphonate had no detectable effect on the growth of any of the cell types studied. Both diphosphonates decreased the cloning efficiency of calvaria cells and fibroblasts. 3. The K+ content of cartilage, calvaria and skin cells was diminished only by the highest (0.25 mM) concentration of dichloromethanediphosphonate. 4. Radioactive dichloromethanediphosphonate and 1-hydroxyethane-1,1-diphosphonate were taken up linearly with time for at least 48 h by calvaria cells and fibroblasts. The diphosphonate concentration in the cells depended on its concentration in the medium. 5. Both diphosphonates, in a dose-dependent fashion, markedly inhibited glycolysis, dichloromethanediphosphonate being more effective than 1-hydroxyethane-1,1-diphosphonate, at drug doses that had no effect on cell growth or cellular K+ content. Calvaria cells were much more sensitive than cartilage cells. When cartilage cells were cultured in an N2 atmosphere, these effects on glucose and lactate metabolism disappeared. 6. As increased acid production appears to be associated with resorption of bone, this decrease in lactate may explain why diphosphonates are effective inhibitors of bone resorption in vivo.
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Collagen and/or procollagen was demonstrated on the surface of monolayers of fibroblasts from normal rat kidney by indirect immunofluorescence with affinity-purified antibodies to collagen. The protein was arrayed in a reticular fashion on the cell surface and, in cells attached to a substratum, was severely restricted in its ability to undergo antibody-induced translational movement in the plane of the membrane. A similar pattern was observed for fibronectin (LETS protein, fibroblast surface antigen). These macromolecules were lost when fibroblasts were dissociated and examined in suspension cultures and were not regained until after the cells were replated. On the basis of the morphological findings, and in view of the likelihood of an interaction between fibronectin and collagen, we propose that these that these proteins form a meshwork on the cell surface. This external protein meshwork may mediate a number of important cellular functions, including attachment to a substratum and other interactions with the extracellular matrix.
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The origin of elements of the focus of aseptic inflammation and the normal subcutaneous connective tissue in the xenogenic (mouse-rat) radiation chimaeras was investigated by means of indirect Coons method with antiserum to the rat bone marrow cells. The cells of the imflammation focus (leucocytes, macrophages, fibroblasts or fibroblast-like cells, polynuclear giant cells of foreign bodies), as well as leucocytes, macrophages and some fibroblasts of the normal subcutaneous connective tissue, were shown to take their origin from the transplanted bone marrow cells of the donor.
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