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PubMed1989

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro.

The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [35S]methionine-labeled polypeptide pattern.

Cell Differentiation↗

PubMed1989

A method for removal of fibroblasts from human tissue culture systems.

The phenomenon of fibroblast overgrowth is one of the major problems encountered during long-term culture of more slowly growing specialized cell types. A cell surface glycoprotein, Thy-1, which was originally found to be present on murine T-lymphocytes and brain cells, is also found to be present on only a few human cell types, mainly fibroblasts and neuronal cells. We have taken advantage of this fact, using a solid-phase immunoadsorption technique termed "panning", to rid our culture system (normal human keratinocytes) of contaminating dermal fibroblasts. A mouse monoclonal antibody raised against human brain Thy-1 was used to attach dermal fibroblasts to a goat anti-mouse immunoglobulin-coated plastic surface. By this method we were able to separate a 1:1 mixture of human dermal fibroblasts and keratinocytes with greater than 97.5% efficiency. Furthermore we have successfully removed dermal fibroblasts from naturally arising contaminated keratinocyte cultures, where the proportion of fibroblasts (less than 10%) was considerably less than that of the artificially mixed populations. These results compare favorably with those expected of the fluorescence-activated cell sorter (FACS) method of cell separation. In addition this technique is comparatively simple and inexpensive and is thought to be of use to other primary tissue culture systems (especially human) where contamination and subsequent overgrowth with fibroblasts remains a problem.

Cell Adhesion↗

PubMed1990

Interactions between lymphocytes and dermal fibroblasts: an in vitro model of cutaneous lymphocyte trafficking.

Cultures of dermal fibroblasts were established from skin biopsies of CBA mice and used to study the interactions with murine T-lymphocytes. Electron microscopy showed that zones of contact developed between the fibroblasts and the T-cells, particularly after mitogenic activation. The adhesion of the lymphocytes was temperature-dependent, and many more lymphoblasts than resting cells attached to the fibroblast monolayers. Flow cytometry analysis of the adherent population showed that the most prominent type of resting lymphocyte was of the CD4 phenotype, which was also observed using a T-helper lymphoid cell line. However, neither the CD4 nor the CD8 (T-cytotoxic) antigens were involved in the binding process, and while the fibroblasts expressed Class I MHC molecules (but not Class II), these also had no role in mediating lymphocyte adhesion. Although the fibroblasts did not express the ligand Mala-2, the murine homologue of human ICAM-1, a monoclonal antibody against LFA-1, its cognate receptor on the lymphocytes, nevertheless effectively inhibited binding. T-cell attachment was also partially prevented by antibody against the lymphocyte CD2 antigen and by RGDS, a protein epitope known to mediate a number of receptor-integrin interactions. Moreover, this peptide also rapidly and preferentially detached T-lymphocytes which had previously adhered to the fibroblast monolayers. Lymphocyte binding was substantially elevated following treatment of the fibroblasts with cytokines such as tumor necrosis factor-alpha and interferon-gamma, but not interleukin-1 alpha. This increase in adhesiveness was, however, almost completely abolished by monoclonal antibodies specific for LFA-1 or for Mala-2. The results of this study show that while lymphocytes recognize fibroblasts normally via a number of constitutively expressed receptor-integrin interactions, their adhesion can also be modulated by cytokine-induced changes in the expression of other surface ligands.

Animals↗

PubMed1991

Collagen gel contraction by fibroblasts requires cellular fibronectin but not plasma fibronectin.

Fibroblasts embedded in three-dimensional lattices of collagen fibrils have been known to require serum constituents to induce a cell-mediated contraction of collagen gels. The gel contraction was studied with human skin fibroblasts cultured in the presence of fetal bovine serum (FBS). Removal of bovine serum fibronectin (sFN) from FBS did not affect the extent of gel contraction. Gel contraction occurred in serum-free defined media. Therefore, it is concluded that sFN is not required for gel contraction. That cellular FN (cFN) synthesized and secreted by fibroblasts plays a crucial role in gel contraction was suggested by the following experiments: (1) We obtained monoclonal antibodies (mAb A3A5) against fibroblast surface antigens, which suppressed the fibroblast-mediated gel contraction. Immunoblot analyses showed that mAb A3A5 recognizes cFN secreted by human fibroblasts and human plasma FN (pFN), but not bovine sFN in FBS used for culture. (2) Addition of rabbit antisera, which recognize human cFN, to a serum-free gel culture inhibited contraction. Uninvolvement of human pFN in gel contraction was further confirmed by the fact that neither pretreatment of fibroblasts with excess amounts of human pFN nor the presence of excess amounts of human pFN in gels affected the extent of gel contraction. This study seems to be the first demonstration of functional difference between cFN and pFN (or sFN) and proposes a novel mode of binding of fibroblasts with collagen fibrils via cFN during cell-mediated collagen morphogenesis.

Antibodies, Monoclonal↗

PubMed1992

Culture of human dermal fibroblasts in collagen gels: modulation of interleukin 1-induced prostaglandin E2 synthesis by an extracellular matrix.

Human dermal fibroblasts, cultured as suspensions in collagen gels and as monolayers, were stimulated with recombinant human interleukin-1 beta (rIL 1 beta) at 72 h, and prostaglandin E2 (PGE2) was assayed 24 h later. Fibroblasts in gels were less responsive to rIL 1 beta than monolayers, PGE2 synthesis increasing from less than 1 ng/microgram DNA without rIL 1 beta to maxima of 11.3 and 32.9 ng/micrograms DNA, with half maximal release occurring at 7.47 and 0.75 pM rIL 1 beta for the gel and monolayer cultures, respectively. Increased PGE2 was first detected 4 h after addition of rIL 1 beta to gels and was inhibited by 10(-5) M indomethacin. The amount of PGE2 synthesized per fibroblast increased with the time the gels had been in culture when stimulated with rIL 1 beta and was proportional to the number of fibroblasts in the gels, but inversely related to the collagen concentration. A common feature of these experiments was significantly greater induction of PGE2 synthesis at higher cell densities in collagen gels. Exogenous 10(-4) M arachidonic acid further increased PGE2 synthesis by rIL 1 beta-stimulated fibroblasts, but the differential in the amount of PGE2 released from fibroblasts at high and low population densities in the gels was maintained. These results are consistent with interleukin 1 (IL 1) stimulating PGE2 synthesis in dermal fibroblasts by increasing cyclooxygenase activity. Furthermore, the results show that dermal fibroblasts have an additional regulatory mechanism, related to the cell population densities or their interactions with an extracellular matrix, to finely modulate the amount of PGE2 synthesized in response to IL 1.

Arachidonic Acid↗

PubMed1987

Modulation of fibroblast proliferation by postsurgical macrophages.

Macrophages and fibroblasts are major components of postsurgical peritoneal repair. In order to understand the interaction between these two cell types, we studied the effects of spent macrophage culture media on fibroblast proliferation. Rabbits underwent resection and reanastomosis of their small intestine. Peritoneal exudative cells (PEC) were then collected from these animals on postoperative Days 4, 7, and 28 and from nonsurgical controls. PEC (5 X 10(5) cells/ml) were cultured in M-199 with 3% fetal calf serum. After 48 hr the spent media from the cultured PEC were harvested, centrifuged (200g for 10 min), and stored (medium: M-D0, D4, D7, D28). A second group of rabbits underwent peritoneal wall abrasion followed by collection of fibroblasts directly from the site of injury on postoperative Days 1, 4, and 7. After Day 7 of culture, fibroblasts were resuspended and seeded into dishes (1 X 10(5) cells in 1 ml medium), to which was added 1 ml of spent PEC culture medium. After 24 hr of incubation, 1 muCi [3H]thymidine was added for an additional 18 hr. Fibroblasts were then collected and the amount of [3H]thymidine incorporated into trichloroacetic acid-precipitable material was quantitated. In one protocol, fresh M-199 with 3% fetal calf serum was used, and in another protocol, "U-medium" (which was previously incubated for 48 hr with fibroblasts) was used. The cytology of the PEC was determined by Wright's staining, nonspecific esterase activity, and phagocytosis. At least 80% of the peritoneal exudative cells were identified as macrophages. Postsurgical Day 7 fibroblasts demonstrated greater [3H]thymidine incorporation compared to fibroblasts from postoperative Days 1 and 4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Effects of exogenous monounsaturated fatty acids on fatty acid metabolism in cultured skin fibroblasts from adrenoleukodystrophy patients.

The conversion of [1-14C]16:0 to very-long-chain saturated fatty acids (VLCSFA) was greater in fibroblasts from adrenoleukodystrophy (ALD) patients than fibroblasts from normal subjects. Added 23:1(n-9) decreased the formation of VLCSFA from [1-14C]16:0 in ALD fibroblasts to the value found in normal fibroblasts. Chain-elongation as well as extensive chain-shortening of added 20:1(n-9), 22:1(n-9), 23:1(n-9) and 24:1(n-9) occurred in both normal and ALD fibroblasts, with chain-shortening being less in ALD than in normal fibroblasts. Added 18:1(n-9) together with 22:1(n-9) reduced the levels of both VLCSFA and total n-6 and n-3 polyunsaturated fatty acids (PUFA) in normal and ALD fibroblasts. The levels of total (n-6) and (n-3) PUFA but not the levels of VLCSFA were readily restored by culturing the cells in the presence of 18:1(n-9), 22:1(n-9), 18:2(n-6) and 18:3(n-3). The results are consistent with added monounsaturated fatty acids reducing levels of VLCSFA in ALD fibroblasts by depressing their biosynthesis from 16:0. They also support the use of oils rich in long chain monoenes as a dietary therapy for ALD patients but caution that the PUFA status of ALD patients should be monitored with a view to dietary supplementation, if necessary, with PUFA.

Adrenoleukodystrophy↗

PubMed1989

Increased glucose transport by human fibroblasts with a heritable defect in insulin binding.

Insulin and IGF-I binding and their regulation of hexose transport were evaluated in skin fibroblasts cultured from a family (Atl) whose proband had leprechaunism, hypoglycemia, and severe insulin resistance. High affinity insulin binding to proband Atl cells was absent, and partially, but equally, impaired in fibroblasts from his related parents. IGF-I binding to his cultured fibroblasts was within the normal range. Cells from proband Atl had insulin receptor mRNAs similar to control fibroblasts. 3-O-Methyl-D-glucose (OMG) transport by proband Atl was threefold higher than in control fibroblasts (37.7 v 7.6-11 nmol/mL/s) and was insulin-insensitive. Proband Atl fibroblasts had a threefold increase in the Vmax for OMG entry and a concomitant increase in the number of D-glucose-inhibitable cytochalasin B binding sites on their plasma membrane. Similar levels of glucose transporter mRNA were observed in control and proband Atl fibroblasts. These results suggest that fibroblasts from patient Atl have a genetically transmitted mutation in the alpha subunit of their insulin receptor. In the homozygous affected proband, this mutation impairs insulin binding and causes elevated, insulin-insensitive glucose transport. The dysfunction resulting from this mutation is similar to that introduced in Chinese hamster ovary cells by transfection with a truncated alpha subunit.

3-O-Methylglucose↗

PubMed1993

Immune-mediated mechanisms and immune activation of fibroblasts in the pathogenesis of eosinophilia-myalgia syndrome induced by L-tryptophan.

To determine the immunopathogenesis of the persistent symptoms of patients with eosinophilia-myalgia syndrome (EMS) induced by L-tryptophan, we performed immunocytochemical studies on 10 muscle and fascia biopsy specimens obtained during the acute disease and the chronic persistent connective tissue sclerosis. A series of monoclonal antibodies was used in a single- or double-immunostaining technique to detect and quantify T-cell subsets, macrophages, major histocompatibility complex antigens, eosinophilic basic protein-positive cells, and resting fibroblasts expressing Thy-I antigen or activated fibroblasts expressing the activation marker F-19. We found inflammatory cells consisting of CD8+ cells (45% +/- 8.9%), T4 cells (36% +/- 10.1%), and macrophages (19% +/- 12%), scattered or perivascularly in the fascia, the perimysium, and the endomysial septae. Only rare granulated or degranulating eosinophils were noted. Many muscle fibers around fascicles or near blood vessels expressed major histocompatibility complex-I antigens. The mean number of fibroblasts in the fascia, the perimysial connective tissue, and the spindle capsule was increased in the EMS patients' specimens compared with the endomysial cells seen in six disease-control muscle biopsy specimens from patients with chronic inflammatory myopathies or dystrophies (P < .01). Up to 70% of the fibroblasts in EMS were activated and up to 30% of them expressed HLA-DR antigen. In the disease controls up to 29% of the fibroblasts were activated but none expressed DR. Repeat muscle biopsy a year later in a patient whose symptoms persisted showed reduced inflammation but an increased number of activated fibroblasts and enhanced DR expression. We conclude that in EMS there is a T-cell-mediated process against components of the extracellular matrix, including fibroblasts, in the fascia and the perimysium that persists even years after the drug is discontinued. Because the fibroblasts are activated and aberrantly express DR antigen, they may be the target cells playing a role in the continuing clinical and histologic signs of tissue sclerosis.

Adult↗

PubMed1979

Age-related cell surface changes in human diploid fibroblasts revealed by lectin-mediated red blood cell adsorption assay: a lectin survey.

Two types of age-related cell surface changes could be demonstrated in human diploid fibroblasts with the two methods of the lectin-mediated red blood cell (RBC) adsorption assay: the fibroblast coating method (in which RBCs are adsorbed to lectin-coated fibroblasts) and the RBC coating method (in which lectin-coated RBCs are adsorbed to fibroblasts). With the fibroblast coating method, concanavalin A and agglutinin L from Phaseolus vulgaris gave a change in RBC adsorption which did not occur throughout the phase II period, but increased with the advance of the phase III period (type I). With the RBC coating method, these lectins gave another type of change in RBC adsorption which increased continuously from early phases of cell passage up through cell senescence (type II). Ricinus communis agglutinin 120 also gave the type I change in RBC adsorption with the fibroblast coating method. On the other hand, even with the fibroblast coating method, Phaseolus vulgaris agglutinin E and wheat-germ agglutinin gave the type II change in RBC adsorption. Soy bean agglutinin and Bauhinia purpurea agglutinin gave only a restricted amount of RBC adsorption. Lens culinaris agglutin, pokeweed mitogen, Dolichos biflorus agglutinin, Lotus tetragonolobus agglutinin, Limulus polyhemus agglutinin and divalent succinylated concanavalin A did not give any RBC adsorption throughout the life span of human diploid fibroblasts.

Cell Membrane↗

PubMed1988

Mononuclear cell-fibroblast interactions in scleroderma.

We studied cell proliferation and collagen biosynthesis in cocultures of dermal fibroblasts with peripheral blood mononuclear cells (MNC) from scleroderma patients and from age-matched normal controls. Autologous one-way mixed MNC-fibroblast cultures revealed that fibroblasts do not stimulate MNC proliferation. Conversely, MNC stimulate autologous fibroblasts from scleroderma patients as well as from normal controls. This effect is increased in cells from scleroderma patients in which it seems to be mediated both by cell-to-cell interaction and through the production of soluble factors by MNC. In normal control cell systems we found no proliferative effect of supernatants of unstimulated cells or from those stimulated in autologous mixed-lymphocyte reactions. Coculture of fibroblasts with autologous MNC resulted in increased [14C]proline incorporation into both collagenic and noncollagenic proteins. This effect was mediated mostly by soluble factors that are released into the culture medium. Protein synthesis by MNC-fibroblast cocultures from scleroderma patients was significantly greater than protein synthesis by those from normal controls. Culture supernatants from unstimulated MNC or from autologous mixed-lymphocyte cultures caused a slight decrease in collagenic protein synthesis by cultured fibroblasts from scleroderma patients but not by those from normal controls. This effect of culture supernatants could be reproduced, and magnified, with purified IL-1 on cells from either patients or controls. Our findings indicate abnormal MNC-fibroblast interactions in scleroderma that could play an important role in the pathogenesis of fibrosis, the hallmark of this condition.

Adult↗

PubMed1992

Effect of coculture of rodent mast cells with murine chronic graft-versus-host disease (cGVHD)-derived fibroblasts.

We investigated whether murine chronic graft-versus-host disease (cGVHD)-derived skin fibroblasts maintain the viability and functional activity of rat peritoneal connective tissue-mast cells (CTMCs) and whether they affect the change in phenotype of mouse bone marrow-derived mast cells (BMMCs). Skin fibroblasts were isolated before the development of fibrosis (day 5), during overt fibrosis (day 28), and after recovery from fibrosis (day 120). cGVHD fibroblasts of days 5 and 28 exhibited enhanced proliferation, a property that was maintained through several subcultures. CTMCs adhered to the same extent, did not divide, and maintained their viability in all the different cultures. When CTMCs were activated with compound 48/80, they released approximately 80% of their histamine content, indicating that coculture with cGVHD fibroblasts did not adversely affect CTMC function. The amount of histamine found in the medium of 8 days CTMC/cGVHD fibroblast coculture was similar to that found in control culture. These findings suggest that the degranulation of dermal MCs, characteristic of cGVHD, is not due to a direct activating effect of the cGVHD fibroblasts on the MCs. BMMCs seeded on cGVHD fibroblasts acquired the capacity for safranin staining and increased their histamine content, indicative of a change to CTMCs. Thus, cGVHD fibroblasts are able to provide a microenvironment adequate for maintaining viability and activity of CTMCs and for promoting maturation and change in phenotype of BMMCs.

Animals↗

PubMed1995

Megakaryocytes and fibroblasts--interactions as determined in normal human bone marrow specimens.

An in vitro study was performed to investigate possible interactions between megakaryocytes and bone marrow fibroblasts, both obtained from healthy donors. We were able to demonstrate that the proliferation of fibroblasts increased significantly by co-culturing these cells with megakaryocytes for 6 days. Addition of neutralizing antibodies for PDGF and TGF beta 1, caused a significant reduction of fibroblast growth. Inhibition of cell to cell contacts via tissue culture inserts generated a conspicuous impairment of fibroblast proliferation compared with megakaryocyte-fibroblast co-cultures, where contact was allowed. Hence, our findings suggest that a close spatial relationship between megakaryocytes and fibroblasts is needed for the activation of growth in normal human bone marrow. Neighbouring of megakaryocytes and fibroblasts seems to be necessary in order to achieve a certain threshold of local growth factor concentration. Our results are in keeping with the assumption that PDGF and TGF beta 1, are secreted by normal human megakaryocytes in very low concentrations and promote significantly fibroblast proliferation.

Adult↗

PubMed1991

Abnormal growth of skin fibroblasts from schizophrenic patients.

Fibroblast cultures were established from skin biopsies of 18 schizophrenic patients (14 on drug and 4 off drug) and 13 normal subjects, and growth parameters (initial growth and rate of growth) and morphology were studied. Fibroblasts from patients took significantly longer to grow than did normal fibroblasts. Cell lines were established within 2 weeks for all normal controls, but for only 6 (33%) of 18 schizophrenic patients. The rate of growth (doubling time) was also significantly longer for fibroblasts from patients than from normals. Neither time to establishment of initial growth nor doubling time was related to age, sex, age at onset, duration of illness, or medication status in the patients. Fibroblasts from normals showed uniform, long (slender), characteristic spindle-like, bipolar appearance, with unidirectional orientation, both while growing from explant as well as after subculturing. By contrast, fibroblasts from patients generally showed random size (shorter and flatter), mostly spiny, multipolar cells with short stubby projections, and an irregular orientation resulting in a criss-cross pattern, and often exhibited poor attachment. Fibroblasts from skin biopsies of patients who were drug free at the time of biopsy showed similar initial growth, doubling time, and morphology to those from patients who were receiving neuroleptic treatment. In vitro challenge of skin biopsies of normal subjects with haloperidol in culture resulted in slight delay in initial growth and marginal increase in doubling time. However, the morphology remained normal. Possible molecular mechanisms that may be associated with abnormal growth of fibroblasts in schizophrenia are discussed.

Adolescent↗

PubMed1995

Prostaglandin E2 production by chronic graft-versus-host disease dermal fibroblasts.

Chronic graft versus host disease (cGVHD) across minor histocompatibility barriers is associated with the development of cutaneous fibrosis, disappearance of mast cells and immunosuppression. The idea, which has been the basis of our previous and present studies, is that fibroblasts are not only a target for modulation in cGVHD, but also have effector roles in this condition. In the present study we investigated the production of prostaglandin E2 (PGE2) and of collagen by cultured dermal fibroblasts obtained from cGVHD and control mice. Early in the development of the disease (Day 8) cGVHD fibroblasts generated constitutively more PGE2 (3-fold) than did control fibroblasts. Thereafter, PGE2 production declined to near normal levels by Day 20 post cGVHD induction. On the other hand, at this time point cGVHD fibroblasts displayed an enhanced synthesis of collagen as compared to the control fibroblasts and to earlier time points. Therefore, PGE2 synthesis appears to inversely correlate with collagen synthesis by cGVHD fibroblasts. We propose that fibroblasts may contribute to the development of immunosuppression, which characterizes the early phase of cGVHD.

Animals↗

PubMed1994

Encapsulated genetically engineered fibroblasts: release of nerve growth factor and effects in vivo on recovery of cholinergic markers after devascularizing cortical lesions.

Genetically engineered rat fibroblasts producing nerve growth factor (NGF) were encapsulated in alginate-polylysine-alginate gels with the objective to produce viable "minifactories" continuously producing and secreting NGF into the rat brain. Microencapsulated fibroblasts (NGF secretors and NGF non-secretors) were placed onto the surface of the lesioned rat cortex (unilateral devascularizing lesion) and animals were sacrificed 30 days after surgery. Fibroblasts NGF-non secreters normally produce tumors after implantation, therefore, they were irradiated prior to encapsulation. Three other experimental groups were studied in parallel: non-lesioned (controls), lesioned rats receiving "empty" alginate spheres and lesioned animals without treatment and microspheres. Biochemical analysis of microdissected brain tissues of lesioned animals treated with encapsulated NGF-secretor fibroblasts showed a significant increase in choline acetyltransferase (ChAT) activity in cortices adjacent to the lesion but not far from it (entorhinal cortex). This may indicate a gradient of concentration of the released NGF and/or differential responsivity of lesioned vs non-lesioned target tissue. ChAT enzymatic activity in the microdissected nucleus basalis magnocellularis (NBM) was significantly decreased (P < 0.05) in all lesioned animals treated with spheres without fibroblasts and those with fibroblasts not secreting NGF. Morphometric analysis of ChAT-IR and low affinity NGF-receptor IR cholinergic neurons in the middle portion of the NBM shows a prevention of neuronal shrinkage and extensive neuropil in animals treated with microencapsulated NGF-secretor fibroblasts. The results of this study demonstrate that NGF from encapsulated genetically engineered fibroblasts can be secreted for at least long enough to prevent degenerative changes of cholinergic neurons in the NBM.

Alginates↗

PubMed1982

Abnormalities of lysosomes in human diploid fibroblasts from patients with Farber's disease.

An accumulation of ceramide associated with the deficiency of acid ceramidase has been demonstrated in cultured diploid skin fibroblasts from a patient with Farber's disease. We extend this observation to investigate the lysosomal localization of accumulated ceramide and the abnormalities of lysosomes caused by this ceramide accumulation in Farber's diseased fibroblasts. We have found that the lysosomal fraction isolated from Farber's diseased fibroblasts by a subcellular fractionation procedure is markedly low in density compared with that of normal fibroblasts and is separated from other subcellular organelles. Ultrastructural studies of the isolated lysosomal fraction from Farber's diseased fibroblasts showed a mixed population of intact and swollen vesicles with a lysosomal appearance. Examination under high magnification clearly demonstrated lysosomal inclusions which contain lamellar and curvilinear membranes and resembled those seen in the intact fibroblasts. Subcellular localization of Farber's fibroblasts showed that the accumulated [3H]ceramide from the culture medium was predominantly localized in the lysosomal fraction with a markedly low density and very little was found to be associated with other cellular membranes. Our finding that ceramide is accumulated in the lysosomal fraction of Farber's fibroblasts and that these cells also show membranous inclusions strongly suggests that the accumulation of ceramide is directly involved in the formation of lysosomal inclusions.

Acid Ceramidase↗

PubMed1996

In vitro models to study wound healing fibroblasts.

Phenotypic and contractile properties of human fibroblasts from dermis and from an experimental wound model were studied in vitro. When cultured in monolayer, dermal fibroblasts had an elongated spindle shape, were small in diameter and grew at a high rate. Wound fibroblasts grew slowly and were large, star shaped and had cytoplasmic stress fibres. Smooth muscle alpha actin was detected in 10 percent of dermal cells, whereas 20-80 per cent of wound fibroblasts contained this protein in their cytoplasm. The contractile property of cells was evaluated using a three-dimensional cell culture model. Our results show that wound fibroblasts contract collagen gels during the first days more strongly than dermal fibroblasts. These results show that, in vitro, wound fibroblasts have greater contractile capacity than dermal cells. The significant proportion of wound fibroblasts containing alpha-smooth muscle actin suggests that alpha-smooth muscle actin ratio may be related to wound contraction.

Cells, Cultured↗