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PubMed1987

Mouse bone marrow-derived mast cells cocultured with fibroblasts. Morphology and stimulation-induced release of histamine, leukotriene B4, leukotriene C4, and prostaglandin D2.

Mouse bone marrow-derived mast cells (BMMC), cultured for 2, 7, or 14 days in WEHI-3 conditioned medium in the absence or presence of mouse 3T3 fibroblasts, were examined morphologically and for their functional responses to IgE-Fc-mediated and calcium ionophore-mediated activation. The 7- and 14-day fibroblast-adherent and non-fibroblast-adherent populations of cocultured BMMC had more granules per cell and the granule contents were more electron dense than non-cocultured BMMC or BMMC cocultured for only 2 days. The adherent cocultured BMMC were usually located within multiple layers of fibroblasts, but did not form junctions with the fibroblasts. When activated immunologically, the adherent cocultured mast cells generally discharged their granules singly, but compound exocytosis was occasionally seen. Both the non-adherent cocultured BMMC and the BMMC that were cultured in the absence of fibroblasts were similar to one another in that they exocytosed 9 to 11% of their histamine when sensitized with anti-dinitrophenyl IgE and challenged with dinitrophenyl-bovine serum albumin and 27 to 29% of their histamine when challenged with calcium ionophore. In contrast, adherent cocultured BMMC exocytosed 27 and 61% of their histamine upon immunologic and calcium ionophore activation, respectively, representing a significant two- to three-fold increase relative to that obtained from the other populations of BMMC. When activated immunologically, BMMC cultured in WEHI-3 conditioned medium alone generated a mean of 12 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 1.6 ng of leukotriene B4 (LTB4), and 1.0 ng of prostaglandin D2 (PGD2)/10(6) cells. The immunologic response of the nonadherent 7-day cocultured BMMC was similar. Fibroblast-adherent cocultured BMMC, on the other hand, generated 56 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 6.4 ng of LTB4, and 5.6 ng of PGD2/10(6) mast cells, representing a significant increase for each product. When calcium ionophore was used as the agonist, the adherent cocultured mast cells also generated significantly more arachidonic acid metabolites than nonadherent cocultured BMMC or BMMC cultured in the absence of fibroblasts. Retention times on high performance liquid chromatography confirmed that the generated immunoreactive products were LTB4, PGD2, and LTC4. Thus, coculture of BMMC with fibroblasts induces an alteration in the composition of the secretory granules of the mast cells, as well as an augmentation of the activation-secretion response of the BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Characterization of fibroblastic stromal cells from murine bone marrow.

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.

Animals↗

PubMed1989

[Abnormalities in the regulation of erythropoiesis by bone marrow fibroblasts in aplastic anemia].

The influence of bone marrow fibroblasts in healthy subjects and patients with aplastic anemia on normal erythroid colony formation was studied using the methylcellulose method. These fibroblasts were treated with methylprednisolone as well. Bone marrow fibroblasts of healthy subjects and patients with aplastic anemia, and the supernatant of their conditioned medium significantly inhibited normal erythroid colony formation. A significantly marked inhibition of normal erythroid colony formation was observed of bone marrow fibroblasts (or the conditioned medium) of the aplastic anemia, when compared with that of the bone marrow of healthy subject fibroblasts (or the conditioned medium). By treating both groups of the bone marrow fibroblasts with methylprednisolone the inhibition was slightly improved. From the above experimental results, it was suggested that the bone marrow fibroblasts and the conditioned medium inhibited normal erythroid colony formation through humoral factors secreted by the fibroblasts, and through contact between the fibroblasts and erythroid colony formation cells.

Adult↗

PubMed1989

The mast cell-committed progenitor. II. W/Wv mice do not make mast cell-committed progenitors and S1/S1d fibroblasts do not support development of normal mast cell-committed progenitors.

We have previously reported that the population of mesenteric lymph node cells from normal BALB/c mice infected 14 days with the rodent nematode Nippostrongylus brasiliensis (Nb-MLN) contains a nongranulated mast cell-committed progenitor (MCCP) which does not require IL-3 for proliferation and differentiation if either a fibroblast monolayer or soluble factors produced by monolayers of 3T3 fibroblasts or embryonic skin are present in the culture. When Nb-MLN were cloned in a methylcellulose culture system using fibroblast conditioned medium as the only source of growth factors, numerous colonies of pure mast cells developed. We wished to determine whether the mast cell deficiency of W/Wv or S1/S1d mice could be explained by the failure of these mice to make either the MCCP or the factor to support proliferation and differentiation of the MCCP. We found that Nb-MLN from W/Wv mice were only able to produce mast cell colonies in response to a source of IL-3 such as conditioned medium from pokeweed mitogen-stimulated spleen cells (CM), and cultures given fibroblast conditioned medium as the only source of growth factors did not produce mast cell colonies. In contrast, Nb-MLN from mast cell deficient S1/S1d mice developed many mast cell colonies in methylcellulose cultures supplemented with either fibroblast conditioned medium or conditioned medium from PWM-stimulated spleen cells. These data suggest that S1/S1d mice but not W/Wv mice produce the mast cell progenitor that responds to fibroblast conditioned medium. To determine if mast cell deficient mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of S1/S1d and W/Wv mice and Nb-MLN from normal BALB/c mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned medium from monolayers prepared from W/Wv but not S1/S1d mice supported development of numerous mast cell colonies. Taken together, these data demonstrate that W/Wv mice are incapable of producing normal MCCP whereas S1/S1d fibroblasts fail to produce the appropriate factor to support the MCCP. In accordance with these data, a candidate for the gene product of each of these mutant alleles is discussed.

Animals↗

PubMed1988

Human C5a and C5a des Arg exhibit chemotactic activity for fibroblasts.

C5a and C5a des Arg are chemotactic factors for inflammatory cells but it is not known whether these agents are chemoattractants for fibroblasts. Accordingly, C5a, purified from zymosan-activated human, and C5a des Arg, prepared by incubating C5a with immobilized porcine carboxypeptidase B, were studied for fibroblast chemotactic activity. We observed that both C5a and C5a des Arg stimulated human skin fibroblasts and fetal bovine ligament fibroblasts to migrate in a concentration-dependent fashion, and that the migratory responses were similar in magnitude to the responses achieved with optimal concentrations of two known fibroblast chemoattractants, platelet-derived growth factor and human fibrinopeptide B. The peak responses to C5a and C5a des Arg occurred at approximately 10(-9)M. With ligament fibroblasts, there was a greater response to C5a des Arg than to C5a, but with human fibroblasts there was no difference. Cochemotaxin, which enhances the chemotactic activity of C5a des Arg for neutrophils, had no effect on C5a des Arg fibroblast chemotactic activity but appeared to increase the fibroblast chemotactic activity of C5a. These results indicate that the effects of C5a and C5a des Arg in vivo may extend to the recruitment of mesenchymal cells. Moreover, the findings represent another example of an activity retained by C5a after removal of its carboxyl terminal arginine.

Antibodies↗

PubMed1987

Paracrine interaction between thyrocytes and fibroblasts.

Cell free supernatants (conditioned medium) of isolated porcine thyroid follicles, stimulated with EGF (5 ng/ml) or TSH (1-1000 microU/ml), were tested for a mitogenic activity for fibroblasts. Whereas TSH-conditioned medium dose-dependently stimulated [3H]thymidine incorporation into DNA of fibroblasts, only a weak stimulation was found with EGF. However, when the changes in cell number were determined, a significant increase was only found with EGF-conditioned medium from thyroid follicles. The cause of this discrepancy is a dose-dependent stimulation of [3H]thymidine incorporation into fibroblasts by cAMP and thyroid hormones. Cyclic AMP, however, does not stimulate growth of fibroblasts. IGF I production is stimulated in fibroblasts by basal as well as EGF stimulated conditioned medium of thyroid follicles. In contrast, TSH-conditioned medium inhibited IGF I production in fibroblasts. Conditioned medium itself is free of detectable IGF I. As IGF I stimulates not only growth of fibroblasts, but also of thyrocytes, we conclude, that conditioned medium from thyrocytes stimulates IGF I production in fibroblasts, which itself stimulates fibroblast and thyrocyte growth.

Animals↗

PubMed1986

Clonal analysis of murine graft-vs-host disease. II. Leukokines that stimulate fibroblast proliferation and collagen synthesis in graft-vs. host disease.

T lymphocyte clones were established from mice with acute and chronic graft-vs-host disease (GVHD) [C57B6/6 (B6) mice transplanted i.v. with LP/J spleen cells] and immune mice (LP/J mice immunized intraperitoneally with B6 spleen cells). To determine the role of leukokines in the increased collagen production that characterizes chronic GVHD, the supernatants of in vitro stimulated clones were assayed for their effect on 1) B6 embryonic fibroblast proliferation, 2) total fibroblast collagen secretion, and 3) collagen secretion per fibroblast. Four of nine chronic GVHD (CG) clones stimulated both total collagen secretion and collagen secretion per fibroblast, but none stimulated fibroblast proliferation. Six of 10 immune (I) clones stimulated fibroblast proliferation, and none stimulated collagen secretion per fibroblast. Acute GVHD (G) clones were heterogeneous; 2/10 G clones stimulated fibroblast proliferation, 5/10 stimulated total collagen secretion, and 4/10 stimulated collagen secretion per fibroblast. I and CG clones may act synergistically in vivo. The presence of CG-like clones in mice with acute GVHD suggest that the immunologic events that culminate in chronic GVHD are initiated early after transplantation.

Acute Disease↗

PubMed1987

Control of muscle differentiation in BC3H1 cells by fibroblast growth factor and vanadate.

We have examined the control of actin isoform synthesis by pituitary-derived fibroblast growth factor and serum in BC3H1 cells, a tumor-derived nonfusing muscle cell line. Under differentiating conditions in BC3H1 cells, the synthesis of beta- and gamma-actin ceases, and the rate of alpha-actin synthesis is increased concomitant with cessation of cell growth. Addition of fetal calf serum to differentiated cells reverses the process, whereas the addition of pituitary-derived fibroblast growth factor inhibits synthesis of alpha-actin but fails to induce the synthesis of beta- and gamma-actin. Analysis of RNA from differentiated BC3H1 cells after the addition of fetal calf serum indicated that the serum-induced increase in beta- and gamma-actin synthesis reflected an increase in their mRNA levels. In contrast, the repression of alpha-actin synthesis by fetal calf serum or fibroblast growth factor appears to reflect the translation efficiency of alpha-actin mRNA. Fibroblast growth factor is a competence factor for BC3H1 cells which allows them to progress from G0 4 h into the G1 phase of the cell cycle. In order to understand the nature of the intracellular signals responsible for the effect of fibroblast growth factor, we treated cells with vanadate, a known inhibitor of tyrosine-specific protein phosphatases. Vanadate fully mimics the action of fibroblast growth on actin synthesis and creatine phosphokinase synthesis and causes BC3H1 cells to exit the G0 portion of the cell cycle, as demonstrated by the induction of the c-fos proto-oncogene following addition of serum, vanadate, or bovine pituitary-derived fibroblast growth factor to these cells. We conclude that repression of alpha-actin synthesis and induction of the synthesis of beta- and gamma-actin are under independent control and that the induction of beta- and gamma-nonmuscle actin synthesis following serum addition is independent from movement into the cell cycle, and dependent on as yet unidentified serum components. The rate of synthesis of alpha-actin can be controlled by a defined mitogenic polypeptide fibroblast growth factor, which in short term experiments primarily affects the rate of translation of alpha-actin mRNA. The repression by fibroblast growth factor is most likely due to activation of a tyrosine specific protein kinase(s).

Actins↗

PubMed1980

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity.

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.

Animals↗

PubMed1981

Metabolism of homologous and heterologous lipoproteins by cultured rat and human skin fibroblasts.

Rat fibroblasts degraded human low density lipoprotein (LDL) very slowly, one-tenth to one-fortieth the rates observed in human fibroblasts. In rat cells, human LDL caused only very small increases in cell cholesterol content and acylCoA:cholesterol acyltransferase (ACAT) activity and caused only small decreases in beta-hydroxy-beta-methylglutaryl CoA (HMG CoA) reductase activity; in human cells, however, human LDL induced very large changes in all three of these parameters, as expected. The binding of human LDL to rat fibroblasts was not reduced by previous incubation with human LDL or with 25-hydroxycholesterol. Thus, in rat fibroblasts there appear to be few, if any, regulated high-affinity receptors that recognize human LDL. Rat LDL fractions (d 1.02-1.05 g/ml), in contrast, were degraded more rapidly than human LDL by rat fibroblasts, caused a significant increase in cell cholesterol content, an increase in ACAT activity, and a significant decrease in HMG CoA reductase activity. Moreover, the degradation of this rat LDL fraction by rat fibroblasts as a function of concentration was biphasic, i.e., there appeared to be a high-affinity component of degradation. Thus, it appears that rat fibroblasts do have a receptor for homologous lipoproteins. However, because both apoprotein B and apoprotein E are present in these rat lipoprotein fractions, the observed effects may relate to recognition of either or both of these apoproteins. The metabolism and metabolic effects of the conventionally defined high density lipoprotein (HDL) fraction of the rat by rat or human fibroblasts resembled those of human LDL in human fibroblasts. It is suggested that rat HDL may, because of its apo E content and higher concentration in rat plasma relative to that of LDL, play an important role in cholesterol homeostasis in vivo.

Animals↗

PubMed1982

Low density lipoprotein receptor binding in aging human diploid fibroblasts in culture.

High affinity cell surface receptors for low density lipoproteins (LDL) are inducible in cultured human lung fibroblasts by the removal of lipoproteins from the cell culture medium. The binding, uptake, and degradation of 125I-LDL by fibroblasts decrease with increasing number of population doublings. The affinity of LDL receptor binding, however, remained unchanged at different population doublings levels. Hence, the difference in LDL binding activity in the aging fibroblasts can be attributed to a reduction in the number of receptor sites on the cell membrane. Cellular uptake of [4-14C]cholesterol and 2-deoxy-D-[1-14C]glucose mediated through mechanisms independent of the LDL receptor pathway revealed no significant difference in early and late passage fibroblasts. This suggests that the alteration in the LDL receptor binding in serially passaged fibroblasts is an "age-related" phenomenon. The late population doubling fibroblasts require more LDL in the culture medium for feedback inhibition of LDL receptor synthesis. Thus, aging fibroblasts are both progressively less inducible and less suppressible in the regulation of their cell membrane LDL receptors. Similar results were also obtained with respect to the regulation of DL-3-hydroxy-3-methyl-glutaryl coenzyme A reductase in the aging fibroblasts in culture; the enzyme has become less inducible and less supressible as the fibroblasts approach the limit of their in vitro lifespan. These age-related alterations in the cellular metabolism of LDL and cholesterol might contribute to our understanding of the increased risk of athlerosclerosis in our aging population.

Cell Survival↗

PubMed1984

The effect of dexamethasone on the growth of bone marrow fibroblasts in aplastic anemia.

To study the characteristics of the microenvironment of human bone marrow, fibroblasts derived from normal and aplastic anemia bone marrow aspirates or core biopsies were grown in culture. Normal bone marrow fibroblasts had a slow logarithmic phase of growth (8 days) with doubling times of 2 to 3 days. Aplastic anemia bone marrow fibroblasts had a rapid logarithmic phase of growth (5 to 6 days) and doubling times of 1 day. Dexamethasone (1 X 10(-8) M) stimulated the growth of normal fibroblasts. Growth of fibroblasts derived from aplastic anemia marrows was, however, inhibited by dexamethasone. Specific binding sites for the hormone were present in the normal fibroblasts (Bmax: 460-770 fmole/mg protein) whereas in the aplastic anemia fibroblasts the amount was very low (Bmax 27-215 fmole/mg protein). These observations suggest that in aplastic anemia, the bone marrow fibroblast, a cell constituent of the marrow microenvironment, has the capacity to develop and grow in vitro. However, this cell is different from normal bone marrow fibroblasts in its growth characteristics and response to dexamethasone.

Anemia, Aplastic↗

PubMed1981

Variation in steroid 5 alpha-reductase activity in cloned human skin fibroblasts. Shift in phenotypic expression from high to low activity upon subcloning.

Steroid 5 alpha-reductase in skin fibroblasts varies in two ways: 1) mean activity in uncloned fibroblasts corresponds to the activity in skin from which fibroblasts are derived (high in genital skin and low in nongenital skin); 2) activity in normal genital skin fibroblasts varies over a wide range (more than 200-fold). Studies of activity in fibroblast clones provides an explanation for this variability. Activity in clones of genital skin fibroblasts varies from the limits of detectability to very high levels, whereas activity in fibroblasts cloned from nongenital skin is uniformly low. The mean value of 5 alpha-reductase in genital skin clones is similar to the value in uncloned fibroblasts from the same explant. This suggests that the numbers of high and low activity cells in a given explant influence the overall enzyme activity. Variability among normal genital skin strains is also due to inherent differences in the range of enzyme activities. Furthermore, high activity genital skin clones give rise to a mixture of both high and low-activity subclones, whereas low activity genital skin and nongenital skin clones give rise to populations with uniformly low activity. Thus, with time genital skin gives rise to high activity fibroblasts that continually undergo shift in phenotypic expression to low activity.

17-Hydroxysteroid Dehydrogenases↗

PubMed1981

Difference between the glycosaminoglycans synthetized by corneal and cutaneous fibroblasts in culture.

Although fibroblasts retain similar morphologic characteristics in various tissues, a body of evidence suggests that these cells possess disparate characteristics in different tissues. Keratan sulfate I, a specific product of the corneal fibroblast, is synthesized by the cornea in vivo and by organ cultures of the cornea in the absence of an associated corneal epithelium and endothelium. Confluent cultures of isolated corneal fibroblasts appear to lose this capacity, and the question of whether they produce any keratan sulfate in culture has remained uncertain, owing to the variable sensitivity and specificity of the different analytical methods employed. This study compares the 35S-sulfate- and 3H-glucosamine-labeled glycosaminoglycans produced by confluent cultures of human corneal and cutaneous fibroblasts with those synthesized by corneal organ cultures with different analytical techniques. Using the analytical method of sequential enzymatic degradation, confluent cultures of corneal fibroblasts, but not cutaneous fibroblasts, were found to synthesize and secrete into the nutrient medium a small quantity of sulfate glycosaminoglycans that was susceptible to keratan sulfate endo-beta-galactosidase (Pseudomonas)--an enzyme that degrades corneal keratan sulfate to oligosaccharides of variable size and sulfation. These difference between isolated corneal and cutaneous fibroblasts support the concept that fibroblasts, although ubiquitous, not only manifest metabolic differences but can retain some of these differences when isolated from other cells. Although cutaneous fibroblasts do not produce significant quantities of sulfated material with the attributes of keratan sulfate, they do incorporate 3H-glucosamine into macromolecules that are susceptible to keratan sulfate endo-beta-galactosidase. Most of the sulfated glycosaminoglycans produced by organ cultures of the cornea, which were susceptible to this enzyme, eluted from Dowex 1-X2(Cl-) with a salt concentration of less than 2 M. This observation, together with the findings of others, indicates that the traditional belief that corneal keratan sulfate elutes from Dowex 1-Cl predominantly in the 3 M NaCl fraction needs to be reevaluated.

Chondroitin Lyases↗

PubMed1981

Evidence for receptor-dependent response to dihydrotestosterone in cultured human fibroblasts.

The effect of dihydrotestosterone on the production of proteins and collagen and the synthesis of DNA was studied in cultured fibroblasts obtained from a normal woman and from a patient with complete testicular feminization. The concentration of dihydrotestosterone binding sites in fibroblasts from vulvar skin of a normal woman and from perineal skin of a patient with complete testicular feminization was 35.9 +/- 1.7 fmoles/mg protein and 4.61 +/- 0.37 fmoles/mg protein, respectively. The apparent dissociation constant was 0.24 +/- 0.015 nM for fibroblasts from a normal woman and 0.21 +/- 0.031 nM for fibroblasts from a patient with complete testicular feminization. Fibroblasts grown to confluence were exposed to DHT in the concentrations of 10, 100 and 1000 ng/ml of the culture medium and cultures were maintained for up to six days thereafter. These hormone treatments brought about a significant elevation in the production of proteins and collagen by fibroblasts from a normal woman but failed to produce any perceivable changes in fibroblasts from a patient with complete testicular feminization. The synthesis of DNA in fibroblasts from either subject was unaffected by hormone treatment. These results suggest that augmented production of proteins and collagen in cultured human fibroblasts which follows stimulation by dihydrotestosterone occurs via a receptor system.

Adult↗

PubMed1994

[Effect of angiotensin II on collagen metabolism in cultured rat cardiac fibroblasts: its relation to cardiac hypertrophy].

Hypertensive cardiac hypertrophy is associated with collagen accumulation in the myocardial interstitium. Myocardial fibrosis accounts for impaired myocardial stiffness and ventricular dysfunction. Although cardiac fibroblasts are responsible for the synthesis of fibrillar collagen, factors that regulate collagen synthesis in cardiac fibroblasts are not fully elucidated. The purpose of this study was to investigate the effect of angiotensin II (Ang II) on collagen synthesis by cultured rat cardiac fibroblasts. A 24-hour stimulation of cardiac fibroblasts with Ang II increased their collagen synthesis in a dose-dependent manner. This effect was blocked by the Ang II receptor antagonist, saralasin. Ang II neither affected the proportion of collagen types nor induced DNA synthesis. Basal collagen synthesis in cardiac fibroblasts from spontaneously hypertensive rats (SHR) was greater than that from normotensive Wistar-Kyoto rats. The responsiveness of collagen production to Ang II was significantly enhanced in cardiac fibroblasts from SHR. These results indicate stimulatory actions of Ang II on collagen synthesis in cardiac fibroblasts, enhanced collagen production in cardiac fibroblasts from SHR, and the hyperresponsiveness of cardiac fibroblasts from SHR to Ang II stimulation. Thus, Ang II may play an important role in collagen accumulation in hypertensive cardiac hypertrophy.

Angiotensin II↗

PubMed1993

Fibroblast migration in fibrin gel matrices.

In healing wounds and many solid tumors, locally increased microvascular permeability results in extravasation of fibrinogen and its extravascular coagulation to form a fibrin gel, with concomitant covalent cross-linking of fibrin by factor XIIIa. Subsequently, inflammatory cells, fibroblasts, and endothelial cells migrate into the gel and organize it into granulation tissue and later into mature collagenous connective tissue. To gain insight into some of the cell migration events associated with these processes, we developed a quantitative in vitro assay that permits the study of fibroblast migration in fibrin gels. Early passage human or rat fibroblasts were allowed to attach to tissue culture dishes and then were overlaid with a thin layer of fibrinogen that was clotted with thrombin. Fibroblasts began to migrate upwards into the fibrin within 24 hours and their numbers and the distance migrated were quantified over several days. The extent of fibroblast migration was affected importantly by the nature of the fibrin clot. Fibroblasts migrated optimally into gels prepared from fibrinogen at concentrations of -3 mg/ml; ie, near normal plasma fibrinogen levels. Migration was greatly enhanced by extensive cross-linking of the fibrin alpha-chains by factor XIIIa, as occurs when clotting takes place in vivo. When fibrinogen was clotted in Dulbecco's modified Eagle's medium, gamma-chains were cross-linked, but alpha-chain cross-linking was strikingly inhibited, and fibroblasts migrated poorly. Gels prepared from factor XIII-depleted fibrinogen exhibited neither alpha-nor gamma-chain cross-linking and did not support fibroblast migration. Further purification of fibrinogen by anion exchange high pressure liquid chromatography depleted fibrinogen of fibronectin, plasminogen, and other impurities; this purified fibrinogen clotted to form fibrin gels that supported reproducible fibroblast migration.

Animals↗

PubMed1996

Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat.

The animal model of hepatic fibrosis induced by bile duct ligation represents an experimental model of human chronic biliary fibrosis. Much attention has been given to the hepatic stellate cell (HSC), or perisinusoidal cell, as the source of the extracellular matrix proteins. However, in the bile duct ligation model, mesenchymal cells other than HSC may be involved in the early stages of fibrosis development. The current study examined, in Sprague-Dawley rats, proliferation in different liver cell subpopulations as well as expression of alpha-smooth muscle (SM) actin and desmin in portal fibroblasts and HSC at 6 hours and 1, 2, 3, and 7 days after bile duct ligation. Kinetics of liver cell proliferation and of phenotypic modulation of portal fibroblasts and HSC (expression of alpha-SM actin and desmin) was evaluated by immunocytochemistry, immunofluorescence, and immunoelectron microscopy using immunogold technique. In sham-operated animals, the evaluation of proliferation in various liver cell subpopulations revealed nonsignificant changes compared with nonoperated rats. alpha-SM actin was detected in vessel walls but was absent in cells of portal tract and parenchyma. Desmin was expressed in vessel walls and in some fibroblastic cells of portal stroma (8.2 cells/unit area) as well as in HSC in acinar Zones 1 and 3 (15.6 cells/unit area and 7.1 cells/unit area, respectively). In bile duct-ligated rats, 24 and 48 hours after ligation, marked proliferations of bile duct epithelial cells (labeling indices 36.8% and 29.5%, respectively) and of periductular fibroblasts (labeling indices 16.7% and 31.0%, respectively) were observed; thereafter, proliferation decreased for both populations (labeling indices at 7 days 12.0% and 11.6%, respectively). HSC proliferation increased gradually until the third day (labeling index 18.6%) and then leveled off. Immunocytochemistry and immunoelectron microscopy revealed a significant number of cells expressing alpha-SM actin 72 hours after bile duct ligation in the stroma adjacent to proliferating ductules. The number of alpha-SM actin-positive cells increased until the seventh day (251.6 cells/unit area). At all times examined, the distribution of alpha-SM actin was restricted to the connective tissue stroma adjacent to proliferating ductules; alpha-SM actin was not expressed in HSC of the lobule. An expansion of desmin expression was noted in fibroblastic cells in stroma surrounding proliferating ductules until 72 hours after bile duct ligation (74.7 cells/unit area) followed by a plateau. At this time, desmin expression increased also in HSC; as in controls, the number of positive cells was greater in Zone 1 (31.8 cells/unit area) than in Zone 3 (18.5 cells/unit area). Double immunofluorescence staining detected by confocal microscopy showed that the majority of portal fibroblastic cells expressing alpha-SM actin was desmin negative 48 hours after bile duct ligation. From 72 hours, portal fibroblastic cells coexpressing alpha-SM actin and desmin appeared, and their proportion increased until 7 days. The present findings indicate that in the early phase of bile duct ligation, there is a marked and transient proliferation of bile duct epithelial cells associated with proliferation of portal periductular fibroblasts, which rapidly express alpha-SM actin. This fibroblastic population may play a dominant role in the early portal fibrosis after bile duct ligation.

Actins↗