Cultured vascular endothelial cells secrete a protein factor(s) that promotes the contraction of collagen lattices made with fibroblasts

Conditioned media collected from arterial endothelial cells contain protein factor(s) that promotes the contraction of collagen lattices made with skin fibroblasts. Based on the lattice contraction-promoting activity, a protein with an apparent molecular weight of 22 kDa was identified. This 22-kDa protein stimulated lattice contraction in both serum-containing and serum-free media. When assayed at a 30% equivalent of the conditioned medium, the contraction-promoting activity of the purified factor was about 50 to 60% of that elicited by the unfractionated conditioned medium. Some contraction-promoting activity was also present in certain subfractions of the conditioned medium generated during the separation of the 22-kDa protein. Taken together, the results indicate that the lattice contraction-promoting activity in the endothelial cell-conditioned medium is probably aided by multiple active principles. The biochemical and biological characteristics indicated that the 22-kDa protein is not a transforming growth factor-beta-related factor nor a fibroblast growth promoter.     

Endothelins produced by endothelial cells promote collagen gel contraction by fibroblasts

Endothelial 1 (E1) is identified as an endothelial cell secreted factor that stimulates collagen gel contraction by fibroblasts. This identification is based on (a) co-localization of stimulatory activity in endothelial cell conditioned media with synthetic E1 in reversed phase analysis; (b) removal of the activity from conditioned media with antiserum directed against E1; and (c) the activity of synthetic E1. Treatment of endothelial cell conditioned media with immobilized anti-E1 antibodies removed 59% of the activity from the pool suggesting that E1 is the major contraction promoter in endothelial cell conditioned medium. The mechanism of action of E1 is shown to be different from serum in that E1-promoted contraction is dependent upon the synthesis of an unknown effector protein. Synthetic E1 is shown to be a potent promoter of gel contraction with half-maximal activity occurring at 32 pM. Two other endothelins, E2 and VIC, are slightly less active than E1. A fourth endothelin species, E3, is substantially less active. A comparison of E1 with other contraction promoting peptides revealed that E1 and platelet-derived growth factor are essentially equal in specific activity, whereas TGF beta is approximately 50-fold more potent.     

Cyclic AMP-induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms

The contraction of collagen lattices made with forskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracelluar cyclic AMP raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-β (TGF-β). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-β, but not by aFGF and insulin, when added simultaneously. Thus, TGF-β may attenuate CT's inhibition on collagen lattice contraction by attenuating CTinduced intracellualr cyclic AMP increse, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMPindependent mechannism. © 1993 Wiley-Liss, Inc.     

Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells

DNA synthesis of adult rat parenchymal hepatocytes alone in primary culture can be stimulated only by the addition of humoral growth factors to the culture medium. However, when parenchymal hepatocytes were cocultured with nonparenchymal liver cells from adult rats, their DNA synthesis was markedly stimulated in the absence of added growth factors or calf serum. DNA synthesis of parenchymal hepatocytes was not stimulated by conditioned medium from nonparenchymal liver cells and was greatest when the parenchymal cells were plated on 24-h cultures of nonparenchymal liver cells. A dead feeder layer of nonparenchymal cells was almost as effective as a feeder layer of viable nonparenchymal cells. These results suggest that the stimulation of DNA synthesis in parenchymal hepatocytes was not due to some soluble factors secreted by nonparenchymal liver cells but to an insoluble material(s) produced by the nonparenchymal liver cells. This insoluble material(s) was collagenase- and acid-sensitive, suggesting that it was a protein containing collagen. The effect of nonparenchymal liver cells was specific: coculture with hepatoma cells, liver epithelial cells, or Swiss 3T3 cells did not stimulate DNA synthesis in parenchymal hepatocytes. Added insulin and epidermal growth factor showed additive effects with nonparenchymal cells in the cocultures. These results suggest that DNA synthesis in parenchymal hepatocytes is stimulated not only by various humoral growth factors but also by cell-cell interaction between parenchymal and nonparenchymal hepatocytes, possibly endothelial cells. This cell-cell interaction may be important in repair of liver damage and liver regeneration.     

Cell-mediated contraction of collagen lattices in serum-free medium: Effect of serum and nonserum factors

Summary This study was conducted to identify a defined, serum-free culture medium that supports cell dependent contraction of a collagen lattice. Collagen lattices were found to contract in cultures containing human foreskin fibroblasts (HFF) or rabbit aortic smooth muscle (RASM) cells incubated in serum-free medium. HFF and RASM cells required different supplements to contract the collagen gels. HFF cultured in Dulbecco’s modified Eagle’s (DME) medium supplemented with bovine serum albumin (BSA) and either endothelial cell growth supplement (EnGS), insulin (In), or platelet derived growth factor (PDGF) supported collagen lattice contraction. Replacement of BSA with casein without the addition of other supplements improved contraction. In contrast, RASM cells supplemented with BSA, EnGS, In, and PDGF were able to contract collagen gels only minimally. Similar to HFF, RASM cells cultured in DME medium supplemented with casein, but without the addition of other supplements, contracted collagen lattices. HFF-mediated collagen contraction was inhibited by prostaglandins E₁ or E₂, fibronectin, or ascorbic acid. The reported serum-free model provides a useful in vitro method to investigate the role of serum and nonserum factors regulating cell mediated-contraction of insoluble collagen fibrils. Presented in part as abstract 1963 in the 1985 Federation of American Societies for Experimental Biology, April 21–26, Anaheim, California, and published in Fed. Proc. (44):747; 1985.     

Vascular Permeability Factor/Vascular Endothelial Growth Factor Inhibits Anchorage-Disruption-Induced Apoptosis in Microvessel Endothelial Cells by Inducing Scaffold Formation

Survival and proliferation of endothelial cells requires both growth factors and an appropriate extracellular matrix to which cells can attach. In the absence of either, endothelial cells rapidly undergo apoptosis. Thus, when human microvascular endothelial cells (HDMEC) are plated on a hydrophobic surface such as untreated polystyrene, they rapidly undergo apoptosis and die. The present study demonstrates that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), an endothelial cell-selective cytokine, inhibits apoptosis of HDMEC cultured on untreated polystyrene and induces these cells to adhere, spread, and proliferate. VPF/VEGF-induced HDMEC adhesion was time-dependent, requiredde novoprotein synthesis, and was inhibited by a soluble RGD peptide but not by an inhibitor of collagen synthesis. Under the conditions of these experiments, VPF/VEGF downregulated expression of collagen IV and fibronectin but did not change collagen I mRNA levels. VPF/VEGF-induced HDMEC adhesion was inhibited by antibodies to αvβ5 and vitronectin but not by antibodies to αvβ3. Other endothelial growth factors and cytokines such as bFGF, HGF, and TGFβ did not reproduce the VPF/VEGF effect. We suggest that VPF/VEGF induces endothelial cells to deposit a scaffolding (likely involving vitronectin) that allows them to attach to and proliferate on an otherwise nonsupportive surface (hydrophobic polystyrene) and in this manner serves as both a survival factor and a growth factor.     

The response of small vessel endothelial cells from fetal rat lung to growth factors

Small vessel pulmonary endothelial cells were obtained from rat fetal lung at day 20 of gestation, and were maintained in culture to passage three for study. Endothelial cells grown on a collagen matrix with Dulbecco's minimal essential medium: Ham's F12 medium (1:1, v/v) supplemented with 20 ml/l fetal bovine serum, bovine pituitary extract (50 mg/l), endothelial cell growth supplement (100 mg/l), hydrocortisone (1 mg/l) and an increased (10 mmol/l) magnesium concentration retained the characteristic endothelial cell marker factor VIII antigen during the third passage in culture. The factors responsible for small vessel growth in the developing fetal lung are unknown. To test the hypothesis that small vessel pulmonary endothelial cells would respond to autocrine or paracrine growth factors the effects of conditioned media from fetal lung endothelial cells, fibroblasts and pneumocytes from lungs of the same gestational age were studied in vitro. None of the tested conditioned media had any effect on endothelial cell DNA synthesis in the presence of 20 ml/l fetal bovine serum. Since no paracrine or autocrine effects of conditioned media were observed, the effect of other growth factors that could be derived from the circulation, or from storage sites in subcellular matrix, were studied for effect. When endothelial cells were studied in the presence of 20 ml/l fetal bovine serum and 100 mg/l endothelial cell growth supplement they had enhanced DNA synthesis in response to the progression-type growth factors insulin (5 mg/l), insulin-like growth factor-I and insulin-like growth factor-II (20 micrograms/l) and epidermal growth factor (10 micrograms/l). In the absence of serum or endothelial growth supplement endothelial cell DNA synthesis was enhanced by the competence-type growth factors acidic and basic fibroblastic growth factors at 100 micrograms/l and platelet derived growth factor at 10 micrograms/l. In the absence of exogenous competence-type growth factors neutralizing antibodies to basic fibroblast growth factor reduce DNA synthesis. Of various cytokines tested only interleukin-1 (1 x 10(3) U/l) and tumor necrosis factor (25 x 10(4) U/l) had an effect on endothelial cell DNA synthesis. Endothelial cell division during fetal lung development may be controlled by progression growth factors present in serum, and by either autocrine release of the competence factor basic fibroblast growth factor or paracrine release of platelet-derived growth factor by other cell types.     

Role of growth factors in the contraction and maintenance of collagen lattices made with arterial smooth muscle cells.

The contraction of collagen lattices made with arterial smooth muscle cells was studied in medium MCDB 107 without serum or supplemented with 1% fetal bovine serum, plus insulin, transferrin, and low-density lipoprotein. Under these conditions, smooth muscle cell mitogens including HBGF-1 (aFGF), PDGF, and EGF stimulated contraction. Stimulation by HBGF-1 was more profound than with other factors tested. HBGF-1 stimulation of lattice contraction was blocked by protein synthesis inhibitors, but not inhibitors of DNA synthesis. Histological observations indicated that HBGF-1 also enhanced the maintenance of healthy cells in the lattice. Taken together, these observations suggest that HBGF-1 stimulates lattice contraction, not by a mitogenic effect, but by stimulating synthesis of specific cellular proteins. Since the greatest effects of HBGF-1 on lattice contraction were seen during the first 72 h following casting, the effects on maintenance of cell viability are probably less important in promoting lattice contraction.     

Cloning of a growth arrest-specific and transforming growth factor beta-regulated gene, TI 1, from an epithelial cell line.

By cDNA cloning and differential screening, five genes that are regulated by transforming growth factor beta (TGF beta) in mink lung epithelial cells were identified. A novel membrane protein gene, TI 1, was identified which was downregulated by TGF beta and serum in quiescent cells. In actively growing cells, the TI 1 gene is rapidly and transiently induced by TGF beta, and it is overexpressed in the presence of protein synthesis inhibitors. It appears to be related to a family of transmembrane glycoproteins that are expressed on lymphocytes and tumor cells. The four other genes were all induced by TGF beta and correspond to the genes of collagen alpha type I, fibronectin, plasminogen activator inhibitor 1, and the monocyte chemotactic cell-activating factor (JE gene) previously shown to be TGF beta regulated.     

Immune modulation of connective tissue functions: studies on the production of collagen synthesis inhibitory factor by populations of human peripheral blood mononuclear cells

It has been shown previously that a soluble factor(s) from human peripheral blood mononuclear cells was capable of specifically suppressing collagen synthesis by normal human dermal fibroblasts (S. A. Jimenez, W. McArthur and J. Rosenbloom, J. Exp. Med.150, 1421, 1979). In this communication, the cell sources and the conditions for synthesis of this collagen synthesis inhibitory factor (CSIF) are identified. CSIF production by mononuclear cells was directly related to the number of cells in culture and was significantly enhanced by a variety of mitogens and by antigens. Homologous serum or bovine serum albumin was required for CSIF production and maximal levels were reached 48 hr after stimulation. Thymus-derived lymphocytes appeared to be the main cells responsible for CSIF synthesis but B lymphocytes also produced the factor in response to proper B-cell mitogens. Preparations of plastic-adherent mononuclear cells were also found to produce increased CSIF but it was not possible to exclude completely the presence of T lymphocytes in these preparations and therefore, the cell source of CSIF in these preparations was not clearly established. Through the use of metabolic inhibitors it was shown that CSIF production required de novo protein synthesis but not cell division. Indo-methacin had no effect on either the production of CSIF or on CSIF-mediated inhibition of collagen synthesis. The results indicate that CSIF has the classic characteristics of a lymphokine and suggest a mechanism by which the immune response could modulate connective tissue function.     

Role of the Src homology 2 domain-containing protein Shb in murine brain endothelial cell proliferation and differentiation.

To study the role of the Src homology 2 (SH2) domain-containing protein Shb in angiogenesis, wild-type Shb and SH2 domain-mutated Shb (R522K Shb) were overexpressed in murine immortalized brain endothelial cells. The wild-type Shb cells exhibited an increased rate of apoptosis on serum withdrawal. Both wild-type Shb and R522K Shb cells exhibited enhanced spreading concomitant with cytoskeletal rearrangements that occurred independently of fibroblast growth factor (FGF)-2 stimulation. However, these effects may partly be caused by altered regulation of Rac1 and Rap1 activation in the Shb cells. The Shb-induced cytoskeletal rearrangements were not dependent on phosphatidylinositol 3' kinase activity, but could be reversed by inhibition of Src family kinases. FGF-2 failed to further enhance migration of wild-type Shb and R522K Shb cells. The R522K Shb cells cultured in collagen gels exhibit diminished tubular morphogenesis when treated with FGF-2, implicating the need for a functional Shb molecule in this process. These data suggest that Shb plays a role in the proliferation and differentiation of endothelial cells and, hence, participates in angiogenesis.     

Transforming growth factor beta induces rosettes of podosomes in primary aortic endothelial cells

Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.     

The response of fenestrations, actin, and caveolin-1 to vascular endothelial growth factor in SK Hep1 cells

To study the regulation of fenestrations by vascular endothelial growth factor in liver sinusoidal endothelial cells, SK Hep1 cells were transfected with green fluorescence protein (GFP)-actin and GFP-caveolin-1. SK Hep1 cells had pores; some of which appeared to be fenestrations (diameter 55 +/- 28 nm, porosity 2.0 +/- 1.4%), rudimentary sieve plates, bristle-coated micropinocytotic vesicles and expressed caveolin-1, von Willebrand factor, vascular endothelial growth factor receptor-2, endothelial nitric oxide synthase and clathrin, but not CD31. There was avid uptake of formaldehyde serum albumin, consistent with endocytosis. Vascular endothelial growth factor caused an increase in porosity to 4.8 +/- 2.6% (P < 0.01) and pore diameter to 104 +/- 59 nm (P < 0.001). GFP-actin was expressed throughout the cells, whereas GFP-caveolin-1 had a punctate appearance; both responded to vascular endothelial growth factor by contraction toward the nucleus over hours in parallel with the formation of fenestrations. SK Hep1 cells resemble liver sinusoidal endothelial cells, and the vascular endothelial growth factor-induced formation of fenestration-like pores is preceded by contraction of actin cytoskeleton and attached caveolin-1 toward the nucleus.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.     

Contraction of fibrillar type I collagen by endothelial cells: A study in vitro

The formation of microvascular sprouts during angiogenesis requires that endothelial cells move through an extracellular matrix. Endothelial cells that migrate in vitro generate forces of traction that compress (i.e., contract) and reorganize vicinial extracellular matrix, a process that might be important for angiogenic invasion and morphogenesis in vivo. To study potential relationships between traction and angiogenesis, we have measured the contraction of fibrillar type I collagen gels by endothelial cells in vitro. We found that the capacity of bovine aortic endothelial (BAE) cells to remodel type I collagen was similar to that of human dermal fibroblasts—a cell type that generates high levels of traction. Contraction of collagen by BAE cells was stimulated by fetal bovine serum, human plasma-derived serum, bovine serum albumin, and the angiogenic factors phorbol myristate acetate and basic fibroblast growth factor (bFGF). In contrast, fibronectin and immunoglobulin from bovine serum, several nonserum proteins, and polyvinyl pyrrolidone (a nonproteinaceous substitute for albumin in artificial plasma) were not stimulatory. Contraction of collagen by BAE cells was diminished by an inhibitor of metalloproteinases (1, 10-phenanthroline) at concentrations that were not obviously cytotoxic. Zymography of proteins secreted by BAE cells that had contracted collagen gels revealed matrix metalloproteinase 2. Subconfluent BAE cells that were migratory and proliferating were more effective contractors of collagen than were quiescent, confluent cells of the same strain. Moreover, bovine capillary endothelial cells contracted collagen gels to a greater degree than was seen with BAE cells. Collectively, our observations indicate that traction-driven reorganization of fibrillar type I collagen by endothelial cells is sensitive to different mediators, some of which, e.g., bFGF, are known regulators of angiogenesis in vivo. © 1996 Wiley-Liss, Inc.     

Cultured mouse marrow stromal cell lines. II. Distinct subtypes differing in morphology, collagen types, myelopoietic factors, and leukemic cell growth modulating activities

A series of stromal cell lines were studied for their growth properties, electron microscopic morphology, cytochemical profile, collagen types, production of myelopoietic factors, and modulation of leukemic cell growth. Three cell types were identified in addition to the previously described macrophages (14M and 14M1) and preadipocytes (14F). MBA-1 cells were found to be fibroblasts by their ability to synthesize collagen types I and III, while the cell line MBA-13 shared properties in common with both fibroblasts and endothelial cells (collagen types I, III, IV, V). The third cell type, represented by the stromal cell line MBA-2, produced mainly collagen types IV and V and exhibited junctional complexes between adjacent cells. All of the cell lines tested produced and secreted a macrophage-colony-stimulating factor, CSF-1. MBA-2 and to a lesser extent, MBA-13, produced an additional activity resistant to anti-CSF-1 antiserum. Trypsin extraction of outer surface components from two clones of the MBA-2 cell line (MBA-2.1 and MBA-2.4) yielded high molecular weight factor(s) that specifically inhibited the growth of a plasmacytoma cell line (MPC-11). Such inhibitory activity was not detected in other stromal cell lines. It is possible that this variability in the nature of stromal cell lines represents corresponding diversity of cell types comprising the hematopoietic microenvironment in vivo.     

Fetal and postnatal sera differentially modulate human dermal fibroblast phenotypic and functional features in vitro

Fetal wounds heal without scar formation, fibrosis, or contracture. Compared with adult wounds, they are characterized by major differences in the extracellular matrix and the absence of myofibroblastic cells. The reasons for these differences are not well known and determination of factors affecting the absence of scarring in the fetus may lead to strategies for controlling adult pathological scarring. In the present study, we have assessed the effects of serum on the behavior of normal human dermal fibroblasts. Using an in vitro approach, we investigated the effects of fetal and adult serum on cell properties such as growth rate, collagen synthesis, gelatinase activities, and differentiation to myofibroblasts using biochemical, morphological, and ultrastructural parameters. We studied the induction of α-smooth muscle (α-SM) actin in fibroblasts, and its correlation with increased collagen gel contraction by the cells. Our results showed that, compared with FBS (fetal bovine serum), postnatal calf serum (PCS) decreased mitogenic activity and collagenase synthesis but not collagen synthesis. Furthermore, cells cultured with PCS differentiated to myofibroblasts with an increase in cell diameter, number of stress fibers, α-SM actin expression, and collagen gel contraction. To characterize the molecules involved in this differentiation process, the amount of transforming growth factor β (TGFβ) in FBS and PCS was determined and the effect of neutralizing anti-TGFβ antibody was evaluated. It was determined that FBS contained more TGFβ than PCS, but that essentially all the TGFβ was latent in both sera. However, results obtained with anti-TGFβ antibody show that active TGFβ is present when human dermal fibroblasts are cultured with medium containing PCS. These results suggest that, in the presence of PCS but not FBS, the cells either produce active TGFβ or an enzyme that is able to activate latent serum TGFβ. Alternatively, sera may contain two different forms of latent TGFβ, the PCS form being activated by the dermal fibroblast cells. A similar mechanism may be involved, at least in part, in skin wound healing and may underlie the appearance of myofibroblasts in postnatal wounds. J. Cell. Physiol. 171:1–10, 1997. © 1997 Wiley-Liss, Inc.     

Decreased PDGF Receptor Kinase Activity in Fibroblasts Contracting Stressed Collagen Matrices

Fibroblasts cultured in mechanically stressed collagen matrices proliferate, whereas cells in floating collagen matrices become quiescent. Previous research indicated that one factor contributing to cell quiescence in floating matrices was reduced receptor autophosphorylation in response to PDGF stimulation (i.e., PDGF receptor desensitization). To learn more about the mechanism of PDGF receptor desensitization, we analyzed changes in PDGF receptor autophosphorylation and receptor kinase activity after stressed collagen matrices were switched to floating conditions, which results in rapid cell contraction and dissipation of mechanical stress. PDGF receptor desensitization occurred during contraction stimulated by serum but not in the absence of serum, and desensitization was prevented by inhibitors of contraction but not by inhibitors of the contraction-activated cyclic AMP signaling pathway. Receptor desensitization resulted from decreased receptor kinase activity rather than from elevated protein tyrosine phosphatase activity, and only receptors unoccupied at the time of contraction were affected. After contraction, radiolabeled PDGF binding to the cells was decreased, which suggested that receptor desensitization resulted from a contraction-dependent change in receptor availability or affinity.     

Mouse embryos contain polypeptide growth factor(s) capable of inducing a reversible neoplastic phenotype in nontransformed cells in culture

A growth-factor-like substance capable of inducing nontransformed mouse AKR-2B, rat NRK, and EGF-receptorless mouse NR6 cells to form progressively growing colonies in soft agar was identified in acid/ethanol extracts of 17-day mouse embryos. This "mouse embryo factor" (MEF) is similar to previously described transforming growth factors in that it is capable of stimulating DNA synthesis and conferring a reversible transformed morphology on nontransformed cells in vitro. Passage of crude embryo extracts over a Bio-Gel P-60 column gave a major peak of soft agar growth-stimulating activity in the 15,000 molecular weight range with a minor peak at about 22,000. This biological activity was sensitive to treatment with either trypsin or dithiothreitol, but was unaffected by heat (56 degrees C for 30 minutes or 100 degrees C for 3 minutes), indicating that the activity is due to a heat-stable polypeptide(s) with disulfide bonds. Separation of these polypeptide(s) by chromatography on carboxymethyl cellulose revealed two peaks of soft agar growth-stimulating activity which did not cochromatograph with a peak of epidermal growth factor receptor-competing activity. The similarities of this mouse embryo-derived growth factor to previously identified transforming growth factors suggest that both fetal development and neoplastic transformation may be affected by similar mechanisms.     

Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor+, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.