Platelets, macrophages, endothelium, and growth factors. Their effects upon cells and their possible roles in atherogenesis.
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Animals↗
Biomedical subjects
D F Bowen-Pope
Publications and source records attributed to D F Bowen-Pope.
Locally acting growth factors for vascular smooth muscle cells: endogenous synthesis and release from platelets.
Release of platelet-derived growth factor (PDGF) from platelets has been postulated to stimulate at least some of the cell proliferation seen at sites of tissue damage, both beneficially (wound healing) and perniciously (during formation of atherosclerotic lesions). Two other growth factors have been localized to the platelet: epidermal growth factor and transforming growth factor. These factors may function synergistically with PDGF in promoting smooth muscle cell proliferation in the injured vessel wall. PDGF-like molecules (PDGF-c) that bind to the PDGF receptor and are at least partially recognized by antiserum against PDGF may also be synthesized by vessel wall cells themselves under certain circumstances. Arterial endothelial cells secrete several mitogens, one of which is a PDGF-c. Release is greatly stimulated by exposure of the cells to physiologic concentrations of thrombin. Also, aortic smooth muscle cells from 2-week-old rats secrete mitogenic levels of PDGF-c. In this case, PDGF-c accounts for all the mitogenic activity in conditioned medium (when assayed on 3T3 cells). Smooth muscle cells obtained from adult rat aortae secrete 150-fold less PDGF-c. In a third example, when adult rat carotid arteries are damaged with a balloon catheter, smooth muscle cells migrate into the intima of the artery and proliferate. By 2 weeks, the number of smooth muscle cells in the artery has doubled. When these intimal smooth muscle cells are cultured, they are found to secrete PDGF-c. These findings suggest that activation of endogenous synthesis of PDGF-c may contribute to the smooth muscle cell proliferation seen in response to vascular injury.
Animals↗
The platelet-derived growth factor receptor.
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Animals↗
Detection of high molecular weight forms of platelet-derived growth factor by sequence-specific antisera.
Antisera to synthetic peptides representing sequences of both chains of platelet-derived growth factor (PDGF) were used to structurally analyze PDGF isolated from outdated human platelets and PDGF-like proteins in normal and transformed cells. Most PDGF isolated from platelets did not contain the carboxyl portion of PDGF-2 in contrast to p20sis, the major form of p28sis detected in simian sarcoma virus-transformed cells. In addition, higher molecular weight forms of molecules containing PDGF-1 and PDGF-2 sequences were detected in all cell lines tested. These lines were heterogeneous with respect to species, cell type, and transforming agent.
Amino Acid Sequence↗
Isolation, characterization, and propagation in vitro of human glomerular endothelial cells.
Human glomerular endothelial cells have been isolated, cloned, and characterized. They appeared as the first outgrowth from human glomeruli in the presence of platelet-derived growth factor, which was also a requirement for continuous growth. By phase microscopy they appeared as monolayers of polygonal cells. Von Willebrand's factor (VWF) was detected in the cytoplasm of all clones. Their intermediate filaments differed antigenically from that present in human umbilical vein endothelial cells. Like other endothelial cells, they demonstrated high levels of membrane-associated angiotensin-converting enzyme (ACE).
Cell Division↗
Ganglioside-mediated modulation of cell growth, growth factor binding, and receptor phosphorylation.
Glycosphingolipids added exogenously in cell culture are slowly incorporated into plasma membranes, inhibit cell growth, and modify growth behavior ( Laine , R. A., and Hakomori, S. (1973) Biochem. Biophys. Res. Commun. 54, 1039-1045; Keenan , T. W., Schmid, E., Franke , W. W., and Wiegandt , H. (1975) Exp. Cell Res. 92, 259-270). With the availability of purified growth factors and serum-free culture conditions in recent years, we have been able to examine this phenomenon in mouse Swiss 3T3 cells in greater detail with the following results. 1) Cell growth (cell number increase) in serum-free medium was specifically inhibited by the presence of GM1 and to a lesser extent by GM3, but not by NeuAcnLc4 , although the gangliosides were incorporated equally well into cell membranes. GM3 inhibited both platelet-derived growth factor (PDGF)- and epidermal growth factor-stimulated mitogenesis determined by thymidine incorporation, while GM1 could only inhibit PDGF-stimulated mitogenesis. NeuAcnLc4 had no effect on mitogen-stimulated thymidine incorporation. 2) The concentration-dependent binding of 125I-PDGF binding to cells indicated that cells whose growth was inhibited by GM1 or GM3 showed an increased affinity for PDGF as compared to cells grown without addition of ganglioside, while the total number of receptors stayed the same. Addition of ganglioside did not affect the binding of 125I-EGF. 3) No direct interaction was observed between gangliosides and growth factors as evidenced by the lack of competition by ganglioside-containing liposomes for cellular binding of 125I growth factors. 4) GM1 and GM3, but neither NeuAcnLc4 nor Gb4 , inhibited the PDGF-stimulated tyrosine phosphorylation by membrane preparations of a 170,000 molecular weight protein, which is probably the PDGF receptor. Thus, the level of gangliosides GM1 and GM3 in membranes may modulate PDGF receptor function by affecting the degree of tyrosine phosphorylation and may alter the affinity of the receptor for PDGF.
Animals↗
Platelet-derived growth factor: morphologic and biochemical studies of binding, internalization, and degradation.
Kinetic studies of binding and internalization of 125I-platelet-derived growth factor (PDGF) demonstrate that up to 15% of membrane-associated radioactivity is internalized within 2 minutes after warming to 37 degrees C in a variety of cell types. The T 1/2 for internalization is approximately 20 minutes. The T 1/2 for the subsequent appearance of degradation products in the culture medium is between 60-90 minutes following initiation of internalization. Internalization and lysosomal association of 125I-PDGF were confirmed by EM autoradiography. Quantitative studies using PDGF adsorbed to colloidal gold (gold-PDGF) demonstrate that 17% of the cell-associated sites are along coated regions of the plasma membrane (1.0 sites/micron), while 82% are associated with noncoated membrane (0.2 sites/micron). There is a significant redistribution of the gold-PDGF complexes upon warming. Within 1-2 minutes at 37 degrees C, gold particles are found within endocytic vesicles, endosomes (0.09-0.3 micron diameter), and lysosomes (greater than 0.2 micron diameter). At this time the vesicle/endosome compartment comprises 15% of the total sites and contains 0.9 sites per micron2 of surface area. The lysosomes account for 8% of the total sites and contain 0.8 sites per micron2 of surface area. Simultaneously, there is an increase in the number of gold-PDGF binding sites within coated-pits (1.6 sites/micron, 18% of the total sites) and a decrease along noncoated regions of the membrane (0.11 sites/micron, 58% of the total sites). After 15 minutes at 37 degrees C, 26% of the total sites (1.4 sites/micron2) are highly concentrated within lysosomes, while sites in the vesicle/endosome compartment remain constant. At the same time, binding sites within coated pits decrease substantially (0.5 sites/micron, 4% of the total sites), while the number of sites along noncoated regions of the membrane remain constant. Gold-PDGF was not observed associated with the Golgi complex at any time up to 120 minutes following warming. We conclude that gold-PDGF is processed via both receptor-mediated and nonspecific endocytosis and follows an intracellular pathway comparable to that followed by some other protein ligands.
Animals↗
Platelet-derived growth factor.
Platelet-derived growth factor (PDGF) is a basic (pI congruent to 10) 30 000 molecular weight protein circulating in normal blood sequestered within the platelet alpha-granules. It binds with high affinity (Kd = 10(-11) M) to a specific cell-surface receptor found on many connective tissue cell types in culture. It is active in stimulating the metabolism and multiplication of connective tissue cells at very low concentrations (ED50 = 10(-11) M). It is likely that PDGF is released from platelets at sites of vascular damage and that it contributes toward the cell proliferation and connective tissue formation seen in healing wounds and in arteriosclerotic lesions. PDGF which does not bind to responsive cells at the wound site is largely inactivated by a plasma binding protein and is rapidly cleared from the circulation.
Adult↗
Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cell-surface receptors.
Evidence is presented that the binding of platelet-derived growth factor (PDGF) to plasma constituents inhibits the binding of PDGF to its cell-surface mitogen receptor. Approximately equivalent amounts of PDGF-binding activity were found in plasma from a number of different species known by radioreceptor assay to contain PDGF homologues in their clotted blood. Activation of the coagulation cascade did not significantly alter the PDGF-binding activity of the plasma components. Three molecular weight classes of plasma fractions that inhibit PDGF binding to its cell-surface receptor were defined by gel filtration: approximately equal to 40,000, 150,000, and greater than 500,000. Specific binding of 125I-labeled PDGF to the highest molecular weight plasma fraction could also be demonstrated by gel filtration. The binding of PDGF to these plasma components was reversible under conditions of low pH or with guanidine X HCl, and active PDGF could be recovered from the higher molecular weight fractions. Immunologic and functional evidence is presented that the highest molecular weight plasma fraction may be alpha 2-macroglobulin. A model is proposed in which the activity of PDGF released in vivo may be regulated by association with these plasma binding components and by high-affinity binding to cell-surface PDGF receptors.
Carrier Proteins↗
Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents.
A series of nontransformed human and murine cells and derivative cell lines transformed by methylcholanthrene; by simian virus 40, Kirsten and Moloney murine sarcoma viruses, simian sarcoma virus, and adenovirus; and by a "spontaneous" event in culture were examined for the expression of receptors for the platelet-derived growth factor (PDGF) and for production of substances able to compete with 125I-labeled PDGF for binding to the cell-surface PDGF receptor. In each case, transformation resulted in a 50-100% decrease in available PDGF receptors. All transformed cells except the methylcholanthrene-transformed mouse cells produce a PDGF competitor into the conditioned medium. Levels of PDGF competitor in conditioned medium at the end of a 48-hr collection were as high as 2 ng/ml--high enough to be measured by radioreceptor assay diluted 1:30 and to maximally stimulate [3H]thymidine incorporation by human fibroblasts. The PDGF competitor activity detected in a radioreceptor assay does not reflect irreversible (e.g., proteolytic) damage to the receptor of test cells since its effects are reversed by acetic acid dissociation. Antiserum against human PDGF neutralizes 20-80% of the PDGF competitor found in conditioned medium from different transformed human cells and 100% of the activity from normal human endothelial cells. The possibility that induction of expression of the cellular PDGF gene may be involved in the mechanism of transformation of PDGF-responsive mesenchymal cells is discussed.
Animals↗
Platelet-derived growth factor in vivo: levels, activity, and rate of clearance.
Platelet-derived growth factor (PDGF) is a potent mitogen for many cultured connective tissue cells. It is present in concentrated form within the platelet alpha-granules and is believed to be released during platelet degranulation at sites of vascular injury. We have used a sensitive radioreceptor assay to measure PDGF levels in whole blood serum from normal humans [17.5 +/- 3.1 (SD) ng/mL] and baboons (2.7 +/- 1.2 ng/mL). PDGF was not detected in plasma from either species. In addition, plasma was found to substantially reduce the ability of added purified PDGF to bind to the cell surface PDGF receptor on cultured cells, suggesting that plasma may contain a PDGF-binding protein that would serve to inactivate PDGF released into plasma. Calculations of PDGF concentrations in serum have been corrected for the effects of the binding protein. 125I-PDGF injected intravenously into normal baboons was cleared rapidly from the plasma (t1/2 = two minutes). The rapid clearance of 125I-PDGF did not result from iodination damage, as purified unlabeled PDGF was cleared with comparable kinetics. The rapid clearance of purified and iodinated PDGF did not result from changes in PDGF structure during purification or from removal of PDGF-associated proteins during purification, as PDGF present in freeze-thaw lysates of fresh platelets was cleared equally rapidly. We conclude that release of PDGF at sites of vascular injury would greatly increase the local concentration of PDGF and that PDGF not localized to the site of injury would be rapidly cleared from the circulation.
Animals↗
Is epidermal growth factor present in human blood? Interference with the radioreceptor assay for epidermal growth factor.
Human whole blood serum reduces the binding of [125I]labeled mouse epidermal growth factor to cultured human fibroblasts as well as does 70-100 ng/ml mouse epidermal growth factor. However, at most 1-2% of the binding inhibitor detected in human whole blood serum is related to epidermal growth factor--the remaining consists of other factors released during preparation of serum, predominantly the platelet-derived growth factor, which are capable of altering the binding properties of the epidermal growth factor receptor. This accounts for much of the differences between values reported for epidermal growth factor concentration in blood by investigators using different assay procedures.
Binding, Competitive↗
Characterization of platelet-derived growth factor-stimulated phosphorylation in cell membranes.
Platelet-derived growth factor (PDGF) stimulated the phosphorylation of a 170,000-Mr protein in membranes prepared from parental and several variant lines of Swiss 3T3 cells as well as from diploid human fibroblasts. The intensity of the phosphorylation of the 170,000-Mr protein paralleled the number of PDGF receptors present on the various cells. The enhanced phosphorylation was the result of an increase in the amount of phosphoserine and phosphotyrosine present in the 170,000-Mr protein. PDGF-stimulated phosphorylation of the 170,000-Mr protein was rapid and showed a dose response to PDGF. Down regulation of the PDGF receptor led to a rapid loss in the PDGF-stimulated phosphorylation of the 170,000-Mr protein. In Swiss 3T3 cell membranes, PDGF as well as epidermal growth factor stimulated the phosphorylation of a synthetic, tyrosine-containing peptide. The effects of the two growth factors were additive, indicating that PDGF and epidermal growth factor stimulate peptide phosphorylation through distinct receptors.
Animals↗
Growth stimulatory precipitates of Ca2+ and pyrophosphate.
Inorganic pyrophosphate (PPi) forms an insoluble precipitate with calcium in growth medium when its concentration exceeds about 0.1 mM. This PPi precipitate can reproduce the effects of 10% calf serum on all cell processes examined in Balb/c 3T3 cells, including hexose uptake and metabolism to lactate, 3H-uridine, and 3H-choline uptake, and the incorporation of 3H-leucine and 3H-thymidine into trichloroacetic acid (TCA)-insoluble material. Concentrations of PPi insufficient to form a precipitate are without effect on cell metabolism. The precipitates are most effective when prepared with concentrations of PPi just sufficient to result in precipitate formation and become considerably less effective as the PPi concentration increases, even though the quantity of precipitate formed continues to increase with PPi concentration up to 1 mM PPi. Precipitates formed at low PPi concentrations consist largely of Ca2+ (81% of cations), PPi (77% of anions), and Pi (23% of anions). Precipitates formed with higher concentrations of PPi contain proportionately less Ca2+ and Pi and more monovalent cations and PPi. We have distinguished cell surface-bound PPi from intracellular PPi by differential extraction. The quantity of surface-bound PPi increases sharply when the PPi concentration reaches the point of precipitate formation. If the precipitate is prevented from binding to the cell surface by inverting monolayer cultures in precipitate-containing medium, the cells are not stimulated. These findings suggest that the binding of PPi precipitate to the cell surface is involved in the stimulation of cell metabolism by PPi. PPi precipitates do not absorb serum mitogens or inhibitors from the culture medium, nor do they affect the binding of 125I-platelet-derived growth factor to its specific cell-surface receptor, suggesting that PPi precipitates do not act directly through either of these mitogen-receptor systems. In analogy to cell stimulation by epidermal growth factor and by antigens, we suggest that PPi may be active only in the form of a precipitate because multivalent binding of receptors with formation of clusters is required for stimulation. The inhibitory effects of high concentrations of PPi may be due to interference by free PPi with formation of active receptor clusters.
Animals↗
Cultured endothelial cells produce a platelet-derived growth factor-like protein.
The platelet-derived growth factor (PDGF) binds specifically to high-affinity receptors on the surface of bovine aortic smooth muscle cells and 3T3 cells. Conditioned medium from cultured bovine aortic endothelial cells (EC) prevents PDGF binding to these receptors in a dose-dependent manner at 4 degrees C. The (125)I-labeled PDGF that is displaced by the conditioned medium shows no increase in trichloroacetic acid solubility or decrease in binding capability to fresh cells. The competitor activity was identified as a protein by ammonium sulfate precipitability and sensitivity to trypsin. The competitor protein also is found in the serum-free conditioned media from porcine aortic EC and human umbilical vein EC but not in media from bovine aortic smooth muscle cells, human neonatal foreskin fibroblasts, or the interleukin-producing thyoma cell line EL-4. The competitor protein, like PDGF, has no effect on the specific 4 degrees C binding of either (125)I-labeled insulin to 3T3 cells or (125)I-labeled epidermal growth factor to human epidermoid A431 cells. Saturation curves of PDGF binding to smooth muscle cells that had been preincubated in the presence and absence of competitor indicate that the concentration for half-maximal binding of (125)I-labeled PDGF to its receptor ( approximately 30 pM) is unchanged by the competitor, whereas the apparent number of available receptor sites or maximal level of binding is greatly diminished. The competitor activity produced by cultured human umbilical vein EC is completely inhibited by antiserum against pure human PDGF, whereas the same PDGF antiserum only partially inhibits the mitogenic activity of the conditioned media. In addition, approximately 7-fold more crude endothelium-derived growth factor is required for half-maximal inhibition of (125)I-labeled PDGF binding as is required for half-maximal stimulation of DNA synthesis. These results suggest that EC secrete a PDGF-like protein that is biochemically distinct from the majority of EC-derived mitogenic activity.
Animals↗
Interactions between the receptors for platelet-derived growth factor and epidermal growth factor.
Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.
Animals↗
Platelet-derived growth factor. II. Specific binding to cultured cells.
We have prepared radioiodinated purified platelet-derived growth factor (125I-PDGF) which retains full mitogenic activity. The binding of 125I-PDGF to Swiss 3T3 cells is saturable and highly competed by whole blood serum, purified unlabeled PDGF, and by material from each stage in the purification of PDGF from platelet-rich plasma. Other purified mitogens and substances tested do not compete. 125I-PDGF binding to fibroblasts, 3T3 cells, and arterial smooth muscle cells shows an apparent dissociation constant of 10(-11) M, comparable to the range in which PDGF is mitogenic. A clone of Swiss 3T3 cells obtained from a population selected repeatedly against mitogenic response to PDGF shows a greatly reduced mitogenic response to PDGF and binds only 5% as much 125I-PDGF/cell. The binding capacity of the different cell types tested ranges from 2,500 binding sites/cell on the poorly responding variant to 390,000 binding sites/cell on one strain of Swiss 3T3 cells. Cell types that do not respond to PDGF do not show specific high affinity binding of 125I-PDGF. At 4 degrees C, 125I-PDGF binding to monolayer cultures is relatively slow. Equilibrium binding of low concentrations of 125I-PDGF is not achieved during 7 h unless the binding medium is constantly mixed. 125I-PDGF binding at 4 degrees C shows a broad pH optimum between 6.3 and 8.0. Binding does not seem to require Ca2+ or Mg2+ but is reduced more than 6-fold if both monovalent and divalent salts are omitted. The initial rate of 125I-PDGF binding is greater at 37 degrees C than at 4 degrees C but cell-associated 125I begins to decline soon after reaching a peak value at 30-60 min. Coincident with this decline, trichloroacetic acid-soluble 125I appears in the medium and the binding capacity of the cells declines. These phenomena suggest that PDGF and its receptor may be internalized and degraded.
Animals↗
Platelet-derived growth factor. III. Identification of a platelet-derived growth factor receptor by affinity labeling.
Two homobifunctional cross-linking reagents have been used to cross-link 125I-platelet-derived growth factor (PDGF) to a cell surface component with an approximate Mr = 164,000 that has many characteristics of a specific PDGF receptor. Excess unlabeled PDGF competed for labeling of this component, while high concentrations of fibroblast growth factor, insulin, epidermal growth factor, low density lipoprotein or acetylated low density lipoprotein had no effect. Preincubation of cells with 125I-PDGF at 37 degrees C reduced specific 125I-PDGF binding (down regulation) and produced a parallel decrease in the amount of the 164,000-dalton receptor available for labeling. The 164,000-dalton component contains at least some protein that is accessible to trypsin in the extracellular medium. A complex of comparable Mr is seen on all PDGF-responsive cell types examined, but not on a nonresponsive cell type. 125I-PDGF does not become covalently cross-linked to this component in the absence of a cross-linking reagent.
Affinity Labels↗