Analysis of the mitogenic effect of fetuin preparations on arterial smooth muscle cells: the role of contaminant platelet-derived growth factor

Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha 2-macroglobulin or "Embryonin" has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha 2-macroglobulin that stimulated smooth muscle cell growth. However, alpha 2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha 2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha 2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha 2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha 2-macroglobulin or "Embryonin" is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.     

Suramin inhibits binding and degradation of platelet-derived growth factor in arterial smooth muscle cells but does not interfere with autocrine stimulation of DNA synthesis

Summary During in vitro culture arterial smooth muscle cells of adult rats are able to produce a platelet-derived growth factor (PDGF)-like protein and to promote their own growth in an autocrine manner. Here, this process has been studied using suramin, a polyanionic drug that has been reported to interfere with the cellular binding of several growth factors. Our results indicate that suramin speeds up the transition of the cells from a contractile to a synthetic phenotype early in primary culture. It inhibits the binding of PDGF to the cells, displaces PDGF bound to the cell surface, and slows down the degradation of PDGF internalized by the cells. It reduces the specific activities of the lysosomal enzymes acid phosphatase, -N-ace-tylglucosaminidase and -glucuronidase, and gives rise to an accumulation of lysosomes with myelin-like inlcusions. It blocks PDGF- and serum-induced DNA synthesis and cellular proliferation in secondary cultures, but lacks a distinct inhibitory effect on DNA synthesis in primary cultures under serum-free conditions. The results suggest that the PDGF-like protein produced by the smooth muscle cells under the latter conditions may bind to its receptor and exert its autocrine effect intracellularly, without prior release into the pericellular space.     

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.     

Pregnancy-stimulated growth of vascular smooth muscle cells: Importance of protein kinase C-dependent synergy between estrogen and platelet-derived growth factor

Dramatic smooth muscle cell (SMC) growth occurs in the uterine artery during pregnancy. The potential for pregnancy-associated growth may also exist at other vascular sites. We tested the hypothesis that increased growth of uterine artery SMC isolated from pregnant (vs. nonpregnant) guinea pigs would be detectable in culture, that pregnancy-associated phenotypic changes would also be found in nonuterine vascular cells (aortic SMC), and that the enhanced growth would be dependent on estrogen, peptide growth factors like platelet-derived growth factor (PDGF), and protein kinase C (PKC). Growth responses were measured by [³H]-thymidine incorporation and cell counts. Uterine artery SMC from pregnant guinea pigs grew to a higher plateau density with serum stimulation, had increased spontaneous DNA synthesis and persistent growth following serum withdrawal, and were more responsive to 3–30 ng/ml PDGF-BB than nonpregnant cells. Aortic SMC from pregnant animals also grew to a higher plateau density and had enhanced responsiveness to PDGF-BB. This increased response to PDGF-BB by pregnant uterine artery and aortic SMC (40–233% increase over nonpregnant PDGF result) was reproduced in nonpregnant cells by pretreatment for 1–24 h with 17-beta(β)-estradiol (30–100 nM). Neither the pregnancy-induced difference nor the estradiol pretreatment was associated with increased PDGF-BB binding activity. The synergistic effect of 17β-estradiol was partially (62%) reproduced with 17-alpha(α)-estradiol, an isomer which does not bind the estrogen receptor. This suggested that 17β-estradiol modulates the PDGF-BB response by both estrogen-receptor- and nonreceptor-mediated mechanisms. To test if the estrogen effects were dependent on PKC, two different antagonist strategies (3 μM dihydrosphingosine and phorbol-ester-induced downregulation) were applied prior to 17α- or β-estradiol and blocked the enhanced responses to PDGF. The synergistic effect of 17β-estradiol on PDGF was then reproduced by 1 h pretreatment with the cell-permeable PKC activator, 10 nM PMA. We conclude that pregnancy stimulates increased growth of uterine and aortic SMC in vitro which is dependent on estrogen, PDGF, and PKC and may be important in vascular remodeling during pregnancy. © 1996 Wiley-Liss, Inc.     

Enhancement of endogenous production of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid in aortic smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF) has a chemotactic effect on smooth muscle cells, which is inhibited by lipoxygenase inhibitor caffeic acid. In order to study the role of endogenous lipoxygenase products of arachidonic acid on the chemotactic action of PDGF, effects of PDGF on the lipoxygenase pathway in smooth muscle cells were examined. Lipoxygenase products were analyzed by high-performance liquid chromatography. 15-, 5- and 12-lipoxygenase activities, in order of magnitude, were found in smooth muscle cell homogenate. However, when the lipoxygenase products were analyzed using intact cells prelabelled with [14C]arachidonic acid, only 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE) was found to be produced endogenously. In addition, 12-HETE was not released into the medium. Treatment of the cells with PDGF increased the endogenous production of 12-HETE. The amounts of intracellular 12-HETE in PDGF-treated cells were 126, 132 and 146% at 1, 3, and 10 hr's after the initiation of PDGF treatment, respectively, when control value at each time point was considered as 100%. Caffeic acid (10(-4) M) completely inhibited the PDGF effect on 12-HETE production. However, PDGF treatment did not significantly alter the 12-lipoxygenase activity. These results suggest that the stimulatory effect of PDGF on 12-HETE production was not mediated by the activation of 12-lipoxygenase activity. Since 12-HETE itself is a potent chemoattractant for smooth muscle cells, the present dat strongly suggest that 12-HETE could be an important intracellular mediator of the chemotactic action of PDGF on aortic smooth muscle cells.     

Cardiac microvascular endothelial cells express and release nerve growth factor but not fibroblast growth factor-2

Endothelial cells (ECs) from different vascular beds not only display common characteristics but are also quite heterogeneous in terms of expression and secretion of neuro-angiogenic factors, which may help explain some of their distinct physiological roles. We investigated by RT-PCR the gene expression, by PC12 bioassay the neurotropic activity, and by ELISAs the levels of NGF and FGF-2 using conditioned medium collected from cultures of ECs derived from myocardial and cerebral capillaries. While NGF was expressed and released by both cell types, FGF-2 was expressed and released solely by the brain but not heart ECs. Oxygen-glucose deprivation (ischemic) insult blocked NGF secretion from heart and brain ECs and inhibited by 70% the secretion of FGF-2 from brain ECs. We propose that the differential expression of NGF and FGF-2 in heart and brain EC cultures reflect heterogeneity on demand of the microcapillary components and the surrounding microenvironment for a proper tissue-specific homeostasis.     

Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle alpha-actin and nonmuscle beta-actin mRNA expression in cultured rat aortic smooth muscle cells

Previous studies have demonstrated that rat aortic smooth muscle cells (SMC) show marked changes in smooth muscle (SM) alpha-actin content and fractional synthesis as a function of cell density and growth (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352; Blank, R., Thompson, M. M., and Owens, G. K. (1988) J. Cell Biol. 107, 299-306). Results of this study show that, although there is a 6-fold increase in SM alpha-actin content in postconfluent density arrested cultures as compared to proliferating subconfluent cultures, SM alpha-actin mRNA levels are not different between these cells. This suggests that the SM alpha-actin gene is constitutively active under both of these conditions and that accumulation of SM alpha-actin in postconfluent cells is due to translational and/or post-translational controls. The relationship between growth and cytodifferentiation was further explored by examining the effects of platelet-derived growth factor (PDGF)- or serum-induced growth on actin expression in postconfluent, quiescent cultures maintained in a defined serum-free media. Although both factors have been shown to stimulate proliferation and decrease fractional SM alpha-actin synthesis (Blank et al., 1988), their effects on actin mRNA levels were quite different. PDGF was found to induce a dramatic drop in SM alpha-actin steady state mRNA level but had no effect on nonmuscle beta-actin mRNA level. In contrast, serum stimulation was shown to increase nonmuscle beta-actin mRNA level, whereas SM alpha-actin mRNA level remained constant. Taken together these results indicate that PDGF is a specific and potent repressor of SM alpha-actin expression in vascular SMC and implicate a possible developmental role for PDGF in control of SMC differentiation. In addition, the observation that the level of SM alpha-actin mRNA is unaltered in serum-stimulated cells indicates that an absolute decrease in SM alpha-actin mRNA is not obligatory for cell cycle entrance.     

Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor

Proliferation of smooth muscle cells from the pulmonary arteries and aortas of fetal calves is inhibited by heparin in vitro. This effect is reversible and dose dependent. Comparisons with effects of other polysaccharides indicate that only extensively sulfated polysaccharides inhibit proliferation of smooth muscle cells but that specific structural features of heparin are required to achieve maximum effect. Heparin-Sepharose chromatography of medium containing fetal calf serum reduces the ability of that medium to promote growth of smooth muscle cells from fetal pulmonary arteries, suggesting that heparin may remove soluble growth factors in serum. However, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is identical in media supplemented either with serum prepared from fetal calf plasma, in which platelet-derived growth factor (PDGF) is not detectable, or with fetal calf serum, which contains relatively abundant PDGF (114 pg/ml). Thus, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is not mediated solely by decreased availability or activity of exogenous PDGF. These studies suggest that morphogenesis of the smooth muscle investment of the pulmonary arteries could be regulated by local production of heparin-like inhibitors of smooth muscle cell growth.     

Insulin, insulin-like growth factor I and platelet-derived growth factor interact additively in the induction of the protooncogene c-myc and cellular proliferation in cultured bovine aortic smooth muscle cells

Vascular smooth muscle cell (SMC) growth is under the influence of various growth factors. We demonstrate that platelet-derived growth factor (PDGF) stimulates DNA synthesis of cultured bovine aortic SMCs by 2.5- to 3.5-fold. PDGF also exhibits additivity with insulin and insulin-like growth factor I (IGF-I) for DNA synthesis and cellular proliferation. Insulin (2 x 10(-6) M), IGF-I (1 x 10(-8) M), and PDGF (1 x 10(-9) M) cause a 60-80% increase in cell numbers over basal, but PDGF with insulin or IGF causes a 40-150% increase over basal. No additivity between insulin and IGF-I is evident. PDGF also induces commitment to DNA synthesis earlier than insulin or IGF-I. After exposure to PDGF for 4 h, SMCs incorporate 3H-thymidine to 60% of maximum (with PDGF alone) levels (achieved after exposure of 12 h or longer). Insulin and IGF-I exposure for 4 h, on the other hand, achieves 3H-thymidine incorporation that is only a 20-30% of maximum (with insulin or IGF-I alone). Insulin, IGF-I, and PDGF increase mRNA levels of the protooncogene c-myc. This induction begins within 30 min of exposure to these growth factors which causes a 4- to 6-fold increase in c-myc mRNA levels. Additivity is also observed between PDGF with insulin or IGF-I, but not between insulin or IGF-I, in c-myc induction. C-myc mRNA levels remain elevated as long as the hormones are present, although there's a tendency for the mRNA levels to fall off with insulin and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous thrombospondin-1 is not necessary for proliferation but is permissive for vascular smooth muscle cell responses to platelet-derived growth factor.

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.     

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.     

Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing

The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.     

Lipid oxidation products and oxidized low-density lipoproteins impair platelet-derived growth factor receptor activity in smooth muscle cells: implication in atherosclerosis

The platelet-derived growth factor receptor-beta (PDGFRbeta) signaling pathway regulates smooth muscle cell (SMC) migration and proliferation in the vascular wall. Oxidized low-density lipoproteins (oxLDLs) and 4-hydroxynonenal (4-HNE) induce a dual effect on PDGFRbeta signaling. Short-term incubation of SMCs with oxLDLs and 4-HNE induced PDGFRbeta activation. Long-term incubation triggered a desensitization of PDGFR to its own agonist, with a progressive inhibition of PDGFRbeta phosphorylation, associated with increased formation of HNE-PDGFR adducts in SMC and in vivo, in the aortae of apoE-deficient mice. Hydralazine used as carbonyl scavenger prevented PDGFRbeta inhibition in vitro and in vivo In conclusion, PDGFRbeta is a target for 4-HNE, acrolein and oxidative stress and its progressive inhibition may contribute to defective SMC proliferation and decrease the stability of a vulnerable plaque.     

Spleen tyrosine kinase modulates the proliferation and phenotypes of vascular smooth muscle cells induced by platelet-derived growth factor

Platelet-derived growth factor BB (PDGF-BB) regulates vascular smooth muscle cells (VSMCs) by activating signaling cascades that promote vasoconstriction and growth, but the underlying mechanisms remain incompletely characterized. In this study, we aimed at investigating the role of spleen tyrosine kinase (Syk) in the proliferation and phenotypes in rat pulmonary arterial VSMCs. Our results demonstrate that PDGF-BB or Syk-adenovirus led to a substantial increase of proliferation of VSMCs and cytoskeleton rearrangement in rat VSMCs. Consistently, these cells underwent phenotype changes. Notably, Syk inhibitor piceatannol significantly inhibited those biological effects induced by PDGF-BB. Thus, we conclude that Syk plays an important role in vascular remodeling through the modulation of proliferation and phenotypes of VSMCs.     

Human arterial smooth muscle cells in culture. Effects of platelet-derived growth factor and heparin on growth in vitro

Human arterial smooth muscle cells (hASMC) were cultured from explants of the inner media of uterine arteries obtained at hysterectomy. The presence of alpha-actin and smooth muscle-specific actin isoforms and the microscopic appearance of the cells in secondary culture established their smooth muscle origin. The hASMC were diploid and had no signs of transformation. Plasma-derived serum failed to stimulate their proliferation in vitro. Their rate of proliferation was, however, proportional to the concentration of whole blood serum in the medium. Anti-PDGF IgG at high concentrations inhibited the stimulatory effect of whole blood serum on cell proliferation. This suggests that hASMC depend on exogenous PDGF for their growth. In PDS or bovine serum albumin cell numbers remained constant for 7 days in culture and the thymidine index was below 1% per 24 h. When reexposed to whole blood serum these cells started to proliferate within 2 days. This indicates that hASMC when deprived of PDGF enter a quiescent state that is fully reversible upon rexposure to the mitogen. Heparin is a powerful growth inhibitor for SMC. In our system, heparin caused a dose-dependent inhibition of cell proliferation despite optimal concentrations of whole blood serum. This inhibition was reversible upon withdrawal of heparin. At heparin concentrations which caused a half-maximal inhibition it was also competed for by increasing concentrations of whole blood serum. Quiescent hASMC expressed the PDGF receptor on their surface as judged from immunofluorescence with a monoclonal antibody. This was true irrespective of whether growth arrest was achieved by serum depletion or by the addition of heparin to serum-containing medium. Cells growing in the presence of whole blood serum did not, however, express the receptor antigen. These observations suggest that heparin may interfere with PDGF or with its binding and further processing at the level of the cell-surface receptor.