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Expression of Acidic and Basic Fibroblast Growth Factors in Human and Bovine Vascular Smooth Muscle Cells Growth Factors Downloaded from informahealthcare.com by University of Toronto on 12/25/14 For personal use only.
HERBERT A. WEIGH'., NIGGI [BERG1,MICHAEL KLAGSBRUN1.L,and JUDAH FOLKMAN' Di.purtnicvit or jiuyy'. CliddrciI'j Hiwpitul, 300 Lonpood Aomur, Boston. .L3assaclrirsrtts. 02175, arid Dryurtinrrrts 01 :Biolopcu/ Clicrnistn, arid Aimtomy arid 'Crllitlur Biology, Hurrurd Mcdicul Scliool. Boston. Mussucliurctfe 011 75
f Rtcrived
Szptembrr 21. 1989; Accepted October ti, 1989)
The expression and synthesis of acidic and basic fibroblast growth factors (aFGF and bFGF) in cultures of bovine and human vascular smooth muscle cells (BSMC and HSMC) was studied. BSMC express and synthesize only bFGF, whereas HSMC express and synthesize both bFGF and aFGF. .The presence of bFGF in BSiMC is shown by the following criteria: (1) the growth factor activity in BSMC lysates binds to a heparin-affinity column and elutes as a single peak at 1 . 5 - 1 . 7 ~ NaCI, characteristic for bFGF (2) this extract is mitogenic for smooth muscle cells; (3) Northern blot analysis demonstrates three distinct bFGF mRNAs of 7.0. 4.0, and 1.9 kb; no aFGF mRNA species were detected. Analysis of human umbilical vein endothelial cells yielded similar results: Heparin-affinity chromatography and Northern blot analysis failed to demonstrate the presence of aFGF despite the detection of bFGF by these techniques. In contrast, HSMC synthesize two growth factor activities: First, they bind to an immobilized heparin column and elute as two separate peaks at 1.2 and 1.5-1.7 M NaCI, characteristic for aFCF and bFGF; and second, Northern blot analysis demsnstrates the expression of aFGF mRNA of 4.6 kb and bFGF mRNAs of 7.0, 4.0 and 1.9 kb. Furthermore. it is shown that aFGF and bFGF are potent mitogens for smooth muscle cells in oitro.
KEYWORDS: smooth muscle cells, aodic FGF.basic FGF,gene expression
INTRODUCTION EndotheIiaI cells and smooth muscle celIs are the two major cell types which form the blood vessels. Under normal physiological conditions, the proliferation of vascular cells is strictly controlled and a near quiescent state is maintained. Exceptions are found during development, in the female reproductive system and in wound healing. Also many pathological conditions are associated with angiogenesis. For example, the formation of new blood vessels is a prerequisite for the growth of solid tumors beyond a self-limiting size (Folkman, 1985a). The proliferation of vascular endothelial and smooth muscle cells is regulated mainly by polypeptide growth factors (Ross and Vogel, 1978; Gospodarowicz et al.. 1981). The most widely studied of these factors is platelet-derived growth factor 'Corresponding author.
(PDGF). Cultured endothelial cells secrete a growth factor which appears to be PDGF (DiCorIeto and Bowen-Pope, 1983). Expression of both the PDGF-A and PDGF-B genes has been demonstrated (CoIIins et al., 1985; Kavanaugh et al., 1988), suggesting that all three PDGF isoforms (AA, BB, AB) may be secreted by endothelial cells. The conditioned medium from endothelial cell cultures has been shown by several criteria to contain PDGF-like activity (DiCorleto and Bowen-Pope, 1983), but the isoforms of the secreted growth factor(s) have not been reported. Although cultured smooth muscle cells (SMC)also secrete a PDGF-like mitogen. this is Iikeiy to be PDGF-A since only the PDGF-A gene is expressed by these cells (Sjdund et al., 1988). Interestingly, although endothelial cells produce PDGF, this cell type is unresponsive to PDGF-like mitogens (Heldin et al., 19811, presumably due to the absence of PDGF receptor expression. On the other hand, SMC.are known to respond mitogenically to PDGF
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(Ross et al., 1974), suggesting that the principal autocrine or paracrine role of this growth factor within the vasculature is the regulation of smooth muscle growth. It is not yet known whether the effect of PDGF an SMC proliferation is dependent on the particular combinations of PDGF jsoforms. Also the distribution of the receptor type for PDGF-A and PDGF-B on vascular SMC has not been reported. Endothelial cells in culture also synthesize and secrete a heparin-like molecule that is a SMC inhibitor (Castellot et al., 1981). They also secrete TGF-p which, after activation, is a potent inhibitor of endothelial cell proliferation (Baird and Durkin, 1986; Frater-Schrbder et al., 1986; Hannan et al., 1968). The effect of TGF-p on SMC proliferation is controversial. It has been shown to both inhibit as well as potentiate the growth of cultured SMC (Owens et al., 1988; Majack, 1987). The effect may be dependent upon cell density or on the presence or absence of other growth regulators. Work over the last few years has also demonstrated that acidic and basic fibroblast growth factors (aFGF, bFGF) are potent regulators of vascular endothelial cells in mtro and in vivo. Both growth factors are not only mitogens for endothelial cells (Moscatelli et al., 1986; Lobb et al., 1986; Schweigerer et al., 1987), but are also chemotactic for endothelial cells and can induce angiogenesis in vifro and in vim (Gospodarowiu et al., 1979; Thomas et al., 1985; Montesano et al., 1986; Sat0 and Riflcin, 1968). An intriguing feature of these factors is that they lack a signal peptide to direct secretion (Abraham et al., 1986a; Jaye et al., 1986) and, in contrast to PDGF, appear to be cell-associated (Vlodavsky et al., 1987b) rather than secreted. aFGF and bFGF bind with high affinity to heparin and can therefore bind strongly to heparan sulfate, a component of the extracellular matrix (Em).bFGF has been localized in the subendothelial ECM and in the basement membrane (Vlodavsky et al., 1987a; FoIkman et al., 1988). The physiological function of this interaction is not clear. Since aFGF and bFGF are stabilized in the presence of heparin or heparan sulfate (Gospodarowicz and Cheng, 1986), they may be protected against denaturation and degradation. They could then be mobilized when needed during remodeIing of the ECM or the basement membrane by hydrolasks (Vlodavsky et al., 1987a). The high affinity of aFGF and bFGF for heparin enabled the purification, sequencing, and molecular cloning of these mitogens (Shing et al., 1984; Lobb
et al., 1986; Baird et al., 1986; Abraham et al., 1986a;b; Jaye et al., 1986). aFGF shows 53% amino acid sequence identity to bFGF (Esch et al., 1985; Abraham et al., 1986a), and both growth factors appear to bind to the same cell surface receptor (Neufeld and Gospodarowicz, 1986). The most abundant source cf,aFGF appears to be neural tissue such as brain or retina (Folkman and Klagsbrun, 1967; Gospodarowicz, 1967), but it is also present in some malignant human gliomas (Libermann et al., 1967). In contrast, bFGF is expressed in many different cells and tissues, as well as in some tumors (Exh et al., 1985; Folkman, 1985b; Klagsbrun et al., 1966; Gospodarowicz, 1967). Although relatively little is known about the distribution of aFGF and bFGF in vascular cells, previous reports have documented the presence of aFGF in human smooth muscle cells (HSMC) (Winkles et al., 1987) and of bFGF in bovine or human endothelial cells (Schweigerer et al., 1967; Hannan et al., 1988). Here we show that both bovine and human SMC express bFGF and that HSMC also express aFGF. The growth factor activity from these cells was partially purified by heparin-affinity duomatography, shawing an elution pattern characteristic of aFGF and bFGF. Cell lysates from bovine smooth muscle cells {BSMC) or purified aFGF or bFGF stimulated the proliferation of BSMC.
METHODS
cell cultures BSMC were explanted from bovine aorta using standard methods (Voyta et al., 1964) and grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, “) containing 10% calf serum (HyClone, Logan, LT). The cells were identified as smooth muscle cells by their ‘hill and valley’ growth pattern (characteristic of confluent SMCs) and by staining with a monoclonal antibody speaf~cfor SMC a-actin (Skalli et al., 1986), which is a differentiation marker for these cells. Human vascular smooth muscle cells (HSMC) were explanted from splenic artery (obtained by R. Hendren, Department of Surgery, Children’s Hospital, Boston, MA). Cells were grown in DMEM with ’10% calf serum. HSMC were identified as described for BSMC. Cells of passage 3 to 6 were used for all experiments.
FIBROBLAST C R O W H FACTORS IN SMOOTH MUSCLE CELLS
Primary human umbilical vein endothelial cells
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column (TSK Heparin-3PW column, 8X75mm.
(HUVE)were provided by R. Smith (Department of TOSOH Corp, Japan) equilibrated with 0.6 M NaCI,
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Surgery. Children’s Hospital, Boston, MA) and S. Kourambanas (Department of Hematology, DanaFarber Cancer Inst., Boston, MA). HUVE cells were cultured on gelatin-coated plates (1.3%, Gibco) in Medium 199 (Gibco), 15% fetal calf serum (HyClone), 100 ,&ml heparin and 25 pglrnl endothelial mitogen (Biomedial Technologies, Inc. Cambridge, MA) and used between passage 6 and
0.02 M Tris-HCI, pH 7.4. After washing with approximately 3 column volumes, growth factors were eluted with a gradient (3OmI) of 0.6-2~NaCl in 0.02 M Tris, pH 7.4, at a flow rate of 1ml/min. Fractions of 0.5 ml were collected and aliquots tested for growth factor activity as described above.
Preparation of RNA and mRNA Blot Hybridization
1’.
Total RNA from cultured cells was prepared as described (Chirgwin et al., 1979), except that the Growth Factor Activity protease digestion step was omitted. Either 10 or Growth factor activity was tested by the stimulation 15 pg total RNA was denaturated in 2.2 M formaldeof 13H]thymidineincorporation into DNA of serum- hyde/5Oo/0 (vol/vol) formamide in the presence of depleted BALB 3T3 cells as described (Klagsbrun ethidium bromide, and RNA gels were prepared by and Shing, 1985;). In this assay a unit is defined as a new surface-tension method (K. Rosen, E. the amount of growth factor required to yield half- tamperti, and L. Vila-Komaroff, manuscript in maximal [Wlthymidine incorporation into DNA. To preparation). Gels were then photographed and measure BSMC proliferation, cells were seeded briefly rinsed in H,O. RNA was transferred to nitrosparsely (3000 ceUs/well in 24-well cluster plates) in cellulose membranes (Schleicher and S c h U Inc., DMEM/100/o CS. About 24hr after seeding, the Keene, NH) using 10 xSSC (1xSSC -0.15 M NaCV medium was replaced by DMEM containing 0.3% 0.015 M sodium citrate). Following transfer, the calf serum (day 0). Growth factor was added every ribosomal RNA bands were marked under UV light other day. At various times after seeding, cells were and the filter baked in a vacuum oven at 8VC for dissociated with trypsin/EDTA and counted in 2 hr. triplicate on a Coulter Counter. Purified human Hybridization with T-labeled cDNA probes was recombinant bFGF was a slft from Takeda Chemical performed as described earlier (Weich et al., 1986). Industries, Japan and purified bovine aFGF was pur- The blots were washed at 65°C in 60 m NaCI/O.l% chased from FGF Inc., San Diego. SDS/2 rn EDTA. The bFGF and aFGF probes (kindly provided by J. Abraham and J. Fiddes, California B i o t h o l o g y , Extraction of Growth Factors from Cells hc.) used were a 1.0-kb Nco I fragment of the Growth factors were extracted from vascular cells as bovine bFGF cDNA clone pJJl1-1 and a 0.46-kb Nco previously described for SK-HEP cells (Klagsbm et I/Eco RI fragment of the human aFGF cDNA clone al., 1986). Briefly, overconfluent multilayered BSMC pJC 3-5 (Abraham et al., 1986a, b). The chicken aand HSMC (1-3 ~10‘cells) or HUVE (4 x10‘ cells) tubulin cDNA was kindly provided by K. Rosen were harvested from cultures by trypsinization, (Department of Neurology, Children’s Hospital, washed with PBS, suspended (10; ceUs/ml) in l u Boston, MA). The 1.3-kb Hind 111 fragment was used NaCI, 0 . 0 2 ~Tris-HCI, pH 7.4, and disrupted by as a probe to estimate sample loading and integrity three cycles of freezing and thawing followed by of RNA samples after feprobing of each blot. Isosonkation 3 x15 sec. Cell extracts were centrifuged lated cDNA fragments were SzP-labeled using a (25,OOOxg, 20 min), and the supernatant was diluted random hexanucleotide priming kit (Boehringer with 0.02 M Tris-HC1, pH 7.4, to lower the concen- Mannheim). Specific activity of the probes was tration of Nafl to 0.5 M and subsequently filtered 0.5-2 x109 dpm/pg DNA. through a 0 . 2 - p filter.
Heparin-AffinityHPLC Heparin-affinity HPLC was performed as dexribed (berg et al., 1989). Briefly, diluted and filtered cell extracts were applied directly to a TSK heparin-
RESULTS
Synthesis of aFGF and bFGF in Vascular Cells Extracts derived from BSMC, HSMC, and HUVE stimulated DNA synthesis in 3T3 cells. To inves-
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tigate aFGF and bFGF activity, these extracts were subjected to heparin-affinity HPLC, and their elution profiles of the biological activity were compared with bovine retinal extract, a rich source of aFGF and bFGF (Courty et a]., 1985). Most of the mitogenic activity from the different cell extracts was retained on the heparin column during the i~itial wash. Subsequent elution was performed using a linear gradient from 0.6 to 2 M NaCl. Extracts from bovine retinas showed two peaks of heparin-binding growth factor activity which eluted at 1.2 M NaCI, characteristic for aFGF (Lobb and Fett, 1964; Bdhlen et a]., 1985), and at 1.5-1.7 M NaCI, characteristic for bFGF (Vlodavsky et a]., 3987b; Schweigerer et a]., 1987) (Fig. 1A). The growth factor activity from the BSMC extract eluted at 1.5-1.7 M NaCl (Fig. lB), characteristic of bFGF. No peak for aFGF could be detected. When cell extracts from HSMC were analyzed, two peaks of activity eluted (Fig. 1C): at 1.2 M NaC1, and 1.5-1.7 M NaCl, characteristic for aFGF and bFGF, respectively. The elution profile of HUVE cell lysate showed a single peak at 1.5-1.7 M NaCf characteristic for bFGF (Fig. 1D). No peak for aFGF was detectable. These data show that, in contrast to BSMC and HUVE where all the cell assodated activity seems to be attributable to bFGF, HSMC produce aFGF and bFGF. A similar elution profile for aFGF and bFGF was found in extracts of human foreskin fibroblasts (data not shown). However, the majority of the heparin-binding activity in bovine and human vascular cells seems to be associated with bFGF rather than with GGF.
Expression of aFGF and bFGF mRNA in Smooth Muscle Cells and in Endothelid Cells The presence of bFGF in all of our cell extracts and of aFGF in HSMC extracts indicates that the expression of these genes is regulated differently in these cells. To quantify this, the various vascular cell types were examined for expression of aFGF and bFGF mRNA. Equal amounts of total cellular RNA were examined by Northern blot analysis using a human aFGF cDNA probe. Bovine retina and HSMC contain significant amounts of 4.6-kb aFGF mRNA (Fig. 2). The level of expression is very similar in both cases. The size of the mRNA is almost identical to other aFGF mRNA transcripts previously detected in human brain stem uaye et al., 19861, human glioma cells (Libermann et a]., 1967), and HSMC
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Frocfion FIGURE I. Heparin-affinity chromatography of different cell extracts. Lysates from confluent cells ( I - S x I O ~ )were filtered and directlv applied to a TSK-heparin column. Growth factor activity was eiuted with a gradient of 0.6-2 M NaCI, and aliquots of each fraction were tested for mitogenic activity in a BALB 3T3 cell lZH]thymidineincorporation assay. Extract or lysate from bovine retina tissue (A), bovine aortic smooth muscle cells (BSMC) (B) human vascular smooth muscle cells (HSMC)(C). and human cndothelial cells (HUVE) (D).
(Winkles et al., 1967). No expression of this gene could be detected in BSM'C or HUVE. Northern blot analysis was also used to exarnine the level of expression of bFGF mRNA in these cell types. Random hexanucleotide primed bFGF cDNA
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FIBROBLAST GROWTH FACTORS IN SMOOTH MUSCLE CELLS
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4
a-tubulin
FlGURE 2. Northern blot analysis of aFGF mRNA expression in different vascular cell types. In each lane, 15pg total RNA was fractionated on a 1.2% agarose gel and transferred to nitrocellulose membranes. The 4.6-kb aFGF transcripts were detected by hybridization to a 3T-labeled 0.46-kb Nco 1-Eco Rl human aFGF cDNA fragment (top panel), specific for the coding region of the aFGF gene: 1, bovine retina tissue; 2, BSMC;3, HSMC;4, bovine aortic endothelial cells (BAE); 5, bovine capillary endothelial cells (BCE); 6, HUVE. To control for sample loading and RNA integrity, radiolabeled DNA was stripped and the filter reprobed using a chicken a-tubulin cDNA fragment (bottom panel).
wcy CJ
31 7
a-tubulin
FIGURE 3. Northern blot analysis of bFGF mRNA expression in different vascular cell types. In each lane, 1Opg total RNA was fractionated on a 1.2% agarose gel and transferred to nitrocellulose membranes. The bFGF transcripts were detected by hybridization to a 32P-labeled I kb Nco I bovine cDNA fragment (top panel). The filter was rehybridized to a 32P-labeled chicken atubulin cDNA probe (bottom panel). For identification of different lanes see Fig. 2.
Effects of aFGF and bFGF on BSMC Proliferation
was used as a probe (Abraham et al., 1986a) for hybridization to mRNA blots containing equivalent amounts of total cellular RNA. As seen in Figure 3, nearly all cell types express bFGF mRNA in similar amounts. In contrast, little expression was detected in long-term cultures of HUVE. Three different bFGF mRNA transcripts were identified having sizes of 7.0, 4.0, and 1.9 kb. These sizes are in general agreement with the 7.0- and 3.7-kb transcripts previously reported (Abraham et al., 1986a) and indicate an additional transcript of 1.9-kb. A very similar transcription pattern was recently reported for human fibroblasts and SK-HEP-1 cells (Sternfeld et al., 1988). The detection of aFGF and bFGF by heparin-affinity chromatography agrees with the level of specific gene expression shown by the Northern blot analysis.
The growth-stimulatory activity of BSMC cell extracts as well as of purified aFGF and bFGF on BSMC proliferation and DNA synthesis was investigated. In oitro SMC were found to divide in response to exogenous aFGF, bFGF, or SMC lysates when initially seeded at low cell densities (Fig. 4). Half-maximal stimulation of 13H]thymidineincorporation into DNA occurs at 0.25 ng/ml bFGF (data not shown). These results demonstrate that aFGF and bFGF are potent mitogens for SMC, as they are for many other cell types.
DISCUSSION Expression and synthesis of aFGF and bFGF in BSMC, HSMC, and. H U E were analyzed by heparin-affinity HPLC and Northern blot analysis. In each case there was a strong correlation between
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Time, duys FlGURE 4. Growth factor-stimul.ted mitogenesis in BSMC. Proliferation assay was done m the presence of 0.5% CS with ( A ) BSMC l p t c (3.2 Unitdwell), (A) purified acidic FGF (33ng/ml), (0) purified basic FGF (5ngld). and (0) no addition. Evay second day, cells were tryp&iA and counted in a Coultex counter. Values are the means d triplicate determiMtiOnS.
the expression of the mRNA for aFGF or bFGF and the detection of these mitogens by heparin-affinity chromatography. We have demonstrated that proliferating HSMC in culture express and synthesize aFGF and bFGF. The majority (-75%) of the cell-associated heparinbinding growth factor activity appeared to be bFGF and the remainder aFGFffhese results 9 similar to those of earlier studies demonstrating that HSMC from abdominal aorta or umbilical vein synthesize aFGF (Winkles et al., 1987) and that fetal HSMC synthesize bFGF (Gospodarowiu et al., 1988). However, this is the first demonstration of the expression and synthesis of both aFGF and bFGF in a single preparation of HSMC. Several nonvascular cell types (e.g., cardiac myocytes) have previously been shown to express both aFGF and bFGF (Weiner and Swain, 1989). However, the functional significance, if any, of producing both factors has yet to be established since both growth factors apparently bind to the same cell surface receptor (Neufeld and Gospodarowicz, 1986). In this context it is interesting that, in contrast to HSMC, BSMC synthesize only bFGF. Although this difference may reflect species diversity or heterogeneity of the vessel type used for SMC isolation, it may suggest
111.
that the same physiological roles are performed by aFGF or bFGF. Heparin-affinity activity profiles of bovine endothelial cell lysates have previously demonstrated that these cells contain bFGF but not aFGF (Schweigerer et al., 1987; Vlodavsky et a]., 1987b). Similarly, our results show that HUVE synthesize bFGF with no apparent production of aFGF. .Taken together, these $indings suggest that endothelial cells produce relatively high levels of bFGF but no detectable aFGF. However, we cannot exclude the possibility that under certain conditions the expression of the aFGF gene may be induced. It is of interest that all bovine vascular cells examined to date appear to produce bFGF but not aFGF (see above). In contrast, we find that the human vaxulature does contain a source of aFGF, namely the SMC,which produces comparable levels of aFGF and bFGF. As discussed above, the physiological importance of such qualitative differences remains to be established. We propose that an autocrine and a paracrine mechanism of action of aFGF and bFGF may play a crucial role in SMC and endothelial cell proliferation. Although much work remains to establish the relative importance of each growth factor for these cells in vim, it may be relevant that successful culture of endothelial cells requires a continuous supply of exogenous FGF (Vlodavsky et al., 1979;Rogelj et al., 1989), whereas SMC may be successfully cultured in its absence. After synthesis, growth factors are normally secreted and interact with specific cell surface receptors in an autocrine or paracrine fashion. However, aFGF and bFGF are mostly cell-associated (Libermann et al., 1987; Vlodavsky et al., 1987b), a relatively unusual feature for polypeptide growth factors. The mechanism by which aFGF and bFGF can be released and could interact with the surface receptor is not known: Both mitogens lack a signal peptide sequence, which is a requirement for secretion (Abraham et al., 1986a; Jaye et al., 1986). Although the FGFs do not appear, therefore, to be secreted in a classical way like many other polypeptide growth factors, it is of interest that bFGF has been found in the ECM (Vlodavsky et al., 1987a). One possible mechanism for this localization might be seen in the high affinity that aFGF and bFGF have for heparan sulfate, a component of the ECM. It is possible that aFGF and bFGF may be transported in physical association with components of the ECM and then stored as an integral part of the ECM in vascular cells. Matrix-bound FGF stored in
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FIBROBLAST GROWTH FACTORS IN SMOOTH MUSCLE CELLS
this way may be released when needed by the activation of extracellular heparinase (Vlodavsky et al., 1987a; Folkman et al., 1988). SMC in culture also produce other growth factors, such as PDGF, insulin-like growth factor-1 (IGF-I) and a smooth muscle cell-derived growth factor (SDGF), which are secreted into the medium (Sj6lund et al., 1988; Clemmons and Van Wyk, 1983; Morisaki et al., 1988). PDGF is a potent mitogen for SMC (Ross et al., 1971) and is synthesized and secreted by these cells (Seifert et al., 1984; SjCilund et al., 1988). Recently, it has been shown, that bFGF in combination with PDGF has a synergistic effect on growth stimulation in BSMC, suggesting that both growth factors together are necessary for a high mitogenic response (Weich and Folkman, 1989). It is possible that cell-associated aFGF and/or bFCF together with PDGF secreted by vascular SMC and endothelial cells have an important role in the autocrine and paracrine regulation of proliferation of these vascular cell types.
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Castellot, 1. J., Addonirio. M. L.. Rosenberg, R. and Karnovsky. M. J. (1981) Cultured endothelid cells produce a heparin-like inhibitor of smooth muxle cell growth. I. Crlf B i d . 90. 32-379. Chirgwin, J. M.,Pryzyba. A. E. MacDonald. R. J. and Ruttner. W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 18,3294-5299. Clemmons, D. R. and Van Wyk, J. J. (1985) Evidence for a functional role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. 1. Clin. lnvcst. 75, 1911-1918.
Colliis, T., Ginsburg, D., Boss, J. M.,Orkin, S. H. and Pober, J. 5. (1985) Cultured human endothelial cells express plateletderived growth factor chain 2 cDNA cloning and structural analysis. Nnturc316,748-750. Courty, J., Loret. C., Moenner, M., Chevallier, B., Lagente. 0.. Courtois. Y. and Barritault. 0. (1985) Bovine retina contains three growth factor activities with different affinity for heparin: eye-derived growth factor I, IX, and In. Biochimic67.263-169. DiCorleto, P. E. and Bowen-Pope, D. F. (1983) Cultured endothelial cells produce a platelet-derived growth factor-like protein. Proc. Nut[. Acnd. Sci. USA 80. 1919-1923. Exh, F.. Baird. A.. Ling, N., Ueno. N.. Hill, F., Denoroy. L.. Klepper, R,Cospodamwicz. D.,'Bdhlen, P. and Guillemin. R. (1985) Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Froc. Nutl. Acud:SEi. USA 82.6507-6511.
Folkman, J. (198fa) Tumor angiogenesis. Ado. Cuncrr Rrs. 43, 175-203.
AW0Wl.EDGMENTS We wish to thank David Brigstock and Kenneth Rosen for helphl suggestions and discussions. H.A.W. is a recipient of a fellowship from the Deutsche Forschungsgemeinxhaft, Grant We 1211-1; N.I.is a recipient of a grant from the Swiss National Science Foundation, Grant 83.497.0.87; and M.K. was supported by NCI Grant CA-37392. This work was also supported by a grant from Takeda Chemical Industries, Ltd. to Harvard University (J.F.).
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Abraham. J. A., Whang, J. L., Tumolo. A., Mergia. A., Friedman, I., G@spodarowicz. D. and Fiddes. I. C. (1986b) Human basic fibroblast growth factor: Nucleotide sequence and genomic organization. EMBO 1. 5,2523-2528. Baird, A. and Durkm, T. (1986) Inhibition of endothelil cell proliferation by type beta-transforming growth factor: interactions with acidic and basic fibroblast growth factors. Biochm. Biophp. R e . Commun. 138,476-482. Baud, A., Exh, F., Monnede, P., Ueno, N., Ling, N., Bbhlen, P.. Ymg, S. -Y.. Wehrenberg, W. B. and Guillemin. R. (1986) Molecular characterization of fibrcXast growth factor: Distribution and biological activities in various tissues. Rccmt Prog. Horm. Rrs. 42. 143-205. Bdhlen, P., Exh. F.. Baird. A. and Gospodarowicz.'D. (1985) Acidic fibroblast growth factor (FCF) from bovine brain: amino-terminal sequence and comparison with basic FGF. EhlBO /. 4, 1951-1956.
Folkman. J. (198%) Toward M understanding of angiqenesis: search and discovery. Perspec. Biol. Mcd. 29.1036. Folkman, j. and Klagbrun, M. (1987) Angiogenic factors. Science 235,442447.
Folkmu\, J., Klrgsbrun, M.. S a m . J., Wadziiski, M.,Ingber, D. and Vlodavsky, I. (1988) A heparin-binding angiogenic protein, basic fibroblast growth factor, is stored within basement membrane. Am. I. Pethol. 130,393-W. Frater-Schrdder, M.. Miiller, C., Birchmeier, W. and WhIen. P. (1986) Transforming growth factor-beta inhibits endothelid cell proliferation. Biochmc. Biophys. Rcs. Commun. l37,295-302. Cospodarowia. D. (1987) purification of bnin and pituitary FGF. Methods Enrymd. Fcptih Growth Fncton 1178.106-117. Cospodarowia, D. and Cheng, J. (1986) Heparin protects basic and acidic FGF from inactivation. 1. Cell. Physiol. 128,4775-484. Gospodamwia, D., Bhlecki, H. and Thakral, T. K. (1979) The angiogenic activity of the fibroblast and epidermal growth factor. kp. .€ye Rcs. Zb,M1-514. Cospodarowia, C.. Hirabayashi. K.. Giguere, L. and Tauber. J. -P. (1981) Factors controUig the proliferation rate, final cell density, and life span of bovine vascular smooth muscle cells in culture. 1. Ccfl Biol. 89, 568-578. Gospodarowicz, D.. Ferrara, N., Haaparanta, T. and Neufeld, G. (1988) 8aric fibroblast growth factm expression in cultured bovine vascular smooth muxle cells. Eur. 1. Cell Biol. 46, 144-151. Hannan. R. L., Kourembanas, S., Flanders, K. C., Rogelj, S.. Roberts, A. 8.. Faller, D. V. and Klagsbrun, M. (1988) Endothelial cells synthesize basic fibroblast growth factor and transforming growth factor beta. Crowfh Fnctors. 1.7-17. Heldin. C. -H.. Westennark. B. and Wasteson, A. (1981) Spedic receptors for platelet-derived growth factor on cells derived from connective tissue and glia. Pmc. Nut!. Acud. Sn. USA 78, 3661-3668. Iberg, N.. Rogelj, S.. Fanning, P. and Klagsbrun, M.(1989) Purification of 18 kDa and 22 kDa forms of basic fibroblast growth factor from rat cells transformed by the rns oncogene. I. Biol. Chem. (in press). laye. M., Howk. R., Burgess, W., Ricca, C. A., Chiu, 1. -M.,
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Ravera. M. W..O'Brien, 5. J., Modi, W. S., Maciag, T. and Droham, W. N. (1986) Human endothelial cell growth factor: Cloning, nucleotide sequence and chromosome localization. Stirnw233.541-545. Kavanaugh, W. M., Harsh, 1. V., Starksen, N. F., Rocco, C. M. and Williams, L. T. (1988) Transcriptional regulation of the A and B genes of platelet-derived growth factor in miuovaxular endothelial cells. 1. Brol. C h n . 263, 8470-8472. Klagsbrun, M. and Shin&, Y. (1985) Heparin affinity of anionic and cationic capillary endothelial cell growth factors: analysis of hypothalamus-derived growth factors and fibroblast growth factors. Proc. Nutl. Acud. So'. USA 82, 805-809. Klagsbrun, M.,Sasse, I., Sullivan, R. and Smith, J. A. (1986) Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast growth factor. Proc. Natl. Acud. Sci. USA 83,2448-2452. Libermann, T. A., Friesel, R., Jaye, M.,. Lyd, R M., Westermark, B., h h a n , W., Schmidt, A., k c i a g , T. and Schlessinger, J. (1987) An angiosenic growth factor is expressed in human glioma 4 s . EMBO].6,1627-1632. h b b . R. R. and Fett, J. W. (19&) F'urification of two distinct growth factors from bovine neural tissue by heparin affinity chromatography. Biochmistry23,6295-6299. Lobb, R. R., Harper, J. W. and Fett, J. W. (1986) Purification of hcprrin-binding growth faaots. A d . B ~ O C ~ O I Is I( . ,1-14. Majack, R. A. (1987) Beb-type transforming growth factor specifies organizational behavior m vascular smooth m u d e cells. 1. Cell B i d . 105,465-471. Montesano, R., Vassali, J. D., Baird, A., Guillemin, R. and Orci, L (1986) Basic FCF induces angiogenesis in vim. Proc. Natl. Acud. Sn'. USA 83,7297-7301. Morisaki, N., Kmzaki, T., Koshikrwa, T., hito, Y. and Yoshida, S. (1988)seartion of a new growth factor, smooth muscle cell derived growth factor, distinct from platekt derived growth factor by cultured rabbit aortic smooth muscle cells. FEBS Lat. 230,186-192. Mosutelli, D., Presta, M. and Rifkin,D. B. (1986) Purification of a factor from human pbcmta that stimulates apiuuY qdoh e l d cell protease production, DNA synthesis urd migration. PTOC. Natl. Aad. Sci. USA 83,2091-2095. NeufeH, G. and Gospodarowia, D. (1986) M c and aodic f i b blast growth factors interact with the same cell surface ncep ton. 1. Biol. Chn. 261, 5631-5637. O W ~ S ,G. K, ceisterfa, A. T., y.ng. Y. w -H. urd Komoriya, A. (1988) Tmsfaming growth factor beta-induced growth inhiition and ceUular hyprtrophy in cultured vascular smooth muscle cells. 1. GZI B i d . 107,77l-780. Rogelj, S., Klagsbmn, M.,Atmron, R., Kurokawa, M.,Haimovitz, A., Fuchs, 2. and Vlodavsky, 1. (3989) Basic hiroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothel*l cells and the differentiation of PC 12 cells. J. Gll Bid.109,823-831. Ross, R. and Vogel, A. (1978) The platelet-derived growth factor. Glf14, 203-210. Ross, R., Glomset, 1.. Kariya, 8.and Harker, L. (1974) A plateletdependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Nutl. Acud. So'. USA n,1207-1210. Sato. Y. and Rifkin, D. B. (1988) Autouine activities of basic fibroblast growth factor regulation of endotheld cell movement, plasminogen activator synthesis and DNA synthesis. 1. Cell B i d . 107, 1199-1205. Schweigerer. L., Neufeld, G., Friedman, J., Abraham, J. A., Fiddes, J. C. and Gospodarowia. D. (1987) CapilIary endo-
thelial cells express basic fibroblast growth factor, a mitogen that promotes their own growth. Nuturt325.257-259. Seifert, R. A,, Schwartr, S. M. and Bowen-Pope, D. F. (1984) Developmentally regulated production of platelet-derived growth factor-like molecules. Nuturt 311, 669-671. Sjblund, M., Hedin, U., Sejersen, T., Heldin, C -H. and Thyberg, J. (1988) Arterial smooth muscle cells express platelet-derived growth factor (PDGF) A chain mRNA, secrete a PDGF-like mitogen, and bind exogenous PDGF in a phenotype- and growth state-dependent manner. 1. Cell B i d . 106,403-413. Skalli, 0.. Ropraz, P., Trzeciak, A.. Benzonana, C.,Cillessen, D. and Gabbiani, G. (1986) A monoclonal antibody against asmooth muscle actin: A new probe for smooth muscle ddferentiation. 1. Cell Biol. 103,2787-2796. Shing, Y., Folkman, 1.. Sullivan., Butterfield. C.. Murray. J. and Klagsbnm, M. (1984) H W affinity: purification of a tumorderived capillary endothelial cell growth factor. Scirncc 223, 1296-1299. Stemfeld. M. D., Hendrickron, J. E., Keeble, W. W.,Rosenbaum, J. T., Robertson, J. E., Pittelkow, M. R. and Shipley, G. D. '(1988) Differential expression of mRNA coding for heparinbinding growth factor type 2 in human cells. ]. Gll. Physiol. 136,297-304. Thomas, K. A., Rios-Candelore, M., Cimmer-Calkgo, G., Disalvo, I., Bennet, C., Rodkey. J. and Fitzpatrick, S. (1985) Pure brain-derived acidic fibroblast growth factor is a potent angiogenic vascular endothelial cell mitogen with sequence homology to interleukin 1. Proc. Nutl. Acud. Sci. USA 82, 6409-6413. Vlodavsky, 1.. Greenburg, G., Johnson, K. and Cospodarowia,D. (1979) Vascular endothelii cells maintained in the absence of fiioblast growth factor undergo structural and functional alterations that are i n m p a t i i l e with their m vivo differentiated pmperties. ]. GI1 B i d . 83, 468-486. Vlodavsky, I., Folkman, 1.. Sullivan, R., Fridman, R., IshaiMichaeli, R, Sasse, I. and Klagsbrun, M. (1987a) Endothelial cell-derived basic fiioblast growth factor: Synthesis and deposition into subendotheld extracellular matrix. Proc. Nut/. Aad. Sd. USA 84,2292-2296. Vlodavsky, I., Fridrmn, R., Sullivan, R., Sasse, J. and Khgsbrun, M. ('198%) Aortic end0theli.l cells synthesize basic fiiroblast growth factor which remains cell associated and plateletderived growth factor-lie protein which is secreted. J. Cell Physiol. l31.402-408. V+, J. C., Via,, D, P., Butterfield, C. E. and Mer, 8. R (1984) Identification and isolation of endothelial cells based on their uptake of acctylated-low density lipoprotein. 1. Cell Biol. 99, 2034-2040. Weich, H. A. and Folkman, J. (1989) Bovine aortic smooth muscle cells: role of different growth factors for cellular replication. 1. Cell Biochn. (Suppl.) 13B, 148. Weich, H. A., Sebaid, W., sdrairer, H. U. and Hoppe, J. (1986) The human osteosarcoma cell line U-2 05 expresses a 3.6 Mobase mRNA which codes for the sequence of the PDGF-B chain. FEBS Lrt!. 198, 344-348. Weiner, H. L. and Swain, J. L. (1989) Acidic fibroblast growth factor mRNA is expressed by cardiac myocytes in culture and the protein is localized to the exhacellular matrix. Proc. Nut/. A u d . So'. USA 86,2683-2687. Winkles, J. A., Friesel, R, Burgess, W. H., Howk, R., Mehlman, T.. Weinstein, R. and kciag, T. (1967) Human vascular smooth muscle cells both express and respond to heparinbinding growth factor I (endothelial cell growth factor). Proc: Nutl. Aad. Sn'. USA 84,7124-7128.
Crorc.tli Fazfors, 1sWO. Vol. 2. pp. 313-320 Reprints available directly trom the publisher Photocopying permitted by license only
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Expression of Acidic and Basic Fibroblast Growth Factors in Human and Bovine Vascular Smooth Muscle Cells Growth Factors Downloaded from informahealthcare.com by University of Toronto on 12/25/14 For personal use only.
HERBERT A. WEIGH'., NIGGI [BERG1,MICHAEL KLAGSBRUN1.L,and JUDAH FOLKMAN' Di.purtnicvit or jiuyy'. CliddrciI'j Hiwpitul, 300 Lonpood Aomur, Boston. .L3assaclrirsrtts. 02175, arid Dryurtinrrrts 01 :Biolopcu/ Clicrnistn, arid Aimtomy arid 'Crllitlur Biology, Hurrurd Mcdicul Scliool. Boston. Mussucliurctfe 011 75
f Rtcrived
Szptembrr 21. 1989; Accepted October ti, 1989)
The expression and synthesis of acidic and basic fibroblast growth factors (aFGF and bFGF) in cultures of bovine and human vascular smooth muscle cells (BSMC and HSMC) was studied. BSMC express and synthesize only bFGF, whereas HSMC express and synthesize both bFGF and aFGF. .The presence of bFGF in BSiMC is shown by the following criteria: (1) the growth factor activity in BSMC lysates binds to a heparin-affinity column and elutes as a single peak at 1 . 5 - 1 . 7 ~ NaCI, characteristic for bFGF (2) this extract is mitogenic for smooth muscle cells; (3) Northern blot analysis demonstrates three distinct bFGF mRNAs of 7.0. 4.0, and 1.9 kb; no aFGF mRNA species were detected. Analysis of human umbilical vein endothelial cells yielded similar results: Heparin-affinity chromatography and Northern blot analysis failed to demonstrate the presence of aFGF despite the detection of bFGF by these techniques. In contrast, HSMC synthesize two growth factor activities: First, they bind to an immobilized heparin column and elute as two separate peaks at 1.2 and 1.5-1.7 M NaCI, characteristic for aFCF and bFGF; and second, Northern blot analysis demsnstrates the expression of aFGF mRNA of 4.6 kb and bFGF mRNAs of 7.0, 4.0 and 1.9 kb. Furthermore. it is shown that aFGF and bFGF are potent mitogens for smooth muscle cells in oitro.
KEYWORDS: smooth muscle cells, aodic FGF.basic FGF,gene expression
INTRODUCTION EndotheIiaI cells and smooth muscle celIs are the two major cell types which form the blood vessels. Under normal physiological conditions, the proliferation of vascular cells is strictly controlled and a near quiescent state is maintained. Exceptions are found during development, in the female reproductive system and in wound healing. Also many pathological conditions are associated with angiogenesis. For example, the formation of new blood vessels is a prerequisite for the growth of solid tumors beyond a self-limiting size (Folkman, 1985a). The proliferation of vascular endothelial and smooth muscle cells is regulated mainly by polypeptide growth factors (Ross and Vogel, 1978; Gospodarowicz et al.. 1981). The most widely studied of these factors is platelet-derived growth factor 'Corresponding author.
(PDGF). Cultured endothelial cells secrete a growth factor which appears to be PDGF (DiCorIeto and Bowen-Pope, 1983). Expression of both the PDGF-A and PDGF-B genes has been demonstrated (CoIIins et al., 1985; Kavanaugh et al., 1988), suggesting that all three PDGF isoforms (AA, BB, AB) may be secreted by endothelial cells. The conditioned medium from endothelial cell cultures has been shown by several criteria to contain PDGF-like activity (DiCorleto and Bowen-Pope, 1983), but the isoforms of the secreted growth factor(s) have not been reported. Although cultured smooth muscle cells (SMC)also secrete a PDGF-like mitogen. this is Iikeiy to be PDGF-A since only the PDGF-A gene is expressed by these cells (Sjdund et al., 1988). Interestingly, although endothelial cells produce PDGF, this cell type is unresponsive to PDGF-like mitogens (Heldin et al., 19811, presumably due to the absence of PDGF receptor expression. On the other hand, SMC.are known to respond mitogenically to PDGF
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(Ross et al., 1974), suggesting that the principal autocrine or paracrine role of this growth factor within the vasculature is the regulation of smooth muscle growth. It is not yet known whether the effect of PDGF an SMC proliferation is dependent on the particular combinations of PDGF jsoforms. Also the distribution of the receptor type for PDGF-A and PDGF-B on vascular SMC has not been reported. Endothelial cells in culture also synthesize and secrete a heparin-like molecule that is a SMC inhibitor (Castellot et al., 1981). They also secrete TGF-p which, after activation, is a potent inhibitor of endothelial cell proliferation (Baird and Durkin, 1986; Frater-Schrbder et al., 1986; Hannan et al., 1968). The effect of TGF-p on SMC proliferation is controversial. It has been shown to both inhibit as well as potentiate the growth of cultured SMC (Owens et al., 1988; Majack, 1987). The effect may be dependent upon cell density or on the presence or absence of other growth regulators. Work over the last few years has also demonstrated that acidic and basic fibroblast growth factors (aFGF, bFGF) are potent regulators of vascular endothelial cells in mtro and in vivo. Both growth factors are not only mitogens for endothelial cells (Moscatelli et al., 1986; Lobb et al., 1986; Schweigerer et al., 1987), but are also chemotactic for endothelial cells and can induce angiogenesis in vifro and in vim (Gospodarowiu et al., 1979; Thomas et al., 1985; Montesano et al., 1986; Sat0 and Riflcin, 1968). An intriguing feature of these factors is that they lack a signal peptide to direct secretion (Abraham et al., 1986a; Jaye et al., 1986) and, in contrast to PDGF, appear to be cell-associated (Vlodavsky et al., 1987b) rather than secreted. aFGF and bFGF bind with high affinity to heparin and can therefore bind strongly to heparan sulfate, a component of the extracellular matrix (Em).bFGF has been localized in the subendothelial ECM and in the basement membrane (Vlodavsky et al., 1987a; FoIkman et al., 1988). The physiological function of this interaction is not clear. Since aFGF and bFGF are stabilized in the presence of heparin or heparan sulfate (Gospodarowicz and Cheng, 1986), they may be protected against denaturation and degradation. They could then be mobilized when needed during remodeIing of the ECM or the basement membrane by hydrolasks (Vlodavsky et al., 1987a). The high affinity of aFGF and bFGF for heparin enabled the purification, sequencing, and molecular cloning of these mitogens (Shing et al., 1984; Lobb
et al., 1986; Baird et al., 1986; Abraham et al., 1986a;b; Jaye et al., 1986). aFGF shows 53% amino acid sequence identity to bFGF (Esch et al., 1985; Abraham et al., 1986a), and both growth factors appear to bind to the same cell surface receptor (Neufeld and Gospodarowicz, 1986). The most abundant source cf,aFGF appears to be neural tissue such as brain or retina (Folkman and Klagsbrun, 1967; Gospodarowicz, 1967), but it is also present in some malignant human gliomas (Libermann et al., 1967). In contrast, bFGF is expressed in many different cells and tissues, as well as in some tumors (Exh et al., 1985; Folkman, 1985b; Klagsbrun et al., 1966; Gospodarowicz, 1967). Although relatively little is known about the distribution of aFGF and bFGF in vascular cells, previous reports have documented the presence of aFGF in human smooth muscle cells (HSMC) (Winkles et al., 1987) and of bFGF in bovine or human endothelial cells (Schweigerer et al., 1967; Hannan et al., 1988). Here we show that both bovine and human SMC express bFGF and that HSMC also express aFGF. The growth factor activity from these cells was partially purified by heparin-affinity duomatography, shawing an elution pattern characteristic of aFGF and bFGF. Cell lysates from bovine smooth muscle cells {BSMC) or purified aFGF or bFGF stimulated the proliferation of BSMC.
METHODS
cell cultures BSMC were explanted from bovine aorta using standard methods (Voyta et al., 1964) and grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, “) containing 10% calf serum (HyClone, Logan, LT). The cells were identified as smooth muscle cells by their ‘hill and valley’ growth pattern (characteristic of confluent SMCs) and by staining with a monoclonal antibody speaf~cfor SMC a-actin (Skalli et al., 1986), which is a differentiation marker for these cells. Human vascular smooth muscle cells (HSMC) were explanted from splenic artery (obtained by R. Hendren, Department of Surgery, Children’s Hospital, Boston, MA). Cells were grown in DMEM with ’10% calf serum. HSMC were identified as described for BSMC. Cells of passage 3 to 6 were used for all experiments.
FIBROBLAST C R O W H FACTORS IN SMOOTH MUSCLE CELLS
Primary human umbilical vein endothelial cells
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column (TSK Heparin-3PW column, 8X75mm.
(HUVE)were provided by R. Smith (Department of TOSOH Corp, Japan) equilibrated with 0.6 M NaCI,
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Surgery. Children’s Hospital, Boston, MA) and S. Kourambanas (Department of Hematology, DanaFarber Cancer Inst., Boston, MA). HUVE cells were cultured on gelatin-coated plates (1.3%, Gibco) in Medium 199 (Gibco), 15% fetal calf serum (HyClone), 100 ,&ml heparin and 25 pglrnl endothelial mitogen (Biomedial Technologies, Inc. Cambridge, MA) and used between passage 6 and
0.02 M Tris-HCI, pH 7.4. After washing with approximately 3 column volumes, growth factors were eluted with a gradient (3OmI) of 0.6-2~NaCl in 0.02 M Tris, pH 7.4, at a flow rate of 1ml/min. Fractions of 0.5 ml were collected and aliquots tested for growth factor activity as described above.
Preparation of RNA and mRNA Blot Hybridization
1’.
Total RNA from cultured cells was prepared as described (Chirgwin et al., 1979), except that the Growth Factor Activity protease digestion step was omitted. Either 10 or Growth factor activity was tested by the stimulation 15 pg total RNA was denaturated in 2.2 M formaldeof 13H]thymidineincorporation into DNA of serum- hyde/5Oo/0 (vol/vol) formamide in the presence of depleted BALB 3T3 cells as described (Klagsbrun ethidium bromide, and RNA gels were prepared by and Shing, 1985;). In this assay a unit is defined as a new surface-tension method (K. Rosen, E. the amount of growth factor required to yield half- tamperti, and L. Vila-Komaroff, manuscript in maximal [Wlthymidine incorporation into DNA. To preparation). Gels were then photographed and measure BSMC proliferation, cells were seeded briefly rinsed in H,O. RNA was transferred to nitrosparsely (3000 ceUs/well in 24-well cluster plates) in cellulose membranes (Schleicher and S c h U Inc., DMEM/100/o CS. About 24hr after seeding, the Keene, NH) using 10 xSSC (1xSSC -0.15 M NaCV medium was replaced by DMEM containing 0.3% 0.015 M sodium citrate). Following transfer, the calf serum (day 0). Growth factor was added every ribosomal RNA bands were marked under UV light other day. At various times after seeding, cells were and the filter baked in a vacuum oven at 8VC for dissociated with trypsin/EDTA and counted in 2 hr. triplicate on a Coulter Counter. Purified human Hybridization with T-labeled cDNA probes was recombinant bFGF was a slft from Takeda Chemical performed as described earlier (Weich et al., 1986). Industries, Japan and purified bovine aFGF was pur- The blots were washed at 65°C in 60 m NaCI/O.l% chased from FGF Inc., San Diego. SDS/2 rn EDTA. The bFGF and aFGF probes (kindly provided by J. Abraham and J. Fiddes, California B i o t h o l o g y , Extraction of Growth Factors from Cells hc.) used were a 1.0-kb Nco I fragment of the Growth factors were extracted from vascular cells as bovine bFGF cDNA clone pJJl1-1 and a 0.46-kb Nco previously described for SK-HEP cells (Klagsbm et I/Eco RI fragment of the human aFGF cDNA clone al., 1986). Briefly, overconfluent multilayered BSMC pJC 3-5 (Abraham et al., 1986a, b). The chicken aand HSMC (1-3 ~10‘cells) or HUVE (4 x10‘ cells) tubulin cDNA was kindly provided by K. Rosen were harvested from cultures by trypsinization, (Department of Neurology, Children’s Hospital, washed with PBS, suspended (10; ceUs/ml) in l u Boston, MA). The 1.3-kb Hind 111 fragment was used NaCI, 0 . 0 2 ~Tris-HCI, pH 7.4, and disrupted by as a probe to estimate sample loading and integrity three cycles of freezing and thawing followed by of RNA samples after feprobing of each blot. Isosonkation 3 x15 sec. Cell extracts were centrifuged lated cDNA fragments were SzP-labeled using a (25,OOOxg, 20 min), and the supernatant was diluted random hexanucleotide priming kit (Boehringer with 0.02 M Tris-HC1, pH 7.4, to lower the concen- Mannheim). Specific activity of the probes was tration of Nafl to 0.5 M and subsequently filtered 0.5-2 x109 dpm/pg DNA. through a 0 . 2 - p filter.
Heparin-AffinityHPLC Heparin-affinity HPLC was performed as dexribed (berg et al., 1989). Briefly, diluted and filtered cell extracts were applied directly to a TSK heparin-
RESULTS
Synthesis of aFGF and bFGF in Vascular Cells Extracts derived from BSMC, HSMC, and HUVE stimulated DNA synthesis in 3T3 cells. To inves-
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tigate aFGF and bFGF activity, these extracts were subjected to heparin-affinity HPLC, and their elution profiles of the biological activity were compared with bovine retinal extract, a rich source of aFGF and bFGF (Courty et a]., 1985). Most of the mitogenic activity from the different cell extracts was retained on the heparin column during the i~itial wash. Subsequent elution was performed using a linear gradient from 0.6 to 2 M NaCl. Extracts from bovine retinas showed two peaks of heparin-binding growth factor activity which eluted at 1.2 M NaCI, characteristic for aFGF (Lobb and Fett, 1964; Bdhlen et a]., 1985), and at 1.5-1.7 M NaCI, characteristic for bFGF (Vlodavsky et a]., 3987b; Schweigerer et a]., 1987) (Fig. 1A). The growth factor activity from the BSMC extract eluted at 1.5-1.7 M NaCl (Fig. lB), characteristic of bFGF. No peak for aFGF could be detected. When cell extracts from HSMC were analyzed, two peaks of activity eluted (Fig. 1C): at 1.2 M NaC1, and 1.5-1.7 M NaCl, characteristic for aFGF and bFGF, respectively. The elution profile of HUVE cell lysate showed a single peak at 1.5-1.7 M NaCf characteristic for bFGF (Fig. 1D). No peak for aFGF was detectable. These data show that, in contrast to BSMC and HUVE where all the cell assodated activity seems to be attributable to bFGF, HSMC produce aFGF and bFGF. A similar elution profile for aFGF and bFGF was found in extracts of human foreskin fibroblasts (data not shown). However, the majority of the heparin-binding activity in bovine and human vascular cells seems to be associated with bFGF rather than with GGF.
Expression of aFGF and bFGF mRNA in Smooth Muscle Cells and in Endothelid Cells The presence of bFGF in all of our cell extracts and of aFGF in HSMC extracts indicates that the expression of these genes is regulated differently in these cells. To quantify this, the various vascular cell types were examined for expression of aFGF and bFGF mRNA. Equal amounts of total cellular RNA were examined by Northern blot analysis using a human aFGF cDNA probe. Bovine retina and HSMC contain significant amounts of 4.6-kb aFGF mRNA (Fig. 2). The level of expression is very similar in both cases. The size of the mRNA is almost identical to other aFGF mRNA transcripts previously detected in human brain stem uaye et al., 19861, human glioma cells (Libermann et a]., 1967), and HSMC
13
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80
Frocfion FIGURE I. Heparin-affinity chromatography of different cell extracts. Lysates from confluent cells ( I - S x I O ~ )were filtered and directlv applied to a TSK-heparin column. Growth factor activity was eiuted with a gradient of 0.6-2 M NaCI, and aliquots of each fraction were tested for mitogenic activity in a BALB 3T3 cell lZH]thymidineincorporation assay. Extract or lysate from bovine retina tissue (A), bovine aortic smooth muscle cells (BSMC) (B) human vascular smooth muscle cells (HSMC)(C). and human cndothelial cells (HUVE) (D).
(Winkles et al., 1967). No expression of this gene could be detected in BSM'C or HUVE. Northern blot analysis was also used to exarnine the level of expression of bFGF mRNA in these cell types. Random hexanucleotide primed bFGF cDNA
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FIBROBLAST GROWTH FACTORS IN SMOOTH MUSCLE CELLS
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u u y& * ,.
4
a-tubulin
FlGURE 2. Northern blot analysis of aFGF mRNA expression in different vascular cell types. In each lane, 15pg total RNA was fractionated on a 1.2% agarose gel and transferred to nitrocellulose membranes. The 4.6-kb aFGF transcripts were detected by hybridization to a 3T-labeled 0.46-kb Nco 1-Eco Rl human aFGF cDNA fragment (top panel), specific for the coding region of the aFGF gene: 1, bovine retina tissue; 2, BSMC;3, HSMC;4, bovine aortic endothelial cells (BAE); 5, bovine capillary endothelial cells (BCE); 6, HUVE. To control for sample loading and RNA integrity, radiolabeled DNA was stripped and the filter reprobed using a chicken a-tubulin cDNA fragment (bottom panel).
wcy CJ
31 7
a-tubulin
FIGURE 3. Northern blot analysis of bFGF mRNA expression in different vascular cell types. In each lane, 1Opg total RNA was fractionated on a 1.2% agarose gel and transferred to nitrocellulose membranes. The bFGF transcripts were detected by hybridization to a 32P-labeled I kb Nco I bovine cDNA fragment (top panel). The filter was rehybridized to a 32P-labeled chicken atubulin cDNA probe (bottom panel). For identification of different lanes see Fig. 2.
Effects of aFGF and bFGF on BSMC Proliferation
was used as a probe (Abraham et al., 1986a) for hybridization to mRNA blots containing equivalent amounts of total cellular RNA. As seen in Figure 3, nearly all cell types express bFGF mRNA in similar amounts. In contrast, little expression was detected in long-term cultures of HUVE. Three different bFGF mRNA transcripts were identified having sizes of 7.0, 4.0, and 1.9 kb. These sizes are in general agreement with the 7.0- and 3.7-kb transcripts previously reported (Abraham et al., 1986a) and indicate an additional transcript of 1.9-kb. A very similar transcription pattern was recently reported for human fibroblasts and SK-HEP-1 cells (Sternfeld et al., 1988). The detection of aFGF and bFGF by heparin-affinity chromatography agrees with the level of specific gene expression shown by the Northern blot analysis.
The growth-stimulatory activity of BSMC cell extracts as well as of purified aFGF and bFGF on BSMC proliferation and DNA synthesis was investigated. In oitro SMC were found to divide in response to exogenous aFGF, bFGF, or SMC lysates when initially seeded at low cell densities (Fig. 4). Half-maximal stimulation of 13H]thymidineincorporation into DNA occurs at 0.25 ng/ml bFGF (data not shown). These results demonstrate that aFGF and bFGF are potent mitogens for SMC, as they are for many other cell types.
DISCUSSION Expression and synthesis of aFGF and bFGF in BSMC, HSMC, and. H U E were analyzed by heparin-affinity HPLC and Northern blot analysis. In each case there was a strong correlation between
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Time, duys FlGURE 4. Growth factor-stimul.ted mitogenesis in BSMC. Proliferation assay was done m the presence of 0.5% CS with ( A ) BSMC l p t c (3.2 Unitdwell), (A) purified acidic FGF (33ng/ml), (0) purified basic FGF (5ngld). and (0) no addition. Evay second day, cells were tryp&iA and counted in a Coultex counter. Values are the means d triplicate determiMtiOnS.
the expression of the mRNA for aFGF or bFGF and the detection of these mitogens by heparin-affinity chromatography. We have demonstrated that proliferating HSMC in culture express and synthesize aFGF and bFGF. The majority (-75%) of the cell-associated heparinbinding growth factor activity appeared to be bFGF and the remainder aFGFffhese results 9 similar to those of earlier studies demonstrating that HSMC from abdominal aorta or umbilical vein synthesize aFGF (Winkles et al., 1987) and that fetal HSMC synthesize bFGF (Gospodarowiu et al., 1988). However, this is the first demonstration of the expression and synthesis of both aFGF and bFGF in a single preparation of HSMC. Several nonvascular cell types (e.g., cardiac myocytes) have previously been shown to express both aFGF and bFGF (Weiner and Swain, 1989). However, the functional significance, if any, of producing both factors has yet to be established since both growth factors apparently bind to the same cell surface receptor (Neufeld and Gospodarowicz, 1986). In this context it is interesting that, in contrast to HSMC, BSMC synthesize only bFGF. Although this difference may reflect species diversity or heterogeneity of the vessel type used for SMC isolation, it may suggest
111.
that the same physiological roles are performed by aFGF or bFGF. Heparin-affinity activity profiles of bovine endothelial cell lysates have previously demonstrated that these cells contain bFGF but not aFGF (Schweigerer et al., 1987; Vlodavsky et a]., 1987b). Similarly, our results show that HUVE synthesize bFGF with no apparent production of aFGF. .Taken together, these $indings suggest that endothelial cells produce relatively high levels of bFGF but no detectable aFGF. However, we cannot exclude the possibility that under certain conditions the expression of the aFGF gene may be induced. It is of interest that all bovine vascular cells examined to date appear to produce bFGF but not aFGF (see above). In contrast, we find that the human vaxulature does contain a source of aFGF, namely the SMC,which produces comparable levels of aFGF and bFGF. As discussed above, the physiological importance of such qualitative differences remains to be established. We propose that an autocrine and a paracrine mechanism of action of aFGF and bFGF may play a crucial role in SMC and endothelial cell proliferation. Although much work remains to establish the relative importance of each growth factor for these cells in vim, it may be relevant that successful culture of endothelial cells requires a continuous supply of exogenous FGF (Vlodavsky et al., 1979;Rogelj et al., 1989), whereas SMC may be successfully cultured in its absence. After synthesis, growth factors are normally secreted and interact with specific cell surface receptors in an autocrine or paracrine fashion. However, aFGF and bFGF are mostly cell-associated (Libermann et al., 1987; Vlodavsky et al., 1987b), a relatively unusual feature for polypeptide growth factors. The mechanism by which aFGF and bFGF can be released and could interact with the surface receptor is not known: Both mitogens lack a signal peptide sequence, which is a requirement for secretion (Abraham et al., 1986a; Jaye et al., 1986). Although the FGFs do not appear, therefore, to be secreted in a classical way like many other polypeptide growth factors, it is of interest that bFGF has been found in the ECM (Vlodavsky et al., 1987a). One possible mechanism for this localization might be seen in the high affinity that aFGF and bFGF have for heparan sulfate, a component of the ECM. It is possible that aFGF and bFGF may be transported in physical association with components of the ECM and then stored as an integral part of the ECM in vascular cells. Matrix-bound FGF stored in
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FIBROBLAST GROWTH FACTORS IN SMOOTH MUSCLE CELLS
this way may be released when needed by the activation of extracellular heparinase (Vlodavsky et al., 1987a; Folkman et al., 1988). SMC in culture also produce other growth factors, such as PDGF, insulin-like growth factor-1 (IGF-I) and a smooth muscle cell-derived growth factor (SDGF), which are secreted into the medium (Sj6lund et al., 1988; Clemmons and Van Wyk, 1983; Morisaki et al., 1988). PDGF is a potent mitogen for SMC (Ross et al., 1971) and is synthesized and secreted by these cells (Seifert et al., 1984; SjCilund et al., 1988). Recently, it has been shown, that bFGF in combination with PDGF has a synergistic effect on growth stimulation in BSMC, suggesting that both growth factors together are necessary for a high mitogenic response (Weich and Folkman, 1989). It is possible that cell-associated aFGF and/or bFCF together with PDGF secreted by vascular SMC and endothelial cells have an important role in the autocrine and paracrine regulation of proliferation of these vascular cell types.
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Castellot, 1. J., Addonirio. M. L.. Rosenberg, R. and Karnovsky. M. J. (1981) Cultured endothelid cells produce a heparin-like inhibitor of smooth muxle cell growth. I. Crlf B i d . 90. 32-379. Chirgwin, J. M.,Pryzyba. A. E. MacDonald. R. J. and Ruttner. W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Biochemistry 18,3294-5299. Clemmons, D. R. and Van Wyk, J. J. (1985) Evidence for a functional role of endogenously produced somatomedin-like peptides in the regulation of DNA synthesis in cultured human fibroblasts and porcine smooth muscle cells. 1. Clin. lnvcst. 75, 1911-1918.
Colliis, T., Ginsburg, D., Boss, J. M.,Orkin, S. H. and Pober, J. 5. (1985) Cultured human endothelial cells express plateletderived growth factor chain 2 cDNA cloning and structural analysis. Nnturc316,748-750. Courty, J., Loret. C., Moenner, M., Chevallier, B., Lagente. 0.. Courtois. Y. and Barritault. 0. (1985) Bovine retina contains three growth factor activities with different affinity for heparin: eye-derived growth factor I, IX, and In. Biochimic67.263-169. DiCorleto, P. E. and Bowen-Pope, D. F. (1983) Cultured endothelial cells produce a platelet-derived growth factor-like protein. Proc. Nut[. Acnd. Sci. USA 80. 1919-1923. Exh, F.. Baird. A.. Ling, N., Ueno. N.. Hill, F., Denoroy. L.. Klepper, R,Cospodamwicz. D.,'Bdhlen, P. and Guillemin. R. (1985) Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Froc. Nutl. Acud:SEi. USA 82.6507-6511.
Folkman, J. (198fa) Tumor angiogenesis. Ado. Cuncrr Rrs. 43, 175-203.
AW0Wl.EDGMENTS We wish to thank David Brigstock and Kenneth Rosen for helphl suggestions and discussions. H.A.W. is a recipient of a fellowship from the Deutsche Forschungsgemeinxhaft, Grant We 1211-1; N.I.is a recipient of a grant from the Swiss National Science Foundation, Grant 83.497.0.87; and M.K. was supported by NCI Grant CA-37392. This work was also supported by a grant from Takeda Chemical Industries, Ltd. to Harvard University (J.F.).
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