0 1992 Harwood Academic Publishers GmbH
Growth Factors, 1992, Vol. 7, pp. 261-266 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Platelet-Derived Growth Factor is Angiogenic In Vivo WERNER RISAU', HANNES DREXLER', VLADIMIR MIRONOV', ANJA SMITS', AGNETA SIEGBAHN3, KEIKO FUNA2and CARL-HENRIK HELDIN' 'Max-Planck-Institut f u r Psychiatrie, Abteiluizg Neurochemie, Am Klopferspitz 18A, 0-8033 Martinsried, FRG; 'Ludwig Institute for Cancer Research, Box 595, ,575124, Uppsala, Sweden; 3Department of Clinical Chemistry, University Hospital, S-75185 Uppsala, Sweden
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
(Received April29 2992, Accepted May 13 1992)
PDGF receptors have recently been found to be expressed in microvascular endothelium in vim under circumstances of endothelial cell activation and angiogenesis suggesting that PDGF may have a direct effect on endothelial cells. We have tested the angiogenic activity of PDGF-AA and -BB homodimers in the chick chorioallantoic membrane in vim. PDGF-BB was found to consistently induce an angiogenic response whereas PDGFAA was less active. Morphological analyses revealed that there was little inflammation associated with this response but an increase in vessel density suggested a direct effect of PDGF on embryonic chorioallantoic endothelial cells. In vitro, PDGF-BB was found to be more potent than PDGF-AA in stimulating the chemotaxis of rat brain capillary endotheIial cells. This is consistent with a direct effect of PDGF on endothelial cells. Thus, this novel angiogenic activity of PDGF has implications for several developmental and pathological events in which PDGF, particularly the B-chain, is expressed. KEYWORDS: Platelet-derived growth factor, angiogenesis, chemotaxis, endothelial cells
INTRODUCTION Platelet-derived growth factor (PDGF) is a potent growth and chemotactic factor for connective tissue and glia-derived cells. It is a well characterized mitogen that consists of a dimer of A- and B-chains. Endothelial cells have generally been considered nonresponsive to PDGF. This is supported by the absence of PDGF binding and PDGF receptor localization to large vessel endothelium (Heldin et al., 1981; Kazlauskas and DiCorleto, 1985). However, more recently, a number of studies have detected PDGF receptors on microvascular endothelium in tissue sections of human glioma (Hermanson et al., 1988) midgut carcinoid tumor (Funa et al., 1989) and rheumatoid synovium (Reuterdahl et al., 1991) and have described biological effects of PDGF and PDGF receptor binding in cultured microvascular endothelial cells (Streeten et al., 1989; Smits et al., 1989; Bar et al., 1989; Beitz et al., 1991; Correspondence to: Werner Risau, Ph.D., Max-PlanckInstitut fur Psychiatrie, Abteilung Neurochemie, Am Klopferspitz 18A, D-8033 Martinsried, FRG. Tel: +49-89-85783625; Fax: +49-89-8578-3749.
Haraguchi et al., 1991) as well as on macrovascular endothelial cells (Cunningham et al., 1992). Since endothelial cells produce both PDGF Aand B-chair. mRNA, these studies suggested a possible autocrine or paracrine activation of microvascular endothelial cells which may be important for endothelial cell functions, e.g. during angiogenesis. We show here that PDGF is an angiogenesis factor in vivo.
METHODS Chick Chorioallantoic Membrane Angiogenesis Assay The chorioallantoic membrane (CAM) assay was performed as described previously (Crum et al., 1985; Risau, 1986). Briefly, lyophilized PDGF-AA or -BB (recombinant material from yeast expression systems; Ostman et al., 1989) were taken u p by a sterile solution of 0.5% methylcellulose, dried and implanted onto the 9-day-old chick embryo CAM using eggs in which a window had been prepared at day 3 of incubation.
26 1
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
262
RISAU et al.
Neutralizing anti-PDGF-AA or anti-PDGF-BB antibodies (Thyberg et al., 1990) or unrelated antibodies (same IgG concentration) were mixed with the growth factors prior to incorporation into methylcellulose. The assays were analyzed daily for two days. The data shown in Table 1 were compiled from 219 CAMs. The morphology of representative CAMS was investigated using semithin sections of tissue specimen embedded in Araldite stained with toluidine blue. CAMs were fixed in situ in 2.5% glutaraldehyde in cacodylate buffer, pH 7.4 for 30 min. The area of the CAM with the methylcellulose pellet was selected and fixed in the same fixative for 2 hr, then dehydrated in a graded series of ethanol, and embedded in Araldite resin. 1 micron sections were cut and stained with toluidine blue on glass slides.
Hank's balanced salt solution and suspended in F-12 medium supplemented with 10% FCS to a final concentration of 2x105cells/ml. The chemotactic response of the cells to PDGF was investigated in a modified Boyden chamber by means of the leading front technique (Nistkr et al., 1988). Micropore filters (pore size 8 pm) were coated in a collagen solution over night and air-dried immediately before use. PDGF-AA and PDGF-BB were diluted in F-12 medium with 10% FCS. The chemotaxis assay was performed as described for fibroblasts (Nistkr et al., 1988). For each experiment, the migration of endothelial cells in F-12 medium with 10% FCS and with the same medium below the filter in the Boyden chamber (chemokinesis), served as a control, and this migration was set as 100%.All experiments were performed in duplicate for each concentration of PDGF.
Chemotaxis Assay Capillary endothelial cells from rat brain were isolated from adult Sprague-Dawley rats (100-150 g; Alab, Sollentuna, Sweden) and cul- RESULTS tured for 10 days as described (Smits et al., 1989). They were then detached with trypsin, washed in The chick chorioallantoic membrane (CAM) was used to test induction of angiogenesis in vivo. Human recombinant PDGF-AA and -BB (100 and 200 ng doses) were embedded into methylcelluTABLE 1 Induction of Angiogenesis in the Chick CAM lose disks and implanted onto the 9-day-oldchick CAM as described (Crum et al., 1985; Risau, Factor Positive Reaction Negative reaction % % 1986). Both PDGF-AA and -BB induced an angiogenic response (Fig. 1) while the BB homodimer PDGF-AA (100 ng) 25 71 elicited a much stronger and consistent response. 7 86 PDGF-AA (100 ng) 1 summarizes the results of at least three Table +anti-PDGF-AA independent experiments for each group. AntiPDGF-AA (200 ng) 43 50 bodies against the respective homodimers abolished almost completely PDGF induced angioPDGF-AA (200 ng) 8 77 genesis showing that PDGF was responsible for +anti-PDGF-AA the activity. Unrelated antibodies did not inhibit PDGF-BB (100 ng) 63 31 PDGF induced angiogenesis (Table 1).Only 43% of the PDGF-AA (200ng) and only 63% of the PDGF-BB (100 ng) 19 78 +anti-PDGF-BB PDGF-BB (200 ng) treated CAMS showed a positive response. This is less than we observed with 63 31 PDGF-BB (200 ng) other angiogenic factors, e.g. bFGF. Higher doses PDGF-BB (200 ng) 46 46 of PDGF have not been used but may be neces+anti-PDGF-BB sary to achieve a higher percentage of induction. The angiogenic respdnse on the CAM was not 19 76 PDGF-BB (200 ngf +unrelated antibody associated with an obvious inflammatory reaction as judged by the absence of opaque regions Control 12 65 and hemorrhage. In addition, morphological A total of 219 CAMs were analyzed. Only clearly negative or positive reactions analysis of semithin sections showed the were used for this table Intermediate results add u p to 100%for each experiment. ~
ANGIOGENIC ACTIVITY OF PDGF
263
presence of only few leukocytes in the angiogenic area but an increase in the number of small blood vessels (Fig. 2). To address the question whether PDGF has a direct effect on capillary endothelial cells related to its angiogenic activity observed in the CAM, we tested PDGF-AA and -BB for chemotactic activity using the Boyden chamber method. Figure 3 shows that PDGF-BB at a concentration of 10-20 ng/ml stimulated the migration of rat brain capillary endothelial cells with a marked migration at 180% of the control, whereas PDGF-AA showed very little activity.
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
DISCUSSION Several growth factors have been shown to stimulate endothelial cell proliferation in vitro and induce angiogenesis iiz vivo. The fibroblast growth factors (FGFs) seem to be the most potent mitogens for endothelial cells in ziitro but also induce the proliferation of many other cell types (for review see: Folkman and Klagsbrun, 1987; Risau, 1990; Klagsbrun and D’Amore, 1991). Vascular endothelial cell growth factor (VEGF) is a more recently identified angiogenic protein whose target cell specificity appears to be restricted to vascular endothelial cells (Ferrara and Henzel, 1989; Leung et al., 1989; Keck et al., 1989). PDGF has not been considered an angiogenic factor. However, coexpression of mRNA for PDGF B-chain and PDGF beta-receptor was observed in endothelial cells of human glioblastomas suggesting an autocrine growth stimulation (Hermansson et al., 1988). Furthermore, the PDGF beta-receptor was found to be expressed in the vasculature of human rheumatoid synovium (Reuterdahl et al., 1991) and carcinoid tumors (Funa et al., 1989). These studies suggested that PDGF could play a role in patho-
FIGURE I. Angiogenesis assay on the chick chorioallantoic membrane. 200 ng human recombinant PDGF-AA (a), 200 ng PDGF-BB (b) and 200ng PDCF-BB mixed with four times molar excess of neutralizing rabbit anti PDGF-BB IgG (c) were incorporated into methylcellulose disks and implanted onto the 9-day-old chick embryo chorioallantoic membrane. The typical spoke wheel pattern of a positive angiogenic response is illustrated in these representative pictures (a and b; other results are summarized in Table 1). The neutralizing antibody inhibits the angiogenic response, but the typical vascular pattern of the chorioallantoic vessels is maintained (c). (See Color Plate at back of issue).
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
264
RISAU cr a].
FIGURE 2. Morphology of the angiogenic response. A cross section of a normal CAM is shown in (a). Most capillaries are present in close proximity to the ectoderm (EC), the connective tissue is avascular except for large vessels (arrows). (b) In response to PDGF-BB the connective tissue is thickened and numerous blood vessels including capillary profiles are observed (a few examples are indicated by arrows). Some extravasated leukocytes are present .
logical angiogenesis during tumor growth and B-chain and beta-receptor, but not A-chain inflammation. Other in vitro studies have also mRNA, with angiogenesis in glioma and rheureported PDGF effects and binding sites on cul- matoid arthritis, and during placental tured endothelial cells (Streeten et al., 1989; Smits development. We have not yet been able to demonstrate et al., 1989; Bar et al., 1989; Beitz et al., 1991; Haraguchi e t al., 1991). A role for PDGF in expression of PDGF receptor mRNA or protein in physiological angiogenesis was suggested by the the chick chorioallantoic membrane because of expression pattern of PDGF and PDGF receptor the lack of specific crossreacting antibodies and during placental development (Holmgren et al., gene probes. These reagents would be valuable 1991). In this study we provide direct evidence tools to investigate PDGF receptor expression that PDGF is an angiogenic factor in vivo. We during embryonic vascular development. Howfound that the BB homodimer was more active ever, the fact that the PDGF receptor has been than the AA homodimer in stimulating endo- observed to be expressed by endothelial cells in thelial chemotaxis in vitro and angiogenesis in v i m supports the view that PDGF can exert a vivo. This is consistent with previous studies direct angiogenic action. It remains to be detercited above that have correlated the expression of mined whether this action is mediated by the
ANGIOGENIC ACTIVITY OF PDGF OPDGFAA
2oo[
8
160
0
ls0:
”-
+PDGFBB
1
1 80
I
1
10
100
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
PDGF, ng/rnl
FIGURE 3. Chemotactic activity of PDGF for rat brain capillary endothelial cells. PDGF-BB (A) gave a strong chemotactic response, whereas PDGF-AA ( 0 )gave very little effect. Data points represent the meanfSD of two experiments.
chemotaxic activity of PDGF-BB or other additional activities. In PDGF receptor binding assays we have obtained variable results using cultured microvascular endothelial cells. Whereas rat brain endothelial cells expressed receptors for PDGF-BB but no -AA and responded to PDGF by proliferation (Smits et al., 1989) and chemotaxis (this study) bovine adrenal cortex and bovine brain capillary endothelial cells showed a variable response and low specific receptor binding. It is possible that PDGF receptor expression in microvascular endothelial cells, like in explanted smooth muscle cells (Terracio et al., 1988), is upregulated by specific physiological (including developmental, e.g. placenta) and pathological stimuli (including tissue culture) but may be absent or expressed at a low level in normal adult microvascular endothelium. However, there may also be differences in PDGF receptor expression by endothelial cells in different organs or different species. It will be interesting to investigate these possibilities in light of the novel activity of PDGF as an angiogenesis factor.
REFERENCES Bar, R. S., Boes, M., Booth, B. A., Dake, B. L., Henley, S. and Hart, M. N. (1989) The effects of platelet-derived growth factor in cultured microvessel endothelial cells. Endocrinology 124,1841-1848. Beitz, J. G., Kim, I. S., Calabresi, P. and Frackelton, A. R. (1991) Human microvascular endothelial-cells express receptors for platelet-derived growth-factor. Proc. Natl. Acad. Sci. U S A 88,2021-2025. Crum, R., Szabo, S. and Folkman, J. (1985) A new class of ster-
265
oids inhibits angiogenesis in the presence of heparin or a heparin fragment. Science 230,1375-1378. Cunningham, L. D., Brecher, P. and Cohen, R. A. (1992) Platelet-derived growth factor receptors on macrovascular endothelial cells mediate relaxation via nitric oxide in rat aorta. J. Clin. Invest. 89,878-882. Ferrara, N. and Henzel, W. J. (1989) Pituitary follicular cells secrete a novel heparin-binding growth-factor specific for endothelial-cells. Biochern. Biophys. Res. Commun. 161, 851-858. Folkman, J. and Klagsbrun, M. (1987) Angiogenic factors. Science 235,442-447. Funa, K., Papanicolaou, V., Juhlin, C., Rastad, J., Akerstrom, G., Heldin, C.-H. and Oberg, K. (1990) Expression of platelet derived growth factor Preceptors on stromal tissue cells in human carcinoid tumors. Cancer Research 50,748-753. Haraguchi, T., Alexander, D. B., King, D. S., Edwards, C. P. and Firestone, G. L. (1991) Identification of the glucocorticoid suppressible mitogen from rat hepatoma-cells as an angiogenic platelet-derived growth factor-A-chain homodimer. J . B i d . Chem. 266,18299-18307. Heldin, C.-H., Westermark, 8. and Wasteson, A. (1981) Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia. Proc. Natl. Acad. Sci. U S A 78,3664-3668. Hermansson, M., Nister, M., Betsholtz, C., Heldin, C.-H., Westermark, B. and Funa, K. (1988) Endothelial-cell hyperplasia in human glioblastoma-coexpression of messengerRNA for platelet-derived growth-factor (PDGF) 8-chain and PDGF receptor suggests autocrine growth-stimulation. Proc. Natl. Acad. Sci. USA 85, 7748-7752. Holmgren, L., Glaser, A,, Pfeifer-Ohlsson, S. and Ohlsson, R. (1991) Angiogenesis during human extraembryonic development involves the spatiotemporal control of PDGF ligand and receptor gene-expression. Development 113, 749-749. Kazlauskas, A. and DiCorleto, P. E. (1985) Cultured endothelial cells do not respond to a platelet-derived growthfactor-like protein in an autocrine manner. Biochim. Biophys. A C ~846,405-412. U Keck, P. J., Hauser, S. D., Krivi, G., Sanzo, K., Warren, T., Feder, J. and Connolly, D. T. (1989) Vascular-permeability factor, an endothelial-cell mitogen related to PDGF. Science 246,1309-1312. Klagsbrun, M. and DAmore, P. A. (1991) Regulators of angiogenesis. Annual Rev. Physiol. 53,217-239. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V. and Ferrara, N. (1989) Vascular endothelial growth-factor is a secreted angiogenic mitogen. Science 246,1306-1309. Nistkr, M., Hammacher, A,, Mellstrom, K., Siegbahn, A., Ronnstrand, L., Westermark, B. and Heldin, C.-H. (1989) A glioma-derived PDGF A-chain homodimer has different functional activities from a PDGF-AB heterodimer purified from human platelets. Cell 52,791-799. Ostman, A,, Backstrom, G., Fong, N., Betsholtz, C., Wernstedt, C., Hellman, U., Westermark, B., Valenzuela, P. and Heldin, C.-H. (1989) Expression of three recombinant homodimeric isoforms of PDGF in Saccaromyces cerevisiae: Evidence for differences in receptor binding and functional activities. Growth Factors 1,271-281. Reuterdahl, C., Tingstrom, A,, Terracio, L., Funa, K., Heldin, C.-H. and Rubin, K. (1991) Characterization of plateletderived growth-factor beta-receptor expressing cells in the vasculature of human rheumatoid synovium. Lab. Invest. 64,321-329. Risau, W. (1986) Developing brain produces an angiogenesis factor. Proc. Natl. Acad. Sci. U S A 83,3855-3859. Risau, W. (1990) Angiogenic growth factors. Progr. Growth Factor Res. 2,71-79.
266
RISAU ei al.
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
Smits, A,, Hermansson, M., Nister, M., Karnushina, I., Heldin, C.-H., Westermark, B. and Funa, K. (1989) Rat brain capillary endothelial cells express functional PDGF B-type receptors. Growth Factors 2,143. Streeten, E. A,, Ornberg, R.,Curcio, F., Sakaguchi, K., Marx, S., Aurbach, G. D. and Brandi, M. L. (1989) Cloned endothelial-cells from fetal bovine bone. Proc. Nutl. Acad. Sci. USA 86,916-920. Ternacio, L., Ronnstrand, L., Tingstrom, A., Rubin, K.,
Claesson-Welsh, L., Funa, K. and Heldin, C.-H. (1988) Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing. I. Cell B i d . 107,1947-1957. Thyrberg, J., Ostman, A,, Backstrom, G., Westermark, B. and Heldin, C.-H. (1990) Localization of platelet-derived growth factor (PDGF) in CHO cells transfected with PDGF A- or B-chain cDNA: retention of PDGF-BB in the endoplasmic reticulum and Golgi complex. 1. Cell Sci. 97,219-229.
Growth Factors, 1992, Vol. 7, pp. 261-266 Reprints available directly from the publisher Photocopying permitted by license only
Printed in the United Kingdom
Platelet-Derived Growth Factor is Angiogenic In Vivo WERNER RISAU', HANNES DREXLER', VLADIMIR MIRONOV', ANJA SMITS', AGNETA SIEGBAHN3, KEIKO FUNA2and CARL-HENRIK HELDIN' 'Max-Planck-Institut f u r Psychiatrie, Abteiluizg Neurochemie, Am Klopferspitz 18A, 0-8033 Martinsried, FRG; 'Ludwig Institute for Cancer Research, Box 595, ,575124, Uppsala, Sweden; 3Department of Clinical Chemistry, University Hospital, S-75185 Uppsala, Sweden
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
(Received April29 2992, Accepted May 13 1992)
PDGF receptors have recently been found to be expressed in microvascular endothelium in vim under circumstances of endothelial cell activation and angiogenesis suggesting that PDGF may have a direct effect on endothelial cells. We have tested the angiogenic activity of PDGF-AA and -BB homodimers in the chick chorioallantoic membrane in vim. PDGF-BB was found to consistently induce an angiogenic response whereas PDGFAA was less active. Morphological analyses revealed that there was little inflammation associated with this response but an increase in vessel density suggested a direct effect of PDGF on embryonic chorioallantoic endothelial cells. In vitro, PDGF-BB was found to be more potent than PDGF-AA in stimulating the chemotaxis of rat brain capillary endotheIial cells. This is consistent with a direct effect of PDGF on endothelial cells. Thus, this novel angiogenic activity of PDGF has implications for several developmental and pathological events in which PDGF, particularly the B-chain, is expressed. KEYWORDS: Platelet-derived growth factor, angiogenesis, chemotaxis, endothelial cells
INTRODUCTION Platelet-derived growth factor (PDGF) is a potent growth and chemotactic factor for connective tissue and glia-derived cells. It is a well characterized mitogen that consists of a dimer of A- and B-chains. Endothelial cells have generally been considered nonresponsive to PDGF. This is supported by the absence of PDGF binding and PDGF receptor localization to large vessel endothelium (Heldin et al., 1981; Kazlauskas and DiCorleto, 1985). However, more recently, a number of studies have detected PDGF receptors on microvascular endothelium in tissue sections of human glioma (Hermanson et al., 1988) midgut carcinoid tumor (Funa et al., 1989) and rheumatoid synovium (Reuterdahl et al., 1991) and have described biological effects of PDGF and PDGF receptor binding in cultured microvascular endothelial cells (Streeten et al., 1989; Smits et al., 1989; Bar et al., 1989; Beitz et al., 1991; Correspondence to: Werner Risau, Ph.D., Max-PlanckInstitut fur Psychiatrie, Abteilung Neurochemie, Am Klopferspitz 18A, D-8033 Martinsried, FRG. Tel: +49-89-85783625; Fax: +49-89-8578-3749.
Haraguchi et al., 1991) as well as on macrovascular endothelial cells (Cunningham et al., 1992). Since endothelial cells produce both PDGF Aand B-chair. mRNA, these studies suggested a possible autocrine or paracrine activation of microvascular endothelial cells which may be important for endothelial cell functions, e.g. during angiogenesis. We show here that PDGF is an angiogenesis factor in vivo.
METHODS Chick Chorioallantoic Membrane Angiogenesis Assay The chorioallantoic membrane (CAM) assay was performed as described previously (Crum et al., 1985; Risau, 1986). Briefly, lyophilized PDGF-AA or -BB (recombinant material from yeast expression systems; Ostman et al., 1989) were taken u p by a sterile solution of 0.5% methylcellulose, dried and implanted onto the 9-day-old chick embryo CAM using eggs in which a window had been prepared at day 3 of incubation.
26 1
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
262
RISAU et al.
Neutralizing anti-PDGF-AA or anti-PDGF-BB antibodies (Thyberg et al., 1990) or unrelated antibodies (same IgG concentration) were mixed with the growth factors prior to incorporation into methylcellulose. The assays were analyzed daily for two days. The data shown in Table 1 were compiled from 219 CAMs. The morphology of representative CAMS was investigated using semithin sections of tissue specimen embedded in Araldite stained with toluidine blue. CAMs were fixed in situ in 2.5% glutaraldehyde in cacodylate buffer, pH 7.4 for 30 min. The area of the CAM with the methylcellulose pellet was selected and fixed in the same fixative for 2 hr, then dehydrated in a graded series of ethanol, and embedded in Araldite resin. 1 micron sections were cut and stained with toluidine blue on glass slides.
Hank's balanced salt solution and suspended in F-12 medium supplemented with 10% FCS to a final concentration of 2x105cells/ml. The chemotactic response of the cells to PDGF was investigated in a modified Boyden chamber by means of the leading front technique (Nistkr et al., 1988). Micropore filters (pore size 8 pm) were coated in a collagen solution over night and air-dried immediately before use. PDGF-AA and PDGF-BB were diluted in F-12 medium with 10% FCS. The chemotaxis assay was performed as described for fibroblasts (Nistkr et al., 1988). For each experiment, the migration of endothelial cells in F-12 medium with 10% FCS and with the same medium below the filter in the Boyden chamber (chemokinesis), served as a control, and this migration was set as 100%.All experiments were performed in duplicate for each concentration of PDGF.
Chemotaxis Assay Capillary endothelial cells from rat brain were isolated from adult Sprague-Dawley rats (100-150 g; Alab, Sollentuna, Sweden) and cul- RESULTS tured for 10 days as described (Smits et al., 1989). They were then detached with trypsin, washed in The chick chorioallantoic membrane (CAM) was used to test induction of angiogenesis in vivo. Human recombinant PDGF-AA and -BB (100 and 200 ng doses) were embedded into methylcelluTABLE 1 Induction of Angiogenesis in the Chick CAM lose disks and implanted onto the 9-day-oldchick CAM as described (Crum et al., 1985; Risau, Factor Positive Reaction Negative reaction % % 1986). Both PDGF-AA and -BB induced an angiogenic response (Fig. 1) while the BB homodimer PDGF-AA (100 ng) 25 71 elicited a much stronger and consistent response. 7 86 PDGF-AA (100 ng) 1 summarizes the results of at least three Table +anti-PDGF-AA independent experiments for each group. AntiPDGF-AA (200 ng) 43 50 bodies against the respective homodimers abolished almost completely PDGF induced angioPDGF-AA (200 ng) 8 77 genesis showing that PDGF was responsible for +anti-PDGF-AA the activity. Unrelated antibodies did not inhibit PDGF-BB (100 ng) 63 31 PDGF induced angiogenesis (Table 1).Only 43% of the PDGF-AA (200ng) and only 63% of the PDGF-BB (100 ng) 19 78 +anti-PDGF-BB PDGF-BB (200 ng) treated CAMS showed a positive response. This is less than we observed with 63 31 PDGF-BB (200 ng) other angiogenic factors, e.g. bFGF. Higher doses PDGF-BB (200 ng) 46 46 of PDGF have not been used but may be neces+anti-PDGF-BB sary to achieve a higher percentage of induction. The angiogenic respdnse on the CAM was not 19 76 PDGF-BB (200 ngf +unrelated antibody associated with an obvious inflammatory reaction as judged by the absence of opaque regions Control 12 65 and hemorrhage. In addition, morphological A total of 219 CAMs were analyzed. Only clearly negative or positive reactions analysis of semithin sections showed the were used for this table Intermediate results add u p to 100%for each experiment. ~
ANGIOGENIC ACTIVITY OF PDGF
263
presence of only few leukocytes in the angiogenic area but an increase in the number of small blood vessels (Fig. 2). To address the question whether PDGF has a direct effect on capillary endothelial cells related to its angiogenic activity observed in the CAM, we tested PDGF-AA and -BB for chemotactic activity using the Boyden chamber method. Figure 3 shows that PDGF-BB at a concentration of 10-20 ng/ml stimulated the migration of rat brain capillary endothelial cells with a marked migration at 180% of the control, whereas PDGF-AA showed very little activity.
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
DISCUSSION Several growth factors have been shown to stimulate endothelial cell proliferation in vitro and induce angiogenesis iiz vivo. The fibroblast growth factors (FGFs) seem to be the most potent mitogens for endothelial cells in ziitro but also induce the proliferation of many other cell types (for review see: Folkman and Klagsbrun, 1987; Risau, 1990; Klagsbrun and D’Amore, 1991). Vascular endothelial cell growth factor (VEGF) is a more recently identified angiogenic protein whose target cell specificity appears to be restricted to vascular endothelial cells (Ferrara and Henzel, 1989; Leung et al., 1989; Keck et al., 1989). PDGF has not been considered an angiogenic factor. However, coexpression of mRNA for PDGF B-chain and PDGF beta-receptor was observed in endothelial cells of human glioblastomas suggesting an autocrine growth stimulation (Hermansson et al., 1988). Furthermore, the PDGF beta-receptor was found to be expressed in the vasculature of human rheumatoid synovium (Reuterdahl et al., 1991) and carcinoid tumors (Funa et al., 1989). These studies suggested that PDGF could play a role in patho-
FIGURE I. Angiogenesis assay on the chick chorioallantoic membrane. 200 ng human recombinant PDGF-AA (a), 200 ng PDGF-BB (b) and 200ng PDCF-BB mixed with four times molar excess of neutralizing rabbit anti PDGF-BB IgG (c) were incorporated into methylcellulose disks and implanted onto the 9-day-old chick embryo chorioallantoic membrane. The typical spoke wheel pattern of a positive angiogenic response is illustrated in these representative pictures (a and b; other results are summarized in Table 1). The neutralizing antibody inhibits the angiogenic response, but the typical vascular pattern of the chorioallantoic vessels is maintained (c). (See Color Plate at back of issue).
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
264
RISAU cr a].
FIGURE 2. Morphology of the angiogenic response. A cross section of a normal CAM is shown in (a). Most capillaries are present in close proximity to the ectoderm (EC), the connective tissue is avascular except for large vessels (arrows). (b) In response to PDGF-BB the connective tissue is thickened and numerous blood vessels including capillary profiles are observed (a few examples are indicated by arrows). Some extravasated leukocytes are present .
logical angiogenesis during tumor growth and B-chain and beta-receptor, but not A-chain inflammation. Other in vitro studies have also mRNA, with angiogenesis in glioma and rheureported PDGF effects and binding sites on cul- matoid arthritis, and during placental tured endothelial cells (Streeten et al., 1989; Smits development. We have not yet been able to demonstrate et al., 1989; Bar et al., 1989; Beitz et al., 1991; Haraguchi e t al., 1991). A role for PDGF in expression of PDGF receptor mRNA or protein in physiological angiogenesis was suggested by the the chick chorioallantoic membrane because of expression pattern of PDGF and PDGF receptor the lack of specific crossreacting antibodies and during placental development (Holmgren et al., gene probes. These reagents would be valuable 1991). In this study we provide direct evidence tools to investigate PDGF receptor expression that PDGF is an angiogenic factor in vivo. We during embryonic vascular development. Howfound that the BB homodimer was more active ever, the fact that the PDGF receptor has been than the AA homodimer in stimulating endo- observed to be expressed by endothelial cells in thelial chemotaxis in vitro and angiogenesis in v i m supports the view that PDGF can exert a vivo. This is consistent with previous studies direct angiogenic action. It remains to be detercited above that have correlated the expression of mined whether this action is mediated by the
ANGIOGENIC ACTIVITY OF PDGF OPDGFAA
2oo[
8
160
0
ls0:
”-
+PDGFBB
1
1 80
I
1
10
100
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
PDGF, ng/rnl
FIGURE 3. Chemotactic activity of PDGF for rat brain capillary endothelial cells. PDGF-BB (A) gave a strong chemotactic response, whereas PDGF-AA ( 0 )gave very little effect. Data points represent the meanfSD of two experiments.
chemotaxic activity of PDGF-BB or other additional activities. In PDGF receptor binding assays we have obtained variable results using cultured microvascular endothelial cells. Whereas rat brain endothelial cells expressed receptors for PDGF-BB but no -AA and responded to PDGF by proliferation (Smits et al., 1989) and chemotaxis (this study) bovine adrenal cortex and bovine brain capillary endothelial cells showed a variable response and low specific receptor binding. It is possible that PDGF receptor expression in microvascular endothelial cells, like in explanted smooth muscle cells (Terracio et al., 1988), is upregulated by specific physiological (including developmental, e.g. placenta) and pathological stimuli (including tissue culture) but may be absent or expressed at a low level in normal adult microvascular endothelium. However, there may also be differences in PDGF receptor expression by endothelial cells in different organs or different species. It will be interesting to investigate these possibilities in light of the novel activity of PDGF as an angiogenesis factor.
REFERENCES Bar, R. S., Boes, M., Booth, B. A., Dake, B. L., Henley, S. and Hart, M. N. (1989) The effects of platelet-derived growth factor in cultured microvessel endothelial cells. Endocrinology 124,1841-1848. Beitz, J. G., Kim, I. S., Calabresi, P. and Frackelton, A. R. (1991) Human microvascular endothelial-cells express receptors for platelet-derived growth-factor. Proc. Natl. Acad. Sci. U S A 88,2021-2025. Crum, R., Szabo, S. and Folkman, J. (1985) A new class of ster-
265
oids inhibits angiogenesis in the presence of heparin or a heparin fragment. Science 230,1375-1378. Cunningham, L. D., Brecher, P. and Cohen, R. A. (1992) Platelet-derived growth factor receptors on macrovascular endothelial cells mediate relaxation via nitric oxide in rat aorta. J. Clin. Invest. 89,878-882. Ferrara, N. and Henzel, W. J. (1989) Pituitary follicular cells secrete a novel heparin-binding growth-factor specific for endothelial-cells. Biochern. Biophys. Res. Commun. 161, 851-858. Folkman, J. and Klagsbrun, M. (1987) Angiogenic factors. Science 235,442-447. Funa, K., Papanicolaou, V., Juhlin, C., Rastad, J., Akerstrom, G., Heldin, C.-H. and Oberg, K. (1990) Expression of platelet derived growth factor Preceptors on stromal tissue cells in human carcinoid tumors. Cancer Research 50,748-753. Haraguchi, T., Alexander, D. B., King, D. S., Edwards, C. P. and Firestone, G. L. (1991) Identification of the glucocorticoid suppressible mitogen from rat hepatoma-cells as an angiogenic platelet-derived growth factor-A-chain homodimer. J . B i d . Chem. 266,18299-18307. Heldin, C.-H., Westermark, 8. and Wasteson, A. (1981) Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia. Proc. Natl. Acad. Sci. U S A 78,3664-3668. Hermansson, M., Nister, M., Betsholtz, C., Heldin, C.-H., Westermark, B. and Funa, K. (1988) Endothelial-cell hyperplasia in human glioblastoma-coexpression of messengerRNA for platelet-derived growth-factor (PDGF) 8-chain and PDGF receptor suggests autocrine growth-stimulation. Proc. Natl. Acad. Sci. USA 85, 7748-7752. Holmgren, L., Glaser, A,, Pfeifer-Ohlsson, S. and Ohlsson, R. (1991) Angiogenesis during human extraembryonic development involves the spatiotemporal control of PDGF ligand and receptor gene-expression. Development 113, 749-749. Kazlauskas, A. and DiCorleto, P. E. (1985) Cultured endothelial cells do not respond to a platelet-derived growthfactor-like protein in an autocrine manner. Biochim. Biophys. A C ~846,405-412. U Keck, P. J., Hauser, S. D., Krivi, G., Sanzo, K., Warren, T., Feder, J. and Connolly, D. T. (1989) Vascular-permeability factor, an endothelial-cell mitogen related to PDGF. Science 246,1309-1312. Klagsbrun, M. and DAmore, P. A. (1991) Regulators of angiogenesis. Annual Rev. Physiol. 53,217-239. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V. and Ferrara, N. (1989) Vascular endothelial growth-factor is a secreted angiogenic mitogen. Science 246,1306-1309. Nistkr, M., Hammacher, A,, Mellstrom, K., Siegbahn, A., Ronnstrand, L., Westermark, B. and Heldin, C.-H. (1989) A glioma-derived PDGF A-chain homodimer has different functional activities from a PDGF-AB heterodimer purified from human platelets. Cell 52,791-799. Ostman, A,, Backstrom, G., Fong, N., Betsholtz, C., Wernstedt, C., Hellman, U., Westermark, B., Valenzuela, P. and Heldin, C.-H. (1989) Expression of three recombinant homodimeric isoforms of PDGF in Saccaromyces cerevisiae: Evidence for differences in receptor binding and functional activities. Growth Factors 1,271-281. Reuterdahl, C., Tingstrom, A,, Terracio, L., Funa, K., Heldin, C.-H. and Rubin, K. (1991) Characterization of plateletderived growth-factor beta-receptor expressing cells in the vasculature of human rheumatoid synovium. Lab. Invest. 64,321-329. Risau, W. (1986) Developing brain produces an angiogenesis factor. Proc. Natl. Acad. Sci. U S A 83,3855-3859. Risau, W. (1990) Angiogenic growth factors. Progr. Growth Factor Res. 2,71-79.
266
RISAU ei al.
Growth Factors Downloaded from informahealthcare.com by University of Otago on 01/13/15 For personal use only.
Smits, A,, Hermansson, M., Nister, M., Karnushina, I., Heldin, C.-H., Westermark, B. and Funa, K. (1989) Rat brain capillary endothelial cells express functional PDGF B-type receptors. Growth Factors 2,143. Streeten, E. A,, Ornberg, R.,Curcio, F., Sakaguchi, K., Marx, S., Aurbach, G. D. and Brandi, M. L. (1989) Cloned endothelial-cells from fetal bovine bone. Proc. Nutl. Acad. Sci. USA 86,916-920. Ternacio, L., Ronnstrand, L., Tingstrom, A., Rubin, K.,
Claesson-Welsh, L., Funa, K. and Heldin, C.-H. (1988) Induction of platelet-derived growth factor receptor expression in smooth muscle cells and fibroblasts upon tissue culturing. I. Cell B i d . 107,1947-1957. Thyrberg, J., Ostman, A,, Backstrom, G., Westermark, B. and Heldin, C.-H. (1990) Localization of platelet-derived growth factor (PDGF) in CHO cells transfected with PDGF A- or B-chain cDNA: retention of PDGF-BB in the endoplasmic reticulum and Golgi complex. 1. Cell Sci. 97,219-229.
