A platelet factor stimulating the proliferation of vascular endothelial cells

Experimental Cell Research 159 (1985) 487-494

A Platelet Factor Stimulating the Proliferation of Vascular Endothelial Cells Partial Purification and Characterization

KOHEI MIYAZONO, TETSURO OKABE, SHUN ISHIBASHI, AKIO URABE and FUMIMARO TAKAKU The Third Department of lnternal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

Piatelets have been shown to contain a novel growth factor that stimulates the proliferation of vascular endothelial cells in vitro. The factor potently stimulated both DNA synthesis and proliferation rate in serum-deprived endothelial cells. Gel exclusion chromatography showed at least two peaks of activity on endothelial cells, the major peak being at an apparent molecular weight of 20000. Isoelectric focusing revealed that the pI of the factor was 4.0--4.8. It was adsorbed to a column of DEAE ion exchange chromatography and eluted with a salt gradient. The factor was heat-labile and trypsin-sensitive. The activity was not destroyed by a reducing agent including dithiothreitol. This factor stimulated the proliferation of vascular endothelial cells but was found to be inactive against normal rat kidney fibroblasts. © 1985AcademicPress, Inc.

Vascular endothelial cells make a functional monolayer between blood and underlying tissue. In a normal vessel wall, endothelial cells are known to exist in a quiescent growth state [1]. Proliferation of endothelial cells is a key component to a number of biological processes, such as wound repair, thrombosis, atherogenesis and tumor growth. Thus, factors that regulate endothelial cell proliferation may play a central role in these conditions. Platelets have been shown to play an important role in the function and the metabolism of vascular endothelial cells [2, 3]. Maca et al. disclosed that intact platelets enhance DNA synthesis and replication of cultured endothelial cells, and that this growth-promoting effect does not occur in leukocytes or erythrocytes but is limited to platelets [4]. Platelets are well known to contain some peptide growth factors, such as platelet-derived growth factor (PDGF) [5-7] and epidermal growth factor (EGF) [8]. However, these factors have been shown to be inactive on endothelial cells [9-11]. We have previously reported that human platelets contain a polypeptide factor mitogenic for vascular endothelial cells [I 1]. The present study demonstrates the partial purification and characterization of a platelet factor that preferentially stimulates the growth of vascular endothelial cells. Exp CellRes 159 (1985)

488

Miyazono et al. MATERIALS

AND

METHODS

Cell Cultures and Media Endothelial cells were collected from a fresh porcine aorta using collagenase digestion as described by Booyse et al. [12]. Cloning of the endothelial cells was performed by single cell platings as described originally by Puck et al. [13]. The endothelial cells were maintained in 25 cm 2 culture flasks in Ham's F-10 medium containing 10% fetal bovine serum (FBS; Flow Laboratories Inc. Va.) and antibiotics, and subcultured using 0.25 % trypsin solution (Gibco) when the cells reached confluency. There was no evidence of transformation or loss of the endothelial monolayer under these conditions for more than 6 months. Normal rat kidney (NRK) fibroblasts were obtained by the method described by Duc-Nguyen et al. [14] and cultured in Dulbecco's modified Eagle medium containing 10% FBS.

Cell Number Determination For growth experiment, endothelial cells were subcultured at a plating density of 20 000 cells/dish in a 35-mm tissue culture dish (Coming) using Ham's F-10 medium containing 10 % FBS. The cells were incubated for 24 h to allow for attachment, and then the medium was changed to the test media. The cells were removed from the dishes by incubating for 15 min in trypsin-EDTA and cell number was determined in duplicate. The cultures were refed with fresh test media on day 4.

Assay for DNA Synthesis Growth-promoting activity was estimated by measuring the incorporation of [3H]thymidine into DNA in serum-deprived cells [15]. Cells were seeded at 10000 per well with 500 ~tl of medium supplemented with 10% FBS in 24-multiwell plates (2 cm 2, Falcon). Cells were incubated for 24 h to allow for attachment, and replaced by media containing 0.5 % FBS. 24 h later test samples were added to the wells, and after 16 h of incubation, wells were pulsed for 8 h with 0.4 ~tCi of [3H]thymidine (6.7 Ci/mM, New England Nuclear, Boston, Mass.) per ml of medium. The cells were then washed and extracted with cold 5 % trichloroacetic acid (TCA). The resulting precipitates were washed twice with ethanol-ether (ratio of vol. 3 : 1) and solubilized with 1.0 N NaOH. Radioactivity was determined with a liquid scintillation counter.

Preparation of Platelet Lysate Blood was collected from normal volunteers in citrate-phosphate-dextrose solution, and fresh platelet-rich plasma was obtained by centrifugation at 160 g for 20 min at 20°C. The upper four-fifths portion of platelet-rich plasma was centrifuged at 1700 g for 20 min at 20°C, and the platelet pellet was suspended in 10 mM Tris HCi (pH 7.4)/150 mM NaCI/0.01% polyethylene glycol (PEG). The platelets were washed twice with the same buffer in the centrifuge and sonicated for 1 rain. By this method, erythrocyte and leukocyte contamination in the last platelet suspension was less than 0.1%. Sonicated platelets were centrifuged at 48 000 g for 30 min at 4°C and the supernatants were used as platelet lysate. About 2 ml of platelet lysate was obtained from 200 ml of whole blood. Further operations were performed at 4°C, unless otherwise specified.

Gel Exclusion Chromatography One milliliter of platelet lysate was applied to a Sephadex G-75 (Pharmacia, Uppsala, Sweden) column (2.0 x 84 cm) equilibrated with 10 mM Tris-HC1 (pH 7.4)/150 mM NaCI/0.01% PEG and eluted at a flow rate of 10.4 ml/h at 4°C. The column was calibrated using bovine serum albumin (Mr 67 000), ovalbumin (Mr 43000) and ribonuclease A (Mr 13700). The effluents were assayed for growthpromoting activity on endothelial cells and NRK fibroblasts.

lsoelectric Focusing Isoelectric focusing was performed in a fiat bed electrophoresis unit using a granular polyacrylamide gel (LKB 2117 Multiphor, LKB, Sweden). The platelet lysate was dialysed overnight at 4°C in a dialysis tubing (size 18/32, Mr cut-off 10 000; Sanko Junyaku, Japan) against distilled water containing 1% glycine and applied to a bed of Ultrodex (LKB) containing 5 % Ampholine (pH 3.5-10; LKB). The

Exp CellRes 159(1985)

Endothelial cell growth factor from platelets

489

electrofocusing was run for 14 h at 4°C at a constant power of 8 W. The gel bed was divided into 15 fractions, and the protein was eluted from the gel with distilled water. Each fraction was analysed for absorbance at 280 nm, pH and mitogenic activity on endothelial cells and NRK fibroblasts, respectively.

Ion Exchange Chromatography Platelet lysate (2.0 ml, 5.0 mg of protein/ml) dialysed against 10 mM Tris HC1 (pH7.4)/25 mM NaC1 was applied to a 2.4-ml column of DEAE-Sepharose CL-6B (Pharmacia, Uppsala, Sweden) equilibrated with the same buffer. After washing with 20 ml of starting buffer, the column was eluted with a linear gradient (80 ml) of NaCI from 25 to 400 mM in 10 mM Tris-HCl at pH 7.4.2.2-ml fractions were collected and were analysed for protein, conductivity and mitogenic activity on endothelial cells.

Characterization Studies The major part of the growth-promoting activity on endothelial ceils (apparent Mr of 20000) obtained by gel exclusion chromatography was mixed prior to testing and subjected to various physical, enzymatic or chemical treatment. Any precipitate formed by these procedures was removed by centrffugation (40 000 g, 10 min). Heat treatment was carded out at temperatures at 56°C for 30 min or 100°C for 10 min. Trypsin sensitivity was tested by incubation of an aliquot of the active fractions with 50 Ixg of trypsin (sp. act. 11700 U/mg; Sigma, St. Louis, Mo.) at 37°C for 2 h, followed by inactivation using 100 ~tg of soybean trypsin inhibitor (Sigma). As a control, 50 ~tg of trypsin was inactivated with 100 Ixg of soybean trypsin inhibitor for 30 min and then incubated at 37°C with an aliquot for 2 h. The active fractions were treated with 4 M urea, 4 M guanidine-HC1, or 1 M acetic acid at 25°C for 60 min. The samples were then dialysed extensively against several changes of 10 mM Tris HC1 (pH 7.4)/150 mM NaCI/0.01% PEG at 4°C and tested for activity. An aliquot was treated with dithiothreitol (DTT). The sample was dialysed against 0.5 M ammonium bicarbonate and was exposed to 5 mM DTT at 25°C for 2 h in the dark. The mixture was then alkylated with 10 mM iodoacetamide and dialysed against 10 mM Tris HC1 (pH 7.4)/150 mM NaCI/0.01% PEG. The dialys~d material was then tested for activity on endothelial cells.

Protein Determination Protein concentration was estimated as described by Lowry et al. [16] with bovine serum albumin as a standard, except for the fractions from the isoelectric focusing and the gel exclusion chromatography. The protein of these fractions was determined at absorbance at 280 nm.

RESULTS

Effects o f Platelet Lysate on the Growth of Endothelial Cells Platelet l y s a t e was e x a m i n e d for the g r o w t h - p r o m o t i n g a c t i v i t y o n e n d o t h e l i a l cells. E n d o t h e l i a l cells were c u l t u r e d with H a m ' s F-10 m e d i u m c o n t a i n i n g 1 % F B S in the p r e s e n c e o r in the a b s e n c e o f p l a t e l e t l y s a t e (100 ~tg o f p r o t e i n / m l ) . T h e cells w e r e c u l t u r e d for u p to 5 days a n d the cell n u m b e r was d e t e r m i n e d in d u p l i c a t e . A s s h o w n in fig. 1, 1 % F B S a l o n e c o u l d s t i m u l a t e cell growth, b u t platelet l y s a t e was a b l e to i n c r e a s e the cell n u m b e r b y 100% a b o v e the c o n t r o l c u l t u r e s w i t h i n 2 days. P l a t e l e t l y s a t e was e x a m i n e d for the a c t i v i t y to s t i m u l a t e i n c o r p o r a t i o n o f [ 3 H ] t h y m i d i n e i n t o D N A o f e n d o t h e l i a l cells. C r u d e p l a t e l e t l y s a t e s t i m u l a t e d D N A synthesis in a d o s e - d e p e n d e n t m a n n e r , maximal stimulation being obtained at m o r e t h a n 100 ~tg o f p r o t e i n p e r ml o f p l a t e l e t l y s a t e (data n o t shown).

Exp CellRes 159(1985)

490 z.o

Miyazono et al. 1

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0,15

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0,4

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Days in Culture

0 "/5

90

105

120 135 150 165 Effluent volume (mr)

180

195

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Fig. 1. Stimulation of aortic endothelial cells by platelet lysate. Endothelial cells were plated at 2x 104 cells/dish in medium containing 10% FBS. Then the medium was changed to the test media at day 1. Medium containing 1% FBS (3--.--(3, and 100 t~g/ml of platelet lysate O---Q, only. Cell number was determined in duplicate. The cultures were refed with fresh test media on day 4. Fig. 2. Sephadex G-75 gel exclusion chromatography of platelet lysate. A column of Sephadex G-75 (2.0x84 cm) was equilibrated with 10 mM Tris HC1 (pH 7.4)/150 mM NaCI/0.01% PEG and eluted at a flow rate of 10.4 ml/h. Arrows indicate elution position of Mr markers. DNA synthesis of 0 - - 0 , endothelial cells; C)---O, NRK fibroblasts. , A2so.

Gel Exclusion Chromatography Platelet lysate was fractionated on a Sephadex G-75 column. As shown in fig. 2, at least two peaks of growth-promoting activity on endothelial cells were observed with apparent molecular weights (Mr) of 60000 and 20000. Growthpromoting activity of the fractionated platelet lysate on NRK fibroblasts was also examined. Activity was seen at an Mr of 30000, while the major peak on endothelial cells with an Mr of 20 000 did not significantly enhance the incorporation of [3H]thymidine into NRK fibroblasts. The major fractions active on endothelial cells (vascular endothelial cell proliferation factor; VEPF) strongly stimulated the incorporation of [3H]thymidine into DNA in a dose-dependent manner, maximal stimulation being observed at more than 6 I~g/ml of the factor (data not shown).

lsoelectric Focusing Fig. 3 shows the isoelectric focusing pattern of the platelet lysate. Growthpromoting activity on endothelial cells appeared in a narrow peak between pH 4.0 and 4.8. Multiple peaks of activity on fibroblast growth were observed by isoelectric focusing, the major peak being at pH >8.8 which may correspond to PDGF. Active fractions on endothelial cells did not significantly stimulate the growth of fibroblasts.

DEAE Ion Exchange Chromatography Fig. 4 shows the elution pattern of the growth-promoting activity when platelet lysate was chromatographed on DEAE ion exchange chromatography. Virtually Exp Cell Res 159 (1985)

Endothelial cell growthfactor from platelets 491 9.0

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Fraction l~umber

5

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15 20 25 30 Fraction Number

35

40

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Fig. 3. Isoelectric focusing of platelet lysate. Isoelectric focusing was performed in a flat bed

electrophoresis unit using a granular polyacrylamide gel. The electrofocusing was continued for 14 h at 4°C at a constant power of 8 W. The gel bed was divided into 15 fractions. DNA synthesis of O - - O , endothelial cells; O---©, NRK fibroblasts. A - - A , A2s0; & - - - A , pH. Fig. 4. DEAE-Sepharose CL-6B chromatography of platelet lysate. A 2.4-ml column of DEAE-Sepharose CL-6B was equilibrated with l0 mM Tris HC1 (pH 7.4)/25 mM NaC1. After washing with 20 ml of starting buffer, the column was eluted with a linear gradient of NaC1 from 25 to 400 mM at pH 7.4. 2.2 ml-fractions were collected and analysed. O---O, DNA synthesis of endothelial cells; ( 3 - - 0 , protein; ---, NaC1 concentration.

all activity on endothelial cells was adsorbed to a column of DEAE-Sepharose and was eluted between 100 and 150 mM NaCI.

Characterization of the Endothelial Cell Growth-PromotingActivity The nature of the stimulating activity on endothelial cells obtained by gel exclusion chromatography was investigated by testing its stability to various treatments (table 1). The activity was destroyed by heat (56°C for 30 min or 100°C for 10 min), incubation with trypsin, 4 M guanidine-HCl, or 1 M acetic acid. About half of the activity was lost by incubation with 4 M urea for 1 h at 25°C. On the other hand, incubation with 5 mM DTT did not destroy the activity. DISCUSSION In the present report, we have shown that freshly isolated platelets have an anionic polypeptide mitogen (VEPF) that preferentially stimulates the growth of vascular endothelial cells. Platelets have been shown to contain several growth factors (table 2). (1) PDGF; a cationic peptide of about 31000 Mr which is heat-stable but is susceptible to reducing agents [5-7]. (2) EGF; an anionic peptide with Mr of 33000 and 6 000 that is resistant to heat but susceptible to reducing agents [8]. (3) Transforming growth factor-fl (TGF-fl); a cationic polypeptide of 25 000 Mr which is a heatstable factor but the activity is destroyed by reducing agents [17]. (4) Hepatocyte growth factor; a cationic peptide with an Mr of 65 000 that is susceptible to heat or reducing agents [18-19]. The apparent size and/or isoelectric point of VEPF is Exp Cell Res 159 (1985)

492

Miyazono et al.

Table 1. Stability of vascular endothelial cell proliferation factor from human

platelets % Maximal stimulationa Treatment

Mean + SE

None

100.0+ 15.4

Heat

56°C, 30 rain IO0°C, 10 rain

16.9+7.7 -12.1+1.0

Enzyme

Trypsin Inactivated trypsin

12.7+2.7 120.8+ 12.0

Chemical

Urea (4 M) Guanidine-HC1 (4 M) Acetic acid (1 M) Dithiothreitol (5 mM)

47.0+ 13.6 0.9+0.6 -5.2+3.9 91.7+2.1

a % Maximal stimulation was calculated as follows: Maximal stimulation ( % ) =

dpm in treated V E P F - d p m in c o n t r o l s - - x 100 %. dpm in not treated V E P F - d p m in controls

clearly distinct from those of the other growth factors in platelets. In addition, PDGF and EGF have been shown to lack the ability to stimulate growth of endothelial cells [9-11], suggesting that VEPF is functionally as well as biochemically distinct from these factors. Peptide growth factors stimulating the proliferation of endothelial cells have been isolated from various kinds of normal tissues [20-24]. Among these substances, endothelial cell growth factor (ECGF) purified from bovine neural tissue is the most exceedingly characterized. It has been isolated as two Mr forms; Mr, more than 70 000 and 17 000-25 0130. Isoelectric focusing showed that both forms possess similar anionic isoelectric points (between pH 5 and 6). This factor is labile in the extreme variation of pH and temperature. However, ECGF stimulates the growth of fibroblasts as well as endothelial cells and ECGF activity is inactivated with reducing agents [20]. Furthermore, ECGF binds to a column of heparin-Sepharose and eluted at more than 0.8 M NaC1, whereas VEPF does not have an affinity for heparin (unpublished observations). These observations suggest that ECGF and VEPF have similar biochemical properties, but they are clearly different in some respects. Recently, King et al. have isolated a cationic ECGF from outdated platelets with an Mr of 65 000 [25, 26]. Our preliminary experiments revealed that activity of VEPF was markedly diminished when the platelet lysate was taken from Exp Cell Res 159 (1985)

Endothelial cell growth factor from platelets 493 Table 2. Comparison of growth factors in platelets Responsiveness demonstrated in

Stability to

Designation

Mr

pI

Heat

Reducing agents

Fibroblasts

Endothelial cells

PDGF

28.00031000

9.8-10.2

+

-

+

-

EGF

33 000 6000

3.8--4.5

+

-

+

-

TGF-fl

25 000

Basic

+

_

+a

?

HGF

65 000

Basic

_

_

?b

?

VEPF

60 000 20 000

4.0-4.8

-

+

+

+

TGF-fl stimulates anchorage-independent growth of normal rat kidney fibroblasts in the presence of EGF. b HGF stimulates the growth of hepatocytes in vitro. a

outdated platelets, suggesting the extreme lability of the factor at room temperature. In addition, we could not find cationic growth-promoting activity on endothelial cells either by isoelectric focusing or by ion exchange chromatography. In gel exclusion chromatography, we also observed growth-promoting activity on endothelial cells in fractions of such a higher Mr region. However, even when fractionated by a Sephacryl S-200 column, the active fractions of the higher Mr region were not separable from the major protein peak usually found in gel exclusion chromatography of platelet lysate, as reported in the case of the purification study on hepatocyte growth factor [18]. We could not chromatograph the platelet lysate under denaturing conditions because of the instability of the factor. Therefore, further purification studies are required with regard to the Mr of the factor. We conclude that human platelets contain a polypeptide mitogen that promotes the proliferation of vascular endothelial cells in culture. The biochemical and biological properties of the factor substantially differ from other growth factors already isolated from platelets. We thank Miss Miho Omote for her excellent technical assistance and Miss Jacqueline Dyck for her help in preparing the manuscript.

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494 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Miyazono et al.

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Received January 25, 1985

Exp Cell Res 159 (1985)