J Neurosurg 76:822-829, t 992

Endothelial cell chemotactic factor derived from human glioma cell lines SItUICH! TAKAKI, M.D., JUN-IcHI KURATSU, M.D., Yosugw MIHARA, M.D., MASAHARU YAMADA, M.D., AND YUKITAKA USHIO, M.D. Department qf Neurosurgery. Kumamoto University Medical School, Kumamoto, Japan t/ The authors repoit a study of the human umbilical vein endothelial cell chemotactic factor derived from human malignant glioma celt lines. The endothelial celt chemotactic activity of serum-free conditioned medium from cultures of U-373MG, U-251MG, or U-105MG cell lines was measured using a 48-well microchemotaxis chamber. The best response was from U-373MG, which was selected for further study. Chemotactic activity was contained in materials unadsorbed and adsorbed to the heparin-affinity column. Because the higher activity was seen in the unadsorbed material, it was used for characterization and partial isolation. The chemotactic activity was decreased under the condition of tumor protein synthesis inhibition. Heating, exposure to acid, and trypsin digestion also decreased the activity. The factor was found to be a protein with a relative molecular weight of greater than 200 kD; it has no mitogenic activity for endothelial cells in vitro and, partially purified, it was not identical to any other known endothelial cell chemotactic or mitogenic factor. Fibronectin was not detected, and anti-fibronectin antibody failed to inhibit the activity of the factor. These results suggest that malignant glioma cells produce a yet unknown endothelial cell chemotactic factor. KEY WORDS

9 glioma

9 endothelial cell

NGIOGENESIS,the formation of new vessels, may be a crucial process in the development or progression of gliomas since prominent neovascularization is observed in these tumors. Generally, two events, migration and the subsequent proliferation of endothelial cells, are required for tumor angiogenesis. 14 The migratory event is thought to be the initial kinetic response in subsequent angiogenesis; initial vascular sprouting occurring from existing endothelial cells prior to cellular proliferation T M together with a nonmitogenic factor produce in vivo angiogenesis, but this process is only chemotactic in vitro. 4"~8In the present study, we focused on the migration of endothelial cells and attempted to identify a glioma cell-derived endothelial cell chemotactic factor. Acidic and basic fibroblast growth factor (FGF) and transforming growth factor-,x (TGF-~) have been demonstrated in glioma cells) ~ These factors may play an important role in neovascularization in gliomas because they have mitogenic and migratory effects on endothelial cells 8`14`j7,35A2`47,53 and because some factors involved in angiogenesis are identical to them. ]5.25,41.43 However, growth factors are multifunctional, 45 so that their effects on glioma cells may not be restricted to angiogenesis. For example, FGF's are present in the

A

822

9 chemotactic factor

9 neovascularization

normal brain in the hypothalamus, pituitary, and brain stem, Hss3 although no continuous angiogenesis takes place. These observations suggest that some mechanism is required to trigger angiogenesis. We hypothesized that the trigger might be a mechanism that induces the migration of endothelial cells and that this mechanism in turn might be induced by some factor derived from glioma cells. We now report that glioma cells produce an endothelial cell chemotactic factor with two characteristic features: it has no affinity for heparin and it is a protein with a relative molecular weight of greater than 200 kD. To our knowledge, no factors identical to this have been described to date. Materials and Methods Cell Lines Human malignant glioma cell lines U-105MG, U251MG, and U373MG, originally established by Pont+n and Westermark, 33-56'57were used. Human umbilical vein endothelial cells* were obtained for the study. * Human umbilical vein endothelial cells obtained from Kurabou, Inc., Osaka, Japan. J. Neurosurg. / Volume 76/May, 1992

Endothelial cell chemotactic thctor in glioma Cell Culture Glioma cell lines were cultured in 150-sq cm flasks in RPMI-1640 medium containing penicillin (100 U/ ml), streptomycin (100 ug/ml). 2 mM k-glutamine, and 10% fetal calf serum (FCS) in a humidified atmosphere of 5% CO2 at 37~ After the cells reached confluence, the medium was changed to an FCS-free solution and incubation was continued for 3 days. Cell-free conditioned medium was obtained by filtration using 0.45~m filters? and frozen until used. The human umbilical vein endothelial cells were cultured in E-GM medium,~ in which 10 ng/ml epidermal growth factor, 1 ug/ml hydrocortisone, 50 ug/ml gentamicin, 0.25 ug/ml amphotericin B, and 2% FCS are present. The fifth through 10th passages of the cells were used for endothelial cell chemotactic activity assay. Endothelial Cell Chemotactic Activity The endothelial cell chemotactic activity was tested by a modified Boyden chamber method using a 48well microchemotaxis chamber. ~ Briefly, human umbilical vein endothelial cells in subconfluent condition were harvested after detaching them with 0.1% trypsin-0.02% ethylenediaminetetra-acetate in Dulbeceo's phosphate-buffered saline (PBS). The cells were washed twice with RPMI-1640 medium containing 10% FCS and incubated for 1 hour at 37*C in a humidified atmosphere of 5% CO> Test fractions of 27.5 ~1 were added to the lower wells of the chamber. The lower wells were covered with a polyvinylpyrrolidone-free polycarbonate filter with pores 12 um in diameter. After incubation for 1 hour, the cells were resuspended in Gey's balanced salt solution supplemented with 2% bovine serum albumin and adjusted to 3 x 105 cells/ ml. After fixation of the upper plate, 50 #1 of this cell suspension were added to the upper well. The preparation was then incubated for 4 hours at 37~ in a humidified atmosphere of 5% CO2, then the cells on the upper surface of the filter were removed by washing with PBS and the filter was fixed and stained using DiffQuick stain.w The chemotactic response of the cells was assessed by counting the number of cells that migrated to the lower surface of the filter per microscopic highpower field (400 • in five random fields. Each assay was performed in triplicate and endothelial cell chemotactic activity was expressed as the number of cells counted. Isolation of an Endothelial Cell Chemotactic Factor Conditioned medium was concentrated 10-fold by ultrafiltration, then dialyzed against 0.01 M PBS containing 0.15 M NaC1, pH 7.5. the material was applied -t Filters manufactured by Nihon Millipore Co., Yonezawa, Japan. :~Culture medium E-GM obtained from Kurabou, Inc., Osaka, Japan. wDiff-Quick stain manufactured by Kokusaisiyaku Co., Kobe, Japan.

J. Neurosurg. / Volume 76/May, 1992

to a 2.5 • 2.0-cm heparin-Sepharose CL-6B column, l[ equilibrated with PBS, and eluted with a gradient of 0.15 to 2.0 M NaC1 at 16 ml/hr. Fractions of 2 ml were collected and absorbance was measured at 280 nm. The fractions eluted with an NaC1 gradient were dialyzed against PBS, then all fractions were taken into the endothelial cell chemotactic activity assay. The fractions passed through the column were dialyzed against 0.05 M Tris-HC1, pH 7.5, then applied to a 5 • 2-cm diethylaminoethyl cellulose column,* equilibrated with 0.05 M Tris-HC1, and eluted with a gradient of 0 to 0.8 M NaCI at 15 ml/hr. Fractions of 2 ml were collected and absorbance was measured. All fractions were dialyzed against PBS and taken into the endothelial cell chemotactic activity assay. The active fractions were concentrated by ultrafiltration to a volume of 5 ml, and the concentrated material was applied to the Sephacryl column? (2 • 80 cm). Thereafter, 3ml fractions were collected and absorbance was measured. The endothelial cell chemotactic activity of all fractions was tested. Aliquots of the active fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) electrophoresis. A modification of the procedure of Laemmli was used. 27 Sodium dodecyl sulfate and 2-mercaptoethanol were used as a dissociating agent and a reducing agent, respectively. Samples were run on a 6% acrylamide-resolving gel in Tris-glycine buffer, and the gel was stained with a sliver stain kit.:]:

Effect of Inhibition of Protein Synthesis To test the effect of inhibition of protein synthesis on the production of endothelial cell chemotactic activity by U-373MG cells, 1.25 to 10 ug of puromycin/ml was added to confluent cell cultures. Cell-free conditioned medium collected on Day 3 was concentrated 10-fold by ultrafiltration, then tested. Ch eckerboa rd Assay To determine whether the migration of endothelial cells was truly chemokinetic or chemotactic, the checkerboard assay was performed. By adding samples at various concentrations, positive and negative concentration gradients were obtained between the upper and lower wells. Characterizalion of Chemotactic Factor Heat Inactivation. An aliquot of U-373MG-conditioned medium unadsorbed to a heparin affinity column was heated at 60*C for 30 minutes to test for heat inactivation. Ir Heparin-Sepharose CL-6B column manufactured by Pharmacia Japan, Inc., Tokyo, Japan. * Cellulose column, Model DE-52, manufactured by Whatman, Ltd., Kent, England. Sephacryl S-300 HR column manufactured by Pharmacia Japan, Inc., Tokyo, Japan. z~Silver stain kit manufactured by Daiichi Pure Chemicals Industries, Ltd., Tokyo, Japan. 823

S. Takaki, et al. nectin antibody (7.5 ug/ml) was added to the sample and activity was measured.

FIG. 1. Graph showing the effect of glioma-conditioned medium on human umbilical vein endothelial cells. The endothelial cell chemotactic activity (ECA) is shown for various concentrations of conditioned medium from cultures of U-105MG (circles), U-25 IMG (triangles), or U-373MG (rectangles) cells on human umbilical endothelial cells. Cell migration was measured using a 48-we11microchemotaxis chamber as described.

Effect oJ'pH. An aliquot of U-373MG-conditioned medium unadsorbed to a heparin affinity column was dialyzed against citrate-phosphate buffer (0.05 M, pH 3.0) or carbonate-bicarbonate buffer (0.05 M, pH 10.5). Dialysis was conducted in 500-fold excess of dialysate for 24 hours at 4~ then all samples were dialyzed in 1000-fold excess of PBS with two buffer changes per period. Effect of Enzymes. The material was treated with 10 #g/ml of trypsin for 3 hours at 37~ then 20 ug/ml of soybean trypsin inhibitor was added. As a control, both 100 ug/ml of trypsin and 200 ug/ml of soybean trypsin inhibitor were incubated together for 2 hours at room temperature, then incubated with the sample for 3 hours at 370C. The material was also subjected to ribonuclease (RNase) and deoxyribonuclease (DNase); 100 ug/ml of each enzyme was incubated with the material for 30 minutes at 37~ Effects of a Partially Purified Chemotactic Factor with Angiogenesis Modifiers on the Endothelial Cells Aliquots of active fraction (Fraction 31 ) obtained by Sephacryl chromatography were tested to evaluate the effects of protamine, suramin, and heparin. Protamine and suramin are angiogenesis inhibitors and heparin enhances the effect of angiogenesis factors. Then 1 #g/ ml of protamine sulfate, 50 ug/ml of suramin, or 10 U/ml of heparin sodium was added to the active fraction and the endothelial cell chemotactic activity was tested in the same manner as before. Endothelial cell chemotactic activity in the presence of polyclonal antihuman fibronectin antibody was also tested: anti-fibro824

In Vitro Endothelial C~4l Proliferative Activity In vitro proliferative activity of endothelial cells was assessed by tritiated thymidine incorporation. Endothelial cells were trypsinized and plated on 96-well microtiter plates in 200 ul of RPMI-1640 medium containing 10% FCS, at a suspension of 2 x 104. Twenty-four hours later, the culture medium was changed to 200 ul of RPMI-1640 containing 2% FCS, and 50 ul of samples was added with 1 uCi/ml of methyl tritiated thymidine. After incubation for 24 hours at 37~ the radioactivity was determined in a liquid scintillation counter. As samples, RPMI-1640 medium containing 10% FCS, E-GM medium containing epidermal growth factor (10 ng/ml), and aliquots of the active fraction from Sephacryl chromatography (Fraction 31) were tested.

Results

Endothelial Cell Chernotactic Activity of GliornaConditioned Medium Conditioned media from cultures of U-373MG, U251MG, or U-105MG cells could mobilize the endothelial cells in vitro (Fig. 1). The greatest activity was shown in U-373MG-conditioned medium and the activity of the crude sample was dose-dependent. The U251MG and U-105MG cells also produced endothelial cell chemotactic activity in a dose-dependent manner. The best response was from U-373MG, which was selected for further study.

Isolation of an Endothelial Cell Chemotactic Factor Crude conditioned medium from U-373MG cells was applied to a heparin-affinity column and eluted with a salt gradient. The majority of the protein was not retained by the column and unadsorbed material had greater activity (60% that of the input) than adsorbed material (Fig. 2 upper left). The gradient elution of the column yielded a single protein peak eluting at 0.3 to 0.5 M NaCI, and these fractions also contained endothelial cell chemotactic activity. A small amount of activity was eluted continuously at 1.1 to 1.8 M NaC1, although there was little protein. Material unadsorbed to heparin was applied to an ion exchange column and eluted with a salt gradient. A single peak of endothelial cell chemotactic activity was seen at 0.2 to 0.3 M NaCI (Fig. 2 upper right). Fractions 44 through 58 containing endothelial cell chemotactic activity were chromatographed on the Sephacryl column. As shown in Fig. 2 lower, almost all activity was detected in fractions with a molecular weight greater than 200 kD. Aliquots of the highly active (Fractions 31 and 38), low-activity (Fraction 43), and inactive (Fraction 46) fractions obtained from Sephacryl chromatography were run on a 6% SDS-PAG. The gel was stained, and lanes of active and inactive fractions were compared.

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Endothelial cell chemotactic factor in glioma

FIG. 2. Graphs showing results of chromatography. UpperLeft. Heparinaffinity chromatography of 10-fold-concentrated crude U-373MG-eonditioned medium containing 0.8 mg/ml of protein. Fractions of 2 ml were collected during sample loading, column washing, and gradient elution. Aliquots of all fractions were tested for endothelial cell chemotactic activity (ECA). Upper Right: Purification of endothelial cell chemotactic factor obtained from material unadsorbed to the heparin-affinity column. After dialysis against 5 mM Tris-HCl (pH 7.5), the material unadsorbed to the heparin-affinity column was chromatographed on an ion exchange column. Fractions of 2 ml were collected during sample loading, washing, and gradient elution. After dialysis of all fractions against phosphate-buffered saline (pH 7.5), aliquots of all fractions were tested for ECA. Lower:Sephac~'l chromatography of ion exchange-purified endothelial cell chemotactic factor. Active fractions obtained from ion exchange chromatography were concentrated and 10 ml of the sample containing 0.8 mg/ml was applied to a Sephacryl column. All fractions of 3 ml were collected and aliquots were tested for ECA.

A band was seen in the lanes of highly active fractions corresponding to a molecular weight of 205 kD (Fig. 3). Since the active fractions from Sephacryl chromatography corresponded to a molecular weight of greater than 200 kD, the band at 205 kD was thought to represent a possible subunit of the active form of the factor.

Effect of Inhibition of Protein Synthesis To assess the effect of protein synthesis inhibition, a sample unadsorbed to the heparin affinity column was tested. The production of endothelial cell chemotactic activity by U-373MG cells was decreased in a dosedependent manner by the addition of puromycin; 1.25 ug/ml of puromycin inhibited the production of activity by up to 75% and more than 5 ug/ml of puromycin inhibited the production of activity completely (Fig. 4).

Checkerboard Assay An unadsorbed sample as described above was tested for the checkerboard assay. The migration of endothelial cells induced by the sample was found to be a chemotactic, not a chemokinetic, reaction (Table 1). A positive gradient of the sample induced greater cell migration than a negative gradient. When the concentrations in the upper and lower wells were identical, the migratory activity of the cells was independent of the concentrations.

J. Neurosurg. / Volume 76/May, 1992

FIG. 3. Chromatograph showing elution of endothelial chemotactic factor from sodium dodecyl sulfate-polyacrylamide gel. Aliquots of highly active, low-activity, and inactive fractions obtained from Sephacryl chromatography were subjected to electrophoresis. Lane A shows Fraction 31, Lane B shows Fraction 38, Lane C shows Fraction 43, and Lane D shows Fraction 46. One band corresponding to an approximate molecular weight of 205 kD in Lanes A and B was thought to represent endothelial cell chemotactic activity. 825

S. Takaki, et al. q-ABLE 1

TABLE 2

Results of the checkerboard a.ssay*

P/Jysiochemical characterization of the endothelial cell chemotacticfactor

Lower Well

Heparin-UnadsorbedChemotactic Factor (Upper Well)

0% 50% 75% 100% 0% 1 3 _ + 4 . 7 4_+2.1 1_+1.3 3_+2.1 50% 20 _+5.0 16 _+4.5 5 _+2.3 5 _+2.9 75% 23 _+6.4 18 _+3.9 4 + 1.5 8 _+3.4 100% 26 _+4.0 11 +-4.3 7 _+3.3 6 + 2.5 * Endothelialcell chemotacticactivitywas tested in various 10-fold concentration gradients of material unadsorbed to heparin-affinity column. Concentrationsare expressedas a percentageof the sample. Values are means _+standard error of the means for triplicate experiments.

Treatment ECA (cells/field)* control 34 _+ 1.7 incubation at 60*C for 30 rain 11 + 5.01 exposure to DNase 25 _+ l.lt RNase digestion 29 + 5.3 exposure acid (pH 3.0) 16 _+5.2t exposure to base (pH 10.5) 33 + 2.3 control for trypsin 31 + 3.4 trypsin digestion 17 + 3.0~ * The endothelialcell chemotacticactivity(ECA)of lO-foldmaterial unadsorbedto heparin was tested after varioustreatments. Values are means _+standard error of the means for triplicateexperiments. t Significantlydifferentcompared to each control (p < 0.01, nonpaired t-test).

In Vitro Endothelial Cell Proliferation Tritiated thymidine incorporation profiles of endothelial cells are shown in Table 4. The proliferative activity of endothelial cells was very low in all groups and there were no significant differences except for cells treated by E-GM medium containing 10 ng/ml of epidermal growth factor (p < 0.10) and the control cells (Table 4). The partially purified factor did not affect the proliferative activity of endothelial cells. FIG. 4. Graph showing the effect of protein synthesis inhibitor. U-373MG-conditioned medium from cultures with the indicated concentration of puromycin was tested. Endothelial cell chemotactic activity (ECA) is expressed as the percentage of ECA in the control sample.

Characterization The endothelial cell chemotactic activity of the unadsorbed sample was decreased significantly (p < 0.01) by 60"C incubation (Table 2). Exposure to acid (pH 3.0), trypsin digestion, and DNase exposure also decreased the activity significantly (p < 0.01) (Table 2). On the other hand, the endothelial cell chemotactic activity was stable upon exposure to high p H (10.5) and to RNase digestion. These results suggest that the factor was inactivated by heat, acid, and trypsin.

Effects of Angiogenesis Modifiers Table 3 shows the effects of angiogenesis modifiers on aliquots of the active fraction obtained from Sephacryl chromatography (Fraction 31). Protamine sulfate and suramin produced significant inhibition (p < 0.01). Heparin appeared to decrease endothelial cell chemotactic activity but there was no significant difference from the control sample. Anti-fibronectin antibody (7.5 ug/ml) did not inhibit the endothelial cell chemotactic activity of the sample.

826

Discussion

Tumor Angiogenesis Gliomas are the most c o m m o n tumors of the brain and are characterized by rich neovascularization accompanied by rapid growth or invasion. Tumor angiogenesis is thought to be due to some activity produced by the tumor cells themselves. Some angiogenic activity has been found in conditioned medium and in extracts from glioma cells, 19'2448 and potent angiogenic factors such as FGF's and T G F - a were reported to exist, or to be expressed, in glioma cells.~~ Indeed, F G F ' s or T G F - a strongly stimulate endothelial cells in vivo and in vitro, 8'~4'~7'35"4~-'47'53but their activity attracts considerable attention to mitogenic activity only. Moreover, in vivo or in vitro mitogenic activity has been demonstrated in almost all studies concerning angiogenesis, so that a proliferative effect on endothelial ceils is thought to be essential for the angiogenic factors. Especially in human gliomas, where endothelial cell proliferation is a striking feature related to their grade, mitogenic activity has drawn much attention. However, angiogenesis in tumors requires two events, endothelial cell migration and proliferation.~4 As to cell migration, Banda, et al.,4 reported that a nonmitogenic chemotactic factor from wound fluid induces in vivo angiogenesis. They suggested that the abundant mitogenic activity already in the vessels could sustain endothelial cell growth, but could not by itself give direction

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Endothelial cell chemotactic factor in glioma TABLE 3 Effects ()/'angiogem,sis modi/iers* Modifier control protamine suramin heparin anti-fibronectin

ECA (cells/field) 33 21 24 27 35

_+ 2.2 + 4.It + 1.0~ 4- 3.1 _+ 1.3

* Aliquots of the active fraction (Fraction 31) obtained from Sephacryl S-300 HR chromatography were tested. VaLues are means _+standard error of the means for triplicate experiments. ECA = endothelial cell chemotaetic activity. "t Significantly different compared to each control (p < 0.0I, nonpaired t-test).

to that growth. Ausprunk and Folkman 2 hypothesized that the primary event in tumor angiogenesis was a chemotactic attraction of endothelial cells from existing capillaries and venules and that mitosis occurred only secondarily as a result of the development of interendothelial gaps in the preformed vessels. These observations suggest that the migration of endothelial cells may be the initial kinetic response in angiogenesis and may play a more important role than has been thought. Endothelial Cell Chemotaxis

The present study has demonstrated endothelial cell chemotactic activity produced by human malignant glioma cells. The majority of the activity had no affinity for heparin, and the active material was a protein with a relative molecular weight of greater than 200 kD. There is little information concerning the chemotactic activity of endothelial cells in gliomas. Most peptide angiogenic factors have an affinity for heparin, and their relative molecular weight is less than 20 kD. ~3-j5' 20.25.37.43,53 As potent angiogenic factors in gliomas, FGF's should be taken into consideration. In our study, a small amount of activity was eluted at I. 1 to 1.8 M NaC1 in heparin-affinity chromatography. Since FGF is adsorbed and eluted at 1.1 to 1.5 M NaCI from the h e p a r i n - a f f i n i t y column, 13,15,2~ it might be present in small amounts. Because of the lack of a signal peptide, FGF's are not able to escape from cells so that their release depends on tumor cell death.~'37 It is reasonable to propose that cell death with subsequent release of FGF's results in angiogenesis because necrosis usually accompanies malignant gliomas. However, FGF's can be excluded from our partially purified factor because of their strong affinity for heparin and their molecular weight. Transforming growth factor-a is angiogenic 42 and is expressed by glioma cell lines, m'28'32but it also should be excluded because it is stable against exposure to heat and acid. Epidermal growth factor has the same biological activity on endothelial cells as TGF-c~ and it also does not bind to heparin. 42'43 However, epidermal growth factor is stable against heat and its molecular weight differs from that of the present factor. TransJ. Neurosurg. / Volume 76/May, 1992

TABLE 4 In vitro prolifi,rative e~'ct on eiMothelial cells* [3H]Thymidine Incorporation (cpm) control 95 _+28.2 10% FCS added 111 4- 21.6 E-GM medium added 161 4- 41.1 Fraction 31~" 87 ___41.0 * Values are the means _+standard error of the means for triplicate experiments. FCS = fetal calf serum. t An aliquot of an active fraction obtained from Sephacry,1S-300 HR chromatographywas tested. ]-reatment

forming growth factor-r could be secreted with a relative molecular weight of about 200 kD 3~ and it produces angiogenesis in mice; 3s however, it usually acts as an inhibitor of angiogenesis 33 although it is a multifunctional cytokine. 3~ We hypothesized that the most plausible candidate for the present factor was fibronectin. It is a component of the extracellular matrix and demonstrates biological activity on endothelial cells in the chemotactic response. 6'1~s2 Some glioma cell lines have been found to produce fibronectin in detectable amounts, s'23'29'33 Although fibronectin binds to heparin, it might have passed through the heparin-affinity column because of its high molecular weight (220 kD); however, it was ruled out by the Western blot method (data not shown). In addition, anti-fibronectin antibody did not inhibit endothelial cell chemotactic activity. Even if an undetectably low level of fibronectin was contained in the factor, it would be insufficient to stimulate endothelial cells because at least 50 ug/ml of fibronectin is required for stimulation. 6 Thus, we concluded that fibronectin should be excluded. Endothelial Cell Chemotactic Factor

The factor demonstrated in the present study exhibited two characteristic features. First, it was nonbinding to heparin. Heparin is structurally 26 and biologically 54 similar to heparan sulfate, the major glycosaminoglycan species on the endothelial cell surface. 7 Therefore, it is reasonable to suggest that a molecule that acts as a stimulator for endothelial cells can bind to heparin. Shing, et al., 43 reported the purification of a tumorderived capillary endothelial cell growth factor and suggested that heparin affinity made it possible to purify angiogenic thctors in a few steps. Therefore, we attempted to purify the factor using heparin-affinity chromatography in the first step. Contrary to our expectation, the factor did not show affinity for heparin. This suggests that the binding site of the factor is different from that of heparin-binding molecules such as FGF's. Protamine blocks the ability of heparin and basic FGF to stimulate the migration of endothelial cells by binding them, and this agent could also inhibit di827

S. Takaki, et al. rectty. 3'3"'4~ In the present study, prolamine decreased endothelial cell chemotactic activity and we are uncertain whether the agent acted directly or indirectly. Suramin, reported to inhibit the migration of endothelial cells directly, 4~ also decreased endothelial cell chemotactic activity. These results suggest that the agents might block the factor from binding to endothelial cells at a site different from that of heparin-binding molecules. Another feature of our factor was its high molecular weight in secreted form. Almost all peptide angiogenic factors, such as FGF, TGF, and epidermal growth factor, have a relatively low molecular weight (< 20 kD), except for platelet-derived endothelial cell growth factor. 22 We considered the possibility of aggregates of low-molecular-weight molecules and attempted to detect basic FGF and TGF-c~ using the Western blot method. Our results were negative (data not shown). Since SDS-PAG electrophoresis revealed one possible band corresponding to 205 kD, fibronectin came to mind but it could not be detected. Based on these results, we were unable to match our factor to a previously reported factor. Clinical Significance

Some studies have reported that in vivo tumor growth can be inhibited by inhibiting tumor-derived angiogenesis, 9"12'21'36'38'39'49-51and it is conceivable that angiogenesis inhibitors will be used clinically to treat highly angiogenic tumors, especially brain tumors. Although the mechanism of angiogenesis inhibitors is not sufficiently understood at present, inhibitory treatment focused on the proliferation of endothelial cells may cause severe side effects such as myelosuppression. We propose that the migration of endothelial cells is the most marked difference between the normal and tumorbearing condition. Therefore, studying the mechanism of endothelial cell migration may open a new window for the treatment of highly vascularized tumors. References

I. Abraham JA, Mergia A, Whang JL, et al: Nucleotide sequence of a bovine clone encoding the angiogenesis protein, basic fibroblast growth factor. Science 233: 545-548, 1986 2. Ausprunk DH, Folkman J: Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 14: 53-65, 1977 3. Azizkhan RG, Azizkhan JC, Zetter BR, et al: Mast cell heparin stimulates migration of capillary endothelial cells in vitro. J Exp Med 152:931-944, 1980 4. Banda MJ, Knighton DR, Hunt TK, et ah Isolation of a nonmitogenic angiogenesis factor from wound fluid. Pruc Natl Acad Sci USA 79:7773-7777, 1982 5. Bigner DD, Bigner SH, Pont~n J, et al: Heterogeneity of genotypic and phenotypic characteristics of fifteen permanent cell lines derived from human gliomas. J Neurnpathol Exp Neurol 40:201-229, 1981 6. Bowersox JC, Sorgente N: Chemotaxis of aortic endothelial cells in response to fibronectin. Cancer Res 42: 2547-2551, 1982 828

7. Buonassisi V, Root M: Enzymatic degradation of heparinrelated mucopolysaccharides from the surface of endothelial cell cultures. Biochem Biophys Acta 385:1 - 10, 1975 8. De Cristan G, Morbidelli L, Allessandri G, et al: Synergism between gangliosides and basic fibroblastic growth factor in favouring survival, growth, and motility of capillau endothelium. J Cell Physiol 144:505-510, 1990 9. Denekamp J: Vasculature as a target for tumour therapy. Prog Appl Microcirc 4:28-38, 1984 10. Ekstrand AJ, James CD, Cavenee WK, et al: Genes for epidermal growth factor receptor, transforming growth factor c~,and epidermal growth factor and their expression in human gliomas in vivo. Cancer Res 51:2164-2172, 1991 11. Falk W, Goodwin RH Jr, Leonard EJ: A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J lmmunoi Methods 33: 239-247, 1980 12. Folkman J: Anti-angiogenesis: new concept for therapy of solid tumors. Ann Surg 175:409-416, 1972 13. Folkman J: How is blood vessel growth regulated in normal and neoplastic tissue? G. H. A. Clowes Memorial Award Lecture. Cancer Res 46:467-473, 1986 14. Folkman J, Klagsbrun M: Angiogenic factors. Science 235:442-447, 1987 15. Gospodarowicz D, Cheng J, Lui GM, etal: Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chromatography: identity with pituitary, fibroblast growth factor. Proc Natl Acad Sci USA 81:6963-6967, 1984 16. Grant DS, Kleinman HK, Martin GR: The role of basement membranes in vascular development. Ann NY Acad Sci 588:61-72, 1990 17. Grotendorst GR, Soma Y, Takehara K, et al: EGF and TGF-alpha are potent chemoattractants for endothelial cells and EGF-like peptides are present at sites of tissue regeneration. J Cell Physiol 139:617-623, 1989 18. Hrckel M, Sasse J, Wissler JH: Purified monecyte-derived angiogenic substance (angiotropin) stimulates migration, phenotypic changes, and "tube formation" but not proliferation of capillary endothelial ceils in vitro. J Cell Physiol 133:1-13, 1987 19. Hokamura K: Endothelial cell growth factor derived from a human glioblastoma cell line and possible association with tumor angiogenesis. Fukuoka Igaku Zasshi 80: 170-178, 1989 20. Imamura T, Engleka K, Zhan X, etal: Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence. Science 249:1567-1570, 1990 21. Ingber D, Fujita T, Kishimoto S, et al: Synthetic analogues of fumagillin that inhibit angiogenesis and supress tumor growth. Nature 348:555-557, 1990 22. lshikawa F, Miyazono K, Hellman U, etal: Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell factor. Nature 338: 557-562, 1989 23. Jones TR, Ruoslahti E, Schold SC, et al: Fibronectin and glial fibfillary acidic protein expression in normal human brain and anaplastic human gliomas. Cancer Res 42: 168-177, 1982 24. Kelly PJ, Suddith RL, Hutchison HT, et al: Endothelial growth factor present in tissue culture of CNS tumors. J Neurosurg 44:342-346, 1976 25. Klagsbrun M, Sasse J, Sullivan R, et al: Human tumor cells synthesize an endothelial cell growth factor that is structurally related to basic fibroblast growth factor. Proc Natl Aead Sci USA 83:2448-2452, 1986 26. Kraemer PM: Heparan sulfates of cultured cells. II. AcidJ. Neurosurg. / Volume 76/May, 1992

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27. 28.

29.

30.

31. 32.

33. 34. 35.

36.

37. 38. 39.

40.

41.

42.

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Endothelial cell chemotactic factor derived from human glioma cell lines.

The authors report a study of the human umbilical vein endothelial cell chemotactic factor derived from human malignant glioma cell lines. The endothe...
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