Biochimica et Biophysica A cta, 1092 (1991 ) 336-340 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 016748899100162V
336
BBAMCR 12936
Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells Kimiko Amanuma * and Youji Mitsui Cell Science and Technology Dioision. Fermentation Research blstitute, Tsukuba City, lbaraki (dapmO
(Received 15 November 1990)
Key words: Endothelial cells; Hyaluronic acid synthesis; Hyaluronic acid synthetase inhibitor; Transformation
The characteristics of glycosaminoglycan (GAG) synthesis in normal and transformed human endothelial cells were analyzed by the incorporation of [3Hlglucosamine and by the activities of GAG synthetases. The GAG synthesized by normal endothelial cells consisted of mainly heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate but little hyaluronic acid (HA) (less than 1%). The characteristics of GAG synthesis by normal cells reflected the synthetic enzyme activities for each individual GAG: the activity of HA synthetase was very low. In spite of this, the activity of HA synthetase inhibitor, induced in growth-retarded fibroblasts with low HA synthetase activity (Matuoka et al. (1987) J. Cell Biol., 104, 1105-1115), was very low in endothelial cells. In contrast to normal cells, transformed endothelial (ECV304) cells synthesized mainly HA (62% of total GAGs). These findings suggest that the regulatory system of GAG metabolism is cell type" ~pecific, and that transformation is accompanied by high levels of HA synthesis in endothelial cells.
Introduction Hyaluronic acid (HA) is a major extracellular glycosaminoglycan (GAG) and has been thought to have some roles in cell adhesion, migration, and proliferation. Many investigators have shown that, among GAGs, HA synthesis is markedly elevated with the stimulation of proliferation of fihroblasts [1-7]. We have also shown that HA synthesis is tightly coupled with cell proliferation [8-10]. Moreover, we have demonstrated the induction of hyaluronic acid synthetase inhibitor (HASI) in the growth-retarded fibroblasts [9]. Endothelial cells, which line the lumen of blood vessel walls, are characterized by formation of a mono-
layer in vitro, and their proliferation is controlled by the interaction of the cell surface [11]. These cells also produce various kinds of sulfated GAGs according to their growth state [12-17], but HA synthesis and its relationship to endothelial cell proliferation has not been thoroughly investigated yet. Furthermore, alteration of GAG synthesis by endothelial cells accompanied with transformation has not been reported. Therefore, we examined GAG synthesis in human umbilical endothelial (HUE) cells and a HUE transformant cell line, ECV304 [18], with attention to HA and HASI. The results are discussed in connection with the role of HA in growth regulation and transformation of endothelial cells. Materials and Methods
Abbreviations: GAG, 81ycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate, DS, dermatan sulfate; HA, hyaluronic acid; HAS, hyaluronic acid synthetase; HASI, hyaluronic acid synthetase inhibitor; HUE, human umbilical endothelial cells; ECGS, endothelial cell growth supplement; GleUA, glucuronic acid; GIcN, gh~cosamine; PBS, phosphate-buffered saline; FBS, fetal bovine serum. * On leave from Maekawa MFG. Co., Ltd. Correspondence: Y. Mitsui, Cell Science and Technology Division, Fermentation Research Institute, Higashi 1-1-3, Tsukuba, Ibaraki 305, Japan.
Materials
[ 3 H]Glucosamine([ 3 H]GIcN; 1.22 T B q / m m o l , TRK.398) and uridine 5'-diphospho-[t4C]glucuronic acid (UDP-[t4C]GIcUA; 11.6 GBq/mmol, TRK.106) were purchased from Amersham (U.K.). MCDB151 medium and Dulbecco's modified Eagle's (DME) medium were from Sigma (U.S.A.), and fetal bovine serum (FBS) was from Cell Culture Laboratories
337 (U.S.A.). Fibronectin from porcine plasma was obtained from Itoham (Japan). Endothelial cell growth supplement (ECGS) was prepared from bovine brain in our laboratory by the method of Burgess et al. [19]. Authentic GAG and G A G lyases were from Seikagaku Kogyo (Japan). High grade agarose was purchased from Takara (Japan).
Cell culture and metabolic labefing H U E cells isolated from human umbilical cord veins were routinely cultured in MCDB151 medium supplemented with 15% FBS, 5 # g / m l heparin, 200 mg/ml MgSO4.7H20 and an optimum concentration of ECGS (growth medium for H U E cells) in flasks coated with fibronectin [20]. ECV304 cells were cultured routinely in MCDB151 medium supplemented with 10% FBS and 200 mg/ml MgSO4.7H20. Prior to labeling, cells were subcultured in the growth medium for H U E cells. WI-38 CT-I cells were cultured in DME medium with 10% FBS. H U E cells (16-17 PDL: approx. 20% of the life span) were seeded into F75 culture flasks (Corning, U.S.A.) at a density of 3.3- 103 ceUs/cm 2. After 3 days, cells in the growing phase received fresh medium containing 5 #Ci/ml of [3H]GlcN, and cultured for 24 h. For growth-reduction, the medium was changed to low ECGS (concentration of ECGS was 1/8 of the growth medium for HUE) on the third day after the seeding. After 24 h of culture, the cells were given fresh low ECGS medium containing 5 /~Ci of [3H]GIcN and cultured for an additional 24 h. Parallel cultures using 12 well plates (Linbro~ Flow Lab., U.S.A.) under the same conditions were performed and the cell number was counted by Coulter counter (Coulter Electronics, U.S.A.). During the labeling period, cell numbers in the growing and growth-reduced culture increased 1.88 and 1.18-times, respectively.
brane in 0.05 M H2SO4 at 1 m A / c m for 120 min [8]. Individual GAG spots stained by Alcian blue were cut and the radioactivity measured by liquid scintillation counting.
Assay of GAG synthetase and HA synthetase inhibitor (HASI) in cell homogenates The activity of the enzyme system synthesizing GAGs was assayed by incorporation of UDP-[]4C]GIcUA into individual GAGs as described previously [9]. The activity of HASI was measured as described previously [9]. Briefly, homogenate of sample cells (HASI source) (1), homogenate of growing WI-38 CT-1 cells (HAS source) (2), and mixture of both homogenates (3) were assayed for HAS, HASI activity was calculated as percent inhibition of HAS: 100. ([A + B] - C ) / A • protein content of (l)/protein content of (2). A, B, a~d C are HAS activities of (1), (2) and (3), respectively. Colony" formation in soft agar 100 cells per 6 mm dish were contained in an upper layer of 1.5 ml growth medium for HUE cells containing 0.165% agarose. The lower layer composed 0.5% agarose in the same medium. After 16 days in culture, the number and the diameters of colonies were measured. Results
Incorporation of [~H]GlcN hlto GAGs by normal endothefial cells To exanaine GAG synthesis by HUE cells, the cells in growth and growth-retarded phases were labeled with [3H]GlcN for 24 h. GAGs were then isolated from the medium, surface (trypsin-releasable) and cell fraction. 0.3
Isolation and analysis of GAGs Medium containing secreted GAGs was removed and the cell layer was washed twice with Ca 2+, Mg 2+ free phosphate buffered saline (PBS). The first wash together with the medium was centrifuged at 1800 x g for 5 min at 4 ° C and the supernatant was pooled as the 'medium' fraction. Cells were harvested by trypsinization and centrifuged at 200 x g for 5 min at 4°C. The 'cell' fraction was obtained by washing the pellet with PBS and by centrifugation. The superrtatant of this step and the wash were combined as 'surface' fraction. GAGs in each fraction were isolated by alkali treatment, pronase digestion, and precipitation with cetylpyridinium chloride as described Matuoka and Mitsui [21]. GAG preparations were dissolved in an appropriate volume of water with authentic GAGs (HA, HS, chondroitin sulfate (CS) and dermatan sulfate (DS)), and then electrophoresed on a cellulose acetate mere-
[] Cell [] Surface
0
.p.r : f¢
W 0 -i
e.j
0.2
"=e ¢: O
0.1
2
O
O ¢)
0.0
GroiNing
Growth.Retarded
Growth Stats
Fig. 1. Incorporation of [3H]GIcN into GAGs by HUE cells• Cells were cultured in the presence of [3H]GIcN for 24 h under sufficient (growing) or low concentrations of ECGS (growth-retarded). Medium. surface (trypsin-releasable). and cell fractions were collected and GAGs were isolated as described in Materials and Methods•
338 MEDIUM
TOTAL
SURFACE
CELL
l i 0
50
100
SO
100 0
50
100 0
7 SO
100
PERCENTAGE OF GAGs
Fig. 2, Change in GAG synthesis by HUE cells with growth-retardation. Labeled GAGs isolated from growing (G) and growth-retarded (G-R) HUE cells as described in Fig, 1 were analyzed by electrophoresis on cellulose acetate membranes. The data were compared with those obtained from normal fibroblasts, WI-38 [8], The order of [3HIGIeN incorporation by both types of cells was similar.
The incorporation into GAGs by cells in the growth phase was higher in every fraction than by cells in the growth-retarded phase in medium containing a low concentration of ECGS (Fig. 1). In both cases, most labeled GAGs were recovered in the medium (above 80~), and [3H]GlcN incorporation into GAGs in surface and cellular GAGs was less than 10~, respectively. The composition of labeled GAGs was analyzed by electrophoresis on cellulose acetate membranes. The results are shown in Fig. 2 in comparison with fibreblasts [8]. In contrast to fibroblasts which produce much HA, little [3H]GlcN was incorporated into HA irrespective of the growth conditions: less than 1~ in the medium and surface fraction and only 2 - 4 ~ in the cell fraction. The percentage of HA in the cell fraction may be overestimated because some GAGs which were being sulfated may have similar mobility to HA on the acetate membrane. Most [3H]GIcN was incorporated into HS and CS/DS instead of HA by HUE cells. With regard to growth-reduction, HA synthesis was reduced in fibroblasts, while synthesis of CS/DS was reduced in endothelial cells.
Activities of GAG synthetic enzymes To ¢xamine whether the characteristics of GAG composRion in cultured cells were due to the activities of GAG synthetic enzymes, we measured the enzyme activities in cell homogenates. As shown in Table I, activi-
GAG synthetase activities in human fibroblasts and endothelial cells
HA HS CS/DS
Change in GA G synthesis by transformation ECV304 is a cell line obtained from HUE cells by spontaneous transformation [18]. This line maintains some characteristics of HUE; for example, it has a similar doubling time (approx. 24 h), and forms a cobblestone-like monolayer. However, this line had a high efficiency of colony formation in soft agar (Table lit). With transformation of fibroblasts, GAGs produced by cells tend to shift from sulfated GAGs to HA [22]. Because of the absence of HA synthesis in normal endothelial cells, we examined whether or not transformed endothelial cells synthesized HA. GAG synthesis in HUE and ECV304 cells was analyzed by incorporation of [3H]GIcN in the growth medium for HUE. Both cell lines in the growing phase incorporated [3H]GIcN to a similar extent ((2.6-2.8). 10 -t dpm/cell TABLE It
Activity of hyaluronic acid synthetase inhibitor (HASI)
TABLE !
GAG species
ties of both HS and CS/DS synthetase were of an order similar to that of normal fibroblasts, while the activity of HAS was very low in HUE cells. With regard to the regulation of HAS activity, we have found an inhibitor of HAS in fibroblasts, in which HA synthesis was reduced [9]. It is possible that the activity of HAS appeared low in HUE cells because of the high activity of HASI. However, when HASI activity was measured, the activity was very low in HUE cells (Table II).
UDP-[14C]GlcUA incorporation (pmol/h per mg protein) fibroblasts (Wl-38 a)
endothelial cells (HUE)
~.5±3.5 14.6±2.5 11.5±2.8
1.2±1.2 9.9±6.4 9.4±2.6
Mean+S.D. a Data from our previous paper [9].
Homogenates of various cell culture were assayed for HA synthetase inhibition by mixing them with homogenate of growing WI-3 8 CT-1 cells as described in Materials and Methods. HASt activity was calculated from duplicate assay. Growth state
Growing Growth-retarded
HASI activity (~) fibroblasts (WI-38 .)
endothelial cells (HUE)
13..3 62.2
5 9b
a Data from our previous paper [9]. b Cell growth was reduced by reduction of ECGS concentration.
339 TOTAL
HUE
,:cvao4
MEOIUM
~ I, s ?s os -'... 0
$0
SURFACE
CELL
I
100
0
S0
100
50
100
50
100
PERCENTAGE O F GAGS
Fig. 3. Change in GAG synthesis by trans~rmedendothelial cells. Normal(HUE) and transformed (ECV304)ceUsin the growing phase were labeled with[3H]GIcN ~ r 2 4 h, and GAGs wereanalyzed as descfibedinMaterialsandMethods.
in this experiment). However, in contrast to normal endothelial cells, ECV 304 cells incorporated [3H]GIcN into HA to a greater extent (6270) (Fig. 3). The incorporation into HS and C S / D S was lower compared with HUE cells (1670 and 1970 of total GAGs, respectively) (Fig. 3).
Discussion In the present work, we examined the changes in GAG synthesis in relation to growth and transformation of human endothelial cells. In fibroblasts, we previously showed that synthesis of HA was prominent in the growing phase and decreased with growth reduction [9] and passage number [10]. In HUE cells, however, synthesis of HA was very low (less than 1%) even in the rapidly growing phase of early passage cells (16-17 pdl, 2070 of life span) (Fig. 2). Porcine aortic endothelial cells also synthesized little HA compared with HS and C S / D S (unpublised observation). Alternative to HA, HUE cells synthesized mainly HS and CS/DS, and a decrease in CS/DS synthesis was marked with growth reduction (Fig. 2). Thus, modulation of GAG synthesis during cell proliferation is quite different in HUE cells from that in fibroblasts. In HUE cells, the absence of HA synthesis is probably due to the absence of HAS activity (Table I). We previously suggested that the activity of HAS is regulated by the inhibitor, HASI [9]. Although HAS activity was lower in HUE cells than in growth retarded fibroblasts (Table I), HASI activity was very low in HUE cells (Table If). These findings suggest that the absence of HAS activity in HUE cells was not due to the activity of its inhibitor but due to the absence of the enzyme itself. We, however, cannot rule out the possibility that tissue specific HASI which does not inhibit the HAS
TABLE lIl
Colony formation in soft agar HUE
ECV304
Efficiency (7o)
0
41
Diameter (ram)
-
+1
0.129 + 0.010
activity of fibroblasts exists in endothelial cells. Concerning species specificity of HASI, we previously found that HASI derived from BALB/3T3 cells [9] and Swiss 3T3 cells (unpublised observation) was active to HAS from human fibroblasts, suggesting that HASI is not species specific. In contrast to normal cells, transformed ECV304 synthesized high levels of HA (Fig. 3). Despite the difference in GAG composition, the growth of ECV304 cells in culture dishes resembled tha: of HUE ~'ells: both cell lines have similar doubling time, form monolayers, and their growth is curbed by contact inhibition. These findings suggest that the absence of HA synthesis in HUE cells did not always correlate with the formation of a monolayer. On the contrary, ECV304 cells showed highly efficient colony formation in soft agar, whereas HUE cells did not (Table IV), suggesting that elevation of HA synthesis in ECV304 has some correlation with the ability of colony formation. This was supported by the finding that PAE-20, an immortalized cell line of porcine aortic endothelial cells [23] with very low levels of HA synthesis did not form colonies in soft agar (unpublised observation). Histochemical studies by Ausprunk et al. showed that glycocalyx on the lumenal coat of the endothelium was not removed by the degradation enzyme specific for HA [24,25], suggesting that HA production by endothelial cells in vivo may be very low. In the present work, we showed that HA synthesis was extremely low (or absent) in normal endothelial cells. The biological significance of this absence in HUE cells has not yet been clarified, but it might be important for maintaining strict integrity of endothelial cell monolayer in blood vessels, because recent study of West and Kumer showed that exogenously added high-molecular-weight HA disrupted newly formed monolayer of endothelial cells in vitro [26]. Furthermore, several reports suggested that HA has some roles in angiogenesis: degradation products of HA stimulated angiogenesis in vivo [26] and in vitro [27], and epithelial tissues synthesizing much HA caused avascularity in limb buds of chick embryo [28]. Thus, the absence of HA synthesis in endothelial cells may be prerequisite for the control of proliferation of these cells by HA. Furthermore, supressed expression of HAS and its inhibitor in normal
340 endothelial cells might be critical to maintain the vascular network. It may therefore be important to elucidate the biological significance of the absence of HA synthesis in normal endothelial cells and induction of HAS in transformed endothelial cells.
Acknowledgments We thank Ms. K. Takahashi (National Defence Medical College) for her kind supply of ECV 304 cells. We also thank Dr. K. Matuoka for useful advice on analysis of GAG synthesis and Dr. T. Imamura for helpful discussion. This work was supported in part by a project grant for Basic Technology for Future Industry from the Ministry of International Trade and Industry of Japan,
Reference I Tomida, M., Koyama, H, and Ono, T.(1975) J. Cell. Physiol. 86, 121-130. 2 Lembach, K,J, (1976) J. Cell, Physiol. 89, 277-288. 3 Moscatelli, D, and Rubin, H. (1977) J. Cell. Physiol. 91, 79-88. 4 Murota, S., Abe, M. and Ohtsuka, K. (1977) Prostaglandins 12, 983-991. 5 Sisson, J.C., Castor, C.W. and Klavons, J.A. (1980) J. Lab. Clin. Med. 96, 189-197. 6 UIIrich, S,J. and Hawkes, S.P. (1983) Exp. Cell Res. 146, 377-386, 7 Heldin, P., Laurent, T.C. and Heldin, C,-H. (1989) Biochem. J. 258, 919-922. 8 Matuoka, K., Mitsui. Y. and Murota, S. (1985) Cell Biol. int. Rep, 9, 577-586.
9 Matuoka, K., Namba, M. and Mitsui, Y. (1987) J. Cell Biol. 104, 1105-1115. 10 Matuoka, K., Hasegawa, N., Namba, M., Smith, G.J. and Mitsui, Y, (1989) Aging l, 47-54, 11 Hasegawa N., Yamamoto, M., Imamura, T., Mitsui. Y. and Yamamoto, K. (1988) Mech. Age. Dev. 46, 111-123. 12 Gamse, G., Fromme, H.G. and Kresse, H. (1978) Biochim. Biophys. Acta 544, 514-528. 13 Oohira, A., Wight, T.N. and Bornstein, P. (1983) J. Biol. Chem. 258, 2014-2021. 14 Humphries, D.E., Silbert, C.K. and Silbert, J.E. (1986) J. Biol. Chem. 261, 9122-9127. 15 Kinsella, M.G. and Wight, T.N. (1986) J. Cell Biol. 102, 679-687. 16 Robinson, J, and Gospodarowicz, D. (1983) J. Cell. Physiol. 117, 368-376. 17 Gordon, R.B., Conn, G. and Hatcher, V.B. (1985) J. Cell. Physiol. 125, 596-607. 18 Takahashi, K., Sawasaki, Y., Hata, J., Mukai, K. and Goto, T. (1990) In Vitro Cell. Dev. Biol. 25, 265-274. 19 Burgess, W.H., Mehlman, T., Friesel, R., Jonson, W.V. and Maciag, T. (1985) J. Biol. Chem. 260, 11389-11392. 20 lmamura, T. and Mitsui, Y. (1987) Exp. Cell Res. 172, 92-100. 21 Matuoka, K. and Mitsui, Y. (1981) Mech. Age. Dev. 15, 153-163. 22 HS0k, M., Kjellen, L., Johansson, S. and Robinson J. (1984) Annu. Rev. Biochem. 53, 847-869. 23 Mitsui, Y., Yamamoto, M. and Yamamoto, K. (1984) in Progress Microcirculation Res. II (Courtice, F.C., Garlick, D.G. and Perry, M.A., ¢ds.), pp. 37-44, The University of New South, Wales. 24 Ausprunk, D.H., Boudreau, C.L. and Nelson D.A. (1981) Am. J. Pathol. 103, 353-366. 25 Ausprunk, D.H., Boudreau, C.L. and Nelson D.A. (1981) Am. J. Pathol. 103, 367-375. 26 West, D.C. and Kumar, S. (1989) Exp. Cell Res. 183, 179-196. 27 West, D.C., Hampson, I.N., Arnold, F. and Kumar, S. (1985) Science 228, 1324-1326. 28 Feinberg, R.N. and Beebe, D.C. (1983) Science 220, 1177-1179.
336
BBAMCR 12936
Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells Kimiko Amanuma * and Youji Mitsui Cell Science and Technology Dioision. Fermentation Research blstitute, Tsukuba City, lbaraki (dapmO
(Received 15 November 1990)
Key words: Endothelial cells; Hyaluronic acid synthesis; Hyaluronic acid synthetase inhibitor; Transformation
The characteristics of glycosaminoglycan (GAG) synthesis in normal and transformed human endothelial cells were analyzed by the incorporation of [3Hlglucosamine and by the activities of GAG synthetases. The GAG synthesized by normal endothelial cells consisted of mainly heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate but little hyaluronic acid (HA) (less than 1%). The characteristics of GAG synthesis by normal cells reflected the synthetic enzyme activities for each individual GAG: the activity of HA synthetase was very low. In spite of this, the activity of HA synthetase inhibitor, induced in growth-retarded fibroblasts with low HA synthetase activity (Matuoka et al. (1987) J. Cell Biol., 104, 1105-1115), was very low in endothelial cells. In contrast to normal cells, transformed endothelial (ECV304) cells synthesized mainly HA (62% of total GAGs). These findings suggest that the regulatory system of GAG metabolism is cell type" ~pecific, and that transformation is accompanied by high levels of HA synthesis in endothelial cells.
Introduction Hyaluronic acid (HA) is a major extracellular glycosaminoglycan (GAG) and has been thought to have some roles in cell adhesion, migration, and proliferation. Many investigators have shown that, among GAGs, HA synthesis is markedly elevated with the stimulation of proliferation of fihroblasts [1-7]. We have also shown that HA synthesis is tightly coupled with cell proliferation [8-10]. Moreover, we have demonstrated the induction of hyaluronic acid synthetase inhibitor (HASI) in the growth-retarded fibroblasts [9]. Endothelial cells, which line the lumen of blood vessel walls, are characterized by formation of a mono-
layer in vitro, and their proliferation is controlled by the interaction of the cell surface [11]. These cells also produce various kinds of sulfated GAGs according to their growth state [12-17], but HA synthesis and its relationship to endothelial cell proliferation has not been thoroughly investigated yet. Furthermore, alteration of GAG synthesis by endothelial cells accompanied with transformation has not been reported. Therefore, we examined GAG synthesis in human umbilical endothelial (HUE) cells and a HUE transformant cell line, ECV304 [18], with attention to HA and HASI. The results are discussed in connection with the role of HA in growth regulation and transformation of endothelial cells. Materials and Methods
Abbreviations: GAG, 81ycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate, DS, dermatan sulfate; HA, hyaluronic acid; HAS, hyaluronic acid synthetase; HASI, hyaluronic acid synthetase inhibitor; HUE, human umbilical endothelial cells; ECGS, endothelial cell growth supplement; GleUA, glucuronic acid; GIcN, gh~cosamine; PBS, phosphate-buffered saline; FBS, fetal bovine serum. * On leave from Maekawa MFG. Co., Ltd. Correspondence: Y. Mitsui, Cell Science and Technology Division, Fermentation Research Institute, Higashi 1-1-3, Tsukuba, Ibaraki 305, Japan.
Materials
[ 3 H]Glucosamine([ 3 H]GIcN; 1.22 T B q / m m o l , TRK.398) and uridine 5'-diphospho-[t4C]glucuronic acid (UDP-[t4C]GIcUA; 11.6 GBq/mmol, TRK.106) were purchased from Amersham (U.K.). MCDB151 medium and Dulbecco's modified Eagle's (DME) medium were from Sigma (U.S.A.), and fetal bovine serum (FBS) was from Cell Culture Laboratories
337 (U.S.A.). Fibronectin from porcine plasma was obtained from Itoham (Japan). Endothelial cell growth supplement (ECGS) was prepared from bovine brain in our laboratory by the method of Burgess et al. [19]. Authentic GAG and G A G lyases were from Seikagaku Kogyo (Japan). High grade agarose was purchased from Takara (Japan).
Cell culture and metabolic labefing H U E cells isolated from human umbilical cord veins were routinely cultured in MCDB151 medium supplemented with 15% FBS, 5 # g / m l heparin, 200 mg/ml MgSO4.7H20 and an optimum concentration of ECGS (growth medium for H U E cells) in flasks coated with fibronectin [20]. ECV304 cells were cultured routinely in MCDB151 medium supplemented with 10% FBS and 200 mg/ml MgSO4.7H20. Prior to labeling, cells were subcultured in the growth medium for H U E cells. WI-38 CT-I cells were cultured in DME medium with 10% FBS. H U E cells (16-17 PDL: approx. 20% of the life span) were seeded into F75 culture flasks (Corning, U.S.A.) at a density of 3.3- 103 ceUs/cm 2. After 3 days, cells in the growing phase received fresh medium containing 5 #Ci/ml of [3H]GlcN, and cultured for 24 h. For growth-reduction, the medium was changed to low ECGS (concentration of ECGS was 1/8 of the growth medium for HUE) on the third day after the seeding. After 24 h of culture, the cells were given fresh low ECGS medium containing 5 /~Ci of [3H]GIcN and cultured for an additional 24 h. Parallel cultures using 12 well plates (Linbro~ Flow Lab., U.S.A.) under the same conditions were performed and the cell number was counted by Coulter counter (Coulter Electronics, U.S.A.). During the labeling period, cell numbers in the growing and growth-reduced culture increased 1.88 and 1.18-times, respectively.
brane in 0.05 M H2SO4 at 1 m A / c m for 120 min [8]. Individual GAG spots stained by Alcian blue were cut and the radioactivity measured by liquid scintillation counting.
Assay of GAG synthetase and HA synthetase inhibitor (HASI) in cell homogenates The activity of the enzyme system synthesizing GAGs was assayed by incorporation of UDP-[]4C]GIcUA into individual GAGs as described previously [9]. The activity of HASI was measured as described previously [9]. Briefly, homogenate of sample cells (HASI source) (1), homogenate of growing WI-38 CT-1 cells (HAS source) (2), and mixture of both homogenates (3) were assayed for HAS, HASI activity was calculated as percent inhibition of HAS: 100. ([A + B] - C ) / A • protein content of (l)/protein content of (2). A, B, a~d C are HAS activities of (1), (2) and (3), respectively. Colony" formation in soft agar 100 cells per 6 mm dish were contained in an upper layer of 1.5 ml growth medium for HUE cells containing 0.165% agarose. The lower layer composed 0.5% agarose in the same medium. After 16 days in culture, the number and the diameters of colonies were measured. Results
Incorporation of [~H]GlcN hlto GAGs by normal endothefial cells To exanaine GAG synthesis by HUE cells, the cells in growth and growth-retarded phases were labeled with [3H]GlcN for 24 h. GAGs were then isolated from the medium, surface (trypsin-releasable) and cell fraction. 0.3
Isolation and analysis of GAGs Medium containing secreted GAGs was removed and the cell layer was washed twice with Ca 2+, Mg 2+ free phosphate buffered saline (PBS). The first wash together with the medium was centrifuged at 1800 x g for 5 min at 4 ° C and the supernatant was pooled as the 'medium' fraction. Cells were harvested by trypsinization and centrifuged at 200 x g for 5 min at 4°C. The 'cell' fraction was obtained by washing the pellet with PBS and by centrifugation. The superrtatant of this step and the wash were combined as 'surface' fraction. GAGs in each fraction were isolated by alkali treatment, pronase digestion, and precipitation with cetylpyridinium chloride as described Matuoka and Mitsui [21]. GAG preparations were dissolved in an appropriate volume of water with authentic GAGs (HA, HS, chondroitin sulfate (CS) and dermatan sulfate (DS)), and then electrophoresed on a cellulose acetate mere-
[] Cell [] Surface
0
.p.r : f¢
W 0 -i
e.j
0.2
"=e ¢: O
0.1
2
O
O ¢)
0.0
GroiNing
Growth.Retarded
Growth Stats
Fig. 1. Incorporation of [3H]GIcN into GAGs by HUE cells• Cells were cultured in the presence of [3H]GIcN for 24 h under sufficient (growing) or low concentrations of ECGS (growth-retarded). Medium. surface (trypsin-releasable). and cell fractions were collected and GAGs were isolated as described in Materials and Methods•
338 MEDIUM
TOTAL
SURFACE
CELL
l i 0
50
100
SO
100 0
50
100 0
7 SO
100
PERCENTAGE OF GAGs
Fig. 2, Change in GAG synthesis by HUE cells with growth-retardation. Labeled GAGs isolated from growing (G) and growth-retarded (G-R) HUE cells as described in Fig, 1 were analyzed by electrophoresis on cellulose acetate membranes. The data were compared with those obtained from normal fibroblasts, WI-38 [8], The order of [3HIGIeN incorporation by both types of cells was similar.
The incorporation into GAGs by cells in the growth phase was higher in every fraction than by cells in the growth-retarded phase in medium containing a low concentration of ECGS (Fig. 1). In both cases, most labeled GAGs were recovered in the medium (above 80~), and [3H]GlcN incorporation into GAGs in surface and cellular GAGs was less than 10~, respectively. The composition of labeled GAGs was analyzed by electrophoresis on cellulose acetate membranes. The results are shown in Fig. 2 in comparison with fibreblasts [8]. In contrast to fibroblasts which produce much HA, little [3H]GlcN was incorporated into HA irrespective of the growth conditions: less than 1~ in the medium and surface fraction and only 2 - 4 ~ in the cell fraction. The percentage of HA in the cell fraction may be overestimated because some GAGs which were being sulfated may have similar mobility to HA on the acetate membrane. Most [3H]GIcN was incorporated into HS and CS/DS instead of HA by HUE cells. With regard to growth-reduction, HA synthesis was reduced in fibroblasts, while synthesis of CS/DS was reduced in endothelial cells.
Activities of GAG synthetic enzymes To ¢xamine whether the characteristics of GAG composRion in cultured cells were due to the activities of GAG synthetic enzymes, we measured the enzyme activities in cell homogenates. As shown in Table I, activi-
GAG synthetase activities in human fibroblasts and endothelial cells
HA HS CS/DS
Change in GA G synthesis by transformation ECV304 is a cell line obtained from HUE cells by spontaneous transformation [18]. This line maintains some characteristics of HUE; for example, it has a similar doubling time (approx. 24 h), and forms a cobblestone-like monolayer. However, this line had a high efficiency of colony formation in soft agar (Table lit). With transformation of fibroblasts, GAGs produced by cells tend to shift from sulfated GAGs to HA [22]. Because of the absence of HA synthesis in normal endothelial cells, we examined whether or not transformed endothelial cells synthesized HA. GAG synthesis in HUE and ECV304 cells was analyzed by incorporation of [3H]GIcN in the growth medium for HUE. Both cell lines in the growing phase incorporated [3H]GIcN to a similar extent ((2.6-2.8). 10 -t dpm/cell TABLE It
Activity of hyaluronic acid synthetase inhibitor (HASI)
TABLE !
GAG species
ties of both HS and CS/DS synthetase were of an order similar to that of normal fibroblasts, while the activity of HAS was very low in HUE cells. With regard to the regulation of HAS activity, we have found an inhibitor of HAS in fibroblasts, in which HA synthesis was reduced [9]. It is possible that the activity of HAS appeared low in HUE cells because of the high activity of HASI. However, when HASI activity was measured, the activity was very low in HUE cells (Table II).
UDP-[14C]GlcUA incorporation (pmol/h per mg protein) fibroblasts (Wl-38 a)
endothelial cells (HUE)
~.5±3.5 14.6±2.5 11.5±2.8
1.2±1.2 9.9±6.4 9.4±2.6
Mean+S.D. a Data from our previous paper [9].
Homogenates of various cell culture were assayed for HA synthetase inhibition by mixing them with homogenate of growing WI-3 8 CT-1 cells as described in Materials and Methods. HASt activity was calculated from duplicate assay. Growth state
Growing Growth-retarded
HASI activity (~) fibroblasts (WI-38 .)
endothelial cells (HUE)
13..3 62.2
5 9b
a Data from our previous paper [9]. b Cell growth was reduced by reduction of ECGS concentration.
339 TOTAL
HUE
,:cvao4
MEOIUM
~ I, s ?s os -'... 0
$0
SURFACE
CELL
I
100
0
S0
100
50
100
50
100
PERCENTAGE O F GAGS
Fig. 3. Change in GAG synthesis by trans~rmedendothelial cells. Normal(HUE) and transformed (ECV304)ceUsin the growing phase were labeled with[3H]GIcN ~ r 2 4 h, and GAGs wereanalyzed as descfibedinMaterialsandMethods.
in this experiment). However, in contrast to normal endothelial cells, ECV 304 cells incorporated [3H]GIcN into HA to a greater extent (6270) (Fig. 3). The incorporation into HS and C S / D S was lower compared with HUE cells (1670 and 1970 of total GAGs, respectively) (Fig. 3).
Discussion In the present work, we examined the changes in GAG synthesis in relation to growth and transformation of human endothelial cells. In fibroblasts, we previously showed that synthesis of HA was prominent in the growing phase and decreased with growth reduction [9] and passage number [10]. In HUE cells, however, synthesis of HA was very low (less than 1%) even in the rapidly growing phase of early passage cells (16-17 pdl, 2070 of life span) (Fig. 2). Porcine aortic endothelial cells also synthesized little HA compared with HS and C S / D S (unpublised observation). Alternative to HA, HUE cells synthesized mainly HS and CS/DS, and a decrease in CS/DS synthesis was marked with growth reduction (Fig. 2). Thus, modulation of GAG synthesis during cell proliferation is quite different in HUE cells from that in fibroblasts. In HUE cells, the absence of HA synthesis is probably due to the absence of HAS activity (Table I). We previously suggested that the activity of HAS is regulated by the inhibitor, HASI [9]. Although HAS activity was lower in HUE cells than in growth retarded fibroblasts (Table I), HASI activity was very low in HUE cells (Table If). These findings suggest that the absence of HAS activity in HUE cells was not due to the activity of its inhibitor but due to the absence of the enzyme itself. We, however, cannot rule out the possibility that tissue specific HASI which does not inhibit the HAS
TABLE lIl
Colony formation in soft agar HUE
ECV304
Efficiency (7o)
0
41
Diameter (ram)
-
+1
0.129 + 0.010
activity of fibroblasts exists in endothelial cells. Concerning species specificity of HASI, we previously found that HASI derived from BALB/3T3 cells [9] and Swiss 3T3 cells (unpublised observation) was active to HAS from human fibroblasts, suggesting that HASI is not species specific. In contrast to normal cells, transformed ECV304 synthesized high levels of HA (Fig. 3). Despite the difference in GAG composition, the growth of ECV304 cells in culture dishes resembled tha: of HUE ~'ells: both cell lines have similar doubling time, form monolayers, and their growth is curbed by contact inhibition. These findings suggest that the absence of HA synthesis in HUE cells did not always correlate with the formation of a monolayer. On the contrary, ECV304 cells showed highly efficient colony formation in soft agar, whereas HUE cells did not (Table IV), suggesting that elevation of HA synthesis in ECV304 has some correlation with the ability of colony formation. This was supported by the finding that PAE-20, an immortalized cell line of porcine aortic endothelial cells [23] with very low levels of HA synthesis did not form colonies in soft agar (unpublised observation). Histochemical studies by Ausprunk et al. showed that glycocalyx on the lumenal coat of the endothelium was not removed by the degradation enzyme specific for HA [24,25], suggesting that HA production by endothelial cells in vivo may be very low. In the present work, we showed that HA synthesis was extremely low (or absent) in normal endothelial cells. The biological significance of this absence in HUE cells has not yet been clarified, but it might be important for maintaining strict integrity of endothelial cell monolayer in blood vessels, because recent study of West and Kumer showed that exogenously added high-molecular-weight HA disrupted newly formed monolayer of endothelial cells in vitro [26]. Furthermore, several reports suggested that HA has some roles in angiogenesis: degradation products of HA stimulated angiogenesis in vivo [26] and in vitro [27], and epithelial tissues synthesizing much HA caused avascularity in limb buds of chick embryo [28]. Thus, the absence of HA synthesis in endothelial cells may be prerequisite for the control of proliferation of these cells by HA. Furthermore, supressed expression of HAS and its inhibitor in normal
340 endothelial cells might be critical to maintain the vascular network. It may therefore be important to elucidate the biological significance of the absence of HA synthesis in normal endothelial cells and induction of HAS in transformed endothelial cells.
Acknowledgments We thank Ms. K. Takahashi (National Defence Medical College) for her kind supply of ECV 304 cells. We also thank Dr. K. Matuoka for useful advice on analysis of GAG synthesis and Dr. T. Imamura for helpful discussion. This work was supported in part by a project grant for Basic Technology for Future Industry from the Ministry of International Trade and Industry of Japan,
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