Gerontology 1992;38(suppl 1):36—42
Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Department of Cardiology, Jichi Medical School, Tochigi, and Department of Medicine and Geriatrics, Kochi Medical School, Kochi, Japan
Key Words Porcine aortic endothelial cells Glycosaminoglycans Transforming growth factors
Human Platelet-Derived Transforming Growth Factor-ß Stimulates Synthesis of Glycosaminoglycans in Cultured Porcine Aortic Endothelial Cells
Abstract ■
Human platelet-derived transforming growth factor-P (TGFp) stimulated the incorporation of [35S]sulfate into glycosami noglycans (GAGs) both in the medium and on the cell surface of cultured aortic endothelial cells in a dose- and time-depen dent manner. TGF-P inhibited the suppression of GAG syn thesis by other cytokines. TGF-p increased the protein synthe sis, while reducing the DNA synthesis by endothelial cells. The proportion of anticoagulant heparan sulfate in total GAGs was reduced. Thus, TGF-p affectd the endothelial GAG me tabolism both quantitatively and qualitatively.
Transforming growth factor-P (TGF-P) has profound effects on all cell types making up the vasculature, including endothelial cells. It plays a prominent role not only in endothelial cell growth or angiogenesis, but also matrix production by endothelial cells. TGF-P has been shown to increase synthesis and to de crease degradation of matrix proteins, such as
fibronectin, laminin and collagen [1], The growth, architecture and migration of endo thelial cells is known to be influenced by the composition of the extracellular matrix on which the cells are attached. On the other hand, pericellular glycosaminoglycans (GAGs), particularly heparan sulfate, another matrix component of endothelial cells, have been implicated in diverse molecular pro cesses during interactions between blood
This work was supported in part by research grants No. 2670398 from the Ministry of Education. Science and Culture, and No. 2A-1 for Cardiovascular Disease from the Ministry of Health and Welfare of Japan.
Kazuyuki Shimada, MD Department of Cardiology Jichi Medical School Minamikawachi, Kawachigun Tochigi 329-04 (Japan)
Introduction
© 1992 S. Karger AG, Basel 0304-324X/92/ 0387-003652.75/0
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Masashi Kobayashia Kazuyuki Shimada'0 Toshio Ozawac
components and the blood vessel wall, includ ing heparin-like anticoagulant properties of the endothelium. Heparin-like compounds in the endothelial cells can interact with anti thrombin III (AT III), thereby accelerating the thrombin (or Xa) inactivation by the protease inhibitor [2], Although TGF-ß is known to enhance the synthesis of extracellular matrix components including proteoglycans in mesenchymal cells, such as vascular smooth muscle cells and Fibroblasts [3, 4], little is known about its effects on GAG synthesis by endothelial cells. We reported earlier that macrophage-derived cytokines, such as human recombimant tu mor necrosis factor (rTNF-a) and interleukin 1 (rIL-lß), suppressed the synthesis of endo thelial cell surface heparin-like compounds, resulting in a reduction in the anticoagulant activity of the cells [5], Since TGF-ß is stored in the a-granules of platelets and released to the site of the vascular injury [6], its effects on the anticoagulant molecules of the endothelial cells are of particular interest. In the present study, we examined the influence of TGF-ß on the synthesis and anticoagulant properties of endothelial GAGs.
concentrations of cytokines. [3H]Leucine or [3H]thymidine incorporation by PAECs was measured as pre viously described [5], Biosynthesis o f Endothelial GAGs. Endothelial GAGs were metabolically labeled by incubating cell cultures with 50 gCi/ml of [35S]sulfate for 24 h in the presence or absence of cytokines. Labeled GAGs and proteoglycans were isolated from cells and medium as previously described [7], Briefly, following removal of free [35S]sulfate in the medium by Column PD-10 (Pharmacia Fine Chemicals, Uppsala, Sweden), la beled macromolecules (‘medium’ fraction) were pre cipitated with cetylpyridinium chloride. The washed cell layers were trypsinized, and resultant cell suspen sions were centrifuged to obtain a cell pellet (‘cell’ frac tion) and a supernatant (‘cell surface’ fraction). [35S]GAGs in cell surface fractions were isolated by cetylpyridinium chloride precipitation before or after treatments with chondroitin ABC lyase, following proteolytic digestion with pronase. Heparan sulfate was determined as chondroitin-ABC-lyase-resistant GAGs. The radioactivity remaining on the plates (‘plate’ fractions) was recovered in 0.1 N NaOH solu tion. The radioactivities in each fraction were mea sured by liquid scintillation counting. The ability of cell surface heparan sulfate to interact with AT III was also investigated by affinity chromatography on the immobilized proteinase inhibitor as previously de scribed [7], Protein contents in the cell fraction were measured according to Lowry et al. [9],
Results Materials. rIL-lp and rTNF-a were from Otsuka Pharmaceutical, Tokushima, Japan, and Genzyme, Boston, Mass., USA, respectively. Purified human platelet-derived TGF-P was purchased from Wako, Osaka, Japan. Porcine AT III was purified as pre viously described [7]. Endothelial Cel! Cultures. Porcine aortic endothe lial cells (PAECs) were cultured in gelatin-precoated 35-mm Petri dishes containing RPMI-1640 medium, supplemented with 30% fetal calf serum and antibiot ics as previously described [8]. For treatment with cytokines, when PAECs were grown close to con fluence, the conditioned medium was replaced with fresh culture medium containing 20% fetal calf serum. Incubations were further continued for the indicated periods of time in the presence or absence of various
The time dependency of the incorporation of 35S radioactivity into GAGs from both cell surface and medium fractions in PAECs treated with 2 ng/ml of TGF-p is shown in fig ure 1. The cell-surface-associated [35S]GAGs were progressively increased with time up to approximately 140% of control by 24 h (fig. la). Further incubations with TGF-P re sulted in a decline in 35S radioactivity toward a control level. [35S]GAGs in the medium fractions were similarly increased up to ap proximately 140% of control (fig. lb). The maximal effect was achieved after 12 h of incubation.
37
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Materials and Methods
tion period. The radioactivity incorporated into total GAGs isolated from cell surface (a) and medium (b) fractions was measured. Data represent means ± SEM of five to six determinations of two separate experi ments.
Table 1. Effect of TGF-P, IL-ip and TNF-a on [35S]sulfate incorporation into GAGs in various frac tions of PAEC cultures
When PAECs were incubated with various concentrations of TGF-P for 24 h, TGF-P caused a dose-dependent increase in the in corporation of radioactivity into cell surface GAGs (Fig. 2a). Maximal stimulation was noted by treatment with 2 ng/ml of TGF-P, resulting in an increase in radioactivities in corporated by approximately 80%. Half-max imal effects were achieved at approximately 0.2 ng/ml of TGF-p. The stimulatory effects on medium GAGs following incubations of PAECs with various concentrations of TGF-P for 12 h was also progressively enhanced with increasing concentrations of TGF-p up to at least 20 ng/ml (fig. 2b). The extent of the stim ulatory effect of TGF-P varied from experi ment to experiment, ranging from 10 to 80% increase in the synthesis of cell surface GAGs. Furthermore, its response to TGF-P was lost when subcultures were repeated more than
Treatment
35S-labeled GAGs
35S-labeled compounds
Control TGF-ß IL-lß TNF-a TGF-ß + IL-lß TGF-ß + TNF-a
387±9 518 ± 5 268 ±26 247 ± 5 355 ± 16 308 + 12
72 ± 6 79 ± 1 56 ± 7 64 ± 1 65 ± 5 70±2
9± 1 19 ± 7 9±3 10 ± 2 9± 1 7±3
PAECs were incubated with 50 pCi/ml of [35S]sulfate in the absence or presence of 2 ng/ml of TGF-p, 100 ng/ml of IL-ip or 5 ng/ml of TNF-a. alone or in combination for 24 h. The radioactivity incorporated into 35S-labeled GAGs or compounds in various frac tions of PAEC cultures was assessed. Data (dpm/pg cell protein) represent means ± SEM of four determi nations of two separate experiments.
38
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Fig. 1. Time- and dose-dependent stimulation of biosynthesis of PAEC GAGs by TGF-p. PAECs were incubated in the absence or presence of 2 ng/ml of TGF-P for the indicated periods of time. 50 pCi/ml of [35S]sulfate was added 6 h before the end of the incuba
o
fe. 0
O)
n
\
1
XJ
ITO
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00
CO
8
T G F - p Concentration (n g /m l)
Fig. 2. PAECs were incubated in the absence or presence of various concentrations of TGF-P for 24 (a) or 12 h (b). 50 pCi/ml of [35C]sulfate was added 6 h before the end of the incubation period. The radioac
tivity incorporated into total GAGs isolated from cell surface (a) and medium (b) fractions was measured. Data represent means and ranges of two determina tions of two separate experiments.
twice after explantation from aorta (data not shown). The incorporation of [35S]sulfate into com pounds in various fractions of PAEC cultures treated with TGF-p, IL-ip and TNF-a was compared with that in control cells (table 1). 35SC>4-labeled compounds from cell surface fractions were increased by treatment with TGF-P alone. On the other hand, treatment with IL-ip or TNF-a alone decreased the amount of 35S04-labeled GAGs from cell sur face fractions. In the presence of TGF-p, how ever, the reduction in cell surface 35S 0 4labeled GAGs in IL-ip- or TNF-a-treated cells was substantially inhibited. The effects of TGF-P on cell numbers, pro tein contents or protein and DNA synthesis were assessed and expressed as percent of control in table 2. The incorporation of [3H]leucine into TCA-insoluble materials was
slightly increased after incubation with TGFP for 24 h. In contrast, [3H]thymidine incor poration into DNA was progressively reduced with time. The proportion of heparan sulfate in total GAGs (fig. 3a) and the proportion of heparan sulfate with high affinity for AT III in total heparan sulfates (fig. 3b) from cell surface fractions were then assessed. The proportion of heparan sulfate was similar in control and TGF-p-treated cells. When 35S-labeled cell surface heparan sulfate was fractionated by affinity chromatography on antithrombin-af finity gel, the proportion of the high-affinity material that specifically adsorbed to the anti thrombin-affinity column (0.5-2 M NaCl fractions) was reduced approximately by 25% in TGF-p-treated cells.
39
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TG F— p Concentration (n g /m l)
95± 4%
Fig. 3. Effect of TGF-[3 on the composition of GAGs and on the proportion of anticoagulant heparan sulfate in total heparan sulfates from cell surface frac tion. PAECs were incubated in the absence or presence of 2 ng/ml of TGF-P for 24 h. 50 gCi/ml of [35S]sulfate was added 6 h before the end of the incubation period, a The proportion of heparan sulfate (chondroitinABC-lyase-resistant materials) in cell surface fractions was expressed as percent of total GAGs (93 ± 4 and 140 ± 2 dpm/gg cell protein, control and TGF-Ptreated cells, respectively). Data represent means ± SEM of five to six determinations of two separate experiments, b 35S-labeled cell surface heparan sulfate was fractionated by affinity chromatography on anti thrombin-affinity gel. The proportion of the highaffinty material that specifically adsorbed to the anti thrombin-affinity column (0.5-2 M NaCl fractions) was expressed as percent of total heparan sulfates (88 ± 4 and 130 ± 2 dpm/pg cell protein, control and TGF-p-treatcd cells, respectively). Data represent means ± SEM of four determinations of two separate experiments.
93 ± 39$
Table 2. Effect of TGF-P on numbers, protein contents, protein synthesis and DNA synthesis of PAECs
Incubation time with TGF-ß, h
Cell number
Protein content
[3H] thymidine [3H]leucine incorporation incorporation
24 49
114 ± 3 91 ± 2
109 ± 8 106 ± 2
140 ±12 106 ±31
65 ± 3 28 ± 6
Discussion
Our data showed that human plateletderived TGF-P exerted a stimulatory effect on GAG synthesis by cultured PAECs, in con trast with the previously reported study [3].
40
TGF-P increased the syntheses of both cellsurface-associated and soluble GAGs. The ex tent of the stimulatory effect of TGF-P was substantially varied in repeated experiments. Furthermore, only the primary and secondpassaged cells responded to TGF-p. These
Kobayashi/Shimada/Ozawa
Endothelial Cell Glycosaminoglycans
Downloaded by: Kings's College London 137.73.144.138 - 11/3/2017 12:12:58 PM
Cell numbers were counted using a hemocytometer. Protein contents and the radioactivities incorporated into proteins or DNAs were mea sured as described in Materials and Methods. Data are expressed as per cent of untreated control cells. Numbers, protein contents and [3H]leucine or [3H]thymidine incorporation of control cells were 7.04-12.1 X 105 cells/dish, 131-201 gg/dish, 11,039-12,000 dpm/dish and 4,194-37,085 dpm/dish, respectively. Each value represents the mean ± SEM of two separate experiments.
results indicate that the receptor activity or intracellular signal transduction in endothe lial cells for TGF-p appears to be influenced by cell culture conditions [10]. This may ex plain the discrepancy observed between our data and previous results [3], which were ob tained in endothelial cells passaged several times. Macrophage-derived cytokines (rIL-ip and TNF-a) reduced the amount of GAGs associated with the cell surface of endothelial cells, in agreement with the previously re ported data [5]. TGF-P appears to antagonize the inhibitory effects of these other cytokines. Thus, various cytokines released from plate lets and macrophages may interact with each other in regulating the GAG metabolism of vascular endothelial cells. The mechanism by which TGF-p stimu lates GAG production is presently unknown. It may be accompanied by an increase in the synthesis of various cellular proteins, includ ing extracellular matrix components [1], The increase in the incorporation of radiolabeled leucine may support this concept. TGF-P ap pears to stimulate the synthesis of various types of GAGs to a similar extent, since the proportion of each GAG on the cell surface was not changed. AT III affinity chromatogra phy, however, showed that TGF-p somewhat reduced the proportion of heparan sulfate
with high affinity for AT III in total heparan sulfates. Although TGF-p increased the amount of heparan sulfate associated with the cell surface, it may not enhance the heparin like anticoagulant activity of endothelial cells. In fact, no significant differences were ob served in AT III binding between control and TGF-p-treated cells (data not shown). Thus, TGF-p may affect the GAG metabolism of endothelial cells both quantitatively and qual itatively [3, 4, 11, 12]. The increased production of GAGs by TGF-p may affect the components of extra cellular matrix of endothelial cells, thereby influencing the growth, migration or architec ture of underlying vascular smooth muscle cells as well as endothelial cells themselves [1], Furthermore, a certain class of TGF-p receptors is known to be related to heparan sulfate proteoglycan itself [ 13], suggesting the potential positive feedback mechanisms. Thus, the physiological significance of the present finding in vascular biology should be determined in the future experiments.
Acknowledgments The authors thank Otsuka Pharmaceutical for gen erously providing human rIL-lp, and Mrs. Miki Kurose and Ms. Minori Hisada for their expert technical assistance.
1 Roberts AB, Sporn MB: Regulation of endothelial cell growth, architec ture, and matrix synthesis by TGFß. Am Rev Respir Dis 1989; 140: 1126-1128. 2 Shimada K, Ozawa T: The anticoag ulant role of heparin-like molecules in the endothelial cell surface in he mostasis. Endothelial heparin-like molecules. Acta Haematol Jpn 1986;49:1604-1609.
3 Chen JK, Hoshi H, McKeehan WL: Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult ar terial smooth muscle cells. Proc Natl Acad Sci USA 1987;84:5287-5291. 4 Bassols A, Massague J: Transform ing growth factor beta regulates the expression and structure of extracel lular matrix chondroitin/dermatan sulfate proteoglycans. J Biol Chem 1988;263:3039-3045.
5 Kobayashi M, Shimada K, Ozawa T: Human recombinant interleukin1(i- and tumor necrosis factor amediated suppression of heparin like compounds on cultured porcine aortic endothelial cells. J Cell Phys iol 1990;144:383-390. 6 Sporn MB, Roberts AB: Transform ing growth factor-beta. Multiple ac tions and potential clinical applica tions. JAMA 1989;262:938-941.
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References
9 Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 10 Gamble J, Vadas MA: Endothelial adhesiveness for blood neutrophils is inhibited by transforming growth factor-p. Science 1988;242:97-99. 11 Breuer B, Schmidt G, Kresse H: Non-uniform influence of trans forming growth factor-p on the bio synthesis of different forms of small chondroitin sulphate/dermatan sul phate proteoglycan. Biochem J 1990;269:551-554.
12 Uhlman DL, Mooradian DL, Furcht LT, et al: The effect of trans forming growth factor-p! on glycos aminoglycan production by human marrow cultures. Exp Hematol 1990;18:1121-1125. 13 Cheifetz S. Andres JL, Massague J: The transforming growth factorbeta receptor type III is a membrane proteoglycan. Domain structure of the receptor. J Biol Chem 1988;263: 16984-16991.
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7 Shimada K. Ozawa T: Modulation of glycosaminoglycan production and antithrombin III binding by cul tured aortic endothelial cells treated with 4-methylumbelliferyl-ß-Z)-xyloside. Arteriosclerosis 1987;7:627— 636. 8 Shimada K, Ozawa T: Evidence that cell surface heparan sulfate is in volved in the high affinity thrombin binding to cultured porcine aortic endothelial cells. J Clin Invest 1985; 75:1308-1316.
Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Department of Cardiology, Jichi Medical School, Tochigi, and Department of Medicine and Geriatrics, Kochi Medical School, Kochi, Japan
Key Words Porcine aortic endothelial cells Glycosaminoglycans Transforming growth factors
Human Platelet-Derived Transforming Growth Factor-ß Stimulates Synthesis of Glycosaminoglycans in Cultured Porcine Aortic Endothelial Cells
Abstract ■
Human platelet-derived transforming growth factor-P (TGFp) stimulated the incorporation of [35S]sulfate into glycosami noglycans (GAGs) both in the medium and on the cell surface of cultured aortic endothelial cells in a dose- and time-depen dent manner. TGF-P inhibited the suppression of GAG syn thesis by other cytokines. TGF-p increased the protein synthe sis, while reducing the DNA synthesis by endothelial cells. The proportion of anticoagulant heparan sulfate in total GAGs was reduced. Thus, TGF-p affectd the endothelial GAG me tabolism both quantitatively and qualitatively.
Transforming growth factor-P (TGF-P) has profound effects on all cell types making up the vasculature, including endothelial cells. It plays a prominent role not only in endothelial cell growth or angiogenesis, but also matrix production by endothelial cells. TGF-P has been shown to increase synthesis and to de crease degradation of matrix proteins, such as
fibronectin, laminin and collagen [1], The growth, architecture and migration of endo thelial cells is known to be influenced by the composition of the extracellular matrix on which the cells are attached. On the other hand, pericellular glycosaminoglycans (GAGs), particularly heparan sulfate, another matrix component of endothelial cells, have been implicated in diverse molecular pro cesses during interactions between blood
This work was supported in part by research grants No. 2670398 from the Ministry of Education. Science and Culture, and No. 2A-1 for Cardiovascular Disease from the Ministry of Health and Welfare of Japan.
Kazuyuki Shimada, MD Department of Cardiology Jichi Medical School Minamikawachi, Kawachigun Tochigi 329-04 (Japan)
Introduction
© 1992 S. Karger AG, Basel 0304-324X/92/ 0387-003652.75/0
Downloaded by: Kings's College London 137.73.144.138 - 11/3/2017 12:12:58 PM
Masashi Kobayashia Kazuyuki Shimada'0 Toshio Ozawac
components and the blood vessel wall, includ ing heparin-like anticoagulant properties of the endothelium. Heparin-like compounds in the endothelial cells can interact with anti thrombin III (AT III), thereby accelerating the thrombin (or Xa) inactivation by the protease inhibitor [2], Although TGF-ß is known to enhance the synthesis of extracellular matrix components including proteoglycans in mesenchymal cells, such as vascular smooth muscle cells and Fibroblasts [3, 4], little is known about its effects on GAG synthesis by endothelial cells. We reported earlier that macrophage-derived cytokines, such as human recombimant tu mor necrosis factor (rTNF-a) and interleukin 1 (rIL-lß), suppressed the synthesis of endo thelial cell surface heparin-like compounds, resulting in a reduction in the anticoagulant activity of the cells [5], Since TGF-ß is stored in the a-granules of platelets and released to the site of the vascular injury [6], its effects on the anticoagulant molecules of the endothelial cells are of particular interest. In the present study, we examined the influence of TGF-ß on the synthesis and anticoagulant properties of endothelial GAGs.
concentrations of cytokines. [3H]Leucine or [3H]thymidine incorporation by PAECs was measured as pre viously described [5], Biosynthesis o f Endothelial GAGs. Endothelial GAGs were metabolically labeled by incubating cell cultures with 50 gCi/ml of [35S]sulfate for 24 h in the presence or absence of cytokines. Labeled GAGs and proteoglycans were isolated from cells and medium as previously described [7], Briefly, following removal of free [35S]sulfate in the medium by Column PD-10 (Pharmacia Fine Chemicals, Uppsala, Sweden), la beled macromolecules (‘medium’ fraction) were pre cipitated with cetylpyridinium chloride. The washed cell layers were trypsinized, and resultant cell suspen sions were centrifuged to obtain a cell pellet (‘cell’ frac tion) and a supernatant (‘cell surface’ fraction). [35S]GAGs in cell surface fractions were isolated by cetylpyridinium chloride precipitation before or after treatments with chondroitin ABC lyase, following proteolytic digestion with pronase. Heparan sulfate was determined as chondroitin-ABC-lyase-resistant GAGs. The radioactivity remaining on the plates (‘plate’ fractions) was recovered in 0.1 N NaOH solu tion. The radioactivities in each fraction were mea sured by liquid scintillation counting. The ability of cell surface heparan sulfate to interact with AT III was also investigated by affinity chromatography on the immobilized proteinase inhibitor as previously de scribed [7], Protein contents in the cell fraction were measured according to Lowry et al. [9],
Results Materials. rIL-lp and rTNF-a were from Otsuka Pharmaceutical, Tokushima, Japan, and Genzyme, Boston, Mass., USA, respectively. Purified human platelet-derived TGF-P was purchased from Wako, Osaka, Japan. Porcine AT III was purified as pre viously described [7]. Endothelial Cel! Cultures. Porcine aortic endothe lial cells (PAECs) were cultured in gelatin-precoated 35-mm Petri dishes containing RPMI-1640 medium, supplemented with 30% fetal calf serum and antibiot ics as previously described [8]. For treatment with cytokines, when PAECs were grown close to con fluence, the conditioned medium was replaced with fresh culture medium containing 20% fetal calf serum. Incubations were further continued for the indicated periods of time in the presence or absence of various
The time dependency of the incorporation of 35S radioactivity into GAGs from both cell surface and medium fractions in PAECs treated with 2 ng/ml of TGF-p is shown in fig ure 1. The cell-surface-associated [35S]GAGs were progressively increased with time up to approximately 140% of control by 24 h (fig. la). Further incubations with TGF-P re sulted in a decline in 35S radioactivity toward a control level. [35S]GAGs in the medium fractions were similarly increased up to ap proximately 140% of control (fig. lb). The maximal effect was achieved after 12 h of incubation.
37
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Materials and Methods
tion period. The radioactivity incorporated into total GAGs isolated from cell surface (a) and medium (b) fractions was measured. Data represent means ± SEM of five to six determinations of two separate experi ments.
Table 1. Effect of TGF-P, IL-ip and TNF-a on [35S]sulfate incorporation into GAGs in various frac tions of PAEC cultures
When PAECs were incubated with various concentrations of TGF-P for 24 h, TGF-P caused a dose-dependent increase in the in corporation of radioactivity into cell surface GAGs (Fig. 2a). Maximal stimulation was noted by treatment with 2 ng/ml of TGF-P, resulting in an increase in radioactivities in corporated by approximately 80%. Half-max imal effects were achieved at approximately 0.2 ng/ml of TGF-p. The stimulatory effects on medium GAGs following incubations of PAECs with various concentrations of TGF-P for 12 h was also progressively enhanced with increasing concentrations of TGF-p up to at least 20 ng/ml (fig. 2b). The extent of the stim ulatory effect of TGF-P varied from experi ment to experiment, ranging from 10 to 80% increase in the synthesis of cell surface GAGs. Furthermore, its response to TGF-P was lost when subcultures were repeated more than
Treatment
35S-labeled GAGs
35S-labeled compounds
Control TGF-ß IL-lß TNF-a TGF-ß + IL-lß TGF-ß + TNF-a
387±9 518 ± 5 268 ±26 247 ± 5 355 ± 16 308 + 12
72 ± 6 79 ± 1 56 ± 7 64 ± 1 65 ± 5 70±2
9± 1 19 ± 7 9±3 10 ± 2 9± 1 7±3
PAECs were incubated with 50 pCi/ml of [35S]sulfate in the absence or presence of 2 ng/ml of TGF-p, 100 ng/ml of IL-ip or 5 ng/ml of TNF-a. alone or in combination for 24 h. The radioactivity incorporated into 35S-labeled GAGs or compounds in various frac tions of PAEC cultures was assessed. Data (dpm/pg cell protein) represent means ± SEM of four determi nations of two separate experiments.
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Endothelial Cell Glycosaminoglycans
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Fig. 1. Time- and dose-dependent stimulation of biosynthesis of PAEC GAGs by TGF-p. PAECs were incubated in the absence or presence of 2 ng/ml of TGF-P for the indicated periods of time. 50 pCi/ml of [35S]sulfate was added 6 h before the end of the incuba
o
fe. 0
O)
n
\
1
XJ
ITO
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00
CO
8
T G F - p Concentration (n g /m l)
Fig. 2. PAECs were incubated in the absence or presence of various concentrations of TGF-P for 24 (a) or 12 h (b). 50 pCi/ml of [35C]sulfate was added 6 h before the end of the incubation period. The radioac
tivity incorporated into total GAGs isolated from cell surface (a) and medium (b) fractions was measured. Data represent means and ranges of two determina tions of two separate experiments.
twice after explantation from aorta (data not shown). The incorporation of [35S]sulfate into com pounds in various fractions of PAEC cultures treated with TGF-p, IL-ip and TNF-a was compared with that in control cells (table 1). 35SC>4-labeled compounds from cell surface fractions were increased by treatment with TGF-P alone. On the other hand, treatment with IL-ip or TNF-a alone decreased the amount of 35S04-labeled GAGs from cell sur face fractions. In the presence of TGF-p, how ever, the reduction in cell surface 35S 0 4labeled GAGs in IL-ip- or TNF-a-treated cells was substantially inhibited. The effects of TGF-P on cell numbers, pro tein contents or protein and DNA synthesis were assessed and expressed as percent of control in table 2. The incorporation of [3H]leucine into TCA-insoluble materials was
slightly increased after incubation with TGFP for 24 h. In contrast, [3H]thymidine incor poration into DNA was progressively reduced with time. The proportion of heparan sulfate in total GAGs (fig. 3a) and the proportion of heparan sulfate with high affinity for AT III in total heparan sulfates (fig. 3b) from cell surface fractions were then assessed. The proportion of heparan sulfate was similar in control and TGF-p-treated cells. When 35S-labeled cell surface heparan sulfate was fractionated by affinity chromatography on antithrombin-af finity gel, the proportion of the high-affinity material that specifically adsorbed to the anti thrombin-affinity column (0.5-2 M NaCl fractions) was reduced approximately by 25% in TGF-p-treated cells.
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TG F— p Concentration (n g /m l)
95± 4%
Fig. 3. Effect of TGF-[3 on the composition of GAGs and on the proportion of anticoagulant heparan sulfate in total heparan sulfates from cell surface frac tion. PAECs were incubated in the absence or presence of 2 ng/ml of TGF-P for 24 h. 50 gCi/ml of [35S]sulfate was added 6 h before the end of the incubation period, a The proportion of heparan sulfate (chondroitinABC-lyase-resistant materials) in cell surface fractions was expressed as percent of total GAGs (93 ± 4 and 140 ± 2 dpm/gg cell protein, control and TGF-Ptreated cells, respectively). Data represent means ± SEM of five to six determinations of two separate experiments, b 35S-labeled cell surface heparan sulfate was fractionated by affinity chromatography on anti thrombin-affinity gel. The proportion of the highaffinty material that specifically adsorbed to the anti thrombin-affinity column (0.5-2 M NaCl fractions) was expressed as percent of total heparan sulfates (88 ± 4 and 130 ± 2 dpm/pg cell protein, control and TGF-p-treatcd cells, respectively). Data represent means ± SEM of four determinations of two separate experiments.
93 ± 39$
Table 2. Effect of TGF-P on numbers, protein contents, protein synthesis and DNA synthesis of PAECs
Incubation time with TGF-ß, h
Cell number
Protein content
[3H] thymidine [3H]leucine incorporation incorporation
24 49
114 ± 3 91 ± 2
109 ± 8 106 ± 2
140 ±12 106 ±31
65 ± 3 28 ± 6
Discussion
Our data showed that human plateletderived TGF-P exerted a stimulatory effect on GAG synthesis by cultured PAECs, in con trast with the previously reported study [3].
40
TGF-P increased the syntheses of both cellsurface-associated and soluble GAGs. The ex tent of the stimulatory effect of TGF-P was substantially varied in repeated experiments. Furthermore, only the primary and secondpassaged cells responded to TGF-p. These
Kobayashi/Shimada/Ozawa
Endothelial Cell Glycosaminoglycans
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Cell numbers were counted using a hemocytometer. Protein contents and the radioactivities incorporated into proteins or DNAs were mea sured as described in Materials and Methods. Data are expressed as per cent of untreated control cells. Numbers, protein contents and [3H]leucine or [3H]thymidine incorporation of control cells were 7.04-12.1 X 105 cells/dish, 131-201 gg/dish, 11,039-12,000 dpm/dish and 4,194-37,085 dpm/dish, respectively. Each value represents the mean ± SEM of two separate experiments.
results indicate that the receptor activity or intracellular signal transduction in endothe lial cells for TGF-p appears to be influenced by cell culture conditions [10]. This may ex plain the discrepancy observed between our data and previous results [3], which were ob tained in endothelial cells passaged several times. Macrophage-derived cytokines (rIL-ip and TNF-a) reduced the amount of GAGs associated with the cell surface of endothelial cells, in agreement with the previously re ported data [5]. TGF-P appears to antagonize the inhibitory effects of these other cytokines. Thus, various cytokines released from plate lets and macrophages may interact with each other in regulating the GAG metabolism of vascular endothelial cells. The mechanism by which TGF-p stimu lates GAG production is presently unknown. It may be accompanied by an increase in the synthesis of various cellular proteins, includ ing extracellular matrix components [1], The increase in the incorporation of radiolabeled leucine may support this concept. TGF-P ap pears to stimulate the synthesis of various types of GAGs to a similar extent, since the proportion of each GAG on the cell surface was not changed. AT III affinity chromatogra phy, however, showed that TGF-p somewhat reduced the proportion of heparan sulfate
with high affinity for AT III in total heparan sulfates. Although TGF-p increased the amount of heparan sulfate associated with the cell surface, it may not enhance the heparin like anticoagulant activity of endothelial cells. In fact, no significant differences were ob served in AT III binding between control and TGF-p-treated cells (data not shown). Thus, TGF-p may affect the GAG metabolism of endothelial cells both quantitatively and qual itatively [3, 4, 11, 12]. The increased production of GAGs by TGF-p may affect the components of extra cellular matrix of endothelial cells, thereby influencing the growth, migration or architec ture of underlying vascular smooth muscle cells as well as endothelial cells themselves [1], Furthermore, a certain class of TGF-p receptors is known to be related to heparan sulfate proteoglycan itself [ 13], suggesting the potential positive feedback mechanisms. Thus, the physiological significance of the present finding in vascular biology should be determined in the future experiments.
Acknowledgments The authors thank Otsuka Pharmaceutical for gen erously providing human rIL-lp, and Mrs. Miki Kurose and Ms. Minori Hisada for their expert technical assistance.
1 Roberts AB, Sporn MB: Regulation of endothelial cell growth, architec ture, and matrix synthesis by TGFß. Am Rev Respir Dis 1989; 140: 1126-1128. 2 Shimada K, Ozawa T: The anticoag ulant role of heparin-like molecules in the endothelial cell surface in he mostasis. Endothelial heparin-like molecules. Acta Haematol Jpn 1986;49:1604-1609.
3 Chen JK, Hoshi H, McKeehan WL: Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult ar terial smooth muscle cells. Proc Natl Acad Sci USA 1987;84:5287-5291. 4 Bassols A, Massague J: Transform ing growth factor beta regulates the expression and structure of extracel lular matrix chondroitin/dermatan sulfate proteoglycans. J Biol Chem 1988;263:3039-3045.
5 Kobayashi M, Shimada K, Ozawa T: Human recombinant interleukin1(i- and tumor necrosis factor amediated suppression of heparin like compounds on cultured porcine aortic endothelial cells. J Cell Phys iol 1990;144:383-390. 6 Sporn MB, Roberts AB: Transform ing growth factor-beta. Multiple ac tions and potential clinical applica tions. JAMA 1989;262:938-941.
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References
9 Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 10 Gamble J, Vadas MA: Endothelial adhesiveness for blood neutrophils is inhibited by transforming growth factor-p. Science 1988;242:97-99. 11 Breuer B, Schmidt G, Kresse H: Non-uniform influence of trans forming growth factor-p on the bio synthesis of different forms of small chondroitin sulphate/dermatan sul phate proteoglycan. Biochem J 1990;269:551-554.
12 Uhlman DL, Mooradian DL, Furcht LT, et al: The effect of trans forming growth factor-p! on glycos aminoglycan production by human marrow cultures. Exp Hematol 1990;18:1121-1125. 13 Cheifetz S. Andres JL, Massague J: The transforming growth factorbeta receptor type III is a membrane proteoglycan. Domain structure of the receptor. J Biol Chem 1988;263: 16984-16991.
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Endothelial Cell Glycosaminoglycans
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7 Shimada K. Ozawa T: Modulation of glycosaminoglycan production and antithrombin III binding by cul tured aortic endothelial cells treated with 4-methylumbelliferyl-ß-Z)-xyloside. Arteriosclerosis 1987;7:627— 636. 8 Shimada K, Ozawa T: Evidence that cell surface heparan sulfate is in volved in the high affinity thrombin binding to cultured porcine aortic endothelial cells. J Clin Invest 1985; 75:1308-1316.
