EXPERIMENTAL
CELL
RESEARCH
194,154-156
Vitronectin-Induced MICHITAKA Department
(1991)
SHORT NOTE Haptotaxis of Vascular Smooth Muscle Cells in Vitro
NAITO,’ TOSHIO HAYASHI, CHIAKI FUNAKI, MASAFUMI KANICHI ASAI, KAZUYOSHI YAMADA, AND FUMIO KUZ~A
of Geriatrics,
Nagoya University
School of Medicine,
65 Tsuruma-cho,
Showa-ku,
MATERIALS Vitronectin, a multifunctional glycoprotein present in the plasma and interstitial tissues, has recently been found to be localized in atherosclerotic lesions. In this study we examined the effects of vitronectin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to fluid-phase vitronectin in a concentration-dependent fashion. The cells also migrated to membrane filter surfaces precoated with vitronectin for a few minutes in the absence of additional vitronectin in the fluid phase, suggesting that this substance binds easily to the filters and stimulates cell migration by haptotaxis under the conditions described. These observations suggest that vitronectin deposited in the intima may be involved in the pathogenesis of atherosclerosis by recruiting smooth muscle cells from the media into the 0 1991 Academic Press. Inc. intima.
INTRODUCTION
Vitronectin, also called S-protein or serum-spreading factor, is a multifunctional glycoprotein found in the plasma and interstitial tissues [ 1,2]. It occurs in human plasma and serum at 200-300 pg/ml [ 21. Vitronectin regulates blood coagulation by preventing the inactivation of thrombin and by reducing the inhibition of factor Xa by antithrombin III [3-51. It also inhibits the activation of the complement system by preventing the insertion of C5b-9 into the cell membrane [6, 71. In uitro, vitronectin promotes cell attachment and spreading on such substrates as plastic and glass and modulates the growth rate and differentiation of various cell types in serum-free conditions [f&-10]. Based on immunohistochemical studies it was recently reported that vitronectin is localized in atherosclerotic lesions [ll-131. In this study we evaluated the effect of vitronectin on the migration of vascular smooth muscle cells (SMCs), the main cell type in atherosclerotic lesions, using a modified Boyden chamber system. r To whom reprint 0014.4827191
$3.00 1991
requests should be addressed.
Copyright 0 by Academic Press, Inc. All rights of reproduction in any form reserved.
KUXJYA, Nagoya 466, Japan
AND
METHODS
Materials. Purified bovine plasma vitronectin was purchased from Yagai Central Institute (Japan). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the purity was >99%. Other materials were obtained from the following sources: bovine serum albumin (BSA) from Sigma; trypsin from GIBCO; Dulbecco’s modified Eagle’s medium (DMEM) from Nissui (Japan); fetal bovine serum (FBS) from Cell Culture Laboratories; and human platelet-derived growth factor (PDGF) from Takara Biomedical (Japan). Cell culture. Fetal bovine aortic SMCs were cultivated as described previously [14]. Bovine fetuses were obtained from a slaughterhouse. The thoracic aorta was isolated aseptically and the inner surface was rubbed gently to remove endothelial cells. After the adventitia was removed carefully, medial SMCs were cultured using the explant method described by Ross [15]. Cells were grown in 25 cm* Aasks (Falcon) in DMEM supplemented with 10% (v/v) FBS, pH 7.4, in a CO, incubator under the conditions of 37”C, 95% sir/5% CO,, and 100% humidity. The cells reached confluence within 4 weeks. SMCs were characterized morphologically using both phase-contrast microscopy and a transmission electron microscopy [16, 171. They were then subcultured by brief treatment with 0.05% trypsin/0.02% EDTA and split in a 1:4 ratio. Cultures were used for the assays within 10 passages. Cell migration assays. Chemotaxis and haptotaxis were assayed as described previously [14, 18, 191. Single cell suspensions of SMCs were obtained by the brief treatment of cultures with 0.05% trypsinl 0.02% EDTA. Harvested cells were washed in serum-free DMEM supplemented with 0.2% (w/v) BSA and were resuspended in this medium. The lower wells containing 27 ~1 of the solution to be assayed were covered with a 5-pm pore size filter made of polyvinylpyrrolidone (PVP)-free polycarbonate (Nuclepore). The resuspended cells were added to the upper wells at a volume of 45 al (2.5 X 10’ cells/well). After a 6-h incubation in a CO, incubator (37”C, 95% air-5% CO,, humidity loo%), the filter was removed from the chamber and the nonmigrated cells were scraped from the upper surface with a wiper blade. Migrated cells on the lower surface were then fixed, stained with Giemsa, and counted under a high power (400X) field (HPF). A total of 4 HPFs were counted per sample and averaged. Precoating of filters with vitronectin. For the evaluation of haptotaxis, filters were precoated by the method of McCarthy et al. [20] as follows. PVP-free polycarbonate filters were coated on the lower or upper surface only by floating the filters, or on both surfaces by immersing the filters in a solution of vitronectin in DMEM containing 0.2% BSA, pH 7.4, overnight at 37°C. In some experiments the precoating procedure was performed during an indicated time. The precoated filters were washed extensively in Dulbecco’s phosphate-buffered saline (PBS) before use. Chambers were then assembled with DMEM containing 0.2% BSA. Statistical analysis. Statistical analysis was performed by Student’s t test. A P-value of to.05 was considered to be significant.
154
SHORT
155
NOTE Vitronectin (pg/ml) Upper / Lower
Migrated cells / HPF 0
10
20
30
40
50
o/o 0
5
10 Vitronectin
15 20 (pg / ml)
25
PDGF
DI /
20 I 0 0 I 20
:: I_ 2 8 PP
605040 30 20 -
.P =
‘0 o5
10 Vitronectm
15 20 (pg / ml)
25
FIG. 1. Dose-response curves for chemotactic (A) and haptotactic (B) responses of SMCs to vitronectin. (A) Increasing concentrations of vitronectin were added to the lower wells and diluted in DMEM supplemented with 0.2% BSA in a volume of 27 ~1. As a positive control we used 10 rg/ml of PDGF, a potent chemoattractant which is not a cell attachment-promoting protein. The chambers were then assembled with filters; 45 ~1 of 5 X lo5 cells/ml in DMEM supplemented with 0.2% BSA was added to the upper wells. Incubation time was 6 h. (B) Filters were precoated on the lower surfaces with a step gradient of increasing concentrations of vitronectin in DMEM containing 0.2% BSA as described under Materials and Methods. DMEM (27 ~1) containing 0.2% BSA was added to the lower wells, the chambers were assembled with the filters, and 45 ~1 of 5 X lo5 cells/ml in DMEM containing 0.2% BSA was added to the upper wells. Incubation time was 6 h. Data are expressed as means f SD. N = 8.
RESULTS
Vitronectin stimulated the migration of SMCs dosedependently in the range between 5 and 100 ccg/ml (Fig. 1A). PDGF, a potent chemoattractant which is not a so-called cell attachment-promoting protein, was used as a positive control. A modified checkerboard analysis showed that the response of SMCs to vitronectin was largely chemotactic in nature (Fig. 2). In the absence of soluble vitronectin SMCs also migrated over substratum-bound vitronectin in a dose-dependent manner (Fig. 1B). A modified checkerboard analysis showed that the migration was mainly directional in nature, namely haptotactic (Fig. 3). The time course of precoating the filter with vitronectin suggested that vitronectin binds on the filter surface within 5 min under the assay conditions (Fig. 4). However, we could not demonstrate directly the binding of the attractant to the filter because of the difficulties in radiolabeling vitronectin. These observations suggested that the directed migration of SMCs to soluble vitronectin shown in Fig. 1 was in fact largely due to substratum-bound vitronectin
11
'** 'I T 1
r-----q+
20 I 20
0
1 7
T
SJJ
FIG. 2. Checkerboard analysis of the response of SMCs to vitronectin. Vitronectin was added to the upper wells, the lower wells, both wells, or neither at 20 rg/ml. The migration assay procedure was performed as described in the legend to Fig. 1A. Results are expressed as means + SD; N = 12; *P < 0.001.
(haptotaxis) as opposed to cell movement in response to fluid-phase vitronectin (chemotaxis). DISCUSSION
The infiltration concept of atherosclerosis was first proposed by Page [21] in 1954 on the basis of the view that atherogenesis is due to a tissue reaction to such substances as lipoproteins and fibrinogen insudated from plasma. The response to injury hypothesis of the pathogenesis of atherosclerosis at first emphasized the importance of endothelial injury and the subsequent aggregation of platelets with the release of various active substances including platelet-derived growth factor [22]. However, endothelial injury, nondenuding or denuding, is not a feature of early atherosclerotic lesions [23]. Endothelial cells tend to adapt to the presence of atherosclerotic lesions and preserve the integrity of the Vitronectin coating Upper I Lower
Migrated cells / HPF 0
10
-/-
B
+/-
B
20
30
40
50
60
Jsl 1
-/+ +/+ FIG. 3. Analysis of the response of SMCs to substrate-bound vitronectin. Filters were precoated with 20 pg/ml vitronectin in DMEM supplemented with 0.2% BSA, as described under Materials and Methods, on the upper surface, the lower surface, both surfaces, or neither surface (control). The procedure for migration assay was performed as described in the legend to Fig. 1B. Results are expressed as means + SD; N = 12; *P < 0.001; NS, not significant.
156
SHORT
60 -
NOTE
gradient in response to a substratum-bound material [25]. In conclusion, vitronectin may play a role in the pathogenesis of atherosclerosis by recruiting SMCs from the media into the intima as well as by restricting the extent of complement activation in atherosclerotic lesions.
50 40 30 20 -
These studies were funded in part by a Grant-in-Aid for Scientific Research B (No. 63480225) from the Ministry of Education, Science and Culture, Japan.
10 OA
1
I
0
1
2
3
4
REFERENCES
J
5'60
Precoating time (min) FIG. 4. Time-response curve for haptotactic response of SMCs to vitronectin. Filters were precoated with 20 aglml vitronectin for the indicated time on the lower surfaces as described under Materials and Methods. The migration assay procedure was performed as described in the legend to Fig. 3.
1. 2. 3. 4.
Hayman, E. G., Pierschbacher, M. D., Ohgren, Y., and Ruoslahti, E. (1983) Proc. Natl. Acad. Sci. USA 80,4003-4007. Barnes, D. W., and Silnutzer, J. (1983) J. Bill. Chem. 268, 12548-12552. Jenne, D., Hugo, F., and Bhakdi, S. (1985) Thromb. Res. 38, 401-410. Podack, E. R., Dahlback, B., and Griffin, J. H. (1986) J. Biol.
Chem. 261,738'7-7392.
endothelial lining [24]. The alteration of endothelial permeability to certain substances may be a key event in the initiation of atherosclerosis. It has been proposed that the directional migration of vascular SMCs from the media into the intima and their proliferation in the intima may be the main process in the pathogenesis of atherosclerosis [23]. Such plasma-derived substances as fibrinogen/fibrin deposited in subendothelial space, particularly to connective tissue components, may recruit medial SMCs into the intima [19]. Vitronectin, now known to be identical to serumspreading factor or S-protein, is a multifunctional glycoprotein involved in the adhesion of cells to the extracellular matrix and in the regulation of complement and coagulation pathways. Recently it has been shown that vitronectin is localized in atherosclerotic lesions [ll131. Immunoelectron-dense specific deposits were found in both intimal thickenings and fibrous plaques associated with elastic fibers, collagen bundles, and cellular debris in the vicinity of elastin, by means of immunoelectron microscopy using an affinity-purified rabbit IgG specific for human vitronectin [ 131.Whether the vitronectin in atherosclerotic lesions had originated in the plasma by insudation or was elaborated by cells in the lesions is still unclear. In this study, we showed that SMCs migrate in response to vitronectin mainly by haptotaxis in vitro, suggesting that vitronectin deposited in the subendothelial matrix may recruit SMCs from the media into the intima. Chemotaxis is an equivocal term that describes the directional migration of cells in response to a gradient of the attractant, usually assumed to be a soluble substance in the fluid phase. On the other hand, haptotaxis refers to the directional cellular movement based on the formation of an adhesion Received October 25, 1990 Revised version received December
27, 1990
5. 6. 7. 8. 9. 10. 11. 12.
Preissner, K. T., and Muller-Berghaus, G. (1987) J. Biol. Chem. 262,12247-12253. Bhakdi, S., and Roth, M. (1981) J. Zmmunol. 127,576-580. Bhakdi, S., and Tanum-Jensen, J. (1983) B&him. Biophys. Acta 737,343-372. Hayman, E. G., Engvall, E., A’Hearn, E., Barnes, D., Pierschbather, M. D., and Ruoslahti, E. (1982) J. CeU Biol. 95, 20-23. Barnes, D. W., and Sato, G. (1979) Nature 281,388-389. Barnes, D., Wolfe, R., Serrero, G., McClure, D., and Sato, G. (1980) J. Supramol. Struct. 14,47-63. Niculescu, F., Rus, H. G., and Vlaicu, R. (1987) Atherosclerosis 66,1-11. Guettier, C., Hinglais, N., Bruneval, P., Kazatchkine, M., Bariety, J., and Camilleri, J.-P. (1989) Virchows Archiu. A Pathol.
Anat. 414,309-313. 13. 14. 15. 16. 17.
Niculescu, F., Rus, H. G., Porutiu, D., Ghiurca, V., and Vlaicu, R. (1989) Atherosclerosis 78.197-203. Naito, M., Hayashi, T., Kuzuya, M., Funaki, C., Asai, K., and Kuzuya, F. (1989) FEBS Z.&t. 247,356-360. Ross, R. (1971) J. Cell Biol. 50, 172-186. Charnley-Campbell, J., Campbell, G. R., and Ross, R. (1979) Physiol. Rev. 59, 1-61. Dilley, R. J., Mcgeachie, J. K., and Prendergast, F. J. (1987)
Atherosclerosis 63,99-107. 18.
Falk, W., Goodwin,
R. H., and Leonard,
E. J. (1980) J. Zmmunol.
Methods 33,239-247. 19. 20.
Naito, M., Hayashi, T., Kuzuya, M., Funaki, C., Asai, K., and Kuzuya, F. (1990) Atherosclerosis 83,9-14. McCarthy, J. B., Palm, S. L., and Furcht, L. T. (1983) J. Cell
Biol. 97, 772-777. 21. 22. 23. 24. 25.
Page, I. H. (1954) Circulation 10, l-27. Ross, R., and Glomset, J. A. (1976) N. En&. J. Med. 295,369377,420-425. Ross, R. (1986) N. Engl. J. Med. 314,488-500. Taylor, K. E., Glagov, S., and Zarins, C. K. (1989) Arterioscbrosis 9,881-894. Carter, S. B. (1965) Nature 208.1183-1187.
CELL
RESEARCH
194,154-156
Vitronectin-Induced MICHITAKA Department
(1991)
SHORT NOTE Haptotaxis of Vascular Smooth Muscle Cells in Vitro
NAITO,’ TOSHIO HAYASHI, CHIAKI FUNAKI, MASAFUMI KANICHI ASAI, KAZUYOSHI YAMADA, AND FUMIO KUZ~A
of Geriatrics,
Nagoya University
School of Medicine,
65 Tsuruma-cho,
Showa-ku,
MATERIALS Vitronectin, a multifunctional glycoprotein present in the plasma and interstitial tissues, has recently been found to be localized in atherosclerotic lesions. In this study we examined the effects of vitronectin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to fluid-phase vitronectin in a concentration-dependent fashion. The cells also migrated to membrane filter surfaces precoated with vitronectin for a few minutes in the absence of additional vitronectin in the fluid phase, suggesting that this substance binds easily to the filters and stimulates cell migration by haptotaxis under the conditions described. These observations suggest that vitronectin deposited in the intima may be involved in the pathogenesis of atherosclerosis by recruiting smooth muscle cells from the media into the 0 1991 Academic Press. Inc. intima.
INTRODUCTION
Vitronectin, also called S-protein or serum-spreading factor, is a multifunctional glycoprotein found in the plasma and interstitial tissues [ 1,2]. It occurs in human plasma and serum at 200-300 pg/ml [ 21. Vitronectin regulates blood coagulation by preventing the inactivation of thrombin and by reducing the inhibition of factor Xa by antithrombin III [3-51. It also inhibits the activation of the complement system by preventing the insertion of C5b-9 into the cell membrane [6, 71. In uitro, vitronectin promotes cell attachment and spreading on such substrates as plastic and glass and modulates the growth rate and differentiation of various cell types in serum-free conditions [f&-10]. Based on immunohistochemical studies it was recently reported that vitronectin is localized in atherosclerotic lesions [ll-131. In this study we evaluated the effect of vitronectin on the migration of vascular smooth muscle cells (SMCs), the main cell type in atherosclerotic lesions, using a modified Boyden chamber system. r To whom reprint 0014.4827191
$3.00 1991
requests should be addressed.
Copyright 0 by Academic Press, Inc. All rights of reproduction in any form reserved.
KUXJYA, Nagoya 466, Japan
AND
METHODS
Materials. Purified bovine plasma vitronectin was purchased from Yagai Central Institute (Japan). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the purity was >99%. Other materials were obtained from the following sources: bovine serum albumin (BSA) from Sigma; trypsin from GIBCO; Dulbecco’s modified Eagle’s medium (DMEM) from Nissui (Japan); fetal bovine serum (FBS) from Cell Culture Laboratories; and human platelet-derived growth factor (PDGF) from Takara Biomedical (Japan). Cell culture. Fetal bovine aortic SMCs were cultivated as described previously [14]. Bovine fetuses were obtained from a slaughterhouse. The thoracic aorta was isolated aseptically and the inner surface was rubbed gently to remove endothelial cells. After the adventitia was removed carefully, medial SMCs were cultured using the explant method described by Ross [15]. Cells were grown in 25 cm* Aasks (Falcon) in DMEM supplemented with 10% (v/v) FBS, pH 7.4, in a CO, incubator under the conditions of 37”C, 95% sir/5% CO,, and 100% humidity. The cells reached confluence within 4 weeks. SMCs were characterized morphologically using both phase-contrast microscopy and a transmission electron microscopy [16, 171. They were then subcultured by brief treatment with 0.05% trypsin/0.02% EDTA and split in a 1:4 ratio. Cultures were used for the assays within 10 passages. Cell migration assays. Chemotaxis and haptotaxis were assayed as described previously [14, 18, 191. Single cell suspensions of SMCs were obtained by the brief treatment of cultures with 0.05% trypsinl 0.02% EDTA. Harvested cells were washed in serum-free DMEM supplemented with 0.2% (w/v) BSA and were resuspended in this medium. The lower wells containing 27 ~1 of the solution to be assayed were covered with a 5-pm pore size filter made of polyvinylpyrrolidone (PVP)-free polycarbonate (Nuclepore). The resuspended cells were added to the upper wells at a volume of 45 al (2.5 X 10’ cells/well). After a 6-h incubation in a CO, incubator (37”C, 95% air-5% CO,, humidity loo%), the filter was removed from the chamber and the nonmigrated cells were scraped from the upper surface with a wiper blade. Migrated cells on the lower surface were then fixed, stained with Giemsa, and counted under a high power (400X) field (HPF). A total of 4 HPFs were counted per sample and averaged. Precoating of filters with vitronectin. For the evaluation of haptotaxis, filters were precoated by the method of McCarthy et al. [20] as follows. PVP-free polycarbonate filters were coated on the lower or upper surface only by floating the filters, or on both surfaces by immersing the filters in a solution of vitronectin in DMEM containing 0.2% BSA, pH 7.4, overnight at 37°C. In some experiments the precoating procedure was performed during an indicated time. The precoated filters were washed extensively in Dulbecco’s phosphate-buffered saline (PBS) before use. Chambers were then assembled with DMEM containing 0.2% BSA. Statistical analysis. Statistical analysis was performed by Student’s t test. A P-value of to.05 was considered to be significant.
154
SHORT
155
NOTE Vitronectin (pg/ml) Upper / Lower
Migrated cells / HPF 0
10
20
30
40
50
o/o 0
5
10 Vitronectin
15 20 (pg / ml)
25
PDGF
DI /
20 I 0 0 I 20
:: I_ 2 8 PP
605040 30 20 -
.P =
‘0 o5
10 Vitronectm
15 20 (pg / ml)
25
FIG. 1. Dose-response curves for chemotactic (A) and haptotactic (B) responses of SMCs to vitronectin. (A) Increasing concentrations of vitronectin were added to the lower wells and diluted in DMEM supplemented with 0.2% BSA in a volume of 27 ~1. As a positive control we used 10 rg/ml of PDGF, a potent chemoattractant which is not a cell attachment-promoting protein. The chambers were then assembled with filters; 45 ~1 of 5 X lo5 cells/ml in DMEM supplemented with 0.2% BSA was added to the upper wells. Incubation time was 6 h. (B) Filters were precoated on the lower surfaces with a step gradient of increasing concentrations of vitronectin in DMEM containing 0.2% BSA as described under Materials and Methods. DMEM (27 ~1) containing 0.2% BSA was added to the lower wells, the chambers were assembled with the filters, and 45 ~1 of 5 X lo5 cells/ml in DMEM containing 0.2% BSA was added to the upper wells. Incubation time was 6 h. Data are expressed as means f SD. N = 8.
RESULTS
Vitronectin stimulated the migration of SMCs dosedependently in the range between 5 and 100 ccg/ml (Fig. 1A). PDGF, a potent chemoattractant which is not a so-called cell attachment-promoting protein, was used as a positive control. A modified checkerboard analysis showed that the response of SMCs to vitronectin was largely chemotactic in nature (Fig. 2). In the absence of soluble vitronectin SMCs also migrated over substratum-bound vitronectin in a dose-dependent manner (Fig. 1B). A modified checkerboard analysis showed that the migration was mainly directional in nature, namely haptotactic (Fig. 3). The time course of precoating the filter with vitronectin suggested that vitronectin binds on the filter surface within 5 min under the assay conditions (Fig. 4). However, we could not demonstrate directly the binding of the attractant to the filter because of the difficulties in radiolabeling vitronectin. These observations suggested that the directed migration of SMCs to soluble vitronectin shown in Fig. 1 was in fact largely due to substratum-bound vitronectin
11
'** 'I T 1
r-----q+
20 I 20
0
1 7
T
SJJ
FIG. 2. Checkerboard analysis of the response of SMCs to vitronectin. Vitronectin was added to the upper wells, the lower wells, both wells, or neither at 20 rg/ml. The migration assay procedure was performed as described in the legend to Fig. 1A. Results are expressed as means + SD; N = 12; *P < 0.001.
(haptotaxis) as opposed to cell movement in response to fluid-phase vitronectin (chemotaxis). DISCUSSION
The infiltration concept of atherosclerosis was first proposed by Page [21] in 1954 on the basis of the view that atherogenesis is due to a tissue reaction to such substances as lipoproteins and fibrinogen insudated from plasma. The response to injury hypothesis of the pathogenesis of atherosclerosis at first emphasized the importance of endothelial injury and the subsequent aggregation of platelets with the release of various active substances including platelet-derived growth factor [22]. However, endothelial injury, nondenuding or denuding, is not a feature of early atherosclerotic lesions [23]. Endothelial cells tend to adapt to the presence of atherosclerotic lesions and preserve the integrity of the Vitronectin coating Upper I Lower
Migrated cells / HPF 0
10
-/-
B
+/-
B
20
30
40
50
60
Jsl 1
-/+ +/+ FIG. 3. Analysis of the response of SMCs to substrate-bound vitronectin. Filters were precoated with 20 pg/ml vitronectin in DMEM supplemented with 0.2% BSA, as described under Materials and Methods, on the upper surface, the lower surface, both surfaces, or neither surface (control). The procedure for migration assay was performed as described in the legend to Fig. 1B. Results are expressed as means + SD; N = 12; *P < 0.001; NS, not significant.
156
SHORT
60 -
NOTE
gradient in response to a substratum-bound material [25]. In conclusion, vitronectin may play a role in the pathogenesis of atherosclerosis by recruiting SMCs from the media into the intima as well as by restricting the extent of complement activation in atherosclerotic lesions.
50 40 30 20 -
These studies were funded in part by a Grant-in-Aid for Scientific Research B (No. 63480225) from the Ministry of Education, Science and Culture, Japan.
10 OA
1
I
0
1
2
3
4
REFERENCES
J
5'60
Precoating time (min) FIG. 4. Time-response curve for haptotactic response of SMCs to vitronectin. Filters were precoated with 20 aglml vitronectin for the indicated time on the lower surfaces as described under Materials and Methods. The migration assay procedure was performed as described in the legend to Fig. 3.
1. 2. 3. 4.
Hayman, E. G., Pierschbacher, M. D., Ohgren, Y., and Ruoslahti, E. (1983) Proc. Natl. Acad. Sci. USA 80,4003-4007. Barnes, D. W., and Silnutzer, J. (1983) J. Bill. Chem. 268, 12548-12552. Jenne, D., Hugo, F., and Bhakdi, S. (1985) Thromb. Res. 38, 401-410. Podack, E. R., Dahlback, B., and Griffin, J. H. (1986) J. Biol.
Chem. 261,738'7-7392.
endothelial lining [24]. The alteration of endothelial permeability to certain substances may be a key event in the initiation of atherosclerosis. It has been proposed that the directional migration of vascular SMCs from the media into the intima and their proliferation in the intima may be the main process in the pathogenesis of atherosclerosis [23]. Such plasma-derived substances as fibrinogen/fibrin deposited in subendothelial space, particularly to connective tissue components, may recruit medial SMCs into the intima [19]. Vitronectin, now known to be identical to serumspreading factor or S-protein, is a multifunctional glycoprotein involved in the adhesion of cells to the extracellular matrix and in the regulation of complement and coagulation pathways. Recently it has been shown that vitronectin is localized in atherosclerotic lesions [ll131. Immunoelectron-dense specific deposits were found in both intimal thickenings and fibrous plaques associated with elastic fibers, collagen bundles, and cellular debris in the vicinity of elastin, by means of immunoelectron microscopy using an affinity-purified rabbit IgG specific for human vitronectin [ 131.Whether the vitronectin in atherosclerotic lesions had originated in the plasma by insudation or was elaborated by cells in the lesions is still unclear. In this study, we showed that SMCs migrate in response to vitronectin mainly by haptotaxis in vitro, suggesting that vitronectin deposited in the subendothelial matrix may recruit SMCs from the media into the intima. Chemotaxis is an equivocal term that describes the directional migration of cells in response to a gradient of the attractant, usually assumed to be a soluble substance in the fluid phase. On the other hand, haptotaxis refers to the directional cellular movement based on the formation of an adhesion Received October 25, 1990 Revised version received December
27, 1990
5. 6. 7. 8. 9. 10. 11. 12.
Preissner, K. T., and Muller-Berghaus, G. (1987) J. Biol. Chem. 262,12247-12253. Bhakdi, S., and Roth, M. (1981) J. Zmmunol. 127,576-580. Bhakdi, S., and Tanum-Jensen, J. (1983) B&him. Biophys. Acta 737,343-372. Hayman, E. G., Engvall, E., A’Hearn, E., Barnes, D., Pierschbather, M. D., and Ruoslahti, E. (1982) J. CeU Biol. 95, 20-23. Barnes, D. W., and Sato, G. (1979) Nature 281,388-389. Barnes, D., Wolfe, R., Serrero, G., McClure, D., and Sato, G. (1980) J. Supramol. Struct. 14,47-63. Niculescu, F., Rus, H. G., and Vlaicu, R. (1987) Atherosclerosis 66,1-11. Guettier, C., Hinglais, N., Bruneval, P., Kazatchkine, M., Bariety, J., and Camilleri, J.-P. (1989) Virchows Archiu. A Pathol.
Anat. 414,309-313. 13. 14. 15. 16. 17.
Niculescu, F., Rus, H. G., Porutiu, D., Ghiurca, V., and Vlaicu, R. (1989) Atherosclerosis 78.197-203. Naito, M., Hayashi, T., Kuzuya, M., Funaki, C., Asai, K., and Kuzuya, F. (1989) FEBS Z.&t. 247,356-360. Ross, R. (1971) J. Cell Biol. 50, 172-186. Charnley-Campbell, J., Campbell, G. R., and Ross, R. (1979) Physiol. Rev. 59, 1-61. Dilley, R. J., Mcgeachie, J. K., and Prendergast, F. J. (1987)
Atherosclerosis 63,99-107. 18.
Falk, W., Goodwin,
R. H., and Leonard,
E. J. (1980) J. Zmmunol.
Methods 33,239-247. 19. 20.
Naito, M., Hayashi, T., Kuzuya, M., Funaki, C., Asai, K., and Kuzuya, F. (1990) Atherosclerosis 83,9-14. McCarthy, J. B., Palm, S. L., and Furcht, L. T. (1983) J. Cell
Biol. 97, 772-777. 21. 22. 23. 24. 25.
Page, I. H. (1954) Circulation 10, l-27. Ross, R., and Glomset, J. A. (1976) N. En&. J. Med. 295,369377,420-425. Ross, R. (1986) N. Engl. J. Med. 314,488-500. Taylor, K. E., Glagov, S., and Zarins, C. K. (1989) Arterioscbrosis 9,881-894. Carter, S. B. (1965) Nature 208.1183-1187.
