ANALYTICAL
BIOCHEMISTRY
190,
39-47
(1990)
An in Vitro Model of Cell Migration: Vascular Endothelial Cell Migration’ K. Jozaki,*
P. T. Marucha,t$§
A. W. Despins,t$
Evaluation
of
and D. L. Kreutzert$?
*Department of Pediatrics, Keio University Medical School, 35 Shinano-machi, Shinjuku-ku, Tokyo, Japan; TDepartment of Pathology, University of Connecticut Health Center, Farmington, Connecticut 06032; *Department of Periodontology, University of Connecticut Health Center, Farmington, Connecticut 06032; and 5 Vision Immunology Center, University of Connecticut Health Center, Farmington, Connecticut 06032
Received
March
26, 1990
In uiuo vascular
endothelial cell (VEC) migration is thought to play a central role in the development of new capillaries as well as the resurfacing of large vessels. Recently, we have developed an in vitro VEC migration assay system based on the ability of VEC to migrate off of tissue culture microcarrier beads. For these studies, bovine pulmonary artery VEC were grown to confluence on Cytodex 3 microcarrier beads (MCB). Next, the confluent VEC covered microcarrier beads were pipetted into 4-cm* wells of a tissue culture plate and incubated at 37”C/5% CO,. At various time intervals, the movement of the VEC off of the MCB onto the tissue culture surface was evaluated microscopically. Using this assay, we have studied the effect of endothelial cell growth supplement and various matrices (i.e., fibronectin, gelatin, and Matrigel) on VEC migration. These studies demonstrated that: (i) gelatin had no effect on normal or mitomycin C-pretreated VEC migration; (ii) fibronectin had no effect on normal VEC migration, but stimulated the relative migration of mitomycin pretreated VEC; and (iii) Matrigel significantly suppressed both normal and mitomycin C-pretreated VEC migration. Endothelial cell growth supplement (ECGS) stimulated both normal and mitomycin C-pretreated VEC migration on fibronectin at concentrations of 10 wglml ECGS. Pretreatment with ECGS had no effect of normal or mitomycin C VEC migration on gelatin. Finally, ECGS stimulated a statistically significant increase in the migration of normal and mitomycin C-pretreated VEC migration on Matrigel. Thus, these data clearly
1 Presented at the 1987 Meeting of the Federation of American Societies of Experimental Biology, Washington, D.C. Supported in part by Grants HL-25015 and EY-04131 from The National Institutes of Health (Bethesda, MD). * Corresponding author: Dr. Donald Kreutzer, Department of Pathology, University of Connecticut Health Center, Farmington, CT 06030. 0003.2697190
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
demonstrate the ability of the MCB assay to detect both enhancements and suppressions of VEC migration in uitro, and therefore this new method should prove a simple and rapid method for the quantitation of VEC mib 1990 Academic heas, Inc. gration in vitro.
Vascular endothelial cell (VEC)3 migration is thought to play a central role in wound healing, embryogenesis, tumor growth, atherosclerosis, and the reendotheliation of large vessel walls. Because of the importance of VEC in these various disease processes, a variety of in vitro assays have been used in an attempt to quantitate VEC migration in vitro. For example, investigators have employed video tracking or gold tracking (l-5) in order to follow the movement of individual cells. Other investigators have used a scrape injury of monolayers of VEC in tissue culture to evaluate VEC migration (6-9). Additionally, many investigators have utilized evaluation of the migration of VEC into porous membranes as a method of quantitating VEC migration (10-13). Recently, we have undertaken in our laboratory to develop a simple, rapid assay of VEC migration. To these ends we present data to support a new assay of VEC migration which involves the quantitation of radial migration of VEC from confluent microcarrier beads to the surface of tissue culture plastic. This assay system utilizes direct microscopic evaluation of the VEC migration and image analysis to speed quantitation. Additional studies are presented which demonstrate the ability of this assay system to evaluate the influence of various soluble factors and matrices on VEC migration.
a Abbreviations used: VEC, vascular endothelial cell; MCB, carrier beads; ECGS, endothelial cell growth supplement; minimum essential medium; MI, migration index.
microMEM,
39
40 MATERIALS
JOZAKI AND
METHODS
Reagents and Materials Valine-free MEM, gentamicin, amphotericin B, nonessential amino acid, glutamine, MEM vitamins (Gibco, New York, NY), D-valine (Sigma, St. Louis, MO), calf serum (Hyclone, Logan, UT), Cytodex 3 beads (Pharmacia, Piscataway, NJ), mitomycin C (Sigma, St. Louis, MO), a spinner flask (Bellco, Vineland, NJ), 12-well plate (Costar, Cambridge, MA), endothelial cell growth supplement, and Matrigel (Collaborative Research, Bedford, MA) were obtained commercially. Fibronectin was obtained from human plasma by affinity chromatography on gelatin sepharose (14,15). Vascular Endotheliul Cell Culture Conditions Primary cultures of bovine pulmonary artery endothelial cells were isolated according to established procedures and maintained in fibroblast-free long-term cultures using a minimum essential media containing D-valine (16). The VEC used for these studies were Factor VIII positive in culture. For the preparation of VECcoated microcarrier beads, the VECs were initially grown to confluence in T-75 flasks (in MEM containing 15% calf serum). Cells were harvested by trypsinization and placed in a spinner flask with 25 mg of Cytodex 3 beads in 50 ml of the same tissue culture media. The cell/bead mixture was then incubated in 5% CO, at 37°C with slow spinning. Generally, the beads were totally covered (confluent) with the VECs in 2 to 5 days. Bead cell confluence was determined by exclusion of an Evans blue dye-albumin complex from the beads. When the beads were totally confluent, they were washed by sedimenting at unit gravity to remove any free cells and cell aggregates, and then used for the migration assay. Effect of Mytomycin
C and ECGS on VEC Proliferation
As mentioned earlier, a number of investigations have suggested the importance of cell proliferation in vascular endothelial cell migration (6,10,17,18). Thus, to begin to determine the contribution of cell proliferation to our migration assays, we determined the effect of mitomycin C and ECGS on VEC proliferation in uitro. For these studies, VECs were harvested from the T-75 flask, counted, and added to plastic 12 X 75 test tubes at cell concentrations from 5 X lo4 to 2 X lo5 VEC/ml. Next, either 1 ml of control tissue culture media or mitomycin C was added to the VEC at final concentration of 25 pg/ml. The cells were then incubated at 37°C for 30 min, washed twice with media, and seeded into a T-25 flask. After 48 and 168 h, cell numbers were determined by trypsinization of cells from the T-25 flasks followed by direct microscopic counts using a hemocytometer. Because of our use of ECGS in migration assays, studies were also undertaken to determine the influence of
ET AL.
ECGS on the proliferation of control or mitomycintreated VECs. For these studies, either control VECs or mitomycin-treated VECs were prepared as described above and seeded on T-25 flasks. Next, ECGS at a final concentration of 100 pg/ml was added to both control and mitomycin C-treated VECs. After 48 and 168 h, the final cell number was determined directly as described above.
VEC Migration The VEC migration assay generally involves evaluation of the radial migration of VECs from the VECcoated microcarrier beads. Mitomycin C pretreatment of the VEC was used to determine the contribution of cell proliferation to VEC migration in this assay. Thus, for the VEC migration assay, the confluent MCB were initially divided into two parts. The resulting pools of VEC received either control media or mitomycin C (25 pglml) and were incubated at 37°C for 30 min. After incubation, the VEC-coated beads were washed twice with media and added to 4-cm2 wells containing either control media or ECGS (O-100 pg/ml). In selected experiments, 4-cm2 wells were precoated with various matrices (see below). The resulting cultures were then incubated at 5% CO, in 37°C for 48 h. VEC migration from the microcarrier was monitored directly using an inverted microscope. After 24-48 h of incubation, the cells were fixed with 3.7% formaldehyde and stained with crystal violet (1% solution for 1 min). The VEC migration from the microcarrier beads was quantitated using image analysis (Optimax 410 image analyzer, Boston, MA) and the data were expressed as a migration index. Generally, 5-10 beads were quantitated for any given culture condition. The migration index represents the sum of the number of cells at any given distance from the MCB multiplied by the distance (in micrometers) migrated by each individual cell. The results of VEC migration was analyzed statistically using the paired t test (31).
Effect of Matrix
on VEC Migration
The importance of various matrices in the control of cell migration and proliferation has been clearly demonstrated. Thus we evaluated the influence of fibronectin, gelatin, or Matrigel on VEC migration in our MCB assay. For these studies, fibronectin- (15 mg/ml), gelatin(15 mg/ml), or Matrigel-coated 4-cm2 wells were incubated for 2 h at 37°C followed by three independent washes with media prior to the addition of the VEC coated microcarrier beads. The influence of the various matrices on the VEC migration was evaluated at 48 h and the data expressed as migration index.
VASCULAR
ENDOTHELIAL
CELL
MIGRATION
IN
VITRO
41
FIG. 1. Phase contrast micrograph of VEC migration from microcarrier beads. As can be noted, the migration of VEC off of the microcarrier beads is seen at 24-48 h, both in the presence and the absence of mitomycin C. (A) VEC migration at 24 h post-seeding in the absenceof mitomycin C. (B) Migration of mitomycin C-pretreated VEC’s, 24 h after plating. (Cl Phase contrast micrographs of the migration of the non-mitomycin C-treated cells at 48 h. (D) The migration response of mitomycin C-pretreated VEC’s evaluated at 48 h in culture.
RESULTS
Effect of Mitomycin in Vitro
C and ECGS on VEC Proliferation
Our initial studies to evaluate the effect of mitomycin C on VEC proliferation indicated that concentrations of 25 kg/ml mitomycin C totally blocked VEC proliferation when compared to media exposed VEC (data not shown). Additionally, we demonstrated that although ECGS could markedly stimulate proliferation of VEC in uitro, it was unable to stimulate proliferation in mitomycin C-pretreated VEC (data not shown). These data thus indicate that 25 pgfml mitomy?in C completely blocks proliferation of VEC in the presence or the absence of ECGS. Evaluation of VEC Migration from Microcarrier
Beads
To begin our studies of the MCB assay of vascular endothelial cell migration, we initially evaluated the movement of non-mitomycin C-treated VEC from the
MCB qualitatively. As can be seen in Fig. 2, within 24 h of plating, a significant number of vascular endothelial cells have migrated from the microcarrier beads to the plastic surface. At 48 h, a significant increase in the number of cells migrated onto the plastic. When the cells were pretreated with mitomycin C, the VEC migration was slightly less than non-mitomycin C-treated VEC, but consistently large numbers of VEC migrated off of the MCB (see Fig. 1). After establishing the qualitative nature of VEC migration from microcarrier beads, we next determined the ability of our assay to quantitate cell migration. As can be seen in Fig. 2A, The radial migration of individual VEC from the center of the microcarrier beads generally ranged from 100 to 500 pm. When VEC were exposed to 25 pg/ml mitomycin C a slightly smaller number of cells migrated from the MCB at distances greater than 225 pm, but generally both the cells types (i.e., mitomycin- vs non-mitomycin-treated) migrated approximately the same distance from the beads. When the VEC migration was quantitated using migration in-
42
JOZAKI
-
W/O MITOMYCIN
C
0
Brn
MIGRATED
3lxlOO' 5
25000-
E
zmm-
2 E
16000
2 (I 3
-
loaoOwoo
ET
AL.
distance of cells migrating from the microcarrier beads when compared to non-ECGS-treated VEC (Fig. 3). As can be seen in Fig. 3, mitomycin C pretreatment of VECs had only slight suppression on VEC migration in the absence of ECGS. Additionally, although mitomycin C pretreatment of VEC reduced the number of cells migrating from the microcarrier beads, it did not dramatically alter the distance the cells migrated radially from the microcarrier beads. Thus, these data suggest that although cell proliferation may influence the number of cells migrating, it does not dramatically alter the distance that migrating cells travel. To further investigate the influence of ECGS on VEC migration from the microcarrier beads we next determined the effect of varying concentrations of ECGS on control or mitomycin C pretreated VEC migration. As can be seen in Fig. 4, ECGS stimulated a dose-dependent increase in VEC migration under both control and mitomycin C conditions. Generally, the ECGS stimulated maximum migration in the 50 to 100 @g/ml concentration.
-
Effects of Matrix
04
CONTROL
MITOMYCIN
C
FIG. 2. Quantitation of VEC migration from microcarrier beads in the presence and absence of mitomycin C. The figure represents the distribution of cells by number versus distance from the microcarrier beads, as well as by migration index.
dex, non-mitomycin C-treated VEC had a mean MI of 21,050 +- 3788. Mitomycin C pretreatment caused a statistically significant decrease in the MI (10,980 f 2370), but a consistently large number of mitomycin C-pretreated VEC did migrate from the MCB. At this time point in non-mitomycin C-treated wells, 1.5 times and 3 times the number of cells are available for migration in control and ECGS wells, respectively, as compared to the mitomycin C-treated wells. This probably accounts for most of the differences in MI between mitomycin C and non-mitomycin C-treated wells.
on VEC Migration
In addition to growth factors, various matrices have also been shown to influence cell proliferation and migration, (6,7,20). Thus, we determined the influence of fibronectin, gelatin, and Matrigel on VEC migration from MCB. As can be seen in Fig. 5A, fibronectin had no significant influence on the migration of VEC in the absence of mitomycin C. Interestingly, fibronectin did enhance VEC migration in the presence of mitomycin C (see Fig. 5B). To further evaluate the influence of matrices on VEC migration, we determined the effect of gelatin on VEC migration. As can be seen in Figs. 5C and 5D, gelatin had no statistically significant effect on the migration of normal or mitomycin pretreated VEC. Evaluation of the influence of Matrigel on VEC migration indicated Matrigel had a significant suppressive effect on normal or mitomycin C-pretreated VEC migration (Figs. 5E and 5F).
Effect of ECGS and Matrices Effect of ECGS on VEC Migration Since various factors have been previously shown to in vitro induce VEC proliferation and migration (8,10,19), we next determined the influence of ECGS on the migration of the vascular endothelial cells from microcarrier beads. As can be seen in Fig. 3, at 48 h after plating the MCB, ECGS (100 pg/ml) significantly increased (i) the number of endothelial cells migrating off of the MCB as well as (ii) the average distance migrated. Clearly, because of the established role of ECGS in stimulating cell proliferation, we then determined the influence of ECGS on mitomycin C-treated cells. Although the VEC response was diminished by mitomycin C, ECGS significantly stimulated both the number and
on VEC Migration
During in vivo VEC migration the influence of both matrix and factors such as ECGS likely occur. Therefore, we evaluated the influence of both matrix and ECGS on VEC migration from MCB. As can be seen in Fig. 6A, Fn plus 10 pg/ml ECGS induced maximum increase in VEC migration relative to the matched non-fibronectin-exposed VEC. Although fibronectin + 100 pg/ml ECGS induced a greater absolute migration index, there was no significant difference between the fibronectin only and fibronectin + 100 pg/ml ECGS exposed VEC. When the effect of fibronectin and ECGS was evaluated using mitomycin C-treated VEC the fibronectin induced a consistent increase in migration index under all conditions (see Fig. 6B). Generally, ECGS had
VASCULAR W/O
ENDOTHELIAL
MITOMYCIN
CELL
MIGRATION
IN
43
VITRO
W1 MITOMYCIN
C
C
1:;
0
150
300
pm
450
600
750
60000-
5 F=
40000-
s 9 a
20000-
150
300
pm
P
BIOCHEMISTRY
190,
39-47
(1990)
An in Vitro Model of Cell Migration: Vascular Endothelial Cell Migration’ K. Jozaki,*
P. T. Marucha,t$§
A. W. Despins,t$
Evaluation
of
and D. L. Kreutzert$?
*Department of Pediatrics, Keio University Medical School, 35 Shinano-machi, Shinjuku-ku, Tokyo, Japan; TDepartment of Pathology, University of Connecticut Health Center, Farmington, Connecticut 06032; *Department of Periodontology, University of Connecticut Health Center, Farmington, Connecticut 06032; and 5 Vision Immunology Center, University of Connecticut Health Center, Farmington, Connecticut 06032
Received
March
26, 1990
In uiuo vascular
endothelial cell (VEC) migration is thought to play a central role in the development of new capillaries as well as the resurfacing of large vessels. Recently, we have developed an in vitro VEC migration assay system based on the ability of VEC to migrate off of tissue culture microcarrier beads. For these studies, bovine pulmonary artery VEC were grown to confluence on Cytodex 3 microcarrier beads (MCB). Next, the confluent VEC covered microcarrier beads were pipetted into 4-cm* wells of a tissue culture plate and incubated at 37”C/5% CO,. At various time intervals, the movement of the VEC off of the MCB onto the tissue culture surface was evaluated microscopically. Using this assay, we have studied the effect of endothelial cell growth supplement and various matrices (i.e., fibronectin, gelatin, and Matrigel) on VEC migration. These studies demonstrated that: (i) gelatin had no effect on normal or mitomycin C-pretreated VEC migration; (ii) fibronectin had no effect on normal VEC migration, but stimulated the relative migration of mitomycin pretreated VEC; and (iii) Matrigel significantly suppressed both normal and mitomycin C-pretreated VEC migration. Endothelial cell growth supplement (ECGS) stimulated both normal and mitomycin C-pretreated VEC migration on fibronectin at concentrations of 10 wglml ECGS. Pretreatment with ECGS had no effect of normal or mitomycin C VEC migration on gelatin. Finally, ECGS stimulated a statistically significant increase in the migration of normal and mitomycin C-pretreated VEC migration on Matrigel. Thus, these data clearly
1 Presented at the 1987 Meeting of the Federation of American Societies of Experimental Biology, Washington, D.C. Supported in part by Grants HL-25015 and EY-04131 from The National Institutes of Health (Bethesda, MD). * Corresponding author: Dr. Donald Kreutzer, Department of Pathology, University of Connecticut Health Center, Farmington, CT 06030. 0003.2697190
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
demonstrate the ability of the MCB assay to detect both enhancements and suppressions of VEC migration in uitro, and therefore this new method should prove a simple and rapid method for the quantitation of VEC mib 1990 Academic heas, Inc. gration in vitro.
Vascular endothelial cell (VEC)3 migration is thought to play a central role in wound healing, embryogenesis, tumor growth, atherosclerosis, and the reendotheliation of large vessel walls. Because of the importance of VEC in these various disease processes, a variety of in vitro assays have been used in an attempt to quantitate VEC migration in vitro. For example, investigators have employed video tracking or gold tracking (l-5) in order to follow the movement of individual cells. Other investigators have used a scrape injury of monolayers of VEC in tissue culture to evaluate VEC migration (6-9). Additionally, many investigators have utilized evaluation of the migration of VEC into porous membranes as a method of quantitating VEC migration (10-13). Recently, we have undertaken in our laboratory to develop a simple, rapid assay of VEC migration. To these ends we present data to support a new assay of VEC migration which involves the quantitation of radial migration of VEC from confluent microcarrier beads to the surface of tissue culture plastic. This assay system utilizes direct microscopic evaluation of the VEC migration and image analysis to speed quantitation. Additional studies are presented which demonstrate the ability of this assay system to evaluate the influence of various soluble factors and matrices on VEC migration.
a Abbreviations used: VEC, vascular endothelial cell; MCB, carrier beads; ECGS, endothelial cell growth supplement; minimum essential medium; MI, migration index.
microMEM,
39
40 MATERIALS
JOZAKI AND
METHODS
Reagents and Materials Valine-free MEM, gentamicin, amphotericin B, nonessential amino acid, glutamine, MEM vitamins (Gibco, New York, NY), D-valine (Sigma, St. Louis, MO), calf serum (Hyclone, Logan, UT), Cytodex 3 beads (Pharmacia, Piscataway, NJ), mitomycin C (Sigma, St. Louis, MO), a spinner flask (Bellco, Vineland, NJ), 12-well plate (Costar, Cambridge, MA), endothelial cell growth supplement, and Matrigel (Collaborative Research, Bedford, MA) were obtained commercially. Fibronectin was obtained from human plasma by affinity chromatography on gelatin sepharose (14,15). Vascular Endotheliul Cell Culture Conditions Primary cultures of bovine pulmonary artery endothelial cells were isolated according to established procedures and maintained in fibroblast-free long-term cultures using a minimum essential media containing D-valine (16). The VEC used for these studies were Factor VIII positive in culture. For the preparation of VECcoated microcarrier beads, the VECs were initially grown to confluence in T-75 flasks (in MEM containing 15% calf serum). Cells were harvested by trypsinization and placed in a spinner flask with 25 mg of Cytodex 3 beads in 50 ml of the same tissue culture media. The cell/bead mixture was then incubated in 5% CO, at 37°C with slow spinning. Generally, the beads were totally covered (confluent) with the VECs in 2 to 5 days. Bead cell confluence was determined by exclusion of an Evans blue dye-albumin complex from the beads. When the beads were totally confluent, they were washed by sedimenting at unit gravity to remove any free cells and cell aggregates, and then used for the migration assay. Effect of Mytomycin
C and ECGS on VEC Proliferation
As mentioned earlier, a number of investigations have suggested the importance of cell proliferation in vascular endothelial cell migration (6,10,17,18). Thus, to begin to determine the contribution of cell proliferation to our migration assays, we determined the effect of mitomycin C and ECGS on VEC proliferation in uitro. For these studies, VECs were harvested from the T-75 flask, counted, and added to plastic 12 X 75 test tubes at cell concentrations from 5 X lo4 to 2 X lo5 VEC/ml. Next, either 1 ml of control tissue culture media or mitomycin C was added to the VEC at final concentration of 25 pg/ml. The cells were then incubated at 37°C for 30 min, washed twice with media, and seeded into a T-25 flask. After 48 and 168 h, cell numbers were determined by trypsinization of cells from the T-25 flasks followed by direct microscopic counts using a hemocytometer. Because of our use of ECGS in migration assays, studies were also undertaken to determine the influence of
ET AL.
ECGS on the proliferation of control or mitomycintreated VECs. For these studies, either control VECs or mitomycin-treated VECs were prepared as described above and seeded on T-25 flasks. Next, ECGS at a final concentration of 100 pg/ml was added to both control and mitomycin C-treated VECs. After 48 and 168 h, the final cell number was determined directly as described above.
VEC Migration The VEC migration assay generally involves evaluation of the radial migration of VECs from the VECcoated microcarrier beads. Mitomycin C pretreatment of the VEC was used to determine the contribution of cell proliferation to VEC migration in this assay. Thus, for the VEC migration assay, the confluent MCB were initially divided into two parts. The resulting pools of VEC received either control media or mitomycin C (25 pglml) and were incubated at 37°C for 30 min. After incubation, the VEC-coated beads were washed twice with media and added to 4-cm2 wells containing either control media or ECGS (O-100 pg/ml). In selected experiments, 4-cm2 wells were precoated with various matrices (see below). The resulting cultures were then incubated at 5% CO, in 37°C for 48 h. VEC migration from the microcarrier was monitored directly using an inverted microscope. After 24-48 h of incubation, the cells were fixed with 3.7% formaldehyde and stained with crystal violet (1% solution for 1 min). The VEC migration from the microcarrier beads was quantitated using image analysis (Optimax 410 image analyzer, Boston, MA) and the data were expressed as a migration index. Generally, 5-10 beads were quantitated for any given culture condition. The migration index represents the sum of the number of cells at any given distance from the MCB multiplied by the distance (in micrometers) migrated by each individual cell. The results of VEC migration was analyzed statistically using the paired t test (31).
Effect of Matrix
on VEC Migration
The importance of various matrices in the control of cell migration and proliferation has been clearly demonstrated. Thus we evaluated the influence of fibronectin, gelatin, or Matrigel on VEC migration in our MCB assay. For these studies, fibronectin- (15 mg/ml), gelatin(15 mg/ml), or Matrigel-coated 4-cm2 wells were incubated for 2 h at 37°C followed by three independent washes with media prior to the addition of the VEC coated microcarrier beads. The influence of the various matrices on the VEC migration was evaluated at 48 h and the data expressed as migration index.
VASCULAR
ENDOTHELIAL
CELL
MIGRATION
IN
VITRO
41
FIG. 1. Phase contrast micrograph of VEC migration from microcarrier beads. As can be noted, the migration of VEC off of the microcarrier beads is seen at 24-48 h, both in the presence and the absence of mitomycin C. (A) VEC migration at 24 h post-seeding in the absenceof mitomycin C. (B) Migration of mitomycin C-pretreated VEC’s, 24 h after plating. (Cl Phase contrast micrographs of the migration of the non-mitomycin C-treated cells at 48 h. (D) The migration response of mitomycin C-pretreated VEC’s evaluated at 48 h in culture.
RESULTS
Effect of Mitomycin in Vitro
C and ECGS on VEC Proliferation
Our initial studies to evaluate the effect of mitomycin C on VEC proliferation indicated that concentrations of 25 kg/ml mitomycin C totally blocked VEC proliferation when compared to media exposed VEC (data not shown). Additionally, we demonstrated that although ECGS could markedly stimulate proliferation of VEC in uitro, it was unable to stimulate proliferation in mitomycin C-pretreated VEC (data not shown). These data thus indicate that 25 pgfml mitomy?in C completely blocks proliferation of VEC in the presence or the absence of ECGS. Evaluation of VEC Migration from Microcarrier
Beads
To begin our studies of the MCB assay of vascular endothelial cell migration, we initially evaluated the movement of non-mitomycin C-treated VEC from the
MCB qualitatively. As can be seen in Fig. 2, within 24 h of plating, a significant number of vascular endothelial cells have migrated from the microcarrier beads to the plastic surface. At 48 h, a significant increase in the number of cells migrated onto the plastic. When the cells were pretreated with mitomycin C, the VEC migration was slightly less than non-mitomycin C-treated VEC, but consistently large numbers of VEC migrated off of the MCB (see Fig. 1). After establishing the qualitative nature of VEC migration from microcarrier beads, we next determined the ability of our assay to quantitate cell migration. As can be seen in Fig. 2A, The radial migration of individual VEC from the center of the microcarrier beads generally ranged from 100 to 500 pm. When VEC were exposed to 25 pg/ml mitomycin C a slightly smaller number of cells migrated from the MCB at distances greater than 225 pm, but generally both the cells types (i.e., mitomycin- vs non-mitomycin-treated) migrated approximately the same distance from the beads. When the VEC migration was quantitated using migration in-
42
JOZAKI
-
W/O MITOMYCIN
C
0
Brn
MIGRATED
3lxlOO' 5
25000-
E
zmm-
2 E
16000
2 (I 3
-
loaoOwoo
ET
AL.
distance of cells migrating from the microcarrier beads when compared to non-ECGS-treated VEC (Fig. 3). As can be seen in Fig. 3, mitomycin C pretreatment of VECs had only slight suppression on VEC migration in the absence of ECGS. Additionally, although mitomycin C pretreatment of VEC reduced the number of cells migrating from the microcarrier beads, it did not dramatically alter the distance the cells migrated radially from the microcarrier beads. Thus, these data suggest that although cell proliferation may influence the number of cells migrating, it does not dramatically alter the distance that migrating cells travel. To further investigate the influence of ECGS on VEC migration from the microcarrier beads we next determined the effect of varying concentrations of ECGS on control or mitomycin C pretreated VEC migration. As can be seen in Fig. 4, ECGS stimulated a dose-dependent increase in VEC migration under both control and mitomycin C conditions. Generally, the ECGS stimulated maximum migration in the 50 to 100 @g/ml concentration.
-
Effects of Matrix
04
CONTROL
MITOMYCIN
C
FIG. 2. Quantitation of VEC migration from microcarrier beads in the presence and absence of mitomycin C. The figure represents the distribution of cells by number versus distance from the microcarrier beads, as well as by migration index.
dex, non-mitomycin C-treated VEC had a mean MI of 21,050 +- 3788. Mitomycin C pretreatment caused a statistically significant decrease in the MI (10,980 f 2370), but a consistently large number of mitomycin C-pretreated VEC did migrate from the MCB. At this time point in non-mitomycin C-treated wells, 1.5 times and 3 times the number of cells are available for migration in control and ECGS wells, respectively, as compared to the mitomycin C-treated wells. This probably accounts for most of the differences in MI between mitomycin C and non-mitomycin C-treated wells.
on VEC Migration
In addition to growth factors, various matrices have also been shown to influence cell proliferation and migration, (6,7,20). Thus, we determined the influence of fibronectin, gelatin, and Matrigel on VEC migration from MCB. As can be seen in Fig. 5A, fibronectin had no significant influence on the migration of VEC in the absence of mitomycin C. Interestingly, fibronectin did enhance VEC migration in the presence of mitomycin C (see Fig. 5B). To further evaluate the influence of matrices on VEC migration, we determined the effect of gelatin on VEC migration. As can be seen in Figs. 5C and 5D, gelatin had no statistically significant effect on the migration of normal or mitomycin pretreated VEC. Evaluation of the influence of Matrigel on VEC migration indicated Matrigel had a significant suppressive effect on normal or mitomycin C-pretreated VEC migration (Figs. 5E and 5F).
Effect of ECGS and Matrices Effect of ECGS on VEC Migration Since various factors have been previously shown to in vitro induce VEC proliferation and migration (8,10,19), we next determined the influence of ECGS on the migration of the vascular endothelial cells from microcarrier beads. As can be seen in Fig. 3, at 48 h after plating the MCB, ECGS (100 pg/ml) significantly increased (i) the number of endothelial cells migrating off of the MCB as well as (ii) the average distance migrated. Clearly, because of the established role of ECGS in stimulating cell proliferation, we then determined the influence of ECGS on mitomycin C-treated cells. Although the VEC response was diminished by mitomycin C, ECGS significantly stimulated both the number and
on VEC Migration
During in vivo VEC migration the influence of both matrix and factors such as ECGS likely occur. Therefore, we evaluated the influence of both matrix and ECGS on VEC migration from MCB. As can be seen in Fig. 6A, Fn plus 10 pg/ml ECGS induced maximum increase in VEC migration relative to the matched non-fibronectin-exposed VEC. Although fibronectin + 100 pg/ml ECGS induced a greater absolute migration index, there was no significant difference between the fibronectin only and fibronectin + 100 pg/ml ECGS exposed VEC. When the effect of fibronectin and ECGS was evaluated using mitomycin C-treated VEC the fibronectin induced a consistent increase in migration index under all conditions (see Fig. 6B). Generally, ECGS had
VASCULAR W/O
ENDOTHELIAL
MITOMYCIN
CELL
MIGRATION
IN
43
VITRO
W1 MITOMYCIN
C
C
1:;
0
150
300
pm
450
600
750
60000-
5 F=
40000-
s 9 a
20000-
150
300
pm
P
