0963-6897/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
Cell Transplantation, Vol. 1, pp, 355-364, 1992 Printed in the USA. All rights reserved.
Original Contribution ENDOTHELIAL CELL SENESCENCE INHIBITS UNIDIRECTIONAL ENDOTHELIALIZATION IN VITRO SATOSHI NIU* AND TAKEHISA MATSUDAt
Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita, Osaka 565, Japan
o Abstract -
many studies showed that endothelial regeneration for animals is completed within several months to years. However, the general observation is that the regeneration for human implantation is limited to nearly the anastomotic sites even upon quite long-term implantation. Cellular aging of the endothelium may be a possible factor which is associated with atherogenesis (28). Nevertheless, little attention has been paid to the effects of individual biological aging and/or cellular senescence of the host vessel on neointimal healing of vascular prostheses implanted in humans. Recent advances in cell culture techniques have made it possible to study the effects of cellular aging on proliferative potential, morphology, and differentiated functions of vascular endothelial cells in vitro (3,5,7,9,14,26). In this paper we studied the effects of cellular aging on in vitro unidirectional tissue formation, simulating the anastomotic endothelialization of vascular prostheses. Bovine endothelial cellswhich have finite life span wereemployed. In addition, we discussed whether and how cellular senescenceof the neoendothelium may result in limitations of unidirectional endothelialization. This was based on the study of the cellular mobility at the endothelializing front of the in vitro model simulating the anastomotic endothelialization.
We investigated the effects of cellular senescence on unidirectional endothelialization in vitro, simulating the anastomotic endothelialization of vascular prosthesis. The experiments were carried out with three different cumulative population-doubling levels (CPDLs) of bovine aortic endothelial cells (ECs), which have finite life span. Young ECs with 22 CPDL, middle aged with 46, and senescent with 70 at the time of inoculation were used. The effect of aging on unidirectional endothelialization, as well as cellular morphology and proliferative and migratory potentials of isolated cells, were qualitatively and quantitatively analyzed. The unidirectional endothelialization rate was determined by our newly designed method to prepare the square monolayer sheet with linear margins between ceO-adhesion and noncelladhesion regions. The results showed that endothelial cell senescence retarded not only proliferation and migration but also unidirectional endothelialization. Time-lapsed videomicroscopic study of unidirectional endothelialization process revealed that ECs at several rows back from the leading edge represented much slower rate of migration than did the ECs at the leading edge. These findings suggest that high cellular mobility observed for the ECs at the leading edge may result in localized excessive cell replication. Thus, atherosclerotic vessels containing senescent or injured ECs may have limited capability of anastomotic endotbelialization.
o Keywords -
Endothelium; Senescence; Anastomotic endothelialization; Migration. INTRODUCTION
A prosthetic graft is implanted to reconstruct diseased vessels involved in atherosclerosis. Although the incompleteness of neointimal regeneration has been clinically noted (l,22), mechanistic understanding of its incompleteness has not been clarified. Experimental and clinical studies have shown that there is a marked difference in the rate of intimal healing of vascular prostheses between human and animals (23). That is,
MATERIALS AND METHODS
Endothelial Cell Culture Endothelial cells (ECs) were harvested from bovine thoracic aortas by the mechanical scraping technique. ECs were routinely cultured at 37°C in a humidified atmosphere in 95070 air and 5% CO 2 on Corning tissue culture flasks (Iwaki Glass, Tokyo, Japan) in Dul-
6/22/92. *Present affiliation: The Second Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.
[To whom correspondence should be addressed.
ACCEPTED
355
356
Cell Transplantation. Volume I, Number 5, 1992
becco's modification of Eagle's medium (DMEM, Flow Laboratories Inc., Irvine, Scotland), supplemented with 15010 fetal bovine serum (HyClone Laboratories Inc., Logan, UT). When the cultured cells became confluent, they were subcultured with 0.25% trypsin treatment after being washed twice with phosphate-buffered saline (PBS, Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) and 0.04% EGTA. All experiments were carried out with ECs at 12, 24, and 36 passages, which were designated as young, rnidd1eaged and senescent ECs, respectively. Since cultures were split 1:4 at every passage, passage X was equivalent to 2(X - 1) cumulative population-doubling levels (CPDLs). These translated to the young ECs with 22 CPDLs, the middle aged with 46, and the senescent with 70. Endothelial Cell Growth and Morphology ECs (2 X 104 cells/well) were seeded in 24-well plates (Cell Wells, Corning Glass Works, Corning,
NY; 2 cmvwell), and the cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for up to 7 days in 1 mL of culture medium. The number of cells, which were detached with trypsinization, was counted with a Coulter counter (Model ZBI, Coulter Electronics, Inc., Hialeah, FL) on days 1,3, and 7. The culture medium was renewed every 2 days. Before harvest, cellular morphology on days 1, 3, and 7 was observed with a phase-contrast microscope (DIAPHOT, Nikon, Tokyo, Japan) and photographed using Polaroid 667 films (Nippon Polaroid, Tokyo, Japan) with a Nikon camera (HFX) connected to a microscope. Endothelialization In Vitro The endothelialization capability of each cell group was quantitatively assess by using our in vitro model of anastomotic endothelialization. The details of the method were described elsewhere (17,18). Briefly described, a 1 cm' EC sheet was prepared on a slide
Fig. 1. Appearance of bovine aortic endothelial cells at different cumulative population-doubling levels (CPDLs) maintained in tissue culture (A-C, young at CPDL 22; D-F, middle aged at CPDL 46; G-I, senescent at CPDL 70). Endothelial cells were plated at an initial cell density of 10,000 cells per 1 em". Cultures were photographed by phase-contrast microscope: 1 day (A,D,G), 3 days (B,E,H), and 7 days (C,F,I) after seeding. Bar = 100/Lrn.
Senescenced endothelialization • S. Nru
glass by the aid of an assembled culture chamber (Belko Glass Inc., Vineland, NJ). This method allows us to prepare a square monolayer sheet with linear edge without any damage of cellular sheet. The growth of the cell sheet was observed with a phase-contrast microscopy and photographed. The cell number per unit area as a function of distance from the original cell sheet edge was determined from photographs. These were translated into a histogram of the cell density-moving distance relation. Two different sets of endothelialized distances were defined on the histogram as follows: one was the position of cells at the leading edge (a distance of the leading edge), the other the point exhibiting the half cell density of the confluent region (a distance of half-confluency). The cell sheets were cultured for 7 days to measure these two indices of endothelialized distance. The details of preparative method and measuring of endothelialized distance should be referred to the first paper of the series (17).
Assay for Isolated Cell Migration Time-lapsed videomicroscopy (LA-500, PIAS, Osaka, Japan) was used to observe cell migratory tracks of isolatedly seeded cells (young, middle-aged, and senescent ECs) and of the cells at the leading edge of a cell sheet. Positions of isolated cells, which were seeded at 8 x 102 cells/em/ of density on 35-mm culture dishes (Terumo, Tokyo, Japan) and were cultured for 1 day, was recorded every 10 min for 2 h with a time-lapsed computerized videomicroscope. Specimens were maintained at 37°C in an atmosphere containing 5OJo CO2 during 2 h of measurements. The position of individual cell nuclei wee digitized on a TV monitor. The distances between the two successive cell positions were calculated and the migratory tracks were illustrated through the use of graphics and database software (Microsoft Excel, Microsoft Corp., Redmont, WA). The migration rate (tl-m/h)was determined from the averaged distance migrated for 2 h. Movements of the cells in the vicinity of the leading edge of unidirectional endothelialization were determined in the same manner as described above. Fluorescent Microscopy To observe the distribution of cytoskeletal fibers of ECs, stress fiber staining was performed. Cell sheets derived from the young ECs at days 3 and 7 were fixed in 4% formaldehyde for 10 min, subsequently treated with 0.5% Triton X-I00 for 5 min. The sheets were then rinsed with PBS, pH 7.4, and treated with a diluted Rhodamine-Phalloidine (Molecular Probes Inc., Eugene, OR) solution for 10 min at room temperature. The cells were observed with a Nikon fluorescent microscope (OPTIPHOT, Nikon) and photographed
AND
T.
357
MATSUDA
with Kodak film (TMAX-4oo, Eastman Kodak, Rochester, NY). RESULTS
ECs used for this study had three different CPDLs (young, 22 CPDL; middle aged, 46 CPDL; senescent, 70 CPDL at the time of inoculation). As shown below, the effect of aging of ECs on morphological features, proliferation, migration, and unidirectional endothelialization in vitro for these ECs were studied.
Cellular Behavior of Singly Adhered Endothelial Cells Figure 1 shows the morphological appearances of ECs with different CPDLs as a function of culture period. The characteristic difference was summarized as follows: The young ECs. On day 1, the young ECs, which have of around 100 tl-m in length in average, adhered well to the substratum. A confluent monolayer was
YCUlg
-6
5
Middle-aged
10
N
-t
senescent
C
..J
4
!:Y 10
>
..J ..J W
0
3
10
1
357
DAYS Fig. 2. Growth curves as a function of the cumulative population-doubling level (CPDL) for bovine aortic endothelial cells. Changes in proliferative rates and confluent densities associated with cellular aging were monitored by comparing growth curves of cell density with time for cells taken from cultures at different CPDLs (young, CPDL 22; middle aged, CPDL 46; senescent, CPDL 70). Cultures were refed with fresh medium every 2 days. Each point is the average of triplicate cultures.
Cell Transplantation. Volume 1, Number 5, 1992
358
obtained 3 days after seeding. Upon further culturing, sprouting cells (24) had appeared. These were a typical observation of nonaged EC culturing as reported by many researchers.
The middle-aged ECs. On the first day after seeding, the middle-aged ECs remained normal in size and shape, similar to the young ECs. The confluency was not achieved even 3 days after seeding. Although some of adhered cells were vacuolated, no enlarged cell was observed. The confluent monolayer was formed on day 7. Vacuolated cells remained spread. The senescent ECs. On the first day, some of adhered ECs possessed very broad veil-like or extremely elongated cytoplasm with vacuolation. These were over 300 J.tm in length. Upon culturing, some of them were doubly nucleated. On day 7, the confluent monolayer was formed, where two major subtypes of EC were observed; one was nearly normal in terms of size and shape, the other giant and bizarre. As shown in Fig. 2, the cells proliferated at different rates, responding to CPDL. Mean doubling time of the young ECs at the logarithmic growth phase from day 1 to day 3 was found to be 12 h. In contrast, those of the middle-aged and the senescent ECs were 20 hand 37 h, respectively. The cell densities at confluency considerably decreased as the cellular aging is forwarded. Concomitantly, adhesional areas per cell, calculated from these cell densities, significantly increased with aging ofthe cells(Table 1). The drastic increase in adhesional area per cell, which was observed for senescent ECs, apparently resulted from highly populated giant and bizarre cells as observed in Fig. 1. The migration rates of isolated cells, measured with the aid of time-lapsed computerized videomicroscopy, also depended on the aging of ECs used (Table 2). The young cells represented the fastest rate of migration, followed by the middle aged. The senescent exhibited the slowest, which is less than 50070 of the young's rate. It is apparent that this reduced migration rate was contributed from giant cells.
Table 2. Effect of cellular senescence on endothelial cell migration
Young cells Middle-aged cells Senescent cells
No. of observation
Migration rate of isolated cells (p.m/h)
16
38 ± 3
19
29 ± 3*
11
18 ± 2t
One day after seeding the migration rates of the cells were measured as described in the Materials and Methods section. Values are the mean ± the standard error of the mean calculated from the measurements of individual cell. *p < 0.05, tp < 0.001, when compared with young cells by Student's r-test.
0 half-connuency ,-.,
~
I. G.I
....G.I
leading edge
e
Q I.
Cell Transplantation, Vol. 1, pp, 355-364, 1992 Printed in the USA. All rights reserved.
Original Contribution ENDOTHELIAL CELL SENESCENCE INHIBITS UNIDIRECTIONAL ENDOTHELIALIZATION IN VITRO SATOSHI NIU* AND TAKEHISA MATSUDAt
Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita, Osaka 565, Japan
o Abstract -
many studies showed that endothelial regeneration for animals is completed within several months to years. However, the general observation is that the regeneration for human implantation is limited to nearly the anastomotic sites even upon quite long-term implantation. Cellular aging of the endothelium may be a possible factor which is associated with atherogenesis (28). Nevertheless, little attention has been paid to the effects of individual biological aging and/or cellular senescence of the host vessel on neointimal healing of vascular prostheses implanted in humans. Recent advances in cell culture techniques have made it possible to study the effects of cellular aging on proliferative potential, morphology, and differentiated functions of vascular endothelial cells in vitro (3,5,7,9,14,26). In this paper we studied the effects of cellular aging on in vitro unidirectional tissue formation, simulating the anastomotic endothelialization of vascular prostheses. Bovine endothelial cellswhich have finite life span wereemployed. In addition, we discussed whether and how cellular senescenceof the neoendothelium may result in limitations of unidirectional endothelialization. This was based on the study of the cellular mobility at the endothelializing front of the in vitro model simulating the anastomotic endothelialization.
We investigated the effects of cellular senescence on unidirectional endothelialization in vitro, simulating the anastomotic endothelialization of vascular prosthesis. The experiments were carried out with three different cumulative population-doubling levels (CPDLs) of bovine aortic endothelial cells (ECs), which have finite life span. Young ECs with 22 CPDL, middle aged with 46, and senescent with 70 at the time of inoculation were used. The effect of aging on unidirectional endothelialization, as well as cellular morphology and proliferative and migratory potentials of isolated cells, were qualitatively and quantitatively analyzed. The unidirectional endothelialization rate was determined by our newly designed method to prepare the square monolayer sheet with linear margins between ceO-adhesion and noncelladhesion regions. The results showed that endothelial cell senescence retarded not only proliferation and migration but also unidirectional endothelialization. Time-lapsed videomicroscopic study of unidirectional endothelialization process revealed that ECs at several rows back from the leading edge represented much slower rate of migration than did the ECs at the leading edge. These findings suggest that high cellular mobility observed for the ECs at the leading edge may result in localized excessive cell replication. Thus, atherosclerotic vessels containing senescent or injured ECs may have limited capability of anastomotic endotbelialization.
o Keywords -
Endothelium; Senescence; Anastomotic endothelialization; Migration. INTRODUCTION
A prosthetic graft is implanted to reconstruct diseased vessels involved in atherosclerosis. Although the incompleteness of neointimal regeneration has been clinically noted (l,22), mechanistic understanding of its incompleteness has not been clarified. Experimental and clinical studies have shown that there is a marked difference in the rate of intimal healing of vascular prostheses between human and animals (23). That is,
MATERIALS AND METHODS
Endothelial Cell Culture Endothelial cells (ECs) were harvested from bovine thoracic aortas by the mechanical scraping technique. ECs were routinely cultured at 37°C in a humidified atmosphere in 95070 air and 5% CO 2 on Corning tissue culture flasks (Iwaki Glass, Tokyo, Japan) in Dul-
6/22/92. *Present affiliation: The Second Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.
[To whom correspondence should be addressed.
ACCEPTED
355
356
Cell Transplantation. Volume I, Number 5, 1992
becco's modification of Eagle's medium (DMEM, Flow Laboratories Inc., Irvine, Scotland), supplemented with 15010 fetal bovine serum (HyClone Laboratories Inc., Logan, UT). When the cultured cells became confluent, they were subcultured with 0.25% trypsin treatment after being washed twice with phosphate-buffered saline (PBS, Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) and 0.04% EGTA. All experiments were carried out with ECs at 12, 24, and 36 passages, which were designated as young, rnidd1eaged and senescent ECs, respectively. Since cultures were split 1:4 at every passage, passage X was equivalent to 2(X - 1) cumulative population-doubling levels (CPDLs). These translated to the young ECs with 22 CPDLs, the middle aged with 46, and the senescent with 70. Endothelial Cell Growth and Morphology ECs (2 X 104 cells/well) were seeded in 24-well plates (Cell Wells, Corning Glass Works, Corning,
NY; 2 cmvwell), and the cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for up to 7 days in 1 mL of culture medium. The number of cells, which were detached with trypsinization, was counted with a Coulter counter (Model ZBI, Coulter Electronics, Inc., Hialeah, FL) on days 1,3, and 7. The culture medium was renewed every 2 days. Before harvest, cellular morphology on days 1, 3, and 7 was observed with a phase-contrast microscope (DIAPHOT, Nikon, Tokyo, Japan) and photographed using Polaroid 667 films (Nippon Polaroid, Tokyo, Japan) with a Nikon camera (HFX) connected to a microscope. Endothelialization In Vitro The endothelialization capability of each cell group was quantitatively assess by using our in vitro model of anastomotic endothelialization. The details of the method were described elsewhere (17,18). Briefly described, a 1 cm' EC sheet was prepared on a slide
Fig. 1. Appearance of bovine aortic endothelial cells at different cumulative population-doubling levels (CPDLs) maintained in tissue culture (A-C, young at CPDL 22; D-F, middle aged at CPDL 46; G-I, senescent at CPDL 70). Endothelial cells were plated at an initial cell density of 10,000 cells per 1 em". Cultures were photographed by phase-contrast microscope: 1 day (A,D,G), 3 days (B,E,H), and 7 days (C,F,I) after seeding. Bar = 100/Lrn.
Senescenced endothelialization • S. Nru
glass by the aid of an assembled culture chamber (Belko Glass Inc., Vineland, NJ). This method allows us to prepare a square monolayer sheet with linear edge without any damage of cellular sheet. The growth of the cell sheet was observed with a phase-contrast microscopy and photographed. The cell number per unit area as a function of distance from the original cell sheet edge was determined from photographs. These were translated into a histogram of the cell density-moving distance relation. Two different sets of endothelialized distances were defined on the histogram as follows: one was the position of cells at the leading edge (a distance of the leading edge), the other the point exhibiting the half cell density of the confluent region (a distance of half-confluency). The cell sheets were cultured for 7 days to measure these two indices of endothelialized distance. The details of preparative method and measuring of endothelialized distance should be referred to the first paper of the series (17).
Assay for Isolated Cell Migration Time-lapsed videomicroscopy (LA-500, PIAS, Osaka, Japan) was used to observe cell migratory tracks of isolatedly seeded cells (young, middle-aged, and senescent ECs) and of the cells at the leading edge of a cell sheet. Positions of isolated cells, which were seeded at 8 x 102 cells/em/ of density on 35-mm culture dishes (Terumo, Tokyo, Japan) and were cultured for 1 day, was recorded every 10 min for 2 h with a time-lapsed computerized videomicroscope. Specimens were maintained at 37°C in an atmosphere containing 5OJo CO2 during 2 h of measurements. The position of individual cell nuclei wee digitized on a TV monitor. The distances between the two successive cell positions were calculated and the migratory tracks were illustrated through the use of graphics and database software (Microsoft Excel, Microsoft Corp., Redmont, WA). The migration rate (tl-m/h)was determined from the averaged distance migrated for 2 h. Movements of the cells in the vicinity of the leading edge of unidirectional endothelialization were determined in the same manner as described above. Fluorescent Microscopy To observe the distribution of cytoskeletal fibers of ECs, stress fiber staining was performed. Cell sheets derived from the young ECs at days 3 and 7 were fixed in 4% formaldehyde for 10 min, subsequently treated with 0.5% Triton X-I00 for 5 min. The sheets were then rinsed with PBS, pH 7.4, and treated with a diluted Rhodamine-Phalloidine (Molecular Probes Inc., Eugene, OR) solution for 10 min at room temperature. The cells were observed with a Nikon fluorescent microscope (OPTIPHOT, Nikon) and photographed
AND
T.
357
MATSUDA
with Kodak film (TMAX-4oo, Eastman Kodak, Rochester, NY). RESULTS
ECs used for this study had three different CPDLs (young, 22 CPDL; middle aged, 46 CPDL; senescent, 70 CPDL at the time of inoculation). As shown below, the effect of aging of ECs on morphological features, proliferation, migration, and unidirectional endothelialization in vitro for these ECs were studied.
Cellular Behavior of Singly Adhered Endothelial Cells Figure 1 shows the morphological appearances of ECs with different CPDLs as a function of culture period. The characteristic difference was summarized as follows: The young ECs. On day 1, the young ECs, which have of around 100 tl-m in length in average, adhered well to the substratum. A confluent monolayer was
YCUlg
-6
5
Middle-aged
10
N
-t
senescent
C
..J
4
!:Y 10
>
..J ..J W
0
3
10
1
357
DAYS Fig. 2. Growth curves as a function of the cumulative population-doubling level (CPDL) for bovine aortic endothelial cells. Changes in proliferative rates and confluent densities associated with cellular aging were monitored by comparing growth curves of cell density with time for cells taken from cultures at different CPDLs (young, CPDL 22; middle aged, CPDL 46; senescent, CPDL 70). Cultures were refed with fresh medium every 2 days. Each point is the average of triplicate cultures.
Cell Transplantation. Volume 1, Number 5, 1992
358
obtained 3 days after seeding. Upon further culturing, sprouting cells (24) had appeared. These were a typical observation of nonaged EC culturing as reported by many researchers.
The middle-aged ECs. On the first day after seeding, the middle-aged ECs remained normal in size and shape, similar to the young ECs. The confluency was not achieved even 3 days after seeding. Although some of adhered cells were vacuolated, no enlarged cell was observed. The confluent monolayer was formed on day 7. Vacuolated cells remained spread. The senescent ECs. On the first day, some of adhered ECs possessed very broad veil-like or extremely elongated cytoplasm with vacuolation. These were over 300 J.tm in length. Upon culturing, some of them were doubly nucleated. On day 7, the confluent monolayer was formed, where two major subtypes of EC were observed; one was nearly normal in terms of size and shape, the other giant and bizarre. As shown in Fig. 2, the cells proliferated at different rates, responding to CPDL. Mean doubling time of the young ECs at the logarithmic growth phase from day 1 to day 3 was found to be 12 h. In contrast, those of the middle-aged and the senescent ECs were 20 hand 37 h, respectively. The cell densities at confluency considerably decreased as the cellular aging is forwarded. Concomitantly, adhesional areas per cell, calculated from these cell densities, significantly increased with aging ofthe cells(Table 1). The drastic increase in adhesional area per cell, which was observed for senescent ECs, apparently resulted from highly populated giant and bizarre cells as observed in Fig. 1. The migration rates of isolated cells, measured with the aid of time-lapsed computerized videomicroscopy, also depended on the aging of ECs used (Table 2). The young cells represented the fastest rate of migration, followed by the middle aged. The senescent exhibited the slowest, which is less than 50070 of the young's rate. It is apparent that this reduced migration rate was contributed from giant cells.
Table 2. Effect of cellular senescence on endothelial cell migration
Young cells Middle-aged cells Senescent cells
No. of observation
Migration rate of isolated cells (p.m/h)
16
38 ± 3
19
29 ± 3*
11
18 ± 2t
One day after seeding the migration rates of the cells were measured as described in the Materials and Methods section. Values are the mean ± the standard error of the mean calculated from the measurements of individual cell. *p < 0.05, tp < 0.001, when compared with young cells by Student's r-test.
0 half-connuency ,-.,
~
I. G.I
....G.I
leading edge
e
Q I.
