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Comparison of pulmonary endothelial cell and fibroblast proliferation using time-lapse cinematographic analysis
TISSU F & CELL 1981 13 (4) 645-650
0040-8166/81/00490645502.00
',(~ 1981 Longman Group Lid
MARLENE ABSHER* and UNA S. R Y A N I
C O M P A R I S O N OF P U L M O N A R Y E N D O T H E L I A L CELL A N D F I B R O B L A S T PROLIFERATION USING T I M E - L A P S E C I N E M A T O G R A P H I C ANALYSIS ABSTRACT. Time-lapse cinematography was used to study and compare the proliferation and migration activity of puhnonary endothelial cells and fibroblasts, two cell types with very different structural and functiomd properties. Endothelial cells were found to have a more rapid growth rate than fibroblasts. Contributing to the shorter population doubling time of the endothelial cells were lower interdivision times and a tendency for these cells to remain in division cycle with successive generadons of growth. Striking differences between endothelial cells and fibroblasts were seen in migration behaviour, Endothelial cells had lower migration rates and tended to remain w/thin a restricted growth area, whereas libroblasts m/grated freely throughout the growth area.
Introduction IN order to improve t m d e r s t a n d i n g o f structure-fttnction correlations in the lungs, we u n d e r t o o k c o m p a r a t i v e studies o f the proliferative behavior o f two cell types with very different structural a n d functional properties, different patterns o f developmetat a n d different responses to injury; n a m e l y ptthaaonary endothelial cells a n d fibroblasts. • For these studies we chose to use timelapse c i n e n m t o g r a p h y (TLC), a uniqttely valuable techniqtte for assessing the proliferative behavior o f cells in a more detailed and quantitative m a n n e r than can be obtained from studies o f populations o f cells. Proliferation o f populations o f cells d e p e n d s on the p r o p o r t i o n o f cells capable o f dividing, the interdivision times o f the cycling cells and the p r o p o r t i o n o f cells that remain in * Department of Medicine. University of Vermont, Burlington, Vermont. t Department of Medicine, University of Miami. Miami, Florida. Send reprint requests to: Dr Marlene Absher, Department of Medicine, Given Build. E-210B, University of Vermont, College of Medicine, Burlington, Vermont 05405. Received 13 August 1981.
cycle with tin'le in culture. T L C allows ttS to delineate these c o m p o n e n t s o f proliferation in a very precise nlanner. H u m a n em bryon ic lung fibroblasts (H E L F } were chosen to c o m p a r e with tb,~ bovine p u l m o n a r y endothelial cells because H E L F have been so well characterized at the popttlation, clonal a n d individttal cell level. T L C studies o1" e m b r y o n i c a n d adult lung libroblasts have revealed few differences in cell division patterns and migration activity (Davis et a L , 1979). Previous T L C studies with lung fibroblasts have s h o w n these cells to be highly heterogeneous with respect t o the above parameters o f proliferation (Absher et al., 1974; A b s h e r a n d Absher, 1976). We were interested in using T L C to s t u d y the proliferative behavior o f p u l m o n a r y endothelial cells in culture a n d to c o m p a r e these cells to lung fibroblasts; the two cell types being subject to very different d e m a n d s d u r i n g the overall functioning o f the lungs both in h e a l t h a n d disease. In the studies reported h e r e , c o m parisons were m a d e o f g r o w t h :rates, interdivision time (IDT); relationship between IDTs o f m o t h e r - d a u g h t e r a n d sister-sister pairs. We also c o m p a r e d the migration
646
ABSHER
AND
RYAN
patterns and spatial relationships in division sites of progeny o f clones o f endothelial cells and fibroblasts. M a r k e d differences were found in growth rates, 1DT and migration patterns but relationship between mother-dauglater and sister-sister I DT pairs were similar for endothelial cells and hmg fibroblasts.
individual cells were traced between division events. The tracings were measured with a map meter and the units converted to micrometers. The distance travelled was divided by the I D T of the cell to derive the mean migration rate (MIG) in/Lm/hr.
Materials and Methods
The overall growth o f the endothelial cells and fibroblasts as expressed by cunaulatl~'e nunaber o f divisions with time in culture is shown in Fig. I. The lag time to the first division was longer for the fibroblasts tlmn for the endothelial cells and the growth rate for endothelial cells was greater than for the fibroblasts. The final n u m b e r of divisions in the endothelial cell culture was 4-5 times that o f the fibroblasts. Comparison o f interdivision time data for endothelial cells vs. fibroblasts also revealed marked differences. As seen in Table ! the mean
Bovine endothelial cells (CPA-6) were derived from the main pulmonary artery by gentle scraping with a scalpel and cultured as described previously (Ryan et aL, 1980). Endothelial cells were characterized by morphological and biochemical criteria (Ryan et aL, 1978; Ryan, 198I). To prepare cells for filming 7-5× 10 a cells in 7.0ml medium were seeded into a 6 0 r a m Lux plastic dish. The cells were cultured in Medium 199 (M199) supplemented with 10% fetal bovine serum (FBS). The cells were filmed at population doubling level 1 I. Embryonic lung fibroblasts, IMR-90, were obtained from the Institute for Medical Research, Camden, NJ (Nichols et aL, 1977). The cells were cultured in Minimal Essential Medium (MEM), supplemented with 10yo fetal bovine serum (FBS) and I00 /~g/ml chlortetracycline. F o r filming, 4.0x 104 cells in 7.0 ml medium were seeded into a 60 mm Lux plastic dish. Tile cells were filmed at population doubling level of 22. For filming the culture dishes were fitted with Cooper dish lids and sealed with sterile petroleum jelly. The T L C apparatus consisted o f a Zeiss microscope equipped with long working distance condensor and 10x phase objective, a Sage model 50I time-lapse control unit fitted w i t h a 1 6 r a m Bolex camera. The stage and culture dish were maintained a t 3 7 ° C , w i t h a Zeiss alrstream incubator. K o d a k Plus X reversal film was used and the filming rate was one frame per minute. The filmed area was approximately 0.7 mm z. The films were edited using a K o d a k Analyst projector to identify all dividing cells; their locations a n d times o f division. Each cell was followed from its p o i n t o f creation to subsequent division. Interdivision time ( I D T ) was calculated from the filming r a t e and the number o f elapsed frames between divisions. F o r migration studies, the trajectories of
Results
Table 1. Comparisolz o f htterdivision thne ( I D T ) h~ endothelial cells attd fibroblasts
Range of IDT (hr) Mean IDT_+s.e.m. Median IDT Distribution of IDTs I0 hr ~<15 hr ~20 hr
Endothelial cells CPA-6
Fibroblasts 1M R-90
7-35 12.4_.+0.16 (n= 542) 11 "2
10-40 17.4_+0.37 (n= 158) 16.4
~o of IDTs 3I .2 84.6 96-5
~ of IDTs 1.0 44.3 86"7
Analysis o f l D T data for all cells dividing cells during culture period of approximately 90 hr. and median IDTs were substantially lower in the endothelial cultures than in tile fibroblasts although the range of I D T values was essentially the s a m e for these two cell types. The major difference can be seen in the distributions o f IDTs (also s h o w n in Table I). In the endothelial c e l l cultures over 30Yo o f t h e I D T values were ~< I0 hr, c o m p a r e d to less than 1 Yo of IDTs being ~<10hr in fibroblast cultures. 84"6~o o f endothelial cell I DTs were ~<15 hr c o m p a r e d to 44-3Yo o f fibroblasts. While I D T is an important factor in the overall growth rate
ENDOTHELIAL
A N D F I B R . O B L A S T GP, O W T l t
647
Table 2. Relationship between poptdatiotl dottblhsg thne (PDT) attd htterdivision time (IDT) hi endothelial cells atM fibroblasts Endothelial cells CPA-6
Fibroblasts I M R-90
Popttlation size group
PDT
I DT* +__s.e.m,
Population size group
PDT
i DT* 4- s.e.m.
51-100 I01-200 201--400
13.4 13-1 14.5
13.2±0.49 12.4±0.38 12.9±0.23
25-50 51-100 101-200
21.1 18.3 27.1
17.1±0.49 17.3± 0.53 16.3 ± 0 . 4 4
* I DT value assigned to population size group in which cell was created. PDT and IDT in hours. o f a culture, the ability o f cells to remain in cycle primarily determines p o p u l a t i o n size. C o m p a r i s o n o f population d o u b l i n g time ( P D T ) and interdivision time ( I D T ) provides a m e a n s o f assessing whether cells are remaining in cycle. If I D T = P D T this indicates cells are in cycle, whereas when 1 D T is less than P D T this indicates that cells are "dropping out" o f cycle. As can be seen in T a b l e 2, the I D T and P D T values for the endothelial cells are very close, whereas in the fibroblast culture I D T is less than P D T at all p o p u l a t i o n doublings. T h e latter data
are consistent with previously r e p o r t e d T L C studies o f diploid fibroblasts ( A b s h e r and Absher, I976; A b s h e r and Sylvester, 1981; A b s h e r et aL, 1981). T h e I D T relationships between m o t h e r d a u g h t e r and sister-sister pairs were n o t different for endothelial cells and fibroblasts as seen in T a b l e 3. N o t e that sister pairs are m o r e highly correlated in each cell type than are m o t h e r - d a u g h t e r pairs. In b o t h
Endothelial Cells
Table 3. Correlations between IDTs o f sister pairs oral mo/her~ltmgh/er pah's b), generation Correlation coefficients (r) for sister pairs Generation ]
2 3 4 All ge~eralions
Endothelial cells 0.35 (n=20) 0"4001-30) 0"23 01=46) 0"59 (n=33) 0.41
Fibroblasts t-" O
Fibroblasts 0'86 0"12 0"33 0.30 0.51
01----12) 0z=lS) (n----21) (n=9)
:~ 1oo .> "~ 50
z
._> t~
"~ io Correlation coefficients (r) for mother-daughter pairs Generation 1 2
Endothelial cells
0.45 --0.06 3 0.03 4 0.03 All generations 0.13
5
Fibroblasts
(n=70) 0-39 (n=45) (n= 103) --0.08 (n=49) Or-----88) --0-16 (u=29) 01=32) 0.47 (n=4) 0-27
n = number of pairs analyzed.
E
zo
40
60
80
I00
Hours in Culture Fig. 1. Growth curve of endothelial cells (CPA-6) and lung fibroblasts (IMR-90) expressed as ¢umulative number of divisions per uni t time in culture.
ABSHER
648
endothelial cell cultures and fibroblasts there is considerable fluctuation fi-om generation to generation in IDT values such that in any generation about half of t h e mother cell IDTs will be < or > the IDT of its progeny in the next generation. Thus, there tends Io be a low overall correlation coefficienl value for I DTs ot" m o t h e r - d a u g h t e r pairs. These results are consistent with previously observed mother: daughter and sister pair i DT relationships in clones of H E L F (Absheret aL, 1974; Absher and Absher, 1976; Absher and Syiwester, 1981). Interesting differences were observed in the spatial relationships and migration patterns o f progeny o f clones o f fibroblasts and endo-
AND
tllelial ceils. Fig. 2 is a diagram denoting where the progeny o f a fibroblast clone and an endothelial celt clone divided in relation to each other and the site of the original division. The migration rate of endothelial cells was lower than that of fibroblasts. The mean itm/hr for" the progeny of endothelial cells, clone 9, was 25.9 +__!.2 s.e.m, compared to 42.8+ 2"0 s.e.m, for the fibroblast, clone 3. The spatial relationships of division sites o f cional progeny depicted in the diagram ira Fig. 2 suggests that endothelial cells identify with a cluster group, and though they may stray to :adjacent cell clusters between division events, they return to their 'own neighborhood" to divide, whereas
Endothelial Cells
Fibroblasis
,
RYAN
®
Fig. 2. Diagrammatic representation o f division sites o f progeny o f a clone of'endothelial cells, clone 9 and o f a clone o f fibroblasts, clone 3. Numbers in circles are I D nun]bets for each cell; A r r o w s indicate direction of" migration from mother cell to progeny.
ENDOTHI"_LIAL AND FIBI[OBLAST GI~OWTH
649
substances, and to provide a smooth gliding surface for the blood. Confluent cultures of fibroblasts show who'~r arrays of cells Discussion with areas of monolayering and multilayerComparison of endothelial cells and libro- ing. Observation of the structuring of the blasts has shown marked differences in cell sheets by these two celt types was posseveral growth characteristics. Endothelial sible with the TLC films. It was observed cells had a higher growth rate than fibro- that endothelial cells do not appreciably blasts. Contributing to the higher growth migrate over or under each other, but mainrate were shorter interdivision times (IDT) tain a strict single layered sheet as itt vivo. for a large portion of endothelial cells and In contrast, fibroblasts migrate fi'eely over the tendency of endothelial cells to remain in and under each other and appear to migrate cycle with successive generations of growth. in random directions, frequently changing Growth characteristics that were similar course. The endothelial cells appear to in endothelial cell and fibroblast cultures identify with a cluster group, and though were the IDT relationships between sibling they do migrate to adjacent cell clusters pairs and mother-daughter pairs. Studies between division events, they return to of fibroblasts and other types of cells have their own clonal or cluster group to divide generally sho~n that sibling I DTs are much (see Fig. 2). Since ht vivo endothelial cells more highly correlated than randomly selec- form single-layered sheets the observed ted pairs of cells' IDTs (Absher and Absher, migration patterns (which includes rate 1976; Absher et aL, 1981 ; Minor and Smith, and direction of movement and maintenance 1974). On the other hand n'totlaerdaughter of a spatially cohesive clonal unit) may I DTs are generally not correlated (Absher represent an economy of movement beneand Absher, 1976; Absher et aL, 1981). ficial to generation:of these cells in Vivo and Comparison of I DT pairs showed that corre- maintenance of the endothelium. This would lation coefficients for sibling pairs were the be of particular importance when repair same for endothelial cells and fibroblasts. of damaged endothelium was required. On Mother-daughter pairs were not correlated the other hand, fibroblasts involved in in eitherendotlaelial cell or fibroblast cultures. connective tissue generation and wound A striking difference in cultural behavior healing must be able to migrate into approbetween endothelial cells and fibroblasts priate areas and also to form multilayers of was in the migration patterns and spatial cells into a tissue matrix, a response clearly relationsliips of division sites of progeny of e,hdent in the progressive thickening of the individual clones, in confluent cultures, interstitium in, for example, fibrotic and endothelial cells form a monolayer of cells immunologic interstitial lung disease (Hance clustered in a cobblestone arrangement and Crystal, 1975). t,Ryan et aL, t978). The monolayer morpllology is a critical feature of the most Acknowledgements important fi.mctions of endothelial cells: to present an extensive surface area for inter- This study was supported by grants HL141212 and HL-21568 from the National action of cell-bound enzymes with circulating vasoactive substrates, to allow a minimal Institutes of Health and the Council for diffusion barrier for the passage of volatile Tobacco Research, USA, Inc. fibroblasts tend to traverse the entire growth area in what appears to be a randon't li~shion.
ABSHER AND RYAN
650
References AnslleR, P. M., ABSHEa, R. G. and BarNEs, W. D. 1974. Genealogies of clones of diploid fibroblasts. Cinemicrophotographic observations of cell division patterns in relation to population age. Expl. Cell Res., 88, 95-104. AnS, ER, P. M. and AaslJEa, R. G. 1976. Ctonal variation and aging of diploid fibroblasts. Cinematographic studies of cell pedigrees. Expl Cell Res., 103, 257-255. A13SHER, M. and SYLW~STER, D. 1981. Effects of silica o51 Iluman lung fibroblasts. Survival data analysis of time-lapse cinematography data. Era,. Res. (in press'). AnsuEI~, M;, SYt.WES'r,£1~,D. and HAItT, B. A. Time-lapse cinematographic analysis of beryUium-lung fibro. blast interactions. Expl Ltotg Res. (submitted). Davis, G. S., MOE.RJNc;, J. M., AI~SHEg, P. M., BROD¢, A. R., Kt~Lt,E~', J., Low, R. B. and GREEN, G. M. 1979. Isolation and Characterization of fibroblasts obtained by pulmonary lavage of human subjects. In lqtro, 12, 619-635. H^NcE, A. J. and CttvsT^t, R. G. 1975. Tile connective tissue of the lung. Am. Rev. Resp. Dis., 112, 657-71 I. Mllqo~t, P. D. and SMrrH, J. A. 1974. Explanations of degree of correlation of sibling generation times in animal cells. Nature, 248, 241-243. NicHoLs, W. W., MuapllY, D. G., CRISTOFALO,V. J., TOJI, L, H., Gp.EENE,A. E. and DWIGttT, S. A. 1977. Characterization of a new human diploid cell strain, 1MR-90. Science, 196, 60-63. RY^N, U. S. 1981. Isolation and cultures of pulmonary endothelial cells. Pulmonary Toxicology (etL G. E. R. Hook). Raven Press. RvAN, U. S., CLE~,tEUTS,E., H^r~LJSTON,D. and RYAN, J. W. 1978. Isolation and culture of pulmonary artery endothelial cells. Tissue & Cell, I0, 535-554. Re^N, U. S., MORTARA, M. and WHrrAKER,C. 1980. Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes. Tissue & Cell, 12, 619-635.
0040-8166/81/00490645502.00
',(~ 1981 Longman Group Lid
MARLENE ABSHER* and UNA S. R Y A N I
C O M P A R I S O N OF P U L M O N A R Y E N D O T H E L I A L CELL A N D F I B R O B L A S T PROLIFERATION USING T I M E - L A P S E C I N E M A T O G R A P H I C ANALYSIS ABSTRACT. Time-lapse cinematography was used to study and compare the proliferation and migration activity of puhnonary endothelial cells and fibroblasts, two cell types with very different structural and functiomd properties. Endothelial cells were found to have a more rapid growth rate than fibroblasts. Contributing to the shorter population doubling time of the endothelial cells were lower interdivision times and a tendency for these cells to remain in division cycle with successive generadons of growth. Striking differences between endothelial cells and fibroblasts were seen in migration behaviour, Endothelial cells had lower migration rates and tended to remain w/thin a restricted growth area, whereas libroblasts m/grated freely throughout the growth area.
Introduction IN order to improve t m d e r s t a n d i n g o f structure-fttnction correlations in the lungs, we u n d e r t o o k c o m p a r a t i v e studies o f the proliferative behavior o f two cell types with very different structural a n d functional properties, different patterns o f developmetat a n d different responses to injury; n a m e l y ptthaaonary endothelial cells a n d fibroblasts. • For these studies we chose to use timelapse c i n e n m t o g r a p h y (TLC), a uniqttely valuable techniqtte for assessing the proliferative behavior o f cells in a more detailed and quantitative m a n n e r than can be obtained from studies o f populations o f cells. Proliferation o f populations o f cells d e p e n d s on the p r o p o r t i o n o f cells capable o f dividing, the interdivision times o f the cycling cells and the p r o p o r t i o n o f cells that remain in * Department of Medicine. University of Vermont, Burlington, Vermont. t Department of Medicine, University of Miami. Miami, Florida. Send reprint requests to: Dr Marlene Absher, Department of Medicine, Given Build. E-210B, University of Vermont, College of Medicine, Burlington, Vermont 05405. Received 13 August 1981.
cycle with tin'le in culture. T L C allows ttS to delineate these c o m p o n e n t s o f proliferation in a very precise nlanner. H u m a n em bryon ic lung fibroblasts (H E L F } were chosen to c o m p a r e with tb,~ bovine p u l m o n a r y endothelial cells because H E L F have been so well characterized at the popttlation, clonal a n d individttal cell level. T L C studies o1" e m b r y o n i c a n d adult lung libroblasts have revealed few differences in cell division patterns and migration activity (Davis et a L , 1979). Previous T L C studies with lung fibroblasts have s h o w n these cells to be highly heterogeneous with respect t o the above parameters o f proliferation (Absher et al., 1974; A b s h e r a n d Absher, 1976). We were interested in using T L C to s t u d y the proliferative behavior o f p u l m o n a r y endothelial cells in culture a n d to c o m p a r e these cells to lung fibroblasts; the two cell types being subject to very different d e m a n d s d u r i n g the overall functioning o f the lungs both in h e a l t h a n d disease. In the studies reported h e r e , c o m parisons were m a d e o f g r o w t h :rates, interdivision time (IDT); relationship between IDTs o f m o t h e r - d a u g h t e r a n d sister-sister pairs. We also c o m p a r e d the migration
646
ABSHER
AND
RYAN
patterns and spatial relationships in division sites of progeny o f clones o f endothelial cells and fibroblasts. M a r k e d differences were found in growth rates, 1DT and migration patterns but relationship between mother-dauglater and sister-sister I DT pairs were similar for endothelial cells and hmg fibroblasts.
individual cells were traced between division events. The tracings were measured with a map meter and the units converted to micrometers. The distance travelled was divided by the I D T of the cell to derive the mean migration rate (MIG) in/Lm/hr.
Materials and Methods
The overall growth o f the endothelial cells and fibroblasts as expressed by cunaulatl~'e nunaber o f divisions with time in culture is shown in Fig. I. The lag time to the first division was longer for the fibroblasts tlmn for the endothelial cells and the growth rate for endothelial cells was greater than for the fibroblasts. The final n u m b e r of divisions in the endothelial cell culture was 4-5 times that o f the fibroblasts. Comparison o f interdivision time data for endothelial cells vs. fibroblasts also revealed marked differences. As seen in Table ! the mean
Bovine endothelial cells (CPA-6) were derived from the main pulmonary artery by gentle scraping with a scalpel and cultured as described previously (Ryan et aL, 1980). Endothelial cells were characterized by morphological and biochemical criteria (Ryan et aL, 1978; Ryan, 198I). To prepare cells for filming 7-5× 10 a cells in 7.0ml medium were seeded into a 6 0 r a m Lux plastic dish. The cells were cultured in Medium 199 (M199) supplemented with 10% fetal bovine serum (FBS). The cells were filmed at population doubling level 1 I. Embryonic lung fibroblasts, IMR-90, were obtained from the Institute for Medical Research, Camden, NJ (Nichols et aL, 1977). The cells were cultured in Minimal Essential Medium (MEM), supplemented with 10yo fetal bovine serum (FBS) and I00 /~g/ml chlortetracycline. F o r filming, 4.0x 104 cells in 7.0 ml medium were seeded into a 60 mm Lux plastic dish. Tile cells were filmed at population doubling level of 22. For filming the culture dishes were fitted with Cooper dish lids and sealed with sterile petroleum jelly. The T L C apparatus consisted o f a Zeiss microscope equipped with long working distance condensor and 10x phase objective, a Sage model 50I time-lapse control unit fitted w i t h a 1 6 r a m Bolex camera. The stage and culture dish were maintained a t 3 7 ° C , w i t h a Zeiss alrstream incubator. K o d a k Plus X reversal film was used and the filming rate was one frame per minute. The filmed area was approximately 0.7 mm z. The films were edited using a K o d a k Analyst projector to identify all dividing cells; their locations a n d times o f division. Each cell was followed from its p o i n t o f creation to subsequent division. Interdivision time ( I D T ) was calculated from the filming r a t e and the number o f elapsed frames between divisions. F o r migration studies, the trajectories of
Results
Table 1. Comparisolz o f htterdivision thne ( I D T ) h~ endothelial cells attd fibroblasts
Range of IDT (hr) Mean IDT_+s.e.m. Median IDT Distribution of IDTs I0 hr ~<15 hr ~20 hr
Endothelial cells CPA-6
Fibroblasts 1M R-90
7-35 12.4_.+0.16 (n= 542) 11 "2
10-40 17.4_+0.37 (n= 158) 16.4
~o of IDTs 3I .2 84.6 96-5
~ of IDTs 1.0 44.3 86"7
Analysis o f l D T data for all cells dividing cells during culture period of approximately 90 hr. and median IDTs were substantially lower in the endothelial cultures than in tile fibroblasts although the range of I D T values was essentially the s a m e for these two cell types. The major difference can be seen in the distributions o f IDTs (also s h o w n in Table I). In the endothelial c e l l cultures over 30Yo o f t h e I D T values were ~< I0 hr, c o m p a r e d to less than 1 Yo of IDTs being ~<10hr in fibroblast cultures. 84"6~o o f endothelial cell I DTs were ~<15 hr c o m p a r e d to 44-3Yo o f fibroblasts. While I D T is an important factor in the overall growth rate
ENDOTHELIAL
A N D F I B R . O B L A S T GP, O W T l t
647
Table 2. Relationship between poptdatiotl dottblhsg thne (PDT) attd htterdivision time (IDT) hi endothelial cells atM fibroblasts Endothelial cells CPA-6
Fibroblasts I M R-90
Popttlation size group
PDT
I DT* +__s.e.m,
Population size group
PDT
i DT* 4- s.e.m.
51-100 I01-200 201--400
13.4 13-1 14.5
13.2±0.49 12.4±0.38 12.9±0.23
25-50 51-100 101-200
21.1 18.3 27.1
17.1±0.49 17.3± 0.53 16.3 ± 0 . 4 4
* I DT value assigned to population size group in which cell was created. PDT and IDT in hours. o f a culture, the ability o f cells to remain in cycle primarily determines p o p u l a t i o n size. C o m p a r i s o n o f population d o u b l i n g time ( P D T ) and interdivision time ( I D T ) provides a m e a n s o f assessing whether cells are remaining in cycle. If I D T = P D T this indicates cells are in cycle, whereas when 1 D T is less than P D T this indicates that cells are "dropping out" o f cycle. As can be seen in T a b l e 2, the I D T and P D T values for the endothelial cells are very close, whereas in the fibroblast culture I D T is less than P D T at all p o p u l a t i o n doublings. T h e latter data
are consistent with previously r e p o r t e d T L C studies o f diploid fibroblasts ( A b s h e r and Absher, I976; A b s h e r and Sylvester, 1981; A b s h e r et aL, 1981). T h e I D T relationships between m o t h e r d a u g h t e r and sister-sister pairs were n o t different for endothelial cells and fibroblasts as seen in T a b l e 3. N o t e that sister pairs are m o r e highly correlated in each cell type than are m o t h e r - d a u g h t e r pairs. In b o t h
Endothelial Cells
Table 3. Correlations between IDTs o f sister pairs oral mo/her~ltmgh/er pah's b), generation Correlation coefficients (r) for sister pairs Generation ]
2 3 4 All ge~eralions
Endothelial cells 0.35 (n=20) 0"4001-30) 0"23 01=46) 0"59 (n=33) 0.41
Fibroblasts t-" O
Fibroblasts 0'86 0"12 0"33 0.30 0.51
01----12) 0z=lS) (n----21) (n=9)
:~ 1oo .> "~ 50
z
._> t~
"~ io Correlation coefficients (r) for mother-daughter pairs Generation 1 2
Endothelial cells
0.45 --0.06 3 0.03 4 0.03 All generations 0.13
5
Fibroblasts
(n=70) 0-39 (n=45) (n= 103) --0.08 (n=49) Or-----88) --0-16 (u=29) 01=32) 0.47 (n=4) 0-27
n = number of pairs analyzed.
E
zo
40
60
80
I00
Hours in Culture Fig. 1. Growth curve of endothelial cells (CPA-6) and lung fibroblasts (IMR-90) expressed as ¢umulative number of divisions per uni t time in culture.
ABSHER
648
endothelial cell cultures and fibroblasts there is considerable fluctuation fi-om generation to generation in IDT values such that in any generation about half of t h e mother cell IDTs will be < or > the IDT of its progeny in the next generation. Thus, there tends Io be a low overall correlation coefficienl value for I DTs ot" m o t h e r - d a u g h t e r pairs. These results are consistent with previously observed mother: daughter and sister pair i DT relationships in clones of H E L F (Absheret aL, 1974; Absher and Absher, 1976; Absher and Syiwester, 1981). Interesting differences were observed in the spatial relationships and migration patterns o f progeny o f clones o f fibroblasts and endo-
AND
tllelial ceils. Fig. 2 is a diagram denoting where the progeny o f a fibroblast clone and an endothelial celt clone divided in relation to each other and the site of the original division. The migration rate of endothelial cells was lower than that of fibroblasts. The mean itm/hr for" the progeny of endothelial cells, clone 9, was 25.9 +__!.2 s.e.m, compared to 42.8+ 2"0 s.e.m, for the fibroblast, clone 3. The spatial relationships of division sites o f cional progeny depicted in the diagram ira Fig. 2 suggests that endothelial cells identify with a cluster group, and though they may stray to :adjacent cell clusters between division events, they return to their 'own neighborhood" to divide, whereas
Endothelial Cells
Fibroblasis
,
RYAN
®
Fig. 2. Diagrammatic representation o f division sites o f progeny o f a clone of'endothelial cells, clone 9 and o f a clone o f fibroblasts, clone 3. Numbers in circles are I D nun]bets for each cell; A r r o w s indicate direction of" migration from mother cell to progeny.
ENDOTHI"_LIAL AND FIBI[OBLAST GI~OWTH
649
substances, and to provide a smooth gliding surface for the blood. Confluent cultures of fibroblasts show who'~r arrays of cells Discussion with areas of monolayering and multilayerComparison of endothelial cells and libro- ing. Observation of the structuring of the blasts has shown marked differences in cell sheets by these two celt types was posseveral growth characteristics. Endothelial sible with the TLC films. It was observed cells had a higher growth rate than fibro- that endothelial cells do not appreciably blasts. Contributing to the higher growth migrate over or under each other, but mainrate were shorter interdivision times (IDT) tain a strict single layered sheet as itt vivo. for a large portion of endothelial cells and In contrast, fibroblasts migrate fi'eely over the tendency of endothelial cells to remain in and under each other and appear to migrate cycle with successive generations of growth. in random directions, frequently changing Growth characteristics that were similar course. The endothelial cells appear to in endothelial cell and fibroblast cultures identify with a cluster group, and though were the IDT relationships between sibling they do migrate to adjacent cell clusters pairs and mother-daughter pairs. Studies between division events, they return to of fibroblasts and other types of cells have their own clonal or cluster group to divide generally sho~n that sibling I DTs are much (see Fig. 2). Since ht vivo endothelial cells more highly correlated than randomly selec- form single-layered sheets the observed ted pairs of cells' IDTs (Absher and Absher, migration patterns (which includes rate 1976; Absher et aL, 1981 ; Minor and Smith, and direction of movement and maintenance 1974). On the other hand n'totlaerdaughter of a spatially cohesive clonal unit) may I DTs are generally not correlated (Absher represent an economy of movement beneand Absher, 1976; Absher et aL, 1981). ficial to generation:of these cells in Vivo and Comparison of I DT pairs showed that corre- maintenance of the endothelium. This would lation coefficients for sibling pairs were the be of particular importance when repair same for endothelial cells and fibroblasts. of damaged endothelium was required. On Mother-daughter pairs were not correlated the other hand, fibroblasts involved in in eitherendotlaelial cell or fibroblast cultures. connective tissue generation and wound A striking difference in cultural behavior healing must be able to migrate into approbetween endothelial cells and fibroblasts priate areas and also to form multilayers of was in the migration patterns and spatial cells into a tissue matrix, a response clearly relationsliips of division sites of progeny of e,hdent in the progressive thickening of the individual clones, in confluent cultures, interstitium in, for example, fibrotic and endothelial cells form a monolayer of cells immunologic interstitial lung disease (Hance clustered in a cobblestone arrangement and Crystal, 1975). t,Ryan et aL, t978). The monolayer morpllology is a critical feature of the most Acknowledgements important fi.mctions of endothelial cells: to present an extensive surface area for inter- This study was supported by grants HL141212 and HL-21568 from the National action of cell-bound enzymes with circulating vasoactive substrates, to allow a minimal Institutes of Health and the Council for diffusion barrier for the passage of volatile Tobacco Research, USA, Inc. fibroblasts tend to traverse the entire growth area in what appears to be a randon't li~shion.
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References AnslleR, P. M., ABSHEa, R. G. and BarNEs, W. D. 1974. Genealogies of clones of diploid fibroblasts. Cinemicrophotographic observations of cell division patterns in relation to population age. Expl. Cell Res., 88, 95-104. AnS, ER, P. M. and AaslJEa, R. G. 1976. Ctonal variation and aging of diploid fibroblasts. Cinematographic studies of cell pedigrees. Expl Cell Res., 103, 257-255. A13SHER, M. and SYLW~STER, D. 1981. Effects of silica o51 Iluman lung fibroblasts. Survival data analysis of time-lapse cinematography data. Era,. Res. (in press'). AnsuEI~, M;, SYt.WES'r,£1~,D. and HAItT, B. A. Time-lapse cinematographic analysis of beryUium-lung fibro. blast interactions. Expl Ltotg Res. (submitted). Davis, G. S., MOE.RJNc;, J. M., AI~SHEg, P. M., BROD¢, A. R., Kt~Lt,E~', J., Low, R. B. and GREEN, G. M. 1979. Isolation and Characterization of fibroblasts obtained by pulmonary lavage of human subjects. In lqtro, 12, 619-635. H^NcE, A. J. and CttvsT^t, R. G. 1975. Tile connective tissue of the lung. Am. Rev. Resp. Dis., 112, 657-71 I. Mllqo~t, P. D. and SMrrH, J. A. 1974. Explanations of degree of correlation of sibling generation times in animal cells. Nature, 248, 241-243. NicHoLs, W. W., MuapllY, D. G., CRISTOFALO,V. J., TOJI, L, H., Gp.EENE,A. E. and DWIGttT, S. A. 1977. Characterization of a new human diploid cell strain, 1MR-90. Science, 196, 60-63. RY^N, U. S. 1981. Isolation and cultures of pulmonary endothelial cells. Pulmonary Toxicology (etL G. E. R. Hook). Raven Press. RvAN, U. S., CLE~,tEUTS,E., H^r~LJSTON,D. and RYAN, J. W. 1978. Isolation and culture of pulmonary artery endothelial cells. Tissue & Cell, I0, 535-554. Re^N, U. S., MORTARA, M. and WHrrAKER,C. 1980. Methods for microcarrier culture of bovine pulmonary artery endothelial cells avoiding the use of enzymes. Tissue & Cell, 12, 619-635.