Biol Cell (1990) 68, 231-238 © Elsevier, Paris
231
Original article
Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor Claude Klein-Soyer 1,, Alain Beretz ~, Jean-Pierre Cazenave 1, Francis Driot 2, Jean-Pierre M a f f r a n d 2 t Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatdriaux, I N S E R M U 311, Centre Rdgional de Transfusion Sanguine, 10 rue Spielmann, 67085 Strasbourg Cedex; 2Sanofi Recherche, 195 route d'Espagne, 31035 Toulouse, France (Received 12 December 1989; accepted l0 April 1990) Summary - Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95070 in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50°70. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50070 of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation. irradiation / heparin / acidic fibroblast growth factor / endothelium / wound repair
Introduction
Injury of the vascular endothelium is of importance in the development of atherosclerosis [30]. The repair of a d e n u d i n g lesion o f c o n f l u e n t e n d o t h e l i u m , like angiogenesis, involves both cell migration and proliferation. Angiogenesis is implicated in the progressive growth of solid tumors and in non-neoplasic processes such as inflammation and wound healing [8, 11, 13, 25]. Angiogenesis during t u m o r growth can be influenced by radiotherapy which not only affects t u m o r growth but also the normal surrounding tissues, especially the blood vessels and capillaries which are susceptible to radiation injury [5, 16, 40]. The stimulation of neovascularization by heparin, a sulfated polysaccharide, has been well described [1, 9]. Heparin stimulates the migration of capillary EC in vivo and in vitro [1, 9], but is chemotactic for EC f r o m large vessels in vitro [34] and inhibits their migration [42]. Furthermore, at low concentrations of serum, heparin inhibits [~H]-TdR uptake in h u m a n proliferating EC or during the repair process after a mechanical injury of confluent endothelium [21, 28]. In addition, heparin has
the capacity to potentiate the growth promoting activity of a F G F during the proliferation of sparsely seeded EC [38] or during the regeneration of a denuded area [21]. We have previously described a model of in vitro mechanical injury of confluent human EC where the cells are detached but the underlying matrix is preserved [18]. The modulation o f the lesion repair by several sulfated polysaccharides including heparin, alone or in combination with b F G F or aFGF, was studied quantitatively [21]. In the present study the cultures were irradiated prior to removal o f EC in order to differentiate between the contributions of cell division and cell migration to the repair process. C o m p u t e r i z e d image analysis coupled to autoradiography specially adapted to the mechanical injury model [26, 41] was used to quantify the wound coverage after irradiation. The repair process in these conditions was modulated by heparin alone or by heparin associated with aFGF. Both substances are implicated in t u m o r growth during metastatic invasion and in wound healing after vascular injury [2, 39].
Materials and Methods
Reagents * Correspondence and reprints Abbreviations: aFGF or bFGF, acidic fibroblast growth factor or basic fibroblast growth factor; EC, endothelial cells. [~H]-TdR,pH]-thymidine; HIV, human immunodeficiencyvirus ; HS, human serum ; HSA, human serum albumin.
Cell culture media (M199 with Hank's salts and 25 mM HEPES, and RPMI 1640), L-glutamine, antibiotics (penicillin, streptomycin) and fungizone were from Gibco, Paisley, Scotland, UK. Petri culture dishes ref 2500 HA-20 were from Coming Glass
232
C Klein-Soyer et al
Works, New York, NY USA. Cellulose polyacetate paper (Sepraphore III, No 51003) was from Gelman, Ann Arbor, MI, USA. Standard heparin (170 U/mg, average molecular weight 15 000, degree of sulfatation = 11.2%) was from Institut Choay, Paris, France. Crystallized bovine serum albumin was from Schwartz/Mann, Orangeburg, NY, USA. [3H]-TdR (37 MBq/ml, 7.4 GBq/mmol) was from Amersham, Les Ulis, France. Autoradiographic emulsion NTB 2 was from Eastman Kodak Company, Rochester, NY, USA.
Endothelial cell culture Human umbilical vein' EC in primary culture or cryopreserved [19] were seeded and cultured in 35-mm diameter Petri dishes precoated with a human plasma fraction enriched in fibronectin (50%) by cryoprecipitation, as already described [17]. The culture medium was M199/RPMI 1640 v/v, 2 mM L-glutamine, 100 U/ml penicillin, 100 ~g/ml streptomycin, 2.5 /~g/ml fungizone and supplemented with 30% pooled hepatitis B virus and HIV free, heat inactivated human serum (HS) prepared as described previously [17].
Acidic fibroblast growth factors Human aFGF a F G F was purified from human brains obtained 24-48 h
post-mortem and stored at - 8 0 ° C as already described [21]. The purity of aFGF was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (one band of M, 16500), by reverse-phase high performance liquid chromatography (one major peak) and N-terminus analysis.
Bovine aFGF Acidic bovine FGF was from British Bio-Technology Limited, Oxford, UK. The purity of bovine aFGF given by the manufacturer was over 95070 as assessed by silver-stained S D S - P A G E and N-terminal sequencing. This is similar to the purity grade of human aFGF [21]. Human and bovine aFGFs are highly homologous (92070 homology) [12] and in our conditions no difference was observed when experiments were performed with either human or bovine aFGF.
Growth promoting activity of FGFs The concentration of FGFs (tested in a range of 10-150 ng/ml) giving optimal proliferation of sparsely seeded human EC was determined in the presence of 5 and 10% HS and assessed by cell quantification and [3H]-TdR incorporation [20] and was found to be 50 ng/ml for both human and bovine aFGF.
center of the lesion. The time course of lesion coverage was followed for 4 d after injury. The speed of repair was calculated by linear regression from the curve representing the covered lesion area versus time as Ts0, ie the time necessary to cover 50070 of the initial lesion [18].
Experimental protocol EC were exposed to radiation in the presence of 30% HS immediately prior to establishment of the lesion. During irradiation the cultures were at room temperature for a maximum of 15 min. The culture medium was removed and the lesions were established at time zero, one lesion per culture dish. Then, depending on the experiment, the culture medium was changed and contained either 30 or 5°7o pooled HS. Standard heparin and aFGF were added to the medium every day; the medium was changed at day 2. Standard heparin solubilized in M199/RPMI 1640 was added at a final concentration of 100 ~g/ml. a F G F diluted in M I 9 9 / R P M I 1640 containing 0.5% bovine serum albumin was added at a concentration of 50 ng/ml, allowing optimal growth of sparse EC. After injury, culture dishes were removed every day or every second day until day 4 and fixed as previously described [21]. Prior to fixation, the EC were pulsed for 18 h with 9.25 kBq/ml ['H]-TdR. The dishes were then processed for autoradiography.
A utoradiography and image analysis An activated autoradiographic method with subsequent computer image analysis has been developed [26, 41]. Briefly, a uniform 15-~m thick layer of liquid emulsion was poured directly on the EC layer with a micropipette. After 24 h exposure, the dishes were submitted to development and stained with May-Grfinwald Giemsa. With this sensitive autoradiographic procedure, labeled cells display countless grains over the nucleus, while unlabeled EC have no more than 2 - 4 grains [41]. The image analysis system and associated software allowed monitoring of the cell density and the labeling index in any area of the culture.
Statistical anaO,sis The effects of irradiation, serum concentration and standard heparin, alone or in combination with aFGF, on the repair process were compared by variance analysis followed by a test of Newman-Keuls using the statistical software STAT-ITCF (ITCF, Boigneville, France).
Results
Irradiation of endothelial cells
Effect o f irradiation on the repair process
Confluent EC were irradiated with an irradiator for blood products IBL 437 C (Commissariat ~ I'Energie Atomique, Saclay, France). The radiation source was ~37Caesium, delivering 10 Gy per min in water.
E C were g r o w n to c o n f l u e n c e in the presence o f 30°70 HS. P r i o r to w o u n d i n g , h a l f o f the s a m p l e s served as c o n t r o l s and the o t h e r h a l f was s u b m i t t e d to a r a d i a t i o n dose o f l0 Gy. This dose o f r a d i a t i o n was c h o s e n for the experiments because it had been shown previously that a dose o f 8.7 G y i n h i b i t e d E C g r o w t h in replicating cells for at least one wk [5] a n d that a dose o f 10 G y d i m i n i s h e d [3H]-TdR u p t a k e by 96o70 in s u b c o n f l u e n t p e r i p h e r a l w o u n d areas [32]. The Ts0 (time necessary to recover 5007o o f initial lesion surface) [18] a n d the lesion c o v e r e d 96 h a f t e r i n j u r y were c o m p a r e d (table I). T h e time required to cover the d e n u d e d areas in i r r a d i a t e d cultures o r controls was not significantly d i f f e r e n t ( P > 0 . 0 5 ) . T h e cell d e n s i t y a n d the labeling index in c o n f l u e n t a r e a s a n d at the leading edge o f the lesion (as defined in Methods) were e x a m i n e d as a f u n c t i o n o f t i m e (fig 1). T h e cell d e n s i t y (54000+_3 300 c e l l s / c m ~) in c o n f l u e n t areas r e m a i n e d c o n s t a n t in c o n t r o l cultures a n d was s i g n i f i c a n t l y
Mechanical injury of confluent endothelial cells and repair process A calibrated circular lesion of 6-mm diameter was made at postconfluence in the center of the cell monolayer with a disk of polyacetate paper as described previously [18]. EC were selectively detached from the underlying matrix in the area of application of the disk. The repair process was quantitatively estimated by measuring the denuded area by image analysis (Biocom, Les Ulis, France). The border between the cell-free matrix and the EC layer was referred to as the leading edge of the lesion, where EC migrate and proliferate concentrically in order to cover the denuded area. Measurements of EC density in the confluent area were usually made at a distance between 5 and l0 mm from the
Irradiation, heparin, aFGF and endothelial repair diminished by 56°70 ( P < 0 . 0 5 ) between 24 and 96 h following injury in irradiated cultures. Similarly, the labeling index in irradiated cultures was reduced by 87070 ( P < 0 . 0 5 ) at day 1 and by 70070 ( P < 0 . 0 5 ) at day 4 after injury as c o m p a r e d to the control (fig 1A). At the leading edge o f the lesion, the cell density in irradiated cultures diminished regularly by 71 079 at day 4. The labeling index at the edge was reduced by 8407o at day 1 and 71070 at day 4 as c o m p a r e d to the controls (fig IB).
Table I. Effect of irradiation on the repair process of damaged endothelium. Before establishment of the lesions, the samples concerned were submitted to a radiation dose of l0 Gy. The repair process occurred in the presence of 3007o HS. The growth curves were obtained at time points 24, 48, 72 h with 2 samples per time point. The Ts0 of the regeneration curves and the areas recovered 96 h after wounding (mean _+SD, n = 4) were compared by variance analysis followed by a test of Newman-Keuls and were not found to be significantly different (P>0.05).
Control
Irradiated cells
Ts0(h )
46.7 _+ 1.9
49.0_+ 2.5
07o of area recovered 96 h after injury (°70 of initial lesion)
90.5 ___I. 1
89.5 -+ 2.8
Effects o f high and low concentrations o f serum EC were submitted to irradiation (10 Gy). A high (30070) and a low (5079) concentration o f HS were tested after establishment o f the lesions, during the repair process. There was no difference in the time required to cover the denuded areas for irradiated cells or control cells for a given serum concentration; the areas recovered 48 and 96 h after injury were not significantly different (table II). The repair o f the lesion was slower and the percentage o f covered area at days 2 and 4 after w o u n d i n g was significantly lower ( P < 0 . 0 5 ) in the presence o f 507o serum as opposed to 30°70 serum. The decrease o f the cell density and o f the labeling index observed in irradiated cultures both in confluent areas and at the leading edge was not significantly different when the lesion was repaired in the presence o f 30 or 507o HS (data not shown).
At post confluence, EC were exposed to 8 different doses o f radiation ranging f r o m 0 to 30 G y prior to wounding. The lesions were then allowed to repair in the presence o f 5°70 HS. This low serum concentration was chosen for further experiments in order to better differentiate the influence o f irradiation, a F G F and heparin. The cells were exposed to [~H]-TdR; then representative samples were fixed and stained 48 and 96 h following injury.
B
A
7r ~
Effects o f increasing doses o f radiation
233
Control
Irrodfotod
Irrodloted
Control
2s 0
?
~oo] 75[
x
25 0 0 24 48 72 96120 0 24 467296120
0 24 4B 72 96120
2,4 ,16 72 96 120
Time after Inltlol injury (hours)
Effect on the speed o f repair The areas recovered 48 h and 96 h after injury were not significantly different in control and irradiated cultures at any dose o f radiation (data not shown).
Effect on the cell density The cell density was considerably diminished in irradiated cultures as c o m p a r e d to control cultures, but not in a manner significantly different for each dose o f radiation from 5 to 30 Gy. The mean cell density in confluent areas for all radiations doses was diminished by 45.3°70 ( P < 0 . 0 5 ) at day 2 and by 67.707o ( P < 0.05) at day 4 following injury (table III). At the lesion margin the decrease in cell density
Fig 1. Labeling index and cell density as a function of time. EC were irradiated (10 Gy) prior to establishment of the lesion. During the repair process, the cells were labeled with [~H]-TdR (9.35 kBq/ml, 18 h). After fixation and autoradiographic processing, the cell density and thelabeling index were determined at the leading edge of the lesion and in confluent areas. A : cell density (cells/cm2 × 10-3) and labeling index (07o of labeled cells) in confluent areas of control and irradiated cultures as a function of time, B: cell density (cells/cm2 x l0 -J) and labeling index (o70 of labeled cells) at the leading edge of the lesion of control and irradiated cultures as a function of time. Results are expressed as mean_95 070 confidence interval (n=4).
Table 11. Effect of serum concentration on the repair process of damaged endothelium. Before establishment of the lesions, representative samples of the cultures were submitted to a radiation dose of 10 Gy. The repair process occurred in the presence of 30 or 5°/0 HS. Samples were taken 48 or 96 h following injury with 2 dishes per time point for each condition. The results were compared by variance analysis followed by a test of Newman-Keuls. Results are expressed as mean_+SD, n = 4 (*P < 0.05).
Control
Irradiated cells
30
5
30
5
°7o of area recovered 48 h after injury
52.3 -+ 3.8
47.5 -+2.3*
50.1 -+ 1.1
47.9 + 1.5"
07o of area recovered 96 h after injury (°70 of initial lesion)
90.5+ 1.2
77.9_+3.3*
89.4+2.8
78.4+6.4*
07o HS
234
c Klein-Soyeret at
was 35.4070 ( P < 0 . 0 5 ) at day 2 and 47.4070 ( P < 0 . 0 5 ) at day 4 (table III). Incorporation o f [JH]-TdR After determination of the cell density by image analysis, EC were solubilized in 2°70 sodium dodecyl sulfate and the incorporated radioactivity counted [17]. The specific activity (expressed as the number of cpm per number of cells per culture dish) as a function of the dose of radiation, was examined (fig 2). Two days after injury, the specific activity decreased rapidly by 80070 ( P < 0.05) between 0 and 5 Gy and by 95070 between 0 and 10 Gy ( P < 0 . 0 5 ) and then remained constant f6r doses greater than 10 Gy. Between day 2 and day 4 after injury [3H]-TdR incorporation diminished by 46070 in the control (no irradiation), although the cell density remained constant (see table III). Effects o f standard heparin and aFGF, alone or in combination with aFGF Modulation o f the speed o f repair Heparin and a F G F were added to the irradiated (10 Gy) or control cultures in medium containing 5070 HS. There was no difference in the time required to cover the denuded areas of irradiated and non-irradiated samples (table VI). Heparin alone slowed down the speed of repair : the areas recovered at days 2 and 4 were reduced as compared to the control, a F G F added alone did not modify the speed of recovery during the linear phase of the repair process when essentially cell proliferation was involved in the wound repair, as shown previously [21]. The areas recovered at day 2 after injury were not different f r o m controls. However, after 4 d of repair the areas recovered in the presence of aFGF were slightly larger than in controls but identical in irradiated and non-irradiated cultures (table IV). When a F G F was associated with heparin, the speed of repair was accelerated: the areas recovered at days 2 and 4 were larger as compared to the control. Effects on the cell density and on the labeling index After autoradiographic processing, the cell density in irradiated and non-irradiated cultures was determined. The EC [~H]-TdR uptake at the leading edge of the lesion in irradiated cultures and the labeling index were diminished by 8307o 2 d after injury (P
231
Original article
Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor Claude Klein-Soyer 1,, Alain Beretz ~, Jean-Pierre Cazenave 1, Francis Driot 2, Jean-Pierre M a f f r a n d 2 t Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatdriaux, I N S E R M U 311, Centre Rdgional de Transfusion Sanguine, 10 rue Spielmann, 67085 Strasbourg Cedex; 2Sanofi Recherche, 195 route d'Espagne, 31035 Toulouse, France (Received 12 December 1989; accepted l0 April 1990) Summary - Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95070 in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50°70. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50070 of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation. irradiation / heparin / acidic fibroblast growth factor / endothelium / wound repair
Introduction
Injury of the vascular endothelium is of importance in the development of atherosclerosis [30]. The repair of a d e n u d i n g lesion o f c o n f l u e n t e n d o t h e l i u m , like angiogenesis, involves both cell migration and proliferation. Angiogenesis is implicated in the progressive growth of solid tumors and in non-neoplasic processes such as inflammation and wound healing [8, 11, 13, 25]. Angiogenesis during t u m o r growth can be influenced by radiotherapy which not only affects t u m o r growth but also the normal surrounding tissues, especially the blood vessels and capillaries which are susceptible to radiation injury [5, 16, 40]. The stimulation of neovascularization by heparin, a sulfated polysaccharide, has been well described [1, 9]. Heparin stimulates the migration of capillary EC in vivo and in vitro [1, 9], but is chemotactic for EC f r o m large vessels in vitro [34] and inhibits their migration [42]. Furthermore, at low concentrations of serum, heparin inhibits [~H]-TdR uptake in h u m a n proliferating EC or during the repair process after a mechanical injury of confluent endothelium [21, 28]. In addition, heparin has
the capacity to potentiate the growth promoting activity of a F G F during the proliferation of sparsely seeded EC [38] or during the regeneration of a denuded area [21]. We have previously described a model of in vitro mechanical injury of confluent human EC where the cells are detached but the underlying matrix is preserved [18]. The modulation o f the lesion repair by several sulfated polysaccharides including heparin, alone or in combination with b F G F or aFGF, was studied quantitatively [21]. In the present study the cultures were irradiated prior to removal o f EC in order to differentiate between the contributions of cell division and cell migration to the repair process. C o m p u t e r i z e d image analysis coupled to autoradiography specially adapted to the mechanical injury model [26, 41] was used to quantify the wound coverage after irradiation. The repair process in these conditions was modulated by heparin alone or by heparin associated with aFGF. Both substances are implicated in t u m o r growth during metastatic invasion and in wound healing after vascular injury [2, 39].
Materials and Methods
Reagents * Correspondence and reprints Abbreviations: aFGF or bFGF, acidic fibroblast growth factor or basic fibroblast growth factor; EC, endothelial cells. [~H]-TdR,pH]-thymidine; HIV, human immunodeficiencyvirus ; HS, human serum ; HSA, human serum albumin.
Cell culture media (M199 with Hank's salts and 25 mM HEPES, and RPMI 1640), L-glutamine, antibiotics (penicillin, streptomycin) and fungizone were from Gibco, Paisley, Scotland, UK. Petri culture dishes ref 2500 HA-20 were from Coming Glass
232
C Klein-Soyer et al
Works, New York, NY USA. Cellulose polyacetate paper (Sepraphore III, No 51003) was from Gelman, Ann Arbor, MI, USA. Standard heparin (170 U/mg, average molecular weight 15 000, degree of sulfatation = 11.2%) was from Institut Choay, Paris, France. Crystallized bovine serum albumin was from Schwartz/Mann, Orangeburg, NY, USA. [3H]-TdR (37 MBq/ml, 7.4 GBq/mmol) was from Amersham, Les Ulis, France. Autoradiographic emulsion NTB 2 was from Eastman Kodak Company, Rochester, NY, USA.
Endothelial cell culture Human umbilical vein' EC in primary culture or cryopreserved [19] were seeded and cultured in 35-mm diameter Petri dishes precoated with a human plasma fraction enriched in fibronectin (50%) by cryoprecipitation, as already described [17]. The culture medium was M199/RPMI 1640 v/v, 2 mM L-glutamine, 100 U/ml penicillin, 100 ~g/ml streptomycin, 2.5 /~g/ml fungizone and supplemented with 30% pooled hepatitis B virus and HIV free, heat inactivated human serum (HS) prepared as described previously [17].
Acidic fibroblast growth factors Human aFGF a F G F was purified from human brains obtained 24-48 h
post-mortem and stored at - 8 0 ° C as already described [21]. The purity of aFGF was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (one band of M, 16500), by reverse-phase high performance liquid chromatography (one major peak) and N-terminus analysis.
Bovine aFGF Acidic bovine FGF was from British Bio-Technology Limited, Oxford, UK. The purity of bovine aFGF given by the manufacturer was over 95070 as assessed by silver-stained S D S - P A G E and N-terminal sequencing. This is similar to the purity grade of human aFGF [21]. Human and bovine aFGFs are highly homologous (92070 homology) [12] and in our conditions no difference was observed when experiments were performed with either human or bovine aFGF.
Growth promoting activity of FGFs The concentration of FGFs (tested in a range of 10-150 ng/ml) giving optimal proliferation of sparsely seeded human EC was determined in the presence of 5 and 10% HS and assessed by cell quantification and [3H]-TdR incorporation [20] and was found to be 50 ng/ml for both human and bovine aFGF.
center of the lesion. The time course of lesion coverage was followed for 4 d after injury. The speed of repair was calculated by linear regression from the curve representing the covered lesion area versus time as Ts0, ie the time necessary to cover 50070 of the initial lesion [18].
Experimental protocol EC were exposed to radiation in the presence of 30% HS immediately prior to establishment of the lesion. During irradiation the cultures were at room temperature for a maximum of 15 min. The culture medium was removed and the lesions were established at time zero, one lesion per culture dish. Then, depending on the experiment, the culture medium was changed and contained either 30 or 5°7o pooled HS. Standard heparin and aFGF were added to the medium every day; the medium was changed at day 2. Standard heparin solubilized in M199/RPMI 1640 was added at a final concentration of 100 ~g/ml. a F G F diluted in M I 9 9 / R P M I 1640 containing 0.5% bovine serum albumin was added at a concentration of 50 ng/ml, allowing optimal growth of sparse EC. After injury, culture dishes were removed every day or every second day until day 4 and fixed as previously described [21]. Prior to fixation, the EC were pulsed for 18 h with 9.25 kBq/ml ['H]-TdR. The dishes were then processed for autoradiography.
A utoradiography and image analysis An activated autoradiographic method with subsequent computer image analysis has been developed [26, 41]. Briefly, a uniform 15-~m thick layer of liquid emulsion was poured directly on the EC layer with a micropipette. After 24 h exposure, the dishes were submitted to development and stained with May-Grfinwald Giemsa. With this sensitive autoradiographic procedure, labeled cells display countless grains over the nucleus, while unlabeled EC have no more than 2 - 4 grains [41]. The image analysis system and associated software allowed monitoring of the cell density and the labeling index in any area of the culture.
Statistical anaO,sis The effects of irradiation, serum concentration and standard heparin, alone or in combination with aFGF, on the repair process were compared by variance analysis followed by a test of Newman-Keuls using the statistical software STAT-ITCF (ITCF, Boigneville, France).
Results
Irradiation of endothelial cells
Effect o f irradiation on the repair process
Confluent EC were irradiated with an irradiator for blood products IBL 437 C (Commissariat ~ I'Energie Atomique, Saclay, France). The radiation source was ~37Caesium, delivering 10 Gy per min in water.
E C were g r o w n to c o n f l u e n c e in the presence o f 30°70 HS. P r i o r to w o u n d i n g , h a l f o f the s a m p l e s served as c o n t r o l s and the o t h e r h a l f was s u b m i t t e d to a r a d i a t i o n dose o f l0 Gy. This dose o f r a d i a t i o n was c h o s e n for the experiments because it had been shown previously that a dose o f 8.7 G y i n h i b i t e d E C g r o w t h in replicating cells for at least one wk [5] a n d that a dose o f 10 G y d i m i n i s h e d [3H]-TdR u p t a k e by 96o70 in s u b c o n f l u e n t p e r i p h e r a l w o u n d areas [32]. The Ts0 (time necessary to recover 5007o o f initial lesion surface) [18] a n d the lesion c o v e r e d 96 h a f t e r i n j u r y were c o m p a r e d (table I). T h e time required to cover the d e n u d e d areas in i r r a d i a t e d cultures o r controls was not significantly d i f f e r e n t ( P > 0 . 0 5 ) . T h e cell d e n s i t y a n d the labeling index in c o n f l u e n t a r e a s a n d at the leading edge o f the lesion (as defined in Methods) were e x a m i n e d as a f u n c t i o n o f t i m e (fig 1). T h e cell d e n s i t y (54000+_3 300 c e l l s / c m ~) in c o n f l u e n t areas r e m a i n e d c o n s t a n t in c o n t r o l cultures a n d was s i g n i f i c a n t l y
Mechanical injury of confluent endothelial cells and repair process A calibrated circular lesion of 6-mm diameter was made at postconfluence in the center of the cell monolayer with a disk of polyacetate paper as described previously [18]. EC were selectively detached from the underlying matrix in the area of application of the disk. The repair process was quantitatively estimated by measuring the denuded area by image analysis (Biocom, Les Ulis, France). The border between the cell-free matrix and the EC layer was referred to as the leading edge of the lesion, where EC migrate and proliferate concentrically in order to cover the denuded area. Measurements of EC density in the confluent area were usually made at a distance between 5 and l0 mm from the
Irradiation, heparin, aFGF and endothelial repair diminished by 56°70 ( P < 0 . 0 5 ) between 24 and 96 h following injury in irradiated cultures. Similarly, the labeling index in irradiated cultures was reduced by 87070 ( P < 0 . 0 5 ) at day 1 and by 70070 ( P < 0 . 0 5 ) at day 4 after injury as c o m p a r e d to the control (fig 1A). At the leading edge o f the lesion, the cell density in irradiated cultures diminished regularly by 71 079 at day 4. The labeling index at the edge was reduced by 8407o at day 1 and 71070 at day 4 as c o m p a r e d to the controls (fig IB).
Table I. Effect of irradiation on the repair process of damaged endothelium. Before establishment of the lesions, the samples concerned were submitted to a radiation dose of l0 Gy. The repair process occurred in the presence of 3007o HS. The growth curves were obtained at time points 24, 48, 72 h with 2 samples per time point. The Ts0 of the regeneration curves and the areas recovered 96 h after wounding (mean _+SD, n = 4) were compared by variance analysis followed by a test of Newman-Keuls and were not found to be significantly different (P>0.05).
Control
Irradiated cells
Ts0(h )
46.7 _+ 1.9
49.0_+ 2.5
07o of area recovered 96 h after injury (°70 of initial lesion)
90.5 ___I. 1
89.5 -+ 2.8
Effects o f high and low concentrations o f serum EC were submitted to irradiation (10 Gy). A high (30070) and a low (5079) concentration o f HS were tested after establishment o f the lesions, during the repair process. There was no difference in the time required to cover the denuded areas for irradiated cells or control cells for a given serum concentration; the areas recovered 48 and 96 h after injury were not significantly different (table II). The repair o f the lesion was slower and the percentage o f covered area at days 2 and 4 after w o u n d i n g was significantly lower ( P < 0 . 0 5 ) in the presence o f 507o serum as opposed to 30°70 serum. The decrease o f the cell density and o f the labeling index observed in irradiated cultures both in confluent areas and at the leading edge was not significantly different when the lesion was repaired in the presence o f 30 or 507o HS (data not shown).
At post confluence, EC were exposed to 8 different doses o f radiation ranging f r o m 0 to 30 G y prior to wounding. The lesions were then allowed to repair in the presence o f 5°70 HS. This low serum concentration was chosen for further experiments in order to better differentiate the influence o f irradiation, a F G F and heparin. The cells were exposed to [~H]-TdR; then representative samples were fixed and stained 48 and 96 h following injury.
B
A
7r ~
Effects o f increasing doses o f radiation
233
Control
Irrodfotod
Irrodloted
Control
2s 0
?
~oo] 75[
x
25 0 0 24 48 72 96120 0 24 467296120
0 24 4B 72 96120
2,4 ,16 72 96 120
Time after Inltlol injury (hours)
Effect on the speed o f repair The areas recovered 48 h and 96 h after injury were not significantly different in control and irradiated cultures at any dose o f radiation (data not shown).
Effect on the cell density The cell density was considerably diminished in irradiated cultures as c o m p a r e d to control cultures, but not in a manner significantly different for each dose o f radiation from 5 to 30 Gy. The mean cell density in confluent areas for all radiations doses was diminished by 45.3°70 ( P < 0 . 0 5 ) at day 2 and by 67.707o ( P < 0.05) at day 4 following injury (table III). At the lesion margin the decrease in cell density
Fig 1. Labeling index and cell density as a function of time. EC were irradiated (10 Gy) prior to establishment of the lesion. During the repair process, the cells were labeled with [~H]-TdR (9.35 kBq/ml, 18 h). After fixation and autoradiographic processing, the cell density and thelabeling index were determined at the leading edge of the lesion and in confluent areas. A : cell density (cells/cm2 × 10-3) and labeling index (07o of labeled cells) in confluent areas of control and irradiated cultures as a function of time, B: cell density (cells/cm2 x l0 -J) and labeling index (o70 of labeled cells) at the leading edge of the lesion of control and irradiated cultures as a function of time. Results are expressed as mean_95 070 confidence interval (n=4).
Table 11. Effect of serum concentration on the repair process of damaged endothelium. Before establishment of the lesions, representative samples of the cultures were submitted to a radiation dose of 10 Gy. The repair process occurred in the presence of 30 or 5°/0 HS. Samples were taken 48 or 96 h following injury with 2 dishes per time point for each condition. The results were compared by variance analysis followed by a test of Newman-Keuls. Results are expressed as mean_+SD, n = 4 (*P < 0.05).
Control
Irradiated cells
30
5
30
5
°7o of area recovered 48 h after injury
52.3 -+ 3.8
47.5 -+2.3*
50.1 -+ 1.1
47.9 + 1.5"
07o of area recovered 96 h after injury (°70 of initial lesion)
90.5+ 1.2
77.9_+3.3*
89.4+2.8
78.4+6.4*
07o HS
234
c Klein-Soyeret at
was 35.4070 ( P < 0 . 0 5 ) at day 2 and 47.4070 ( P < 0 . 0 5 ) at day 4 (table III). Incorporation o f [JH]-TdR After determination of the cell density by image analysis, EC were solubilized in 2°70 sodium dodecyl sulfate and the incorporated radioactivity counted [17]. The specific activity (expressed as the number of cpm per number of cells per culture dish) as a function of the dose of radiation, was examined (fig 2). Two days after injury, the specific activity decreased rapidly by 80070 ( P < 0.05) between 0 and 5 Gy and by 95070 between 0 and 10 Gy ( P < 0 . 0 5 ) and then remained constant f6r doses greater than 10 Gy. Between day 2 and day 4 after injury [3H]-TdR incorporation diminished by 46070 in the control (no irradiation), although the cell density remained constant (see table III). Effects o f standard heparin and aFGF, alone or in combination with aFGF Modulation o f the speed o f repair Heparin and a F G F were added to the irradiated (10 Gy) or control cultures in medium containing 5070 HS. There was no difference in the time required to cover the denuded areas of irradiated and non-irradiated samples (table VI). Heparin alone slowed down the speed of repair : the areas recovered at days 2 and 4 were reduced as compared to the control, a F G F added alone did not modify the speed of recovery during the linear phase of the repair process when essentially cell proliferation was involved in the wound repair, as shown previously [21]. The areas recovered at day 2 after injury were not different f r o m controls. However, after 4 d of repair the areas recovered in the presence of aFGF were slightly larger than in controls but identical in irradiated and non-irradiated cultures (table IV). When a F G F was associated with heparin, the speed of repair was accelerated: the areas recovered at days 2 and 4 were larger as compared to the control. Effects on the cell density and on the labeling index After autoradiographic processing, the cell density in irradiated and non-irradiated cultures was determined. The EC [~H]-TdR uptake at the leading edge of the lesion in irradiated cultures and the labeling index were diminished by 8307o 2 d after injury (P
