THROMBOSIS RESEARCH Printed in the United
BRIEF
Vol. States
7, pp. 807-812, Pergamon Press,
19'75 Inc.
COMMUNICATION
EFFECTS OF PLATELETS AND CERTAIN PLATELET COMPONENTS ON GROWTH OF CULTURED HUMAN ENDOTHELIAL CELLS S.R. Saba and R.G. Mason Department of Pathology University of North Carolina Chapel Hill, N.C. 27514
(Received
5.9.1975.
Accepted
by
Editor
M.I.
Barnhart)
INTRODUCTION The recent successful growth of human and nonhuman endothelial cells 1-6 in tissue culture in several laboratories has opened the way for a study of factors that may influence the rate of replication of these cells.
One
laboratory has reported that the presence of platelets in the nutrient medium 1 enhances the growth of endothelial cells. We have confirmed this report and have extended these observations to include the effects of certain platelet components on endothelial cell replication.
The importance of these
studies lies in the fact that platelets and certain of their components might influence the rate at which endothelial cells grow in vivo as well as in vitro. METHODS Growth of Human Endothelial Cells in Tissue Culture. Techniques for recovery of endothelial cells from human umbilical cord 2 Isolated endothelial cells
vein by use of collagenase have been reported.
were washed once in modified (Ca++ and Mg++ free Tyrode's solution (MT'S))and
twice in medium 199 containing 20% fetal calf serum. to 96% viable by the erythrosin B dye exclusion test.
Isolated cells were 83 Cells were isolated
from umbilical cord vein under strict sterile conditions.
Cell cultures were
carried out in polystyrene culture flasks (Falcon Plastics, Los Angeles, Cal.) 2 with a growth area of 25 cm. Culture flasks were seeded with approximately 500,000 cells per flask with the cells at a final:concentration of 500 per mm3.
807
808
ENDOTHELIAL
GROWTH
AND
PLATELETS
Vo1.7,No.5
Isolated Human Platelets. Sterile human Platelets were prepared from normal human blood anti_ coagulated with an acid-citrate-dextrose mixture (formula A) These platelets were washed 2 times in a mixture of 5.4 x 10 -3 M sodium citrate and 1.46 x -1 M sodium chloride , pH 6.5, and once in medium 199 and were resuspended 10 finally in N'S or in medium 199 containing 20% fetal calf serum; control experiments indicated that both suspension media produced similar results. The pH of the final platelet suspension was 7.2.
Platelet concentration was
determined through direct counts carried out by phase contrast microscopy. Medium 199 and fetal calf serum were obtained from Grand Island Biological co., Grand Island, N.Y. Other Test Agents, Adenosine diphosphate (ADP), epinephrine, and serotonin were obtained from Sigma Chemical Company, St. Louis, Missouri.
Solutions were made
using MIS as solvent and were passed through a 0.22 micron Millipore Filter before use in tissue culture experiments.
Solutions of each of these test
agents were made fresh on the day of use. Techniques for Demonstration of Possible Effects of Test Agents on Growth of Endothelial Cells Controls and tests were carried out with isolated endothelial cells from the same umbilical cord vein in each experiment.
Test mixtures
consisted of isolated endothelial cells at a final concentration of 500 cells/mm3 and test agent.
Control mixtures consisted of endothelial cells
at a final concentration of 500 cells/mm3 plus MTS.
The pH of the nutrient
medium in both test and control flasks was in the range of 7.2 to 7.4. Initial experiments with mixtures of endothelial cells and isolated platelets indicated that a final platelet concentration in the nutrient medium of 25,000 platelets/mm3 was optimal, with lower concentrations of platelets producing lesser degrees of enhancement of endothelial cell growth, while higher concentrations of platelets did not produce proportionally greater degrees of enhancement.
In experiments with other test agents, ADP was
present at final concentrations of 10V5M, 10s6M, or 10e7M while epinephrine -6 was present at final concentrations of 10 M or 10m7M and serotonin at a final concentration of 10B6M. Test and control tissue culture flasks were incubated at 37'C in 5% Cog. Every third day the growth medium was replaced by an identical mixture consisting of medium 199 with 20% fetal calf serum and either the test agent in MTS or MTS alone.
The endothelial cells in test flasks generallY
grew
to
Vol.7,No.
EXDUIXELIAL
j
GROWTH
AND
809
PLATELETS
confluence in 7 to 9 days, while cells in control flasks grew at the same rate or somewhat more slowly.
Whenever the endothelial cells in either test or
control flasks had grown to apparent confluence, the cells were harvested by first removing the growth medium, then washing the adherent cells with 5 ml of a 0.2% solution of trypsin in 0.05% ethylenediamine tetraacetic acid in 0.154 M NaCl.
Detached cells were collected in 10% fetal calf serum in MTS Counts were performed in duplicate.
and counted by phase contrast microscopy.
Hence, endothelial cell growth is expressed as number of cells recovered. RESULTS Isolated platelets, ADP, and serotonin were each shown to enhance the growth of endothelial cells in tissue culture, while epinephrine had no discernible effect.
Results are shown in Table 1.
Of the agents tested,
isolated platelets produced the greatest enhancement of growth of endothelial cells.
The growth enhancing effects of ADP were clearly concentration depend-
ent, since optimum enhancement was produced by ADP at a concentration of 10-6M.
At equimolar concentrations, serotonin produced less of an enhancing
effect on the growth of endothelial cells than did ADP.
Fpinephrine at two
different concentrations produced no discernible effect on the growth of endothelial cells in tissue culture. TAHLF I
Effects of Platelets and Certain Platelet Components on the Growth of Endothelial Cells
Test Agent Isolated Platelets
Final Concentration
Epinephrine
Serotonin
* = mean values
% Change in Number Cells Recovered* of ?Zxperi (Test vs. Control) ments
3056
1638
+87
5
10-5,
1900
1392
+37
5
10%
2100
1392
+51
6
10-7M
1533
1392
+10
6
10-6M
1500
1531
-2
7
10-7M
1625
1668
-3
4
10-6M
1592
1183
+35
6
25,000/mm3
ADP
Count of Endothelial Cells/mm3 Recovered* Test Control
810
ENDOTHELlXL
GROWTH
AND
PLATELETS
Vo1.7,No.5
DISCUSSION From time to time in the scientific literature there have been reports and suggestions that platelets interact with and support endothelial cells.738 Direct evidence for these contentions has been sparse and not entirely convincing.
Evidence for a direct effect of platelets on the growth of
endothelial cells could have far reaching implications with respect to thrombosis and atherosclerosis. Our studies began initially as an attempt to substantiate a preliminary report that platelets added to endothelial cells in tissue culture stimulated the growth of these cel1s.l
We were successful in that attempt.
Following
this, we embarked on a study designed to identify the component or components within platelets that might produce this growth stimulation.
Initial results
are reported above and indicate that at least two platelet components, ADP and serotonin, can be shown to exert an enhancing effect on the growth of endothelial cells in tissue culture.
Further, this enhancing effect can be
shown to be concentration dependent in the case of ADP.
Concentration
dependence could be shown also in studies with isolated platelets, although these results were not as clear-cut as was the case in studies with ADP.
The
concentration range of isolated platelets that enhanced the growth of endothelial cells in tissue culture was broad, and results were not as reproducible as with studies of effects of ADP.
Finally, platelets have
been found to enhance the growth of smooth muscle cells in tissue culture' but the active factor was felt to be a molecule of large size. Possible mechanisms by which isolated platelets, ADP, and serotonin exert their effects on endothelial cell growth may be diverse.
Isolated
platelets contain numerous components in addition to ADP that might affect the energy metabolism of growing cells.
On the other hand, serotonin likely
does not produce its effect on endothelial cell growth through a direct enhancement of cell metabolism.
Epinephrine
neither inhibited nor enhanced
the growth of endothelial cells in tissue culture. Similarly, epinephrine 10 does not cause contraction of endothelium in vivo. Platelets are known to play an important role in the formation of thrombi 11 Thrombi may damage endothelium by in both the arterial and venous systems. 12 The loss of pressure effects and by production of endothelial cell anoxia. endothelium removes one of the barriers protecting the arterial wall and appears to permit the accumulation of various plasmatic substances including
13 Thus platelets, while they participate in the
lipids within the intima.
formation of thrombi and indirectly damage endothelium, may well play a role
Vo1.7,No.5
ENDOTHELIAL
GROWTH
AND
811
PLATELETS
in the healing of vascular lesions by enhancing the growth of endothelial cells that cover the wound area.
On the other hand, numerous other agents
14
are known to damage endothelium.
Knowledge of the growth enhancing effects
of platelets on endothelial cells may permit reinterpretation of the effect of infused platelets and platelet components on the hemostatic potential of thrombocytopenic patients and animals.
This newly discovered growth
enhancing function of platelets on endothelial cells may have far reaching implications in our understanding of the mechanisms of hemostasis, thrombosis, and atherosclerosis. ACKNOWLEDGEMENT Supported in part by Contract PH 43-68-977with the Artificial Kidney Chronic Uremia Program of the National Institute for Arthritis, Metabolism, and Digestive Diseases and by grant 939 from the Council for Tobacco Research, USA, Inc. Requests for reprints should be addressed to R.G. Mason, M.D., PhD. Dept. of Pathology, The Memorial Hospital, Pawtucket, R.I., 02860
REFERENCES 1.
BOOYSE, F.M. Development of a new concept in the control and mechanisms of platelet-endothelial interactions. Circul. 45, 11-33, 1972, and personal communication.
2.
SABA, S.R., ZUCKER, W.H., and MASON, R.G. Some properties of endothelial cells isolated from human umbilical cord vein. Ser. Hemat. 6, 456, 1973.
3. JAFFE, E.A., NACHMAN, R.L., BECKER, C.G., and MINICK, C.R. human endothelial cells derived from umbilical veins.
Culture of J. Clin. Invest.
52, 2745, 1973. 4. LEWIS, L.J., HOAK, J.C., MACA, R.D., and FRY, G.L. Replication of human endothelial cells in culture. Science 181, 453, 1973. 5. GIMBRONE, M.A., COTRAN, R.S., and FOLKMAN, J. Human vascular endothelial cells in culture. J. Cell Biol. 60, 675, 1974. 6. SHEPRO, D., BATLOUTA, J.C., ROBBLEE, L.X., CARSON, M.P., and BEI&+lARICH, F.A. Serotonin transport by cultured bovine endothelium. Circ. Res. 36, 799, 1975. 7. CRONKITE, Z.P., BOND, V.P..,FLIEDNER, T.M., PAGLIA, D.A., and ADAMICK, E.R. Studies on the origin, production, and destruction of platelets. in: Blood Platelets. JOHNSON, S.A., et al. (Eds.) Boston, Little Brown co., 1961, P 595.
8. JOHNSON, S.A., BALBOA, R.S., DESSEL, B.H., MONTO, R.W., SIEGESMUND, K.A., and GREENWALT, R.J. The mechanisms of the endothelial supporting function of intact platelets. Exp. Molec. Path. 3, 115, 1964.
812
9.
10.
ENDOTHJ3LIALGROWTI-I AND PLATELETS
Vo1.7,No.5
ROSS, R., GLOMSET, J., KARIYA, B., and KARKER, L. A platelet dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Nat. Acad. Sci. 71, 1207, 1974.
MAJNO, G., RYAN, G.B., GABBIANI, G., HIRSCHEL, B.J., IRLE, C., and JORIS, I. Contractile events in inflammation and repair. In: Inflsmmation. Mechanisms and Control. LEPOW, I.H. and WARD, P.A. (Eds.1, N.Y. Academic Press, 1972, p. 13.
11.
RODMAN, N.F., Thrombosis. In: The Inflammatory Process. et al. (Eds.), N.Y., Academic Press, 1973, p. 363.
12.
JORGENSEN, L.
13.
SPAET, T.H., GAYNOR, E., and ST-, M.B. and endothelium. Am. Heart J. 87, 661, 1974.
14.
MAJNO, G. Ultrastructure of the vascular membrane. 3, 2293, 1965.
Mechanisms of thrombosis.
ZWEIFACH, B.W.
Pathobiol. Ann. 1, 139, 1971. Thrombosis, atherosclerosis,
Handbook Pbysiol.
BRIEF
Vol. States
7, pp. 807-812, Pergamon Press,
19'75 Inc.
COMMUNICATION
EFFECTS OF PLATELETS AND CERTAIN PLATELET COMPONENTS ON GROWTH OF CULTURED HUMAN ENDOTHELIAL CELLS S.R. Saba and R.G. Mason Department of Pathology University of North Carolina Chapel Hill, N.C. 27514
(Received
5.9.1975.
Accepted
by
Editor
M.I.
Barnhart)
INTRODUCTION The recent successful growth of human and nonhuman endothelial cells 1-6 in tissue culture in several laboratories has opened the way for a study of factors that may influence the rate of replication of these cells.
One
laboratory has reported that the presence of platelets in the nutrient medium 1 enhances the growth of endothelial cells. We have confirmed this report and have extended these observations to include the effects of certain platelet components on endothelial cell replication.
The importance of these
studies lies in the fact that platelets and certain of their components might influence the rate at which endothelial cells grow in vivo as well as in vitro. METHODS Growth of Human Endothelial Cells in Tissue Culture. Techniques for recovery of endothelial cells from human umbilical cord 2 Isolated endothelial cells
vein by use of collagenase have been reported.
were washed once in modified (Ca++ and Mg++ free Tyrode's solution (MT'S))and
twice in medium 199 containing 20% fetal calf serum. to 96% viable by the erythrosin B dye exclusion test.
Isolated cells were 83 Cells were isolated
from umbilical cord vein under strict sterile conditions.
Cell cultures were
carried out in polystyrene culture flasks (Falcon Plastics, Los Angeles, Cal.) 2 with a growth area of 25 cm. Culture flasks were seeded with approximately 500,000 cells per flask with the cells at a final:concentration of 500 per mm3.
807
808
ENDOTHELIAL
GROWTH
AND
PLATELETS
Vo1.7,No.5
Isolated Human Platelets. Sterile human Platelets were prepared from normal human blood anti_ coagulated with an acid-citrate-dextrose mixture (formula A) These platelets were washed 2 times in a mixture of 5.4 x 10 -3 M sodium citrate and 1.46 x -1 M sodium chloride , pH 6.5, and once in medium 199 and were resuspended 10 finally in N'S or in medium 199 containing 20% fetal calf serum; control experiments indicated that both suspension media produced similar results. The pH of the final platelet suspension was 7.2.
Platelet concentration was
determined through direct counts carried out by phase contrast microscopy. Medium 199 and fetal calf serum were obtained from Grand Island Biological co., Grand Island, N.Y. Other Test Agents, Adenosine diphosphate (ADP), epinephrine, and serotonin were obtained from Sigma Chemical Company, St. Louis, Missouri.
Solutions were made
using MIS as solvent and were passed through a 0.22 micron Millipore Filter before use in tissue culture experiments.
Solutions of each of these test
agents were made fresh on the day of use. Techniques for Demonstration of Possible Effects of Test Agents on Growth of Endothelial Cells Controls and tests were carried out with isolated endothelial cells from the same umbilical cord vein in each experiment.
Test mixtures
consisted of isolated endothelial cells at a final concentration of 500 cells/mm3 and test agent.
Control mixtures consisted of endothelial cells
at a final concentration of 500 cells/mm3 plus MTS.
The pH of the nutrient
medium in both test and control flasks was in the range of 7.2 to 7.4. Initial experiments with mixtures of endothelial cells and isolated platelets indicated that a final platelet concentration in the nutrient medium of 25,000 platelets/mm3 was optimal, with lower concentrations of platelets producing lesser degrees of enhancement of endothelial cell growth, while higher concentrations of platelets did not produce proportionally greater degrees of enhancement.
In experiments with other test agents, ADP was
present at final concentrations of 10V5M, 10s6M, or 10e7M while epinephrine -6 was present at final concentrations of 10 M or 10m7M and serotonin at a final concentration of 10B6M. Test and control tissue culture flasks were incubated at 37'C in 5% Cog. Every third day the growth medium was replaced by an identical mixture consisting of medium 199 with 20% fetal calf serum and either the test agent in MTS or MTS alone.
The endothelial cells in test flasks generallY
grew
to
Vol.7,No.
EXDUIXELIAL
j
GROWTH
AND
809
PLATELETS
confluence in 7 to 9 days, while cells in control flasks grew at the same rate or somewhat more slowly.
Whenever the endothelial cells in either test or
control flasks had grown to apparent confluence, the cells were harvested by first removing the growth medium, then washing the adherent cells with 5 ml of a 0.2% solution of trypsin in 0.05% ethylenediamine tetraacetic acid in 0.154 M NaCl.
Detached cells were collected in 10% fetal calf serum in MTS Counts were performed in duplicate.
and counted by phase contrast microscopy.
Hence, endothelial cell growth is expressed as number of cells recovered. RESULTS Isolated platelets, ADP, and serotonin were each shown to enhance the growth of endothelial cells in tissue culture, while epinephrine had no discernible effect.
Results are shown in Table 1.
Of the agents tested,
isolated platelets produced the greatest enhancement of growth of endothelial cells.
The growth enhancing effects of ADP were clearly concentration depend-
ent, since optimum enhancement was produced by ADP at a concentration of 10-6M.
At equimolar concentrations, serotonin produced less of an enhancing
effect on the growth of endothelial cells than did ADP.
Fpinephrine at two
different concentrations produced no discernible effect on the growth of endothelial cells in tissue culture. TAHLF I
Effects of Platelets and Certain Platelet Components on the Growth of Endothelial Cells
Test Agent Isolated Platelets
Final Concentration
Epinephrine
Serotonin
* = mean values
% Change in Number Cells Recovered* of ?Zxperi (Test vs. Control) ments
3056
1638
+87
5
10-5,
1900
1392
+37
5
10%
2100
1392
+51
6
10-7M
1533
1392
+10
6
10-6M
1500
1531
-2
7
10-7M
1625
1668
-3
4
10-6M
1592
1183
+35
6
25,000/mm3
ADP
Count of Endothelial Cells/mm3 Recovered* Test Control
810
ENDOTHELlXL
GROWTH
AND
PLATELETS
Vo1.7,No.5
DISCUSSION From time to time in the scientific literature there have been reports and suggestions that platelets interact with and support endothelial cells.738 Direct evidence for these contentions has been sparse and not entirely convincing.
Evidence for a direct effect of platelets on the growth of
endothelial cells could have far reaching implications with respect to thrombosis and atherosclerosis. Our studies began initially as an attempt to substantiate a preliminary report that platelets added to endothelial cells in tissue culture stimulated the growth of these cel1s.l
We were successful in that attempt.
Following
this, we embarked on a study designed to identify the component or components within platelets that might produce this growth stimulation.
Initial results
are reported above and indicate that at least two platelet components, ADP and serotonin, can be shown to exert an enhancing effect on the growth of endothelial cells in tissue culture.
Further, this enhancing effect can be
shown to be concentration dependent in the case of ADP.
Concentration
dependence could be shown also in studies with isolated platelets, although these results were not as clear-cut as was the case in studies with ADP.
The
concentration range of isolated platelets that enhanced the growth of endothelial cells in tissue culture was broad, and results were not as reproducible as with studies of effects of ADP.
Finally, platelets have
been found to enhance the growth of smooth muscle cells in tissue culture' but the active factor was felt to be a molecule of large size. Possible mechanisms by which isolated platelets, ADP, and serotonin exert their effects on endothelial cell growth may be diverse.
Isolated
platelets contain numerous components in addition to ADP that might affect the energy metabolism of growing cells.
On the other hand, serotonin likely
does not produce its effect on endothelial cell growth through a direct enhancement of cell metabolism.
Epinephrine
neither inhibited nor enhanced
the growth of endothelial cells in tissue culture. Similarly, epinephrine 10 does not cause contraction of endothelium in vivo. Platelets are known to play an important role in the formation of thrombi 11 Thrombi may damage endothelium by in both the arterial and venous systems. 12 The loss of pressure effects and by production of endothelial cell anoxia. endothelium removes one of the barriers protecting the arterial wall and appears to permit the accumulation of various plasmatic substances including
13 Thus platelets, while they participate in the
lipids within the intima.
formation of thrombi and indirectly damage endothelium, may well play a role
Vo1.7,No.5
ENDOTHELIAL
GROWTH
AND
811
PLATELETS
in the healing of vascular lesions by enhancing the growth of endothelial cells that cover the wound area.
On the other hand, numerous other agents
14
are known to damage endothelium.
Knowledge of the growth enhancing effects
of platelets on endothelial cells may permit reinterpretation of the effect of infused platelets and platelet components on the hemostatic potential of thrombocytopenic patients and animals.
This newly discovered growth
enhancing function of platelets on endothelial cells may have far reaching implications in our understanding of the mechanisms of hemostasis, thrombosis, and atherosclerosis. ACKNOWLEDGEMENT Supported in part by Contract PH 43-68-977with the Artificial Kidney Chronic Uremia Program of the National Institute for Arthritis, Metabolism, and Digestive Diseases and by grant 939 from the Council for Tobacco Research, USA, Inc. Requests for reprints should be addressed to R.G. Mason, M.D., PhD. Dept. of Pathology, The Memorial Hospital, Pawtucket, R.I., 02860
REFERENCES 1.
BOOYSE, F.M. Development of a new concept in the control and mechanisms of platelet-endothelial interactions. Circul. 45, 11-33, 1972, and personal communication.
2.
SABA, S.R., ZUCKER, W.H., and MASON, R.G. Some properties of endothelial cells isolated from human umbilical cord vein. Ser. Hemat. 6, 456, 1973.
3. JAFFE, E.A., NACHMAN, R.L., BECKER, C.G., and MINICK, C.R. human endothelial cells derived from umbilical veins.
Culture of J. Clin. Invest.
52, 2745, 1973. 4. LEWIS, L.J., HOAK, J.C., MACA, R.D., and FRY, G.L. Replication of human endothelial cells in culture. Science 181, 453, 1973. 5. GIMBRONE, M.A., COTRAN, R.S., and FOLKMAN, J. Human vascular endothelial cells in culture. J. Cell Biol. 60, 675, 1974. 6. SHEPRO, D., BATLOUTA, J.C., ROBBLEE, L.X., CARSON, M.P., and BEI&+lARICH, F.A. Serotonin transport by cultured bovine endothelium. Circ. Res. 36, 799, 1975. 7. CRONKITE, Z.P., BOND, V.P..,FLIEDNER, T.M., PAGLIA, D.A., and ADAMICK, E.R. Studies on the origin, production, and destruction of platelets. in: Blood Platelets. JOHNSON, S.A., et al. (Eds.) Boston, Little Brown co., 1961, P 595.
8. JOHNSON, S.A., BALBOA, R.S., DESSEL, B.H., MONTO, R.W., SIEGESMUND, K.A., and GREENWALT, R.J. The mechanisms of the endothelial supporting function of intact platelets. Exp. Molec. Path. 3, 115, 1964.
812
9.
10.
ENDOTHJ3LIALGROWTI-I AND PLATELETS
Vo1.7,No.5
ROSS, R., GLOMSET, J., KARIYA, B., and KARKER, L. A platelet dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Nat. Acad. Sci. 71, 1207, 1974.
MAJNO, G., RYAN, G.B., GABBIANI, G., HIRSCHEL, B.J., IRLE, C., and JORIS, I. Contractile events in inflammation and repair. In: Inflsmmation. Mechanisms and Control. LEPOW, I.H. and WARD, P.A. (Eds.1, N.Y. Academic Press, 1972, p. 13.
11.
RODMAN, N.F., Thrombosis. In: The Inflammatory Process. et al. (Eds.), N.Y., Academic Press, 1973, p. 363.
12.
JORGENSEN, L.
13.
SPAET, T.H., GAYNOR, E., and ST-, M.B. and endothelium. Am. Heart J. 87, 661, 1974.
14.
MAJNO, G. Ultrastructure of the vascular membrane. 3, 2293, 1965.
Mechanisms of thrombosis.
ZWEIFACH, B.W.
Pathobiol. Ann. 1, 139, 1971. Thrombosis, atherosclerosis,
Handbook Pbysiol.
