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Heparan sulfate as a venostatic endothelial stabilizing factor
THROMBOSIS RESEARCH 68; 459-4651992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
HEPARAN
SULFATE
AS A VENOSTATIC FACTOR
J. Hladovec
Medicine
and
ENDOTHELIAL
STABILIZING
F. Kornalik
Angiological Research Laboratory of the 4th Dept. of and Institute of Pathophysiology, Medical Faculty, Charles University, Prague
(Received
10.9.1992;
accepted
in revised form 8.10.1992
by Editor J.E. Dyr)
ABSTRACT A humoral transfer of a factor inducing a decrease of circulating endothelial cells (CEC) released during venostasis was demonstrated in rats. The possibility to block its activity by an in-vitro addition of protamine suggests its identity with an endogenous heparan sulfate possessing an inhibitory effect on endothelial turnover. This was supported by an analogous effect of intravenously administered heparan sulfate-related agent.
Heparin is known, in addition to being an anticoagulant, to posses other activities as well. One of them which shifts now into the focus of attention is the antiproliferative activity particularly in with connection the arterial reocclusion after angioplasty and other procedures renewing the vessel wall patency (l-4). However, heparin is not present under physiological conditions in the blood or on the vascular surface. Instead, heparin-like several substances may be present, particularly heparan sulfate The in-vivo (6). activity of this not completely defined agent is not easy to study.
keywords:
Venostasis, heparan sulfate, endothelium, antiproliferative effect
459
460
HEPARAN SULFATE AND VENOSTASIS
Vol. 68, No. 6
For this purpose we have used our method of the invivo study of endothelial stability based on counting detached endothelial cell carcasses in blood (circulating endothelial cells - CEC) (7,8).
Female Wistar rats (180-220 g) were divided into groups of 8% animals. Venous occlusion was produced in anesthetized animals (urethane 1900 mg/kg s.c.) by a tight binding up of both hind extremities above the knee joint by rubber tubes for 10 min. The effectiveness of venostasis was controlled by the palpation of arterial pulse, discoloration of extremities and increase in dorso-plantar before the Just paw diameter. release of occlusion blood was collected from the aorta with citrate (3.8 % trisodium citrate 1 : 10) into a siliconized collecting material in the volume of at least 3 ml.In transfer experiments, titrated plasma obtained after centrifugation at 2100 g from normal or venostasis animals was injected to recipient animals by the tail vein in the volume of 1 m1/200g body weight. Blood was collected 5 min after the injection. In some animals protamine sulfate Roche 0.05 mg/kg was added in the volume of 0.1 ml to 1 ml plasma before the transfer to recipient rats. Manetti-Roberts, Heparan sulfate was supplied by It was administered intravenously dissolved in 1 Florence. ml/200 g of saline 5 min before the blood collection. circulating Endothelial cell carcasses representing samples were endothelial cells counted in plasma (CEC) (7). In method described according to the previously principle, the method is based on the removal of platelets platelet-rich with ADP and from plasma by aggregation They isolation of elements by differential centrifugation. results were counted in Burker's were chamber the and expressed as the number of elements in 9 pl plasma (i.e. the plasma chamber corrected for volume of one platform concentration). The results were statistically evaluated by two-tailed Student's t-test.
RESULTS After venostasis in both hind extremities for 10 min an about 50% decrease in CEC was repeatedly observed in the blood collected from the aorta immediately before the release of venostasis (Fig.1). In an attempt.to explain this decrease as mediator, humoral endogenous release of a possible an platelet-poor plasma obtained, from this blood was injected to a group of recipient animals (lm1/200 g) intravenously (Fig.2). The same experiment was carried out with plasma of from plasma Whereas venostasis. not subjected to rats untreated animals had no effect:on the normal CEC level which (s&SD), plasma in six independent ex.periments was 2.120.38
Vol. 68, No. 6
461
HEPARAN SULFATE AND VENOSTASIS
P’ 0.001
3
0
??
0
CONTROL (Physiol.
Sal.1
VENOUS OCCLUSION (10 min 1
Effect of venostasis rats (Z&SD).
FIG.1 in both hind
before VENOSTASIS 10 min
legs
for 10
min
on CEC
in
+ 0.01 mg/ml
'a
FIG.2 designated to representation of the experiment Schematic demonstrate the humoral transfer of the venostatic endothelial stabilizing factor.
462
HEPARAN SULFATE AND VENOSTASIS
ko,Ol
b ir
4
“<0,01
I
n.s.
E i3 d a .-c
Vol. 66, No. 6
n--8
T 2
Recipient plasma plasma plasma plasma obtained : normal after after normal venost. venost. + prota proia - mine mine FIG.3 The results of the experiment demonstrating the humoral transfer of the venostatic endothelial stabilizing factor in rats (Z&SD).
I
’
0
I
0,2
HEPARAN
I
0;4
1
I
0,8
SULF. - mg I kg i. v.
FIG.4 Effect of heparan sulfate-related drug on CEC in rats after intravenous administration. 3i_+SD,fullcircle: control CEC levels after physiological saline i.v.
Vol. 68, No. 6
HEPARAN
SULFATE
AND VENOSTASIS
collected from animals with venostasis produced a significant decrease of CEC in recipients (Fig.3). Evidently, a humoral agent was transferred with the venostatic plasma. In order to sulfate was added to partially identify this agent, protamine without and from animals with plasma collected both venostasis. Protamine completely abolished the CEC-decreasing some insignificant In addition, effect of venostatic plasma. effect was observed with the tendency to a CEC-increasing Normal plasma with venostatic plasma with added protamine. added protamine had some effect of its own but in the opposite direction, i.e. it decreased the CEC in comparison with an untreated control (Fig.3). heparan sulfate 5 min The intravenous administration of before blood collection at the dose of 0.8 mg/kg decreased the CEC significantly under the basal level (Fig.4).
DISCUSSI~ rats an ligation of arteries induced in Whereas repeatedly after increased CEC, a decrease was observed venostasis in animal and clinical studies (9,lO). Similar were previously observed after agents inhibiting decreases endothelial turnover such as corticoids and cytostatics (11). Meanwhile, the CEC is a good indicator of the endothelial turnover. Heparin and heparin-like agents have been shown to posses a stabilizing effect on endothelium against various desquamation-inducing agents (12,13). After high doses of heparin-like agents decreased CEC was repeatedy observed even probably the under the basal level (13). This effect reflected heparin was antiproliferative effect of and particularly pronounced after low-molecular-weight heparin (Fraxiparin) and the preparation analogous to heparan sulfate. It was therefore suggested that during venostasis endogenous heparan sulfate is released from the endothelial surface producing a decreased CEC by suppressing proliferative activity. This effect of heparan sulfate is probably connected with the ability of heparin-like agents to bind to some growth factors present in the vessel wall and it was previously suggested that heparan sulfate might posses a modulating effect on endothelial proliferation in cultures The identification of (3). endogenous heparan sulfate as the humoral factor transferred with venostatic plasma was supported by the possibility to suppress its activity by the addition of protamine. However, in the present study protamine might have some effect of its own even though in the opposite direction, i.e. more or less in the same one as heparin sulfate. This latter effect is still unexplained. The preparation closely related to heparan sulfate was tested previously in a series of thrombosis models (13). It was effective also in the endothelial model preventing an increase in CEC after a standard endothelial perturbation. The effective dosage was already 0.2 mg/kg i.v. In the present study the preparation was effective even without the preliminary provocation. At the dose of 0.6-0.8 mg/kg it
463
464
HEPAFiAN SULFATE AND VENOSTASIS
Vol. 68, No. 6
decreased the basal CEC level reflecting most probably an inhibition of endothelial turnover. The CEC method makes study of possible the antiproliferative effect of heparin-like in-vivo. agents Another implication of this study is the possibility that venostasis causes the release of heparan sulfate from the vascular wall resulting in a decrease of the local antithrombotic potential. This effect, together with the well known release of plasminogen activator resulting in a local decrease of the thrombolytic potential might contribute to the explanation of the prothrombotic effect of venostasis.
1. WILSON,N.V.,SALISBURY,J.R. and KAKKAR,V.V. Effect of low molecular weight heparin on intimal hyperplasia. Brit. J.Surq. 18, 1381-1383, 1991 2.
PUKAC,L.A.,HIRSCH,G.M.,LORMEAU,J.-C.,PETITOU,M.,CHOAY,J.and KARNOVSKY,M.L. Antiproliferative effect of novel, nonanticoagulant heparin derivatives on vascular smooth muscle cells in vitro and in vivo. Amer.J.Path. 1501-1509, 1991.
3. ROSENBAUM,J.,TOBELEM,G.,MOLHO,P.,BARZU,T.and CAEN,J.P. Modulation of endothelial cell growth induced by heparin. cell Riol. Intwt. Reuorts UT, 437-446, 1986. 4. LOBB,R.R. Clinical application of heparin-binding growth factors. Eur.J.Clin.Invest. LB, 321-336, 1988. 5. MULLER,D.W.M.,ELLIS,S.G.and TOPOL,E.J. Experimental models of coronary artery stenosis. J.Am.Coll.Cardlol.lj., 418-432, 1992. 6. JALKANEN,M. Biology of cell surface heparan sulfate proteoglycans. Medical BioL a, 41-47, 1987 7. HLADOVEC,J. and ROSSMANN,P. Circulating endothelial cells isolated together with platelets and the experimental modification of their counts in rats. Thromb.Res. 3, 665-674, 1973. 8. HLADOVEC,J. Antithrombotic Druas Press, Boca Raton,F1.,1989. 9.
in Thrombosis
Models.
Circulating endothelial cells as a HLADOVEC,J. vessel wall lesions. EBysiol.Rohemoslov. 22, 1978.
CRC
sign of 140-144,
Vol. 68, No. 6
465
HEPARAN SULFATE AND VENOSTASIS
10. HLADOVEC,J. and PREROVSK!?,I. The effect of venostasis on several laboratory parameters in patients with predisposition to venous thromboembolism. Cor Vasa 27, 214-219, 1985. and MALEK,P. Decrease of 11. HLADOVEC,J.,JIRKA,J.,PREROVSK~,I. endothelaemia during immunosuppression. Biomedicia & 204-206, 1976. 12. HLADOVEC,J.The effect of heparin on endothelial Thromb Res . 3, 347-352, 1984.
stability.
13. HLADOVEC,J.and KORNALfK,F. Antithrombotic effects of heparin and related agents. Cor Vasa a, 68-74, 1991.
HEPARAN
SULFATE
AS A VENOSTATIC FACTOR
J. Hladovec
Medicine
and
ENDOTHELIAL
STABILIZING
F. Kornalik
Angiological Research Laboratory of the 4th Dept. of and Institute of Pathophysiology, Medical Faculty, Charles University, Prague
(Received
10.9.1992;
accepted
in revised form 8.10.1992
by Editor J.E. Dyr)
ABSTRACT A humoral transfer of a factor inducing a decrease of circulating endothelial cells (CEC) released during venostasis was demonstrated in rats. The possibility to block its activity by an in-vitro addition of protamine suggests its identity with an endogenous heparan sulfate possessing an inhibitory effect on endothelial turnover. This was supported by an analogous effect of intravenously administered heparan sulfate-related agent.
Heparin is known, in addition to being an anticoagulant, to posses other activities as well. One of them which shifts now into the focus of attention is the antiproliferative activity particularly in with connection the arterial reocclusion after angioplasty and other procedures renewing the vessel wall patency (l-4). However, heparin is not present under physiological conditions in the blood or on the vascular surface. Instead, heparin-like several substances may be present, particularly heparan sulfate The in-vivo (6). activity of this not completely defined agent is not easy to study.
keywords:
Venostasis, heparan sulfate, endothelium, antiproliferative effect
459
460
HEPARAN SULFATE AND VENOSTASIS
Vol. 68, No. 6
For this purpose we have used our method of the invivo study of endothelial stability based on counting detached endothelial cell carcasses in blood (circulating endothelial cells - CEC) (7,8).
Female Wistar rats (180-220 g) were divided into groups of 8% animals. Venous occlusion was produced in anesthetized animals (urethane 1900 mg/kg s.c.) by a tight binding up of both hind extremities above the knee joint by rubber tubes for 10 min. The effectiveness of venostasis was controlled by the palpation of arterial pulse, discoloration of extremities and increase in dorso-plantar before the Just paw diameter. release of occlusion blood was collected from the aorta with citrate (3.8 % trisodium citrate 1 : 10) into a siliconized collecting material in the volume of at least 3 ml.In transfer experiments, titrated plasma obtained after centrifugation at 2100 g from normal or venostasis animals was injected to recipient animals by the tail vein in the volume of 1 m1/200g body weight. Blood was collected 5 min after the injection. In some animals protamine sulfate Roche 0.05 mg/kg was added in the volume of 0.1 ml to 1 ml plasma before the transfer to recipient rats. Manetti-Roberts, Heparan sulfate was supplied by It was administered intravenously dissolved in 1 Florence. ml/200 g of saline 5 min before the blood collection. circulating Endothelial cell carcasses representing samples were endothelial cells counted in plasma (CEC) (7). In method described according to the previously principle, the method is based on the removal of platelets platelet-rich with ADP and from plasma by aggregation They isolation of elements by differential centrifugation. results were counted in Burker's were chamber the and expressed as the number of elements in 9 pl plasma (i.e. the plasma chamber corrected for volume of one platform concentration). The results were statistically evaluated by two-tailed Student's t-test.
RESULTS After venostasis in both hind extremities for 10 min an about 50% decrease in CEC was repeatedly observed in the blood collected from the aorta immediately before the release of venostasis (Fig.1). In an attempt.to explain this decrease as mediator, humoral endogenous release of a possible an platelet-poor plasma obtained, from this blood was injected to a group of recipient animals (lm1/200 g) intravenously (Fig.2). The same experiment was carried out with plasma of from plasma Whereas venostasis. not subjected to rats untreated animals had no effect:on the normal CEC level which (s&SD), plasma in six independent ex.periments was 2.120.38
Vol. 68, No. 6
461
HEPARAN SULFATE AND VENOSTASIS
P’ 0.001
3
0
??
0
CONTROL (Physiol.
Sal.1
VENOUS OCCLUSION (10 min 1
Effect of venostasis rats (Z&SD).
FIG.1 in both hind
before VENOSTASIS 10 min
legs
for 10
min
on CEC
in
+ 0.01 mg/ml
'a
FIG.2 designated to representation of the experiment Schematic demonstrate the humoral transfer of the venostatic endothelial stabilizing factor.
462
HEPARAN SULFATE AND VENOSTASIS
ko,Ol
b ir
4
“<0,01
I
n.s.
E i3 d a .-c
Vol. 66, No. 6
n--8
T 2
Recipient plasma plasma plasma plasma obtained : normal after after normal venost. venost. + prota proia - mine mine FIG.3 The results of the experiment demonstrating the humoral transfer of the venostatic endothelial stabilizing factor in rats (Z&SD).
I
’
0
I
0,2
HEPARAN
I
0;4
1
I
0,8
SULF. - mg I kg i. v.
FIG.4 Effect of heparan sulfate-related drug on CEC in rats after intravenous administration. 3i_+SD,fullcircle: control CEC levels after physiological saline i.v.
Vol. 68, No. 6
HEPARAN
SULFATE
AND VENOSTASIS
collected from animals with venostasis produced a significant decrease of CEC in recipients (Fig.3). Evidently, a humoral agent was transferred with the venostatic plasma. In order to sulfate was added to partially identify this agent, protamine without and from animals with plasma collected both venostasis. Protamine completely abolished the CEC-decreasing some insignificant In addition, effect of venostatic plasma. effect was observed with the tendency to a CEC-increasing Normal plasma with venostatic plasma with added protamine. added protamine had some effect of its own but in the opposite direction, i.e. it decreased the CEC in comparison with an untreated control (Fig.3). heparan sulfate 5 min The intravenous administration of before blood collection at the dose of 0.8 mg/kg decreased the CEC significantly under the basal level (Fig.4).
DISCUSSI~ rats an ligation of arteries induced in Whereas repeatedly after increased CEC, a decrease was observed venostasis in animal and clinical studies (9,lO). Similar were previously observed after agents inhibiting decreases endothelial turnover such as corticoids and cytostatics (11). Meanwhile, the CEC is a good indicator of the endothelial turnover. Heparin and heparin-like agents have been shown to posses a stabilizing effect on endothelium against various desquamation-inducing agents (12,13). After high doses of heparin-like agents decreased CEC was repeatedy observed even probably the under the basal level (13). This effect reflected heparin was antiproliferative effect of and particularly pronounced after low-molecular-weight heparin (Fraxiparin) and the preparation analogous to heparan sulfate. It was therefore suggested that during venostasis endogenous heparan sulfate is released from the endothelial surface producing a decreased CEC by suppressing proliferative activity. This effect of heparan sulfate is probably connected with the ability of heparin-like agents to bind to some growth factors present in the vessel wall and it was previously suggested that heparan sulfate might posses a modulating effect on endothelial proliferation in cultures The identification of (3). endogenous heparan sulfate as the humoral factor transferred with venostatic plasma was supported by the possibility to suppress its activity by the addition of protamine. However, in the present study protamine might have some effect of its own even though in the opposite direction, i.e. more or less in the same one as heparin sulfate. This latter effect is still unexplained. The preparation closely related to heparan sulfate was tested previously in a series of thrombosis models (13). It was effective also in the endothelial model preventing an increase in CEC after a standard endothelial perturbation. The effective dosage was already 0.2 mg/kg i.v. In the present study the preparation was effective even without the preliminary provocation. At the dose of 0.6-0.8 mg/kg it
463
464
HEPAFiAN SULFATE AND VENOSTASIS
Vol. 68, No. 6
decreased the basal CEC level reflecting most probably an inhibition of endothelial turnover. The CEC method makes study of possible the antiproliferative effect of heparin-like in-vivo. agents Another implication of this study is the possibility that venostasis causes the release of heparan sulfate from the vascular wall resulting in a decrease of the local antithrombotic potential. This effect, together with the well known release of plasminogen activator resulting in a local decrease of the thrombolytic potential might contribute to the explanation of the prothrombotic effect of venostasis.
1. WILSON,N.V.,SALISBURY,J.R. and KAKKAR,V.V. Effect of low molecular weight heparin on intimal hyperplasia. Brit. J.Surq. 18, 1381-1383, 1991 2.
PUKAC,L.A.,HIRSCH,G.M.,LORMEAU,J.-C.,PETITOU,M.,CHOAY,J.and KARNOVSKY,M.L. Antiproliferative effect of novel, nonanticoagulant heparin derivatives on vascular smooth muscle cells in vitro and in vivo. Amer.J.Path. 1501-1509, 1991.
3. ROSENBAUM,J.,TOBELEM,G.,MOLHO,P.,BARZU,T.and CAEN,J.P. Modulation of endothelial cell growth induced by heparin. cell Riol. Intwt. Reuorts UT, 437-446, 1986. 4. LOBB,R.R. Clinical application of heparin-binding growth factors. Eur.J.Clin.Invest. LB, 321-336, 1988. 5. MULLER,D.W.M.,ELLIS,S.G.and TOPOL,E.J. Experimental models of coronary artery stenosis. J.Am.Coll.Cardlol.lj., 418-432, 1992. 6. JALKANEN,M. Biology of cell surface heparan sulfate proteoglycans. Medical BioL a, 41-47, 1987 7. HLADOVEC,J. and ROSSMANN,P. Circulating endothelial cells isolated together with platelets and the experimental modification of their counts in rats. Thromb.Res. 3, 665-674, 1973. 8. HLADOVEC,J. Antithrombotic Druas Press, Boca Raton,F1.,1989. 9.
in Thrombosis
Models.
Circulating endothelial cells as a HLADOVEC,J. vessel wall lesions. EBysiol.Rohemoslov. 22, 1978.
CRC
sign of 140-144,
Vol. 68, No. 6
465
HEPARAN SULFATE AND VENOSTASIS
10. HLADOVEC,J. and PREROVSK!?,I. The effect of venostasis on several laboratory parameters in patients with predisposition to venous thromboembolism. Cor Vasa 27, 214-219, 1985. and MALEK,P. Decrease of 11. HLADOVEC,J.,JIRKA,J.,PREROVSK~,I. endothelaemia during immunosuppression. Biomedicia & 204-206, 1976. 12. HLADOVEC,J.The effect of heparin on endothelial Thromb Res . 3, 347-352, 1984.
stability.
13. HLADOVEC,J.and KORNALfK,F. Antithrombotic effects of heparin and related agents. Cor Vasa a, 68-74, 1991.