47
Atherosclerosis, 83 (1990) 47-51 Elsevier Scientific Publishers Ireland. Ltd.
ATHERO
04490
Heparin protects cultured arterial endothelial cells from damage by toxic oxygen metabolites Linda M. Hiebert Department
of Physiology,
University of Saskatchewan,
and Ji-min Liu Saskatoon, Saskatchewan,
S7N 0 WO (Canada]
(Received 5 August, 1989) (Revised, received 6 March, 1990) (Accepted 8 March, 1990)
Toxic oxygen metabolites can damage endothelial cells and may play an important role in the initiation and progression of atherosclerotic lesions. Since the antithrombotic drug heparin, interacts with endothelium, we wished to determine if heparin would protect endothelial cells from free radical injury. Endothelial cell injury was produced by the addition of xanthine and xanthine oxidase to cultured cells and assessed by changes in cell viability and release of lactate dehydrogenase (LDH) to the media. Pretreatment with heparin 24 h prior to addition of xanthine and xanthine oxidase significantly decreased cell damage. We suggest that heparin (and related compounds) can protect endothelium from free radical damage, and is therefore prophylactic for ischemic and inflammatory injury, and the development and progression of atheroma.
Key words: Endothelial injury; Heparin; Oxygen metabolites
Introduction Endothelial injury and dysfunction are believed to play an important role in the initiation and development of atheroma [l]. Recent observations showed toxic oxygen metabolites can contribute to endothelial damage and alterations in function [2]. Free radicals can be generated following anoxia/ reoxygenation associated with myocardial ischemia
Correspondence to: Dr. L.M. Hiebert, Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N OWO, Canada. 0021-9150/90/$03.50
[3], during inflammation when reactive species are produced by activated neutrophils and monocytes [4], during oxidative modification of low density lipoproteins [5], and from xenobiotics [6]. Free radical injury of endothelial cells may be important for initiation and progression of atherosclerotic lesions and exacerbation related complications such as thrombosis, and vascular occlusion. Heparin, a glycosaminoglycan, is used clinically as an antithrombotic agent and in vascular surgery. Despite evidence in experimental and clinical studies that heparin is beneficial in the treatment of atherosclerosis, few clinicians have used heparin
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
48 as an anti-atherogenic drug [7]. Heparin interacts directly with the endothelial suface and uptake of heparin by endothelium has been shown following in vivo and in vitro administration [8-lo]. A few studies suggest that heparin is protective of the endothelium, but the mechanisms involved have not been determined (11,121. Since oxygen free radicals can damage endothelium, our aim was to investigate if one of the protective actions of heparin was prevention of endothelial injury caused by toxic oxygen metabolites. We have produced a model of endothelial injury by adding xanthine and xanthine oxidase to media of cultured endothelial cells. Our results show that in the presence of heparin, endothelial damage is decreased as assessed by changes in cell viability and release of lactate dehydrogenase (LDH) to the media. Methods Growth of cells Endothelial cells were derived from porcine aorta by collagenase digestion according to the method of Gotlieb [13]. Cells were grown in 35mm culture dishes with Medium 199, 5% fetal bovine serum and then placed in an incubator at 37 a C with 5% CO,. Confluent cultures were used for all procedures. Damage of endothelial cells Xanthine oxidase (lot No. 11161227-86, Boehringer Mannheim) was added to medium of confluent cultures in concentrations of O.Ol-0.4U/ml medium. Xanthine (Sigma Chemical Co. St. Louis, MO) prepared in Dulbecco’s phosphate-buffered saline (DPBS) was added in concentrations of 0.00-l pM/ml medium. Procedure of addition was fresh medium added to cultures, xanthine added to medium and mixed by agitation of the culture dish, xanthine oxidase immediately added with mixing as above. Addition of heparin Heparin (Upjohn Pharmaceuticals) was bovine lung lot No. 722EH with 151 USP U/mg. Heparin, prepared in DPBS, was added to media at a concentration of 50 pg/ml, 24 h prior to the addition of xanthine and xanthine oxidase.
Cell viahiliQ Cells were harvested using 0.025% trypsin in 0.01% EDTA and suspended in 1.5 ml medium. Cell viability was determined by trypan blue exclusion [14]. 0.5 ml cell suspension was added to 50 ~1 4% trypan blue. After 3 n-tin cells were counted in a hemocytometer chamber with the light microscope. Lactate dehydrogenase (LDH) determination The assay for LDH was performed on medium samples using Sigma Diagnostic Kit No. 228-UV. This is based on the measurement of NADH formed from NAD plus lactate. 50 ~1 medium was added to 500 ~1 reagent. LDH was quantitated by the change in absorbance at 340 nm at room temperature. The LDH released was calculated as LDH released per cell. LDH found in experimental medium was expressed as a percentage of that found in control medium from untreated cells. Results Development of a model of arterial endothelial cell damage by xanthine, xanthine oxidase Xanthine and xanthine oxidase were added to medium of cultured porcine endothelial cells. Cell injury was indicated by a significant decrease in cell viability accompanied by a significant increase in release of lactate dehydrogenase (LDH) to the medium as compared to medium from control cells. Cell damage was not apparent in our system when the concentration of xanthine oxidase was below 0.1 U/ml. The extent of cell damage at higher concentrations of xanthine oxidase was dependent on the concentration of xanthine. Fig. 1, shows endothelial cell damage at varying concentrations of xanthine and xanthine oxidase. As the concentrations of xanthine and xanthine oxidase added to the medium increased cell viability decreased and LDH released into the medium increased. A standard protocol was devised for subsequent experiments in which a dose of xanthine oxidase and xanthine was required which gave reproducible moderate cell injury when added to cells for 24 h. The concentrations selected were xanthine at 0.01 PM/ml medium and xanthine oxidase at 0.2U/ml medium. This treatment resulted in a 60% decrease in cell viability
looQ”
*
.
X0
0.2
U/ml
0
x0
0.4
Ulrnl
6070 60-
p
\
0
50 40 -
P ‘\
30 -
I
20 lo-
, 0.001
,\ 0.01
p-\_ 0.1
6
12
16
20
24
4
6
12
16
20
24
z
13OOr
900
4 700
1 i :
600
z
500 1
-
TIME
o.001
0.01
XANTHINE
CONCENTRATION
1 pM/ml
Fig. 1. Cell viability and release of LDH when varying concentrations of xanthine (X) and xanthine oxidase (X0) are added to media of endothelial cells. A dose-dependant decrease in cell viability and increase in LDH release is seen with increasing concentrations of X and X0. Means+ SEM are shown. Cell viability for untreated cells is 94.6 + 0.6 SE.
and a 300% increase in LDH released as compared to control. Effects of heparin pretreatment on arterial endothelial cell damage Heparin was added to cultured cells 24 h prior to the addition of xanthine, xanthine oxidase. Cell viability and release of LDH to the medium was examined from 1 to 24 h after the addition of xanthine, xanthine oxidase. As shown in Fig. 2, changes in cell viability and LDH release were seen within 1 h after the addition of xanthine and xanthine oxidase. A progressive decrease in viable cells and increase in release of LDH occurred over
AFTER
ADDITION
OF
X + X0
(hr)
Fig. 2. The protective effect of heparin on endothelial cells treated with xanthine (X) and xanthine oxidase (X0). Heparin was added to medium at 50 as/ml, 24 h prior to the addition of X+X0. Means& SEM of 3 cultures per group are shown. All cultures used in this experiment were taken from the same cell population and were in passage 4. This experiment was performed in duplicate and in quadruplicate at the 24-h time interval with similar results. *P i 0.01 (I test).
a 24-h period. When heparin was present in the medium, cell damage was significantly less. This protective effect was seen within 1 h as judged by a significant increase in cell viability in the heparin-treated versus the untreated group. This difference became more apparent at 6 and 24 h. The protective effect of heparin on the release of LDH to the medium was not seen for 6 h but continued for 24 h. Discussion The addition of xanthine and xanthine oxidase to cultured endothelial cells has provided us with a reproducible and useful model of endothelial cell damage. Similar models have been used by others to study free radical injury of endothelial
50
cells [6,15,16]. Although some cell injury from contaminating proteases cannot be entirely ruled out, the observation that damage from xanthine oxidase increased in the presence of xanthine indicates that endothelial damage is dependent on generation of free radicals. Our results show that in a culture system endothelial cell injury caused by the addition of xanthine/xanthine oxidase to cell medium can be prevented by prior exposure to heparin. This is the first observation of heparin protection of cells in vitro from free radical injury. This study is supported by in vivo evidence of Hladovec [17] who observed a decrease in circulating endothelial cells (endothelaemia) when heparin was injected 5 mm prior to hydrogen peroxide and sodium citrate as compared to the administration of hydrogen peroxide and sodium citrate alone. Others have suggested that heparin may be involved in protection from free radical damage. Karlsson and Marklund [18] have shown that intravenous injection of 200 U heparin/kg body weight releases extracellular superoxide dismutase (EC-SOD) from the endothelial cell into plasma which may help in the control of generated free radicals. Grant et al. [19] have hypothesized that heparin and heparan sulfate may serve as antioxidants possibly by binding ions of transition metals which are necessary for generation of the OH radical. Studies are currently underway in our laboratory to observe the protective effects of other glycosaminoglycans and to determine the mechanisms involved. Heparin is commonly added to endothelial cells in culture to promote cell growth [20]. The protective action of heparin observed in our results is not due to cell proliferation since cell numbers were not significantly increased in heparin-treated groups (data not shown). This is supported by a decreased release of LDH in heparin-treated cultures versus those not treated with heparin. The observation that heparin prevents damage from reduced oxygen species suggests a role for heparin in the mast cell. The release of heparin from mast cell granules during an inflammatory reaction may serve to protect surrounding endothelium from damage due to neutrophil, mast cell or macrophage generated oxygen metabolites. Carr [21] has shown that intravenous heparin has an anti-inflammatory effect and decreases vascular
permeability induced by histamine, bradykinin and prostaglandin E. This anti-inflammatory activity was presumed to occur at the level of the endothelial cell. This preliminary observation provides evidence that heparin and related compounds such as heparan sulfates may play an important role in protection of the endothelium from damage by free radicals produced during inflammation, ischemic episodes, and metabolism of lipoproteins. This further supports the concept that heparin and related compounds deserve wider use as anti-atherogenic drugs as proposed by others [7]. Moreover heparin may be important in the treatment of ischemic episodes and inflammatory related disorders in addition to its use as an antithrombotic agent. Acknowledgements This work was supported by a grant from the Saskatchewan Health Research Board. We thank Dr. A. Richardson and members of the Department of Anatomy, University of Saskatchewan for aid in growth of endothelial cell cultures and Dr. N. McDuffie for helpful comments. References 1 Reidy, M.A., Biology of a disease: a reassessment of endothelial injury and arterial lesion formation. Lab. Invest., 53. (1985) 513. 2 Ryan, U.S., Activation of endothelial cells. Ann. N.Y. Acad. Sci., 516 (1987) 22. 3 Simpson, P.J. and Lucchesi, B.R.. Free radicals and myocardial ischemia and reperfusion injury, J. Lab. Clin. Med., 110 (1987) 13. 4 Harlan, J.M.. Leukocyte-endothelial interactions, Blood, 65 (1985) 513. 5 Steinbrecher, U.P., Parthasarathy, S.. Leake, D.S., Witzturn, J.L., and Steinberg, D.. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids, Proc. Natl. Acad. Sci., 81 (1984) 3883. 6 Halliwell, B. and Gutteridge, J.M.C., Free Radicals in Biology and Medicine, Clarendon Press, Oxford, 1985. 7 Engelberg, H., Heparin and the Prevention of Atherosclerosis: Basic Research and Clinical Application, Alan R. Liss, New York, 1990, in press. 8 Hiebert, L.M. and Jaques, L.B., The observation of heparin on endothelium after injection, Thromb. Res.. 8 (1976) 195. 9 Barzu, T.. Molho, P.. Tobelem, G., Petitou. M. and Caen, J.. Binding and endocytosis of heparin by human endo-
51
10
11
12 13
14 15
16
thelial cells in culture, B&him. Biophys. Acta, 845 (1985) 196. Hiebert, L.M. and McDuffie, N.M., The intracellular uptake and protracted release of exogenous heparins by cultured endothehal cells, Artery, 16 (1989) 208. Engelberg, H., Heparin, heparin fractions, and the atherosclerotic process, Semin. Thromb. Hemost., 11 (1985) 48. Jaques, L.B. and Hiebert, L.M., The close relationship of heparin and the vessel wall, Artery, 16 (1989) 140. Gotlieb, AL and Spector, W., Migration into an in vitro experimental wound. A comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation., Am. J. Pathol., 108 (1981) 271. Patterson, M.K., Methods in Enzymology, Academic Press, London, 1979, p. 141. Andreoli, S.P., Mallett, C.P. and Bergstein, J.M., Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury, J. Lab. Clin. Med., 108 (1986) 190. Ratych, R.E., Chuknyiska, R.S. and Bulkley, G.B. The
17
18
19
20
21
primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells, Surgery, 102 (1987) 122. Hladovcc, J., Protective effect of oxygen-derived free radical scavengers on the endothelium in viva, Physiol. Bohemosl., 35 (1986) 97. Karlsson, K. and Marklund, L., Heparin-induced release of extracellular superoxide dismutase to human blood plasma, B&hem. J.. 242 (1987) 55. Grant, D., Long, W.F. and Williamson, F.B., Pericellular heparans may contribute to the protection of cells from free radicals., Med. Hypotheses, 23 (1987) 67. Rosengart, T.K.. Johnson, W.V., Friesel, R.. Clark, R. and Maciag, T., Heparin protects heparin-binding growth factor-l from proteolytic inactivation in vitro, B&hem. Biophys. Res. Commun., 152 (1988) 432. Car-r, J., The anti-inflammatory action of heparin: heparin as an antagonist to histamine, bradykinin and prostaglandin E, Thromb. Res., 16 (1979) 507.
Atherosclerosis, 83 (1990) 47-51 Elsevier Scientific Publishers Ireland. Ltd.
ATHERO
04490
Heparin protects cultured arterial endothelial cells from damage by toxic oxygen metabolites Linda M. Hiebert Department
of Physiology,
University of Saskatchewan,
and Ji-min Liu Saskatoon, Saskatchewan,
S7N 0 WO (Canada]
(Received 5 August, 1989) (Revised, received 6 March, 1990) (Accepted 8 March, 1990)
Toxic oxygen metabolites can damage endothelial cells and may play an important role in the initiation and progression of atherosclerotic lesions. Since the antithrombotic drug heparin, interacts with endothelium, we wished to determine if heparin would protect endothelial cells from free radical injury. Endothelial cell injury was produced by the addition of xanthine and xanthine oxidase to cultured cells and assessed by changes in cell viability and release of lactate dehydrogenase (LDH) to the media. Pretreatment with heparin 24 h prior to addition of xanthine and xanthine oxidase significantly decreased cell damage. We suggest that heparin (and related compounds) can protect endothelium from free radical damage, and is therefore prophylactic for ischemic and inflammatory injury, and the development and progression of atheroma.
Key words: Endothelial injury; Heparin; Oxygen metabolites
Introduction Endothelial injury and dysfunction are believed to play an important role in the initiation and development of atheroma [l]. Recent observations showed toxic oxygen metabolites can contribute to endothelial damage and alterations in function [2]. Free radicals can be generated following anoxia/ reoxygenation associated with myocardial ischemia
Correspondence to: Dr. L.M. Hiebert, Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N OWO, Canada. 0021-9150/90/$03.50
[3], during inflammation when reactive species are produced by activated neutrophils and monocytes [4], during oxidative modification of low density lipoproteins [5], and from xenobiotics [6]. Free radical injury of endothelial cells may be important for initiation and progression of atherosclerotic lesions and exacerbation related complications such as thrombosis, and vascular occlusion. Heparin, a glycosaminoglycan, is used clinically as an antithrombotic agent and in vascular surgery. Despite evidence in experimental and clinical studies that heparin is beneficial in the treatment of atherosclerosis, few clinicians have used heparin
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
48 as an anti-atherogenic drug [7]. Heparin interacts directly with the endothelial suface and uptake of heparin by endothelium has been shown following in vivo and in vitro administration [8-lo]. A few studies suggest that heparin is protective of the endothelium, but the mechanisms involved have not been determined (11,121. Since oxygen free radicals can damage endothelium, our aim was to investigate if one of the protective actions of heparin was prevention of endothelial injury caused by toxic oxygen metabolites. We have produced a model of endothelial injury by adding xanthine and xanthine oxidase to media of cultured endothelial cells. Our results show that in the presence of heparin, endothelial damage is decreased as assessed by changes in cell viability and release of lactate dehydrogenase (LDH) to the media. Methods Growth of cells Endothelial cells were derived from porcine aorta by collagenase digestion according to the method of Gotlieb [13]. Cells were grown in 35mm culture dishes with Medium 199, 5% fetal bovine serum and then placed in an incubator at 37 a C with 5% CO,. Confluent cultures were used for all procedures. Damage of endothelial cells Xanthine oxidase (lot No. 11161227-86, Boehringer Mannheim) was added to medium of confluent cultures in concentrations of O.Ol-0.4U/ml medium. Xanthine (Sigma Chemical Co. St. Louis, MO) prepared in Dulbecco’s phosphate-buffered saline (DPBS) was added in concentrations of 0.00-l pM/ml medium. Procedure of addition was fresh medium added to cultures, xanthine added to medium and mixed by agitation of the culture dish, xanthine oxidase immediately added with mixing as above. Addition of heparin Heparin (Upjohn Pharmaceuticals) was bovine lung lot No. 722EH with 151 USP U/mg. Heparin, prepared in DPBS, was added to media at a concentration of 50 pg/ml, 24 h prior to the addition of xanthine and xanthine oxidase.
Cell viahiliQ Cells were harvested using 0.025% trypsin in 0.01% EDTA and suspended in 1.5 ml medium. Cell viability was determined by trypan blue exclusion [14]. 0.5 ml cell suspension was added to 50 ~1 4% trypan blue. After 3 n-tin cells were counted in a hemocytometer chamber with the light microscope. Lactate dehydrogenase (LDH) determination The assay for LDH was performed on medium samples using Sigma Diagnostic Kit No. 228-UV. This is based on the measurement of NADH formed from NAD plus lactate. 50 ~1 medium was added to 500 ~1 reagent. LDH was quantitated by the change in absorbance at 340 nm at room temperature. The LDH released was calculated as LDH released per cell. LDH found in experimental medium was expressed as a percentage of that found in control medium from untreated cells. Results Development of a model of arterial endothelial cell damage by xanthine, xanthine oxidase Xanthine and xanthine oxidase were added to medium of cultured porcine endothelial cells. Cell injury was indicated by a significant decrease in cell viability accompanied by a significant increase in release of lactate dehydrogenase (LDH) to the medium as compared to medium from control cells. Cell damage was not apparent in our system when the concentration of xanthine oxidase was below 0.1 U/ml. The extent of cell damage at higher concentrations of xanthine oxidase was dependent on the concentration of xanthine. Fig. 1, shows endothelial cell damage at varying concentrations of xanthine and xanthine oxidase. As the concentrations of xanthine and xanthine oxidase added to the medium increased cell viability decreased and LDH released into the medium increased. A standard protocol was devised for subsequent experiments in which a dose of xanthine oxidase and xanthine was required which gave reproducible moderate cell injury when added to cells for 24 h. The concentrations selected were xanthine at 0.01 PM/ml medium and xanthine oxidase at 0.2U/ml medium. This treatment resulted in a 60% decrease in cell viability
looQ”
*
.
X0
0.2
U/ml
0
x0
0.4
Ulrnl
6070 60-
p
\
0
50 40 -
P ‘\
30 -
I
20 lo-
, 0.001
,\ 0.01
p-\_ 0.1
6
12
16
20
24
4
6
12
16
20
24
z
13OOr
900
4 700
1 i :
600
z
500 1
-
TIME
o.001
0.01
XANTHINE
CONCENTRATION
1 pM/ml
Fig. 1. Cell viability and release of LDH when varying concentrations of xanthine (X) and xanthine oxidase (X0) are added to media of endothelial cells. A dose-dependant decrease in cell viability and increase in LDH release is seen with increasing concentrations of X and X0. Means+ SEM are shown. Cell viability for untreated cells is 94.6 + 0.6 SE.
and a 300% increase in LDH released as compared to control. Effects of heparin pretreatment on arterial endothelial cell damage Heparin was added to cultured cells 24 h prior to the addition of xanthine, xanthine oxidase. Cell viability and release of LDH to the medium was examined from 1 to 24 h after the addition of xanthine, xanthine oxidase. As shown in Fig. 2, changes in cell viability and LDH release were seen within 1 h after the addition of xanthine and xanthine oxidase. A progressive decrease in viable cells and increase in release of LDH occurred over
AFTER
ADDITION
OF
X + X0
(hr)
Fig. 2. The protective effect of heparin on endothelial cells treated with xanthine (X) and xanthine oxidase (X0). Heparin was added to medium at 50 as/ml, 24 h prior to the addition of X+X0. Means& SEM of 3 cultures per group are shown. All cultures used in this experiment were taken from the same cell population and were in passage 4. This experiment was performed in duplicate and in quadruplicate at the 24-h time interval with similar results. *P i 0.01 (I test).
a 24-h period. When heparin was present in the medium, cell damage was significantly less. This protective effect was seen within 1 h as judged by a significant increase in cell viability in the heparin-treated versus the untreated group. This difference became more apparent at 6 and 24 h. The protective effect of heparin on the release of LDH to the medium was not seen for 6 h but continued for 24 h. Discussion The addition of xanthine and xanthine oxidase to cultured endothelial cells has provided us with a reproducible and useful model of endothelial cell damage. Similar models have been used by others to study free radical injury of endothelial
50
cells [6,15,16]. Although some cell injury from contaminating proteases cannot be entirely ruled out, the observation that damage from xanthine oxidase increased in the presence of xanthine indicates that endothelial damage is dependent on generation of free radicals. Our results show that in a culture system endothelial cell injury caused by the addition of xanthine/xanthine oxidase to cell medium can be prevented by prior exposure to heparin. This is the first observation of heparin protection of cells in vitro from free radical injury. This study is supported by in vivo evidence of Hladovec [17] who observed a decrease in circulating endothelial cells (endothelaemia) when heparin was injected 5 mm prior to hydrogen peroxide and sodium citrate as compared to the administration of hydrogen peroxide and sodium citrate alone. Others have suggested that heparin may be involved in protection from free radical damage. Karlsson and Marklund [18] have shown that intravenous injection of 200 U heparin/kg body weight releases extracellular superoxide dismutase (EC-SOD) from the endothelial cell into plasma which may help in the control of generated free radicals. Grant et al. [19] have hypothesized that heparin and heparan sulfate may serve as antioxidants possibly by binding ions of transition metals which are necessary for generation of the OH radical. Studies are currently underway in our laboratory to observe the protective effects of other glycosaminoglycans and to determine the mechanisms involved. Heparin is commonly added to endothelial cells in culture to promote cell growth [20]. The protective action of heparin observed in our results is not due to cell proliferation since cell numbers were not significantly increased in heparin-treated groups (data not shown). This is supported by a decreased release of LDH in heparin-treated cultures versus those not treated with heparin. The observation that heparin prevents damage from reduced oxygen species suggests a role for heparin in the mast cell. The release of heparin from mast cell granules during an inflammatory reaction may serve to protect surrounding endothelium from damage due to neutrophil, mast cell or macrophage generated oxygen metabolites. Carr [21] has shown that intravenous heparin has an anti-inflammatory effect and decreases vascular
permeability induced by histamine, bradykinin and prostaglandin E. This anti-inflammatory activity was presumed to occur at the level of the endothelial cell. This preliminary observation provides evidence that heparin and related compounds such as heparan sulfates may play an important role in protection of the endothelium from damage by free radicals produced during inflammation, ischemic episodes, and metabolism of lipoproteins. This further supports the concept that heparin and related compounds deserve wider use as anti-atherogenic drugs as proposed by others [7]. Moreover heparin may be important in the treatment of ischemic episodes and inflammatory related disorders in addition to its use as an antithrombotic agent. Acknowledgements This work was supported by a grant from the Saskatchewan Health Research Board. We thank Dr. A. Richardson and members of the Department of Anatomy, University of Saskatchewan for aid in growth of endothelial cell cultures and Dr. N. McDuffie for helpful comments. References 1 Reidy, M.A., Biology of a disease: a reassessment of endothelial injury and arterial lesion formation. Lab. Invest., 53. (1985) 513. 2 Ryan, U.S., Activation of endothelial cells. Ann. N.Y. Acad. Sci., 516 (1987) 22. 3 Simpson, P.J. and Lucchesi, B.R.. Free radicals and myocardial ischemia and reperfusion injury, J. Lab. Clin. Med., 110 (1987) 13. 4 Harlan, J.M.. Leukocyte-endothelial interactions, Blood, 65 (1985) 513. 5 Steinbrecher, U.P., Parthasarathy, S.. Leake, D.S., Witzturn, J.L., and Steinberg, D.. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids, Proc. Natl. Acad. Sci., 81 (1984) 3883. 6 Halliwell, B. and Gutteridge, J.M.C., Free Radicals in Biology and Medicine, Clarendon Press, Oxford, 1985. 7 Engelberg, H., Heparin and the Prevention of Atherosclerosis: Basic Research and Clinical Application, Alan R. Liss, New York, 1990, in press. 8 Hiebert, L.M. and Jaques, L.B., The observation of heparin on endothelium after injection, Thromb. Res.. 8 (1976) 195. 9 Barzu, T.. Molho, P.. Tobelem, G., Petitou. M. and Caen, J.. Binding and endocytosis of heparin by human endo-
51
10
11
12 13
14 15
16
thelial cells in culture, B&him. Biophys. Acta, 845 (1985) 196. Hiebert, L.M. and McDuffie, N.M., The intracellular uptake and protracted release of exogenous heparins by cultured endothehal cells, Artery, 16 (1989) 208. Engelberg, H., Heparin, heparin fractions, and the atherosclerotic process, Semin. Thromb. Hemost., 11 (1985) 48. Jaques, L.B. and Hiebert, L.M., The close relationship of heparin and the vessel wall, Artery, 16 (1989) 140. Gotlieb, AL and Spector, W., Migration into an in vitro experimental wound. A comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation., Am. J. Pathol., 108 (1981) 271. Patterson, M.K., Methods in Enzymology, Academic Press, London, 1979, p. 141. Andreoli, S.P., Mallett, C.P. and Bergstein, J.M., Role of glutathione in protecting endothelial cells against hydrogen peroxide oxidant injury, J. Lab. Clin. Med., 108 (1986) 190. Ratych, R.E., Chuknyiska, R.S. and Bulkley, G.B. The
17
18
19
20
21
primary localization of free radical generation after anoxia/reoxygenation in isolated endothelial cells, Surgery, 102 (1987) 122. Hladovcc, J., Protective effect of oxygen-derived free radical scavengers on the endothelium in viva, Physiol. Bohemosl., 35 (1986) 97. Karlsson, K. and Marklund, L., Heparin-induced release of extracellular superoxide dismutase to human blood plasma, B&hem. J.. 242 (1987) 55. Grant, D., Long, W.F. and Williamson, F.B., Pericellular heparans may contribute to the protection of cells from free radicals., Med. Hypotheses, 23 (1987) 67. Rosengart, T.K.. Johnson, W.V., Friesel, R.. Clark, R. and Maciag, T., Heparin protects heparin-binding growth factor-l from proteolytic inactivation in vitro, B&hem. Biophys. Res. Commun., 152 (1988) 432. Car-r, J., The anti-inflammatory action of heparin: heparin as an antagonist to histamine, bradykinin and prostaglandin E, Thromb. Res., 16 (1979) 507.
