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Alterations in prostaglandin metabolism of cultured human umbilical vein endothelial cells affected by smoking during pregnancy
Trophoblast Research 7:147-155, 1993
ALTERATIONS IN PROSTAGLANDIN METABOLISM OF CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS AFFECTED BY SMOKING DURING PREGNANCY Brigitta K a r b o w s k i , H a n s - J o c h e n Bauchl, a n d H e r m a n n Peter G e r h a r d S c h n e i d e r Department of Obstetrics und Gynecology University of Mfinster 1Institute for Arteriosclerosis Research at the University of Miinster 4400 Mfinster, Germany INTRODUCTION
During pregnancy maternal smoking is often associated with chronic placental insufficiency characterized by both insufficient fetal nutrition and impaired oxygen supply. Depending on the degree of intrauterine hypoxemia, fetal growth retardation, or even intrauterine death occurs with a high incidence of perinatal or neonatal morbidity and mortality (Ounsted et al., 1981). Even after infancy, such children show intelligence defects and late neurological damage (Naeye und Peters, 1984). Changes in the endothelial cell function could contribute to the increased vascular resistance and chronic placental insufficiency that develops in smokers. We therefore compared endothelial cells from the umbilical veins of smokers with those from veins of non-smokers for their abilities to synthesize prostaglandins and also to grow in culture. The vascular endothelium plays a major role in the regulation of this functional unit and controls the metabolic exchange from the intravascular to the extravascular space. Moreover, the vascular endothelium influences the regulation of vascular tonus and the formation of an anti-thrombogenic luminal vascular surface (Busse and Bassenge, 1985; Weksler et al., 1988). Thus, irregular endothelial metabolism may lead to reduced placental perfusion. The vascular endothelium and blood cells exhibit several biological functions which are of great importance in hemodynamics and therefore in placental perfusion (Schroer, 1984). These functions are influenced by metabolites of arachidonic acid, in particular, prostaglandins. We studied the influence of smoking on the metabolism of vascular endothelium and used cultured human umbilical endothelial cells (HUVEC) as an in vitro model. In addition, we investigated whether prostaglandin metabolism is altered in the umbilical vein endothelium of smoking mothers in comparison to healthy non-smoking controls.
147 01993 Universlty of R o c h ~ " Press
148
Karbowski et al.
MATERIALS AND METHODS Endothelial Cell Culture HUVEC were obtained from human umbilical cord veins. Eighteen mothers who smoked more than 20 cigarettes per day during pregnancy and 20 healthy nonsmoking control mothers were included in this study. Umbilical cords were obtained immediately after delivery and stored in cord buffer, pH 7.2, at 4~ prior to isolation of endothelial cells. Primary cultures were prepared according to the method of Jaffe et al. (1973). Cells were suspended in medium 199 containing 20% heat-inactivated pooled human serum, counted with a hemocytometer, and seeded in petri dishes 5 cm.in diameter at a density of 250,000 cells per dish in 5ml of medium. The cells were kept at 37~ in an atmosphere containing 5% CO2. _ During the proliferation experiments all cell numbers were monitored using a hemocytometer. For statistical evaluations the U-test according to Mann-Whitney was used. Incubation of HUVEC with SH-Arachidonic Acid The culture medium was supplemented with 10 uCi 3H-arachidonic acid (spec. act. 210 Ci/mmol, Amersham) 24 hours after seeding. Cells were incubated with the tracer for 24 hours and the culture medium was collected. Isolation of Prostaglandins f~om Culture Media
Prostaglandins were isolated from culture supernatants using a liquid extraction procedure described by Salmon and Flower (1982). Briefly, proteins were separated by acetone precipitation and lipids were extracted thereafter with n-hexane. The pH of the remaining aqueous liquid was adjusted to 3.5-4.0 with I M citric acid and the liquid was extracted three times with 50 ml of chloroform. Chloroform extracts were dried over Na2SO4 and evaporated under nitrogen. The residues were reconstituted in 300 Ill of acetonitrile and 60 ttl aliquots were applied to analytical silica gel thin-layer plates (0.2 mm) for further analysis. Separation of Prostaglandins by Thin-layer Chromatography and Their Quantitative Determination Thin,layer plates were developed in e t h y l a c e t a t e / i s o - o c t a n e / a c e t i c acid/water = 150/60/30/150 (organic phase). After airdrying, the plates were sprayed with 3H-enhancer (New England Nuclear) and dried slightly. They were exposed to Xray films (Kodak) at -70~ for 4 or 14 days. The amount of radioactivity in the separated compounds was determined by laser-densito metric analysis of the X-ray films.
Smoking and Chronic Placental Insufficiency
149
Prostaglandins were identified by co-chromatography using authentic samples as a reference. Authentic samples were visualized by spraying with 3,5% phosphomolybdic acid yielding blue spots on a yellow background. In all experiments PGI 2synthesis was measured by monitoring the formation of its stable metabolite 6-oxoPGFla. RESULTS Influence of Smoking on Proliferation and Growth of Cultured HUVEC HUVEC from smoking mothers showed decreased proliferation compared with cells from non-smoking normal individuals (Figure 1). Our statistical studies on the influence of smoking on cell growth support the hypothesis that the decreased proliferation of cells in culture may be related to a high number of growth inhibited or even necrotic cells. The impaired proliferation observed may be caused by the long in vivo exposure to the risk factor smoking. We observed significantly reduced cell growth in HUVEC from smoking mothers (p < 0,001) compared with normal controls (Figure 2), 1000"
800
600 (,} I0 X
t
400
E
200t 2'4 48 time of culture ( h )
72
9'6
9 Nonmanmkingmoth,re n = 2 0 9 ~noking ~ n=18 p < O,O01 Figure 1. Influence of the risk factor smoking on proliferation of cultured HUVEC. 9 Non-smoking mothers, n = 20; 9 Smoking mothers, n = 18; P < 0.001.
150
K a r b o w s k i e t al.
800.
700.
eGO.
(0I o!-.
500
400
l
300
o
200
1 0 0 84
i I~
- smolO~ mothers n : 20
Smoking moem~ n : 18 p < 0,0001
Figure 2. Effect of exposure to the risk factor smoking on growth of cultured HUVEC. There is a significant difference (P < 0.0001) between groups. Vasoactive hormones such as epinephrine or norepinephrine are involved in the regulation of hemodynamic processes. These substances affect the permeability and activity of endothelial cells (Bauch et al., 1987c). Therefore, we studied the influence of epinephrine on the proliferation of HUVEC. When 10.7 M epinephrine was added to the culture medium, the cells were activated, and they proliferated more rapidly than controls. This reaction was dose-dependent. A 10.7 M concentration of epinephrine in the culture medium exerted an optimal mitogenic effect. Endothelial cells from smoking mothers responded more readily to epinephrine than did control cells (Figure 3). Influence of Smoking on l'rosta~andin Metabolism in Cultured HUVEC HUVEC from smoking mothers showed a prostaglandin metabolism different from that of HUVEC from non-smoking individuals. The synthesis of PGI2, PGE2, and PGF2a was decreased compared with that of controls (Figure 4).
Smoking and Chronic Placental Insufficiency
151
170" 160"
150140130120 110 100 90
i
8O 7O 60 50 iS 40 30 20 10 0
I
I
J
10-13
I
10-11
I
1'0"-9' 110.7' 10-5
I
I
10-3
Ep~Npiv~ (M) Figure 3. Influence of epinephrine on growth of cultured HUVEC from smoking and nonsmoking mothers. 12-
10-
POX2
P|[,~
c
s
N9
S
Figure 4. Synthesis of pmstaglandin derivatives in HUVEC from smoking mothers (S) and non-smoking mothers (C).
152
Karbowski et al.
DISCUSSION Our results indicate that cells from the vascular endothelium become functionally differentiated as a result of prolonged in vivo exposure to the risk factor smoking. Smoking is known to lead to chronic placental insufficiency. Functional differentiation of HUVEC in culture was expressed as decreased cell proliferation and growth, increased sensitivity to a vasoactive substance, e.g. epinephrine, and decreased synthesis of the vasodilatory and platelet anti-aggregatory prostaglandins PGI2 and PGE2. Stress of the vascular endothelium due to smoking in vivo caused impaired cell proliferation in vitro. This may indicate that exposure to these risk factors in vivo reduces the ability of the vascular endothelium to regenerate. This hypothesis is supported by the work of Bottarro et al. (1985) on vascular endothelial regeneration. A perturbation of the ability of the vascular endothelium to regenerate favors thrombogenesis (Lam et al., 1987). The increased vascular sensitivity to the vasoactive hormone epinephrine would enhance the thrombogenic tendencies of the luminal vascular surface. This supposition is in agreement with findings of Booyse et al. (1975). Our results could possibly be of clinical significance, because elevated plasma concentrations of epinephrine are found in heavy smokers (Bauch et al., 1987b). Chronic placental insufficiency is characterized by decreased placental perfusion, disturbance of vascularization, edema and placental infarction (Asmussen and Kjeldsen, 1975; Naeye, 1977). Moreover, smoking depresses the synthesis of the vasodilatory and antiaggregatory prostaglandins PGI2 and PGE2 in the vascular endothelium. Since both PGI2 and PGE2 are important in the regulation of fetal hemodynamics (Pomerantz et al., 1978; Rankin, 1978), the decreased synthesis of these prostaglandins may be responsible for hemodynamic imbalance and decreased placental perfusion in smoking individuals. Our results complement the data of Stuart et al. (1981) who emphasized decreased PGI2-synthesis in human umbilical arteries as a charateristic phenomenon of chronic placental insuffiency syndromes, such as hypertension, preeclampsia, and intrauterine growth retardation. Moreover, PGI2 and PGE2 control several biological cell functions that play a central role in the pathogenesis of arteriosclerosis (Moncada, 1982; Nilsson und Olsson, 1984; Schroer, 1985; Smith, 1986; Bauch et al., 1987a). Arteriosclerosis, a chronic vascular disease, is associated with the same risk factors as chronic placental insufficiency, i.e. smoking, hypertension, or diabetes mellitus. Arteriosclerotic vascular changes have been detected in umbilical veins of placental vessels in smoking mothers (Asmussen, 1979). These results support the hypothesis that the initial events in the pathogenesis of placental insufficiency are comparable to the early changes in the pathogenesis of arteriosclerosis. Functional differentiation of the vascular endothelium, i.e., the non-specific mesenchymal reaction, is a key event in early atherogenesis (Hauss, 1979; Schwartz et al., 1981; Ross, 1986). SUMMARY Many endothelial cell functions that exert a regulatory influence on fetal hemodynamics and placental perfusion are modulated by metabolites of arachidonic acid, in particular prostaglandins. We used an in vitro model system to study the possible role of the vascular endothelium in the pathogenesis of chronic placental insufficiency. We examined cell growth behavior and prostaglandin metabolism in cultured umbilical vein endothelial cells from mothers exposed to the risk factor smoking during pregnancy (n = 18) and compared them with healthy non-smoking
Smoking and Chronic Placental Insufficiency
153
control mothers (n = 20). Endothelial cells from smoking mothers grew less readily than control cells. The synthesis of the prostaglandins PGI2 and PGE2, both potent vasodilators and platelet aggregation inhibitors, was reduced significantly in endothelial cells from smoking mothers. Thus, if this change also occurs in the placenta, reduced prostaglandin synthesis could contribute to impaired placental perfusion. Our results suggest that prostaglandins may play an important role in the etiology and pathogenesis of chronic placental insufficiency. ACKNOWLEDGEMENTS The technical assistance of Ms. G. B6rgeling and Ms. M. K/ise is gratefully acknowledged. REFERENCES
Asmussen, J. and Kjeldsen, K. (1975) Intimal ultrastructure of human umbilical arteries. Circ. Res. 50, 583-588. Asmussen, I. (1979) Fetal cardiovascular system as influenced by maternal smoking. Clin. Cardiol. 2, 246-256. Bauch, H.-J., Vischer, P., Kehrel, B., and Hauss, W.H. (1987a) Atherosclerotic risk factors and their influence on prostaglandin metabolism in cultured endothelial and smooth muscle cells. In: Proceedings of the 3rd International Symposium on Prostaglandins Inc. (ed.) A.R. Liss, New York, pp. 329-336. Bauch, H.-J., Kelsch, Ul., Grotemeyer, K.-H., Buchwalsky, R., and Hauss, W.H. (1987b) Plasmakatecholamine als m6glicher Risikofaktor bei der Atherogenese. In: Friihverilnderungen bei der Atherogenese. (ed.) E. Betz, Mfinchen, Bern, Wien, San Francisco. Zuckschwerdt, pp. 147-155. Bauc~ H.-J., Grfinwald, J., Vischer, P., Gerlach, U., and Hauss, H.W. (1987c) A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis. Exp. Pathol. 31, 193-204. Booyse, F. M., Bell, S. Sedlak, B., and Rafelson M. E. (1975) Development of an in vitro vessel wall model for studying certain aspects of platelet-vessel (endothelium) reactions. Artery 1, 518-539. Bottarro, D., Shepro, E., Peterson, S., and Hechtman, H.B. (1985) Serotonin, histamine and norepinephrine mediation of endothelial and vascular smooth muscle cell movement. Am. J. Physiol. 248, 252-257. Busse, R. and Bassenge, E. (1985) Endothelial control of vascular tone. Z. Kardiol. 74, 99-106. Hauss, W. H. (1979) The role of arterial wall cells in atherogenesis. Cardiovasc. Res. Center. Bull. 17, 75-110.
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Jaffe, E.A., Nachman, R.L., Becker, C.G., and Minick, C.R. (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphometric and immunologic criteria. J. Clin. Invest. 52, 2745-2756. Lam, J.Y.T., Chesebro, J.H., Steele, P.M., Badimon, L., and Fuster, V. (1987) Is vasospasm related to platelet deposition? Relationship in a porcine preparation of arterial injury in vivo. Circulation 75, 243-248. Moncada, G. (1982) Prostaglandin and arterial wall biology. Arteriosclerosis 2, 193-207. Naeye, R.L. (1977) Placental infarcation leading to fetal or neonatal death. Obstet. Gynecol. 50, 583-588. Naeye, R.L. and Peters E.C. (1984) Mental development of children whose mothers smoked during pregnancy. Obstet. Gynecol. 64, 601-607. Ntis,son, J. and Olsson, A. G. (1984) Prostaglandin E1 inhibits DNA synthesis in arterial smooth muscle cells stimulated with platelet derived growth factor. Atherosclerosis 53, 77-82. Ounsted, M., Moar, V., and Scott, W. (1981) Perinatal morbidity and mortality in small-for-date-babies. Early Hum. Dev. 5, 367-375. Pomerantz, K., Sintetos, A., and Ramwell, P. W. (1978) The effect of prostaglandins on the human umbilical artery. Prostaglandins 15, 1035-1039. Rankin, J.H.G. (1978) Role of prostaglandins in the maintenance of the placental circulation. Adv. Prostaglandin Thromboxane Res. 4, 261-268. Ross, R. (1986) The pathogenesis of atherosclerosis. N. Engl. J. Med. 314, 488-500. Salmon, J.A. and Flower, R.J. (1982) Extraction and thin-layer-chromatography of arachidonic acid metabolites. In: Methods in Enzymatology, Vol. 86, Prostaglandins and arachidonate metabolites, (eds.) W.E.M. Lands and W.L. Smith, pp. 477-493. Schroer, K. (1984) Prostaglandine und verwandte Verbindungen. Bildung, Funktion und pharmakologische Beeinflussung. Stuttgart: Thieme. Schroer, K. (1985) Prostaglandins, other eicosanoids and endothelial cells. Bas/c. Res. Cardiol. 80, 502-514. Schwartz, S.M., Gajdusek, C.M., and Selden, S.C. (1981) Vascular wall growth control: The role of the endothelium. Arteriosclerosis 1, 107-126. Smith, W.L. (1986) Prostaglandin biosynthesis and its compartimentation in vascular smooth muscle and endothelial cells. Ann. Re~. PhysioL 48, 251-261. Smart, M.J., Sunderji, S.G., Yambo, T., Clark, D.A., Allen, J.B., and Elrad, H. (1981) Decreased prostacyclin production: A characteristic of chronic placental insufficiency syndromes. Lancet, ii, 1126-1028.
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Weksler, B.B., Marcus, A.J., and Jaffe, E.A. (1988) Synthesis of prostaglandin 12 (prostacyclin) by cultured human and bovine endothelial cells. Cell. Biol. 9, 3922-3926.
ALTERATIONS IN PROSTAGLANDIN METABOLISM OF CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS AFFECTED BY SMOKING DURING PREGNANCY Brigitta K a r b o w s k i , H a n s - J o c h e n Bauchl, a n d H e r m a n n Peter G e r h a r d S c h n e i d e r Department of Obstetrics und Gynecology University of Mfinster 1Institute for Arteriosclerosis Research at the University of Miinster 4400 Mfinster, Germany INTRODUCTION
During pregnancy maternal smoking is often associated with chronic placental insufficiency characterized by both insufficient fetal nutrition and impaired oxygen supply. Depending on the degree of intrauterine hypoxemia, fetal growth retardation, or even intrauterine death occurs with a high incidence of perinatal or neonatal morbidity and mortality (Ounsted et al., 1981). Even after infancy, such children show intelligence defects and late neurological damage (Naeye und Peters, 1984). Changes in the endothelial cell function could contribute to the increased vascular resistance and chronic placental insufficiency that develops in smokers. We therefore compared endothelial cells from the umbilical veins of smokers with those from veins of non-smokers for their abilities to synthesize prostaglandins and also to grow in culture. The vascular endothelium plays a major role in the regulation of this functional unit and controls the metabolic exchange from the intravascular to the extravascular space. Moreover, the vascular endothelium influences the regulation of vascular tonus and the formation of an anti-thrombogenic luminal vascular surface (Busse and Bassenge, 1985; Weksler et al., 1988). Thus, irregular endothelial metabolism may lead to reduced placental perfusion. The vascular endothelium and blood cells exhibit several biological functions which are of great importance in hemodynamics and therefore in placental perfusion (Schroer, 1984). These functions are influenced by metabolites of arachidonic acid, in particular, prostaglandins. We studied the influence of smoking on the metabolism of vascular endothelium and used cultured human umbilical endothelial cells (HUVEC) as an in vitro model. In addition, we investigated whether prostaglandin metabolism is altered in the umbilical vein endothelium of smoking mothers in comparison to healthy non-smoking controls.
147 01993 Universlty of R o c h ~ " Press
148
Karbowski et al.
MATERIALS AND METHODS Endothelial Cell Culture HUVEC were obtained from human umbilical cord veins. Eighteen mothers who smoked more than 20 cigarettes per day during pregnancy and 20 healthy nonsmoking control mothers were included in this study. Umbilical cords were obtained immediately after delivery and stored in cord buffer, pH 7.2, at 4~ prior to isolation of endothelial cells. Primary cultures were prepared according to the method of Jaffe et al. (1973). Cells were suspended in medium 199 containing 20% heat-inactivated pooled human serum, counted with a hemocytometer, and seeded in petri dishes 5 cm.in diameter at a density of 250,000 cells per dish in 5ml of medium. The cells were kept at 37~ in an atmosphere containing 5% CO2. _ During the proliferation experiments all cell numbers were monitored using a hemocytometer. For statistical evaluations the U-test according to Mann-Whitney was used. Incubation of HUVEC with SH-Arachidonic Acid The culture medium was supplemented with 10 uCi 3H-arachidonic acid (spec. act. 210 Ci/mmol, Amersham) 24 hours after seeding. Cells were incubated with the tracer for 24 hours and the culture medium was collected. Isolation of Prostaglandins f~om Culture Media
Prostaglandins were isolated from culture supernatants using a liquid extraction procedure described by Salmon and Flower (1982). Briefly, proteins were separated by acetone precipitation and lipids were extracted thereafter with n-hexane. The pH of the remaining aqueous liquid was adjusted to 3.5-4.0 with I M citric acid and the liquid was extracted three times with 50 ml of chloroform. Chloroform extracts were dried over Na2SO4 and evaporated under nitrogen. The residues were reconstituted in 300 Ill of acetonitrile and 60 ttl aliquots were applied to analytical silica gel thin-layer plates (0.2 mm) for further analysis. Separation of Prostaglandins by Thin-layer Chromatography and Their Quantitative Determination Thin,layer plates were developed in e t h y l a c e t a t e / i s o - o c t a n e / a c e t i c acid/water = 150/60/30/150 (organic phase). After airdrying, the plates were sprayed with 3H-enhancer (New England Nuclear) and dried slightly. They were exposed to Xray films (Kodak) at -70~ for 4 or 14 days. The amount of radioactivity in the separated compounds was determined by laser-densito metric analysis of the X-ray films.
Smoking and Chronic Placental Insufficiency
149
Prostaglandins were identified by co-chromatography using authentic samples as a reference. Authentic samples were visualized by spraying with 3,5% phosphomolybdic acid yielding blue spots on a yellow background. In all experiments PGI 2synthesis was measured by monitoring the formation of its stable metabolite 6-oxoPGFla. RESULTS Influence of Smoking on Proliferation and Growth of Cultured HUVEC HUVEC from smoking mothers showed decreased proliferation compared with cells from non-smoking normal individuals (Figure 1). Our statistical studies on the influence of smoking on cell growth support the hypothesis that the decreased proliferation of cells in culture may be related to a high number of growth inhibited or even necrotic cells. The impaired proliferation observed may be caused by the long in vivo exposure to the risk factor smoking. We observed significantly reduced cell growth in HUVEC from smoking mothers (p < 0,001) compared with normal controls (Figure 2), 1000"
800
600 (,} I0 X
t
400
E
200t 2'4 48 time of culture ( h )
72
9'6
9 Nonmanmkingmoth,re n = 2 0 9 ~noking ~ n=18 p < O,O01 Figure 1. Influence of the risk factor smoking on proliferation of cultured HUVEC. 9 Non-smoking mothers, n = 20; 9 Smoking mothers, n = 18; P < 0.001.
150
K a r b o w s k i e t al.
800.
700.
eGO.
(0I o!-.
500
400
l
300
o
200
1 0 0 84
i I~
- smolO~ mothers n : 20
Smoking moem~ n : 18 p < 0,0001
Figure 2. Effect of exposure to the risk factor smoking on growth of cultured HUVEC. There is a significant difference (P < 0.0001) between groups. Vasoactive hormones such as epinephrine or norepinephrine are involved in the regulation of hemodynamic processes. These substances affect the permeability and activity of endothelial cells (Bauch et al., 1987c). Therefore, we studied the influence of epinephrine on the proliferation of HUVEC. When 10.7 M epinephrine was added to the culture medium, the cells were activated, and they proliferated more rapidly than controls. This reaction was dose-dependent. A 10.7 M concentration of epinephrine in the culture medium exerted an optimal mitogenic effect. Endothelial cells from smoking mothers responded more readily to epinephrine than did control cells (Figure 3). Influence of Smoking on l'rosta~andin Metabolism in Cultured HUVEC HUVEC from smoking mothers showed a prostaglandin metabolism different from that of HUVEC from non-smoking individuals. The synthesis of PGI2, PGE2, and PGF2a was decreased compared with that of controls (Figure 4).
Smoking and Chronic Placental Insufficiency
151
170" 160"
150140130120 110 100 90
i
8O 7O 60 50 iS 40 30 20 10 0
I
I
J
10-13
I
10-11
I
1'0"-9' 110.7' 10-5
I
I
10-3
Ep~Npiv~ (M) Figure 3. Influence of epinephrine on growth of cultured HUVEC from smoking and nonsmoking mothers. 12-
10-
POX2
P|[,~
c
s
N9
S
Figure 4. Synthesis of pmstaglandin derivatives in HUVEC from smoking mothers (S) and non-smoking mothers (C).
152
Karbowski et al.
DISCUSSION Our results indicate that cells from the vascular endothelium become functionally differentiated as a result of prolonged in vivo exposure to the risk factor smoking. Smoking is known to lead to chronic placental insufficiency. Functional differentiation of HUVEC in culture was expressed as decreased cell proliferation and growth, increased sensitivity to a vasoactive substance, e.g. epinephrine, and decreased synthesis of the vasodilatory and platelet anti-aggregatory prostaglandins PGI2 and PGE2. Stress of the vascular endothelium due to smoking in vivo caused impaired cell proliferation in vitro. This may indicate that exposure to these risk factors in vivo reduces the ability of the vascular endothelium to regenerate. This hypothesis is supported by the work of Bottarro et al. (1985) on vascular endothelial regeneration. A perturbation of the ability of the vascular endothelium to regenerate favors thrombogenesis (Lam et al., 1987). The increased vascular sensitivity to the vasoactive hormone epinephrine would enhance the thrombogenic tendencies of the luminal vascular surface. This supposition is in agreement with findings of Booyse et al. (1975). Our results could possibly be of clinical significance, because elevated plasma concentrations of epinephrine are found in heavy smokers (Bauch et al., 1987b). Chronic placental insufficiency is characterized by decreased placental perfusion, disturbance of vascularization, edema and placental infarction (Asmussen and Kjeldsen, 1975; Naeye, 1977). Moreover, smoking depresses the synthesis of the vasodilatory and antiaggregatory prostaglandins PGI2 and PGE2 in the vascular endothelium. Since both PGI2 and PGE2 are important in the regulation of fetal hemodynamics (Pomerantz et al., 1978; Rankin, 1978), the decreased synthesis of these prostaglandins may be responsible for hemodynamic imbalance and decreased placental perfusion in smoking individuals. Our results complement the data of Stuart et al. (1981) who emphasized decreased PGI2-synthesis in human umbilical arteries as a charateristic phenomenon of chronic placental insuffiency syndromes, such as hypertension, preeclampsia, and intrauterine growth retardation. Moreover, PGI2 and PGE2 control several biological cell functions that play a central role in the pathogenesis of arteriosclerosis (Moncada, 1982; Nilsson und Olsson, 1984; Schroer, 1985; Smith, 1986; Bauch et al., 1987a). Arteriosclerosis, a chronic vascular disease, is associated with the same risk factors as chronic placental insufficiency, i.e. smoking, hypertension, or diabetes mellitus. Arteriosclerotic vascular changes have been detected in umbilical veins of placental vessels in smoking mothers (Asmussen, 1979). These results support the hypothesis that the initial events in the pathogenesis of placental insufficiency are comparable to the early changes in the pathogenesis of arteriosclerosis. Functional differentiation of the vascular endothelium, i.e., the non-specific mesenchymal reaction, is a key event in early atherogenesis (Hauss, 1979; Schwartz et al., 1981; Ross, 1986). SUMMARY Many endothelial cell functions that exert a regulatory influence on fetal hemodynamics and placental perfusion are modulated by metabolites of arachidonic acid, in particular prostaglandins. We used an in vitro model system to study the possible role of the vascular endothelium in the pathogenesis of chronic placental insufficiency. We examined cell growth behavior and prostaglandin metabolism in cultured umbilical vein endothelial cells from mothers exposed to the risk factor smoking during pregnancy (n = 18) and compared them with healthy non-smoking
Smoking and Chronic Placental Insufficiency
153
control mothers (n = 20). Endothelial cells from smoking mothers grew less readily than control cells. The synthesis of the prostaglandins PGI2 and PGE2, both potent vasodilators and platelet aggregation inhibitors, was reduced significantly in endothelial cells from smoking mothers. Thus, if this change also occurs in the placenta, reduced prostaglandin synthesis could contribute to impaired placental perfusion. Our results suggest that prostaglandins may play an important role in the etiology and pathogenesis of chronic placental insufficiency. ACKNOWLEDGEMENTS The technical assistance of Ms. G. B6rgeling and Ms. M. K/ise is gratefully acknowledged. REFERENCES
Asmussen, J. and Kjeldsen, K. (1975) Intimal ultrastructure of human umbilical arteries. Circ. Res. 50, 583-588. Asmussen, I. (1979) Fetal cardiovascular system as influenced by maternal smoking. Clin. Cardiol. 2, 246-256. Bauch, H.-J., Vischer, P., Kehrel, B., and Hauss, W.H. (1987a) Atherosclerotic risk factors and their influence on prostaglandin metabolism in cultured endothelial and smooth muscle cells. In: Proceedings of the 3rd International Symposium on Prostaglandins Inc. (ed.) A.R. Liss, New York, pp. 329-336. Bauch, H.-J., Kelsch, Ul., Grotemeyer, K.-H., Buchwalsky, R., and Hauss, W.H. (1987b) Plasmakatecholamine als m6glicher Risikofaktor bei der Atherogenese. In: Friihverilnderungen bei der Atherogenese. (ed.) E. Betz, Mfinchen, Bern, Wien, San Francisco. Zuckschwerdt, pp. 147-155. Bauc~ H.-J., Grfinwald, J., Vischer, P., Gerlach, U., and Hauss, H.W. (1987c) A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis. Exp. Pathol. 31, 193-204. Booyse, F. M., Bell, S. Sedlak, B., and Rafelson M. E. (1975) Development of an in vitro vessel wall model for studying certain aspects of platelet-vessel (endothelium) reactions. Artery 1, 518-539. Bottarro, D., Shepro, E., Peterson, S., and Hechtman, H.B. (1985) Serotonin, histamine and norepinephrine mediation of endothelial and vascular smooth muscle cell movement. Am. J. Physiol. 248, 252-257. Busse, R. and Bassenge, E. (1985) Endothelial control of vascular tone. Z. Kardiol. 74, 99-106. Hauss, W. H. (1979) The role of arterial wall cells in atherogenesis. Cardiovasc. Res. Center. Bull. 17, 75-110.
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