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Stem villous arteries as regulators of fetal placental blood flow
Trophoblast Research 12:403-408, 1998
STEM VILLOUS ARTERIES AS REGULATORS FETAL PLACENTAL BLOOD FLOW - A W o r k s h o p Report -
OF
John Kingdom ~ and Lucilla Poston 2 1Department of Obstetrics and Gynaecology University College Hospital London WCIE 6HX, United Kingdom 2Department of Obstetrics and Gynaecology United Medical and Dental Schools St.Thomas' Hospital London SE1 7EH, United Kingdom This workshop aimed to provide an update of the physiology and pathophysiology of the stem villous arteries in relation to fetal placental blood flow. The first two presentations provided a general update and were followed by six invited speakers whose subjects of interest covered areas of current research. John Kingdom outlined the clinical importance to obstetricians of umbilical artery Doppler in the assessment and management of high-risk pregnancies. Stem arteries are not innervated, and thus fetoplacental blood flow is under endocrine and paracrine control (Kingdom et al., 1997). The initial studies o n placentae from pregnancies complicated by intrauterine growth restriction (IUGR) and abnormal umbilical artery Doppler suggested a reduction in the density of small stem arteries as compared to control placentae. More recent application of systematic random sampling of the entire placenta has facilitated a stereological analysis of the composition of the IUGR placenta and cast doubt on the observations of reduced numbers of stem arterioles. Some studies have suggested that the tunica media of stem arteries is hypertrophied in IUGR, or that the intima and endothelium are abnormal. Delayed fixation and immersion fixation methods may have created artefacts in these studies and thus further work, employing modern sampling methods and perfusion-fixation techniques, is required to clarify the significance of these earlier observations. Finally, although much data exists on the physiological control of fetoplacental blood flow using dual-circuit placental perfusion, few data are available regarding abnormal placentae; while such studies are attractive from a theoretical standpoint, they partially address only the problem of vessel selection bias in myography, since only functioning lobules can be perfused. Gaby Kohnen (Glasgow, Scotland) gave an elegant didactic presentation on the developmental biology and structure of stem villi. This clarified the developmental sequence from primary, through secondary and tertiary villi into immature intermediate villi, which gradually differentiate into the stem villi. Immature intermediate villi differentiate proximaUy into stem villi (with a regressing perivascular capillary network) and eventually differentiate into mature intermediate villi, which in turn make terminal
403
9
of Rochester
404
Kingdom and Poston
villi through non-branching angiogenesis. The proliferative rate and differentiation pathway of the immature intermediate villi are crucial to the final construction of the stem arterial tree. The second part of this talk introduced the concept of specialized contractile extravascular stromal cells outside the fetal blood vessels (Kohnen at al., 1996), also termed the perivascular contractile sheath (Graf et al., 1995). Different subpopulations of extravascular stromal cells can be distinguished according to the coexpression patterns of cytoskeletal proteins. Proliferating mesenchymal cells are located just below the trophoblast while stromal cells close to the central stem vessels develop immunohistochemical features of myofibroblasts and smooth muscle cells (Kohnen et al., 1996). The results obtained indicate a clearly defined spatial differentiation gradient with increasing cytoskeletal complexity in human placental stromal cells from the superficial trophoblast towards the blood vessel in the center of the stem villus. Gaby Khonen also suggested some functional relevance of these contractile cells. Contraction or relaxation could compress or widen the extravascular space and could enable the fetus to gain some "control" over maternal blood flow. Renate Graf (Berlin) continued to deal with the perivascular sheath, outlining a proposed signal transduction pathway between cells of the perivascular sheath and media smooth muscle cells. Dr. Graf also described the procedure of selective dissection and isolation of the perivascular contractile sheath (Graf et al., 1995), which provides an excellent model to study cells and to compare with smooth muscle cells of the media. As mentioned by Gaby Kohnen, this speaker observed that the longitudinal arrangement of the fibers and their predominance in anchoring villi increases the possibility that they control the dimensions of the intervillous space. However, in the subsequent discussion, it was remarked that the potential for tension development was very small in relation to that of the vascular media, so this view was challenged. It was concluded that much more work was needed to define the functional importance of these fibers. Stephen Burrell (London UK) described the evolution of a pre-embedding technique for placental terminal villi, choosing the cellular distribution of type III nitric oxide synthase enzyme (or ecNOS) as his example. Staining of syncytiotrophoblast, Hofbauer cells and endothelium was demonstrated, but the majority of cytotrophobast cells were negative for type III NOS, consistent with earlier studies (Myatt et al., 1993). Endothelial cell staining was heterogeneous, as previously reported in other vascular beds, though light microscopy studies thus far have failed to demonstrate type III NOS in terminal villous capillaries. Some workshop participants did not accept the concept of heterogeneity, preferring the alternative suggestion that peripheral cytoplasm of endothelial ceils may be negative due to the distance from the nucleus. The data are preliminary, but illustrate the current dangers of over-interpretation of light immunohistochemistry in the placenta. The importance of this technique is the clarity by which the cellular distribution of epitopes may be studied. David Somerset (Birmingham, UK) outlined immunohistochemical and molecular studies addressing the role of hepatocyte growth factor (HGF). HGF, via its receptor, c-met, has been shown to cause trophoblast proliferation in vitro. HGF is secreted by cells of mesenchymal origin, whereas c-met is expressed by epithelial and endothelial cells. Knockout studies in mice have shown that an absence of either HGF or c-met leads to intra-uterine death secondary to severe IUGR with small placentae. In situ hybridization pictures were elegant, and showed HGF mRNA to be located primarily around developing stem vessels, suggesting expression in developing vascular smooth muscle of the stem villi, with weaker hybridization in the villous stroma and no signal
Fetal Placental Blood Flow
405
from the trophoblast. This pattern was similar throughout gestation. The distribution of c-met mRNA was confined to undifferentiated cytotrophoblast in the first trimester villi (but absent in extravillous trophoblast). At later gestation the signal from the perivillous trophoblast was weaker, reflecting the reduced proportion of undifferentiated cytotrophoblast to syncytium. These findings are consistent with a role for HGF/c-met in controlling mesenchymal-epithelial interaction in the human placenta, and in the promotion of normal villous trophoblast development. Helle Clausen (Hvidovre, Denmark) presented detailed studies of the stem villous arteries in heavy cigarette smokers (>15/day) and in non-smokers. Although pertinent worldwide, this study is of is particular relevance to Danish women, as cigarette smoking in pregnancy is very common in Denmark, where about 30% of all pregnant women continue to smoke during pregnancy. In this study, using a small vessel myograph, circumference/tension relationships and responses to constrictor and dilator agonists in stem villous arteries were characterized. In addition, immunocytochemical techniques were used to evaluate the distribution and relative expression of endothelin-1, NO synthase (type III NOS) and several endothelial cell markers in the stem villous arteries. Lastly, using the disector technique, vascular smooth muscle number in the artery wall was calculated to determine any influence of smoking on small artery myocyte proliferation. Arteries from the smokers were more sensitive to the constrictor effects of endothelin-1 and demonstrated greater expression of the peptide, suggestive of a mechanism which may contribute to reduced fetoplacental blood flow. Conversely, a reduction in vascular tone might arise from the increased expression of type III NOS observed in the endothelium, although the lack of an abnormality in the constrictor response to the NOS inhibitor L-NNA would suggest that the enhanced expression was insufficient to be of functional relevance. For a given transmural pressure (i.e., a given degree of stretch) the arteries of the smokers were less responsive to a depolarizing stimulus than those of controls. This could reflect an alteration in one of the many elements of the signal transduction pathway or a structural alteration in the contractile filament assembly, perhaps as result of sustained constrictor activity. Dr. Clausen's laboratory had taken advantage of the local expertise in stereological methodology provided by Dr. H.J.G. Gundersen in the Stereological Research Laboratory of Aarhus University. The volume and number of cells in the small stem villus arteries were calculated and then the number per unit area, by counting the nuclei (stained by Toluidine blue) using the optical disector and customized software. No difference in endothelial cell or myocyte number was found between the smoking and non-smoking groups, but this study showed clearly the potential for this technique, the strengths of which were also elaborated by Dr. Gundersen in his lecture in one of the plenary sessions (Clausen et al., 1998). In addition to Dr. Clausen's elegant study showing subtle alterations in function of the fetoplacental vasculature, one other report at the meeting highlighted the potential dangers of cigarette smoking to this vascular bed. Krebs and colleagues perfused the placental blood vessels of smokers with liquid plastic and used scanning electron microscopy of the resulting cast (after digestion of all nonvascular tissue) to show a significant increase in capillary branching, probably indicative of a cigarette induced hypoxic environment (Krebs et al., 1997). In Helle Clausen's study, arteries from both smokers and non-smokers were found not to respond to corticotrophin releasing hormone (CRH) previously shown to be a potent dilator in the isolated perfused placental cotyledon. This lack of response in isolated arteries has also been shown by another group (Dixon et al., 1996). The role of CRH as a dilator in the fetoplacental circulation was the next topic to be dealt with at this workshop.
406
K i n g d o m and Poston
CRH in the maternal plasma rises during gestation and probably originates from the trophoblast. In pregnancies complicated by IUGR or preeclampsia, maternal CRH rises further. CRH is recognized to be a vasodilator as it causes hypotension when infused into animals or man and reduces vascular tone in isolated arteries or isolated perfused vascular beds from some circulations. Vicki Clifton (Newcastle, Australia), the next speaker, has shown that CRH is fifty times more potent than prostacyclin in effecting vasodilation of the perfused human placental cotyledon (Clifton et al., 1994) and that the relaxation is mediated by nitric oxide (Clifton et al., 1995). This has led to the suggestion that CRH, through vasodilation, may help to preserve fetoplacental blood flow in some complicated pregnancies. However, Vicki Clifton showed new data from which it was clear that the IUGR placenta is less responsive to CRH than the placenta from a normal pregnancy. Potentially, this may offset some of the benefit gained from the raised CRH. In agreement with the observation that isolated stem villous arteries from normal pregnancies fail to respond to CRH, Dr. Clifton has also shown that isolated fetoplacental arteries do not relax in response to CRH. The discrepancy between responses to CRH in the isolated artery and perfused cotyledon could indicate that CRH acts indirectly through release of a trophoblast derived vasodilator, perhaps nitric oxide. Further experiments showed that the response to CRH is sensitive to the ambient oxygen tension. If the cotyledon was perfused with physiological buffer gassed with low 02, there was no relaxation, indeed a slight increase in perfusion pressure occurred, indicative of vasoconstriction. The question was raised as to whether this implied that CRH was also a vasoconstictor in the fetoplacental circulation in which the O 2 tension is also low. There followed a discussion of the difficulty of knowing whether the use of a low pO 2 in a physiological buffer, in the absence of erythrocytes, faithfully reproduces the oxygen carrying capacity of whole blood. This discussion highlighted one of the recurrent problems of in vitro preparations. Free radical induced vascular damage is implicated in many diseases of the cardiovascular system. In pregnancies associated with preeclampsia, there is increasing evidence that free radicals, possibly of placental origin, cause dysfunction of the maternal vascular endothelium. This in turn may underlie the propensity to intravascular coagulation and to vasoconstriction and hypertension. Leslie Myatt (Cincinnati, USA), the last speaker of the workshop, has recently published evidence which suggests that interaction between nitric oxide and free radicals may lead to damage of the fetoplacental circulation in preeclampsia and in IUGR (Myatt et al., 1996). In normal pregnancies, the placental vasculature appears to have very considerable dependence upon NO for the maintenance of low resistance, and type III NOS is widely distributed in placental structures. Moreover, type III NOS expression increases in preeclampsia. Whereas this may be of benefit to the maintenance of blood flow, it also increases the potential for the synthesis of peroxynitrite. Peroxynitrite is formed by the interaction of NO and the superoxide radical. Peroxynitrite, which has been implicated in the early stages of atherosclerosis, is cytotoxic and will cause non-specific cellular dysfunction. Interaction with tissue leads to the formation of nitrotyrosine residues. In a previous study (Myatt et al., 1996) enhanced staining for nitrotyrosine was observed in the endothelium and media of small placental arteries from women with preeclampsia a n d / o r IUGR. In the workshop Leslie Myatt presented data showing that a similar deposition of nitrotyrosine residues is apparent in the placentae of women whose pregnancies are complicated by diabetes. Oxidative stress is implicated increasingly in the vasculopathies associated with diabetes (Tribe and Poston, 1996) and it is of interest that, despite the good control of diabetes in the patients he studied, marked nitrotyrosine staining was apparent. The potential role of oxidative stress in complications of diabetic pregnancies is further
Fetal Placental Blood Flow
407
emphasized by recent studies in rats which have shown that antioxidant supplement (vitamin E) can drastically reduce the incidence of diabetic embryopathy (Siman and Eriksson, 1997). In view of these studies and the added possibility that dysfunction induced in utero of the fetal vasculature may program for adult disease, there would seem to be an increasing rationale for the evaluation of antioxidant supplementation by randomized controlled trial in diabetic pregnancies. REFERENCES
Clausen, H.V., Larsen, L.G. and Gundersen, H.J.G. (1998) Simple and efficient stereological quantitation of some placental structures. Trophoblast Res. 12:41-56. Clifton, V.L., Read, M.A., Boura, A.L.A., Robinson, P.J. and Smith, R. (1994). Corticotropin-releasing hormone-induced vasodilatation in the human fetal placental circulation. J. Clin. Endocrinol. Metab. 79, 666-669. Clifton, V.L., Read, M.A., Leitch, I.M., Giles, W.B., Boura, A.L.A., Robinson, P.J. and Smith, S. (1995) Corticotropin-releasing hormone induced vasodilatation in the human fetal-placental circulation: Involvement of the nitric oxide-cyclic guanosine 3'5'-monophosphate-mediated pathway. J. Clin. Endocrinol. Metab. 80, 2888-2893. Dixon, W.G., Tribe, R.M., Palmer, A.M., Linton, E,A. and Poston, L (1996) Corticotropin releasing hormone (CRH) does not relax isolated human fetoplacental resistance arteries. J. Soc. Gynecol. Invest. 3, 235A. Graf, R., Schonfelder, G., Muhlberger, M. and Gutsmann, M. (1995) The perivascular contractile sheath of human placental stem villi; its isolation and characterisation. Placenta 16, 57-66. Kingdom, J.C.P., BurreU, S.J. and Kaufmann, P. (1997) Pathology and clinical implications of abnormal umbilical artery Doppler waveforms. Ultrasound Obstet. Gynecol. 9, 271-286. Kohnen G., Kertschanska, S., Demir, R. and Kaufmann P. (1996) Placental villous stroma as a model system for myofibroblast differentiation. Histochem. Cell Biol. 105, 415429. Krebs, C., Kingdom, J.C.P., Macara, L. and Leiser, R. (1997) Smoking alters term human fetal placental vasculature. Placenta 18, A34. Myatt, L., Brockman,D.E., Eis, A.L.W. and Pollock, J.S. (1993). Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta 14, 487-495. Myatt, L., Rosenfield, R.B., Eis, A.W., Brockman, D.E., Greer, I. and Lyall. F. (1996) Nitrotyrosine residues in placenta. Evidence of peroxynitrite formation and action. Hypertension 28, 488-493. Siman, C.M. and Eriksson, U.J. (1997) Vitamin E decreases the occurrence of malformations in the offspring of diabetic rats. Diabetes 46, 1054-1055.
408
Kingdom and Poston
Tribe, R.M. and Poston, L. (1996) Oxidative stress and lipids in diabetes: A role in endothelium vasodilator dysfunction. Vas. Med. 1, 195-206.
STEM VILLOUS ARTERIES AS REGULATORS FETAL PLACENTAL BLOOD FLOW - A W o r k s h o p Report -
OF
John Kingdom ~ and Lucilla Poston 2 1Department of Obstetrics and Gynaecology University College Hospital London WCIE 6HX, United Kingdom 2Department of Obstetrics and Gynaecology United Medical and Dental Schools St.Thomas' Hospital London SE1 7EH, United Kingdom This workshop aimed to provide an update of the physiology and pathophysiology of the stem villous arteries in relation to fetal placental blood flow. The first two presentations provided a general update and were followed by six invited speakers whose subjects of interest covered areas of current research. John Kingdom outlined the clinical importance to obstetricians of umbilical artery Doppler in the assessment and management of high-risk pregnancies. Stem arteries are not innervated, and thus fetoplacental blood flow is under endocrine and paracrine control (Kingdom et al., 1997). The initial studies o n placentae from pregnancies complicated by intrauterine growth restriction (IUGR) and abnormal umbilical artery Doppler suggested a reduction in the density of small stem arteries as compared to control placentae. More recent application of systematic random sampling of the entire placenta has facilitated a stereological analysis of the composition of the IUGR placenta and cast doubt on the observations of reduced numbers of stem arterioles. Some studies have suggested that the tunica media of stem arteries is hypertrophied in IUGR, or that the intima and endothelium are abnormal. Delayed fixation and immersion fixation methods may have created artefacts in these studies and thus further work, employing modern sampling methods and perfusion-fixation techniques, is required to clarify the significance of these earlier observations. Finally, although much data exists on the physiological control of fetoplacental blood flow using dual-circuit placental perfusion, few data are available regarding abnormal placentae; while such studies are attractive from a theoretical standpoint, they partially address only the problem of vessel selection bias in myography, since only functioning lobules can be perfused. Gaby Kohnen (Glasgow, Scotland) gave an elegant didactic presentation on the developmental biology and structure of stem villi. This clarified the developmental sequence from primary, through secondary and tertiary villi into immature intermediate villi, which gradually differentiate into the stem villi. Immature intermediate villi differentiate proximaUy into stem villi (with a regressing perivascular capillary network) and eventually differentiate into mature intermediate villi, which in turn make terminal
403
9
of Rochester
404
Kingdom and Poston
villi through non-branching angiogenesis. The proliferative rate and differentiation pathway of the immature intermediate villi are crucial to the final construction of the stem arterial tree. The second part of this talk introduced the concept of specialized contractile extravascular stromal cells outside the fetal blood vessels (Kohnen at al., 1996), also termed the perivascular contractile sheath (Graf et al., 1995). Different subpopulations of extravascular stromal cells can be distinguished according to the coexpression patterns of cytoskeletal proteins. Proliferating mesenchymal cells are located just below the trophoblast while stromal cells close to the central stem vessels develop immunohistochemical features of myofibroblasts and smooth muscle cells (Kohnen et al., 1996). The results obtained indicate a clearly defined spatial differentiation gradient with increasing cytoskeletal complexity in human placental stromal cells from the superficial trophoblast towards the blood vessel in the center of the stem villus. Gaby Khonen also suggested some functional relevance of these contractile cells. Contraction or relaxation could compress or widen the extravascular space and could enable the fetus to gain some "control" over maternal blood flow. Renate Graf (Berlin) continued to deal with the perivascular sheath, outlining a proposed signal transduction pathway between cells of the perivascular sheath and media smooth muscle cells. Dr. Graf also described the procedure of selective dissection and isolation of the perivascular contractile sheath (Graf et al., 1995), which provides an excellent model to study cells and to compare with smooth muscle cells of the media. As mentioned by Gaby Kohnen, this speaker observed that the longitudinal arrangement of the fibers and their predominance in anchoring villi increases the possibility that they control the dimensions of the intervillous space. However, in the subsequent discussion, it was remarked that the potential for tension development was very small in relation to that of the vascular media, so this view was challenged. It was concluded that much more work was needed to define the functional importance of these fibers. Stephen Burrell (London UK) described the evolution of a pre-embedding technique for placental terminal villi, choosing the cellular distribution of type III nitric oxide synthase enzyme (or ecNOS) as his example. Staining of syncytiotrophoblast, Hofbauer cells and endothelium was demonstrated, but the majority of cytotrophobast cells were negative for type III NOS, consistent with earlier studies (Myatt et al., 1993). Endothelial cell staining was heterogeneous, as previously reported in other vascular beds, though light microscopy studies thus far have failed to demonstrate type III NOS in terminal villous capillaries. Some workshop participants did not accept the concept of heterogeneity, preferring the alternative suggestion that peripheral cytoplasm of endothelial ceils may be negative due to the distance from the nucleus. The data are preliminary, but illustrate the current dangers of over-interpretation of light immunohistochemistry in the placenta. The importance of this technique is the clarity by which the cellular distribution of epitopes may be studied. David Somerset (Birmingham, UK) outlined immunohistochemical and molecular studies addressing the role of hepatocyte growth factor (HGF). HGF, via its receptor, c-met, has been shown to cause trophoblast proliferation in vitro. HGF is secreted by cells of mesenchymal origin, whereas c-met is expressed by epithelial and endothelial cells. Knockout studies in mice have shown that an absence of either HGF or c-met leads to intra-uterine death secondary to severe IUGR with small placentae. In situ hybridization pictures were elegant, and showed HGF mRNA to be located primarily around developing stem vessels, suggesting expression in developing vascular smooth muscle of the stem villi, with weaker hybridization in the villous stroma and no signal
Fetal Placental Blood Flow
405
from the trophoblast. This pattern was similar throughout gestation. The distribution of c-met mRNA was confined to undifferentiated cytotrophoblast in the first trimester villi (but absent in extravillous trophoblast). At later gestation the signal from the perivillous trophoblast was weaker, reflecting the reduced proportion of undifferentiated cytotrophoblast to syncytium. These findings are consistent with a role for HGF/c-met in controlling mesenchymal-epithelial interaction in the human placenta, and in the promotion of normal villous trophoblast development. Helle Clausen (Hvidovre, Denmark) presented detailed studies of the stem villous arteries in heavy cigarette smokers (>15/day) and in non-smokers. Although pertinent worldwide, this study is of is particular relevance to Danish women, as cigarette smoking in pregnancy is very common in Denmark, where about 30% of all pregnant women continue to smoke during pregnancy. In this study, using a small vessel myograph, circumference/tension relationships and responses to constrictor and dilator agonists in stem villous arteries were characterized. In addition, immunocytochemical techniques were used to evaluate the distribution and relative expression of endothelin-1, NO synthase (type III NOS) and several endothelial cell markers in the stem villous arteries. Lastly, using the disector technique, vascular smooth muscle number in the artery wall was calculated to determine any influence of smoking on small artery myocyte proliferation. Arteries from the smokers were more sensitive to the constrictor effects of endothelin-1 and demonstrated greater expression of the peptide, suggestive of a mechanism which may contribute to reduced fetoplacental blood flow. Conversely, a reduction in vascular tone might arise from the increased expression of type III NOS observed in the endothelium, although the lack of an abnormality in the constrictor response to the NOS inhibitor L-NNA would suggest that the enhanced expression was insufficient to be of functional relevance. For a given transmural pressure (i.e., a given degree of stretch) the arteries of the smokers were less responsive to a depolarizing stimulus than those of controls. This could reflect an alteration in one of the many elements of the signal transduction pathway or a structural alteration in the contractile filament assembly, perhaps as result of sustained constrictor activity. Dr. Clausen's laboratory had taken advantage of the local expertise in stereological methodology provided by Dr. H.J.G. Gundersen in the Stereological Research Laboratory of Aarhus University. The volume and number of cells in the small stem villus arteries were calculated and then the number per unit area, by counting the nuclei (stained by Toluidine blue) using the optical disector and customized software. No difference in endothelial cell or myocyte number was found between the smoking and non-smoking groups, but this study showed clearly the potential for this technique, the strengths of which were also elaborated by Dr. Gundersen in his lecture in one of the plenary sessions (Clausen et al., 1998). In addition to Dr. Clausen's elegant study showing subtle alterations in function of the fetoplacental vasculature, one other report at the meeting highlighted the potential dangers of cigarette smoking to this vascular bed. Krebs and colleagues perfused the placental blood vessels of smokers with liquid plastic and used scanning electron microscopy of the resulting cast (after digestion of all nonvascular tissue) to show a significant increase in capillary branching, probably indicative of a cigarette induced hypoxic environment (Krebs et al., 1997). In Helle Clausen's study, arteries from both smokers and non-smokers were found not to respond to corticotrophin releasing hormone (CRH) previously shown to be a potent dilator in the isolated perfused placental cotyledon. This lack of response in isolated arteries has also been shown by another group (Dixon et al., 1996). The role of CRH as a dilator in the fetoplacental circulation was the next topic to be dealt with at this workshop.
406
K i n g d o m and Poston
CRH in the maternal plasma rises during gestation and probably originates from the trophoblast. In pregnancies complicated by IUGR or preeclampsia, maternal CRH rises further. CRH is recognized to be a vasodilator as it causes hypotension when infused into animals or man and reduces vascular tone in isolated arteries or isolated perfused vascular beds from some circulations. Vicki Clifton (Newcastle, Australia), the next speaker, has shown that CRH is fifty times more potent than prostacyclin in effecting vasodilation of the perfused human placental cotyledon (Clifton et al., 1994) and that the relaxation is mediated by nitric oxide (Clifton et al., 1995). This has led to the suggestion that CRH, through vasodilation, may help to preserve fetoplacental blood flow in some complicated pregnancies. However, Vicki Clifton showed new data from which it was clear that the IUGR placenta is less responsive to CRH than the placenta from a normal pregnancy. Potentially, this may offset some of the benefit gained from the raised CRH. In agreement with the observation that isolated stem villous arteries from normal pregnancies fail to respond to CRH, Dr. Clifton has also shown that isolated fetoplacental arteries do not relax in response to CRH. The discrepancy between responses to CRH in the isolated artery and perfused cotyledon could indicate that CRH acts indirectly through release of a trophoblast derived vasodilator, perhaps nitric oxide. Further experiments showed that the response to CRH is sensitive to the ambient oxygen tension. If the cotyledon was perfused with physiological buffer gassed with low 02, there was no relaxation, indeed a slight increase in perfusion pressure occurred, indicative of vasoconstriction. The question was raised as to whether this implied that CRH was also a vasoconstictor in the fetoplacental circulation in which the O 2 tension is also low. There followed a discussion of the difficulty of knowing whether the use of a low pO 2 in a physiological buffer, in the absence of erythrocytes, faithfully reproduces the oxygen carrying capacity of whole blood. This discussion highlighted one of the recurrent problems of in vitro preparations. Free radical induced vascular damage is implicated in many diseases of the cardiovascular system. In pregnancies associated with preeclampsia, there is increasing evidence that free radicals, possibly of placental origin, cause dysfunction of the maternal vascular endothelium. This in turn may underlie the propensity to intravascular coagulation and to vasoconstriction and hypertension. Leslie Myatt (Cincinnati, USA), the last speaker of the workshop, has recently published evidence which suggests that interaction between nitric oxide and free radicals may lead to damage of the fetoplacental circulation in preeclampsia and in IUGR (Myatt et al., 1996). In normal pregnancies, the placental vasculature appears to have very considerable dependence upon NO for the maintenance of low resistance, and type III NOS is widely distributed in placental structures. Moreover, type III NOS expression increases in preeclampsia. Whereas this may be of benefit to the maintenance of blood flow, it also increases the potential for the synthesis of peroxynitrite. Peroxynitrite is formed by the interaction of NO and the superoxide radical. Peroxynitrite, which has been implicated in the early stages of atherosclerosis, is cytotoxic and will cause non-specific cellular dysfunction. Interaction with tissue leads to the formation of nitrotyrosine residues. In a previous study (Myatt et al., 1996) enhanced staining for nitrotyrosine was observed in the endothelium and media of small placental arteries from women with preeclampsia a n d / o r IUGR. In the workshop Leslie Myatt presented data showing that a similar deposition of nitrotyrosine residues is apparent in the placentae of women whose pregnancies are complicated by diabetes. Oxidative stress is implicated increasingly in the vasculopathies associated with diabetes (Tribe and Poston, 1996) and it is of interest that, despite the good control of diabetes in the patients he studied, marked nitrotyrosine staining was apparent. The potential role of oxidative stress in complications of diabetic pregnancies is further
Fetal Placental Blood Flow
407
emphasized by recent studies in rats which have shown that antioxidant supplement (vitamin E) can drastically reduce the incidence of diabetic embryopathy (Siman and Eriksson, 1997). In view of these studies and the added possibility that dysfunction induced in utero of the fetal vasculature may program for adult disease, there would seem to be an increasing rationale for the evaluation of antioxidant supplementation by randomized controlled trial in diabetic pregnancies. REFERENCES
Clausen, H.V., Larsen, L.G. and Gundersen, H.J.G. (1998) Simple and efficient stereological quantitation of some placental structures. Trophoblast Res. 12:41-56. Clifton, V.L., Read, M.A., Boura, A.L.A., Robinson, P.J. and Smith, R. (1994). Corticotropin-releasing hormone-induced vasodilatation in the human fetal placental circulation. J. Clin. Endocrinol. Metab. 79, 666-669. Clifton, V.L., Read, M.A., Leitch, I.M., Giles, W.B., Boura, A.L.A., Robinson, P.J. and Smith, S. (1995) Corticotropin-releasing hormone induced vasodilatation in the human fetal-placental circulation: Involvement of the nitric oxide-cyclic guanosine 3'5'-monophosphate-mediated pathway. J. Clin. Endocrinol. Metab. 80, 2888-2893. Dixon, W.G., Tribe, R.M., Palmer, A.M., Linton, E,A. and Poston, L (1996) Corticotropin releasing hormone (CRH) does not relax isolated human fetoplacental resistance arteries. J. Soc. Gynecol. Invest. 3, 235A. Graf, R., Schonfelder, G., Muhlberger, M. and Gutsmann, M. (1995) The perivascular contractile sheath of human placental stem villi; its isolation and characterisation. Placenta 16, 57-66. Kingdom, J.C.P., BurreU, S.J. and Kaufmann, P. (1997) Pathology and clinical implications of abnormal umbilical artery Doppler waveforms. Ultrasound Obstet. Gynecol. 9, 271-286. Kohnen G., Kertschanska, S., Demir, R. and Kaufmann P. (1996) Placental villous stroma as a model system for myofibroblast differentiation. Histochem. Cell Biol. 105, 415429. Krebs, C., Kingdom, J.C.P., Macara, L. and Leiser, R. (1997) Smoking alters term human fetal placental vasculature. Placenta 18, A34. Myatt, L., Brockman,D.E., Eis, A.L.W. and Pollock, J.S. (1993). Immunohistochemical localization of nitric oxide synthase in the human placenta. Placenta 14, 487-495. Myatt, L., Rosenfield, R.B., Eis, A.W., Brockman, D.E., Greer, I. and Lyall. F. (1996) Nitrotyrosine residues in placenta. Evidence of peroxynitrite formation and action. Hypertension 28, 488-493. Siman, C.M. and Eriksson, U.J. (1997) Vitamin E decreases the occurrence of malformations in the offspring of diabetic rats. Diabetes 46, 1054-1055.
408
Kingdom and Poston
Tribe, R.M. and Poston, L. (1996) Oxidative stress and lipids in diabetes: A role in endothelium vasodilator dysfunction. Vas. Med. 1, 195-206.