LOCAL CONTROL OF HUMAN UMBILICAL VESSEL TONE.
Thesis submitted to the University of London
for the degree of Doctor of Philosophy
in the Faculty of Science.
by
Anita-Jane Sexton B.Sc. (Hons.)
Department of Anatomy and Developmental Biology
University College London. ProQuest Number: 10017796
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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT
This thesis examines the influence of the vascular endothelium on the local control of human feto-placental vascular tone and how these influences may vary with gestation. Electron microscopic studies have revealed that human umbilical vessels are devoid of nerves throughout gestation. Structural changes in the arterial and venous wall could be detected throughout gestation.
The presence of a subpopulation of endothelial cells containing nitric oxide synthase (NOS)-like immunoreactivity was identified only in late pregnancy. The umbilical artery and vein, from both early and late pregnancy, did not relax to acetylcholine (ACh), adenosine 5-triphosphate (ATP), substance P (SP) and calcium ionophore A23187, known to be endothelium-dependent vasodilators, but did relax to sodium nitroprusside (SNP).
Using the method of in vitro pharmacology, it was shown that 5- hydroxytryptamine (5-HT), histamine and endothclin-1 (ET-1 ) arc all vasoconstrictors in early and late pregnancy, acting independently of the endothelium in late pregnancy. 5-HT was shown to mediate constriction of the human umbilical artery and vein by an action on 5-HT^ and 5-HTo receptors. This contraction was found to be significantly more potent in early pregnancy. Evidence for endothelial cells as a source of 5-HT, histamine and ET-1 was found only in late pregnancy and is presented with electron immunocytochemical localization of these substances in the endothelial cells of the human umbilical artery and vein. A physiological role for these substances is suggested since exogenous addition of these substances had an effect on isolated vessel preparations. ATP and a,y5-methylene ATP (a,y5-MeATP) induced vasconstriction in the human umbilical artery from early and late pregnancy, acting independently of the endothelium in late pregnancy. 2-Methylthio ATP (2-MeSATP) and ATP did not initiate vasodilatation of the umbilical artery or vein from either early or late pregnancy. Receptor binding studies and autoradiography have identified the presence of P 2X"purinoceptors on the smooth muscle of umbilical arteries and veins from early and late pregnancy.
The results reported here contribute to the understanding of the local control
mechanisms regulating feto-placental vascular tone. Of particular interest is the fact
that umbilical endothelial cells do not directly modify blood flow throughout
gestation, but substances shown to be localised in umbilical endothelial cells may be
released to act in an autocrine/paracrine manner. ACKNOWLEDGEMENTS
I would like to thank my supervisor, Professor Geoffrey Bumstock, for his advice and encouragement during this studentship and in the preparation of this thesis.
I wish to thank all those who have collaborated in this work; in particular, the excellent nursing staff of the Labour Suite, University College London, for never tiring of my persistence; Dr. L. Wong from the MRC Tissue Bank, Hammersmith
Hospital for help in obtaining cords; Dr. John Kingdom for help in obtaining cords, as well as advice and friendship; Doreen Bailey and Dr. P. Bodin for their continual help and sharing of experimental tissue; Mark Turmaine, for carrying out the electron microscopy in Chapter 3; Dr. A. Loesch who performed the electron immunocytochemistry in Chapters 4 and 5; Dr. X. Bo and Dr. M. Zhao for the autoradiography in Chapter 6 ; Jane Pendjiky and Chris Syms for the excellent photographic assistance; Dr. D. Christie for all her editorial assistance; 1 would also like to thank my past and present colleagues, for help, friendship and encouragement over the last four years.
1 would like to thank my parents and grandparents for their endless support during my years as a student, and the friends who have shared the anxieties of the last four years with me. Finally, a special thank-you goes to Dr Gill Knight, for her never ending patience, time and friendship.
This work was supported by the Anatomical Society of Great Britain and
Ireland. CONTENTS
PAGE
Abstract ...... 2
Acknowledgements ...... 3
Contents ...... 5
List of Tables and Figures ...... 8
Publications Arising from this Thesis ...... 9
List of Abbreviations ...... 11
Preface ...... 13
Chapter 1 HISTORICAL INTRODUCTION
1.1 THE FETO-PLACENTAL CIRCULATION ...... 16
1.1.1 The discovery of the placental circulation ...... 16
1.1.2 Anatomy of the feto-placental circulation ...... 17
1.2 LOCAL CONTROL OF FETO-PLACENTAL
VASCULAR TONE ...... 24
1.2.1 Prostanoids ...... 26
1.2.2 Endothelium-derived relaxing factor ...... 28
1.2.3 Endothelin ...... 30
1.2.4 Atrial natriuretic peptide ...... 31
1.2.5 Renin-angiotensin system ...... 32
1.2.6 ...... 1.2.7 Arachidonic acid ...... 33
1.2.8 Purines ...... 34
1.3 ENDOTHELIUM AS A SOURCE OF VASOACTIVE
SUBSTANCES ...... 35
1.4 CLINICAL IMPLICATIONS ...... 36
Chapter 2 METHODS AND MATERIALS
2.1 COLLECTION OF TISSUE ...... 39
2.2 ELECTRON MICROSCOPY ...... 39
2.3 IN VITRO PHARMACOLOGY OF ISOLATED
BLOOD VESSELS ...... 40
2.3.1 Preparation of tissue ...... 40
2.3.2 Application of agonists ...... 40
2.3.3 Application of antagonists ...... 41
2.3.4 Analysis of results ...... 42
2.4 HISTOCHEMISTRY ...... 42
2.4.1 Endothelial cell studies ...... 42
2.4.2 Electron immunocytochemistry ...... 43
2.5 RADIOLIGAND BINDING STUDIES ...... 45
2.6 AUTORADIOGRAPHY ...... 45
2.6.1 Drugs ...... 46
2.6.2 Statistical analysis ...... 46
2.7 SOURCES OF DRUGS, PEPTIDES, ANTIBODIES
AND CHEMICALS ...... 47 EXPERIMENTAL RESULTS
Chapter 3 A Ultrastructural Study of the Developing
Human Umbilical Vessels ...... 49
Chapter 4 Electron Immunolabelling of Vasoactive Substances
in Human Umbilical Endothelial Cells and Their
Actions From Early and Late Pregnancy ...... 75
Chapter 5 Nitric Oxide and Human Umbilical Vessels:
Pharmacological and Immunohistochemical
Studies ...... 100
Chapter 6 Autoradiographic and Pharmacological Evidence for the
Presence of a P 2x~P*Jrinoceptor in Human
Umbilical Vessels from Early and Late
Pregnancy ...... 133
GENERAL DISCUSSION ...... 148
REFERENCES ...... 160 LIST OF TABLES AND FIGURES.
Page Page
Chapter 1 Chapter 5
Figure 1.1 . 23 Figure 5.1 109
Figure 5.2 . 111
Chapter 3 Figure 5.3 113
Figure 3.1 . 60 Figure 5.4 115
Figure 3.2 . 62 Figure 5.5 117
Figure 3.3 .. 64 Figure 5.6 119
Figure 3.4 . 66 Figure 5.7 121
Figure 3.5 .. 6K Figure 5.8 123
Figure 3.6 .. 70 Figure 5.9 125
Figure 5.10 127
Chapter 4
Figure 4.1 .. 83 Chapter 6
Figure 4.2 85 Figure 6.1 140
Figure 4.3 .. 87 Figure 6.2 142
Figure 4.4 .. 80 Figure 6.3 144
Figure 4.5 .. 91
Figure 4.6 .. 93
Figure 4.7 .. ...95 PUBLICATIONS ARISING FROM THESIS
Gai, W.Q., Bodin, P., Loesch, A., Sexton, A. & Bumstock, G. (1992) Localisation of
vasoactive substances in the endothelial cells of the human term umbilical blood
vessels. The Histochem. J. 24, 60.
Cai, W.Q., Bodin, P., Sexton, A., Loesch, A. & Bumstock, 0. (1993) Localisation of
neuropeptide Y and atrial natriuretic peptide in the endothelial cells of human
umbilical blood vessels. Cell Tiss. Res. 272, 175-181.
Cai, W.Q., Bodin, P., Loesch, A., Sexton, A. & Bumstock, G. (1993) Endothelium of
human umbilical blood vessels: ultrastructural immunolocalisation of
neuropeptides. J. Vase. Res. 30, 348-355.
Sexton, A.J. & Bumstock, G. (1993) Vasoconstrictor effects of 5-hydroxytryptamine,
histamine and endothelin on umbilical vessels from second and third trimesters of
human pregnancy. Placenta, 14, A70.
Sexton, A., Loesch, A., Minh, S., Turmaine, M. & Bumstock. G. (1995)
Nitric oxide and human umbilical vessels: pharmacological and
immunocytochemical studies. Placenta 16, 277-288.
Sexton, A.J., Loesch, A. & Bumstock, G. (1993) Pharmacological actions of various
vasoactive substances contained in the endothelial cells of human umbilical
vessels. 7. Anat. 184, 205. Sexton, A., Turmaine M., Cai, W.Q. & Bumstock G. (In press) A developmental
study of the human umbilical cord. J. Anat.
Sexton, A.J., Loesch, A., Minh, S., Turmaine, M. & Bumstock, G. (In press)
Pharmacological actions of various vasoactive substances contained in the
endothelial cells of human umbilical vessels. Cell Tiss. Res.
Sexton, A.J., Bo, X., Zhao, M., & Bumstock, G. (submitted) Autoradiographical and
pharmacological evidence for the presence of a ? 2X“Purinoceptor in human
umbilical vessels from early and late pregnancy. Euro. J. Pharmacol.
10 LIST OF ABBREVIATIONS
AA arachidonic acid
ACh acetylcholine
AChE acetylcholinesterase
ADP adenosine 5'-diphosphate
Ag angiotensin
AN? atrial natriuretic peptide
L-ARG L-arginine
ATP adenosine 5'-triphosphate
AVP arginine vasopressin
Bk bradykinin
ESA bovine serum albumin
CGRP calcitonin gene-related peptide
CRC concentration response curve
EC endothelial cell
EDCF endothelium-derived constrictor factor
EDRF endothelium-derived relaxing factor
EET eicosatrienoic acid
ELISA enzyme-linked immunosorbant assay
ER endoplasmic reticulum
ET endothelin
GMP guanosine monophosphate
HETE hydroxyeicosatetraenoic acid
5-H T 5-hydroxytryptamine/serotonin lEL internal elastic lamina lUGR intrauterine growth retardation
11 KCl potassium chloride
HUVECs human umbilical vein endothelial cells a,/?-MeATP a,^-methylene ATP y5,y-MeATP /?,y-methylene ATP
2-M eSATP 2-methylthio ATP mRNA messenger ribonucleic acid
L-NAME L-N^-nitroarginine methyl ester
NO nitric oxide
NOS nitric oxide synthase
NPY neuropeptide Y
OSO4 osmium tetroxide
PAP peroxidase-antiperoxidase
PBS phosphate buffered saline
PG prostaglandin
RER rough endoplasmic reticulum
S.E.M. standard error of the mean
SER smooth endoplasmic reticulum
SMC smooth muscle cell
SNP sodium nitroprusside
SP substance P
Tx thromboxane
VIP vasoactive intestinal polypeptide
12 PREFACE
The feto-placental circulation is essentially part of the fetal circulation, linking the fetus to the mother. It is the only means of transfer of substances essential for metabolism and nutrients and for the removal of the by-products of metabolism, since these functions are not fully established until birth. The placenta is, therefore, the main interface between the fetus and the outside world.
Since the discovery in 1980 by Furchgott and Zawadzki of the essential role played by the endothelium in mediating vasomotor relaxant responses to ACh, an increasing number of agents have been shown to exert their effects on vascular tone via the endothelium. More recently, the demonstration that some of these substances may be synthesised in the endothelium itself has highlighted the importance of endothelial cells as a source of vasoactive agents, introducing a new dimension to the concept of regulation of vascular tone.
The endothelium is especially important in the regulation of vascular tone in the feto-placental circulation, since the human umbilical cord and placenta lack innervation and are not subject to the reflex vasodilator and vasoconstrictor mechanisms which influence fetal body vascular tone via the autonomic nervous system. Therefore the endothelium may act in an autocrine/paracrine manner to regulate vascular tone. Up until 20 weeks of gestation there is very little difference in the development of a normal healthy fetus and placenta and a fetus from a pregnancy that is complicated by, for example, pre-eclampsia or intrauterine growth retardation
(lUGR). Since vascular impedance continually decreases with advancing gestation, it is therefore important to study all available fetal vascular tissue throughout gestation, since in normal pregnancy vascular tone is continually changing.
13 The aim of this thesis is to examine aspects of local regulation of vascular tone with particular emphasis on the influence of the endothelium. The possibility that endothelial cells are a source of vasoactive agents is also investigated. Furthermore, since certain physiological and pathological conditions associated with pregnancy may alter vascular reactivity, this study aims in particular to investigate changes in the regulation of vascular tone that occur as a consequence of gestation.
The background to this work is given in the Historical Introduction (Chapter
1). After a description of the general methods employed (Chapter 2) the experimental results are presented. The experimental results are presented in four chapters. The first
(Chapter 3) looks at the structure of the unique human umbilical vessels throughout gestation. Chapters 4 & 5 look at the pharmacology of various vasodilators and vasoconstrictors to investigate the role of the endothelium of the umbilical vessels during early and late pregnancy. From the information obtained, the presence of these vasoactive substances in the endothelial cells of the umbilical arteries and vein from early and late pregnancy is examined. In Chapter 5, the pharmacology of 5-HT is also investigated on the umbilical vessels throughout gestation, using antagonists to see if there are any changes in receptor density. Chapter 6 examines the effect of ATP and some of its analogues on the human umbilical vessels from early and late pregnancy, and the autoradiographic localization of the P 2x~P’Jrinoceptor throughout gestation is investigated. The results of the experimental work are discussed at the end of each chapter and then assessed in a wider context in the General Discussion (Chapter 7).
14 Chapter 1
GENERAL INTRODUCTION
15 This chapter is a review of the literature relevant to this thesis, discussing the general features of the feto-placental circulation, its structure and its regulation. This chapter is divided into three main parts: 1.1 the feto-placental circulation; 1.2 local control of the feto-placental vascular tone; 1.3 endothelium as a source of vasoactive substances; 1.4 clinical implications.
1.1 THE FETO-PLACENTAL CIRCULATION
1.1.1 THE DISCOVERY OF THE PLACENTAL CIRCULATION
The "afterbirth" has been referred to as far back in ancient literature as the
Old Testament. The early Egyptians believed it to be the seat of the "external soul"
(Murray, 1930) and they treated it with a special respect. Long (1963) describes folklores associated with the placenta, umbilical cord and fetal membranes, discussing the traditions, associated beliefs and superstitions including those that have persisted into the present century. For example, some African tribes attach a special significance to the fate of the placenta and its bearing on the future welfare of the individual to whom it was attached. Neither the Greeks nor the Romans had a specific name for this organ. The term placenta was not used with its present connotation until it was introduced in 1559, by Realdus Columbus (1516-1559). He introduced the
Latin word for a circular cake as a simile in his description of the thick part of the human fetal membranes. The term was generally adopted as the technical name for the human organ and was later extended to include equivalent structures in the other mammalian species; eventually analogous structures were found in all other mammalian groups.
No detailed descriptions of the placenta were given until the early 16th century and then many of these proved to be incorrect. Following Harvey's (1578-
16 1615) discovery of the circulation of the blood, John Mayow (1643-1679) then demonstrated the transfer of particles from the maternal to the fetal circulation.
Lavoiser (1743-1794) suggested this was air or oxygen. William Hunter (1743) was the first investigator to give a detailed description of the human placenta describing the existence of two distinct circulations within the placenta and the uterine lining, which he called the decidua (see de Witt, 1959). John Hunter (1786) described for the first time the intervillous space, although the term was introduced at a later date
(Comer, 1963).
Knowledge of placental development is inextricably interwoven into the history of embryology (de Witt, 1959; Needham, 1959; Corner, 1963). The understanding of the development and nature of the placental villi took place over many years. The true nature of the placental villi were first described in a paper published in 1842 by Dalrymple. By 1880, the basic knowledge of the circulation of the intervillous space had been established. However, two other important aspects of placental structure were finally clarified in the late 19th century. The first was the demonstration of the exact nature of the membranes covering the chorionic villi, and secondly, the basis for placental circulation (see Boyd & Hamilton, 1970).
1.1.2. ANATOMY OF THE FETO-PLACENTAL CIRCULATION
The umbilical cord.
As the tail of the embryo develops, the embryonic disc lies on the ventral
(anterior) aspect of the embryo, and the body stalk is now next to the remains of the yolk sac. The amniotic cavity expands within the extra-embryonic coelom and the amnion and chorion fuse, which results in the amnion enclosing the body stalk and yolk sac with their vessels to form the tubular umbilical cord. The space bound by the
17 junction of the amnion with the anterior body wall of the embryo will become the umbilicus. The mesenchymal core of the cord is converted into a loose connective tissue called Whartons Jelly and embedded in this are the remains of the yolk sac and allantois, the vittelline duct and umbilical vessels. The umbilical cord is a twisted, tortuous structure, which increases in length until the end of pregnancy. The umbilical vessels of humans usually consist of two arteries showing thick muscular walls without an elastic interna or externa, but they do have fibrils diffusely scattered throughout the muscular wall. They carry deoxygenated blood from the fetus to the placenta. In the adult these are represented by the "obliterated umbilical arteries" which are branches of the internal illiac arteries. Two umbilical veins convey oxygenated blood from the placenta to the fetus, one of which, the right vein, disappears at an early stage of development. In the adult, the left vein is represented by the ''li^cimcntum teres", which runs from the umbilicus to join the left branch of the cord. Close to the fetus remains a small part of the extra-embryonic coelom, this will be used to accommodate the developing gut, which will be later withdrawn into the abdominal cavity. The human umbilical vessels are unique in that there are some anatomical peculiarities not seen in other blood vessels including spiral twisting of the vessels; valves of Hoboken; knots in the cord; and the lack of a nerve supply.
i) Innervation of the umbilical cord.
The presence or absence of nerve fibres in the human umbilical cord has been debated for many years and as early as 1825 Home observed nerve bundles in various mammalian umbilical cords. There are other nineteenth century accounts alleging the presence of such bundles and much later there has been further descriptions of nerves in the cord, placenta and chorionic villi. In 1946, Spivak drew attention to a number of investigators who described nerve fibres distributed to the extra-abdominal portion of the umbilical vessels. She was unable, however, to identify any nerve fibres herself, but she concluded that there was a rich innervation
18 of the intra-abdominal vessels. Later, Jacobson & Chap 1er (1967) confirmed the presence of cholinergic neuronal elements in the placenta of human and monkey using methylene blue. Also, using methylene blue. Fox & Jacobson (1969) demonstrated nerves in the human cords of both term babies and those from first trimester therapeutic terminations. Ehinger et al. (1968) and Boyd & Hamilton (1970), however, have also failed to demonstrate evidence of extra-abdominal innervation, except for a few nerve fibres and nerve bundles ending blindly in the first centimetre of the cord. Pearson & Sauter (1970) traced nerve fibres along the umbilical artery into the umbilical cord, with the richest innervation within first few inches of the cord and then rapidly decreasing in number into the cord. Ellison (1971) showed acetylcholine esterase (AChE) positive nerves in rat umbilical cords after 21 days gestation and full term human umbilical cords revealed extrafetal AChE positive nerves. Nadkarni (1970) was unable to demonstrate nervous tissue in sections examined at the light microscopic level, whereas, at the electron microscope level he showed a myelinated nerve structure within the smooth muscle cells of extrafetal segment of the umbilical artery. Reilly & Russell (1977) have also failed to demonstrate preganglionic and postganglionic sympathetic and parasympathetic nerves in the human placenta and umbilical cord.
More recently, Baljet & Drukker (1981) demonstrated nerve plexuses extending along the blood vessels of the umbilical cord and urachus up to 30mm from the umbilicus. Matsubara & Tamada (1988) positively stained large bundles of AChE nerve fibres in the fetal side (approximately 10 cm) of the umbilical cord. No AChE fibres were found on the maternal side of the cord, placenta or fetal membranes.
Kawano & Mori (1989) showed that catecholaminergic fibres occurred chiefly in the interarterial and surrounding region of the umbilical arteries, longitudinally running catecholaminergic fibres forming a periarterial plexus near the umbilical ring was also
19 described and in 1990 they demonstrated adrenergic nerve fibres in the region surrounding the umbilical artery at the fetal end of the cord; the umbilical vein had no adrenergic innervation. Fox & Khong (1990) using monoclonal and polyclonal antibodies to neuron-associated markers have also failed to demonstrate nerve fibres in human umbilical cords examined throughout gestation.
The placenta.
The vessels of the umbilical cord continue onto the fetal surface of the chorionic plate. Exact central insertion of the umbilical cord is usually the exception rather than the rule. Most umbilical cords are paracentrally inserted and some are inserted at the periphery (battledore insertion). Placental circulation starts as the umbilical arteries anastomose and divide in a dichotomous manner across the surface of the chorionic plate of the placenta. Chorionic plate arteries, like the umbilical arteries, are highly muscularised, with vertical branches penetrating the chorionic plate. Together with a corresponding vein, each artery forms the central part of the villous tree or truncus chorii (Boyd & Hamilton, 1970), dividing many times to form thousands of ramuli chorii (Leiser et al., 1991). The placenta itself is formed of syncytiotrophoblast which becomes vacuolated as it enlarges forming a lacunar system engulfing maternal capillaries in its tracks. Radial outgrowths of cytotrophoblast cells then invade the syncytiotrophoblast to form anchoring primary villi and the trophoblastic shell (Hertig & Rock, 1945). This controlled invasion is precisely regulated. During the first trimester invasive cytotrophoblast cell line increases its expression of type IV collagen and receptors for fibronectin, laminin and collagen. With advancing gestation, these enzymes and receptors are down regulated, thus modifying the invasive process (Lim et a i, 1993). By the 14th day after conception, the extraembryonic mesenchyme derived from the embryonic disc
(Luckett & Beier, 1978)j invades the cytotrophoblast forming secondary villi. The latter are vascularised from within (Demir et a i, 1989) and receive blood from the
20 developing fetal circulation from the 6 th week of gestation. At this stage, there is no
formal intervillous circulation (Burchill, 1967; Schaaps & Hustin, 1988) and the
developing placenta is therefore effectively a barrier between the mother and fetus.
The developing placenta and embryonic tissues are hypoxic relative to the mother
(Hustin & Schaaps, 1987). The subsequent perfusion of the intervillous space, once
maternal spiral arterioles are breached around 10-12 weeks following fertilisation
(Hustin et al., 1988) establishes the usual haemochorial arrangement between the
maternal and fetal blood. Oxygen tension in the placenta then appears to match more
closely that of the maternal blood. By 18-20 weeks, the structure of the placental villi
changes. Both the number of capillaries within each villous and the volume of each
villous occupied by capillaries rises dramatically (Jauniaux et a!., 1991; Jackson et al.. 1992) thus increasing oxygen diffusion conductance (Mayhew et al., 1993).
Maturation of the placental vasculature is necessary to cope with the
metabolic activities of the rapidly growing fetus. This is achieved by two
mechanisms; 1) development of the low resistance uteroplacental circulation by
trophoblastic invasion of maternal spiral arteries (Brosens et al., 1967) and 2) through
growth and maturation of the fetal-placental vascular tree (Jackson et al., 1992).
The functional unit of the placenta is the placentome, a villous tree which
arises from a penetrating branch of the chorionic artery and which is suspended in the
intervillous space and perfused by a central spiral arteriole. The branching
arrangement of the villi and the vessels within these is complex but important
(Castellucci et al., 1989; Leiser et al., 1991) and is illustrated in Figure 1.
21 Figure 1.1
(a, b) Diagrammatic representation of the branching patterns of the villous tree. ChP
= chorionic plate; BP = basal plate; T = truncus chorii; I-IV = four generations of rami; 1- 11= eleven generations of ramuli. The vessels are shown within the villous tree, (c) Fetal vessels architecture of an ending stem villous of the last ramulus
(hatched line) and a terminal convolute composed of one mature intermediate villus
(slightly point shaded) together with its terminal villi (unshaded). Arterioles are point shaded, capillaries are black and venules unshaded. Note the long capillary loops supplying several terminal villi in series.
Figure 1.1 is reprinted with permission of Dr. J.C.P. Kingdom
22
The chorionic artery penetrates through the chorionic plate into the truncus chorii, this divides to produce about 4 divisions of rami chorii, each of which divides dichotomously to form a further 3-30 generations of ramuli chorii. Rami and ramuli chorii represent the stem villi which are characterised by the presence of fetal arteries and veins and of fetal arterioles and venules. In the preterm placenta, the smallest ramuli chorii give rise to immediate intermediate villi. With maturation of the placenta, however, mature intermediate villi are formed in preference. From these arc formed the terminal villi, the final ramifications of the villous tree and the sites of maximal feto-maternal exchange.
1.2 LOCAL CONTROL OF FETO-PLACENTAL VASCULAR TONE
Normal increases in blood flow are achieved in both circulations of the placenta by a combination of altered vascular anatomy and in both vascular resistance and perfusion pressure, which depend on systemic cardiovascular changes. In normal pregnancy, there is a progressive increase in umbilical end diastolic velocity and thus a steady fall in resistance in the fetal-placental circulation (Erskine & Ritchie, 1985).
A number of groups (Davignon ct al., 1965; Somlyo et aL, 1965; Gokhale et al.,
1966; Eltherington, e ta l, 1968; Lewis, 1968; Altura et al., 1972) have studied the reactivity of umbilical arteries, using a variety of methods; perfused artery and perfused placenta (von Euler, 1938; Eiliasson & Âstrôm, 1955; Âstrôm & Samelius,
1957; Panigel, 1962), perfused cord (Gokhale et al., 1966), isolated artery (Davignon et al., 1965; Lewis, 1968) and with very little agreement between groups and even within a set of experiments. The main point of interest in the earliest studies was the effect of various drugs on the umbilical vasculature, von Euler (1938) showed adrenaline to act as a vasoconstrictor, Eiliasson and Âstrôm (1955) showed both vasoconstriction and vasodilatation, whereas Gokhale et al. (1966) said it effected all or some preparations. This also applied to ACh. The effect of drugs on the human
24 umbilical artery using isotonic contraction (Somylo et aL, 1965; Eltherington et aL,
1968) again show little agreement. Eltherington et al. (1968) using the perfused artery preparation found bradykinin (Bk) to be fifteen times more contractile than 5-HT, whilst Altura et al. (1972), using strips found that 5-HT was more potent than Bk. At the time when these studies where carried out, the importance of the endothelium in modulating the action of the smooth muscle had not been realised and no mention is made by these workers on the preservation of the endothelium. Since the human fetal-placental circulation is unusual in that it lacks innervation (Spivak, 1943; Reilly
& Russell. 1977; Fox & Khong, 1990), vascular resistance must therefore depend on local physiochemical factors or on vasoactive substances produced or conveyed through the blood stream.
The endothelium is a continuous monolayer of cells covering the inner surface of all blood vessels. Their anatomical position between the circulating blood and the vascular smooth muscle makes it appropriate that they should play an important role in the control of vascular smooth muscle tone. They have a variety of functions including, prevention of the adherence of platelets, leukocytes and monocytes, the production of factors involved in blood clotting, capillary transport and exchanges between blood and tissue, activation (conversion of angiotensin (Ag) I to Ag II by endogenous Ag converting enzyme) and inactivation (degradation of noradrenaline (NA), 5-HT, Bk and adenosine 5'-diphosphate (ADP)) of circulating hormones and other plasma constituents, synthesis and secretion of vasodilator
(endothelium-derived relaxing factor(s) (EDRF), prostacyclin (PGI 2)) and vasoconstrictor substances (endothelium-derived constricting factor(s) (EDCF), prostanoids, Ag, histamine) (Vane et al., 1987; Rubanyi, 1991; Ryan & Rubanyi,
1992). Endothelial cells of the human umbilical vessels have been shown to produce the eicosanoids PGI 2 and thromboxane (TXA 2) (Tuvemo etal., 1976; Tuvemo, 1980;
25 Bjcro, 19X6). Van de Voorde ct a/.(1987) and Chaudhuri et al. (1991) have shown the release of EDRF/nitric oxide (NO) in human umbilical vessels. More recently binding sites for ET-1 were demonstrated in membranes prepared from whole human placenta
(Nakajo et al., 1989) and of stem villous vessels (Mondon et al., 1990).
1.2.1 PROSTANOIDS
Arachidonic acid (AA) and its metabolites are extensively located within
endothelial cells and have been implicated in both endothelial-dependent contraction
and endothelial-dependent relaxation (for reviews see Vane et al., 1990; Ralevic &
Burnstock, 1993). The human umbilical artery has been shown to synthesise several
prostaglandins (Mitsuhashi & Kuwabara, 1994), and both the human umbilical artery
and vein release PGIo, TxAo, prostaglandin Eg (PGEg) and prostaglandin p 2^
(PGFg^) (Tuvemo et al., 1976, Tuvemo, 1980, Bj0ro, 1986). The amount of PGI 2 produced by human umbilical arteries and veins (Benedetto et al., 1987) is much
lower than other vessels (Kawano & Mori, 1983; Benedetto et al., 1985). A gradient
in production of PGI 2 by umbilical vessels exists, such that there is a higher production by umbilical arteries and veins close to the fetus compared with vessel
rings taken close to the placenta (Benedetto, et al., 1987). This may reflect progressive changes in the properties of the endothelium. The umbilical artery and vein have an increased rate of PGI 2 production compared with placental vessels
(Kawano & Mori, 1983). The rate of 6-keto-PGF^:TxA 2 is constant in the umbilical vessels but the ratio is four times greater in the umbilical artery than the mammary vessels (Benedetto, et al., 1985). This may reflect an antithrombotic mechanism, important for fetal development.
The role of PGM in mediating local placental vasodilatation has been questioned, since inhibition of its synthesis has no effect on the perfusion pressure in
26 dually perfused human placental cotyledons (Jacobson ct ai, 1991) or isolated umbilical artery (Templeton ct al., 1991a). In the isolated cotyledon in vitro, Myatt et at. (1992a) were unable to show vasodilatation to prostaglandin (PGEj^), PGE 2 and PGIg suggesting that the resting preparation is maximally dilated. Whereas,
Glance ct al. (1986) were able to see weak vasoconstrictions to prostaglandin D 2
(PGDo) and PGP 2^. U46619, a Tx mimetic is also a potent vasoconstrictor (Mak et al., 1984; Howard ct al., 1986). Tx receptors have been described in placental tissue
(Hedberg ct al., 1989). When the fetal-placental circulation was constricted in vitro with a bolus injection of Ag II (0.5yWg), PGD 2, PGE^ and the PGI2 analogue were able to cause vasodilatation in a concentration-dependent manner (5 ng - 50 yUg). The rank order of potency suggests a they may all be acting through a PGI 2 receptor
(Glance ct al.. 1986). During Ag II vasoconstriction there is an increase in the release of PGlo and a PGE-like substance, whereas the release of Tx and PGE 2 was unchanged (Glance ct al., 1985). Inhibition of prostaglandin synthesis with indomethacin (1 /xM) caused a potentiation of Ag II vasoconstriction (Glance ct al.,
1985), but infusion of indomethacin did not affect resting pressure in the perfused cotyledon suggesting that eicosanoid synthesis is not the major determinant of resting tone (Glance ct al., 1985). The relative potency suggests that Tx is a major vasoconstrictor of the placental circulation. PGIo appears to be acting to attenuate the actions of the vasoconstrictors. The umbilical artery and umbilical vein appear to have a greater capacity to produce PGM than the chorionic plate arteries and veins suggesting PGM has a greater role in the umbilical vessels (Kawano & Mori, 1983).
Locally within the placenta, the trophoblast produces Tx and 5-HT, each of which constricts the feto-placental circulation in vitro (Mak ctal., 1984)
Changes in the p 0 2 and pCO^ may affect umbilical-placental blood flow, leading to the differential release of PGM and TxAo. Isolated human umbilical vessel
27 preparations contract when exposed to increasing oxygen tensions (16-120 mmHg)
(Templeton ct al., 1991b). Tx appears to mediate enhancement of the contractile effects of low concentrations of 5-HT by high oxygen tensions in segments of umbilical artery, but not umbilical veins, constricted by an increase in external oxygen tension, which were inhibited by indomethacin (McGrath et al., 1986), supporting the hypothesis that the occlusion of the umbilical cord at birth by high oxygen tensions (Oberhansli-Weiss ct al., 1972) is mediated via the release of eicosanoids (McGrath et al., 1986). However, oxygen-induced contractions have not been reported in the villous vasculature. Acute hypoxia is reported to cause reversible vasoconstriction in the placental cotyledon perfused in vitro, and was prevented by lipoxygenase inhibitors, suggesting a mechanism involving the release of lipoxygenase product(s) (Howard et al., 1987). In vivo animal models gave only a small rise in umbilical vascular resistance at very high pCOn levels. Whereas, extremely high concentrations of adrenergic and cholinergic agents are necessary to
I effect the vessel tone (Myatt, 1993^. This suggests that there is little or no effect, or -
that the effect of these agents in vivo may be secondary to changes in systemic
pressure or heart rate.
1.2.2. ENDOTHELIUM-DERIVED RELAXING FACTOR
In 1980, Furchgott & Zwadazki demonstrated that the relaxation of arterial vascular smooth muscle by ACh is dependent on the presence of an intact endothelium. Since then, many additional substances have been shown to produce endothelium-dependent relaxation of arterial preparations. It was suggested that the activation of specific receptors on the endothelium induced the release of an EDRF(s) which acts on the smooth muscle to cause relaxation, now known to be NO (Palmer et al., 1987), generated from L-Arg (L-arginine) (Palmer et al., 1988) in the endothelial cells (for reviews see Calver et al., 1993; Moncada & Higgs, 1993; Ralevic &
Burnstock, 1993).
28 Van de Voorde et al. (1987) and Chaudhuri et al. (1991) have both demonstrated the release of EDRF from human umbilical vessels. Van de Voorde et al.
(1987) showed the release of EDRF from human umbilical arteries, veins and from endothelial cells in culture by the calcium ionophore A23187 and histamine, but not by ACh. However, they did not observe a relaxant effect with histamine on umbilical preparations mounted for isometric tension-readings in the presence and absence of indomethacin and preconstricted with 5-HT. Izumi et al. (1994), using the same cascade bioassay technique as Van de Voorde et al. (1987), found the amount of
EDRF and the sensitivity of smooth muscle to EDRF decreases with the progression of gestation. It has been reported that in endothelium intact human umbilical vessels, histamine and Bk both cause a minor or transient relaxation followed by a large increase in tension (Clymann, 1957; Gokhale et al., 1966; Myatt et al., 1991a; Pinto er al., 1991 ). Histamine has been shown to increase the cellular concentration of cyclic guanosine monophosphate (cGMP) (Clymann, 1957) whilst producing a large contraction of the human umbilical artery. Despite many groups finding a lack of cndothelial-mediated relaxation in human umbilical vessels, the NO donors, glycerol trinitrate and .9-nitrosoacetyl penicillamine are able to relax the human umbilical artery (Chaudhuri et al., 1991). These results suggest that possibly the human umbilical vessels produce less EDRF, or are less sensitive than other vessels to the action of EDRF, or that cGMP accumulation is associated with contraction rather than relaxation in the umbilical vessels at term. Using the cascade bioassay system, first described by Vane (1964), Chaudhuri et al. (1991) were able to characterise the properties of human umbilical EDRF. NOS has now been located by immunocytochemistry both in the feto-placental vascular endothelium and also in the villous syncytiotrophoblast (Myatt et al., 1993a; Buttery et al., 1994), and identified as the constitutive calcium-dependent isoform of NOS, corresponding to the type III calcium-calmodulin dependent endothelial isoform (Conrad et al., 1993; Myatt et al..
29 1993b). Conrad et al. (1993) have found that the syncytiotrophoblast expresses mRNA for NOS. The regulation of EDRF production within the placenta in vivo is not understood at present but mechanical stretch of endothelial cells and vasoconstrictor mechanisms are likely to be important factors.
1.2.3. ENDOTHELIN
In addition to the release of EDRF(s), the endothelium has been shown to mediate vasoconstriction via the production of an EDCF(s) (Rubanyi, 1988).
Although the nature of EDCF(s) appears to be different in different blood vessels, at least three classes of endothelial vasoconstrictor substances have been recognised:
metabolites of AA oxygen-derived free radicals; a diffusible factor released from anoxic/hypoxic endothelial cells; and ET. ET is a 21 amino acid peptide (Yanagisawa ct a!., 1988) with three isotypcs (Inoue et al., 1989), produced from prepropeptides
(D'Orlcans-Juste et al., 1990). ET causes a slow-developing, long lasting vasoconstriction in almost all arteries and veins (Yanagisawa et al., 1988). This suggests that ET is likely to be a local rather than systemic regulating factor (for reviews see Masaki, 1989; Yanagisawa & Masaki, 1989; Ralevic & Burnstock, 1993).
The placenta is a rich source of ET receptors (Fischli et al., 1989). The receptor for ET-1 has been characterised in homogenates of isolated tissue from the human placenta (Fischli et al., 1989; Nakajo et al., 1989; Wada et al., 1990; Svane et al., 1993), and more recently in membrane preparations of both stem villous vessels and chorionic plate vessels (Robaut et al., 1991), thus making the placenta a potent target tissue. Kilpatrick et al. (1993) has demonstrated that the ET receptor concentration is highest in the first trimester placenta and decreases progressively towards term. Several groups have shown ET-1 to be a potent vasoconstrictor of the human placental circulation (Wilkes et al., 1990; Myatt et al., 1991b), producing the
30 characteristically long-acting vasoconstriction, which can be attenuated by NO
(Myatt et al., 1992b). ET-3 also constricts the perfused human placenta, but is less potent than ET-1, despite its maximal response being greater (Gude et at., 1991;
Myatt et al., 1992c). The mRNA for preproendotheiin-1 and preproendothelin-3
(Samson et al., 1990; Benigni et al., 1991) have been found in the human placenta as well as the mRNA for ET-1 and ET-3 (Myatt et al., 1992c) and Myatt et al. (1992c) have shown the presence of the ET-converting enzyme in the placenta. The human placenta appears to produce equivalent amounts of big ET-1, ET-1 and ET-3, but less E T -2 (Benigni et al., 1991). Release of ET-1 from human umbilical vein endothelial cells (HUVECs) has also been demonstrated (Clozel et al., 1989; Fischli et al., 1989). Localisation of ET in the vessels and the syncytiotrophoblast of the villi of the human term placenta has been demonstrated, as well as in the decidua and the extra villous cytotrophoblast of the basal plate (Malassinc et al., 1993) and in cultured trophoblast cells. All these results strongly suggest a role for ET's in the placental circulation, but no physiological stimuli that releases ET's has been described in this system (Kingdom etal., 1993).
1.2.4. ATRIAL NATRIURETIC PEPTIDE
Specific receptor sites for atrial natriuretic peptide (ANP) have been characterised in placental stem vessels (Salas et al., 1991; McQueen et al., 1993).
However, ANP does not appear to be synthesised within the placenta (Inglis et al.,
1993) suggesting that endocrine activity emanates from peptide released into the circulation from the fetal heart (Wharton et al., 1985). However, Cai et al. (1993a) recently showed that human umbilical vein endothelial cells contain ANP mRNA, implying synthesis of the peptide. Umbilical cord levels of ANP are higher than corresponding maternal levels and are in the range that would stimulate smooth
31 muscle contraction (Kingdom et al., 1992). A similar concentration of ANP has been shown to antagonise the vasoconstrictor effects of Ag II in the perfused cotyledon
(McQueen et a i, 1991) suggesting that it has a role in promoting smooth muscle relaxation under resting conditions. ANP levels increase following intravascular transfusion suggesting that ANP-mediated vasodilatation may increase to assist with blood volume expansion (Kingdom et al., 1991). Also other peptides, such as parathyroid hormone, a peptide related to ANP may have endocrine or paracrine actions to regulate smooth muscle tone in the placenta (Mansager et al., 1993) and the
umbilical cord (Ferguson et al., 1994).
1.2.5. RENIN-ANGIOTENSIN SYSTEM
Both the umbilical vessels and villous tree of the placenta are constricted by components of the renin-angiotcnsin system. Using the isolated dually perfused single cotyledon preparation it has been shown that Ag I, Ag II and Ag III all cause a concentration-dependent constriction of the fetal-placental vasculature (Glance et al.,
1984). Inhibition of the conversion of Ag I to Ag II, implies that the converting enzyme is present in the placental vasculature. The actions of all three Ag's can be inhibited in vitro by using a Ag II receptor antagonist suggesting that they all act through the Ag II receptor. High affinity Ag II receptors have been characterised in placental tissue (Wilkes et al., 1985) and placental resistance arteries (McQueen et al.,
1990). Tulenko (1979) has shown that Ag II is more potent on vascular segments of resistance vessels of the villous tree than on the chorionic plate or umbilical vessels in vitro and was more potent than 5-HT. This has also been observed in the perfused cotyledon by Abramovich et al. (1983) and Mak et al. (1984). 5-H T is more potent on umbilical or chorionic plate vessels than on the villous stem vessels (Tulenko,
1979; Mak et al., 1984; MacLean et al., 1992) suggesting that 5-HT may have a major role in occlusion of the cord at birth. Prentice et al. (1987) has shown that the
32 majority of 5-HT is destroyed by the time it reaches the cotyledonary circulation. Renin and thus Ag II are produced in the developing kidney indicating an endocrine mechanism of action within the feto-placental circulation (Graham et ai, 1992).
1.2.6. BRADYKININ
Bk is a systemic vasodilator, but is a vasoconstrictor of umbilical arteries and umbilical veins in vitro with a greater potency than Ag II (Mak et al., 1984; White,
1989). In the perfused cotyledon Bk is a vasoconstrictor, but is less potent than Tx
(Wilkes & Mento, 1988), probably due to the rapid inactivation of Bk by the angiotensin converting enzyme (kinase II) in this circulation (Prentice et al., 1987).
Al-Obaid ct al. (1993) has suggested that Bk-induced contractions of the human umbilical artery are not attributable to either Bk| or Bko receptor activation, nor are the contractions likely to be mediated by cyclo-oxygenase/lipo-oxygenase metabolites of AA..
1.2.7. ARACHIDONIC ACID
AA products of the lipoxygenase pathway, such as, leukotriene B 4 and the peptidyl leukotrienes C 4 and D 4 have been found to be vasoconstrictors of the human placental cotyledon in vitro (Thorp et al., 1992). Their potency is very low relative to that of Tx. In addition to formation of leukotrienes, AA metabolised by the lipoxygenase pathway can form a variety of hydroxyeicosatetraenoic acids (HETE's), which do not have an effect on the fetal-placental vasculature (Myatt, 1992a). The eicosatrienoic acids (EET's), products of the arachidonic pathway do not have a vasoactive effect on this system, whereas they have been shown to be potent vasodilators in other systems (Fitzpatick & Murphy, 1988).
33 1.2.8. PURINES
The cardiovascular effects of purines were first reported by Drury and Szent-
Gyorgyi (1929), and subsequently have been found to have dual effects on a wide variety of vascular beds; ATP produces vasoconstriction via on the vascular smooth muscle and vasodilation via P 2Y”Purinoceptor on endothelial cells (leading to the release of NO) or directly on the smooth muscle of some vessels.
Adenosine produces vasodilation by acting directly on smooth muscle purinoceptors (for review see Burnstock, 1990, 1993). While adenosine vasodilation has been demonstrated in many vascular beds, there is little indication that it
\ asodilates the intact human placental vascular bed. In the human umbilical artery,
Leuotsakos ct al. (1989) found that adenosine and some of its agonists elicited relaxation in only some of the vessels examined, however, in the sheep it has been shown that the placental vasculature responds to systemic adenosine infusion in a biphasic manner; the immediate response to adenosine is a transient vasconstriction followed by a long lasting vasodilation. Membrane receptors which bind adenosine
have been characterised in the human placental tissue (Fox & Kurpis, 1983; Schockcn &
Schneider, 1986) and Revivego ct al. (1990) has suggested that similar populations of
A |- and Ag-receptors exist in the chorionic vessels of the human placenta, mediating relaxation by a cyclic adenosine monophosphate (cAMP)-independent mechanism. In the human placental circulation, adenosine has been associated with vasoconstriction during hypoxia (Kitagawa ct a!., 1987), since the response was blocked by the
adenosine antagonist theophylline (Howard et at., 1987; Slegal et at., 1988).
ATP has been shown to have a contractile effect in the dually perfused human cotyledon. Dobronyiet al. (1992) has suggested thatATP elicits vasodilatation via
P2Y“P*Jrinoceptor-mediatedNO release and vasoconstriction via the actionP2X” at
34 purinoccptor in the feto-placental bed. Read et al. (1993a) have also suggested that
ATP vasodilatation via the Poy-purinoceptor in the human placenta is endothelium- dependent and linked to the formation of NO. They have also suggested that the vasodilation caused by the ATP may mask an accompanying vasoconstriction effect mediated via a Po^-purinoceptor in the villous vascular smooth muscle. Bax
(1992) found that the pattern of response in cultured human trophoblast cells was typical of a Pg-purinoceptor rather than a P^-purinoceptor.
1.3 ENDOTHELIUM AS A SOURCE OF VASOACTIVE SUBSTANCES
There arc various possibilities as to the source of vasoactive substances.
Hormones, catecholamines, rcnin-Ag, and vasopressin are all present in the circulation, whilst others may be produced locally, such as, thrombin, AA, ATP, ADP and 5-HT from platelets. It is unlikely that transmitters released from periarterial nerves act on endothelial receptors, at least in large vessels. For example, ACh would be partially degraded by AChE and ATP by Mg/Ca"^"^ activated ATPase and 5'- nucleotidase (Burnstock, 1985, 1987).
The first study to implicate the endothelium as a source of vasoactive substances was by Pamavelas et al. (1985). These workers demonstrated using electron immunocytochemical techniques the presence of the enzyme choline acetyl transferase (ChAT), the enzyme responsible for the conversion of ACh, inside the endothelial cells lining the capillaries and small blood vessels of the rat cortex. This method has since been used to show the presence of ChAT, SP, 5-HT, A VP and Ag II within the endothelial cells of a variety of blood vessels including femoral, mesenteric, renal, pulmonary and coronary vessels (Burnstock et al., 1988; Loesch
35 and Burnstock, 1988; Milner et al., 1989; Lincoln et al., 1990; Loesch et al., 1991;
Tomlinson et al., 1991). The presence of ANP mRNA in the endothelial cells of the umbilical artery (Cai et al., 1993a) has also been demonstrated, implying that the presence of ANP is due to intracellular synthesis rather than a result of uptake from the circulation.
Various studies have been addressed to determine under what physiological conditions these substances localised in endothelial cells may be released. 5-HT, SP,
ATP and ACh, have been shown to be released from the guinea-pig and rat heart during periods of hypoxia (sec Ralevic & Burnstock, 1993). Since ATP, 5-HT, SP and ACh have been shown to elicit coronary vasodilatation via specific receptors on the coronary endothelium (Cohen et al., 1983; Hopwood et al., 1986; Stewart et al.,
1987) it is likely that these substances are involved in mechanisms of hypoxic vasodilatation. It has also been demonstrated that during increased flow SP is released from the endothelial cells of the rat hind-limb (Ralevic et al., 1990) and from cultured endothelial cells (Lincoln & Burnstock, 1990). Various vascular beds release
ATP during the shear stress induced by increased flow (Milner et al., 1990a, 1990b).
Shear stress is also an important mechanism in regulating the release of peptides such as ET-1 and ANP. These studies suggest that physicochemical stimuli may cause release of vasoactive agents from endothelial cells to give rise to flow-induced dilatation, when increase blood flow is necessary to maintain vascular homeostasis.
1.5 CLINICAL IMPLICATIONS
The human placenta and umbilical cord are unique in that they lack innervation, so other mechanisms regulating vascular tone must exist. However, the human placenta is the organ least well understood by most pathologists. This is a
36 situation for which there are a number of good reasons; unlike other organs, the human placenta is almost never available for study in an intact state in its normal position in the body, and it is almost never available at all stages of gestation. The maternal vascular connections are extensively disrupted during delivery so that it is difficult to determine the important spatial relationships between maternal and fetal circulations when the placenta is received in the laboratory. As the human placenta differs radically in structure from that of the commonly available species (rats, rabbits, guinea pigs, sheep) there is no readily available experimental model for the study of placental pathology comparable to that of the human placenta.
The overlap which occurs between healthy babies bom small for gestational age (SGA) and babies with lUGR has hampered our understanding of the latter conditions. SGA is relatively easy to identify by biometry but reliably identifying the subgroup of fetuses which are also growth retarded may be difficult. During the first trimester, the umbilical circulation is characterised by absent end diastolic flow velocity (AEDFV), as detected by Doppler ultrasound. However by 20 weeks, end diastolic flow velocity is consistently present in the fetal umbilical circulation, which suggests a low distal impedance. This is a physiological event in early pregnancy, characteristic of normal pregnancy and intimately related to the development of the mature placenta. Some growth retarded fetuses demonstrate reduced or even reversed flow during! diastole(Pardi et al., 1993), and this is usually associated with perinatal morbidity and death (Burke et al., 1990; Fairlie et al., 1991). Therefore there is a need to understand the mechanisms regulating vascular tone of the normal feto-placental circulation before investigating the abnormal situation. The possibility that substances synthesised, stored and released from umbilical endothelial cells are involved in the physiology of the fetus also needs to be studied.
37 C hapter 2
METHODS AND MATERIALS
38 2.1 COLLECTION OF TISSUE
Human umbilical cords were obtained from legal terminations, 8-17 weeks
(early pregnancy group) from the MRC Tissue Bank, Royal Marsden Hospital and the
Elizabeth Garrett Anderson Hospital, and as soon as practically possible from normal term vaginal deliveries 38-41 weeks (late pregnancy group) from University College
Hospital and processed accordingly. Cords from term deliveries were clamped immediately after delivery.
2.2 ELECTRON MICROSCOPY
Sections of umbilical cords, 2 cm in length were taken from the placental up to the mid region of the cord and were fixed as soon as practically possible, for 2 h in a 4% glutaraldchyde, 2.5% paraformaldehyde in 0.1 M cacodylate buffer. The vessels were then carefully dissected out from the surrounding tissue, cut into 0.5 cm segments and fixed overnight at 4 °C. The vessels were subsequently postfixed in a
1% osmium tetroxide solution withO.l M cacodylate buffer for 1 hr; some of the samples were stained for 45 min in aqueous 2% uranyl acetate solution at 4 °C. All samples were then dehydrated using graded ethanol. The samples were then finally treated with propylene oxide and embedded in Araldite. Semi-thin sections (1 fjm thick) were cut using a diamond knife and stained with toluidine blue for observation under a light microscope. Ultrathin sections of the vessels in cross-section were then counterstained with uranyl acetate and lead citrate prior to examination under a JEOL
1010 electron microscope.
39 2.3 IN VITRO PHARMACOLOGY OF ISOLATED BLOOD VESSELS
2.3.1 PREPARATION OF TISSUE
Ring preparations of umbilical arteries and veins, approximately 5 mm in
length, were dissected free of surrounding tissue and mounted horizontally in 5 ml
organ baths containing a modified Kreb's solution of the following composition
(mM): NaCl 118, KCl 4.7, NaH 2P0 ^ 1.0, NaHCOg 25, Mg 2S0 ^ 1.2, glucose
11.1, CaCW 2.5, bovine serum albumin (BSA) 2.5 mgl“ ^, and bacitracin (1 000
units/ml), pH 7.4, continually aerated with 95% O 2 / 5% CO2 and maintained at 37
°C. The endothelium of some preparations was removed by gentle rubbing of the
intimai surface with a pair of serrated forceps (see section 2.4.1). Preparations were
mounted horizontally in the organ baths by inserting 2 tungsten wires through the
lumen of the vessel, attached to a rigid support and a Grass force displacement
transducer FTO 3C. Mechanical activity was displaced on a Grass ink-writing
oscillograph. An initial load of 2 g was applied and the tissues were allowed to
equilibrate for 2 h, resulting in an approximate tension of 1.5 g.
2.3.2 APPLICATION OF AGONISTS
(a) Application of Vasoconstrictor Agents
Vasoconstrictor agents were added to the organ bath in a cumulative manner
in order to ascertain the maximal contractile response, and the pD 2 value ( -log EC^q) (see below for EC 5Q definition).
(i) 5 -H T
A cumulative concentration-response curve (CRC) for 5-HT (10 nM - 30
^M) was established on each segment of umbilical artery and vein in order to
ascertain the maximal contractile response, the EC^q (the concentration of agonist
40 required to produce 50% of the maximal contraction) and the EC '75 (the concentration required to produce 75% maximal contraction).
(ii) Potassium chloride (KCl)
KCl (120 mM) was added to the organ bath. Contractions to other agents were expressed as a percentage of the KCl-induced contraction.
(b) Application of Vasodilator Agents
Before tissues were exposed to any vasodilator, they were contracted to approximately 75% of their maximal tension with 5-HT. After contractions reached a plateau, vasodilator agents were administered cumulatively and subsequent relaxations were expressed as a percentage of the initial tone.
(c) Application of peptides
Peptides were added to the organ bath as a single concentration , each at 30 min intervals. The peptides were washed out of the the Krebs' solution after the response had plateaued.
2.3.3 APPLICATION OF ANTAGONISTS
(i) 5-H T antagonists
Methysergide (10 ^M), ketanserin (10 ywM) and ondansertron (10 fjM) were equilibrated for 20 min prior to repeating dose-response curves to 5-HT.
41 (ii) o,l3-MeATP
a,/5-MeATP was used to desensitise P 2X”Purinoceptors. Total desensitisation was achieved by exposing the vessel to repeated applications of a f i -
MeATP (3 yuM), until no further contractile response was seen.
2.3.4 ANALYSIS OF RESULTS
Results were expressed as a mean ± standard error mean (S.B.M.). Results were analysed by paired or unpaired Student's f-test as appropriate and a probability
(P) of less than 0.05 was taken as significant.
2.4 HISTOCHEM ISTRY
2.4.1 ENDOTHELIAL CELL STUDIES
The endothelium of the umbilical artery and vein was removed by the gentle rubbing of the intimai surface with a pair of serrated forceps. To demonstrate the successful removal of the endothelium, rubbed and unrubbed preparations were opened longitudinally and stained with silver nitrate (AgNOg) as described by Caplan et al., (1974). The preparations were immersed successively in the dark at room temperature, in: (1) HEPES (20 mM) buffered (pH 7.4) solution containing 4.6% glucose for 150 s; (2) 0.4% AgNOg in 4.2% glucose solution for 60 s, and (3) 4.6% glucose solution for 60 s. The vessels were then fixed at room temperature in 0.1 M sodium cacodylate containing 7.5% sucrose and examined under the light microscope.
42 2.4.2 ELECTRON IMMUNOCYTOCHEMISTRY
(a) Pre-embedding Immunocytochemistry
Segments of umbilical cords, from the mid to placental region, were immersed into fixative containing 4% paraformaldehyde and 0.1% gluteraldehyde in
0.1 M phosphate buffer at pH 7.4 and stored overnight. The following day the umbilical arteries and veins were dissected free of surrounding tissue, washed in phosphate buffer and cut longitudinally to produce about 3-5 mm long strips. These were then processed for immunocytochemistry according to the peroxidase- antiperoxidase (PAP) method of Stemberger (1979) using the same steps of the procedure as previously reported (Loesch & Burnstock, 1993a; Loesch et al., 1993b,
1994). Initially, however, the strips were exposed to 0.3% HoOi in 30-50% methanol for 30 min (for the blocking of endogenous peroxidases). After immunoprocedure, the specimens were postfixed in 1% OsO^, dehydrated in a graded series of ethanol and embedded in Araldite. The ultrathin circumferential sections were stained with uranyl acetate and lead citrate and subsequently examined with a JEM-1010 transmission electron microscope. The rabbit NOS antiserum used in chapter 4 was used at a
1:1000 dilution. The specimens in chapter 5 were incubated for 48 h at 4 °C with rabbit polyclonal antibodies to: 5-HT (immunogen: 5-HT conjugated to BSA with paraformaldehyde); histamine (immunogen: histamine conjugated to BSA) and ET
(immunogen: synthetic human ET-1) at a dilution of 1:1000.
(b) Controls for NOS
The rabbit polyclonal NOS antiserum was raised against soluble NOS Type 1
(neuronal) purified from rat cerebellum and tested for specificity as described previously in detail (Schmidt et al., 1992). Anti-NOS 1 antibody reacted with a single
43 160 kDa protein band in rat cerebeila 105 000 g supernatant fractions. In all tissues examined (rat: cerebellum, kidney, pancreas, lung, uterus) the relative intensity of the cross-reacting protein band (150-165 kDa) coincided with NOS 1 activity (Schmidt etal., 1992). Preabsorption (22 h at 4 °C) of the NOS antiserum (diluted 1:1000) with soluble NOS Type 1 purified from rat cerebellum resulted in elimination of positive labelling in endothelial cells and perivascular nerves in the pre-embedding PAP immunoprocedure (Loesch & Burnstock, 1993a; Loesch et at., 1993b, 1994). The specificity of the immunolabelling was tested by omission of the NOS antiserum and anti-rabbit immunoglobulin G serum independently, as well as by using non-immune normal goat serum or non-immune normal rabbit serum (Nordic Immunology,
Tilberg, The Netherlands) at a dilution of 1:1000 instead of NOS antiserum. No labelling was observed in these control experiments.
(c) Controls for 5-HT, Histamine and ET-1
The specificity of the immunolabelling was tested by the omission of the 5-
HT, histamine and ET-1 antiserum and goat anti-rabbit immunoglobin G serum independently, as well as by using non-immune normal goat serum or non-immune normal rabbit serum at a dilution of 1:1000 instead of 5-HT, histamine and ET-1 antiserum. Preabsorption (overnight at 4 °C) of the antisera with their respective substances (5-HT, histamine and ET-1) prevented positive labelling. No labelling was observed in these control experiments.
44 The following protease inhibitors were used, 1 mM EGTA, 1 mM benzamidine hydrochloride, 0.1 mM phenylmethylsulphonyl fluoride, 0.001% (w/v) bacitracin and
0.002% (w/v) soybean tiy^psin inhibitor. 2.5 RADIOLIGAND BINDING ASSAY
Human umbilical artery and vein from late pregnancy were thawed at room
temperature, and dissected free of adventitia. They were minced and homogenised in
50 mM Tris-HCl buffer with several types of protease inhibitors (pH 7.4, 20 °C). The
homogcnate was passed through double layers of nylon mesh, centrifuged at 105 000
g for 50 min. The supernatant was discarded and the pellet was suspended in 50 mM
Tris-HCl buffer. Protein concentration was measured using the method of Lowry et
fl/. (1951) and BSA was used as a standard. For the saturation experiments, serial
concentrations of [^H]a,^-MeATP were used. The reaction mixture contained
[^H|a,/3-McATP, tissue sample and buffer to a total volume of 0.5 ml. Non-specific
binding was determined with 100 ^M /0,Y-methylene ATP (5,y-MeATP). The
incubation was carried out at 4 °C for 120 min. At the end of the incubation the
reaction was terminated by quick filtration of the mixture through Whatman GF/B
filters presoaked in 20 mM disodium pyrophosphate solution. The filters were washed
with two aliquots of 5 ml ice-cold Tris-HCl buffer. Each experiment was carried out
in duplicate. Preliminary kinetic experiments had been carried out to ensure that the
combination of the incubation time and temperature allowed the binding process to
reach equilibrium.
2.6 AUTORADIOGRAPHY
Cyrostat sections of human umbilical vessels were cut (14 /um) and thaw-
mounted onto acid-washed, gelatin-coated slides. Slide-mounted sections were
preincubated in 50 mM Tris-HCl buffer (pH 7.40) at 30 °C for 10 min to remove
endogenous ligands and embedding matrix. They were then transferred to 50 mM
Tris-HCl buffer (pH 7.40) containing 10 nM [^H]a,/?-MeATP and incubated at 30
°C for 15 min. Non-specific binding was determined in the presence of 100 ywM ^,y-
45 MeATP or 50 yuM j5,Y~MeATP and 50 juM 2-MeSATP. At the end of the incubation, the slides were washed in ice-cold Tris-HCl buffer and ice-cold double-distilled water. Sections were dried under cold airflow. The time and temperature for the incubation and the wash had been optimised in preliminary experiments. Emulsion- coated coverslips (Ilford K5, diluted 1:1 with distilled water)j were attached to the section-mounted slides and the assemblies were exposed for 3 weeks at 4 °C. The emulsion was developed and the sections were stained with 0.5% toluidine blue. The autoradiographs were examined under a photomicroscope and both the bright- and dark-field images of the same view were photographed for comparison.
2.6.1 Drugs
[^HI a,/?-Me ATP was synthesised by Amersham International (Amersham, UK).
Specific activity was 19.2 Ci/mmol with the chemical purity of 98-99%. y3,y-MeATP was purchased from Sigma Chemicals (Poole, UK)
2.6.1 Statistical Analysis
Vasoconstrictor responses are expressed as a % contraction of KCl (120 mM)
± standard error (s.e.) and statistical significance was determined using a unpaired student r-test. A probability of P<0.05 was taken as significant. The binding data were analysed with a computer programme RADLIG (Biosoft, Cambridge, UK). The choice of binding model was based on the results of an F test on the weighted residual sums of squares and standard errors (Munsen et al. 1980).
46 2.7 SOURCES OF DRUGS, PEPTIDES, ANTIBODIES AND CHEMICALS
acetylcholine chloride Sigma antibodies to ET-1 Cambridge Research Chemicals
histamine Biogenesis
5-H T INCSTAR
NOS Abbott Laboratories
L-ascorbic acid: Sigma
ATP, disodium salt: Sigma bacitracin: Sigma
BSA: Sigma cacodylate buffer: BDH dimethyl sulfoxide: Sigma epoxy resin (Araldite): TAAB UK
Hanks': Gibco
HEPES: Sigma histamine dihydrochloride: Sigma histo-clear: National Diagnostics hydrogen peroxide: BDH
5-HT, creatine sulphate: Sigma glutaraldehyde: BDH ketanserin: Sigma lead citrate: BDH a,/?-MeATP, lithium salt: Sigma
[3H]a,^-MeATP: Amersham
/?,y-MeATP: Sigma
2-MeSATP RBI methysergide Sandoz
47 p-nitrophenylphosphate Sigma ondansertron Glaxo osmium tetroxide Johnson Matthey Chemicals parafomialdehyde TAAB UK
- ET CRB
-SP CRB
- VIP CRB propylene oxide: BDH silver nitrate: BDH sodium azide: Sigma sodium cacodylate Sigma sodium nitroprusside: Sigma suramin Gift of ICI Ltd.
Tris-HCl bufffcr BDH uranyl acetate: Agar Scientific
All drugs were dissolved in distilled water except for: 5-HT - dissolved in 0.1 mM ascorbic acid and methysergide - dissolved in DMSO.
48 C hapter 3
A STUDY OF THE ULTRASTRUCTURE OF DEVELOPING
HUMAN UMBILICAL VESSELS
49 3.1 SUMMARY
1 Electron microscopic techniques were used to examine the ultrastructure of the developing human umbilical artery and vein (8-12, 13-17 and 37-40 weeks gestational age).
2 These studies show that with an increase in age there is: (1) an increase in the size of the lumen and the thickness of the media; ( 2) an increase in the ratio of contractile smooth muscle phenotypic cells; (3) an increase in the myofilament content of the smooth muscle cells and the number of Weibel-Palade bodies; (4) a decrease in the glycogen content; (5) an appearance of microvilli on the luminal surface of the endothelium. Lipid vesicles, nerves and vasa vasorum were not observed in any region of the umbilical vein or artery.
50 3.2 INTRODUCTION
Recognition of the importance of blood vessel endothelium in the control of vascular tone has greatly increased, since the demonstration by Furchgott & Zawadzki in 1980, that ACh only relaxes precontracted vascular tissue in the presence of an intact endothelium. It is now known that the action of a variety of vasodilators and vasoconstrictors are dependent upon an intact endothelium (Angus & Cocks, 1989).
The umbilical vessels have been shown to be devoid of nerves (Spivak, 1943;
Reilly & Russell, 1977; Fox & Khong, 1990), suggesting that a non-neuronal control of the fcto-placcntal circulation may be particularly important in these vessels. Thus the endothelium may play a paracrine role in the regulation of the umbilical blood flow. Though there are many publications to date describing the ultrastructure of human umbilical vessels at term (Roach, 1973; Asmussen & Kjeldsen, 1975; Takagi ct a!.. 1985), there are few published studies comparing the ultrastructure of these vessels during gestation. Parry & Abramovich (1972) studied the endothelium of the human umbilical vessels through pregnancy and found marked differences in the appearance of the rough endoplasmic reticulum (RER) between 10 weeks and full term, but did not study other parts of the vessel wall.
It is of great importance to investigate the feto-placental circulation early in gestation, since during the first three months the placental circulation is separate from the maternal circulation (Hustin & Schaaps, 1987) and hence develops in a hypoxic environment. Following the establishment of the intervillous circulation at the beginning of the second trimester (Jauniaux et al., 1992), a continuous decrease in vascular resistance with advancing pregnancy is seen (Macara et al., 1993).
51 The purpose of this study was to determine the ultrastructure of the vasculature within the umbilical cord during pregnancy, to see if this can aid in the understanding of the mechanisms regulating feto-placental circulation.
52 3.3 METHODS
Human umbilical cords were obtained from legal terminations of pregnancy at two gestational age ranges, 8-12 weeks (n=7), and 13-17 weeks (n=9), and from normal vaginal deliveries (gestational age, 37-40 weeks, n=4) (see Chapter 2, Section
2.1) and processed for electron microscopy (see Chapter 2, Section 2.2).
53 3.4 RESULTS
3.4.1 LIGHT MICROSCOPY
Throughout gestation, the intima of the umbilical arteries and vein both had a single layer of endothelial cells (EC) (figure 3.1). Initially, from 8-15 weeks, the media appeared to consist mainly of circular bands of smooth muscle cells (SMC), but by 16 weeks (figures 3.Id, i) other orientations of SMC could be observed. There was no clear adventitial layer and no nerves were present.
3.4.2 ELECTRON MICROSCOPY
(a) 8-12 weeks
(i) Endothelial cells
\ The endothelium of the umbilical arteries and vein consisted of a single layer of
small, rounded, nucleate cells (intima). The number of organelles varied in section but in general, the nucleus appeared to be dominant (figure 3.2a), irregular in shape with a very prominent nucleoli. The nucleoplasm was finely granular with a few condensations of chromatin attached to the nuclear envelope. The number of organelles: RER, smooth endoplasmic reticulum (SER), multivesicular bodies (MVB) and mitochondria varied in sections but seemed to have increased by 12 weeks. The
Weibel-Palade bodies (Weibel & Palade, 1962) were more numerous in the vein at 12 weeks but were still present in the arteries. Golgi apparatus were well developed, indicating the synthetic activity of the cell. The EC of the arteries contained more organelles, but the RER appeared more prominent in the vein, because of its more dilated appearance, (figure 3.26, 3.4a). Large accumulations of glycogen were observed in only the endothelium of the arteries (figure 3.46) and were more
54 prominent on the luminal side. Cell contact between EC consisted of tight and gap junctions, with gap junctions appearing every 2-3 cells in the arteries and less frequently in the vein. The contact between the endothelium and SMC was initially extensive in both vessels, arising from long, thin processes, generally originating from both the endothelium and SMC (figure 3.2c, d). However, towards 12 weeks of gestation, the processes become fewer, shorter and fatter, and appeared to originate predominately from the SMC (figure 3.4/?). Whole body contact was observed between a few EC and SMC. The EC appeared to sit on a patchy basal lamina, with some areas becoming continuous with the extracellular matrix of the SMC.
(ii) Smooth muscle, cells
The wall of the arteries appeared as an ordered structure, but with an uneven thickness. At 9.5 weeks the arteries consisted of approximately 8-9 and the vein 5-6 circular layers of SMC around the endothelium (figure 3.3a). The muscle cells appeared as individual circular bands becoming less distinct towards 12 weeks, with groupings of smaller cells in the outer regions. There was much contact between the bands via small processes. SMC appeared very active (figure 3.3/?), in that it was abundant in synthetic organelles such as RER, SER, mitochondria, plasmalemmal vesicles, free ribosomes, Golgi apparatus and glycogen. Mitotic figures were observed throughout the muscle layer and were not restricted to any one region. Apart from the
SMC of the outer region of the muscle layer, SMC were uniform in appearance.
Glycogen was rich in the SMC of both artery and vein, granules were scattered around the organelles and gathered in large accumulations throughout the cytoplasm (figure
3.4/?, c). Dense bodies (electron dense structures scattered in the cytoplasm, see
Gabella, 1981) and caveolae were occasionally seen. An intact basement membrane was observed around most SMC.
55 The SMC furthest from the lumen appeared as a mixed population, with some cells lacking the distinct characteristics of SMC and were more fibroblast-like in appearance (figure 3.3c). The cytoplasm contained an increased number of mitochondria, Golgi apparatus, SER and RER, but lesser amounts of glycogen were observed. The filament content was reduced resulting in patchy condensations. Whilst not common, dense bodies, dense bands (reinforcements on the cytoplasmic side of the membrane found between calveloe, see Gabella, 1981) and caveolae were present.
The basement membrane was incomplete. The extracellular matrix consisted of collagen, evenly distributed as small bundles, which become denser further from the lumen. Elastic fibres, which appeared to have an amorphous core surrounded by fibrils with a diameter of 10-11 nm, were distributed throughout the muscle wall as small fibres, and closely associated with collagen and the basement membrane.
(b) 13-17 weeks
(i) Endothelial cells
EC were similar in appearance to those obtained at 8-12 weeks. However there were some differences: 1) the number of organelles and amount of glycogen had increased; 2) the Weibel-Palade bodies appeared to have decreased in the arteries; 3) the contact between the EC and SMC had decreased but was greater in the arteries than the vein; 4) invaginations of the SMC by processes from EC were reduced and 5) the basement membrane was now more obvious and the internal elastic lamina (lEL) was now noticable.
(ii) Smooth muscle cells
Both the artery and vein had greatly increased in size (figure 3.1), such that by 13 weeks the wall of the vein was approximately 12-14 cells thick and increasing to an average of 24-28 cells by 16 weeks. The arteries had increased from 19-21 cells
56 thick at 13 weeks to 27-45 cells thick by 16 weeks. The lEL of the vein was now clearly distinguishable, though more distinct in the vein and was approximately 200-
300 nm thick. The amount of endothelial-muscle cell contact was reduced. The SMC appeared to have a less organised structure in size and orientation. Compared with the samples from 8-12 weeks, there was a greater variability in size, with the cells in the middle of the media being the largest. Several SMC throughout the vessel wall appeared slightly more active but with a decrease in the number of contractile filaments (figure 3.5a, b). Fibroblast-like cells were observed in the outer media and seen in the outer region of the vessel wall.
(c) 37-40 weeks (Term)
(i) Endothelial c ells
The intima of the arteries and vein in the term samples consisted of a single layer of EC as in the 8-12 and 13-17 week vessels. These were however larger in size and more elongated in shape. Many EC, mainly in the vein, contained large microvilli-like structures that projected into the lumen (figure 3.6a), which were rich in RER, mitochondria and ribosomes. Lysosomes and Wiebel-Palade bodies were not uniformly present. Although fine filaments were present in all endothelial cells they were more numerous in some. Venous endothelial cells appeared to contain more organelles than arterial EC. Although contact existed between endothelial cells and the underlying muscle cells in the arteries, contact was greatly reduced in the vein
(figure 3.6a, h). The lEL of the vein was large and fibrous with a few gaps allowing contact between the EC and the underlying SMC. The presence of elastic fibres in the artery was observed. Occasionally, in the vein, a second lEL was observed further from the lumen which was also fibrous and sporadically broken (figure 3.6c).
57 (ii) Smooth muscle cells
The intimai/medial border of the arteries appeared to contain a second cell type which was difficult to identify. These cells resembled SMC but were very rich in
RER and other organelles. They also showed prominent dense bands, a lower filament content and a few caveolae were found, predominantly under the intima. Contact existed between these cells and with normal muscle cells (figure 3.6b).
The SMC of the media now appeared to be randomly distributed and not in distinct bands or bundles. Their general structure appeared very irregular with branching of the cytoplasmic membrane. When the arteries or vein were in a state of contraction, the branches of many SMC lacked all cell content, also glycogen was not observed within any region of the branches (figure 3.6cl). Although contact between individual SMC was high, no gap junctions were observed. Further into the media, the
SMC became less dense with an increase in the branching of the cytoplasmic membranes. An increase in the space around the nucleus was also observed. An external clastic lamina was seen but this was not as distinct as the lEL, being more prominent in the vein but not always continuous.
The border between the media and adventitia was unclear. This region consisted mainly of individual collagen fibres, with the occasional fibroblast-like cell, fibroblast and single SMC scattered within the connective tissue. The presence of membrane whirls on the medial border were apparent. Nerves and vasa vasorum were not observed in this or any other region of either the artery or vein.
58 FIGURE 3.1
Light micrograph showing the patterns of increase in the size of the wall of the umbilical artery (a-c) and umbilical vein (f-j) at 8 weeks (a and f), 9.5 weeks {h and g), 14.8 weeks (r and h), 16 weeks (d and i) and 39 weeks (e and j). Bar represents 100/^m.
59 / •' 'wV
. i t
1a y i : I f i '
' %
■ * ;
1b 1 9 ' ■
\T'
1c
*1. ' f-fc* FIGURE 3.2
Electron micrographs of (a) small, rounded, closely opposed EC of the umbilical vein (9.5 weeks) showing variation in activity and in the proportion of dilated cistemea. Bar represents 1 /^m; (h) an EC of the umbilical vein (12 weeks) with dilated RER, Golgi apparatus, pinocytotic vesicles and mitochondria and a tight junction (arrow). Bar represents 500 nm. The umbilical vein at 9.5 weeks (c) showing the contact between EC and SMC as a long, thin process arising from an endothelial cell (EC) making contact (arrow) with the underlying smooth muscle cell
(SMC). Bar represents 200nm. In (d) a process from a SMC invaginating an EC
(arrow). Bar represents 200 nm.
61 À «a
T vf?i4L FIGURE 3.3.
(a) Low power micrograph showing the arrangement of SMC in orderly bands around the lumen (Lu) in the umbilical vein (9.5 weeks). The large areas of extracellular space contains small groupings of collagen fibres. Bar represents 2 jum.
(h) Dilated peripheral region (asterisk) of one of two SMC from the umbilical artery
(10.7 weeks) in a transition state between the two phenotypes, synthetic and contractile. Bar represents 1 juni. (c) Fibroblast-like cell in the umbilical vein (10.7 weeks) from the outer region of the media. Dense bodies and caveolae are seen, but lack a continuous basement membrane, making the distinction between SMC and fibroblast cell type difficult. Clear regions within the cytoplasm are artifactual, due to the extraction of glycogen during sample preparation. Bar represents 1 jum.
63 ■ if .r .'S FIGURE 3.4
{a) EC from an umbilical artery (12 weeks) with dilated RER and a high content of organelles. Contact with the underlying SMC mainly originates from the muscle
(arrow) and the processes are short and fat, compared to those at 9.5 weeks (see figure 3.3c, d). The clear patches represent glycogen, extracted during the post fixation procedure. Bar represents 1 fjm. (b) EC from an umbilical artery (12 weeks) with preserved glycogen (G), achieved by omitting the uranyl acetate step of the post fixation procedure. Large accumulations of glycogen can be seen around the nucleus (Nu). Bar represents l^wm. (c) SMC of umbilical vein (15.4 weeks) found in the outer region of the media. There are large accumulations of glycogen (Gl) around the nucleus (Nu) and a few smaller accumulations in the peripheral cytoplasm. Bar represents 1 ^m.
65
FIGURE 3.5.
Electron micrograph of umbilical artery (15 weeks) illustrating the heterogeneity of the SMC. (a) SMC of the synthetic phenotype located 2-3 cells into the media. It has a low filament content, dilated RER, mitochondria, free ribosomes, (h) SMC of the contractile phenotype located in the outer region of the media. Note it has a greater filament content and fewer organelles. A basement membrane can be seen and collagen can be seen in bundles, but elastic fibres are not observed. Bar represents I /am.
67 m FIGURE 3.6.
{a) Umbilical vein EC with luminal projections (term), showing a well-developed
Golgi apparatus, RER, but not all dilated. A distinct basement membrane is present.
The elastic lamina is not always in continual contact with the basement membrane
(arrow). Collagen is present in the interstitial space. Bar represents 2 fm\. (h)
Electron micrograph of an EC of the umbilical artery showing a fragmented basement membrane and contact with the cytoplasmic processes of the smooth muscle. A cell type (asterisk) restricted to the endothelial/medial border of the artery, appeared rich in synthetic organelles with a low filament content. Bar represents l^m. (c) Low power electron micrograph of the smooth muscle layer of the umbilical vein (term) showing the elastic lamina as a fibrous, fragmented band, the extracellular space is full of cellular processes. There is variable activity between cells. A second elastic lamina (arrow) can be seen deeper in the media. Bar represents 2 //m. (d) SMC from the outer region of the media; glycogen can be observed around the nucleus, but is reduced in the peripheral cytoplasm. "Blebbing" of the cytoplasmic membrane of the SMC contains a matrix but lacks filaments. Bar represents 1 fum.
69 • i.'A %
'mm 3.5 DISCUSSION
Structurally, the vessel wall of both the arteries and vein were similar at 8-12 weeks of gestation; both consisted of a single layer of active EC, which progressed from small rounded cells in early gestation, to a more stellate appearance in later gestation. The organelle content, particularly the dilated RER and Weibel-Palade bodies were more prominent in vessels from 8-16 weeks. This implies that endothelial cell function may vary throughout pregnancy. Dilated RER has also been described by Parry & Abramovich (1972) in endothelial cells from early gestation, with maximum dilation at 15 weeks. Asmussen & Kjeldsen (1975) observed dilated
RER in EC from term arteries of smoking mothers, as has Dadak et al. (1984) in the umbilical artery of babies bom to pre-eclamptic mothers.
The EC of the arteries and vein at term remain very active suggesting a contribution to the regulation of the fetal-placental circulation by the possible synthesis and release of vasoactive substances. It has been shown that human term umbilical arteries and vein endothelial cells contain many vasoactive substances, such as 5-HT, histamine, ET, (Chapter 5), neuropeptide Y (NPY), ANP, (Cai et al.,
1993b), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide
(CGRP), SP and AVP (Cai et al., 1993c). Under shear stress they are also capable of releasing vasoactive substances such as ATP, SP and ACh (Milner et al., 1990a). The presence of the microvilli-like projections on the luminal side of the term EC observed in the present study, seems to increase the surface area thereby aiding any secretory role. In samples of umbilical vein EC before 17 weeks, the basal accumulations of vesicles may possibly contain and secrete the components of the internal elastic lamina.
71 Arterial EC were full of glycogen, unlike the vein, throughout gestation and in very reduced amounts in the vein at term. Glycogen was identified as occupying the space around the nucleus in both the EC and SMC. Glycogen has been shown to dissolve during the uranyl acetate block staining step of the fixation procedure and in transmission electron microscopy is identified by the existence of vacant areas within the cell cytoplasm (Zhou & Komuro, 1992).
There has been various suggestions as to the orientation of the SMC. Spivak
(1943) suggested that two distinct layers existed; an internal longitudinal layer surrounded by an external circular layer. Boyd & Hamilton (1970) suggest that the arrangement is mainly longitudinal with intermittent circular layers, whereas
Gcbranc-Youncs ct al. (1986) proposed a predominant helicoidal layer in term vessels. In the present study, it has been shown that in the 8-12 week artery and vein the circular layer was dominant; however in the term vessels there are clearly several orientations of the SMC layers although the precise arrangement is difficult to define, because of regional variations. This may be achieved using 3-dimensional reconstructions of the vessel from serial sections of confocal optical slices.
The SMC showed a change from the "synthetic/secretory/proliferative" cell phenotype to the "contractile" cell phenotype during gestation. A few synthetic SMC were observed at term in the muscle layer just below the EC. These cells may be undifferentiated cells remaining from early development, or a result of phenotypic modulation (a reversal of the contractile state) which occurs prior to proliferation, or as a result of arterial repair, hormonal stimulus, or because of alterations in the cell's environment (Campbell et al., 1981).
72 Moving away from the lumen, to the outer region of the vessel wall, the presence of fibroblast-like cells were observed in all vessels. These cells have been described in detail by Takechi et al. (1993), as "myofibroblasts" the cells of Whartons jelly, which act as an adventitial layer around all 3 vessels of the umbilical cord.
Myofibroblasts have also been identified in the large vessels of the chorionic plate and stem villi of the human placenta by the presence of the same intermediate filaments; desmin, vimentin and keratin (Beham et ai, 1988; Khong et al.y 1986;
Bradbury & Ockleford, 1990; Takechi et al., 1993). These intermediate filaments have also been found in the mesenchymal cells of the cores of the chorionic villi of the human placenta. It has also been suggested that myofibroblasts may play a role in contractility or motility, affecting both maternal and fetal-placental circulation
(Ockleford et a!., 1993). Kohen et al. (1993) has suggested that these
"myofibroblasts" arc components of the extravascular contractile system as first described by Spanner in 1936.
Numerous membrane whirls were seen in the outer medial region in the term vessels, compared to a few in earlier vessels. These are probably caused by the retraction of the cytoplasmic membranes after the muscle cell had been stretched during relaxation, these were also observed in samples before 17 weeks but were very much reduced in number. They are unlikely to arise due to degeneration since they were observed in this particular region only.
Throughout the whole of this investigation, neither nerves nor vasa vasorum were observed. If the umbilical vessels lack both of these components, common in all other blood vessels, the question arises as to how the smooth muscle cells are activated. Gebrane-Younes et al. (1986) suggested that the umbilical vessel structure is compatible with a liquid movement, therefore enabling substances to easily permeate through the vessel wall.
73 In summary, the vessel wall of both artery and vein are similar in structure, both containing a large and active endothelial layer, which seems likely to be synthesising substances that are used in the local regulation of feto-placental circulation.
74 C hapter 4
NO AND HUMAN UMBILICAL VESSELS:
PHARMACOLOGICAL
AND IMMUNOHISTOCHEMICAL STUDIES
75 4.1 SUMMARY
1 In this study we examined the pharmacological affects of various substances, known to produce endotheiial-mediated vasodilatation in many blood vessels, on human umbilical arteries and veins from early and late pregnancy.
2 ACh, ATP, the calcium ionophore A23187 and SP had no effect on raised vascular tone.
3 SNP relaxed 5-HT preconstricted umbilical artery and vein from both early and late pregnancy.
4 L-N^-Nitroarginine methyl ester (L-NAME) had no effect on basal tone or on tone raised by 5-HT.
5 Localization of NOS type 1 (neuronal) was examined in the same umbilical vessels using electron immunocytochemistry. No NOS-immunoreactive EC were observed in the umbilical vessels taken during early pregnancy. However, the percentage of
NOS-immunoreactive EC in umbilical artery and vein from late pregnancy was 3 and
10%, respectively.
6 These results suggest that NO does not contributes little, if anything to the local control of umbilical blood flow throughout pregnancy, despite the presence of NOS-IR in a subpopulation of EC in late pregnancy.
76 4.2 INTRODUCTION
The human umbilical and placental circulation both lack innervation (Spivak,
1943; Reilly & Russell, 1977; Fox & Khong, 1990). Therefore the endothelium may play an important role in the regulation of vascular tone. EDRF was first demonstrated in 1980 by Furchgott & Zawadzki. It has since been identified as NO
(Palmer et ai, 1987), and is produced within endothelial cells, from the amino acid
L-Arg by the enzyme NOS (Palmer et al., 1988). NO appears to be an autocrine/paracrine signalling agent synthesised in many tissues including the endothelium, cerebellum, platelets, hepatocytes and macrophages, where it exhibits diverse actions including vasodilatation (Palmer cf a/., 1988).
In 1987, Van de Voorde and colleagues, demonstrated that histamine, the calcium ionophore A23187 and ATP but not ACh or 5-HT released a substance from the umbilical artery and vein with an intact endothelium which caused relaxation in endothelium denuded pre-contracted rat aortic rings. This substance was identified as
NO. Later in 1991(a), Myatt et al. also demonstrated the presence of EDRF in the human preconstricted dually perfused cotyledon, and Tsukahara et al. (1993) has shown that HUVECs release NO on stimulation by several agonists.
The aim of this study was to investigate endothelium-dependent relaxation in umbilical vessels from different stages of gestation, and to observe whether the endothelial cells of these umbilical vessels contain NOS.
77 4.3 METHODS
Human umbilical cords from legal terminations of pregnancy (early pregnancy group, mean gestational age, 15 (8-17) weeks, n=12) and from normal term vaginal deliveries (late pregnancy group, mean gestational age 39 (38-41) weeks, n=12) (see Chapter 2, Section 2.1) were placed into a modified Kreb's solution at and were used within 12 hours for isolated vessel pharmacology (Chapter 2,
Section 2.3) and segments of the same cords were processed for the immunocytochemical localization of NOS (see Chapter 2, Section 2.4.2a and b).
78 4.4 RESULTS
4.4.1 PRECONSTRICTION BY 5-HT
5-H T (10 nM - 30 /uM) induced concentration-dependent constriction of the human umbilical artery and umbilical vein with pD 2 values of 6.9 ± 0.1 (n=12) in the artery, 6.9 ± 0.2 (n=12) in the vein from early pregnancy and 7.0 ±0.1 (n=12) in the artery and 6.9 ± 0.1 (n=12) in the vein from late pregnancy. Vasoconstriction was completely reversible in both artery and vein after washout to remove 5-HT.
4.4.2 ENDOTHELIUM-DEPENDENT RELAXATION IN THE HUMAN
UMBILICAL ARTERY AND VEIN
Umbilical artery and umbilical vein preparations from early (n=12) and late pregnancy (n=12) with an intact endothelium constricted by 5-HT did not relax in response to ACh (0.1 ^M - 300 ywM), ATP (0.1 fuM - 300 /wM), SP (0.01 nM - 30 nM) and calcium ionophore A23187 (10 nM - 30 /WM) (see figures 4.1 & 4.2). SNP
(0.1 //M - 300 ^M) was able to relax preconstricted artery and vein preparations from both early and late pregnancy (see figure 4.3 & 4.4). This was 87.9 ± 3.8% (n=6) and
95.0 ± 6.8% (n=5) of the established tone, for artery and vein, respectively from early pregnancy and 78.8 ± 6.9% (n=6) and 81.6 ± 8.7% (n=6) for the artery and vein respectively of the established tone for late pregnancy (figure 4.4).
79 4.4.3 EFFECT OF L-NAME
No change in basal or raised vessel tone was observed on incubation for up to 4 h of umbilical artery and vein preparations from both early and late pregnancy, with 100fiM of L-NAME.
4.4.4 ELECTRON IMMUNOCYTOCHEMISTRY
By using the technique of pre-embedding immunocytochemistry, EC displaying NOS (type 1) immunorcactivity were observed in both umbilical artery and vein of late pregnancy (figure 4.5). Examination of numerous sections that were taken at different levels of the specimens showed a different percentage of immunorcactivity in the umbilical artery and umbilical vein. Analysis of 490 arterial
EC revealed that only 3% of these cells were positive for NOS (15 positive cells out of the 490 cells examined), whereas analysis of 566 venous showed NOS- immunoreactive in 10% of cells (56 positive cells out of the 566 cells examined). In the labelled cells the immunoprecipitate was distributed throughout the cell cytoplasm, although the density of immunostaining varied from cell to cell. Labelled cells contained a large, unlabelled nucleus and numerous organelles, especially swollen mitochondria and endoplasmic reticulum.
Labelling to NOS was also observed outside the endothelium
(extracellularly). In the umbilical vein, immunoprecipitate was seen in structures difficult to identify as illustrated in figure 4.6a. In the umbilical artery NOS- immunoreactivity was associated with the material covering the endothelial cells from the luminal side which showed NOS-immunoreactivity, as shown in figure 4.6b.
Labelling to NOS was not observed in the control preparations of umbilical arteries and veins from early and late pregnancy (figure 4.6c), nor was any NOS-
80 immunoreactivity observed in endothelial cells from umbilical arteries and veins from early pregnancy (figure 4.7).
81 FIGURE 4.1
Original trace from experiments showing the effect of {a) control, ip) ACh (0.1 fjM
- 300 //M), (c) ATP (0.1 //M - 300//M), and (d) SNP (0.1 juM - 300//M) on human
umbilical artery (a,b) and vein (c,d) from late pregnancy.
82 Control
b ACh 30 100300pM
2g 5-HT;
2min
ATP 0.3 100 300 mM ig 5-HT
2min
0.3 2g HT
2min 3 lOpM FIGURE 4.2
Original trace from experiments showing the effect of (a) ACh (0.1 fjM - 300 ywM),
{h) ATP (0.1 fjM - 300 fjM), (c) Ca^'*' ionophore (0.01 - 30 //M), and (d) SNP
(0.1 //M - 300 //M) on human umbilical artery (a,b,d) and vein (c) from early pregnancy.
84 ACh 300pM 0.1 0.3 30 100
0.5g 5-HT
2min
ATP 0.1 100 300 pM 5-HT
2min
0.01 0.03 0.1 0.3 5-HT 30 pM
2min
d SNP 0.1 0.3 ig 5-HT
1min 30 100 HGURE 4.3
Cumulative concentration-response curve to SNP on 5-HT (EC-y^ concentration) preconstricted umbilical vessels, ©umbilical artery (n= 6 ) and ©umbilical vein
(n=5) from early pregnancy. Symbols represent mean % relaxation ± s.e. (unless masked by symbol).
86 % Relaxation
100 ^
90
80
70
60 _
50 _
40 _
30 _
20 _
10 _
T------1------r 7 6.5 6 5.5 5 4.5 4 3.5
-LogCSNP] FIGURE 4.4
Cumulative concentration-response curve to SNP on 5-HT (EC '75 concentration) preconstricted umbilical vessels. # umbilical artery (n= 6 ) andH umbilical vein
(n= 6 ) from late pregnancy. Symbols represent mean % relaxation ± s.e. (unless masked by symbol).
88 Relaxation
100 ^
90
70 _
50
40 .
20 .
— I ■ I 6.5 5.5 5 4.5 3.5 -Log [SNP] FIGURE 4.5
Electron micrographs of umbilical vessels in late pregnancy immunolabelled for
NOS: (a) umbilical artery and (b, c) umbilical vein immunolabelled for NOS. (a) A fragment of artery displays the presence of one EC with positive cytoplasmic immunoreactivity for NOS (white star). Note the unlabelled nucleus (N). The neighbouring cell profiles (black asterisks) are NOS-negative. /w-lumen. xSOOO. (b)
A fragment of vein shows at least two NOS-positive neighbouring EC. ^/-elastic lamina; .sm-smooth muscle. x7400. (c) Note the two NOS-positive EC showing a difference in the intensity of cytoplasmic immunolabelling. Endoplasmic reticulum
(er), mitochondria (m) and filaments (f) are seen in intense labelled cell, nw-nucleus. xllOOO.
90 1
* m FIGURE 4.6
Electron micrographs of (a) umbilical vein (term) and (b) umbilical artery (term) immunolabelled for NOS, and (c) an example of control specimen (term), (a) Note the intense NOS-immunolabelling in a membrane bound profile (large white star) localised below the NOS-negative cell {black asterisk). The neighbouring EC (small white star) is NOS-positive. A^-cell nucleus; cr-endoplasmic reticulum; / 7 7- mitochondria; ^/-elastic lamina; 6 /77 -smooth muscle; /w-lumen. xlOOOO. (b) Note the intense NOS-positive material (arrows) associated with the luminal surface of unlabelled EC. xSOOO. (c) Control preparation (omission of NOS-antiserum) showing absence of NOS-immunolabelling. x6200.
92 ê
f FIGURE 4.7
Electron micrograph of the (a) umbilical artery and (b) umbilical vein, from early pregnancy following immunolabelling for NOS. No NOS-positive EC were seen.
jV-cell nucleus; cr-endoplasmic reticulum; m-mitochondria; .v«fe-subendothelial
zone; .vm-smooth muscle; /w-lumen. a x 8400. b x 10500
94 ! 4.5 DISCUSSION
Our pharmacological studies have failed to demonstrate any significant evidence of endothelial-dependent relaxation in the human umbilical artery and umbilical vein, either at early or late pregnancy. This is in accordance with the reports of other authors; Monsuko et al. (1990) did not see any relaxation to ACh or A23187.
Griggs & McLaughin (1989) demonstrated this lack of endothelial-dependent relaxation in the chorionic plate arteries of the term placenta, and proposed that this was due to the insufficient release of EDRF/NO to have an effect on the underlying smooth muscle. According to Hull and colleagues (1993) human umbilical vessels do not relax to ACh, ADP, Bk, histamine, SP or A23187. It has been suggested that this lack of relaxation results from a near maximal production of EDRF in the placental arteries, leaving no capacity for increased release to secondary stimulation. However,
Van de Voorde ct ai. (1987) and Chaudhuri et al. (1991) like ourselves, have demonstrated the lack of relaxation due to basal release of NO. Klockenbusch and co workers (1992) have also seen a lack of relaxation due to basal NO release, despite a significant basal NO generation, as seen from cyclic GMP generation. He also rules out the possibility that this could be due to a deficiency of endogenous substrate i.e.
L-Arg, since histamine induced an increase in cyclic GMP accumulation, which was suppressed by L-nitroarginine.
It has been shown that HUVECs are capable of releasing NO when stimulated by the agonists, thrombin, Bk, L-Arg and ionomycin (Tsukahara et al.,
1993). Cascade experiments with human umbilical vessels, have shown that they release a substance capable of dilating the endothelium denuded, ring preparations of rat aorta and bovine pulmonary artery (Van de Voorde et al., 1987; Chaudhuri et al.,
1991). Using the same techniques Izumi et al. (1994) suggests that the amount of
96 EDRF and the sensitivity of smooth muscle to EDRF decreases with gestation. NO synthesised from L-Arg acts through the stimulation of the soluble guanylate cyclase to increase cyclic GMP levels in vascular smooth muscle to produce relaxation.
(Moncada & Palmer, 1991; Nathan, 1992). Renowden etal. (1992), have shown that the umbilical artery is unable to relax significantly to ANP, SNP and A23187, which activate cyclic GMP and forskolin which activates cyclic AMP, when using maximal concentrations of 5-HT. These results imply that the umbilical artery and vein smooth muscle are not responsive to endothelial derived NO, although it is curious that SNP, which is usually taken to mimic the actions of NO, was shown to produce relaxation of these vessels. However, it has been demonstrated that changes in flow and shear stress maybe a stimulus for NO release (Hecker et al., 1993).
The present study shows that no NOS (type I, neuronal)-immunoreactive EC are present in human umbilical artery and vein EC from early pregnancy. By contrast,
EC from umbilical artery and vein in late pregnancy are a heterogeneous population for NOS-immunoreactivity, with, although still small, a greater percentage of NOS- immunoreactive EC in the umbilical vein. A low percentage of NOS-immunoreactive
EC has also been observed in other vessels, for example, 6% of EC were immunoreactive for NOS in the rabbit aorta (Loesch & Burnstock, 1993a) and 1% of
EC were immunoreactive for NOS in the rat basilar artery (Loesch et al., 1994). Both these vessels are known to exhibit strong endothelium-dependent relaxations. Present results are in contrast to the relatively high percentage of immunoreactive cells for the peptides ANP and NPY, 32% and 44%, respectively, as observed by Cai et al.
(1993b) in human umbilical vessels. This appearance of NOS-immunoreactivity within EC may be a physiological process through which an increase in feto-placental blood flow is facilitated as gestation advances (Macara et al., 1993).
97 Our results on neuronal NOS-immunoreactivity differ from that of Myatt et al. (1993a) and Pollock et al. (1993), where both groups found a strong immunoreactivity to NOS in umbilical artery and vein using a monoclonal antibody
"H32" to an endothelial isoform of NOS (NOS type III). The results of these studies, however, indicate that 50% of veins examined with light microscopy did not display any NOS-immunoreactivity (Myatt et al., 1993a), whereas Pollock et al. (1993) found "strong immunoreactivity" to NOS in both umbilical artery and vein. Springall et al. (1993) have demonstrated that polyclonal antibodies to rat brain NOS (type I) enzyme react with the human equivalent and also with other isoforms in human endothelium, and have found positive immunolabelling in the human umbilicus and trophoblast of the human placenta. Although, a 57% sequence homology has been identified between NOS type 1 (neuronal isoform) and NOS type III (endothelial isoform) (Brcdt ct al., 1991; Lamas et al., 1992), it cannot be ruled out that the polyclonal antibody used in this study has not recognised all isoforms of NOS that may be present in human umbilical endothelium, labelling for example only those cells which contained neuronal NOS type I. It is striking, however, that our results differ from those of Dikranian et al. (1994), showing that in the human umbilical vein at the light microscopic level, high proportions of EC (97%) immunoreact with an antibody to neuronal NOS (the same antibody as that used in our study). The localization of neuronal NOS in vascular endothelium may not be surprising, since it has already been found that endothelial isoform of NOS and neuronal isoform of NOS may occur in the same neuronal cell populations in the brain (Dinerman et al., 1994).
Our data shows that the primary antibody can be adsorbed onto the luminal surface of the umbilical artery and vein, as has previously been observed in the rabbit aorta (Loesch & Burnstock, 1993a), where the labelling appeared in the sub- endothelial matrix. We also observed heavy labelling on the luminal surface of the EC from both umbilical artery and vein, despite the use of strong blockers to inhibit
98 endogenous peroxidases. This may produce misleading immunocytochemical results at the light level, as at this level, such material can easily account for the endothelium.
The possibility cannot be excluded that this phenomenon was partially responsible for high proportions of immunolabelled cells at the light microscopic level as seen by
Dikranian et al. (1994). Luminal labelling appears to be only in the term vessels, suggesting the physical properties of the endothelial luminal surface may be different in late pregnancy. At this stage it is difficult to explain this phenomenon, but in situ hybridisation studies would clarify this issue. We can only suggest that it is possibly a material from the blood trapped between the EC, when the vessels constrict/collapse at birth, or possibly a material secreted from the EC, which is rich in NOS and/or peroxidases.
This study demonstrates the inability of the umbilical artery and vein to relax to known endothelium-dependent vasodilators. This is despite demonstrating a small sub-population of NOS-immunoreactive EC, and a functional cyclic GMP system in the smooth muscle, which is responsible for the action of NO. Previous studies have demonstrated the release of NO from human umbilical EC from artery and vein, and shown its effect on the vascular smooth muscle from other animals. This could imply that the amount of NO released may be insufficient to affect smooth muscle locally, or that the mechanism coupling NO with soluble cyclic GMP may be impaired.
Therefore it is unlikely that NO is involved in the local regulation of umbilical blood flow throughout pregnancy.
99 Chapter 5
ELECTRON MICROSCOPIC IMMUNOLABELLING OF
VASOACTIVE SUBSTANCES IN HUMAN UMBILICAL ENDOTHELIAL
CELLS AND THEIR ACTIONS IN EARLY AND LATE PREGNANCY.
100 5.1 SUMMARY
1 Immunocytochemistry of human umbilical vessels indicates that 5-HT, histamine and ET were localised in subpopulations of endothelial cells of both artery and vein from late pregnancy, but not early pregnancy.
2 The pharmacological effects of 5-HT, histamine and ET were examined in umbilical arteries and veins from legal terminations (gestational age 8-17 weeks, n=12) and normal term vaginal deliveries (gestational age 38-41, n=12).
3 5-HT (10 nM - 30 ^M) caused sustained concentration-dependent contractions in all vessels from early and late pregnancy; there is some indirect evidence for 5-HT endothelial-dependent vasodilatation of umbilical arteries, but not veins, in late pregnancy. Histamine (0.1 jM - 30 mM) caused sustained contractions in all vessels from late pregnancy, whereas only 21% of arteries and 41% of veins from early pregnancy responded. ET (10 pM - 30 nM) caused slow, long-lasting contractions in all vessels from early and late pregnancy. ANP (10 pM - 30 nM) and NPY (10 pM -
30 nM) had no effect on basal or raised tone.
4 These results show that although the responses to 5-HT, histamine and ET are not mediated via the endothelium, the endothelium may play an autocrine/paracrine role, possibly by synthesising and/or releasing these substances in late pregnancy to influence feto-placental blood flow.
101 5.2 INTRODUCTION
Furchgott & Zawadzki (1980) demonstrated that the relaxations evoked by
ACh in the aorta of the rabbit are dependent on the presence of an intact endothelium.
It has since been shown that receptors for many substances on endothelial cells, when occupied, result in the release of EDRFs or EDCFs. Many vasoactive substances have been shown to act through the endothelium (Angus & Cocks 1989) and several of these substances have been shown to be located in the endothelial cells of a variety of blood vessels from different species (Ralevic & Bumstock, 1993).
Human umbilical vessels are not innervated (Spivak, 1943; Reilly & Russell,
1977; Fox & Khong, 1990), therefore it is most likely that the regulation of vascular tone in the umbilical vessels is dependent on local vasoactive substances produced by the endothelium (Ralevic & Bumstock, 1993). The production and mechanism of action of endothelium-derived vasoactive substances may act in an autocrine and/or paracrine manner to regulate vascular tone. Little is known about the endothelial cell constituents of non-innervated blood vessels. It has been shown that ATP, SP and
ACh can be released from HUVECs by increased sheer stress (Milner et al., 1990a).
ANP and NPY and other peptides have been localised in the endothelial cells of human umbilical vessels (Cai et al., 1993b, 1993c), which strongly suggests that the umbilical endothelial cells themselves may store and/or synthesis these vasoactive substances. HUVECs have been shown to produce large quantities of prostanoids and release EDRF in response to histamine, bradykinin and the calcium ionophore
A23187, but not ACh, ATP or SP. ACh is released from HUVECs during increased flow (Van de Voorde et al., 1987).
102 The aim of this study is to examine whether certain vasoactive substances, such as 5-HT, histamine and ET, are located in the endothelial cells of human umbilical vessels and to see whether these substances and also ANP and NPY effect | have an effect on vessel tone in the presence and absence of an intact endothelium from early and late pregnancy.
103 5.3 METHODS
Human umbilical cords from legal terminations (early pregnancy group, mean gestational age 15 (8-17); n=12) and from normal term vaginal deliveries (late
! pregnancy group, mean gestational age 391 weeks (38-41);n=12) (see Chapter 2, Section
2.1). The cords were placed into a modified Kreb's solution at 4 °C and used within
12 hours for isolated vessel pharmacology (see Chapter 2, Section 2.3) and segments of the same cords were used for immunocytochemical localisation of 5-HT, histamine and ET (see Chapter 2, Section 2.4.2a and c).
104 5.4 RESULTS
5.4.1 ELECTRON IMMUNOCYTOCHEMISTRY
The technique of pre-embedding immunocytochemistry was used to detect
EC displaying 5-H T-, histamine- and ET-l-like immunoreactivityj and were observed in both umbilical artery and vein of late pregnancy (figure 5.1 - 5.3). Endothelial staining was not observed in any vessels from early pregnancy and in controls (figure
5.4). Examination of numerous sections that were taken at different levels of the specimens showed a different percentage of immunoreactivity in the umbilical artery and vein. Immunopositive and immunonegative EC were counted directly on examination with an electron microscope. Analysis of 845 arterial EC revealed that approximately 13%, 2% and 24% of these cells were immunopositive for 5-HT, histamine and ET-1, respectively, whereas, analysis of 804 venous EC showed that approximately 14%, 40% and 17% were immunopositive for 5-HT, histamine and
ET-1, respectively.
Immunoreactivity to 5-HT, histamine and ET in arterial and venous EC was associated with the cytoplasm as well as with the membrane of ER, mitochondria and cytoplasmic vesicles (figure 5.1 - 5.3). Intensity of immunoreactivity to 5-HT, histamine and ET varied from cell to cell. Relatively discrete 5-HT immunoreactivity was observed in the umbilical artery (figure 5.1a), but strong immunoreactivity was seen in the umbilical vein (figure 5.1b). In addition, to the typical intracellular organelles, such as endoplasmic reticulum, mitochondria and cytoplasmic vesicles, the cytoplasm of immunopositive cells displayed large bundles of filaments, as observed in 5-H T- and ET-positive venous endothelial cells (figure 5.1c, 5.3c). In these filament-rich cells, the cistemae of the ER were substantially enlarged (figure
5.1c, 5.3c).
105 Extracellular endothelial immunostaining for either of the substances examined has also been observed in both the umbilical artery and vein from late pregnancy. The 5-HT positive labelling of material associated with the luminal surface of EC as well as with the subendothelial matrix was prominent (figure 5.4a,b).
In contrast, in all (Control! preparations, no immunocytochemical staining was observed in the EC of umbilical artery and vein. Examples of control preparations are illustrated in figures5.4c and 5.4d.
5.4.2 VASOCONSTRICTION IN THE UMBILICAL ARTERY AND VEIN
5-HT (10 nM - 30 /^M) induced concentration-dependent constriction in all vessels studied, with pD 2 values from second trimester of 6.8 ± 0.2 (n=10) in the artery and 6.9 ± 0.1 (n=10) in the vein, and from third trimester, 6.9 ± 0.2 (n=10) in the artery and 6.9 ± 0.3 (n=10) in the vein. There was no significant difference in the pD2 values for 5-HT between early and late pregnancy. However, the maximum contractions to 5-HT in early pregnancy (270% relative to KCl) were significantly greater than those of late pregnancy (100% relative to KCljf The contraction-response curve to 5-HT in all vessels from early and late pregnancy were shifted to the right in the presence of ketanserin (10 f/M, n=5), a 5-HT^ selective antagonist, and methysergide (10 ^M, n=5), a non-specific 5-HT antagonist, and the pDo values were significantly altered (see Appendix, figure 5.11 for the effect of methysergide on vessels from late pregnancy). Ondansertron (10 ywM, n=4), a 5-HTg antagonist, had no effect on the dose-response curve to 5-HT or on the pD 2 values of the umbilical vessels from early and late pregnancy. Histamine (0.1 /M - 30 mM) also caused sustained constrictions in all vessels from late pregnancy, but only 27% of arteries and
41% of veins from early pregnancy constricted (see figure 5.7 & 5.8). ET-1 (10 pM -
30 nM) caused transient contractions in all vessels tested from early and late pregnancy (see figure 5.9 & 5.10). pD 2 values were not calculated for ET-1 since maximum contractions were not reached. * 10^
* see Appendix, figure 5.12. ANP (10 pM - 30nM) and NPY (10 pM - 30nM) had no effect on either raised or base tone. The removal of the endothelium, did not have any effect on responses in vessels from late pregnancy (figure 5.5 - 5.10). However, the removal of the endothelium from vessels from early pregnancy was problematical; it was not possible without damaging the vessel and abolishing all responses, even to KCl (120 mM).
107 FIGURE 5.1
Electron micrographs of the umbilical artery (a) and vein (b) from late
pregnancy immunolabelled for 5-HT. (a) A fragment of artery displays the
presence of two EC with discrete cytoplasmic immunoreactivity for 5-HT
(black asterisks). The neighbouring cell profile (black star) is 5-H T -
negative. JV-nucleus, m-mitochondria, /w-lumen. x7400. (b) A fragment of
vein shows one 5-HT-positive and one 5-HT-negative endothelial cell.
Note intense cytoplasmic 5-HT immunolabelling. ^/-elastic lamina. x6000.
(c) Note bundles of filaments (fi) and enlarged endoplasmic reticulum (er) in
5-HT-positive endothelial cells. x9000.
108 ¥ FIGURE 5.2
Electron micrographs of umbilical artery (a) and vein (b, c) in late pregnancy
for histamine, (a) A fragment of artery displays the presence of three EC with
positive cytoplasmic immunoreactivity for histamine. One cell profile (black
star) is histamine-negative /i-nucleus, 5m-smooth muscle, /w-lumen. x7800.
(b) A fragment of vein shows at least two histamine-positive and one
histamine-negative neighbouring EC. m-mitochondria, e/-elastic lamina.
x8800. (c) A magnified portion of a histamine-positive EC illustrated in (b)
to show the intense cytoplasmic immunolabelling .cr-endoplasmic reticulum,
v-cytoplasmic vesicles. x30800.
110 s m FIG U RE 5.3
Electron micrographs of umbilical artery (a) and vein (b, c) from late
pregnancy immunolabelled for ET (a) A fragment of artery displays the
presence of one EC with positive immunoreactivity for ET The neighbouring
cell profile (black star) is ET-negative. n-nucleus, /u-lumen. xlSOOO. (b) A
fragment of vein shows one ET-positive and ET-negative neighbouring EC.
m-mitochondria, ^/-elastic lamina. xl5700 (c) Note the ET-positive EC
showing bundles of filaments (fi) and enlarged cistemae of granular ER (er). X13300.
112 m
»
© FIGURE 5.4
An example of intense extracellular immunolabelling for 5-H T (a, b) and
control specimens (c, d). (a) Umbilical artery, note the labelling (arrows^
associated with the luminal surface. M-nucleus, /w-lumen. xl3700. (b)
Umbilical vein, note the labelling (white asterisk) localised below the 5-HT-
negative EC. e/-elastic lamina, j/M-smooth muscle. x6500. (c) Control
preparation of an artery (incubation with non-immune normal rabbit serum)
showing an absence of immunolabelling. exm-extracellular matrix. x5700.
(d) Control preparation of a vein (omission of antiserum) showing an absence
of immunolabelling. x7600.
114 îli' LUX0 FIGURE 5.5
Cumulative concentration-response curves to 5-H T on the human umbilical
artery from late pregnancy in the absence (Q ) and presence (® ) of an intact
endothelium. Symbols represent % contraction ± S.E.M. (unless masked by
symbol).
116 % Contraction
140
120 _
100 _
80 _
60 _
40 _
20
0 J
10 9 8 7 6 5 4 - Log C5-HT) HGURE 5.6
Cumulative concentration-response curves to 5-H T on the human umbilical
vein from late pregnancy in the absence (Q ) and presence ( 0 ) of an intact
endothelium. Symbols represent % contraction ± S.E.M. (unless masked by
symbol).
118 % Contraction
140 _
120 _
100 _
80 _
60 _
40
20 _
0 J
r~ 10 8 7 6 5 4 Log (5-HT] FIGURE 5.7
Cumulative concentration-response curves to histamine on the human
umbilical artery from late pregnancy in the absence (Q ) and presence ( D
of an intact endothelium. Symbols represent % contraction ± SEM (unless
masked by symbols).
120 % Contraction 100 .
90 -
80 _
70 -
60 _
50 _
40 _
30 -
20 _
10 -
0 J
7.5 7 6.5 6 5.5 5 4.5 4 3.5 - Log (Histamine) FIGURE 5.8
Cumulative concentration-response curves to histamine on the human
umbilical vein from late pregnancy in the absence ([[]) and presence (H ) of
an intact endothelium. Symbols represent % contraction ± SEM (unless
masked by symbols).
122 % Contraction
140 _
120 _
100 _
80 _
60 _
40 _
20 _
0 J
7.5 6.5 6 5.5 5 4.5 3.5
- Log [Histamine) FIGURE 5.9
Cumulative concentration-response curves to ET on the human umbilical
artery from late pregnancy in the absence ( ^ ) and presence (J^) of an intact
endothelium. Symbols represent % contraction ± SEM (unless masked by
symbols).
124 % Contraction
90 _
80 _
70 _
60 _
50 _
40 _
30 _
20 _
10 _
r" ~ r 10 9.5 9 8.5 8 7.5
- Log CET-1) FIGURE 5.10
Cumulative concentration-response curves to ET on the human umbilical
vein from late pregnancy in the absence (/V) and presence ( ^ ) of an intact
endothelium. Symbols represent % contraction ± SEM (unless masked by
symbols).
126 % Contraction 100 _
90 _
80 _
70 _
60 _
50 .
40 _
30 _
20 _
10 _
0 J
9.5 8.5 8 7.5
- Log CET-1] 5.5 DISCUSSION
Our electron immunocytochemical studies have demonstrated
immunoreactivity for 5-HT, histamine and ET in the endothelial cells (and partially in the neighbouring extracellular space) of both umbilical artery and
vein from late pregnancy, although no staining was present in vessels from
early pregnancy. However, since contractions for 5-HT and ET could be
elicited in early pregnancy, the lack of 5-HT and ET in the umbilical EC of
early pregnancy suggest they may not be the only source of 5-HT and ET
that can cause contraction of these vessels. Koren et al. (1966) showed that
the 5-HT content of the placenta shows a gradual increase as pregnancy
advances, which is consistent with the lack of 5-HT immunoreactivity in EC
from early pregnancy observed in this study. Various authors have
demonstrated that placental vessels are very much less sensitive to 5-HT than
umbilical arteries (Panigel, 1962; Mak et a/., 1984; MacLean et al., 1992),
which implies that the main role for 5-HT is in the umbilical vessels.
Responses to 5-HT in the feto-placental circulation have been demonstrated
by several authors (McGrath et al., 1990; Breslin, 1991).
McGrath et al. (1988) have reported that there was no significant
difference in response to 5-HT between rubbed (in which the endothelium is
removed) and unmbbed preparations of the umbilical artery and vein from
late pregnancy. Abramovich (1983) have also demonstrated
differences in the responsiveness of cotyledonary and umbilical vessels and
claim that these responses do not appear to depend upon the integrity of the
endothelium. Cocks & Angus (1983) and Cohen et al. (1983) have both'
demonstrated that 5-HT is a significantly more powerful vasoconstrictor in
the absence of an intact endothelium in the pig and dog coronary arteries.
128 These results were taken to suggest that 5-HT can be an endothelial-mediated vasodilator in these vessels. In the present study, we have shown some potentiation (although not statistically significant) of constriction to 5-HT in the human umbilical artery in the absence of an intact endothelium (figure
5.5). This suggests that human umbilical arteries in late pregnancy may be capable of endothelial-mediated relaxation to 5-HT. NOS has been localised in these endothelial cells, although most known endothelial-dependent vasodilators have no effect on these vessels (Chapter 4). Attenuation or loss of the endothelial vasodilator response may be significant in certain pathological conditions, where the loss of, or the damage of the endothelial cells affects their ability to produce and release the vasodilator signal, such as in pre-eclampsia or lUGR, where increased vascular resistance is observed
(Macara et aL, 1993).
When compared with late pregnancy vessels, the early pregnancy vessels exhibited almost a 3-fold greater maximal response to 5-HT, when expressed as a percentage of the KCl (120 mM) contractions, despite there being no significant difference between the pÜ 2 values. The similarity in pD 2 values between early and late pregnancy may be due to a change in receptor density, rather than differential receptor expression. The use of 5-HT antagonists showed that this response to 5-HT in umbilical vessels from early and late pregnancy is probably due to a difference in receptor density, which could be further clarified by the use of autoradiography and radioligand
binding studies. However, the possibility that other 5-HT receptor classes are present cannot be ruled out since it is now known that at least four classes of
5-HT receptors exist (Zifa & Pillion, 1992).
129 The results from this study demonstrate the presence of both 5-HT^ and S-HTo, but not 5-HTg receptors on the umbilical vessels from both early and late pregnancy. MacLennan et al. (1989) also demonstrated that 5-HT contractions were antagonised by ketanserin (0.1 yuM), suggesting a 5-HT^ or
5-HT 2 receptor, whereas methysergide behaved as a partial agonist. Increases in oxygen tensions that occur at birth is believed to assist in closing down the umbilical circulation by inducing a profound vasoconstriction of the umbilical arteries. MacLennan et at. (1989) showed that at higher p02 values, a heterogeneous population of 5-HTj^ and 5-HT2 receptors mediate constriction to 5-HT, and at a low p02 5-HT-mediated contraction is via a homogeneous population of 5-H T| receptors. Our results support this observation, in that we demonstrated that both ketanserin and methysergide antagonised the response to 5-HT at high oxygen tensions. It would therefore be interesting to compare the maximal contractile responses of early and late pregnancy to 5-HT at low oxygen tensions. Increased oxygen tensions have been shown to stimulate TXA 2 production which results in an increase in the sensitivity of the umbilical arteries to 5-HT (Templeton et at., 1991b). These results all indicate that 5-HT may have an important role in constriction of the umbilical vessels at birth. 5-HTg receptors have been described only on neurones (Bradley et at., 1986; Humphrey & Feniuk, 1987), since human umbilical vessels are devoid of nerves (Fox & Khong, 1990) an involvement of a 5-HT 3 receptor in human umbilical vessel constriction to 5-HT is unlikely. It is not surprising, therefore, that the 5-HTg antagonist, ondansertron, had no effect on any of the responses to 5-HT in umbilical vessels from early and late pregnancy.
Dyer et at. (1972) found histamine to be an effective constrictor on all umbilical vessels studied from late pregnancy and like 5-HT, umbilical
130 blood vessels have been shown to be more sensitive to the actions of histamine than placental vessels (Dyer et aL, 1972). The response to histamine in early pregnancy may not be fully developed, which may suggest why we see only a percentage of preparations responding to the agonist.
Lewis (1968) also documented that only 25% of ovine umbilical arteries responded to histamine. Ueda et al. (1992) have shown ultrastructural localisation of histamine in endothelial cells of the human umbilical vein.
This staining was associated with Weibel-Palade bodies (Weibel & Palade,
1964); Ueda and colleagues (1992) suggested that these structures may be a reservoir of histamine and may contribute to the regulation of vascular tone hemostasis during vascular injury. In our study immunoreactivity to histamine and also to 5-HT and ET were distributed in the cell cytoplasm and in association with the membranes of intracellular structures. It is also interesting that there was a far greater percentage of histamine immunopositive cells in the umbilical vein than in the artery. It is possible that the umbilical veini may be a substantial source of histamine for the fetus.
ET-like immunostaining has been demonstrated at the light microscope level in the endothelial cells of umbilical artery and vein
(Haegerstrand et aL, 1989; Hémsen et aL, 1991; Salamonsen et aL, 1992); ET has been shown to have potent presser actions (Wilkes et aL, 1990) and it has been proposed that ET is involved in the regulation of fetal-placental blood flow (Nakamura et aL, 1990; Haugen & Stray-Pederson, 1991).
Cai et al. (1993b) showed the presence of ANP and NPY in subpopulations of EC from the human umbilical vein and artery. ANP mRNA has also been located in the endothelial cells of umbilical vessels (Cai et aL,
131 1993a) suggesting that synthesis rather than uptake of the peptide is occurring. However, we have shown in the present study that ANP and NPY had no effect on vessel tone. We suggest therefore that umbilical endothelial cells may be a source for these peptides to reach the fetus and perhaps influence its physiology.
The results of this study suggest that there are possibly two groups of vasoactive substances that are localised in the endothelial cells of human umbilical vessels; those that have a local affect by acting directly on smooth muscle such as 5-HT, histamine and ET-1, and substances such as ANP,
NPY and NO (Cai et aL, 1993b; Chapter 4) which appear to have no direct local effect on umbilical vessels. It is suggested that these substances have an endocrine role, in that they may be released from the umbilical vessels to have an effect on the placenta or the fetus depending on whether they are released from the umbilical artery or vein.
132 Chapter 6
PHARMACOLOGICAL AND AUTORADIOGRAPHICAL
EVIDENCE FOR THE PRESENCE OF A P2X-PURINOCEPTOR IN HUMAN
UMBILICAL VESSELS FROM EARLY AND LATE PREGNANCY.
133 6.1 SUMMARY
1 Adenine nucleotides were examined for pharmacological activity in the human umbilical artery and vein from early and late pregnancy.
2 Autoradiography and radioligand binding studies with [^H] a^-M eATP demonstrated the presence of a P 2X“purinoceptor on the smooth muscle of the umbilical artery and vein from late pregnancy.
3 ATP and a,^-MeATP caused concentration-dependent contractions of the umbilical artery and vein at basal tone, from early and late pregnancy. However, ATP and 2-MeSATP did not cause vasodilatation on preparations with raised tone.
Constrictions to ATP were inhibited by a,)5-MeATP desensitization.
4 The results from this study suggest that P 2X“Purinoceptors, mediating vasoconstriction, are found in the human umbilical vessels from early and late pregnancy.
134 6.1 INTRODUCTION
The cardiovascular effects of adenosine compounds were first reported by
Drury and Szent-Gyorgyi (1929), and subsequently have been shown to have a dual effect on a wide variety of vascular beds (see Burnstock et al., 1990): ATP produces vasoconstriction via P 2x~purinoceptor on the vascular smooth muscle and vasodilation via P 2Y“Purinoceptors on endothelial cells or directly on smooth muscle of some vessels; adenosine produces vasodilation by acting directly on smooth muscle
P^-purinoceptor. With the use of autoradiographical and radioligand binding techniques, P2X“purinoceptors have been localised in a variety of vessels (including the rabbit femoral artery, rat saphenous artery and the guinea-pig mesenteric artery),
(Bo & Burnstock, 1993), which is in agreement with results from pharmacological experiments, in that, in vessels where potent P 2x~purinoceptors-mediated responses have been demonstrated, a dense specific labelling of ^^-MeATP was observed
(Burnstock, 1990).
There are brief reports of the responses of isolated placental vessels and the perfused human placenta, suggesting the presence of both P2X"^"^ ^2Y” purinoceptors j (Dobronyi eial., 1992; Dobronyi et aL, 1993; Read et at., 1993a).
During normal gestation there is a characteristic fall in vascular resistance (Macara et at., 1993), hence the present study was undertaken to investigate the role of ATP on human umbilical vessels throughout gestation.
135 6.2 METHODS
Human umbilical cords from legal terminations of pregnancy (early pregnancy group, gestational age, 15 (8-17) weeks, n=6) and from normal term vaginal deliveries (late pregnancy group, gestational age 39 (38-41) weeks, n=6) (see
Chapter 2, Section 2.1). The cords were placed into a modified Krebs' solution at 4°C and used within 12 hrs for pharmacological studies (see Chapter 2, section 2.2).
Vessels from segments of the same cords were dissected free of surrounding tissue and frozen in liquid nitrogen for radioligand binding studies and autoradiography (see
Chapter 2, sections 2.5 and 2.6).
136 6.3 RESULTS
6.3.1 RADIOLIGAND BINDING ASSAY
The binding of [^H]a^-MeATP to the membrane preparation of the human umbilical vessels from late pregnancy was saturable (figure 6.1 & 6.2). Curve-fitting revealed that a high-affinity binding site existed in the preparations (figure 6.1). The maximum density of high affinity binding sites was 634 ± 237 fmol/mg protein (n=4) for the umbilical artery and 947 ± 308 (n=3) for the umbilical vein, with the apparent dissociation constant (Kj) of 2.77 ± 1.10 nM and 3.23 ± 1.22 nM, respectively. Both the binding site density and dissociation constant were not significantly different between the artery and vein preparations. Umbilical vessels from early pregnancy were found too be to small to form membrane preparations, therefore the radio-ligand binding assay was not used on these vessels.
6.3.2 Autoradiography
Most of the vessels in this study were labelled after exposure for 2 weeks, which confirms the existence of a [^H]a,/3-MeATP binding site in these vessels, as shown for term vessels in figure 6.3. The specific [^H]a,^-MeATP labelling was observed over the smooth muscle of the vessels, but not over the surrounding connective tissues. As to whether the endothelial cells have been labelled, it is difficult to make out because the resolution of light microscopy autoradiography is not high enough to distinguish a single layer of cells.
137 6.3.3 PHARMACOLOGICAL EFFECTS
At basal tone, ATP and its stable analogue a,^-MeATP caused concentration dependent transitory contractions of the human umbilical artery and vein from early and late pregnancy. 2-MeSATP had no effect. Concentration-response curves were constructed for ATP and a,^-MeATP in the absence and presence of an intact endothelium in the artery and vein from late pregnancy (see Appendix, figure 6.4 &
6.5). Since the concentration-response curves for the individual analogues did not always reach a maximum it was not possible to calculate either the pDo values or the mean maximum tension developed. The P? antagonist suramin (up to a concentration of ImM) was tested against ATP and a,)5-MeATP and had no inhibitory effect.
However, suramin did cause constriction of a number of vessels. Contractions to ATP were abolished by desensitization with a,y5-MeATP. Indomethacin and L-NAME had no inhibitory effect on the concentration response curves. On tone raised by 5-HT,
ATP and 2 -MeSATP had no vasodilator activity.
138 Figure 6.1
A representative saturation curve of the [^H]a^-MeATP binding to the membrane preparation of the human umbilical artery from late pregnancy. Incubation was carried out at 4 °C for 120 min. Non-specific binding was determined in the presence of 100 /M ^,g-MeATP.
139 Bound (fmol/mg protein)
K) 00 o O o -g O o O o o o o 8 § O
to o w X p I 2 O t o B Ia H’
o\ o
o00 Figure 6.2
The plot of the specific binding fitted by the computer programme EBDA-
LIGAND. One binding site model was chosen based on the results of the F test of the weighed residual sum of squares.
l 4l 0 . 020-1
0.015-
0 . 010 - T3 § O PQ
0.005 -
0.000 0 200 400 600 800 1000 1200 1400
[ H]a,p-Methylene ATP Bound (fmol/mg protein) Figure 6.3
Autoradiographs of the human umbilical vessels. A-D Umbilical artery and E-H
Umbilical vein from late pregnancy. A and E Bright-field view of the vascular
sections (14 thick, stained with toluidine blue). B and F, Dark-field view of A
and E respectively, which shows overall distribution of [^H]a,j5-MeATP binding
sites in the sections. C and G, Dark-field views of the adjacent sections of A and E
respectively, which show the non-specific binding sites of [^H]cc,/3-MeATP in the
sections. D and H Dark-field views of adjacent sections of A and E respectively,
which show the non-specific binding sites in the presence of 50 j5y-MeATP and
50 yWM a,^-M eAT. Incubation was carried out at 30 °C for 15 min.
143 '■' - •> ;r
1 6.4 DISCUSSION
The results of both the radioligand binding assay and autoradiography on late pregnancy vessels demonstrate that human umbilical vessels possess an abundance of
P2x~purinoceptors. The localization of the receptors is in agreement with the pharmacological experiments, in that the human umbilical artery and vein demonstrated potent P 2x~purinoceptor-mediated responses via the smooth muscle and dense, specific labelling of [^H]a,^-MeATP has also been observed on the smooth muscle.
All the autoradiographs (figure 6.2) showed that the grains representing specific [^H]a,/?-MeATP binding sites, were distributed over the smooth muscle cells, where pharmacological studies have proposed that P 2X"P^J^^i^oceptors should be located. This is an important point because ATP and its binding proteins exist within every cell type, whereas P2x~purinoceptor are only found extracellularly in restricted cell types. Valera et al. (1994) has localised cellular P 2X"Purinoceptor mRNA in the smooth muscle layer of the rat umbilical artery, confirming our results from the autoradiographic study.
Our pharmacological studies indicate the presence of P 2X”purinoceptors on the smooth muscle cells of human umbilical arteries and veins from early and late pregnancy, mediating constriction. The receptor identified in this study is a classical P2X“Purinoceptor because a,Me ATP caused desensitisation of the response to ATP. The constrictions observed were transitory, which are characteristic of both ATP and a,j6-MeATP. ATP and a,/5-MeATP mediate transitory contractions via the stimulation of ligand-gated ion channels which are permeable to Na"^, K"^ and Ca"^"^, with an associated rapid increase in cytosolic calcium (Dubyak & El-Moatassim, 1993). Petit & Bélisle (1995) have demonstrated
145 an increase in intracellular calcium concentrations in human term placental cells, when stimulated by ATP.
No vasodilator role for ATP was observed in vessels from both early and late pregnancy. Maguire & Dobronyi (1992) suggested that ATP elicits vasodilatation via action on P 2Y~purinoceptors mediating NO release, and vasoconstriction via action on P 2X“pu^‘inoceptors in the human fetal-placental bed. Read et aL (1993a) have also suggested that ATP vasodilatation via P 2Y”P^rinoceptors is endothelium- dependent and linked to the formation of NO in the human placenta. Although a vasodilator role has been described in the human placenta for ATP (Read et al.y
1993a), this was not the case for human umbilical vessels. A possible role for ATP may be in the closure of the umbilical vessels at birth, since neuronal mechanisms are not involved as these vessels are not innervated (Fox & Khong, 1990). Irestedt et al.
(1989) have shown a rise in purine concentration in umbilical cord blood samples immediately post-partum. Prostanoids have been shown to mediate vasoconstriction in some vascular preparations, for example the rabbit aorta via PGEj^ and PGF 2^
(Khairallahet aL, 1967; Strong & Bohr, 1967). However, in this investigation prostanoids were not found to have a role in vasoconstriction. L-NAME was used to examine any dilatory response to ATP via NO, although the removal of the endothelium did not cause the potentiation of ATP and a,/5-MeATP, which implies that there is no vasodilatation via ATP.
The P2“purinoceptor antagonist suramin had no inhibitory effect on ATP and a,^-MeATP constrictions. Suramin also acts as an inhibitor of ecto-ATPases
(Hourani & Chown, 1989; Ziganshin et aL, 1995), which may account for its lack of effect on ATP, although an effect on û:,/5-MeATP may still be expected, since it is resistant to degradation. The occasional vasoconstriction observed in some vessels
146 after the application of suramin is somewhat unusual; however, a hyperpolarization was observed in the guinea pig superior cervical ganglia on application of suramin at equivalent concentrations (Reekie & Burnstock, 1994).
In summary, these results demonstrate the presence of a P 2x~purii^oceptor on the smooth muscle of the umbilical arteries and veins from late human pregnancy.
147 Chapter 7
DISCUSSION
148 In the General Introduction (Chapter 1), the main objective was to investigate the role of the endothelium in the non-innervated human umbilical artery and vein. In this general discussion, certain topics have been selected for more detailed consideration, (1) to discuss the results and their relation to those of others in a section entitled "Control of vessel tone by endothelial cells"; (2) to consider the
"Clinical implications of local control of vascular tone by endothelial cells" and finally (3) to discuss possible "Future directions of research".
9.1 Control of vessel tone by endothelial cells
The endothelium has been shown to be of great importance for the regulation of peripheral resistance and local blood flow (Furchgott & Zawadzki, 1980). In general, endothelial cells appear to act as sensors to a wide range of vasoactive substances via specific endothelial receptors to both locally released agents and circulating hormones
(see Ralevic & Burnstock, 1993).
Particularly exciting however, is the accumulating evidence that endothelial cells may store and release vasoactive substances. The various substances produced and/or released from the endothelium which can effect vascular tone include; PGI 2 and other prostanoids, Ag II, EDRF(s) and EDCF(s) (see Ralevic & Burnstock, 1993).
From the results of this and other studies it appears that an additional category should be added to this list to incorporate substances such as 5-HT, histamine, ET, NO and others. The first study to specifically suggest that endothelial cells might contain vasoactive substances of this category came from electron microscopical localization of ChAT, the ACh synthesising enzyme, in the endothelial cells of rat brain arteries
(Parnevelas et al., 1985) which strongly suggested that ACh is contained within these cells. Since this discovery, electron immunocytochemistry has been employed to demonstrate the presence of a variety of vasoactive substances in the endothelial cells
149 of a number of different vessels, for example, SP and 5-HT in the rat femoral and mesenteric arteries (Loesch & Burnstock, 1988).
Chapters 3, 4 and 5 demonstrate that the endothelial cells from the umbilical
arteries and vein from both early and late pregnancy contain a higher number of
organelles, with a synthetic appearance, implying that these endothelial cells are very
active and highly synthetic.
Electron immunocytochemical studies have shown the human umbilical endothelium to be a source of a number of peptides including ANP, NPY, VIP,
CGRP, A VP and SP in the artery and vein from late pregnancy (Cai et aL, 1993b,
1993c), and in this study the presence of 5-HT, histamine, ET and NOS in both the umbilical artery and vein from late pregnancy was demonstrated. This study has consistently demonstrated that the human umbilical venous endothelium is more active; in appearance (Chapter 1) and in containing a larger sub-population of endothelial cells containing ANP, NPY (Cai et al., 1993a) and other peptides (Cai et al., 1993c), NOS (Chapter 4) and histamine (Chapter 5). Therefore the umbilical vein may play an important role in fetal physiology.
While these studies (Chapters 4 and 6) demonstrate that various substances (5-
HT, histamine, ET and NOS) are localised in the endothelial cells, they do not
indicate whether these substances are present because they are synthesised within
endothelial cells or because they are taken up from the surrounding enviroment.
However, Cai et al. (1993a) have shown the presence of mRNA for ANP in the
endothelial cells of both the human umbilical artery and vein from late pregnancy,
which suggests that the umbilical endothelial cells of the artery and vein are both
150 capable of synthesising their own source of ANP.
In this study, despite the presence of NOS in endothelial cells local vasodilator control of the non-innervated human umbilical vessels does not appear to be endothelial-mediated. The results of McCarthy et al. (1994) do not support a major role for receptor-mediated release of EDRF in the main stem villous arteries of the human. However, in contrast, Xie & Triggle (1994) have shown ACh to relax human umbilical artery preparations, by an endothelial-independent, non-NO, non prostanoid and possibly a non-receptor mechanism. Endothelium-dependent responses to ACH has been shown to be low or negligible in fetal pig pulmonary arteries, but the response develops within 1-3 days of birth (Liu et al., 1989; Zellers
& Vanhoutte, 1991). Therefore the variability in responses observed by various authors (see Chapter 1) to ACh may possibly reflect developmental differences.
Although some authors have shown NO to have a role in the placental circulation, it's role in the umbilical vessels is not clear. These studies do not support a role for receptor-mediated release of NO in the human umbilical vessels throughout gestation, despite a small subpopulation of NOS-containing endothelial cells in late pregnancy. No relaxation was elicited to ACh, SP and ATP, in contrast to other systems. The lack of response to A21387, a calcium ionophore, which directly increases intracellular calcium in endothelial cells is also suprising. However, relaxation was induced by SNP, an NQdonor. Electron immunocytochemistry showed a subpopulation of endothelial cells staining positive for NOS in the artery and vein from only late pregnancy. It may be possible that the expression of NOS is developmentally regulated, as in the case of the pulmonary vessels in the ovine fetus
(Shaul et al., 1993). Antagonist studies of EDRF synthesis and action has demonstrated an increase in perfusion pressure in the human placental lobule (Myatt et al., 1991a), though this was not the case following inhibition of prostacyclin
151 production with aspirin (Jacobson et al., 1991), suggesting that EDRF, unlike prostacyclin, may predominate over smooth muscle relaxation within the feto placental circulation. While there appears to be a role for EDRF in the placenta, the role of EDRF in the umbilical vessels appears to be very different. The umbilical vessels do not appear to dilate by a receptor mediated mechanism, it may be that the amount of EDRF released is not enough to stimulate the umbilical vessels, or that the mechanism linking NO to dilatation is impaired, or even possibly that the cGMP mechanism is linked with constriction rather than dilatation in this system. The amount of EDRF released may not be sufficient to cause relaxation of adjourning vascular smooth muscle (Griggs et at., 1989). The physiological role of EDRF in these conduit vessels may be to prevent platelet aggregation and adhesion to endothelial cells. It is even possible that NO may be stimulated by an alternative mechanism, such as shear stress and/or an increase in fast flow as has been demonstrated in other vasculature systems, eg the rabbit femoral artery (Hecker et al.,
1993). However, NO release from human umbilical endothelial cells may depend on a number of factors; Dr. P. Bodin and colleagues (personnel communication) demonstrated a synergistic effect between fast flow and hypoxia in HUVECs.
HUVECs will only release ATP when stimulated by both fast flow and hypoxia, separately, fast flow and hypoxia do not cause the release of ATP. Hypoxia is known to cause constriction of the placental vessels (Howard et al., 1987). Therefore, EDRF may be released to counteract the hypoxia.
These studies have also demonstrated that the endothelial cells contain the vasoactive substances 5-HT, histamine and ET. All these substances had a local
effect causing vasoconstriction independently of the endothelium. However, in this
study we have shown that indirectly 5-HT may induce the relaxation of the human
umbilical artery from late pregnancy. Although the results were not significant there
152 was a clear tendency for the response to 5-HT to be attenuated in the absence of an intact endothelium.
Coceani et al. (1989) has demonstrated a role for ET in the mechanism that regulates the closure of the ductus arterious. ET-1 induces dose-related constriction in vitro of strips of ductus arterious from mature fetal lambs. ET-1 constricts the umbilical vein at a concentration lOOOx lower than PGF 2q (Haegerstrand et al.y
1989). ET may also be acting as a hormone in the fetal-placental circulation combating dehydration as it has been shown to induce the release of vasopressin
(Mitchell 1990). The vasoactive substances ANP and NPY which have also been localised in the endothelial cells of human umbilical vessels from late pregnancy (Cai et al., 1993b) had no effect on local vessel tone.
ANP and NPY may have an endocrine role rather than an autocrine/paracrine role suggested for 5-HT, histamine, ET and NO. ANP mRNA has been localised in situ in the endothelial cells of the human umbilical vessels, implying synthesis rather than uptake. ANP has been found to be important in the fetus and the placenta. Cardiac levels of ANP have been found to be higher than umbilical vein levels suggesting release from the fetal heart. No evidence has been found to suggest a paracrine action of ANP in the placenta, thus ANP appears to have a purely endocrine action within the fetal-placental circulation.
153 9.2 CLINICAL IMPLICATIONS OF LOCAL CONTROL OF VASCULAR
TONE BY ENDOTHELIAL CELLS
Pre-eclampsia complicates 6% - 8% of human pregnancies and is one of the major causes of maternal and fetal mortality and morbidity. The placenta is thought to play a key role in the genesis of pre-eclampsia, since the symptoms disappear following delivery of the placenta (Roberts et al., 1989). In pre-eclampsia and lUGR there is an increase in vascular impedance in the placenta, it is also thought that endothelial cell function may be impaired in the placenta and EDRF production has been shown to be impaired in the endothelial cells of women with pre-eclampsia
(Pinto et al., 1991), although mounted stem villous arterioles from pre-eclamptic placentas did not demonstrate impaired endothelium-dependent relaxation (McCarthy ct al., 1993). PGIo production by the umbilical artery has been shown to be decreased in pregnancies complicated by pre-eclampsia (McLaren et al., 1987) and lUGR
(Stuart et al., 1981). Inayatulla etal. (1993) found that pre-eclampsia is not associated with an increased activity of vasoconstrictors or a decreased activity in vasodilators, but placental arteries showed increased oscillations in pre-eclamptic women. Read et al. (1993b) found that changes in oxygen tension affects endothelial derived NO, more so than vascular reactivity to NO. This may be important in conditions were endothelial cell damage occurs, in conditions such as pre-eclampsia.
Placental ischemia and hypoxia associated with pre-eclampsia may further compromise endothelial cell function.
Pregnancy induced hypertension (PIH) is primarily a disease of the mother, however there is uneqivocal evidence of functional abnormalities of fetal endothelial cells (Remuzzi et al., 1992). Umbilical artery velocity waveforms in PIH, diabetic I pregnancies! and indicate an increase in impedance to blood flow and probably have impaired distendability of vessels (Erskine & Ritchie, 1985; Trudinger et al..
154 1985; Bracero et al., 1986). Diminished formation or action of endothelial-derived vasodilators or increased formation of endothelial-derived vasoconstrictors by umbilical or placental vessels could partially explain events seen (Chaudhuri et at.,
1991). Some of the pathological changes observed within the feto-placental circulation from complicated pregnancies may possibly develop in the fetal systemic circulation; thus an adverse prenatal environment may induce specific changes in fetal physiology which programmes the fetus to develop diseases in later life. Animal studies show that such programming can occur (Swenna et al., 1987).
Epidemiological studies suggest that similar events may occur in human fetuses, for example, low birth weight may be associated with the development of hypertension and cardiovascular motility as an adult (Gennser et al., 1988; Williams et al., 1992;
Barker et al., 1990). Pathological processes which might link low birth weight and
adult hypertension are not clear. However, chronic fetal hypoxia may activate the fetal
renin-angiotensin system, and since Ag II is a vascular growth factor it could induce
the hypertrophy seen in stem villous arterioles of the lUGR placenta, thus altering the
mechanical properties of the stem villous arteries (Lever, 1986). It has also been
shown that the inhibition of AG II production during early neonatal life can
reprogramme blood pressure in the spontaneously hypertensive rat (Morton et al.,
1992). The possibility of determining the development of cardiovascular disease as an
adult in early fetal life illustrates the importance of understanding more precisely the
pathological processes which result in low birth weight, and to do this we must
understand the mechanisms governing the normal healthy feto-placental vasculature.
Hypoxia is demonstrable in a portion of pregnancies complicated by lUGR
with abnormal Doppler findings (Nicolaides et al., 1988), which, if chronic in nature,
may indirectly influence endothelial cell function. Hypoxia has recently been shown
to stimulate endothelial cell expression of endothelin (Douglas et al., 1991). Fetal
155 levels of ET are thought to be elevated in lUGR particularly if abnormal umbilical
Doppler S/D ratios are present (Hartikainen-Sorri et al., 1991b; McQueen et al.,
1993). The link between lUGR and increased feto-placental impedance may be due to increased levels of endothelin. Hypoxia has also been shown to increase cultured endothelial cell production of ET-1 (Heida et al., 1993). However, we and others have failed to demonstrate the release of ET in the perfused umbilical vein
(unpublished observation) and in the perfused placental cotyledon under hypoxic and acidotic conditions (Kingdom et al., 1993). Endothelial-derived NO inhibits the formation of ET via a cGMP-dependent mechanism (Boulanger & Lüscher, 1990).
This may have important implications in vessels with impaired endothelial-derived
NO release, leading to enhanced ET production and increase vascular impedance.
Systemic regulation of the feto-placental circulation by fetal endocrine signals may also be affected in pregnancies complicated by lUGR. Elevated levels of ANP in the umbilical vein have been shown in pregnancies complicated by lUGR (Kingdom et al., 1992) and in pre-eclampsia (Hatjis et al., 1989). Functional vascular receptor expression for ANP in feto-placental vessels have been shown to be increased in pregnancies complicated with lUGR (Kingdom et al., 1994).
9.3 Future directions for research
This investigation has studied the influence of the vascular endothelium on
the local control of human feto-placental vascular tone and how these influences may
vary with gestation. The individual studies have raised some novel and interesting
points, which have revealed a number | of directions in which this investigation could
follow, to further examine the role of the endothelium during pregnancy.
The development of in situ methods to study autocrine/paracrine and
156 endocrine endothelial cell function is advancing our understanding of the control of the human feto-placental circulation. Such methods allow us to study these events in an undisturbed manner. In situ methods will also allow the interpretation of molecular signals alongside any derangement of vascular anatomy (eg. smooth muscle hypertrophy or endothelial cell damage). Immunocytochemistry demonstrates the presence of a particular substance within a cell, for example 5-HT, histamine, ET,
NOS, ANP, NPY, VIP, AVP, CGRP and SP in human umbilical endothelial cells (see
Chapter 4 & 5, Cai et al., 1993b, 1993c). What it does not show is whether it is there because of uptake or synthesis. Cai et al. (1993a) has demonstrated ANP mRNA in the endothelial cells of HUVECs, implying that the synthesis of ANP occurs in
HUVECs. It would be interesting to repeat this for the vasoactive substances studied in this thesis and for any vasoactive substance located, in human umbilical endothelial cells. During normal pregnancy, the placenta produces equivalent amounts of Tx and
PGI2 (Walsh, 1985), however, Walsh (1985) has demonstrated that in pre-eclamptic pregnancies the placenta produces seven times more Tx than PGI 2. Maternal plasma endothelin levels have been shown to be increased in pre-eclamptic pregnancies
(Nova et al., 1991) and fetal endothelin levels are thought to be elevated in lUGR
(Hartikainen-Sorri et al., 1991b) and Taniguchi et al. (1994) have demonstrated
higher levels of 5-HT in umbilical cord plasma from pre-eclamptic pregnancies. It would therefore be of great interest to compare quantitatively mRNA in endothelial
cells from normal pregnancies with pregnancies complicated by lUGR and pre
eclampsia. Computer-assisted image analysis will facilitate comparison of such data
between placenta from normal and abnormal pregnancies.
Inadequate invasion of maternal spiral arteries by extravillous trophoblast is
probably of fundamental relevance to any disorder of the feto-placental circulation,
thus hypoxia could therefore be viewed as a potential trigger for any changes
157 observed in the mechanisms which govern vasomotor regulation within feto-placental
circulation. The influence of oxygen tension on terminal villous development may
thus be a crucial process to understand since this site may influence overall vascular
impedance.
McCarthy et al. (1994) have found that relaxation to endothelial-dependent
vasodilator SNP was reduced in high oxygen tensions. Van de Voorde et al. (1987)
and Chaudhuri et al. (1991) have both suggested, but not proved that high oxygen
tensions may contribute to poor relaxation to vasodilators in umbilical vessels. Fetal p0 2 is normally very low, so that the fetus is hypoxic relative to the mother during
gestation, with the placenta forming an important barrier in preventing a rise in p0 2
in the fetal circulation. However, in these studies all experiments were carried out at
95% Oo/ 5% COo, it would be interesting to perform these experiments at
physiological oxygen tensions. However, initial studies have failed to demonstrate
endothelial-dependent relaxation at physiological oxygen tensions.
The flow of blood to the maternal side of the placenta is of vital importance
to the fetus because it provides for delivery of nutrients and oxygen necessary for the
survival and growth, as well as the removal of the waste products of fetal metabolism.
Reduced uteroplacental blood flow is associated with lUGR and increased fetal
mortality (Woods, 1993). During pregnancy both maternal vascular resistance and
smooth muscle contractility are reduced, whilst blood flow is redistributed (Peeters et
al., 1980; Weiner et al., 1989) and Weiner et al. (1994) have shown that NOS activity
rises in early pregnancy. The maternal circulation is continually j undergoing changes
throughout pregnancy, it is of great importance to consider these changés when
investigating the feto-placental circulation, and vice versa.
158 It is of particular interest that no vasoactive response was observed to the peptides ANP and NPY (Chapter 5), although they have been localised in the endothelial cells from human umbilical artery and vein (Cai et al., 1993b). In the case of ANP, the mRNA has also been localised, implying synthesis of this peptide. ANP has been identifiedj in umbilical cord blood samples (Yamaji et at., 1986) and there is evidence to show that the placenta is impermeable to peptide hormones (Adam et al.,
1972; Winters et al., 1974), suggesting that ANP is a circulating hormone, produced by the fetus. Cheung & Brace (1988) have found in sheep that acute hypoxia was the most potent stimulus for fetal ANP release. Arginine vasopressin, another peptide localised in human umbilical endothelial cells (Cai et al., 1993c) and with no vasoactive effect on umbilical vessels (unpublished observation), has been shown to augment the Atrial natriuretic factor response to hypoxia in the ovine fetus (Cheung,
1992). It would be interesting to study the umbilical vessels as a possible source of vasoactive substances for the fetus and to investigate the influence of these substances on one another.
In conclusion, this thesis has extended current information on the regulation of fetal-placental vascular control throughout pregnancy.
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190 APPENDIX Figure 5.12
Concentration response curves for 5-HT on early (Q) and late pregnancy (^ ). (a)
Umbilical artery and (b) umbilical vein. Symbols show mean % contraction of KCl
(120 mM) ± S.E.M. (unless masked by symbol). After Student r-test (unpaired) P <
0.05 was taken as significant. * indicates significance. Va oururaciion
270 , * * *
240 .
210 _
180 .
150 _
120 _
90 .
60 _
3 0 .
0 J
r 10 8 7 6 - Log (5-HT) % Contraction 270 _ * *
240 _
210 _
180 _
150 _
120 _
90 _
60 _
30 _
0 J
10 9 8 7 6 5 4 - Log [5-HT) FIGURE 5.11
(a) Cumulative concentration-response curves to 5-H T on the human umbilical artery from late pregnancy in the absence ( ^ ) and presence ( Q ) of methysergide
(10 yWM). (b) Cumulative concentration-response curves to 5-H T on the human umbilical vein from late pregnancy in the absence and presence ( A ) of methysergide (10fiM). Symbols represent% contraction ± S.E.M. (unless masked by symbol)..pa 110 -1
100 - 90 80 c o '4-» 70 o «g k_ 4—' 60 c O ü 50
ü 40
X 30 20 10 0
10 9.5 9 8.5 8 7.5 7 6.5 5.5 4.5 - Log [5-HT] 110 n 100 - 90 - 80 - c o 70 - u (O 60 - o ü 50 - ü 40 -
« 30 -
20 - 10 - 0
10 9.5 8.5 8 7.5 7 6.5 5.5 4.5 - Log [5-HT] n G U R E 6.4
(a) Cumulative concentration-response curves to ATP on the human umbilical artery from late pregnancy in the absence (Q ) and presence ( ^ ) of an intact endothelium.
(b) Cumulative concentration-response curves to ATP on the human umbilical vein from late pregnancy in the absence ( ^ ) and presence (Æ ) of an intact endothelium.
Symbols represent% contraction ± S.E.M. (unless masked by symbol). 90 80 c o ’♦-> 70 o (O L- 4—' 60 c o 50 o 40 30 20 10 0
6.5 5.5 5 4.5 3.5 - Log [ATP] a 100-1 90 - 80 - I 70 H o S 60 H c o O 50 - ^ 40 H 30 -
20 -
10 -
0 -
6.5 5.5 5 4.5 3.5 - Log [ATP] FIGURE 6.5
(a) Cumulative concentration-response curves to cc^-MeATP on the human umbilical artery from late pregnancy in the absence ( Q ) and presence ( ^ ) of an intact endothelium, (b) Cumulative concentration-response curves to Oj^-MeATP on the human umbilical vein from late pregnancy in the absence ( ^ ) and presence
of an intact endothelium. Symbols represent % contraction ± S.E.M. (unless masked
by symbol). fi) % KCI Contraction
-k r\) w ^ ui G) -nI 00 co O o o o o o o O o o o o L 1 I I 1 J 00 - ,
a i
-
o P cQ en
Q %
> ^ H "D en en
en -
en % KCI Contraction O"
ro w cn O) -> i 00 CO o o o o o o o o o o
0 0 - I
O l
-
o P CO cn
Q 0 0 I > o > - H 13
CJl cn
cn -
4 ^ cn