Proc. Indian Acad. Sci. (Chem. Sci.), Vol. 111, No. 1, February 1999, pp. 207-213
© Indian Academy of Sciences
Collagen of the umbilical cord and its alteration in EPH-gestosis (preeclampsia)
E D W A R D BAI(IKOWSI(I
Department of Biochemistry, Medical Academy of Bialystok, ul.
Mickiewicza 2, 15-230 Biatystok-8, Poland email: edward @ amb.ac.bialystok.pl
Abstract. Edema, proteinuria, hypertension (EPH)-gestosis, known in the literature also as preeclampsia, is the most common, pregnancy-associated pathological process.
It is accompanied by significant alterations in the extracellular matrix of the umbilical cord. In particular, a distinct (two-fold) increase in collagen content and alterations between the proportional amounts of type I : type III collagens have been tbund in the umbilical cord arteries (UCAs). An accumulation of collagen in the UCAs of newborns delivered by mothers with EPH-gestosis is accompanied by a significant reduction in the amount of elastin in the arterial walls. Metabolic studies have demonstrated that changes in collagen and elastin biosynthesis and degradation rates are responsible for these phenomena. These alterations co-exist with early replacement of hyaluronic acid by sulphated glycosaminoglycans, both in the arterial walls and in the surrounding Wharton's jelly. Such phenomena correspond to
"premature ageing" of the extracellular matrix of the umbilical cord tissues in fetuses of mothers with EPH-gestosis. It may be the case that similar changes occur in other fetal arteries also. This may result in an increase in peripheral resistance as weU as in blood pressure in the fetal vascular system. The raised blood pressure may persist after birth. One suspects that EPH-gestosis is a factor which evokes the initiation of hypertension in utero and sustains its amplification throughout childhood and adult life.
Keywords. Collagen; umbilical cord; EPH-gestosis; preeclampsia.
1. Introduction
The umbilical cord forms the connection between the placenta and the fetus. In cross- section, the umbilical cord is shown to have a characteristic gross morphology consisting o f one vein and two arteries surrounded by Wharton's jelly and covered by simple amniotic epithelium. Wharton's jelly is modelled as mucous connective tissue, consisting of mesenchymal cells, immersed in a ground substance. This substance is rich in fine collagenous fibrils and hyaluronic acid and contains some sulphated glycosaminoglycans.
Some prenatal pathological processes may be caused by biochemical and morphological alterations in the umbilical cord. Stricture of the umbilical cord, usually accompanied by torsion and characterised by fibrosis of Wharton's jelly and a thickening o f the vascular wall which obstructs the fetoplacental circulation, leads to anoxia and fetal death 1. Absence of Wharton's jelly in the area of the cord involved is believed to be the etiologic factor in constriction and subsequent torsion 2. Gestational diabetes is accompanied by alterations in the pattern of distribution of collagen fibres in Wharton's jelly with large empty spaces occurring amongst them. It is suggested that gestational 207
diabetes exerts a deleterious effect on umbilical vessels and the connective tissue component of Wharton's jelly 3. According to Silver et al 4 a shortage of Wharton's jelly in the umbilical cord increases the risk of cord compression in prolonged pregnancy.
Umbilical cord anomalies, e.g. absence of Wharton's jelly may result in antenatal fetal death 5. It has been suggested that pathology of the umbilical cord occurs frequently but often goes unreported by obstetricians due to the fact that umbilical cords are not carefully examined in all cases of stillbirth 1.
Edema, proteinuria, hypertension (EPH)-gestosis, known in the literature also as preeclampsia, is the most common, pregnancy-associated pathological process. For these reasons studies on the extracellular matrix of the umbilical cord and its alterations in EPH-gestosis are very important.
2. Collagen of Wharton's jelly
Ultrastructural studies of Wharton's jelly cells have been performed by Takechi et al 6. It was found that modified fibroblasts or myofibroblasts are the main cellular components of this tissue. Immunohistochemically, these myofibroblasts stained positive for actin, non- musclemyosin, vimentin and desmin. It was suggested that they may contribute to the elasticity of Wharton's jelly by synthesising collagen fibres. It was also suggested that they may participate in the regulation of umbilical blood flow by virtue of their contractile properties 1. The presence of mast cells has been found in Wharton's jelly, most frequently in close proximity to the three blood vessels 7. Mast cells are known to produce some sulphated glycosaminoglycans 8.
The extracellular matrix of Wharton's jelly is very abundant and contains significant amounts of hyaluronic acid and some sulphated glycosaminoglycans immobilized in an insoluble collagen fibril network 9-11.
Wharton's jelly is a collagen-rich tissue. The amount of collagen in this tissue is four times higher than that contained in the wall of the umbilical cord artery 12. It constitutes 3.6% of wet weight 13 and above 50% of the weight of defatted, dry tissue x2. The collagen of Wharton's jelly demonstrates some characteristic features. First of all, it is very insoluble in neutral salt (1 M NaC1) and in slightly acidic solution (0-15 M citrate, pH 3.7) and appears to be resistant to the action of depolymerizing agents 12. It is known that polymeric collagen treated with pepsin in acetic acid becomes soluble in acidic solutions 14. Almost the total amount of collagen of the UCA was solubilised in these conditions. In contrast to this, despite long-lasting digestion with pepsin, less than half of Wharton's jelly collagen became soluble in 0.5 M acetic acid lz
The extremely low solubility of Wharton's jelly collagen is an unexpected phenomenon. It is common for large proportions of collagen to be extracted from the tissues of young subjects. A decrease in collagen solubility is usually associated with the ageing process. It is attributed to age-related increases in covalent and noncovalent cross- links between collagen subunits. It seems rather unlikely that the collagen of Wharton's jelly is a highly cross-linked material. However, stabilisation of collagen fibres may occur by other means. An increase in the number of weaker forces that stabilise macromolecules and their aggregates, such as van der Waals bonds, ionic interactions, hydrophobic bonds, and a combination of such forces, could account for changes in the physical and chemical properties of collagen fibres. High concentrations of glycosaminoglycans and proteoglycans surrounding the collagen fibres may also affect the solubility of this protein. The negatively charged sulphated glycosaminoglycans are able to develop strong
Collagen of the umbilical cord 209 ionic bonds with the positively charged amino acids on the surface of collagen fibres, particularly lysine, hydroxylysine and arginine 15"16. Such complexes may make the collagen less soluble and the jelly more compact. Furthermore, the low solubility may be evoked by a relatively high proportion in the amount of type III collagen. It is well-known that collagen of this type demonstrates lower solubility than type I collagen 14. The low susceptibility of Wharton's jelly collagen to the solubilising action of pepsin may result from limited access of the enzyme (pepsin) to the substrate (polymeric collagen) surrounded by polysaccharides.
Fractionation of pepsin-solubilised collagen by differential salt precipitation has made it possible to separate four collagen fractions. Three of them were identified by polyacryl- amide gel electrophoresis (PAGE) as type I, type III, and type V collagen and the proportional relationship between them was determined to be 47%, 40% and 12%, respectively. The fourth (unidentified) fraction constituted about 1% of the total collagen 12
It is of interest that the proportional amount of type III collagen in Wharton's jelly is very high. It is a rule that type I and type III collagens co-exist in most human soft tissues.
Type I always dominates over type III 17 Only the liver contains almost equal amounts of both 18
Ryynanen et a111 reported that the fibroblast-like ceils, isolated from Wharton's jelly are able to synthesise type VII collagen (at least in vitro). Type VII collagen is a minor collagen found in anchoring fibrils. It is expressed predominantly by epithelial cells, mainly by keratinocytes. These findings demonstrated that cells other than keratinocytes are able to express the type VII collagen gene. Furthermore, ultrastructural studies of Wharton's jelly, performed by Franc et a119, have demonstrated the presence of type VI collagen. It may be suggested that the unidentified collagen fraction ~2 contains pepsin- degraded type VI or type VII collagens.
The extracellular matrix of umbilical Wharton's jelly is very rich in striated collagen fibrils with diameters ranging from 30 to 60 nm. They are organised in more or less large spiral bundles according to the collapsed or distended state of the cord. In each bundle, the fibrils are associated with a finely particulate material rich in hyaluronic acid. Among the fibrillar collagen, many microfibrils are present. They are either dispersed or gathered in bundles, sometimes embedding striated collagen fibrils. Each microfibril has an average diameter of 10-12 nm and sometimes appears tubular in cross-section. Typical microfibrils of collagen VI with beads at distances of 100 nm have also been observed in fibrillar collagen 19. The extensive distribution of type VI collagen may be of functional significance. It could play a role in the anchorage of stroma cells or mediate the linkage between the main fibrillar matrix components and hyaluronic acid 19.
In addition, other microfibrils with no specific banding pattern have been observed.
These may constitute an organisation of type V collagen different from the one which is generally assembled in heterotypic fibrils with type I collagen 19
According to Vizza et a120, Wharton's jelly appears as a spongy network of interlacing collagen fibres and small woven bundles apparently arranged at random and forming a continuous soft skeleton that encases the umbilical vessels. The collagen fibrillar network shows the presence of a wide system of interconnected cavities consisting of canalicular- like structures as well as cavernous and perivascular spaces. The presence of cleft-like territories occupied by homogeneous ground substance has also been described by Nanaev et al 2,. This system of cavities might play a mechanical role, allowing the storage of the ground substance of the jelly and assisting its diffusion during twisting or
compression. Furthermore, it may have an important role in facilitating the penetration of diffused materials (i.e. water and trophic metabolites) throughout the jelly, either from or to the umbilical vessels and the amniotic cavity, thus overcoming the lace of a proper vasculature with the jelly 20.
EPH-gestosis does not affect the collagen content in Wharton's jelly and does not change the mutual quantitative relationship between collagens of various types 22. On the other hand premature replacement of hyaluronic acid by sulphated glycosaminoglycans has been observed. This leads to suspicion that EPH-gestosis-induced changes in the glycosaminoglycans composition may alter the fibrillogenesis conditions in Wharton's jelly.
Microscopic observation has demonstrated that bundles of collagen fibres and numerous chaotically composed microfibrils are more compactly arranged in the Wharton's jelly of newborns delivered by mothers with EPH-gestosis in comparison to a control group of newborns 22.
3. Collagen of the umbilical cord arteries (UCAs)
The collagen composition of the umbilical cord artery is similar to that of other arteries 23.24. It is of interest that the total amounts of collagen in EPH-gestosis UCAs are more than twice as high as those in control arteries. Both control UCAs and those taken from newborns delivered by mothers with EPH-gestosis contain types I, III, IV, V and probably VI collagens though significant quantitative differences between them have been observed. Type I and type ]II collagens were found to be the most abundant of the umbilical cord arterial collagens. In control arteries they constitute about 63.5 and 25.5%
of total collagen respectively. Type IV, V and VI collagen contents were distinctly lower.
They constitute 3.0, 3.6 and 4.4% of total collagen respectively. The walls of arteries taken from the newborns of patients with EPH-gestosis contains a significantly higher percentage of type III collagen (35%) than the control material. At the same time the proportional amount of type I collagen is distinctly lower with a value of 50.4% 24.
An increase in type III collagen in EPH-gestosis UCAs is in agreement with morphological studies 24. Significantly more reticulin fibres have been found in the arterial walls of newborns delivered by mothers with EPH-gestosis in comparison to normal cord arteries. It is known that type 1II collagen is the main constituent of reticulin fibres 14
An increase in the collagen content of the umbilical cord arteries of newborns delivered by mothers with EPH-gestosis is accompanied by a decrease in its solubility 24.
This phenomenon is probably connected with an increase in the degree of collagen polymerisation or may be evoked by an increase in the amount of type III collagen which is less soluble in comparison to type I collagen 14.
A significant increase in the collagen content of the UCAs of newborns delivered by mothers with EPH-gestosis may result from increased biosynthesis, decreased degradation or by a combination of both phenomena. It was therefore decided to evaluate the biosynthesis of this protein by isotopic methods and to investigate its degradation by
"pulse chase" experiments 25
It is well-known that collagen is the major human protein containing hydroxyproline.
In contrast to other proteins it is specifically digested by bacterial collagenase. For these reasons the assays of hydroxyprolyl residues and collagenase-sensitive proteins allow evaluation to be made of the amount of collagen in the tissues under investigation.
Collagen of the umbilical cord 211 Incubation of arterial tissue slices.with radioactive proline results in incorporation of this amino acid both into collagen and noncollagenous proteins. It was found that the amount of radioactive proline, incorporated into total proteins synthesised by EPH- gestosis UCAs was distinctly higher in comparison to that of controls zs.
Digestion of radioactive proteins with collagenase made it possible to evaluate the amount of newly synthesised collagen and to determine the proportional relationships between collagen and noncollagenous proteins. It was found that the arterial tissue under investigation synthesised 52% more collagen in comparison to the control. Collagen constituted about 22% of the radioactive proteins in the control and much more (almost 27%) in the experimental material z5
A similar conclusion can be drawn from the evaluation of proline hydroxylation (another index of collagen biosynthesis). It was found that EPH-gestosis results in a significant increase (about 63%) of radioactive hydroxyproline formation in the UCAs 25.
The "pulse chase" experiment demonstrated a rapid degradation of collagen synthesised by the slices of control UCAs. During the chase period, the amount of radioactive collagen in the control samples significantly decreased. At the beginning of the chase period collagen constituted 21-9% of total radioactive proteins whereas after two hours it had decreased to 11.4%. In contrast to this, the UCA slices taken from newborns delivered by mothers with EPH-gestosis demonstrated only a slight (about 15%) decrease in radioactive collagen content during the chase period. In comparison to collagen, the radioactivity of non-collagenous proteins did not change much during the chase period either in the control or experimental material 25.
It was found that in the control UCAs more than half (53%) of the newly synthe- sised collagen was degraded to low molecular products during the 2 h chase period. Over the same period in EPH-gestosis UCAs only 14% of newly synthesised collagen was degraded.
Degradation of collagen may occur both intracellularly (before its secretion) or extracellularly. The "pulse chase" experiments performed by Bienkowski et a126 demonstrated that the amount of intracellularly degraded collagen attained a value equal to approximately 20% of the amount synthesised during the labeling period. Similar observations have been reported by Andersson and Warburton z7.
It may be concluded from our studies that EPH-gestosis-associated increase in the collagen content of the umbilical cord arteries may result both from enhanced biosynthesis and decreased intracellular and extracellular degradation of newly synthesised collagen. A combination of these two phenomena has also been described by Bishop et al 2s. Arterial hypertension seems to be a factor which enhances both processes.
They found that the experimentally induced pressure overload in the pulmonary artery of rabbits (reducing the diameter of this artery by 50%) results in right ventricular hypertrophy accompanied with a six-fold increase in collagen synthesis and a decrease (by half) in the collagen degradation rate in the right heart ventricle. This resulted in more than a two-fold accumulation of collagen in the myocardium after 14 days of the experiment. Over the same period no significant changes were seen in the left ventricle 26.
It would seem possible that an EPH-gestosis associated increase in the collagen content of the umbilical cord artery may be the result of a similar process. An increase in blood pressure in the UCA may enhance the biosynthesis of collagen and decrease its degradation. This may result in an increase in collagen content of the arterial wall 24.
The increase in type III collagen was proportionally higher in comparison to type I collagen 24. This leads to the conclusion that the stimulation of collagen biosynthesis and
the decrease in degradation of newly synthesised collagen are more pronounced in the case of type III than type I collagen.
It is of interest that EPH-gestosis-induced increase in collagen content of the UCAs is accompanied by a decrease in the amount of elastin. In contrast to collagen, the reduction of the elastin content in the umbilical cord arteries is a result of a decrease in the biosynthesis rate and accelerated degradation of this protein 29.
4. Clinical implications o f EPH-gestosis-induced alterations in the extracellular matrix o f the umbilical cord
The EPH-gestosis-associated changes in the extracellular matrix of the umbilical cord arteries and in Wharton's jelly may be responsible for various disturbances of the arterial- wall function. A decrease in hyaluronic acid content in the UCAs and in Wharton's jelly with a simultaneous increase in the collagen content and reduction in the amount of elastin in the UCAs may decrease the hydration of the umbilical cord tissues and reduce the elasticity of the umbilical cord arteries. It would seem possible that similar alterations might occur in other fetal arteries. This may result in an increase in the peripheral resistance as well as an increase in blood pressure in the fetal vascular system. Blood pressure in the fetal circulation might be' raised as a method of maintaining placental perfusion, and the raised pressures may persist after birth 30. One might expect that the EPH-gestosis-associated changes in the arterial walls could affect the vascular system in adulthood.
EPH-gestosis is a syndrome which results in reduction in the birth weight of newborns.
In fetuses with retarded growth, Doppler ultrasound has shown evidence of increased peripheral resistance to blood flow in the descending aorta 3°. According to Gennser et a131 some cases of intrauterine growth retardation are associated with maternal hypertension.
It is apparent from studies by several authors that being born small for gestational age may be a predictor of raised blood pressure in childhood and adult life
31-34.
One suspects that EPH-gestosis is a factor that is involved in the initiation of hypertension in utero which, in turn, maintains its amplification throughout childhood and adult life.References
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