916 J3 Clin Pathol 1996;49:916-919 Increase in the relative level of type V during development and ageing of the placenta J Clin Pathol: first published as 10.1136/jcp.49.11.916 on 1 November 1996. Downloaded from

M Iwahashi, A Ooshima, R Nakano

Abstract cells, and in preventing the loss of amniotic Aim-To obtain some insight into the fluid.8 In the present study, we attempted to in the placenta, investigate relative concentrations of type V changes in the composition of collagen in extracts of placental tissue in the during placental development were inves- second and third trimester of pregnancy. tigated. Methods-Collagen was extracted from placentas (group 1, 25-30 weeks, n = 21; Methods group 2, 31-36 weeks, n = 32; and group 3, This study was approved by the Committee on 37-41 weeks of gestation, n = 40) and the Investigations Involving Human Subjects, relative concentrations of various colla- Wakayama Medical College. Informed consent gens were evaluated by SDS-PAGE. was obtained from each subject after the Results-The ratio of the intensity of the purpose and nature of the study had been al (III) band to that ofal (I) chain collagen explained fully. in group 3 placentas were lower than those in group 1 placentas. In contrast, the ratio ofthe intensity ofthe al(V) band to that of TISSUE al(I) chain collagen in group 3 placentas Ninety three placentas (group 1, 25-30 weeks, were higher than those in group 1 and n = 21; group 2, 31-36 weeks, n = 32; and group 2 placentas. group 3, 37-41 weeks of gestation, n = 40), Conclusions-These results suggest that obtained at vaginal delivery from women aged might play an important 19-39 years with uncomplicated pregnancies, role in the function of the placenta and were investigated. The chorion and amnion that an increased relative concentration of were removed, and specimens were cut from type V collagen might be closely associ- three separate central zones. Necrotic, in- ated with the development and ageing of farcted and haemorrhagic areas were excluded. http://jcp.bmj.com/ the placenta. (_ Clin Pathol 1996;49:916-919) SODIUM DODECYL SULPHATE POLYACRYLAMIDE Keywords: type V collagen, placenta, ageing. GEL ELECTROPHORESIS (SDS-PAGE) OF PEPSIN SOLUBILISED COLLAGENS Minced samples of placenta were washed over- night in cold distilled water to remove any on October 2, 2021 by guest. Protected copyright. As the placenta grows and ages, certain types blood. Tissues were homogenised with a Poly- of histological change occur. Such changes tron homogenizer in 50 volumes of 0.5 M ace- include a decrease in the thickness of the tic acid containing 1 mg/ml pepsin (Sigma, St syncytium, partial disappearance of Langer- Louis, Missouri, USA). Collagens were ex- hans cells, a decrease in the stroma, thickening tracted with constant stirring for 24 hours at of the of capillaries and 4°C. The solutions were centrifuged at 39 000 Department of trophoblasts, obliteration of certain vessels, x g for one hour at 4°C. Collagens were Obstetrics and Gynaecology, and deposition of fibrin on the surface of the re-extracted from the pellets under the same Wakayama Medical villi. I conditions over 48 hours. The respective Coliege, Wakayama, The extracellular matrix (ECM) is consid- supernatants were then combined and colla- Japan ered to play an important role in the stability of gens were precipitated by addition of 4.0 M M Iwahashi tissue structure and in the regulation of cell NaCl to a final concentration of 2.0 M. Each R Nakano growth and differentiation.2" The distribution precipitate was dissolved in 0.5 M acetic acid Department of of components of the ECM, such as various and the solution was dialysed against 0.02 M Pathology collagens, fibronectin, and , in the pla- Na2HPO4. Precipitated collagens were redis- A Ooshima centa has been studied by immunohistochemi- solved in 0.5 M acetic acid, dialysed exhaus- cal methods.6 However, little is known about tively against 0.05 M acetic acid and finally Correspondence: Dr R the change in the composition of the ECM in lyophilised. The solubility ofthe tissue collagen Nakano, Department of Obstetrics and Gynaecology, the placenta during development and ageing. from each placental sample was estimated by Wakayama Medical College, Among the various collagens, type V collagen comparing the hydroxyproline content of the 27 Shichibancho, was originally described as a component of initial homogenate with that of the final Wakayama 640, Japan. chorionic and amniotic membranes.7 It is solution of collagen.9 Type V collagen was iso- Accepted for publication thought to play a major role in maintaining a lated by salt precipitation from pepsin digests 12 August 1996 barrier against pathogens and inflammatory of placental tissues, as described elsewhere.'01' Increase in the relative level of type 'collagen drl'izng development anid age'ing of the placenzta 917

The extracted type V collagen was also 1.2 - lyophilised. Estimations of the relative abun- A 10(111) dance of the c1l(III) and ol(V) chains were U.1 (1) J Clin Pathol: first published as 10.1136/jcp.49.11.916 on 1 November 1996. Downloaded from made by interrupted gel electrophoresis, as 1.0 described by Sykes et al.'2 Electrophoresis was performed in an 8% polyacrylamide gel slab (Sigma); 0.1 M phosphate buffer, pH 7.2, con- 0.8 taining 0. 1% SDS (Nacalai Tesque, Kyoto, Japan), was used to bathe the gel and electrode, 0 as described by Laemmli.' Lyophilised sam- c 0.6 ples of placental collagens and type V collagen were dissolved at a concentration of 0.2 mg/ml and denatured by heating in the gel buffer con- 0.4 taining 1'S. SDS at 60°C for 30 minutes. Ali- quots of 25 ml solutions of denatured collagens and 5 ml denatured type V collagen were 0.2 loaded onto the gel and subjected to electro- phoresis at 80 mA. After 90 minutes the 0.0 current was switched off and sample wells were Group 1 Group 2 Group 3 filled with a 20% solution of 3-mercap- 0.4 toethanol (Wako, Osaka, Japan) in gel buffer, B ul1 (V) b,c which was allowed to diffuse into the gel for Ux1 (1) one hour to cleave the intramolecular disul- phide bonds of type III collagen [ul(III)] 3. Electrophoresis was then resumed and allowed 0.3 to continue for another hour. Each collagen (x chain was stained with Coomassie brilliant a blue (Sigma) and intensities of bands were .2_0 quantitated by densitometry. The relative m 0.2 amounts of ul (lIl) or cl (V) chains were calculated by dividing the intensities of band areas under densitometric peaks of (xl (III) and al (V) by that of x I (I). 0.1

STATISTICAL ANALYSIS The ratios of ocl(III) to a 1(I) chains and of 0.0 al (V) to cx1(I) chains, as estimated by Group uroup 2 uroup 3

densitometry, are expressed as mean (SEM). Figure 2 Relative abuniidanice ol/ tlc (A) c(1 (II) and(l (13) http://jcp.bmj.com/ Results were analysed by analysis of variance ce1(J collagei chalinl.s comiipa-red zwit/i theli (I) cliilii i'l groIup I (71 = 36), grouip 2 (ni = 42), anid grouip 3 placentas and unpaired t tests. (n = 53). Data are expressed as miteani (SEM). Iv'hies Tverc analysed for statistical siglificane (of dffereiices amiionig the three groups bv analNsis of var-ianice aiid unlpaired t tests: wp Results < 0. 05 versusgrouip I placentas; p < 0. 01 versIs group 1 Although the relative concentrations of ocl(I) placenitas; p < 0. 05 versus giouip 2 placeiitas. were similar in the three those of groups, od (V) increased and those of cxl (III) decreased on October 2, 2021 by guest. Protected copyright. during placental development and ageing from the second to the third trimester of pregnancy (fig 1). The ratios of intensities of bands of ocl (III) to ocl (I) (fig 2A) were 0.95 (0.17), 0.68 (0.21) and 0.41 (0.14); and those of uf I(V) to ocl(I) (fig 2B) were 0.12 (0.01), 0.18 (0.04) and 0.30 (0.06) for groups 1, 2 and 3, respec- tively. The mean ratio of the intensity of the cx1 (III) band to that of a I (I) in group 3 placentas was significantly lower than that in group 1 placentas (p < 0.05). By contrast, the mean ratio of the intensity of the a 1 (V) band to that of (xl (I) in group 3 placentas was significantly higher than that in group 1 (p < 0.01) and group 2 (p < 0.05) placentas.

Discussion In the present study, we investigated changes in the composition of collagens in placentas dur- ing the second and third trimesters of preg- nancy. We were able to solubilise 70-85' of Figure 1 SDS-PAGE ofpepsin solubilised collagens. Laize 1, 25 zveek old placenta; lanze 2, 33 zveek old placenta; lane collagen in the human placental tissues, as 3, 40 week old placeiita; laiie 4, purified type V collageii. measured by reference to hydroxyproline Iwahashi, Ooshima, Nakano 918 concentrations (data not shown). Therefore, interactions of this collagen with throm- we postulated that the extracted collagen might bospondin and heparan sulphate might be

accurately reflect the entire complement of important in the assembly of the ECM and in J Clin Pathol: first published as 10.1136/jcp.49.11.916 on 1 November 1996. Downloaded from collagen in the sample tissues. the regulation of its biological functions. Although type I and type III collagens are Therefore, it is suggested that increased commonly found in combination, the ratios of relative concentrations of al (V) chains or type type III to in group 1 placentas V collagen in the placenta might provide a bio- were significantly higher than those in group 3 chemical basis for the functional role of the placentas. Changes in the ratio of type III to placenta in the development of the foetus. type I collagen have been demonstrated in skin In conclusion, the placenta at term seems to on ageing" and during the development of be characterised by increased relative concen- atherosclerosis.415 A possible cause of changes trations of type I and type V collagens. Our in the composition of the ECM in the placenta results suggest that alterations in the composi- might be an alteration in the hormonal tion of collagen during placental development environment. Another possible cause might be and ageing might play important roles in the an alteration in the density of cells in the invasion of trophoblastic cells, the supply of placenta. Recently, cell density dependent nutrients to the developing foetus and in main- effects have been reported in the various cell taining pregnancy. types, such as mesangial cells,16-'8 endothelial cells,'9 vascular smooth muscle cells,2"22 fibro- blasts,2324 and primitive mesenchymal cells.25 It 1 Cunningham FG, MacDonald PC, Gant NF, Leveno KJ, has been suggested that cell density might Gilstrap LC (eds). Type V collagen in the placenta. In: Wil- modulate biological behaviour, with changes in liams Obstetrics. 19th edn. Norwalk, CT: Appleton and Lange, 1993:111-38. signal transduction responses to hormonal 2 Lin CQ, Bissell MJ. Multi-faceted regulation of cell stimulation, in growth, in the synthesis and differentiation by extracellular matrix. FASEB Jf 1993;7: 737-43. composition of the ECM, and in the synthesis 3 Madri JA, Basson MD. Extracellular matrix-cell inter- of specific proteins.' '5 Wolthuis et al'8 re- actions: dynamic modulators of cell, tissue and organism structure and function. Lab Invest 1992;66:519-21. ported that mesangial cells synthesised rela- 4 Blankenship TN, King BF. Developmental changes in the tively more type I collagen per cell at higher cell cell columns and trophoblastic shell of the macaque placenta: an immunohistochemical study localizing type IV densities, whereas rates of synthesis of type III collagen, laminin, fibronectin and . Cell Tissue and type IV collagens in each cell did not Res 1993;274:457-66. 5 Nanaev AK, Milovanov AP, Domogatsky SP. Immunohisto- depend on cell density. chemical localization of extracellular matrix in perivillous The distribution of type V collagen in the fibrinoid of normal human term placenta. Histochemistry 1993;100:341-6. placenta has been determined by immunohis- 6 Earl U, Estlin C, Bulmer JN. Fibronectin and laminin in the tochemical staining.5 Changes in the relative early human placenta. Placenta 1990;11:223-31. 7 Burgeson RE, El Adri FA, Kaitila II, Hollister DW. Fetal concentration of this collagen during placental membrane collagens: identification of two new collagen development have not been clarified fully. In alpha chains. Proc Natl Acad Sci USA 1976;73:2379-83. 8 Modesti A, Kalebic T, Scarpa S, Togo S, Grotendorst G, the case of type V collagen, the purified protein Liotta LA, et al. Type V collagen in human amnion is a extracted from placentas by differential salt 12-nm fibrillar component of the pericellular interstitium. http://jcp.bmj.com/ EurJ Cell Biol 1984;35:246-55. precipitation was composed of axl(V), a2(V) 9 Kivirikko KI, Prockop DJ. Hydroxylation of proline in syn- and a3(V) chains, with al (V) chains predomi- thetic polypeptides with purified protocollagen hydroxy- lase. J Biol Chem 1967;242:4009-12. nating. This finding is consistent with previ- 10 Furuto DK, Miller EJ. Isolation of a unique collagenous ously reported data.26 Therefore, the relative fraction from limited pepsin digests of human placental tis- sue. J Biol Chem 1980;255:290-5. concentrations of al (V) were calculated in 11 Miller EJ, Rhodes RK. Preparation and characterization of terms of acl (I). A notable increase in the inten- the different types of collagen. Methods Enzymol 1982;82:

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