Distribution and characterization of anionic sites in trophoblast and capillary basal laminas of human placental villi.код для вставкиСкачать
THE ANATOMICAL RECORD 21263-68 (19851 Distribution and Characterization of Anionic Sites in Trophoblast and Capillary Basal Laminas of Human Placental Villi BARRY F. KING Department of Human Anatomy, School of Medicine, Uniuersity o f California, Dauis, CA 95616 ABSTRACT The distribution of anionic sites was studied in the trophoblastic and fetal capillary basal laminas of developing human placental villi with the cationic stain ruthenium red. At 7-12 weeks of gestation the trophoblastic basal lamina (TBL) contained ruthenium red-positive granules in a quasi-regular array throughout the lamina densa or sometimes concentrated a t the interstitial surface of the lamina densa. The capillary basal lamina (CBL) (and anionic sites) were not present a t this age. Anionic sites were also associated with collagen or reticular fibrils. At term, the TBL was largely devoid of anionic sites except for some distributed along its interstitial surface. The CBL was present in later gestation and sometimes had arrays of anionic sites. In order to characterize the anionic sites, minced pieces of villi were incubated in the presence or absence of either chondroitinase ABC, heparitinase, neuraminidase, or Streptomyces hyaluronidase in appropriate buffer systems. Incubation of early villi with heparitinase resulted in the disappearance of the TBL-associated sites. Chondroitinase ABC appeared to reduce staining of collagen-associated sites. In term villi, heparitinase removed those few sites still associated with the TBL but did not affect sites associated with the CBL or collagen. Chondroitinase ABC resulted in the disappearance of all anionic sites. In later gestation, a number of developmentally important macromolecules are transported across the trophoblast and enter the fetal capillaries. We conclude that the absence of a n array of polyanionic sites from the term placenta TBL and the reduction in the amount of extracellular matrix intervening between the trophoblast and capillaries are adaptations to enhance the exchange of macromolecules across the placenta. Basal laminas are sheets of extracellular matrix materials that characteristically underlie epithelia and endothelia and envelop certain other cell types (e.g., smooth muscle, fat) (Vracko, 1974). Biochemical studies of certain basal laminas have shown that they are composed of a variety of collagenous and noncollagenous glycoproteins and proteoglycans (Szarfman et al., 1982).For most tissues the function of the basal laminas is incompletely understood. They are generally thought to provide structural support and compartmentalization of tissues, and to serve as a substrate for the attachment of epithelial or endothelial cells (Vracko, 1974). They also appear to have important roles in morphogenesis by virtue of their position at the interface of epithelia and mesenchyme (Toole, 1981; Sanders, 1983). Another function that has been established for certain basal laminas is to act as a size- and charge-selective barrier to macromolecules. This function has been extensively studied in glomerular basement membrane (Farquhar et al., 1982) and has been proposed to function similarly in certain capillaries (Charonis and Wissig, 1983). The charge-selective barrier is attributable to an array of polyanionic proteoglycans associated with the basal lamina (Kanwar and Farquhar, 1979). 0 1985 ALAN R. LISS, INC. Human placental villi are covered by a layer of trophoblastic epithelium that is separated from the connective tissue core of the villus by a relatively thick basal lamina. The connective tissue core contains, among other things, the developing fetal capillary bed. During the latter part of gestation the fetal endothelial cells are associated with a thin basal lamina. During this same period, certain maternal serum proteins, most notably IgG, are endocytosed by trophoblast and transported to the fetal circulation (King, 1982).In fact, fetal IgG levels may exceed maternal serum levels (Kohler and Farr, 1966).This observation suggested to us that, unlike glomerular basement membrane, the basal laminas in placental villi may be specially adapted to permit the passage of macromolecules. As a first step toward determining this, we have examined developing placental villi for the presence and distribution of anionic sites in trophoblastic and capillary basal laminas. Received August 15,1984;accepted December 5, 1984 64 B.F. KING MATERIALS AND METHODS lar and interstitial surfaces of the lamina densa (Fig. 6). Other ruthenium red-positive sites were associated with interstitial collagen or reticular fibrils (Fig. 2). Incubation of minced villi in the presence of heparitinase removed most of the anionic sites associated with the TBL but had no effect on sites associated with the collagen fibrils (Fig. 3). Control specimens incubated under identical conditions, but in the absence of hepariRuthenium Red Staining tinase, always had stained sites (Fig. 4). Incubation of In order to determine if anionic sites were present in villi in the presence of chondroitinase ABC resulted in the basal laminas of trophoblast and fetal capillaries, reduced staining of anionic sites associated with intersome pieces of villi were immersed in a solution of 1% stitial collagen fibrils, but it had no effect on anionic ruthenium red (Polysciences, Inc., Warrington, PA) in sites associated with the TBL (Fig. 5 ) .Control specimens McIlvane’s citrate-phosphate buffer (pH 5.6) with or incubated identically, but in the absence of chondroitiwithout 0.2-1.0% Triton X-100 for 1hr at 22°C (Vaccaro nase ABC, showed a typical staining pattern (Fig. 6). and Brody, 1981). As noted by Vaccaro and Brody, the Incubation in the presence of hyaluronidase or neuraTriton damages the cells but allows the ruthenium red minidase had no apparent effect on staining. to penetrate and stain the basal lamina- and collagenTerm Villi associated proteoglycans. Tissue was then fixed overIn placental villi from normal term placentas in which night in 5% glutaraldehyde in citrate-phosphate buffer tannic acid but not ruthenium red was added to the containing 1% ruthenium red. The next day the tissue was washed three times in buffer containing ruthenium fixatives, the TBL was still finely fibrillar and of unired and post fixed in 1%osmium tetroxide containing form density, but it had increased in thickness compared 0.75% ruthenium red. Tissue was then dehydrated in a to early villi (Fig. 7). In addition, the fetal capillaries series of acetones and embedded in an Epon-araldite were now surrounded by a distinct basal lamina (CBL) epoxy resin mixture. Thin sections were cut on an ul- (Fig. 7). In villi treated with ruthenium red and Triton tramicrotome and subsequently stained with lead cit- X-100, anionic sites were absent from the TBL except rate and uranyl acetate. Sections were examined in a for occasional sites associated with the interstitial side of the lamina densa (Figs. 8-10]. Anionic sites were Philips 400 electron microscope. usually associated with the fetal capillary lamina densa (Figs. 8, 10) and with interstitial collagen fibrils (Figs. Enzymatic Digestions To demonstrate the nature of the anionic sites, minced 9, 10).In some cases, it was difficult to detect ruthenium pieces of villi were incubated for 45 min a t 37°C with red-positive sites in either lamina densa even though one of the following enzymes: chondroitinase ABC (Miles nearby collagen fibril-associated sites did stain (Fig. 9). Incubation of minced term villi in the presence of Laboratories, Kankakee, lL)1unit/ml in 0.1 M Tris-HC1 heparitinase removed those few anionic sites associated buffer, pH 8.0, + 0.1 M NaC1; Streptomyces hyaluronidase (Miles) 100 unitdm1 in 0.1 M acetate buffer, pH 6.0; with the interstitial surface of the TBL, but it had little heparitinase (Miles) 3 unitsiml in phosphate-buffered effect on staining of collagen and CBL anionic sites (Fig. saline, pH 7.3; neuraminidase (Worthington Diagnostic 11). Control specimens had a typical staining pattern Systems, Freehold, NJ) 1unit/ml in 0.1 M acetate buffer, (Fig. 12). Villi incubated in the presence of chondroitipH 5.4,+ 0.1 M NaC1. Controls lacking the enzymes nase ABC showed no staining of TBL, CBL, or collagenwere processed identically. After incubation, the tissue associated sites (Fig. 13).Control villi, incubated identiwas fixed in 5% glutaraldehyde in citrate-phosphate cally but in the absence of the enzyme, had a typical buffer containing 1% ruthenium red and subsequently staining pattern (Fig. 14). Incubation of villi in the presence of hyaluronidase or neuraminidase did not appear processed as previously described. to affect the anionic sites demonstrable with ruthenium For comparison, some villi were fixed in 3% glutaraldehyde in 0.1 M cacodylate-HC1 buffer with 1%w/v red. tannic acid added immediately before use. Tissue was DISCUSSION , subsequently washed in buffer, postfixed in 0 ~ 0 4 and processed as previously described. Proteoglycans (PGs) are recognized as common components of basement membranes, although the glycosaRESULTS minoglycan (GAG) composition and arrangement vary Early Villi from tissue to tissue (Hay, 1981;Toole, 1981; Timpl and In placental villi from early pregnancies (7-12 weeks) Martin, 1982; Charonis and Wissig, 1983). Anionic sites in which tannic acid (but not ruthenium red) was added have been localized in the basal laminas of several emto the fixative, the trophoblastic basal lamina (TBL) bryonic epithelia (Trelstad et al., 1974; Hay and Meier, underlying the cyto- and syncytiotrophoblast has a finely 1974; Hay, 1978; Reeves et al., 1980; Meyer et al., 1981; fibrillar, uniform density (Fig. 1).The fetal capillaries Grant et al., 1983). The anionic sites are about 10-20 a t this stage were often at some distance from the base nm in diameter and tend to occur in a quasi-regular of the trophoblast. Even when the capillaries were more array on both sides of the lamina densa. In most cases closely associated with the trophoblast, a basal lamina the PG is predominantly chondroitin sulfate or heparan was usually absent around the endothelial cells (Fig. 1). sulfate (Toole, 1981; Hay, 1981); sometimes hyaluronate In villi treated with ruthenium red, electron-dense, and chondroitin sulfate predominate (Cohn et al., 1977). ruthenium red-positive sites were distributed throughAnionic sites have also been demonstrated in the basal out the thickness of the lamina densa of the TBL (Fig. lamina of a variety of adult epithelia (Katsuyama et al., 2) or sometimes were associated mainly with the cellu- 1977; Kanwar and Farquhar, 1979; Gordon and BernPlacentas from five early pregnancies (7-12 weeks) and five term pregnancies were collected immediately. Small pieces of placental villi were rinsed briefly in Earle’s balanced salt solution to remove maternal blood and processed in several ways, basically following the techniques of Vaccaro and Brody (1979, 1981). ANIONIC SITES IN PLACENTAL BASAL LAMINAS Fig. 1. Low-magnification micrograph of trophoblast and fetal capillary wall at 10 weeks gestation. No ruthenium red treatment. Trophoblast (TI has a thick basal lamina (TBL). The fetal endothelium (FE) does not have a basal lamina at this age. x 18,400. 65 removed, although large granules deeper in the extracellular matrix are stained. ~ 5 7 , 0 0 0 . Fig. 4. TBL region, early placenta, control for heparitinase treatment. Anionic sites are associated with the TBL and collagen. ~57,000. Fig. 2. Trophoblastic basal lamina (TBL) from early placenta after Triton permeabilization and ruthenium red staining. Anionic sites are associated with the TBL and with interstitial collagen fibrils (C). ~57,000. Flg. 5. TBL region, early placenta, after chondroitinase ABC treatment. Anionic sites are still associated with the TBL, although sites associated with collagen (C) are reduced. x57,OOO. Fig. 3. TBL region from early placenta after treatment with heparitinase. Virtually all of the TBL-associated anionic sites have been Flg. 6. TBL reg'on, early placenta, chondroitinase ABC control preparation. Normal staining of anionic sites in TBL is seen. ~ 5 7 , 0 0 0 . field, 1980; Vaccaro and Brody, 1979, 1981; Brody et al., 1982; Pino et al., 1982; Essner and Pino, 1982; Charonis and Wissig, 1983; Vernier et al., 1983). Where it has been investigated, these basal laminas are rich in heparan sulfate PG (glomerulus, lung alveoli) or hyaluronate and heparan sulfate (mammary gland). In general, the anionic sites are similar in size and distribution to those described for embryonic epithelia. Anionic sites have also been demonstrated in the basal lamina surrounding a variety of blood vessels (Wight and Ross, 1975; Katsuyama et al., 1977; Vaccaro and Brody, 1981; Ausprunk et a]., 1981; Brody et al., 1982; Simionescu et al., 1982; Charonis and Wissig, 1983)and in the pericel- lular basal laminas of muscle, fat, and Schwann cells (Charonis and Wissig, 1983). In placental villi, we have demonstrated a developmental change in the pattern of anionic sites in both the trophoblastic basal lamina and in that of the fetal capillaries. In the case of the TBL, anionic sites decreased with development and were virtually absent by term. In the case of the CBL the basal lamina (and anionic sites) were essentially absent in the early material and were moderately, but not consistently, well developed by term. Selective enzymatic digestion demonstrated that the anionic sites associated with the first-trimester TBL probably consist mainly of heparan sulfate. At term, the few 66 Fig. 7. Base of trophoblast (T) and fetal capillary wall in term placenta. No ruthenium red treatment. TBL is quite thick and the fetal endothelium (FE) now has a thin basal lamina (CBL). A few collagen fibrils are interposed between the two basal laminas. ~57,000. Fig. 10. BL region of term placenta after Triton and ruthenium red treatment. Sites associated with collagen fibrils are large and heavily stained. A small number of sites are associated with the interstitial side of the TBL (arrows), and with the CBL (arrowheads). ~57,000. Fig. 8. TBL region of term placenta after Triton and ruthenium red treatment. TBL lacks anionic sites except for occasional ones associated with the interstitial surface of the lamina densa (arrows). The capillary basal lamina iCBL) does have an array of anionic sites iarrowheads). ~57,000. Fig. 11. BL region of term placenta after heparitinase treatment. Anionic sites are still present in the CBL (arrowheads) and to some extent with collagen fibrils (arrows)but not with the TBL. ~57,000. Fig. 9. Micrograph of BL region of term placenta after Triton and ruthenium red treatment, showing a region of close apposition of TBL and CBL. Anionic sites associated with either BL are rare, even though anionic sites associated with a nearby collagen fibril are stained (arrowheads). ~57,000. Fig. 12. Term placenta, heparitinase control. A few anionic sites are associated with the interstitial surface of the TBL (arrows) and with the CBL (arrowheads).T, trophoblast; FE, fetal endothelium. ~57,000. ANIONIC SITES IN PLACENTAL BASAL LAMINAS 67 Fig.13. Term placenta, chondroitinaseABC treatment. Anionic s i b have been removed from the CBL, collagen (C). and interstitial surface of the TBL. ~57.000. Fig. 14. Term placenta, chondroitinase ABC control. Anionic sites awxiated with the CBL, collagen (arrow). and interstitial surface of the TBL are stained. T,trophoblast, FE, fetal endothelium. ~57.000. sites associated with the TBL were removed by either heparitinase or chondroitinase ABC. Those sites associated with the CBL were selectively removed with the chondroitinase ABC, indicating chondroitin sulfate or dermatan sulfate. Biochemical and histochemical studies have shown many different GAGSin human placenta (Lee et al., 1973; Wasserman et al., 1980,1983a,b)These studies also indicate that dermatan sulfate and chondroitin sulfate are the most abundant GAGS near term and that a higher proportion of dermatan sulfate is associated with the placental blood vessels (Wasserman et al., 1983b). Thus, our results are consistent with the biochemical studies, although our observations are restricted to the small capillary exchange vessels, whereas the biochemical studies also included many larger-caliber vessels. At the present time, we do not have a complete understanding of the different functional roles that the TBL may have during development. Presumably it plays a supportive role as in other tissues (Vracko, 1974) and may have a role in branching morphogenesis as in some other embryonic systems (Bedield, 1981). In some other tissues, it has been shown that the basal lamina can act as a charge-selective permeability barrier to macromolecules. This property of basal laminas has been most extensively studied in the kidney glomerulus, where the glomerular basement membrane has a heparan sulfaterich PG layer that presents a negatively charged interface that retards filtration of anionic macromolecules (Farquhar et al., 1982). Our observation that there is a virtual absence of ruthenium red-positive anionic sites in the TBL in later gestation may be of great functional importance in relation to maternal-fetal transfer of macromolecules. A number of developmentally important polypeptides and proteins are transported from tho maternal blood, across the trophoblast, and enter the fetal capillaries. These include maternal IgG, from which the fetus obtains its passive immunity (see King, 1982, for references), and to a lesser extent albumin (Gitlin et al., 1964). The absence of an array of polyanionic sites later in gestation in the TBL suggests that this structure, even though it is quite thick, probably presents no significant permeability barrier to macromolecules. In a number of experimental or pathological situations it has been shown that the absence or enzymatic removal of heparan sulfate PG renders the basal lamina permeable to proteins (Kanwar et al., 1980; Rohrbach et al., 1982; Myndersse et al., 1983). In the case of TBL, our observations suggest the reduction in anionic sites during development assures that macromolecules transported by the trophoblast do not encounter a permeability barrier a t this level, and have access to the fetal capillary bed. In the case of the fetal capillary basal lamina, we were able to detect at least some anionic sites, although the number and arrangement did not always appear to be as regular as in some other continuous capillary beds (Charonis and Wissig, 1983). Charonis and Wissig (1983) reported a regular array of anionic sites around the continuous capillaries of muscle, although these sites were not as negatively charged as similar sitcs around fenestrated capillaries. These sites are apparently heparan sulfate PG (Charonis et al., 19831,but in the fetal placental capillaries these sites appear to be mainly chondroitin sulfate or dermatan sulfate. The effect of the relatively poorly developed array of anionic sites around fetal placental capillaries on the permeability of the capillaries to macromolecules is difiicult to assess. If placental capillaries are compared to the continuous capillaries of muscle, there might be some discrimination based on charge, although the barrier would be incomplete, since macromolecules do escape from and enter muscle capillaries (Renkin, 1978; Noer and Lassen, 1979). Overall, the distribution of anionic sites in TBL and CBL of the term placenta is such that facilitation of macromolecular transport is accomplished. Finally, near term the fetal capillaries become closely apposed to the TBL, eliminating most or all of the intervening collagen and ground substance (e.g., Fig. 9). This developmental change, by reducing additional anionic sites, would also be expected to facilitate macromolecular transport. Transport of proteins from mother to fetus is relatively low during early gestation (Dancis et al., 1961; Gitlin and Biasucci, 1969). We observed the presence of an array of heparan sulfate PG in the early TBL, and usually a greater amount of extracellular matrix separating the TBL from the fetal capillaries. Both these f i ~ t o r s would be expected to retard exchange of macromolecules at this age, although the primary b d e r to exchange be at the level Of~OPhObld. ACKNOWLEDGMENTS I wish to thank John Mais for excellent technical assistance and Meg Svetlichny and Clarrise Northern for typing the manuscript. I also wish to thank Mr. Harry Lowe and the staff of Sutter Memorial Hospital, and Dr. Alton Curtis for assistance in obtaining the tissue. This 68 B.F. KING work was supported by National Institutes of Health grant HD 11658. LITERATURECITED Ausprunk, D.H., C.L. Boudreau, and D.A. Nelson (1981) Proteoglycans in the microvasculature. I. 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