Thomas N. Blankenship

Comparative placental structure.

The primary function of all placentas is to act as an interface between the mother and fetus that allows, and even promotes, appropriate metabolic exchanges. This function is accomplished by bringing maternal and fetal blood into close apposition while maintaining separation of the maternal and fetal circulatory systems. Despite the common physiological functions shared by placentas, however, examination of placental morphology from different animal groups reveals a remarkable diversity of species-specific structural organization.The separation of fetal and maternal blood is always maintained by an elaboration of extraembryonic fetal tissues that cover fetal blood vessels. In some species the outermost layer of this fetal tissue, the trophoblast, is in direct contact with maternal blood. In many other species uterine tissues also contribute to the selective barrier separating the two blood systems. In addition to morphological variation among placentas of different animal groups, placentas undergo substantial structural modifications during pregnancy in a single species. In some animals different types of placentas function successively, or concurrently during a single pregnancy.As a result of these myriad details of placental structure, effective evaluation of fetal-maternal transfer of drugs must consider not only the components of the interhemal barrier of the fully developed placenta characteristic for each species, but also the placental structures functioning at each gestational stage of the fetus.

Trophoblastic invasion and the development of uteroplacental arteries in the macaque : immunohistochemical localization of cytokeratins, desmin, type IV collagen, laminin, and fibronectin

The processes by which trophoblast cells invade and modify the walls of the uteroplacental arteries of macaques during the course of gestation were examined. Antibodies to cytokeratins were employed to identify trophoblast, anti-desmin antibody to identify smooth muscle, and antibodies to type IV collagen, laminin, and fibronectin to examine changes in extracellular matrix distribution in the arterial wall. During early gestation, endovascular trophoblast adhered to the arterial wall, often in an asymmetrical distribution. As trophoblast cells moved outwardly into the tunica media, the basement membrane underlying the endothelium was lost, as indicated by gaps in the layer when stained for type IV collagen and laminin. Trophoblast cells became sequestered in the vessel wall where they hypertrophied and became surrounded by a capsule containing type IV collagen and laminin. As the trophoblast cells became established in the vessel wall, the muscular layer of the artery became discontinuous. Throughout gestation it was common for trophoblast cells to invade the vessel intimal layer and share the lining of the artery with typical endothelial cells. This general disposition of endovascular and intramural trophoblast persisted into late gestation. In addition, and contrary to the results of earlier studies of macaques, we identified trophoblastic invasion and modification of myometrial segments of the uteroplacental arteries in later gestation. We also found evidence of interstitial trophoblast cells among the stromal cells of the endometrium, especially during early gestation.

Trophoblastic invasion and modification of uterine veins during placental development in macaques

Trophoblast cells invade and modify the uterine vasculature to provide circulation of maternal blood through the placenta. Although evidence indicates fundamental differences between trophoblast modification of arteries and veins, interactions between trophoblast cells and uterine veins have not been addressed. In this report we describe the processes by which trophoblast cells invade and restructure uterine veins during placentation in the macaque. Antibodies were used to identify trophoblast, endothelium, and basement membranes. During early gestation, trophoblast migrated from the trophoblastic shell and, by intravasation, replaced portions of the wall and endothelium of veins in the vicinity of the shell; this is in contrast to invasion by extravasation reported for the arteries in this species. These areas had discontinuous endothelial basement membranes and the endothelial cells were variably hypertrophied. Deeper portions of veins were not invaded; this too is in contradistinction to the spiral arteries where trophoblastic modification extends to the myometrial segments. Later in gestation, those portions of veins interacting with trophoblast were contained within the trophoblastic shell or situated such that one side abutted the shell. These regions of the veins were lined by endothelium, but it could not be determined whether this represented re-endothelialization of regions formerly lined by trophoblast or if these endothelial cells were never displaced.

MODIFICATION OF ENDOMETRIAL ARTERIES DURING INVASION BY CYTOTROPHOBLAST CELLS IN THE PREGNANT MACAQUE

Fetal trophoblast cells invade endometrial blood vessels and gain access to maternal blood within two days after the onset of blastocyst implantation in macaques. Soon thereafter, cytotrophoblast cells migrate well into the lumina of arteries and subsequently invade arterial walls. Using electron microscopy and light microscopy we investigated the interactions between invasive cytotrophoblast cells and the cellular and extracellular components in the walls of endometrial arteries. The placentas and adjacent endometrium of 22 macaques (GD 17 to term) were examined. Spiral arteries containing migratory cytokeratin-labeled cytotrophoblast cells were identified at all stages examined. Early modification of each artery showed that a plug of intraluminal cytotrophoblast cells temporarily filled the arterial lumen in the vicinity of the trophoblastic shell. Distal to this plug the group of cells tapered as a continuous mass, filling only a portion of the lumen. Endothelial cells were displaced from their basal lamina by closely apposed cytotrophoblast cell processes. Soon thereafter these processes penetrated the basal lamina and achieved contact with smooth muscle cells of the tunica media. As cytotrophoblast cells infiltrated the arterial wall they hypertrophied and secreted extracellular matrix, thereby differentiating into intramural cytotrophoblast. The patent lumen of the artery was reestablished concomitant with the migration of intraluminal cytotrophoblast cells through the arterial tunica intima and into the tunica media. The presence of clusters of cytotrophoblast cells in the arterial wall results in discontinuity of the tunica media and dispersion of the smooth muscle. The combined changes result in expanded circumferences of invaded arteries as well as diminished ability to contract. In portions of arteries adjacent to the trophoblastic shell cytotrophoblast usually occupied the entire perimeter and thickness of the artery wall, while in areas distal only a portion of the wall was invaded. Despite extensive arterial modification, evidence of cell death among the fetal and maternal tissues involved was rare. By later gestation only a few intraluminal cytotrophoblast cells were seen. Intramural cells were surrounded by a thick layer of matrix, but maintained contact with adjacent cells through cytoplasmic processes, some of which formed gap junctions. Maternal cellular and connective tissue elements were excluded from the cytotrophoblast-matrix pads and the cytotrophoblast cells retained attributes of glycoprotein producing cells to term. Spiral arteries were modified well into the spongiosum layer of the endometrium, and some were modified into the myometrium.

TROPHOBLAST CELL-MEDIATED MODIFICATIONS TO UTERINE SPIRAL ARTERIES DURING EARLY GESTATION IN THE MACAQUE

A specialized subset of invasive embryonic cytotrophoblast cells gains access to maternal uterine arteries early in the gestation of higher primates. These cells continue to migrate extensively within the lumina of spiral arteries, converting them to the highly modified uteroplacental arteries of pregnancy. Although trophoblast cell-mediated modifications are considered critical to the progress of normal pregnancy, few studies have addressed the cellular interactions between maternal arteries and embryonic cells in situ. Macaque placentas and endometrial tissues were collected from 12 animals from day 14 of gestation (blastocyst implantation begins on day 9) to day 49. Standard indirect immunoperoxidase methods were used to identify matrix metalloproteinases (MMP-1, MMP-3, MMP-9), cathepsin B, cathepsin D, platelet-endothelial cell adhesion molecule, cytokeratins, smooth muscle actin, CD68, and factor VIII-related antigen. Cytotrophoblast cells were located deep within spiral arteries in each of the specimens examined. In some examples tightly packed clusters of cytotrophoblast occluded the lumina of invaded arteries. Initial penetration of arterial tunica intima was revealed by discontinuities in the staining pattern for factor VIII and cytotrophoblast intrusion was indicated by cytokeratin staining of the trophoblast cells. Continued cytotrophoblast intrusion into the tunica media was apparent by gaps in the smooth muscle. MMP-1, MMP-3, and MMP-9 were localized within intraluminal and intramural cytotrophoblast. By contrast, neither cathepsin B nor cathepsin D were present, although both were seen in uterine macrophages and stromal cells. Upon reaching the surrounding uterine stroma the cytotrophoblast cells ceased migration. As cytotrophoblast accumulated in the arterial wall the vascular lumen expanded. Evidence of cell death was rarely encountered in associated maternal or embryonic tissues. We conclude that intra-arterial cytotrophoblast cells express several proteinases with substrate specificities sufficient to permit independent remodeling of the extracellular matrix comprising uterine artery walls. The remodeling of the arteries, which involves extensive displacement of maternal endothelium and smooth muscle, in addition to degradation and synthesis of extracellular matrix, is accomplished with little evidence of cell death or loss of the integrity of the arteries. This process provides an interesting example of cooperation between different types of interacting tissues from genetically distinct individuals.

Expression of Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM) by Macaque Trophoblast Cells During Invasion of the Spiral Arteries

Placental development in higher primates is characterized by the invasion of uterine blood vessels by trophoblast cells. These cells proceed to migrate within uterine spiral arteries, opposite to the direction of normal blood flow. Observations indicate adhesion of intra‐arterial trophoblast to endothelium as well as to adjacent trophoblast cells.

Distribution of laminin, type IV collagen, and fibronectin in the cell columns and trophoblastic shell of early macaque placentas

SummaryThe cytotrophoblastic cell columns and trophoblastic shell of macaque placentas accumulate progressively greater amounts of intercellular material during early gestation. We studied the composition of this material in placentas collected from 22–34 days of gestation by using immunoperoxidase techniques directed to the extracellular matrix molecules fibronectin, type IV collagen, and laminin. These antigens co-localized within the intercellular deposits at all stages studied. At day 22 the proximal cell columns were composed of cells with narrow interstices and which lacked immunoreactivity for the 3 antigens. Distally the cells were vacuolated and the intercellular spaces increased in size and contained dense matrix deposits. The trophoblastic shell consisted of closely packed, non-vacuolated cytotrophoblast cells with only a delicate meshwork of matrix. By day 27 the matrix deposits of the distal cell columns increased markedly in size. The trophoblastic shell contained larger numbers of vacuolated cells and was occupied by accumulations of matrix. By 34 days the matrix deposits of the cell columns expanded substantially along the longitudinal axes of the columns. These deposits were often continuous with a matrix-dense, cell-deficient layer in the trophoblastic shell. This matrix-rich zone lay between a cellular layer adjacent to the intervilous space and a similar, but discontinuous, cell layer that formed the junctional zone with the endometrium.

Identification of 72-kilodalton type IV collagenase at sites of trophoblastic invasion of macaque spiral arteries

The walls of uterine spiral arteries are invaded by extravillous trophoblast cells and are thereby converted to the uteroplacental arteries of pregnancy. The mechanisms by which this invasion occurs are not understood, but the extracellular matrices that are breached suggest participation by specific proteinases. In this report we describe the immunohistochemical localization of 72-kd type IV collagenase (gelatinase A or MMP-2) among intra-arterial trophoblast cells and endometrial cells during development of macaque uteroplacental arteries. Cytokeratin-positive trophoblast cells were identified within arteries at each stage studied (between days 22-128 of gestation). Many of these cells, whether located in the arterial lumen or within the vessel wall, were immunoreactive for the proteinase. Early in the invasive process these trophoblast cells were associated with discontinuities of the endothelial basement membrane and later became interspersed with smooth muscle cells of the tunica media. While trophoblast cells comprised the entire thickness of the arterial wall in many locations, typically only a subset of these cells expressed the proteinase. Many endometrial stromal cells were also immunoreactive for the proteinase, as were some arterial endothelial and smooth muscle cells. It is concluded that this, and probably other, proteinases are active throughout gestation in the restructuring of uterine spiral arteries and other endometrial tissues as necessary to accommodate the development of the fetus.

Effect of shear stress on migration and integrin expression in macaque trophoblast cells

During fetal development, trophoblast cells enter endometrial capillaries, migrate within the uterine vasculature, and eventually reside within spiral arteries of the uterus. This invasive activity is accompanied by upregulation of trophoblast beta1 integrin expression. Fluid mechanical shear stress regulates migration and expression of adhesion molecules in vascular endothelial cells, but nothing is known about the effects of shear stress on trophoblast cells. We tested the hypothesis that shear stress regulates the motility and beta1 integrin expression of trophoblast cells. Early gestation macaque trophoblast cells were cultured in 1 x 1-mm square cross-section capillary tubes within which the flow field was determined using three-dimensional computational fluid dynamic simulations. Trophoblast cells in the capillary tubes were exposed to a steady shear stress of 7.5, 15, or 30 dyn/cm2 for up to 24 h. In the absence of flow, trophoblast cells were highly dynamic with constant nondirectional positional shifts but with no net cell migration. Exposure of the cells to shear stress within 24-72 h of cell plating significantly increased the level of this activity and led to net cell migration in the direction of flow. Shear stress also increased the expression and altered the topography of beta1 integrin. These results suggest that shear stress regulates trophoblast motility and beta1 integrin expression in vitro.

Developmental changes in the cell columns and trophoblastic shell of the macaque placenta : an immunohistochemical study localizing type IV collagen, laminin, fibronectin and cytokeratins

Developmental changes in the organization of cells and extracellular matrix in the cell columns and trophoblastic shell of macaque placentas have been examined between 37 days of gestation and term. Between 37 and 53 days a thickened basement membrane developed between the trophoblast cells of the proximal cell columns and the mesenchymal cores of contiguous anchoring villi. This layer stained strongly for type IV collagen and laminin, but weakly for fibronectin. Large “lakes” of extracellular matrix immunoreactive for all 3 of these antigens were present in the distal columns, while smaller amounts were distributed between cells of the proximal columns. During this period the trophoblast cells in the proximal shell reorganized, forming strands of cells that were separated by bands of matrix immunoreactive for type IV collagen, laminin, and fibronectin. Staining for these antigens decreased abruptly at the junction between fetal and maternal tissues. Between 66 and 104 days the thick basement membrane of the proximal columns persisted, but stained only weakly for each of the 3 extracellular matrix antigens. The large lakes of matrix in the distal columns characteristic of earlier stages gradually disappeared. The cell columns became progressively shorter and the tips of the anchoring villi became embedded in the trophoblastic shell. The matrix of the shell decreased in immunostaining intensity except for narrow rims around the trophoblast cells. Gestational ages later than 104 days showed few additional changes in the distribution of the matrix antigens or cell organization of the columns and shell. The thick basement membrane-like layer persisted to term although it continued to stain weakly for the 3 matrix antigens. The distal ends of most anchoring villi were embedded in the trophoblastic shell. The developmental changes in the organization of the columns and shell may be related to changes in placental growth rate.