Intussusceptive Growth of Vascular Bed in Human Placenta

Objective: Normal embryonic and fetal development is strictly bound to maternal health and functioning placenta. Besides the invasion and differentiation of trophoblastic cell lineage; development of effective vasculature is crucial for the function of placenta. Placental vessels first arise by vasculogenesis in early development of villi and then succeeded by angiogenesis during fetal life. In the recent decades a new form of angiogenesis, “intussusceptive angiogenesis”, besides classical sprouting angiogenesis is well documented. The presence of intussusception was shown at multiple organs but in placenta, in recent literature. We aimed to determine whether intussusceptive angiogenesis is present in human placenta to obtain further evidence on the development of vascular bed. Methods: The term placenta samples were obtained from 10 healthy pregnancies following caesarean sections. Tissues were processed using routine plastic embedding technique; thin sections were contrasted with uranyl acetate & lead citrate; observed and photographed by transmission electron microscope. Results: Our examinations revealed that both sprouting and intussusceptive angiogenesis is present in floating villi of term placenta. Phases of intussusception were documented in various samples. Conclusion: The presence of intussusceptive angiogenesis will help our understanding of microvascular bed remodeling during pregnancy. We believe that this new finding will help us to determine the relation of microvascular bed development in normal and abnormal placentas.


INTRODUCTION
Placenta represents one of the highly vascularized organs to establish a critical efficient feto-maternal interface for the exchange of nutrients and waste products for normal development.
Formation of new blood vessels (neovascularization) is essential to achieve a functional microvascular bed thus many investigators focused on the issue. Classically it is described that first blood vessels together with primitive erythrocytes arise within extraembryonic mesoderm de novo from mesenchymal cells during early embryonic life starting from 2nd week of development and this process is termed as vasculogenesis. Such blood vessels in developing placenta are first detected in tertiary villi during the 3rd week (1). Once primitive capillary network is formed and their lumen become prominent, another mechanism for the enlargement of capillary bed is on charge termed angiogenesis (formation of new blood vessels from existing ones) (2)(3)(4)(5)(6). During early stages of vessel development, proliferated endothelial cells establish intercellular junctions (desmosomes, tight junctions) sealing a narrow cleft-like lumen both in vasculogenesis and angiogenesis (7). By the time lumina of young vessels gradually enlarge and become filled with blood. Several investigators introduced a number of terms trying to describe the details of the formation of new vessels like branching angiogenesis, non-branching angiogenesis, sprouting angiogenesis, nonsprouting angiogenesis and finally intussusceptive angiogenesis (5-7). Branching angiogenesis term is used to describe formation of new short capillary loops from existing capillaries to enlarge vascular bed during 5th to 25th weeks of development. It is distinguished by the presence of numerous branching hemangioblastic cords induced by vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). By 25th week an abrupt decrease on VEGF level and diminished PlGF level results in a switch from branching angiogenesis to non-branching angiogenesis. This latter term is used to describe not the formation but also the elongation of capillary loops during mid to late pregnancy interval. Sprouting angiogenesis describes the migration of endothelial cells to form side branches along the lateral walls of parent capillaries and non-sprouting angiogenesis is used just for the elongation of migrated endothelial cells in side branches without proliferation or migration (8)(9)(10)(11)(12)(13)(14)(15).
In addition to all angiogenesis types described above an additional mechanism for the development of new capillaries is also described as "intussusceptive angiogenesis". In this process of angiogenetic expansion of vascular bed, partition then elongation of existing capillaries takes place as reviewed by Djonov et al in detail (16). First findings on intussusception were reported by Short et (17)(18)(19). In the following years Caduff et al studied expansion of vascular bed in rat lung postnatally using scanning electron microscope and they described this phenomenon as intussusceptive microvascular growth (20). Burri and Tarek also studied this mechanism in rat lung and described four phases during this process as: phase I, creation of a zone of contact between opposite capillary walls (formation of an interendothelial bridge); phase II, reorganization of the intercellular junctions of the endothelium, with central perforation of the capillary layer; phase III, formation of an interstitial post core, with successive invasion by cytoplasmic extensions of myofibroblasts, pericytes and finally interstitial fibers; and phase IV, growth of the slender pillar to a normal full size capillary mesh (21).
However, this process is not studied in placenta representing a major organ where intensive neovascularization takes place. Besides its biological significance and therapeutic potential our understanding on this process is still limited as it cannot be easily observed by light microscopy (40). Thus we planned to study human term placentas at electron microscopic level to determine whether signs of intussusceptive angiogenesis and other angiogenetic mechanisms are present or lacking to improve our understanding on expansion and remodeling of microvascular bed in this special organ.

METHODS
In this study 10 placenta samples that were obtained from healthy term pregnants (age 20-40) who were followed in Department of Gynecology and Obstetrics, Baskent University Faculty of Medicine by cesarean section (C/S) with their informed concent approved by the Review Board and Ethical Committee of Baskent University (No: 14/56; dated 04/09/2014). The placental tissue samples were taken immediately after delivery and placed in 2% glutaraldehyde solution as first step. Samples were cut in to small pieces (1 mm 3 ) and kept in 2% glutaraldehyde solution for 24 hours. Following primary fixation samples were washed with Sorenson phosphate buffer, and post-fixed in 1% osmium tetroxide for about an hour. Tissue samples were then dehydrated using graded ethanol series, substituted by propylene oxide and embedded in epoxy resin. Ultrathin sections (70-90 nm) were taken on copper grids using an ultramicrotome (Leica) and precipitated with uranyl acetate and lead citrate. The specimens were observed and photographed by LEO 906E (Zeiss, Germany) electron microscope.

RESULTS
We focused on the capillaries and related structures/cells in the floating villi of term placentas. All villi were rich in capillaries most of which exhibiting the fine structural features of mature capillaries with slender endothelial cells connected to each other by intercellular junctions, continuous basal lamina and associated pericytes.

Sprouting angiogenesis
In some capillaries sprouting angiogenetic endothelial cell clusters were present with a cleft-like narrow lumen. These cuboidal endothelial cells significantly rich in cytoplasmic filaments were connected to each other by tight junctions at their basal compartments ( Figure 1). In most cases we observed cross sections of these sprouts with similar structural features though it was not always possible to outline their original communication with parent capillaries due to section plane. These were frequently observed along the trophoblastic basal lamina at the peripheral compartments of floating villi. Their basal lamina was laminated in some cases, and some were associated with pericytes ( Figure 2). More mature forms of these newly developing capillaries with a broader lumen were also observed. The thickness of endothelial cells in this vasculature gradually diminished surrounding a moderately developed lumen. However, these maturing endothelial cells were still relatively rich in cytoplasmic microfilaments. Lamination of the basal lamina surrounding these vessels and external lamina of pericytes were less prominent and lumina of some of them were occupied by red blood cells (Figure 3).

Intussusceptive angiogenesis
Besides sprouting angiogenetic figures described above we also observed structural features of intussusceptive angiogenesis in many examples representing phase I to IV of this remodeling process. In Phase-I samples, endothelial cells were observed to be extending towards the opposite wall some of which are already attached to each other by intercellular junctions forming pillars. In such newly established contact areas, endothelial cells were richer in cytoplasmic filaments like the endothelial cells of angiogenetic sprouts ( Figure 4). In other samples, processes of pericytes were projected between the attached endothelial cells crossing the original lumen of the parent capillaries representing a further stage of intussusception (Phase II-III). Endothelial cells crossing the lumen were similarly rich in cytoplasmic filaments reflecting a sign in their change in shape ( Figure 5). We also determined migrating pericytes in intussusception areas surrounded by tiny intercellular matrix components resulting in relocation of intercellular junctions to allow this enlargement of the pillar area ( Figure 6). Vascular bed growth Ayran Fidan et al.

DISCUSSION
Growth and remodeling of placental vasculature is critical for normal fetal development. A newer form of angiogenesis, "intussusceptive angiogenesis" is recently described and attracted the attention of a number of researchers to this field in the last decades. We could not find a report about intussusception in human placenta. For this reason, we hypothesized that both sprouting and intussusceptive angiogenesis are efficient processes in human placenta and studied human placenta at electron microscopic level to test this hypothesis. We focused on the capillary sections in detail and obtained structural evidences of both angiogenetic mechanisms.
As efficient and sufficient maternofetal exchange is critical for normal development, many investigators studied the development and vascularization of placenta previously (2)(3)(4)(5)(6)(8)(9)(10). It is generally agreed that neovascularization starts with vasculogenesis which is followed by angiogenesis (2,3,7,9). Several investigators introduced a number of terms like branching, non-branching sprouting etc. to define the formation of new blood vessels during further development (9)(10)(11). Intussusceptive angiogenesis attracted the attention of researchers thus several authors reported intussusceptive angiogenesis in a number of physiological conditions in lung, skeletal muscle, chorio-allantoic membrane (CAM), myocardium, retina, kidney, endometrium and also in some pathological conditions like the human colon adenoma cancer, tumor angiogenesis but there is no report in human placenta (17-21, 23-25, 27-29, 31-33, 35, 36, 40, 41). Djonov et al. previously suggested that intussusceptive mechanisms have several benefits for the organ. They stated that intussusceptive angiogenesis permits rapid expansion of capillary plexus and provides extensive endothelial surface for metabolic changes with a relatively little cost. They also reported that as remodeling occurs in capillary network, it provides reproduction and pruning of vessels according to metabolic needs of that special site (16).
Intussusceptive angiogenesis, which can be described as existing vessel proliferation in itself, is thought to be a more economic method for tissue when compared with new endothelial cell proliferation and sprouting from blood vessels method.
In the literature, changes of molecules (VEGF-A, PIGF, b-FGF, eNOS, O2, HIF, Tie1, Tie2, Ang-1, Ang-2) during vascularization in placenta is evaluated in both physiological and pathological pregnancies (5,12,14,31,42). But most of these studies are conducted in early placental development stages and they are focused on molecules rather than neovascularization types with the exception of a few studies. Mayhew et al., detected an increase in branching angiogenesis in pathologies like iron-deficiency anemia, high-altitude, hypoxia (11). Similarly, Soma et al., observed new vessel formation with angiogenesis during chronic hypoxia (43). Zhang et al., examined adaption, changes in diameter and perivascular cells of placenta vessels during pregnancy (14). Some authors identified intussusceptive angiogenesis and proposed that expansion of pillar is a mechanism that occurs for pruning useless/inefficient vessels (40,44,45). In our study; we observed capillaries in all stages of intussusception in all of the samples examined. We focused on the floating (terminal) villi in which fetomaternal exchange of placenta is most represent the first report of intussusceptive angiogenesis in human term placenta. In conclusion; our findings reveal that further comparative studies in the placentas of maternal or fetal pathologies will be great value to understand the role of intussusception in the etiopathogenesis of a number of placental disfunctions.