RESULTSDetermination of Vasculogenic Stages

Using semithin sections and CD31 immunohistochemistry, vasculogenic and angiogenic areas were determined and classified as described previously. Development and recruitment of hemangioblastic progenitor cells, formation of hemangioblastic cell cords, differentiation of primitive endothelium, formation of primitive lumen, endothelial tube elongation, and angiogenic branching were determined. In mesenchymal villi, islands of hemangioblastic progenitor cells containing two to four cells were determined morphologically and by using CD31 immunoreactivity from 22 to 48 dpc (Fig. 1, a and b). We observed cells elongating from one island to others (Fig. 1, c and d). In addition, main vascular patterns including angiogenic cords with or without primitive lumen, angiogenic branching, and connecting tubes were observed in the sem-ithin and CD31-stained placental sections of villi (Fig. 1, e and f). The hemangioblastic and hematopoietic aspects of the cells in the villous core were further confirmed according to the intensity of immunoreactivity with anti-CD34 antibody (Fig. 1, g and h). canadian neighbor pharmacy online

Molecular Evidence for Apoptosis During Vasculogenesis

To detect the apoptotic cells in vasculogenic areas at the molecular level, TUNEL-CD31 double-staining was used. No TUNEL-positive cell was observed in hemangioblastic progenitor cells observed alone. Moreover, cell islands, formed by recruitment of hemangioblastic progenitor cells, also were negative for TUNEL but positive for CD31 (Fig. 2a). When the later stages of vasculogenesis were analyzed with TUNEL at the stage of primitive vascular lumen formation, some cells located at the center of the primitive lumen were TUNEL-positive (Fig. 2, b-d). Interestingly, some of the stromal cells located between vasculogenic areas during the endothelial tube elongation and angiogenic branching also were TUNEL-positive (Fig. 3, a-c). However, in reverse correlation to vessel maturation, significantly fewer apoptotic nuclei were observed (Fig. 3d).

To confirm these findings further, morphological indicators of apoptosis, such as cell shrinkage, nuclear segmentation, and nuclear bodies, were analyzed in slides stained with hematoxylin-eosin. The morphological findings and localization for apoptotic cells were similar to the results obtained from TUNEL-CD31 double-labeling (Fig. 4).

Ultrastructural Evidence for Apoptosis During Vasculogenesis

Electron-microscopic findings supported the light-microscopic data presented above. During the early stages of placental vasculogenesis, groups of or single hemangiogenic cells derived from undifferentiated mesenchymal cells were noticed in the core of mesenchymal villi. The hemangi-ogenic cells attached to each other and formed cell islands and hemangioblastic cell cords close to the cytotrophoblas-tic layer (Fig. 5a). Moreover, the primitive lumen of the developing vessel structure emerging from the dilating intercellular space of hemangioblastic cells also was observed (Fig. 5b). Interestingly, during the lumen formation, noticeable signs of apoptosis, such as chromosome condensation, nuclear shrinkage, and apoptotic bodies, were detected (Fig. 5c). Also, some cell debris that likely belonged to the very late stage of apoptosis was observed around the main vascular pattern, with hemopoietic cell series at different stages of maturation (Fig. 5d).

Quantitative Analysis of Apoptotic Cell Index

Two villous types, mesenchymal villous and immature-intermediate villous, were analyzed for apoptotic cell ratio. The ratio was calculated in four different areas of villi classified as follows: the angiogenic cell cords, the main vascular pattern (at the stage of lumen formation), in the vasculature with advanced lumen, and the stromal cells among the branching vasculogenic areas. All four areas were analyzed in the immature-intermediate villi, whereas only angiogenic cell cords and main vascular pattern areas were analyzed in the mesenchymal villi. The highest ratio for apoptotic cells was found in the villous core among the vascular branching areas and then in the primitive capillary lumen (Fig. 6). These values are significantly different from those of angiogenic cell cords and vasculatures with advanced lumens in the immature-intermediate villi (P < 0.05) (Fig. 6). Moreover, a significantly higher apoptotic index was found in the main vascular patterns of the immature-intermediate villi than those of mesenchymal villi (P < 0.05). However, no significant difference for the ap-optotic index was found between the angiogenic cell cords of mesenchymal and immature-intermediate villi (Fig. 6). Furthermore, no significant difference was detected in total apoptotic cell index when compared according to the day of pregnancy. When the index was analyzed according to the vasculogenic stages across time, the apoptotic index showed no significant increase in angiogenic cell cord, main vascular pattern, and vasculature with advanced lumen stages as pregnancy advanced (Fig. 7). However, the index was increased significantly in vascular branching areas starting from Pregnancy Week 6 toward Pregnancy Weeks 7-9 (Fig. 7).
Fig1Apoptosis Contributes to Vascular-1FIG. 1. Morphological and molecular organization of human placental vasculogen-esis in mesenchymal and immature intermediate villi as determined by semithin sections (a, c, and e) and CD31 immuno-reactivity (b, d, and e). Angiogenic cell islands (arrows in a and b) as well as bridge-like connector cells and/or microvessel-connecting tubes (arrowheads in c and d) between main vascular pattern (MVP) are seen. Branching angiogenic areas (arrows in e and f) and microvessel-connecting tubes (stars in e and f) also are seen, as is CD34 immunoreactivity in g and h. Hemangioblastic (arrows) and hemopoietic progenitor cells (arrowheads) are positive for CD34 (g and h), but the differentiated cells (star) in the center of the vasculature with advanced lumen is seen without CD34 expression (h). Magnification X100 (a and c), X50 (b, e, and f) and X25 (d, g, and h).

Fig2Apoptosis Contributes to Vascular-2FIG. 2. Detection of TUNEL-positive cells at different stages of vasculogenesis during early placentation using CD31-TUNEL labeling. Reddish and brownish colors indicate the DNA fragmentation (TUNEL-positivity) and CD31-positive cells, respectively. Hemangioblastic cell islands have strong CD31 immunoreactivity (arrowheads), but no sign of apoptosis was detected in these areas (a).TheTUNEL-positive cells (arrows) are observed during primitive vascular lumen formation (b-d). Magnification X250.

Fig3Apoptosis Contributes to Vascular-3FIG. 3. Detection of TUNEL-positive cells at different stages of branching angiogenesis during early placentation using CD31-TUNEL double-labeling. A TUNEL-positive stromal cell (arrow) is seen on the way to forming an elongating primitive vascular structure (a). Several TUNEL-positive stromal cells (arrows) are observed on the branching arms of the main vascular patterns (MVP; b) and around bridge-like connector cells and/or microvessel-connecting tubes (c). No TUNEL-positive cells are seen around the large vessel with a clearly defined lumen (d). A representative picture for negative CD31-TUNEL doublelabeling also is shown (e). Magnification X100 (a and b), X50 (c and d), and X25 (e).

Fig4Apoptosis Contributes to Vascular-4FIG. 4. Morphological detection of apo-ptosis at different stages of vasculogenesis during early placentation in hematoxylin-eosin-stained sections. Hemangioblastic cell islands (with circle) do not have any signs of morphological criteria of apoptosis (a). However, several apoptotic bodies are seen during the later stages of primitive lumen formation (b), vascular elongation (c), and angiogenic branching stages (d and e). The inset of d represents a higher-magnifi-cation view of the labeled area, and an apoptotic cell nucleus (arrow) is seen. Magnification X100 (a-c and inset in d), X25 (d), and X50 (e).

Fig5Apoptosis Contributes to Vascular-5FIG. 5. Representative electron micrographs that show ultrastructurally the process of apoptosis at different stages of vasculogenesis during early placentation. Angiogenic cell islands (circle) are seen (a). Cell islands that newly start to form a primitive lumen do not have signs of morphological criteria of apoptosis (b). Apoptotic bodies (circle) and cell debris (arrow) are seen during the advanced stages of primitive lumen formation, respectively (c and d, respectively). Hemopoietic cell series (HCS) of fetal blood at the different maturation stages also are seen (d). CT, Cytotrophoblast; E, endothelial cell; N, nucleus; Sn, syncytiotrophoblast; St, villous core. Magnification, X1200 (a), X5000 (b), X6300 (c), X3000 (d); X4000 (e), and X2000 (f).

Fig6Apoptosis Contributes to Vascular-6FIG. 6. Quantification of apoptotic cells at different stages of placental vasculogenesis. Analysis of the apoptotic index was performed according to villous types and vasculogenesis stages. Main vascular patterns (MVP) are seen with a significantly increased apoptotic index compared to angiogenic cell cords (ACC) in both mesenchymal villi (MV) and immature intermediate villi (IIV). The MVP also have a higher apoptotic index than that in the vasculature with advanced lumen (VAL) of IIV. Moreover, the stromal cells surrounded by the branching vascular areas (VBA) have the highest apoptotic ratio. Bars represent the mean ± SEM. *P < 0.05.

Fig7Apoptosis Contributes to Vascular-7FIG. 7. Quantification of apoptotic cells at different stages of placental vasculogenesis. Analysis of the apoptotic index was performed according to vasculogenesis stages across time in angiogenic cell cords (ACC; a), main vascular patterns (MVP; b), vasculature with advanced lumen (VAL; c), and the branching vascular areas (VBA; d). Except for the index obtained from VBA, no significant changes were observed as pregnancy advanced. Bars represent the mean ± SeM. *P < 0.05.