Placental vasculogenesis is a process of de novo vascular formation involving molecular and morphological differentiation processes that lead to the development of tertiary villi from secondary villi. It is widely accepted that pluripotent mesenchymal cells are the source of cells that participate in vasculogenesis. Thereafter, these differentiated, multipotent vasculogenic cells initiate the first placental angiogenesis via increased proliferative and mi-grational steps. Several investigators have indicated that conventional angiogenesis initiated by endothelial cells is different from placental angiogenesis. For instance, placental angiogenesis involves a cell type with pluripotent properties for differentiation, proliferation, and migration. Little information is available regarding the exact mechanisms on how placental vasculogene-sis and angiogenesis occur, how angiogenic precursor cells differentiate, or which signals are inducing differentiation of these cells.

Demir et al. showed that angiogenic cells and cell cords are established as ‘‘main vascular pattern-like structures,’’ and these structures form the main route for vessel segments and new blood vessel tubes to attach. Furthermore, the growth occurs by cell budding and outgrowths in the longitudinal axis of the villous growth. Numerous microvessels and vasculogenic areas interconnect by various bridge-like connector cells and/or microvessel connecting tubes and participate in generating the early placental vascular network.

In the classical steps of placental vasculogenesis, emergence of the first precursors of fetal endothelium in the villous core, the so-called hemangioblastic cell cords, can be demonstrated as early as 15-21 dpc. These cells have no cellular extensions, but a few organelles form string-like aggregates of polygonal cells. Desmosomes or band-like junctions that resemble tight junctions bridge the narrow intercellular spaces between these cells. All these steps are confirmed by our present findings, even though we observed no signs of apoptosis either morphologically or molecularly. By 28 dpc, a formation of long, polygonal lumen with surrounding endothelial cells occurs. During this phase, hematopoietic stem cells become visible in the capillary lumen. These cells are not yet circulating, because no anatomical connection exists via the cord. Our findings showing the presence of apoptotic nuclei and TUNEL-positive cells at the center of the capillary lumen suggest that some of the differentiating cells in the islands undergo apoptosis so that the primitive lumen of the vessel may form. The establishment of intercellular spaces between hemangiogenic cell cords and the connection to and fusion with each other to form the presumptive vessel lumen involve various steps. In the present study, we showed that programmed cell death, apopto-sis, takes place in this process as a novel approach for lumen formation during vasculogenesis. All findings obtained from electron microscopy, immunohistochemistry, and TU-NEL analysis support this hypothesis.

After the assembly of primordial vessels, the primitive vascular network, vasculogenesis advances to angiogenesis. In normal human pregnancy, capillary growth has a biphasic property. It involves an initial phase of branching angiogenesis, followed by a phase of nonbranching angiogenesis. In other words, villous morphology partly reflects the underlying angiogenic processes. The endothelial tube segments formed by vasculogenesis are transformed into primitive capillary networks, which occurs by the balanced interaction of two parallel mechanisms: elongation of preexisting tubes by nonbranching angiogenesis, and ramification of these tubes by lateral sprouting (sprouting angiogenesis). The TUNEL-positive cells that we have observed on the elongation sites of the primitive tubes and among the primitive tubes that are likely to connect with each other suggest a possible mechanism for apoptosis that would open physical spaces. Similar results from electron microscopy and hematoxylin-eosin staining further enhance this possibility.

Many growth factors, such as basic fibroblast growth factor, vascular endothelial growth factor, placental growth factor, TNFa, and their receptors, have been reported to stimulate vasculogenesis and angiogenesis in the early placental development. None of these molecules has been proposed for the apoptotic signaling cascade except for TNFa. However, we do not know whether the TNFa-related apoptotic cascade is involved in these processes. On the other hand, several studies have suggested that increased apoptosis in endothelial cells inhibits angiogenesis. The p38 a mitogen-activated protein kinase mutant placentas display a lack of vascularization and increased rates of apoptosis, suggesting a defective placental angiogenesis. Moreover, the regulation of endothelial cell apoptosis is considered to be a potential therapeutic target because endothelial cell apoptosis diminishes neovascularization in the adult organism. The induction of endothelial cell apoptosis may limit unwanted neovascularization of tumors. In contrast, the prevention of endothelial cell apoptosis may improve angiogenesis and vasculogenesis in patients with ischemia. However, according to our findings, it should be considered that placental vasculogenesis requires apoptosis to have a normal and vigorous vessel development. Beck et al. have shown that Ang-2 induction may cause endothelial cell proliferation or apoptosis depending on the presence or absence of vascular endothelial growth factor during angio-genesis. Those authors suggest that this apoptotic stimulus of Ang-2 is an inhibitory signal for angiogenesis.

In conclusion, the present study revealed, to our knowledge for the first time, that apoptosis is involved in the very early stages of placental vasculogenesis and angio-genesis. Although the present study divulges a new approach that suggests a role for apoptosis in these processes, it is not known which signaling mechanisms are involved or how the apoptotic cascades are triggered. Further molecular studies should be performed to answer these questions.