**3. The Influence of Fluid Shear Stress on Trophoblast Turnover and Di**ff**erentiation**

Human placenta development relies on a tightly controlled villous trophoblast turnover, which involves proliferation, differentiation, and fusion of mononucleated cytotrophoblasts with the overlying syncytiotrophoblast [14]. This process guarantees that the syncytiotrophoblast is continuously supplied with cytoplasm and organelles derived from the fusing cytotrophoblasts. Acquisition of newly incorporated cell components is balanced by a concomitant release of apoptotic material as syncytial knots from the syncytiotrophoblast surface into the maternal circulation [30]. Effects of flow and fluid shear stress on trophoblast turnover, in particular on cell differentiation, are manifested by changed cell morphology. Fluid shear stress is suggested to activate signaling pathways involved in trophoblast differentiation and syncytialization by increasing levels of intracellular cyclic adenosine monophosphate (cAMP) and activated cAMP response element-binding protein (CREB) (Table 2, [21]). Activation of cAMP signaling induces upregulation of transcription factor glial cell missing 1 (GCM1) and its downstream targets syncytin-1 (ERVW-1) and syncytin-2 (ERVFRD-1), both of which well-accepted fusogens involved in trophoblast syncytialization [30]. Concurrent to syncytialization is the loss of epithelial junctional- and cytoskeletal proteins, such as E-cadherin, desmoplakin, and α-fodrin [31]. While previous flow culture experiments clearly showed a network of continuous and well-defined junctional complexes in unstimulated trophoblasts after three days perfusion (Table 2, [29]), knowledge on cytoskeleton remodeling upon syncytialization under flow rates is rather limited.

Increased wall shear stress is suggested to act at the villous surface in the inflow regions of the intervillous space, where high wall shear stress could damage the villous trophoblast or at least affect its cellular turnover. This disturbance may be reflected in enhanced trophoblast shedding and elevated levels of free fetal DNA in the maternal circulation [32]. Paradoxically, increasing shear stress has been suggested to have a protective effect against induced apoptotic death in trophoblast cell lines [18]. This has been shown in mononucleated (i.e., undifferentiated) JAR and SGHPL-4 cells, which underwent less apoptosis when cultured under 3 dyn/cm<sup>2</sup> than those in 0.5 dyn/cm<sup>2</sup> cultures [18]. Moreover, trophoblasts have been shown to have a survival advantage over endothelial cells. Trophoblasts cultured on human umbilical vein endothelial cells (HUVECs) monolayers at 0.5 or 3 dyn/cm<sup>2</sup> significantly induced apoptosis in directly adjacent HUVECs, by Fas/Fas-ligand mediated mechanisms [18].

In addition to apoptosis, fluid shear stress is suggested to influence trophoblast fusion. Previous studies with BeWo cells and rabbit trophoblastic stem cells (rTSCs) showed increased cell fusion under fluid shear stress. This has been demonstrated for rabbit trophoblastic stem cells at flow rates of 0.1 ml/min, 0.2 ml/min, and 0.5 ml/min, respectively. The authors of the study described that fusion of rabbit trophoblasts occurred between more than two cells, while in the case of BeWo it was mainly a fusion of only two cells (Table 2, [17]). However, at this point it should be noted that BeWo cells occasionally contain two nuclei, and that intercellular fusion must be distinguished from endoreduplication [33], which represents replication of the nuclear genome in the absence of mitosis, and therefore results in an elevated nuclear gene content and polyploidy. However, besides syncytialization, other signs of trophoblast differentiation have been observed when cells were cultured under fluidic flow. According to Miura et al., BeWo cells and human villous trophoblasts react on fluid shear stress by abundant formation of microvilli, which vary in lengths depending on the flow rate (Table 2, [16]). At the center of the chamber, where the shear stress was low (0.001 dyn/cm<sup>2</sup> ), microvilli were long (>2 µm); whereas they were shortened (<2 µm) in the area at the inlet or outlet of the chamber with high shear stress (0.1 dyn/cm<sup>2</sup> ). In agreement with this observation, ezrin—a member of the ezrin-radixin-moesin (ERM) family, which plays a major role in formation and/or maintenance of actin-based cell surface structures—was predominantly detected at the apical membrane of the cells.

The observation of fluid shear stress-induced microvilli formation has recently been confirmed in rabbit trophoblast stems cells, which were cultured on a collagen gel in the presence of flow (Table 2, [17]). Subsequent transcriptome analysis of the rabbit trophoblasts, showed enrichment in pathways regulating actin cytoskeleton and sphingolipid metabolism, which has been suggested to account for the increased formation of microvilli during differentiation [17].

### **4. The Influence of Fluid Shear Stress on Trophoblast Metabolism**

While the transfer of gases and some other solutes occurs by flow limited diffusion, nutrients, and waste products have to be actively transported across the placental barrier. The extent of nutrient transfer and, hence, of fetal supply is determined by many factors, including placental morphology as well as uteroplacental and fetoplacental blood flow [34]. Perazzolo et al. suggested that the relationship between maternal blood flow and villous structure affects the efficiency of placental uptake and transfer, and moreover, that flow rate may be the major determinant of it [35]. However, besides uteroplacental blood flow, barrier thickness, and concentration gradients, factors such as transporter expression and metabolism of the villous trophoblast influence the dynamics of placental transfer. In fact, a growing body of evidence suggests substantial differences in metabolism in cells cultured under flow, when compared to static conditions. Accordingly, fluidic flow increased the accumulation and size of lipid droplets in rabbit trophoblasts [17], suggesting that morphological differentiation was accompanied by metabolic changes. Consistent with morphological differentiation of rabbit trophoblasts and their increased lipid droplet accumulation, a number of genes of the peroxisome proliferator-activated receptor (PPAR) signaling pathway were upregulated in response to shear stress. Amongst these genes, perilipin 2 (PLIN2), cytochrome P450 1B1 (CYP1B1), and angiopoietin-like 4 (ANGPTL4) are reported to be involved in lipid metabolism, transport and storage (Table 2, [17]), suggesting enhanced metabolic turnover in trophoblasts cultured under flow. In addition to its importance for the formation of lipid droplets, PLIN2 is necessary for trophoblast viability when exposed to fatty acids. This has been shown by overexpression of PLIN2 in human primary term trophoblasts that were exposed to a mixture of linoleic acid and oleic acid [36].

Along with lipid metabolism, trophoblastic glucose uptake is affected by flow, as shown by significantly increased uptake of the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) into BeWo cells exposed to fluid shear stress. The increased glucose uptake could be explained by slightly increased mRNA expression of the glucose transporter type 1, GLUT1 (encoded by *SLC2A1*), which predominantly localized to the apical membrane of cells and cell–cell contact regions after overnight exposure to fluid shear stress [16].
