Sanjay Basak

Insulin-dependent, glucose transporter 1 mediated glucose uptake and tube formation in the human placental first trimester trophoblast cells

During early gestation, hypoxic condition is critically maintained by optimal glucose metabolism and transporter activities. Glucose is readily available energy nutrient required for placentation. However, limited data are available on glucose uptake and its transporters during first trimester placentation processes. To this end, effects of glucose and the roles of glucose transporters (GLUTs) were investigated during hypoxia on trophoblast migration and placental angiogenesis processes using early gestation-derived trophoblast cells, HTR8/SVneo, and first trimester human placental explant tissues. Exogenously added glucose (25xa0mM) significantly increased tube formation (in vitro angiogenesis) in HTR8/SVneo cells with concomitant activation of AKT-PI3K pathway and increased expression of vascular cell adhesion molecule 1 (VCAM1) compared with those in the presence of 11xa0mM glucose. Cobalt chloride (CoCl2)-induced hypoxia also significantly increased glucose uptake and GLUT1 expression along with tube formation and migration of HTR8/SVneo cells. During hypoxia, addition of glucose further stimulated HIF1α expression than by hypoxia alone. Cytochalasin B (cyt-B) inhibited the glucose uptake both in the presence of 11xa0mM and 25xa0mM glucose. Insulin (1xa0ng/ml) stimulated GLUT1 expression and tube formation and up-regulated the expression of VEGFR2 in HTR8/SVneo cells. Insulin and glucose-stimulated tube formation was inhibited by cyt-B but had no effect on hypoxia-induced tube formation. Silencing of GLUT1 inhibited the glucose and insulin-stimulated tube formation as well as glucose uptake. However, fatty acid-stimulated tube formation was not affected in GLUT1 knockdown cells. All these data suggest that glucose uptake, glucose-stimulated tube formation, and insulin-stimulated glucose uptake of the first trimester trophoblast cells, HTR8/SVneo, are mediated in part via GLUT1.

Fat-Soluble and Antioxidant Vitamins and Minerals: Their Roles in Placentation

Vitamins and minerals, often termed as micronutrients, serve essential roles in cellular metabolism, maintenance, and growth throughout life. Micronutrient deficiency is critical in development stages that determines long-term disease outcome. Micronutrients are also central components of many enzymes and transcription factors. The need for optimum amounts of key micronutrients at critical stages starting well before conception through ovulation, placentation, and fetus development has been documented recently. Both excesses and deficiencies of micronutrients can have long-term effects on many fetal tissues and organs due to subclinical deficiency of vitamins in the mother. Micronutrient imbalance of the developing fetus may not reflect any quantitative changes at the time of nutritional insult, but may have a permanent scar on genome imprint due to altered development circuit that manifests later in life (Hovdenak and Haram 2012). Unfortunately, supplementary correction of micronutrients later in gestation or during postnatal life cannot rescue or completely revert the detrimental effects of earlier micronutrient imbalance. In that context, periconceptional supplements and life course nutrition are being given a priority at this moment. The imbalance can affect pregnancy outcome through alterations in maternal and fetus metabolism, as a consequence of their essential role in enzymes and transcription factors and through their involvement in signal transduction pathways that regulate development. Considering wide range of micronutrients which affects development, the number of developmental stages involved, diverse biological pathways associated, and types of tissue affected, it is challenging to pool all the micronutrients in this chapter. In this chapter, the risk associated with pregnancy due to excess or little of micronutrients in periconception and preconception periods that determine fetal health and development will be discussed. The chapter will discuss the functional aspect of fat-soluble vitamins and their implication in human development involving the placenta. Overall the chapter will emphasize the functional role of the fat-soluble vitamins and key minerals in feto-placental growth and development.

Gene Regulation, microRNA, and Placentation

Trophoblast invasion into the decidua and inner myometrium is essential for the establishment of proper implantation, maternal–fetal exchange, and immunological tolerance of the feto-placental allograft. Unlike villous trophoblasts, extravillous trophoblasts (EVT) are unique in their capacity to invade the maternal decidua and myometrium. Despite resemblance to metastasis, trophoblast invasion is tightly regulated as there are several factors involved in ensuring appropriate invasion. Like tumor cells, invasiveness is a feature of trophoblasts; however, unlike tumor invasion, trophoblast invasion is a strictly controlled physiological event. The placenta is a critical organ for the development of the fetus in utero. Trophoblast invasion into the uterine spiral arteries and the rebuilding of the arteries are crucial for normal placental development (Hunkapiller and Fisher 2008; Knofler and Pollheimer 2012). Imperfect uterine spiral artery rebuilding and inadequate trophoblast invasion have been associated with a range of pregnancy-related diseases. The placenta upregulates a variety of angiogenic factors and downregulates anti-angiogenic factors in order to stimulate angiogenesis. Spiral artery remodeling appears to be a multistage process involving spiral artery preliminary remodeling (characterized by vacuolization and apoptosis of resident endothelial cells), trophoblast migration and invasion of the decidua and myometrium, incorporation of EVTs into the spiral arteries vessel walls, and acquisition by the endovascular EVTs of an EC-like phenotype. The trans-differentiation of EVTs into EC-like cells results in the downregulation of epithelial cell-type markers, E-cadherin and integrin α6β4, and the induction of the expression of endothelial adhesion molecules such as VE-cadherin; PECAM; NCAM; as well as integrins α5β1, α1β1, and αVβ3 (Damsky and Fisher 1998). It has been consistently reported that in early onset preeclampsia, the remodeling of the spiral arteries is impaired (Red-Horse et al. 2004). This leads to reduced perfusion of the developing placenta, subsequent intermittent hypoxia-reoxygenation episodes, reactive oxygen (ROS) production, cell injury, inflammation, and liberation of placental factors and debris into the maternal circulation.

Glucose and Amino Acid and Their Roles in Placentation

The “Fetal origins hypothesis” states that individual born small due to maternal malnutrition is predisposed to adult disease. The hypothesis is skewed more towards malnourished mothers and does not specify to include placental factor. In contrast, poor placentation mainly due to inadequate vascular adaptation at the utero-placental interface is far more reasonable cause of reduced fetal growth in adequately nourished populations (Henriksen and Clausen 2002). Now, substantial evidences exist that suboptimal maternal nutrition can regulate newborn in both the cases. Immediate impact of optimum maternal diet or its deviation on placental growth and fetal birth weight would be the key to understand their beneficial effect on placentation process. The availability and supply of nutrients to the developing fetus depend on maternal nutritional status which in turn depends on their nutrient stores, dietary intake, and obligatory requirements during pregnancy (Ramakrishnan et al. 2012). During development, there are critical periods when system and organs undergo maturation; those stages are susceptible to be programmed. The pregnancy-specific stages that span from the preconception to the birth are subjected to be influenced by macro and micronutrients that affect both maternal health and fetus development subsequently. Data so far highlighted the importance of nutrition during pregnancy typically focusing on the second and/or the third trimester by which time major organogenesis would have been completed. However, nutritional impact just before conception and/or during first trimester, when women are typically unaware of their pregnancy status, is scanty. It is likely that preconception, conception, implantation, organogenesis, and placentation will be influenced by status of the maternal periconceptional nutrition. These effects may lead to regulate overall health of childbearing women, their reproductive potential, and birth outcome.

Early Placentation Processes

The process of embryo implantation is a highly sophisticated and synchronized event between activated implanted embryo and a receptive endometrium. Proper development of embryo and implantation of blastocyst largely depends on optimal endometrial receptivity. The process of embryo implantation occurs within restricted period of time known as “window of implantation” which is highly regulated by two prominent hormones such as estrogen (E2) and progesterone (P4). Endometrial receptivity for implantation occur independent of embryo activation. The precise mechanism of each of these hormones on regulation of implantation and endometrium receptivity is not clear. Endometrium is composed of many cell types that include epithelial, endothelial, stromal, and nonresident immune cells. Stromal cells play crucial roles during embryo invasion to the uterine walls.

Placentation as a Predictor of Feto-Placental Outcome: Effects of Early Nutrition

The placenta transfers nutrients, gases, and waste products between maternal and fetal circulations. Placental transfer capacity is determined by a wide range of factors such as placental surface area and thickness, the abundance of transporters, the gradient of concentrations between both maternal and fetal compartments, placental metabolism and utero-placental flows, and other environmental stimuli. Substances need to cross several cellular layers, among them the trophoblast, through different transport mechanisms such as simple or facilitated diffusion or active transport. Early placentation, which is very critical for optimum placental capacity, depends on the extensive remodeling of the maternal uterine vasculature producing low-resistance blood vessels that facilitate the exchange of nutrients and wastes between the mother and the fetus (Zhong et al. 2010). Proper placental development is critical for fetal growth and development (Myatt 2002). The shallow invasion of the spiral arterioles and the maternal decidual stroma by the extravillous trophoblasts (EVTs) results in poor maternal blood flow to the feto-placental unit. Consequently, this reduces oxygen and nutrients’ delivery to the fetus. Concomitantly, ischemia–reperfusion injury may follow inappropriate vasoconstriction of untransformed arteries, increasing oxidative stress, syncytiotrophoblast shedding, and maternal systemic vascular inflammation. All these are important features of preeclampsia (PE). In addition, the compromised placental growth and development may affect fetal growth and development due to the result of insufficient placental transfer of maternal nutrients such as lipids, glucose, amino acids, minerals, and vitamins. There are several other factors that affect the placental transport function such as interrelationships of maternal food intake, availability of nutrients in the maternal circulation, and ability of the placenta to efficiently transport substrates to the fetal circulation.

Endocrine Factors and Their Effects on Placentation

The endocrinology of human pregnancy involves endocrine and metabolic changes that result from physiological alterations at the boundary between mother and fetus. Known as the feto-placental unit, this interface is a major site of protein and hormone production and secretion. Many of the endocrine and metabolic changes that occur during pregnancy can be directly attributed to hormonal signals originating from this unit. Maternal adaptations to hormonal changes that occur during pregnancy directly affect the development of the fetus and placenta. Gestational adaptations that take place in pregnancy include establishment of a receptive endometrium, implantation and the maintenance of early pregnancy, modification of the maternal system in order to provide adequate nutritional support for the developing fetus, and preparation for parturition and subsequent lactation. As described already, a successful human pregnancy requires cytotrophoblasts from the fetal portion of the placenta to adopt tumor-like properties. So, migration and invasion of cytotrophoblasts into the maternal endometrium are key events in human placentation. Trophoblast infiltration of maternal decidua and spiral arteries is regulated by a fine balance between the production of multiple metalloproteinases (MMP) such as MMP1–MMP16 (gelatinases, collagenases, and stromelysins), their physiological activators, and their inhibitors (TIMP1 and TIMP2) (Cohen and Bischof 2007). Many factors have been shown to regulate invasive capacities of EVTs. Some cytokines, such as adiponectin, interleukin (IL) IL-6, IL-10, and IL-11, leukemia inhibitory factor, CCNs, epidermal growth factor, angiotensin II, and leptin, have recently been described to modulate synthesis and release of one or more of these MMPs and TIMPs (Castellucci et al. 2000; Gonzalez et al. 2001; Qiu et al. 2004). In this chapter we will describe the key endocrine/cytokine factors involved in early placentation.

B Vitamins and Their Role on Trophoblast Growth and Development

Folic acid is critical for growing infant particularly relevant to preterm and low birth weight in addition to neural tube defects (NTDs) and should be given as supplement for low birth weight (Orzalesi and Colarizi 1982). Folic acid is also known as vitamin B9 and acts as a coenzyme in the biosynthesis of purine and pyrimidine precursors of nucleic acids, which are critically important for DNA synthesis of the growing fetus during pregnancy. Its natural form is known as folate, metabolized differently than its synthetic oxidized form of folic acid. Pregnant women are at risk for folate insufficiency because for its increased demand for rapid fetal growth, placental development, and enlargement of the uterus. Inadequate folate status may cause fetal malformations, impaired fetal growth, preterm delivery, and maternal anemia. Maternal folic acid deficiency during pregnancy has been associated with neural tube defects (NTDs), miscarriage, placental abruption, IUGR, and PE. Folic acid has the potential to affect the closure of the neural tube and several epigenetic mechanisms within the placenta and the fetus. As fetal growth is totally dependent on maternal folic acid reserve, therefore, pregnant women are advised to receive folic acid supplement starting from periconceptional period. It is possible that folic acid plays other growth-promoting activities in early development such as implantation and development of the placenta and in improving endothelial function. Due to these indispensable functions, folic acid supplements are considered at the late first trimester or early second trimester (Fekete et al. 2010). Folic acid is increasingly required for rapid growth and cell proliferation during the initial phases of pregnancy. Folic acid deficiency is critical in development stages that determines long-term disease outcome. The chapter emphasizes the functional aspect of folic acid and its implication in human development during early placentation. This review discusses well-designed intervention trials to provide a latest update on the output of the efficacy of folic acid supplementation in mitigating and preventing disease etiology. The information is also obtained from observational studies where a large number of samples are associated. The priority is given to human clinical studies; if not available, animal data are reviewed; and in vitro studies are included to understand its mechanism on cellular function. Evidences are now accumulating that periconceptional folic acid supplement starting before 12 weeks of conception has several benefits on the pregnancy outcome through improved feto-placental growth and development activities (Cawley et al. 2016b) In this review, the impact of folic acid supplements on placental growth and development activities is discussed with special emphasis on their effects in early pregnancy.

Dietary Fatty Acids and Placentation

Essential fatty acids (EFAs) belong to the n − 6 (omega-6) and n − 3 (omega-3) families, starting with the precursors; linoleic acid (LA), 18:2n − 6; and alpha-linolenic acid (ALA), 18:3n − 3. The n − 6 series of fatty acids contain two or more double bonds, with the first double bond on the sixth carbon from the methyl end of the molecule; the n − 3 fatty acids contain three or more double bonds, with the first double bond on the third carbon atom from the methyl end. N − 3 fatty acids and n − 6 fatty acids play crucial biological roles that include structure and function of cell membranes, providing substrates for the production of signaling molecules such as eicosanoids and modulating expression of genes involved in cell homeostasis (Smith 1989; Innis 1991; Dutta-Roy 1994). Dietary LA can be converted to AA in the body, mostly in the liver. The three main n − 3 fatty acids are ALA, docosahexaenoic acid (DHA), 22:6n − 3, and eicosapentaenoic acid (EPA), 20:5n − 3. Through the same desaturase and elongase enzymes, the n − 3 fatty acids, ALA can be converted into EPA and DHA. The enzymes responsible for the metabolism of both n − 6 fatty acids and n − 3 fatty acids are the COX, lipoxygenases (LOXs), and cytochrome P450 (CYP 450). Several eicosanoids derived from the n − 6 fatty acids promote tumor angiogenesis, such as the prostaglandins (PGH2, PGE2, PGI2), leukotrienes (4-series LTs), thromboxanes (TXA2), and hydroxyeicosatraenoic acids (12-HETE, 15-HETE)] (Smith 1989; Nie et al. 2000; Hoagland et al. 2001; Pai et al. 2001; Bagga et al. 2003; Pola et al. 2004; Jin et al. 2009; Kamiyama et al. 2006). These eicosanoids make the tumor microenvironment more favorable for neoplasms and metastasis by encouraging the transcription of angiogenic growth factors, increasing the rate of endothelial cell migration and proliferation, and increasing the rate of vascularization. In contrast, n − 3 fatty acid metabolism produces leukotrienes and prostaglandins that attenuate excess vascularization. N − 3 and n − 6 fatty acids compete each other for incorporation into the cell membrane in addition to enzymes for eicosanoid production, including COX-2 and 5-LOX. Thus, high levels of tissue n − 3 fatty acids can reduce angiogenesis through decreased production of pro-angiogenic AA-derived eicosanoids, membrane receptor-ligand interactions, and through n − 3 fatty acid’s intrinsic antitumor properties (Nie et al. 2000; Pai et al. 2001; Bagga et al. 2003; Pola et al. 2004; Kamiyama et al. 2006; Yuan et al. 2009). Furthermore, n − 3 fatty acids have been found to downregulate expression of angiogenic growth factors such as VEGF, PDGF, IL-6, and MMP-2 (Kang and Weylandt 2008; Spencer et al. 2009). N − 3 fatty acids are only found in marine fish and certain vegetables and nuts, whereas corn and soybean oils, processed foods containing these oils, and grain-fed meat contain high levels of n − 6 fatty acids. Now, the ratio of n − 6/n − 3 fatty acids is approximately 15:1 or higher; our bodies may not be accustomed to utilizing such high levels of n − 6 fatty acids. This is considered to be one of many factors responsible for the relatively recent rise in chronic diseases, predominantly those associated with inflammation including cancer, heart disease, arthritis, and diabetes (Simopoulos 2002). The maternal, fetal, and neonatal EFA/LCFA status is an important determinant of health and disease in infancy and later life (Innis 1991; Uauy et al. 2001). In fact, fetal brain and the retina are very rich in AA and DHA (Innis 2007). The numerous studies have demonstrated a positive effect of supplementation with DHA in pregnant women in terms of less premature birth and in the child; in terms of complex brain performance, like visual acuity, attention spans, and intelligence; and in terms of mother’s overall health. In addition, DHA may reduce the incidence of preeclampsia and postpartum depression by stimulating placental angiogenesis.

Placental Epigenetics and Its Importance in Placental Development

Epigenetics, literally “on” genes, refers to all modifications to genes other than changes in the DNA sequence itself. Mechanisms of epigenetic inheritance include methylation of DNA, modification of histones, binding of transcription factors to chromatin, and the timing of DNA replication. Epigenetic regulation of gene expression is necessary for the correct establishment of developmental programs and for the maintenance of cell fates. Importantly, epigenetic processes are thought to be at the interface between gene regulation and the environment, such that external influences can have a major and potentially long-term impact on gene expression (Reik 2007). In fact, nutrition plays an important role in the epigenetic repertoire. Maternal diet during pregnancy is very important in fetal development but in ways that are not yet fully understood. Additionally, the maternal reproductive tract’s metabolic and physiologic characteristics can also modulate the zygote’s development through all embryonic stages. A strong relationship was also observed between the epigenetic alterations in the placenta and diseases of gestation and early life. These findings have critical implications for diet and other environmental factors during pregnancy (Sandovici et al. 2012).