Aberrant retinoic acid production in the decidua: Implications for pre-eclampsia.

Numerous studies have shown that an excess of anti-angiogenic sFLT1 leads to PE pathology. A seminal finding by Maynard et al., in 2003 showed that experimental overexpression of sFLT1 in pregnant rats produced the classic phenotype of PE, namely increased blood pressure, proteinuria and renal dysfunction. 66 Following this report, sFLT1 levels have been analyzed in multiple groups of pregnant women across the globe and its association with the disease has been validated and accepted. The hypothesis is that excess circulating sFLT1 contributes to maternal endothelial dysfunction by binding VEGF and PLGF and preventing their functions. Recent studies have targeted sFLT1 as a treatment for PE, using plasmapheresis to reduce their circulating levels. 67 Currently, there is very limited information regarding the regulation and expression of sFLT1 within the placenta or endometrium. Our recent publication showed that in vitro decidualization of ESC is associated with a dramatic (>90%) inhibition of sFLT1 protein production and secretion. 68 Similar effects were observed in cultured decidual stromal cells (DSC) from term placenta that are induced to re-decidualize in vitro . Since decidualization is a prerequisite for successful implantation, this observation suggests that repression of maternal sFLT1 at the maternal-fetal interface may be necessary for optimal implantation. This thesis is intuitive given the critical need for brisk angiogenesis to establish maternal blood flow to the developing placenta by the 10–12th week of gestation. In addition to the effects of low RA levels on sFLT1 production via effects on decidualization, we have determined that RA also has a direct inhibitory effect on sFLT1 transcription and secretion from ESC and DSC 8 ( Fig. 5 ). Thus, reduced RA levels by these multiple mechanisms can promote overexpression of sFLT1, with its anti-angiogenic mechanisms that further encourage shallow implantation and reduced spiral arteriole modification, leading to serious gestational consequences. The mechanisms involved in the regulation of sFLT1 have not been addressed in placenta, especially during decidualization. Earlier studies established cAMP response elements (CRE) and ETS1 sites in the FLT gene promoter that are essential for active FLT gene transcription in endothelial cells. 69 – 72 Studies also suggest the presence of a negative regulatory element in the first exon of the FLT gene. 72 FLT transcriptional regulation during pregnancy and decidualization is not known. Our comprehensive analysis of the FLT gene promoter sequence using MotifMap 73 , 74 showed an RXR/RAR binding element at −522 and an activator protein 2 (TFAP2A) at −603. Both of these motifs are regulated by RA ( Fig. 6 ). It is presently unknown whether inhibitory action by RA vs. cAMP-induced decidualization is governed by the same or different regulatory elements in the FLT promoter. By cDNA array analysis, we have identified a number of transcription factors and accessory proteins expressed in ESC (both decidualized and nondecidualized) known to be involved in RA- and cAMP-mediated gene regulation and, correspondingly, have cognate response elements located in the immediate 5’-promoter of the FLT gene ( Fig. 6 ). These factors include: CRABP1 and 2; RARα, β and γ; FABP5, PPARβ/δ, RXRα, β and γ; HIF1A; Ets1 and 2; CREB1; CREBBP; NCoR; TFAP2A and SMRT and are currently being characterized.

Our studies have determined the importance of RA in promoting healthy decidua through suppression of sFLT1 and regulation of decidualization via Cx43. We propose the following steps by which RA is involved in early placentation and fetal development and how abnormal RA signaling may promote the development of PE ( Fig. 7 ): The cells that are primarily responsible for the production of RA are M2 macrophages – step (1) in schematic. These cells secrete RA (2) into the surrounding uterine tissue that includes ESC (3), the precursors of decidual cells, as well as a variety of immune cells, including T-lymphocytes (4). By stimulating Treg differentiation (5) and activating the PPARβ/δ molecular pathway (6), RA may control immune tolerance (7) and augment the decidualization response of ESC in combination with ovarian hormones (8). RA production and signaling appear to be defective in PE, as indicated by reduced RA levels in decidua dissected from PE placentas compared with normal pregnancy tissues. This defect may be at least partly responsible for maternal hyper-inflammatory responses and suboptimal decidualization of ESC, the latter resulting in the continued production and secretion of the anti-angiogenic factor sFLT1 (9). High concentrations of sFLT1 in the vicinity of decidualized stromal cells (DSC) during embryo attachment block the action of VEGF and PLGF (10), preventing new blood vessel formation and modification, resulting in an inadequate vascular network necessary for healthy placental development and growth. This scenario gives rise to smaller placentas, with less capacity for nutrient and gaseous exchange necessary to support a normal pregnancy. Hypoxic conditions in the placenta result from these processes, which adversely affect the development of spiral arteries that further enhance the severity of the PE condition. Maternal compensation gives rise to the symptoms of PE (high blood pressure, protein in the urine, liver and kidney problems). This scenario suggests the possibility that PE might be reversed or prevented by treatment with potentially ‘safe’ PPARβ/δ activators to counteract inadequate RA production and/or activity.

It is known that estradiol (E2) and progesterone (P4) are the key steroid hormones involved in the processes of decidualization and regulation of maternal immune tolerance to the developing fetus; E2 and P4 alone can transform the fibroblastic uterine stroma to establish a vascularized, nutritive and immune-privileged secretory platform that permits nidation of the allogeneic blastocyst. 9 However, it is now understood that the myriad regulatory cellular actions of E2 and P4 on the endometrium are mediated in part by elaboration of local growth factors, 10 eicosanoids, 11 vitamins and retinoids 12 that are synthesized or critically metabolized in situ . In this regard, our data and those of others have shown that RA plays a fundamental role regulating optimal decidualization required for effective trophoblast implantation. 13 – 15 RA is regulated by the enzymatic activity and expression of retinol dehydrogenase (RDH) and retinal dehydrogenase (RALDH) enzymes. 16 As demonstrated in a variety of tissues, RA concentrations can be regulated in vivo through the activity of RALDH enzymes that catalyze the oxidation of retinal to RA 17 – 20 ( Fig. 1 ). RALDH expression is limited to select cell types and induced by various stimuli, including RA itself. 17 , 18 , 21 A primary source of RALDH-expressing cells in the small intestinal mucosa are dendritic cells (DC) that reside in the Peyer’s patches, lamina propria and mesenteric lymph nodes. 20 – 22 RA produced from these gut-associated DC is required for intestinal leukocyte tropism, differentiation to IgA secreting cells, and determination of Treg induction. The latter serves to inhibit proinflammatory Th17 differentiation. 19 , 22 – 25 Gut-associated DC from patients with inflammatory bowel diseases (e.g. Crohn’s disease, ulcerative colitis) express lower RALDH and have reduced RA production and action. 26 Based on our understanding of the role of RA in gastrointestinal and other tissues, we have posited that the hyper-inflammatory state occurring in the decidua of PE subjects reflects suppressed RA levels. This hypothesis is consistent with the finding that the concentration and function of Tregs are reduced in PE. 27 , 28 Figure 2a shows that RA in decidual tissue is reduced by almost 30% in pregnancies affected by PE. In addition to DCs, other cell types produce RA, including epithelial cells, stromal cells as well as monocytes, macrophages and T-cells. To determine the major cellular sources of RA in term decidua, we utilized an Aldefluor assay coupled with flow cytometry. 20 As shown in cytograms of Figure 2b , approximately 24% of macrophages/monocytes (CD14+ cells) showed positive RALDH activity compared with less than 4% of stromal cells (CD10+) or T lymphocytes (CD3+). In fact, the subpopulation with the highest RALDH activity shares CD14 and CD163 typical of anti-inflammatory macrophages (so-called ‘M2’ macrophages) and thus appear to be the major source of RA production in the decidua ( Fig. 2c ). 33 Interestingly, the frequency of decidual M2 cells is reduced in PE compared with normal pregnancies. 34 , 35 Whether the paucity of decidual M2 cells and reduced RA production in PE are responsible for impaired maternal immune tolerance in PE has yet to be determined.

Pre-eclampsia (PE) is a life threatening complication of gestation, involving 5–7% of all pregnancies and conferring considerable morbidity and mortality to the mother and infant. 1 – 3 Additionally, 30% of PE pregnancies result in growth-restricted babies, which is another contributor to neonatal morbidity. 4 The prevalence of PE appears to have increased ~20% over the past decade and is 60% more likely to occur in African–American than white mothers. Treatments center on managing hypertension and the associated sequelae of the condition and in severe cases, delivery of the preterm infant is the only option to restore the mother’s health. Studies have shown that in addition to medical complications experienced during pregnancy, women with PE have a 7–8-fold greater risk of death and an increased risk of life-long cardiovascular diseases compared to counterparts with normal pregnancies. 5 In addition to the consequences of prematurity, children and adolescent offspring of PE pregnancies also display increased cardiovascular health risks. The etiology of PE still remains elusive; some prominent theories include those implicating hypoxia resulting in poor placentation, an overexuberant maternal inflammatory response to pregnancy, and an anti-angiogenic state at the fetomaternal interface. 6 , 7 To this end, multiple lines of evidence support the idea that circulating and local anti-angiogenic factors (particularly soluble fms-like tyrosine kinase-1, sFLT1 and soluble endoglin, sENG) are involved in the genesis of the PE syndrome. 7 The pathology of PE includes shallow penetration of fetal trophoblasts into the maternal endometrium (decidua) and fewer spiral arteries are remodeled to supply blood to the growing placenta. As a result, placentas in PE pregnancies are smaller, with less capacity for nutrient and gaseous exchange. A deeper understanding of the causes and predictors of PE may afford improved preventive strategies and inform future treatments, thereby leading to improved pregnancy and long-term health for women and infants. Although the placenta’s role in PE has been extensively studied, contributions from the maternal side of the fetomaternal interface (decidua) have been a lesser focus of investigation. Two compelling lines of investigation regarding the maternal contribution to PE involve defects in decidualization and a heightened maternal immune response directed against the fetus, each of which can lead to inadequate development of blood vessels on both the maternal and fetal sides of the placenta. In recent years, we started exploring events at the maternal–fetal interface to understand the mechanisms of placental pathology leading to diseases like PE. Our studies uncovered certain original and unique events that are dys-regulated at the decidual layer in women who develop this disease. These include reduced levels in the decidua of PE women of certain steroid/retinoid compounds, including the active vitamin A metabolite, all-trans retinoic acid (RA). 8 By utilizing decidual cells obtained from the fetomaternal interface of the placenta, these studies have also revealed various aspects of the maternal decidualization process that is abnormal in PE. We highlight these novel findings in this review.