Nitric oxide (NO), the main vasodilator in the placenta, is involved in the regulation of feto-placental vascular reactivity, placental bed vascular resistance, trophoblast invasion and apoptosis, and platelet adhesion and aggregation in the intervillous space [¹–³]. Placental vascular development is a crucial process required for adequate fetal development [¹,⁴]. Placenta insufficiencies such as in gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR), and preeclampsia are related to vascular dysfunction of the placenta [⁵].

Plasma asymmetric dimethylarginine (ADMA) is recognized as a biomarker of endothelial disorders, cardiovascular disorders, hypercholesterolemia, and stroke [⁶–⁸]. Maternal plasma ADMA levels are reduced in a normal pregnancy but increase as the gestational age increases [⁹,¹⁰] and is increased in compromised pregnancies such as those with preeclampsia [⁹,¹⁰].

The placenta plays a central role in fetal programming by directly regulating blood flow, transporter activity, fetal nutrient supply and fetal growth [¹¹,¹²]. Epigenetic mechanisms play a critical role during placental maturation and development [¹³]. NO is a known epigenetic molecule that plays a role in epigenetic fetal programming [¹⁴,¹⁵].

It has been suggested that therapeutic agents, which target placental blood flow and vascular development, ameliorate fetal growth restriction [²,⁵]. Thus, manipulation of the ADMA-NO pathways may have a therapeutic potential to rescue placental blood flow and improve long-term outcomes in patients with placental insufficiencies.

NO is synthesized in every cell type by nitric oxide synthases (NOSs), namely nitric oxide synthase 1 (NOS1 or nNOS), nitric oxide synthase 2 (NOS2 or iNOS), and nitric oxide synthase 3 (NOS3 or eNOS). NOS1 and NOS3 are considered constitutive NOS. NO is formed from its precursor, L-arginine, by a family of NOSs in the presence of oxygen and the cofactor tetrahydrobiopterin, with the production of L-citrulline. NO produced in endothelial cells relaxes vascular smooth muscle via the cyclic guanosine monophosphate-dependent pathway and also has a potent vasodilatation effect. The exchange of amino acids between the intracellular environment and the plasma is facilitated by specific transporter systems. NOS activities depend on the ability of endothelial cells to take up their specific substrate L-arginine via a variety of membrane transport systems. In human endothelial cells, these membrane transport systems include y⁺, y⁺ L, b^0,+, and B^0,+[⁷,¹⁶].

ADMA is formed when arginine residues in proteins are methylated by the action of types I and II protein arginine methyltransferases (PRMT). ADMA is a competitive inhibitor of L-arginine for all 3 NOS isoforms. Approximately 20% of ADMA is excreted by the kidneys, whereas the other 80% is metabolized by two dimethylarginine dimethylaminohydrolases (DDAH-1 and -2) to L-citrulline and dimethylamine. System y⁺ transporters mediate ADMA uptake by neighboring cells and conform a family of proteins known as cationic amino acid transporters (CATs) that includes CAT-1, CAT-2A, CAT-2B, CAT-3, and CAT-4 isoforms [⁷]. The CATs are the main determinant of the ADMA distribution between the cytosol and the extracellular fluid. Intracellular ADMA can inhibit CAT, block NOS activity, and limit the cellular uptake of L-arginine, thereby contributing to oxidative stress and further NO biogenesis inhibition. The NO and ADMA synthesis and metabolism pathways are presented in Figure 1.

In preterm infants, plasma ADMA levels remain relatively constant during the first week of life but increase from 0.66 μM to 0.95 μM by the fourth week [¹⁷]. During childhood, ADMA levels slowly decrease from birth until around 25 years of age, with a mean decrease of 15 nM per year [¹⁸]. In adults, plasma ADMA concentrations tend to increase with age, with the mean concentration in the range of 0.4–0.6 μM [¹⁹].

During pregnancy, the mother and fetus have a complex anatomical and functional interaction. The placenta acts as the interface between the mother and the fetus and maintains fetal homoeostasis. The placenta originates from trophoblasts that differentiate into cytotrophoblasts and syncytiotrophoblasts, which form the primary villi. Villous cytotrophoblasts are specialized epithelial cells that anchor the fetus to the mother and establish blood flow to the placenta. The syncytiotrophoblasts, which comprise the transporting epithelium of the placenta, contain many transport proteins in the plasma membranes of both the mother and the fetus. These transport systems deliver nutrients to the fetus and provide a substrate for syncytiotrophoblast metabolism. Placental nutrient transport is dependent on vascular development; therefore, NO plays a critical role.

As pregnancy continues, NOS3 expression increases, primarily in the syncytiotrophoblasts [²⁰]. In addition, NOS3 activity is present in the intermediate trophoblastic cells in the cell columns of the anchoring villi and in the trophoblastic cells at the implantation site [²¹]. On the other hand, NOS2 activity increases throughout pregnancy that peaks around mid-gestation [²²]. Suzuki et al. proposed that NO plays a supportive role in promoting embryo survival [²³]. Purcell et al. suggested that after implantation, both NOS3 and NOS2 might play a role in tissue remodeling, immunosuppression, and vasoregulation [²⁴].

Plasma nitrates/nitrites have also been reported to be elevated in human pregnancy [²⁵]. A study involving sheep showed that the pregnancy-associated increase in endothelium-dependent relaxation of the uterine arteries is predominantly regulated by the upregulation of NO release that results in decreased intracellular free calcium in the smooth muscle [²⁶]. The human feto-placental vasculature lacks autonomic innervation, and therefore, NO confers autocrine and/or paracrine effects, playing an important role in the regulation of feto-placental blood flow. NO is the main vasodilator in the placenta and is involved feto-placental vascular reactivity regulation, placental bed vascular resistance, trophoblast invasion and apoptosis, and platelet adhesion and aggregation in the intervillous space [¹].

Adequate morphology and function of the vascular tree are dependent on environmental cues such as blood flow, nutrient supply, and oxygen levels. Placenta vasculogenesis is the process of vessel formation from mesenchymal-derived hemangioblasts that differentiate into endothelial cells [²⁷,²⁸]. Differentiation of embryonic stem cells toward the endothelial lineage is an important step in vasculogenesis, and vascular endothelial growth factor (VEGF) is an important factor implicated in this process. In general, the initiation of vasculogenesis requires VEGF expression, which are effects mediated by NO [²⁹]. The yolk sac plays a crucial role in embryo and placental vascular development. A study of mice yolk sacs showed that vasculogenesis is initiated by NO [³⁰]. Furthermore, the spatio-temporal expression patterns of NOS2 and NOS3 were related to vasculogenesis in the yolk sac [³⁰].

Angiogenesis, the formation of functional capillaries from pre-existing vasculature, helps drive villous development and differentiation [²⁷]. In this process, single vessels are formed because endothelial precursor cells differentiate into endothelial cells. NO is a critical downstream mediator of potent angiogenic agents such as VEGF, basic fibroblast growth factor (FGF), and angiopoietin-1 [³¹]. The role of NO is more established in angiogenesis than in vasculogenesis [³²].

The critical role of NO in angiogenesis has been shown in NOS3 knockout mice [³³]. Likewise, NOS inhibition is accompanied by angiogenesis deficiencies as is exemplified by deficient vascular sprouting [³⁴]. Interestingly, NO may also act upstream of angiogenic growth factors [³⁵] because the effect of NO on VEGF production may be mediated through hypoxia-inducible factor-1.

Maternal plasma ADMA levels are reduced in a normal pregnancy but increase as the gestational age increases [⁹,¹⁰]. Holden et al. reported that the mean plasma ADMA concentratio in 20 nonpregnant women was 0.82 μmol/L [¹⁰]. The mean plasma ADMA concentration was 0.40 μmmol/L in 33 first-trimester pregnancies, 0.52 μmol/L in 50 second-trimester pregnancies, and 0.56 μmmol/L in 44 third- trimester pregnancies [¹⁰].

In early pregnancy, the reduction in ADMA and concomitant increase in NO may lead to hemodynamic adaptation, a higher need of organ perfusion in pregnancy, and uterine relaxation to allow for undisturbed intrauterine growth of the fetus. In late pregnancy, physiologically increased ADMA levels thus help prepare the uterine muscle fibers for the higher contractile activity that is necessary for successful delivery by antagonizing NO-induced uterine relaxation. This is reflected by the higher ADMA level after cesarean birth compared with vaginal birth [³⁶]. After birth, ADMA levels fall, which may contribute to decreased NO production and bioavailability in neonatal vascular beds [³⁶].

NO regulates feto-placental vascular reactivity and placental blood flow. It has been suggested that placental angiogenesis abnormalities and angiogenic factor expression are associated with a variety of compromised pregnancies such as IUGR, GDM, and preeclampsia [³⁷,³⁸].

In an ewe model, circulating NO and its metabolites were elevated in pregnancies with multiple fetuses compared with singletons [³⁹]. NOS3 can interact with the VEGF system and can enhance its production and function in placental tissues during early pregnancy [⁴⁰]. In this context, placental expression of NOS3 is reduced in compromised pregnancies, including those of humans [⁴¹].

A normal pregnancy is accompanied by increasing metabolic activity of the placental mitochondria throughout gestation. The placenta thus generates reactive oxygen species (ROS) and reactive nitrogen species (RNS) in normal pregnancies and increased levels of the same in compromised pregnancies. In parallel, altered expressions of amino acid transporters in the placenta are also found in compromised pregnancies [¹]. In this regard, Khullar et al. showed that NO⁻- and superoxide (O^{2 −})-derived free radicals impair placental syncytiotrophoblast amino acid uptake and increase Na⁺ permeability in vitro[⁴²].

Due to their abundance in the placenta, superoxide and NO can interact to generate peroxynitrite (ONOO⁻), a potent pro-oxidant. Peroxynitrite formation will alter the placental NO level, which in turn will affect physiological function. The presence of nitrative stress was associated with diminished vascular reactivity of the fetal placenta, a situation that could be recapitulated in vitro by peroxynitrite treatment [⁴³]. Roberts et al. found many nitrated proteins in the placenta [⁴⁴]. Nitration of the placental proteins is found in normal pregnancies but at increased levels in pathologic pregnancies [⁴⁵]. For instance, p38 mitogen-activated protein kinase could be nitrated with peroxynitrite at the tyrosine residues and could be associated with the loss of catalytic activity [⁴⁵].

Recent studies have suggested that the plasma concentrations of ADMA may serve as a risk biomarker for endothelial dysfunction, hypercholesterolemia, preeclampsia, stroke, and cardiovascular disease [⁶]. Declining ADMA levels is observed in a normal pregnancy with increasing gestational age [⁹]. In compromised pregnancy such as preeclampsia, ADMA levels rise to levels greater than that seen in normal pregnancy [¹⁰]. Holden et al. showed that the mean plasma ADMA concentration of the 18 third-trimester preeclamptic subjects was 1.17 μmol/L, significantly higher than both the normotensive gestational age-matched and the nonpregnant control groups [¹⁰]. The association of abnormal uterine artery Doppler waveforms with high ADMA concentrations [⁴⁶] in pregnancy supports the role of endogenous NOS inhibitors adversely affecting maternal vasodilation and blood pressure. Consistent with these findings, increased vasoconstriction of human stem villous feto-placental arteries [⁴⁷] and increased ovine umbilical vascular resistance [⁴⁸] are caused by an NOS inhibitor, N^G-monomethyl-L-arginine. Suzuki et al. showed that N^G-nitro-L-argininemethyl ester, an NOS inhibitor, caused apoptosis in the decidua, suggesting that NO in the decidua is essential to cell survival and the maintenance of uterine formation [⁴⁹].

Placental nutrient transport is dependent on vascular development, which determines blood flow to the placenta. NO regulates placental blood flow. ADMA has been associated with uterine artery flow disturbances. Substantial experimental evidence has reliably supported the roles of NO and ADMA in normal pregnancy and placenta insufficiency as well as the link between placenta insufficiency and epigenetic fetal programming. With expanded knowledge on the mechanisms of the impaired ADMA-NO pathway, additional targets for therapeutic intervention will be identified. A multifaceted approach to restoring NO bioavailability will be required to prevent compromised pregnancies and fetal programming.

fn2-ijms-13-14606Conflict of Interest

The authors declare no conflict of interest.

The study was supported in part by grants from Chang Gung Memorial Hospital, Kaohsiung, Taiwan, CMRPG8B0111 to Li-Tung Huang, and grant CMRPG8B0171 and grant NSC 98-2314-B-182A-006-MY3 from National Science Council, Taiwan to You-Lin Tain. We thank the center for Translation Research in Biomedical Sciences and center for Gene Proteomics Research, Kaohsiung Chang Gung Memorial hospital for supports.