Fetal growth restriction (FGR) is a pregnancy complication associated not only with adverse perinatal outcomes but also with increased risk of cardiovascular diseases in adult life of offspring (1,2). The unborn baby has an estimated fetal weight (EFW) below the 10th percentile by gestational age as determined by prenatal ultrasound evaluation (3).

The etiology of FGR is multifactorial. However, one of the most common contributing factors is placental insufficiency (4), a condition that also serves a role in the pathogenesis of hypertensive disorders of pregnancy (HDP), including preeclampsia (5). In a recent study, it was demonstrated that FGR-HDP, a condition with significant obstetric morbidity and mortality, exhibits maternal vascular malperfusion of the placental bed, abnormal feto-placental Doppler parameters and signs of oxidative stress of the syncytiotrophoblast (6). Other recent studies show that extracellular vesicles derived from placental tissue influence endothelial cell function, explaining the relationship between placental insufficiency and hypertensive disorder (7,8).

There is widespread evidence for endothelial dysregulation of the fetoplacental vascular tone in FGR, which compensates for restricted blood flow (9-12). In this regard, nitric oxide production by the endothelium, which is key in maintenance of blood flow in the fetal placental bed during normotensive pregnancy (10) may be disordered in pregnancy with FGR and/or preeclampsia (11,12). Nitric oxide is produced by nitric oxide synthases, including endothelial nitric oxide synthase isozyme whose activity is markedly increased when intracellular Ca²⁺ concentration ([Ca²⁺]_i) rises (13). The activation of this endothelial isozyme depends on conformational changes induced by its interaction with the Ca²⁺-calmodulin complex (14). The vascular endothelium responds to agonists by increasing [Ca²⁺]_i. This response depends on different elements, including the type of receptor activated, release of Ca²⁺ from intracellular stores and store-operated Ca²⁺ entry (SOCE). The latter mechanism is activated by depletion of internal Ca²⁺ stores (15).

Our previous studies demonstrated that bradykinin, histamine, ATP and α-7 nicotinic acetylcholine receptors agonists increase [Ca²⁺]_i in endothelial cells (16-18). In human umbilical vein endothelial cells (HUVECs), ATP-induced Ca²⁺ signal typically consists of an initial transient phase followed by a prolonged sustained phase (18). ATP is an important autacoid and paracrine molecule that exerts dual control of vascular tones by being released from perivascular nerves and endothelial cells in response to changes in blood flow (shear stress) and hypoxia (19). Among purinergic receptors reportedly involved in ATP-induced Ca²⁺ responses in HUVECs are the metabotropic P2Y2(20) and ionotropic P2X4 receptors (21). The P2Y2 receptor, in addition to inducing rapid Ca²⁺ response, promotes nitric oxide production and appears to be the purinergic receptor that contributes most to the ATP-induced Ca²⁺ signal (20). P2X4 receptor seems to be overexpressed in pathological conditions and involved in production of reactive oxygen species and pro-inflammatory activators but not in nitric oxide synthesis (22).

The present study aimed to record clinical data of newborns and mothers from normotensive and pathologic pregnancies and investigated Ca²⁺ signals induced by ATP and SOCE in HUVECs.

During gestation, there is an active metabolic exchange between the fetus and mother, a process in which the efficiency depends on adequate development of the fetoplacental unit. Alterations in the efficacy of this exchange trigger hypoxia, which is associated with fetal distress, perinatal mortality and a potential risk of cardiovascular diseases in offspring (1,2,36). Both FGR and HDP are associated with placental insufficiency and are aggravated by placental ischemia, a condition accompanied by oxidative stress, wherein nitric oxide is unable to compensate for this impairment (37). Recent studies show that during hypoxia, placenta releases extracellular vesicles, which carry cytokines and microRNA that alter the function of endothelial cells (7,8,38). This phenomenon worsens pregnancy pathologies such as FGR and HDP (39).

The present study analyzed clinical characteristics of 11 single pregnant patients with FGR-HDP, a complication that has the highest rates of obstetric morbidity and mortality and elevated incidences of low gestational age at delivery, cesarean section and neonatal death compared with other types of pregnancy disorder (6). Despite attending to ~3,000 pregnant patients annually at Gustavo Fricke Hospital, only 11 met the inclusion criteria FGR-HDP and consented to participate. Given the small sample size, it is difficult to generalize the present findings to the broader population. Future research should employ larger and more representative samples to ensure generalizability of results.

The present study excluded cases of multiple pregnancies as this constitutes an independent risk factor for HDP (40), regardless of chorionicity or zygosity (41-43). In terms of fetal outcomes, moderately elevated blood pressure in multiple pregnancies increases blood flow to the placenta, contributing to decreased risk of preterm birth and low birth weight (43). Multiple pregnancies have other risks, the major one being prematurity, which is associated with adverse outcomes such as respiratory morbidity, intraventricular hemorrhage, necrotizing enterocolitis, and metabolic disorders (23,44). However, the risk of future cardiovascular disease is not increased (45), which contrasts with single pregnancies, where HDP is a risk factor for future cardiovascular disease (23,46,47).

In addition, other maternal, placental or fetal risk factors for abnormal placentation may result in placenta-mediated FGR. Therefore, in the present study these other co-existing factors were excluded due to their potential interference with normal fetal growth and effect on outcomes (23). To the best of our knowledge, however, there is no evidence that the combination of risk factors predicts the presence FGR (48,49). FIGO does not recommend using multiparameter algorithms (combining ultrasound and biochemical markers) for universal screening. This recommendation is based on the lack of sufficient validation of the effectiveness of these models in predicting FGR (23).

Here, gestational age and birth weight were significantly lower and neonatal intensive care admission was higher in the FGR-HDP group compared with the healthy group. The death of a newborn in the FGR-HDP group with an EFW in the 10th percentile and a 5-min Apgar score of 3 suggests that factors beyond these metrics play a critical role in determining the risk of neonatal mortality (50). This case highlights the greater prognostic value of the 5-min Apgar score compared to the 1-min score in predicting neonatal morbidity and mortality (26).

A total of 64% (7/11) of neonates from FGR-HDP pregnancies had abnormal UA Doppler, with 57% (4/7) exhibiting absent end diastolic flow; of these 75% (3/4) had oligohydroamnios. The use of Doppler velocimetry evaluation, especially of the UA, has been studied and reviewed in cases of FGR (30,31,51). A recent study showed that compared with other pregnancy complications, FGR-HDP has higher values of UA Doppler velocimetry, and maternal vascular malperfusion (6). A progressively increasing pulsatility index in UA corresponds to increased fetal artery resistance, which generates a progressive decrease of the placental area available for gas and nutrient exchange (52,53). FGR is associated with a dysfunction of the feto-placental vasculature involving endothelial cells. Compensatory upregulation of the nitric oxide system in feto-placental endothelial cells has been observed in FGR (12).

In accordance with the recommendations outlined by FIGO (23) for managing FGR, pregnant patients affected by FGR should be monitored using biophysical assessments and cardiovascular tests, to determine the timing of delivery. Many studies have focused on the prevention and treatment of HDP and/or FGR (23,54). To the best of our knowledge, however, there is currently no effective treatment to reverse the course of FGR and improve fetal growth (54). For future pregnancies, patients with a history of FGR should be counseled on risk of recurrence, considering the timing of onset, severity of FGR and placental histopathological findings (23). If the placenta is available, histopathological examination may provide valuable insights for counseling in future pregnancies (23).

HUVECs are used as a model to study cardiovascular diseases (55). They are also considered as potential predictors of cardiovascular risk in offspring of pregnancies involving preeclampsia (56). In this regard, HUVECs from preeclampsia pregnancies reportedly display impaired functional capacity, such as migration and tubule formation (57,58). As [Ca²⁺]_i signals are involved in these processes, the present study investigated how they were altered in FGR-HDP.

ATP-induced [Ca²⁺]_i signals were altered in HUVECs from the FGR-HDP group, including slower t_p and lower sustained phase. The initial phase of the ATP-[Ca²⁺]_i signal is primarily mediated by P2Y2 receptors (20), whose activation generates inositol triphosphate (IP₃) and Ca²⁺ release from Ca²⁺ stores, whereas the sustained phase of the [Ca²⁺]_i signals is determined by SOCE (59). The present result suggest that the kinetics of the IP₃-induced Ca²⁺ release and SOCE-mediated Ca²⁺ influx are diminished in FGR-HDP cells. The sustained phase of histamine-induced [Ca²⁺]_i signal is decreased in preeclampsia HUVECs (60,61), which agrees with the present results in FGR-HDP HUVECs, where the delayed phase of the ATP-induced [Ca²⁺]_i signal was also diminished, suggesting that HDP influences endothelial dysfunction.

To understand how Ca²⁺ dynamics is altered by FGR-HDP, components of the ATP-induced Ca²⁺ signals were separated (18). There were no significant changes in the Ca²⁺ signal induced by ATP in the absence of extracellular Ca²⁺, suggesting that the P2Y2 receptor mediated response was not affected in HUVECs from FGR-HDP group. However, the Ca²⁺ signal induced in presence of 10 mM Ca²⁺ was significantly higher in HUVECs from FGR-HDP compared with control HUVECs. This Ca²⁺ signal is associated with SOCE, a mechanism that depends on the Ca²⁺ sensor stromal interaction molecule-1, which senses Ca²⁺ depletion in the endoplasmic reticulum and activates Ca²⁺ release-activated Ca²⁺ channel protein 1 at the plasma membrane (15). This mechanism involves transient receptor potential and connexin channels and mitochondria (59,62). A recent study demonstrate that mitochondrial Ca²⁺ uniporter regulates SOCE at different levels, including Ca²⁺ store replenishment and cytosolic Ca²⁺ buffering systems, and that deletion of the mitochondrial uniporter increases SOCE-mediated [Ca²⁺]_i signals (63). Mitochondrial Ca²⁺ uniporter is impaired in hypertension and cardiovascular disease generating high cytosolic [Ca²⁺]_i levels (64,65).

An unexpected finding of the present study was that whereas the sustained phase of ATP-induced [Ca²⁺]_i signals was diminished in HUVECs from the FGR-HDP group, Ca²⁺ peak amplitude induced with 10 mM Ca²⁺ following depletion of internal Ca²⁺ stores was significantly higher. This result was also different from that observed by Steinert et al (60) using a similar protocol but applying a lower extracellular Ca²⁺ concentration (1 mM) to induce the second peak. High extracellular Ca²⁺ concentration (10 mM) may saturate the Ca²⁺ buffering mechanisms and induce dysfunction. In physiological conditions, the sustained phase of the response induced by agonists such ATP or histamine stimulates the synthesis of nitric oxide, which favors vasodilation (66). However, dysfunction of Ca²⁺ buffering mechanisms might cause overload of cytosolic Ca²⁺ levels, resulting in deleterious cellular effects such as oxidative stress, which contribute to FGR (67).

In conclusion, the present study found that FGR-HDP resulted in impaired UA resistance and altered Ca²⁺ responses to ATP regulated by SOCE in HUVECs. The present results provide better understanding of the mechanisms that regulate [Ca²⁺]_i dynamics in fetal endothelial cells and how they are altered in FGR-HDP. As dysfunction of HUVECs is a potential predictor of cardiovascular risk in offspring (56), the present study also provides an in vitro model to assess novel therapeutic approaches for decreasing or preventing cardiovascular disease in adulthood.