Maternal uterine microvascular adaptations during pregnancy are critical for optimal intrauterine growth, fetal survival (Creeth and John, 2020), and postnatal life. Placenta, a specialized transient organ during pregnancy, is responsible for keeping up with fetal nutritional demands and oxygen, molecule, and hormone feto–maternal exchanges (Guttmacher et al., 2014; Martín-Estal et al., 2019). During the formation of the hemochorial placenta, de novo maternal vascularization is dependent on not only normal development and function of trophoblast cells but also adequate angiogenesis in decidual tissue. During decidualization, early maternal vascularization involves physiological modification, branching, and growth of the uterine artery to transform into spiral arterioles (SAs). The maternal angiogenesis and remodeling of the decidual vascular network, consisting of low-resistance vessels to transport maternal blood with high volume flow at low velocity and pressure, is coordinated by decidual stromal cells, including uterine natural killer (uNK) cells and macrophages (Rätsep et al., 2015). The initiation of SA and matrix remodeling coincides with leukocyte infiltration and residence in the decidua. Decidual NK cells and macrophages, of great significance in early angiogenesis and placental development to pregnancy maintenance (Qin et al., 2023), express relevant factors, including metalloproteinases (MMPs) (Choudhury et al., 2019) and nitric oxide synthases (NOSs), which are some of the most important regulatory complex pathways of angiogenic-remodeling mechanisms of decidual tissue during early gestation.

Imbalanced expression, activity, and release of angiogenic and vasoactive mediators can lead to inadequate spiral artery angiogenesis, such as low decidual vascular remodeling and vasoconstriction of the spiral arteries during the early placentation period, which subsequently reduce perfusion and increase pressure in the placental feto–maternal interface (Gyselaers, 2023). An abnormal maternal angiogenesis and vascular function consequently can induce placental vascular malperfusion, insufficiency, hypertension, vasculopathy, and anatomical placental disorders, leading to gestational complications, recurrent spontaneous abortion, preeclampsia, and preterm labor (Reynolds et al., 2006; Khong et al., 2016; Gyselaers, 2023). Currently, placental abnormalities are associated with perinatal fetal malnutrition, morbidity, mortality, and fetal growth restriction (Sharma et al., 2016; Lacko et al., 2017; Perez-Garcia et al., 2018; Woods et al., 2018) and predispose to programming diseases in adults, such as coronary heart disease, type 2 diabetes mellitus, hyperinsulinemia, and the risk of developing adult-onset degenerative diseases (McMillen and Robinson, 2005; Hanson and Gluckman, 2008).

Different reports suggest that fetal and postnatal defects induced by alcohol use and abuse in gestation, including “fetal alcohol spectrum disorders” (FASDs) (Gupta et al., 2016; Hoyme et al., 2016), adulthood obesity, metabolic syndromes, and cardiovascular disease, among many others, may be due to placental abnormalities (Bada et al., 2005; Burd et al., 2007; Aliyu et al., 2008; 2011; Gundogan et al., 2008; 2010; 2015; Meyer-Leu et al., 2011; Patra et al., 2011; Salihu et al., 2011; Ramadoss and Magness, 2012; Avalos et al., 2014; Lui et al., 2014; Zhu et al., 2015; Carter et al., 2016; Linask and Han, 2016; Davis-Anderson et al., 2017; Tai et al., 2017; Ohira et al., 2019; Orzabal et al., 2019; Kwan et al., 2020; Odendaal et al., 2020). Some studies, both in human and animal models, have focused on the effects of maternal alcohol ingestion on the vascularization of the definitive placenta. Gestational alcohol exposure can alter maternal systemic and reproductive circulatory adaptations during pregnancy, affecting uteroplacental vascular hemodynamics with induced vascular resistance, among other gestational variables. Gestational alcohol intake produces the “alcohol-related placental-associated syndrome” (Salihu et al., 2011) that includes miscarriage, hypertension, preeclampsia, preterm birth, placenta previa, placenta accreta, and placental hemorrhage (Gundogan et al., 2010; Avalos et al., 2014; Carter et al., 2016; Tai et al., 2017; Ohira et al., 2019; Orzabal et al., 2019; Odendaal et al., 2020). Moreover, a high risk of placental abruption was observed after the consumption of 7–21 drinks per week (a mean of two drinks per day and BAC of 5–100 mg/dL) (Burd et al., 2007). Chronic gestational alcohol in rats (37% of caloric content) impairs the physiological remodeling of the maternal placental vasculature (Ramadoss and Magness, 2012). Related to this, some altered processes proposed to explain uteroplacental vascular dysfunction following gestational alcohol consumption were the disruption of trophoblastic function, such as inadequate or poor differentiation, migration/invasion (Han et al., 2012), and defective remodeling/endothelial replacement of maternal vessels by these cells (Radek et al., 2005; Rosenberg et al., 2010; Ramadoss and Magness, 2012b; 2012c; Subramanian et al., 2014; Orzabal et al., 2019). Gundogan et al. (2008, 2010) reported, in an animal model, that one major placental abnormality due to chronic gestational ethanol exposure is the failure of maternal decidual spiral artery remodeling by invasive trophoblasts, thus leading to altered placental blood flow and nutrient exchange. A moderate or high dose of ethanol intake during gestation also reduces labyrinthine development (Gundogan et al., 2008; 2010; 2015). During the third trimester of gestation, alcohol affects the uteroplacental vascular function (Rosenberg et al., 2010; Subramanian et al., 2014; Orzabal et al., 2019) by impairment of uterine spiral artery remodeling, angiogenesis, and vasodilation (Radek et al., 2005) via altered endothelial angiogenic gene expression (Ramadoss and Magness, 2012b; 2012c). Table 1 summarizes the present background of altered placental vascularization and some mechanisms involved, following gestational alcohol exposure in rat, mouse, and human models. However, although early angiogenesis and vessel remodeling are major contributing processes defining normal placental circulation, at present, there are few studies examining the earlier etiology and molecular mechanisms involved in maternal alcohol-induced vasculopathy of the placenta in the mouse model.

However, maternal alcohol consumption is often associated with chronic alcohol ingestion by women prior to and up to early gestation (around 4–6 weeks after human conception). It is common to find a high proportion of female consumers that continue to drink moderate quantities of alcohol (200 mL/day of wine containing ethanol 11%) up to early gestation, while being unaware that they are pregnant. In this regard, some data indicate that approximately 47% of women drink around conception, and 15%–39% cases reported consumption of high doses of alcohol (more than five standard drinks on one occasion) (Colvin et al., 2007). In this context, periconceptional alcohol exposure in the highly susceptible peri-implantatory and early organogenic periods may be affected and can lead to placentopathy at term (Gualdoni et al., 2022b). In view of these periods of consumption, we established a mouse model of perigestational alcohol consumption starting before pregnancy and continuing up to early gestation (mouse gestational days (GD) 4, 5, 7, 8, and 10) to study the embryo development (Cebral et al., 2007; 2011; Coll et al., 2011; 2017; Perez-Tito et al., 2014; Gualdoni et al., 2022a). We have shown that moderate oral ethanol intake for 2 weeks before pregnancy and up to peri-implantational stages affect embryo differentiation and growth and leads to retardation during implantation (Pérez-Tito et al., 2014). In addition, PAC up to early mouse organogenesis (day 10 of gestation) induces delayed embryo development, reduces viability, produces dysmorphogenesis of the neural tube, deregulates the embryonic cadherin expression (Coll et al., 2011), alters the arachidonic acid metabolic pathways (Cebral et al., 2007), and generates embryo oxidative stress (Coll et al., 2017), among other effects. Nevertheless, to date, little is known about the effects of PAC on early maternal vascularization during placentation. Recently, we suggested that one cause of mouse placental abnormalities found at term after PAC (Gualdoni et al., 2022b) may be associated with alterations in decidual and trophoblast tissue development during exposure up to organogenesis (Coll et al., 2018; Ventureira et al., 2019; Gualdoni et al., 2021). Apart from imbalances in the endothelial vascular endothelial growth factor (VEGF) system, defects in MMP and NOS expression/activity and levels of NO may also partially explain the early abnormal placentation (Gualdoni et al., 2021). However, at present, there is little evidence, in animal models, of the deleterious effects of maternal alcohol exposure on early decidual angiogenesis–vascularization and the involvement of MMP and NOS/NO factors as mechanisms responsible for triggering late-stage placental abnormalities.

Considering the background and importance of early maternal vascularization of the placenta for optimal fetal growth and maintenance and successful pregnancy at term, and subsequent life course, in this review, we first overview the current knowledge of early decidual vascular angiogenesis and remodeling involved in normal and abnormal mouse early placentation, focusing on the roles of MMP and NOS/NO systems. Then, we propose hypothetical altered decidual cellular and MMP and NOS/NO mechanisms involved in early maternal abnormal vascularization of the placenta in a PAC experimental mouse model. Overall, this review highlights the importance of adequate early gestational balances of decidual cells and MMPs and NO in the physiology and pathophysiology of maternal vascular development during placentation.

During the early period of mouse pregnancy, decidual capillaries and arterioles develop by angiogenesis, the process of new blood vessel formation and growth from pre-existing vessels. Angiogenesis involves 1) the expression of angiogenic factors, 2) vascular growth stimulation by hypoxia, 3) secretion of essential proteases for tissue remodeling, 4) migration of endothelial cells, and 5) appropriate proliferation of endothelial cells to secure the outgrowth of a new vessel. Disruption of the balance between angiogenic factors and their inhibitors may result in early miscarriage or, alternatively, defective placentation, thereby increasing the risk of pregnancy-related disorders.

The processes of neovascularization require the response to a complex paracrine network of factors (Adams and Alitalo, 2007; Bryan and D’Amore, 2007; Hellstrom M et al., 2007), of which one of the most important is the expression and activation of the VEGF system that involves in a proper gradient necessary to stimulate sprouting and branching of maternal vessels (Bautch, 2012; Gualdoni et al., 2022b). Imbalances of the VEGF lead to aberrant placental vascular morphogenesis (Roberts and Escudero, 2012; Li et al., 2014).

In arteriolar vascular smooth muscle and periendothelial cells of capillaries, the VEGF can act through three receptors: VEGF-R1 (FLT-1), VEGF-R2 (KDR/Flk-1), and VEGF-R3 (FLT-3). The main physiological VEGF effects are via the activation of KDR (Chung and Ferrara, 2011), which induces downstream activation of signaling cascades that stimulate the production of at least 11 angiogenic factors (Lima et al., 2014; Apte et al., 2019), including the placental angiogenic MMP and eNOS regulators (Kimura and Esumi, 2003).

MMPs are multigenic proteolytic zinc-dependent enzymes composed of six classes: collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs, among others (Amălinei et al., 2007; Cui et al., 2017; Henriet and Emonard, 2019). The expressions of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13, and MMP-19 are increased by the activation of the VEGF system (Chen and Khalil, 2017). VEGF-KDR activation upregulates MMP-2 and MMP-9 expressions in human umbilical vein endothelial cells (Heo et al., 2010), and FLT-1 can modulate KDR to regulate the expression of MMP-9 to prevent excessive angiogenesis (Wang and Keiser, 1998). MMPs, secreted as zymogens, are strictly controlled at transcription, secretion, and activation/inhibition levels by exogenous and endogenous factors, such as cytokines, growth factors, hormones, their inhibitors (tissue inhibitors of metalloproteinases (TIMP), oxidative stress, phosphorylation, hypoxia-re-oxygenation, transcription factors, and others (Nuttall et al., 2004; Amălinei et al., 2007; Jacob-Ferreira and Schulz, 2013; Cui et al., 2017). Activation of MMP-2 can occur by non-proteolytic post-translational modifications of the full-length zymogen, by S-glutathiolation, S-nitrosylation, and phosphorylation (Jacob-Ferreira and Schulz, 2013). Post-translational activation of latent forms of MMPs is generally processed in the extracellular compartment (Henriet and Emonard, 2019). Three tissue inhibitors for MMP (TIMP-1, TIMP-2, and TIMP-3) regulate MMP activity. TIMP-1 forms complexes specifically with MMP-9 and TIMP-2 is involved in the regulation of MMP-2 activity. Interestingly, TIMP-3 supports the activation of MMP-2 via membrane-type MMP, as well as inhibition (Alexander et al., 1996). On the other hand, TGFβ1, expressed in the mouse endometrium during implantation and decidualization, has a potential role in the regulation of MMP-9 gene expression (Bany et al., 2000).

MMPs have multiple roles, such as tissue and extracellular matrix (ECM) remodeling, proliferation, apoptosis, migration, differentiation, invasion (Hamutoğlu et al., 2020), cell–matrix and cell–cell interactions, and activation or inactivation of autocrine or paracrine signaling molecules (Amălinei et al., 2007). In addition, proteolysis of the ECM could release matrix-bound growth factors and their receptors. MMPs can directly activate growth factors, and MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 activate TNFα (Hulboy et al., 1997).

Placental hemodynamics and adaptations require extensive structural and functional modifications in the maternal blood vessels of the placenta. In normal pregnancy, remodeling of maternal vessels needs secretion of MMPs by trophoblast and decidual stromal cells to mediate proteolysis and degradation, vascular and matrix remodeling, and angiogenesis (Staun-Ram et al., 2004; Gualdoni et al., 2022; Rusidzé et al., 2023). Through their proteolytic activity, MMPs may mediate detachment of pericytes from the vessels, release ECM-bound angiogenic growth factors, expose cryptic proangiogenic integrin-binding sites in the ECM, generate promigratory ECM component fragments, and cleave endothelial cell–cell adhesions (Rundhaug, 2005). The most important gelatinases with a preponderant role in maternal tissue remodeling, degradation of the basement membrane and ECM (Cui et al., 2017), vasodilatation, and uterine expansion during normal pregnancy in humans and mice are MMP-2 and MMP-9 (Staun-Ram et al., 2004; Fontana et al., 2012; Chen and Khalil, 2017). Specifically, MMP-9, which degrades type IV, V, and IX collagens, gelatin, and elastin, is involved in numerous processes, including implantation, placentation, and embryogenesis (Silvia and Serakides, 2016; Espino et al., 2017; Quintero-Fabián et al., 2019; Timokhina et al., 2020). MMP-9 has been shown to be involved in endometrial remodeling in mouse invasion of trophoblasts (Zhang et al., 2020), development of mouse decidua, vascularization of the implantation site during early organogenesis (Fontana et al., 2012; Gualdoni et al., 2021), and remodeling of the feto–maternal interface during advanced stages of mouse placentation (Rusidzé et al., 2023). However, little has been reported about the localization, expression, and activation of MMP-2 and -9 in the early mouse implantation site prior to the formation of the placenta. In mice, by GD6 and GD8, MMP-9 immunoreactivity was found mainly in the antimesometrial non-decidualized endometrium and in trophoblast giant cells (Alexander et al., 1996). However, deciduomas, in the absence of trophoblast cells, were shown to contain the same level of MMP-9 activity as decidua (Bany et al., 2000), suggesting that this protease can be present in decidua derived from other than decidual cells. Moreover, maternal expression of MMP-9 was demonstrated to be increased in the uterus during decidualization and in cultured mouse endometrial stromal cells from uteri sensitized for decidualization. At mid-gestation, MMP-9 transcripts and protein were found in trophoblast cells (Teesalu et al., 1999; Gualdoni et al., 2021), stromal cells, and the ECM of mesometrial decidua (Fontana et al., 2012), suggesting its role in the angiogenesis of maternal tissue during mouse organogenesis. On the other hand, at mouse gestational day 6, MMP-2 is expressed and activated in the endometrium (Alexander et al., 1996; Bany et al., 2000); by day 8, it was detected in stromal cells at the mesometrial pole of the mouse implantation site and also in the area of the primary trophoblast giant cells, while from mouse mid-pregnancy (GD10), MMP-2 was expressed in trophoblast and decidual tissue (Teesalu et al., 1999; Heo et al., 2010; Gualdoni et al., 2021).

Altered MMP-2 and MMP-9 expression/activity could lead to the release of inflammatory cytokines, hypoxia-inducible factor, and reactive oxygen species, which may target the ECM and endothelial and vascular smooth muscle cells, causing inadequate remodeling of spiral arteries, generalized vascular dysfunction, increased vasoconstriction, and placental ischemia, in turn contributing to the pathogenesis of abnormal gestational outcome, such as premature birth, hypertension, and complications of pregnancy (Jacob-Ferreira and Schulz, 2013; Chen et al., 2020). Particularly, MMP-9 deficiency in homozygous MMP-9 knockout mice was associated with reduced and abnormal development of ectoplacental cones at GD 7.5, with impaired trophoblast differentiation and reduced invasion (Plaks et al., 2013). Also, in MMP-9 null mice, MMP-9 deficiency was associated with a decrease in the number of pregnancies and a reduction in litter size (Dubois et al., 1999; Dubois et al., 2000).

Nitric oxide, the other relevant angiogenic–vasoactive factor, is produced by the oxidation of L-arginine catalyzed by three NOS enzyme isoforms: neuronal (nNOS or NOS1), inducible (iNOS or NOS2), and endothelial (eNOS or NOS3) (Hefler et al., 2001; Förstermann and Sessa, 2012; Qian and Fulton, 2013). The nNOS and eNOS isoforms are frequently expressed constitutively, and their activities are regulated by calcium availability, whereas iNOS is independent of the intracellular calcium concentration and generates a high flow of NO (Förstermann and Sessa, 2012; Eelen et al., 2017). nNOS and iNOS are predominantly cytosolic, whereas eNOS can be either cytosolic or localized in membrane caveolae of endothelial cells (Eelen et al., 2017). In normal pregnancy, regulated by the VEGF by increasing the endothelial calcium signaling, eNOS is the most relevant enzyme in NO production (Moncada and Higgs, 2006). Additionally, endothelial shear stress, produced by flowing blood, can stimulate endothelial NO release and increase endothelial intracellular free Ca²⁺ concentration (Vanhoutte et al., 2016). Under basal conditions, iNOS is normally not expressed. However, different stimuli, including immunologic and inflammatory cues, can induce iNOS expression in various cell types under changes in the cellular environment (Eelen et al., 2017).

Endothelial NO, acting on vascular smooth muscle cells (VSMCs), is a major local regulator of vasodilatation and blood flow (Chen and Khalil, 2017). In the endothelium, the primary function of NO is to relax vascular smooth muscle tissue and regulate arterial blood pressure (Waker et al., 2023). However, NO also plays roles in angiogenesis, such as endothelial cell (EC) migration, control of microvascular volume, vascular permeability, platelet aggregation, and thrombosis (Krause et al., 2011; Aban et al., 2013). During gestational vascularization, new EC function depends on NO release from pre-existing ones, while the vascular smooth muscle responds to released NO levels paracrinally (Esper et al., 2006). Under controlling by oxygen tension, hormones, and oxidative stress, among others, NO supports placental vascularization (Krause et al., 2011) to maintain vascular vasodilatation and low vascular resistance (Boeldt et al., 2011) and to attenuate the effects of vasoconstrictors at the feto–placental interface (Possomato-Vieira and Khalil, 2016).

Relevant balance and gradual increase of the levels of NO are needed for normal gestation, as demonstrated in eNOS knockout mice, where the spiral artery remodeling was dysregulated, the labyrinth was reduced, and uteroplacental hypoxia was induced (Kulandavelu et al., 2012; Kusinski et al., 2012; Walker et al., 2023 ).

In this review, we focused on and summarized the relevant process of early maternal vascularization for placental development and the roles of MMPs and NO as regulators of maternal angiogenesis in normal and abnormal placental conditions in early mouse pregnancy. We provide knowledge to understand better the importance of the research approach of these complex networks of molecular and cellular angiogenic mechanisms affected by maternal exposure to alcohol up to the early stages of pregnancy. Although it is well known that maternal alcohol ingestion is a significant risk factor for placentopathy induction, its association with fetal defects and, potentially, FASD is unclear. At present, few mouse models for studying the gestational and/or perigestational effects of alcohol consumption in placentation are available. In an experimental mouse model, here, we presented hypothetical mechanisms of alterations in early maternal vascularization–angiogenesis produced by perigestational administration of alcohol up to early gestation. This could be useful to explain, in part, the effects of maternal alcohol consumption until the first 2 months of human pregnancy on the feto-placental development at term.

Early failures of SA remodeling and primary maternal mechanisms of angiogenesis–vascularization due to PAC up to early stages of gestation can cause placental pathogenesis, which may underlie pregnancy complications, IUGR, FASD, and/or programming postnatal diseases. Alterations in MMP and NOS/NO mediators may modify placental functions, leading to pathological conditions of pregnancy. In this regard, based on our experience and literature exploration, few studies have described the maternal angiogenic-remodeling events at each stage of early development of the mouse implantation site. Bearing in mind that the murine model of placental development is not totally equal to the development of the human placenta, their similarities mean that a slightly more detailed description of early maternal angiogenesis–vascularization in the mouse model constitutes a contribution to the understanding of various similar physiopathological processes of angiogenesis and cellular and molecular mechanisms that are executed during vascular formation of the definitive placenta. In this sense, the goal of this review is to provide a theoretical–conceptual tool in the mouse experimental model for the analysis of the roles of decidual stromal and leukocyte cells and some angiogenic relevant molecules, such as MMPs and NO, in normal or altered vascularization during early pregnancy under alcohol exposure. However, there is a need to increase the knowledge of mouse experimental models that recapitulate the human gestation to better understand the etiology and pathogenesis of abnormal processes related to human placentopathies and their implications on fetal development and growth, produced by maternal alcohol consumption, at least, up to early gestation.

Studies on relevant angiogenic systems, such as the VEGF/R, MMPs, and NOS/NO, among others, in animal models should be continued, highlighting the importance of analyzing changes in angiogenic–vasoactive molecules in the normal placentation and their roles in altered pathways as etiological mechanisms of placental defects and risk for abnormal fetal growth due to maternal alcohol consumption.