Introduction

Brain functions require adequate and constant blood supply of nutrients and oxygen, which is provided by a complex vascular network [1]. These continuous and non-fenestrated vessels ensure the homeostatic balance and a certain immune privilege to the brain [2]. This is not a one-man job, but rather a team effort among vascular cells, such as brain endothelial cells (BECs), mural cells including pericytes (PCs) and vascular smooth muscle cells (VSMCs), and astrocytes. Together, they form a tightly regulated and dynamic interface known as the blood–brain barrier (BBB) [3,4,5].

BECs are the first frontier between the periphery and the brain. Tight junction proteins, like claudins and occludins, seal the gaps between BECs, effectively blocking potentially harmful peripheral substances such as toxins, pathogens, and immune cells from entering the central nervous system (CNS) ([6, 7], see [8] for review). The brain endothelium is also equipped with specialized transport systems, enabling precise regulation of nutrient and water exchange to maintain brain homeostasis ([7, 9, 10], see [2] for review). Vessel ensheathment by PCs further promotes differentiation and maturation of BECs. In addition, PCs enhance BBB integrity [11, 12], by initiating astrocytic polarization and attachment of their endfeet to the endothelium ([13], see [8] for review). Astrocyte foot processes establish contacts with BECs and mural cells where they contribute to BBB integrity by modulating tight junction expression and regulating water transport to the brain through aquaporin-4 channels [14,15,16,17,18,19].

The BBB is supported by the neurogliovascular unit (NVU), which also includes microglia and neurons in addition to the specialized BEC, PCs and astrocytic endfeet. The basement membrane additionally regulates structural integrity and intercellular crosstalk between the cells of the NVU, as it covers both BECs and PCs and is surrounded by astrocytes endfeet [20]. The synchronized activity of NVU cells ensures efficient regulation of cerebral blood flow (CBF) to respond to metabolic demands in response to neuronal activation, in a process called neurovascular coupling [21,22,23]. It also allows waste clearance and neuroimmune responses [24, 34]. Barrier formation is further carried out by the Wnt/β-catenin signaling pathway, which supports formation of tight junctions, elimination of fenestrations, and restriction of pinocytosis [35]. To support the growing metabolic demands in nutrients, transporters are functional as soon as the BBB emerges in early stages of development [36]. In mice, this transport system has appeared by E13.5 allowing clearance of waste and toxic substances from the brain without preventing the entrance of nutrients and metabolites ([36], see [8] for review). The interval encompassing E13-E18 witnesses the association of pericytes with develo** blood vessels. These cells play a pivotal role in conferring structural stability to the emerging cerebrovasculature [37]. Then, additional cell types are recruited to initiate barrier maturation into the adult BBB (Fig. 1A). After birth, cerebrovascular development continues through the postnatal period. In mice, this extends from postnatal day 0 (P0) to P25, while in humans, it spans from birth into infancy [38, 39]. During this phase, the existing vasculature further matures [40, 41]. For example, astrocytes continue to contribute to the structural and functional aspects of the BBB [42, 43], by upregulating tight junction protein expression through Shh signaling in BECs [32], a crucial step to stabilize the vasculature and promote BBB integrity (see [44] for review).

Fig. 1
figure 1

Cerebrovascular development and corresponding circulating levels of sex hormones throughout life in mice A Schematic timeline illustrating cerebrovascular development and neurogliovascular unit cells assembly events. Cerebrovascular development in mice begins during the embryonic stage ~ E9-E10. Brain endothelial cells (BECs) start to invade the neuroectoderm in response to local gradients of factors like vascular endothelial growth factor (VEGF). This invasion initiates the formation of primitive blood vessels within the develo** brain. Between E11-E14, angiogenesis involves the sprouting of new blood vessels from pre-existing ones, contributing to the expansion of the brain vascular network. Blood–brain barrier (BBB) formation occurs between E12 and E17. During this period, tight junctions seal BECs, restricting the movement of substances across the endothelial cell layer. Between E13 and E17, pericytes wrap around BECs, contributing to the cerebrovasculature structural stability, while a basement membrane arises around blood vessels, providing additional support. After birth, brain capillary networks expand and mature, with astrocyte endfeet hel** to maintain BBB integrity and actively supporting the cerebrovascular system throughout life. B Schematic illustration (not to scale) of sexual differentiation and circulating sex hormone levels from embryogenesis until adulthood. Man and woman characteristics start to develop ~ E10 (gray rectangle), via progressive elevations of testosterone in male mice (blue) and estrogen in female mice (orange) embryos. After birth, levels quickly decrease, to rise again around postnatal day 2 (P2), a period referred to as mini-puberty. In both sexes, gonadal hormone levels slowly escalate until puberty, which is characterized by monthly estrogenic cycling in women until menopause while in men, testosterone peaks then slowly decline yearly throughout adulthood. Testosterone cycling in women and estrogen cycling in men were not represented for simplicity. Early sexual differentiation coincides with several steps of cerebrovascular development, raising questions regarding potential interactions underlying sexually dimorphic cerebrovascular development (gray triangles)

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Effective bidirectional communication between the neuronal and vascular system during brain development is essential for proper maturation [45,46,47,48]. Unlike the adult CNS, the neonatal brain displays absent or even inverted hemodynamic responses to neuronal activity [49]. While neuronal activity generally consumes oxygen, local vascular responses are insufficient to supply additional oxygen in the develo** brain. Hemodynamic responses gradually adapt postnatally [49], coincidently with expansion of the capillary network [40], development of VSMCs contractility capacities [41] and a steady increase of CBF [40, 50, 51]. To this day, the exact mechanisms underlying establishment of hemodynamic responses in the neonatal brain remain poorly understood.

Neurodevelopmental disorders: how does sex influence cerebrovascular development?

Brain vascular dysfunctions are increasingly explored in the onset and progression of neurodevelopmental disorders, with mounting evidence linking BBB disruption to conditions such as schizophrenia [52], autism spectrum disorder (ASD) [53] and attention deficit hyperactivity disorder (ADHD) ([54], see [55,56,57] for review). While it is generally accepted that women are protected from vascular dysfunction (e.g. stroke, cardiovascular diseases, etc.) throughout reproductive years due to sex hormones, little is known regarding the mechanisms underlying estrogen-mediated vascular protection, and very little emphasis has been put to decipher the effects of sex hormones on cerebrovascular development. Considering that several neurodevelopmental conditions, including ASD and ADHD predominantly affect men, it is reasonable to suggest that specific mechanisms come into play during cerebrovascular development to sustain this sex bias.

In this review, we first describe processes occurring during early sexual differentiation and how they could modulate important stages of cerebrovascular development. We specifically highlight two major signaling pathways involved in angiogenesis and BBB development, namely the VEGF and Wnt/β-Catenin pathways, with a specific focus on their interaction with gonadal hormones. We next discuss the role of these relationships in vascular dysfunction observed in two major neurodevelopmental conditions, ASD and ADHD. We chose to focus on findings related to BECs, however steroid hormones can also act directly on other NVU components such as mural and glial cells, suggesting an indirect impact on cerebrovascular function ([58,59,60], see [61,62,63] for review). Due to limited amounts of literature available, we could only speculate about associations and propose hypotheses regarding hormone-mediated effects on cerebrovascular development, both in the context of health and disease. We shed light on pitfalls of current research, as we firmly believe that advancing knowledge in these areas is crucial for the development of novel screening tools, personalized standardized diagnostic tests, and next-generation pharmacological therapies.

Sex hormones and cerebrovascular function

Gonadal development and early sexual differentiation

The hypothalamic-pituitary-gonadal (HPG) axis regulates gonadal hormone production and secretion. In women, the predominant hormones are progesterone and estrogens. Progesterone interacts with the progesterone receptor (PGR) [64], while estrogen acts on BECs through three main receptors: estrogen receptor alpha (Erα), beta (Erβ) and 7-transmembrane spanning G-protein coupled receptor 1 (GPER1, also known as GPR30) [65,66,67]. In men, androgens like testosterone bind to the androgen receptor (AR) and represent the main hormonal effectors. Gonadal hormones are small, lipophilic molecules that can either be endogenously produced in the brain or diffuse through the BBB [68], (see [69] for review). Once in the brain, binding of gonadal hormones to their receptors activate downstream pathways regulating genomic and non-genomic actions [70, 71].

During the fetal period, HPG axis is most active around mid-gestation, before being silenced prior to birth leading to a decline of gonadal hormone levels (Fig. 1B). At birth, the axis is reactivated, leading to the release of gonadotropins by the pituitary gland to allow sex hormone secretion from gonads, in both sexes [72,73,74,75]. This early postnatal period, referred to as mini puberty, occurs concomitantly with rapid brain development. These hormonal fluctuations could participate in sex differentiation of the brain, and shape underlying morphology and behavior, (see [76, 77] for review). Indeed, significant differences in brain volume and region-specific dimorphisms between sexes have been reported in adults, particularly in the cortex [78]. Clustering of sexual dimorphism in the adult brain is observed mainly in areas involved in early sexual differentiation and known to express high levels of gonadal hormone receptors during critical periods of development [78]. Thus, factors implicated in in utero and early postnatal sexual differentiation may underlie region-specific differences in the adult brain of men and women [78] emphasizing that sex as a biological factor should be carefully considered when studying brain development, function, and behavioral regulation [79].

Although beyond the scope of this review, it is important to acknowledge that sex hormones might not be uniquely responsible for brain differentiation according to sex. For example, Dewing et al. [80] performed microarray analysis of male and female mouse brain tissue at 10.5 days post-coitum, a stage prior to gonadal formation. They identified several genes differentially expressed, suggesting that sex determining genetic factors (i.e. XX or XY chromosomes) may underlie brain sexual differentiation prior to gonadal influence [80].

The brain endothelium as a target for sex hormones: what about the develo** brain?

Brain blood vessels are known to be a target for sex hormones, as they express Erα, Erβ, GPeR1 and ARs ([59, 81, 82], see [83] for review). Numerous in vitro and adult rodent studies have confirmed the capacity of estrogens and androgens to modulate angiogenesis, BBB permeability and vascular tone (see [83, 84] for review). These effects are highly age- and dose-dependent, as outlined in Table 1. Estrogen, in particular, is widely recognized for its protective effects on the vasculature, especially while estrogen levels are high during the reproductive years ([85, 86], see [87, 88] for review). This may contribute to sex differences in the prevalence of several disorders. Neuroanatomical studies performed in rodents revealed differential distribution of brain ARs and ERs from early postnatal to prepubertal stages [89,90,91]; however, the functional role of these receptors and their specific expression in brain vasculature during brain development have been poorly defined and relevance to humans is undetermined.

Table 1 Evidence of protective vascular effects of sex hormones in vitro and in adult rodent studies
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Sex differences in cerebral blood flow might arise early in life

CBF is at the root of brain function providing oxygen and nutrients through modulation of capillary perfusion in response to neuronal activation [92]. In humans, CBF gradually increases during the neonatal period to reach a peak during youth, then it starts decreasing with age [93]. This reduction is felt particularly by the default mode and executive networks, which are brain circuits highly involved in cognitive control, mood regulation and behavioral modulation, among others [94]. Sex differences in CBF have been reported in adolescence, with young healthy women showing 11–15% higher CBF than healthy young men, an effect suggested to arise, at least in part, via gonadal hormones ([94, 95], see [87] for review).

Investigation of sex differences in fetal and neonatal CBF is lacking, making it challenging to pinpoint their origin. It has been reported that estrogen receptor concentrations rise shortly (4 to 6 days) in the neonatal rat forebrain prior to rise of nitric oxide synthase (NOS) [96]. This enzyme produces nitric oxide, a potent vasodilator which regulates vascular tone and CBF, activity within the brain [97]. In vitro evidence suggests that estrogen signaling can mediate upregulation of NOS [98], suggesting a potential role for estrogen signaling in regulating NOS-associated developmental pathways [96]. Still, we can only speculate about a potential link between estrogen and the regulation of CBF through NOS changes during early postnatal stages, a critical time point for BBB maturation and establishment of hemodynamic responses [49]. The functional relevance of this relationship should be thoroughly examined, as it could contribute to our understanding of sexually dimorphic patterns of brain development and potentially offer insights into CBF disturbances in neurodevelopmental disorders (see Sects. "Sex hormones, cerebral blood flow and angiogenesis: unraveling sex differences in autism spectrum disorder" and "Sex hormones, cerebral blood flow and angiogenesis: unraveling sex differences in attention deficit hyperactivity disorder (ADHD)").

Sex hormones, angiogenesis and barriergenesis signaling: current knowledge and hypotheses on sex-specific mechanisms underlying cerebrovascular development

VEGF signaling

VEGF is crucial for embryonic angiogenesis and is also identified as a neurotrophic factor capable of stimulating neurogenesis [99,

Table 2 In vitro and adult rodent evidence of sex hormone-mediated effects on Vegf
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VEGF signaling modulates functions in other cell types, such as oligodendrocyte progenitor cells (OPCs) migration [120]. Although not directly part of the NVU, OPCs migrate along the vascular scaffold of newly forming blood vessels during development [121] and support integrity of the mature BBB [122]. Sex hormones have been found to interact with OPCs, through long-lasting actions of androgens during early postnatal development in rodents. Indeed, higher oligodendrocyte density and thicker myelin sheaths were observed in the brain of male vs female mice. This difference emerges between P0 and P10 precisely when a late wave of OPCs arise and start differentiating driven by dihydrotestosterone (DHT), a biologically active androgen-related metabolite derived from testosterone with potent AR affinity [123].

As mentioned earlier, the impact of sex hormones in CNS angiogenesis, BBB formation and VEGF signaling during fetal and neonatal development remains largely unexplored. However, findings in vitro or in adult rodent models, using constitutive gene knockdown approaches summarized in Table 2, suggest that sex hormones can directly promote VEGF signaling and angiogenesis, in a time- and dose-dependent manner. As such, it would be interesting to investigate if similar mechanisms are at play in the develo** vasculature, since early sexual differentiation coincides with barriergenesis in rodents (Fig. 1). The cells from the NVU might be sensitive to hormonal fluctuations in early development, which could further contribute to sex differences in vascular development and BBB formation including through Vegf-related signaling.

WNT signaling

The canonical Wnt/β-catenin pathway is crucial for BBB maturation [35]. This pathway is extraordinarily complex, comprising 19 different Wnt ligands signaling through 10 members of the Frizzled (Fzd) family of G-protein coupled receptors, as well as through Low-density lipoprotein receptor-related proteins (LRP), in particular LRP5 and LRP6 [124]. During mouse embryogenesis, neuronal progenitor cells express Wnt7a and Wnt7b in the develo** forebrain, ventral brain regions and intermediate spinal cord. Conversely, Wnt1, Wnt3, Wnt3a and Wnt4 are expressed in the dorsal spinal cord and hindbrain [35]. Wnt ligands bind to Fzd receptors on the vascular endothelium, leading to β-catenin stabilization, subsequent nuclear translocation and transcription of target genes involved in cell proliferation, adhesion, morphogenesis, and other developmental processes [125, 126]. While expressed in a variety of tissues, loss of Wnt/β-catenin signaling is only detrimental for BECs. Wnt7b knockout is embryonic lethal (between E11.5 and 12.5), causing severe brain hemorrhaging and abnormal vessel morphology [35]. Progressive downregulation of canonical Wnt signaling occurs during postnatal development; however, this pathway remains critical to promote barrier maturation, tight junction formation and maintenance of BBB integrity throughout life [124, 127, 128]. In the adult CNS, dysregulation of Wnt/β-catenin signaling has been implicated in several disorders with neurodevelopmental and post-neurodevelopmental origins, such as ASD, ADHD, and schizophrenia as well as Alzheimer’s disease and multiple sclerosis [128,129,130,131,132,133]. Thus, modulation of this pathway has attracted therapeutic interest for some years [134].

Wnt signaling and sex hormones: potential mechanisms favoring BBB maturation

To our knowledge, no evidence of sex differences in the Wnt/β-catenin pathway regarding BBB development has been reported. However, sparse evidence of hormonal interactions with Wnt ligands allows us to propose hypotheses on sex-specific mechanisms underlying BBB development. For example, Wnt4 is expressed in the mesenchyme (an embryonic connective tissue precursor of many cell types) of both sexes and has been pinpoint as an essential factor for sex determination. In mice, Wnt4 promotes the development of females characteristics but suppresses male reproductive system [135]. On the contrary, Wnt4 is downregulated in male mice testes around E11.5, as gonads emerge [135]. Its critical importance in embryonic ovarian development is supported by evidence of masculinization in Wnt4-mutant female mice [135]. In turn, overexpression of Wnt4 in male mice leads to defective androgen synthesis and lower testicular levels of testosterone. Again, this period coincides with barriergenesis in rodents (~ E10, Fig. 1), suggesting a potential influence of early sex determination on BBB formation. To support this idea, Wnt4 has been linked to proper choroid plexus development of the mammalian brain [136], which is imperative for formation and integrity of the CNS [137]. However, it remains unclear if sex- and cell-specific levels of Wnt4 during gonadal development could affect BBB development and function.

Dikkopf (Dkk) proteins have been identified as Wnt pathway regulators. Dkk1, recognized as a potent inhibitor of Wnt signaling [138], binds to LRP5/6 receptors, thus competing with Wnt binding and providing modulation of its transcription [138]. In the develo** neurovasculature, Dkk1 levels are tightly controlled to allow proper neovascularization [139]. At later stages of embryonic development and postnatally, Dkk1 is expressed in the aorta [140] and can be detected in differentiating human endothelial cells in vitro. Interestingly, a transcriptomic study of human placenta revealed sexually dimorphic gene expression and positive correlation between placental estrogen/testosterone ratio and LRP6 [141]. Further analysis identified an ERα response element at the LRP6 gene promoter, highlighting estrogen’s capacity to modulate LRP6 and to potentially interact with Wnt signaling during fetal development [141].

Overall, the findings presented in this section only provide a loose and conjectural connection between sex hormones-mediated modulation of Wnt/β-catenin signaling, and we could not find any studies specifically focusing on the brain or develo** vasculature. However, some associations have been proposed in neurodevelopmental diseases like ASD and ADHD (see Sects. "Sex hormones, cerebral blood flow and angiogenesis: unraveling sex differences in autism spectrum disorder" and "Sex hormones, cerebral blood flow and angiogenesis: unraveling sex differences in attention deficit hyperactivity disorder (ADHD)"), highlighting the need to focus on mechanisms underlying sex differences in healthy cerebrovascular development.