In mammals, the VEGF system mainly includes five secreted ligands (VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor) and three primary tyrosine kinase receptors (VEGF-R1, VEGF-R2, VEGF-R3) [13]. The VEGF system also includes the cell-surface proteins, heparan sulfate proteoglycans and neuropilin-1 and -2, which operate as VEGF coreceptors [14,15].

VEGFR-1 and VEGFR-2 are expressed in vascular endothelial cells, while VEGF-R3 seems to be prominently expressed in lymphatic endothelial cells [16]. VEGF ligands have different affinities for one of the three VEGF-R. As tyrosine kinases receptors, upon dimerization by a VEFG ligand, the VEGF-Rs auto-phosphorylate, a phenomenon which in turn activates downstream signaling pathways including mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-3 kinase (PI3K-AKT) pathway, and the phospholipase-C-γ pathway. Those pathways drive various intracellular effects in endothelial cells, such as migration, proliferation, and cell survival. The activation of phospholipase-C-γ pathway via VEGF-A-VEGFR-2 binding was reported to be a key signal for endothelial proliferation [17].

In 2001, a new VEGF was identified, the endocrine-gland-derived VEGF (EG-VEGF). This ligand does not show any structural homology to the VEGF family, but displays several biological similarities to VEGF ligands, including hypoxic regulation and ability to induce fenestration in target cells. Moreover, EG-VEGF expression is restricted to steroidogenic tissues (adrenal, ovary, testis and placenta) and its effects seem to be restricted to endothelial cells derived from these organs [18].

Ang-Tie signaling pathway regulates vascular permeability and remodeling during tumor angiogenesis and metastasis. Ang/Tie signaling seems to complement the VEGF signaling pathway by controlling later stages of angiogenesis and by being involved in vascular maturation (Figure 1) [19].

The angiopoietin family includes two type 1 transmembrane protein receptors: Tie1 and Tie2 and four ligands: Ang1, Ang2, Ang3 and Ang4. Ang1 and Ang2 have been identified as the main ligands for Tie receptors, while the Ang3 and Ang4 biological function is still poorly characterized [20,21,22].

Ang1 binds and activates Tie2 resulting in Tie2 internalization and ligand release. Then it leads to Tie2 tyrosine residues phosphorylation that in turn recruits adaptor proteins and ignites PI3K/Akt and MAPK signaling pathways, promoting pro-survival, anti-permeability, and anti-inflammatory effects on endothelial cells [23]. Tie2 is not required for the endothelial cells’ differentiation but is rather reported as necessary for cell maintenance [24].

Ang2 that shares approx. 60% amino acid homology with Ang1, binds to Tie2 with a similar affinity as Ang1. Ang2 seems to block the Ang1-induced Tie2 phosphorylation. Ang2 is upregulated during tumor angiogenesis and so was considered as a potential antiangiogenic target. However, recent studies found Ang2 to have a dual function, acting as a Tie2 antagonist in the presence of Ang1 or acting as a Tie2 agonist in the absence of Ang1 [25]. Different studies reported that it is unlikely that Tie2 can act differently when binding to Ang1 and Ang2, since both angiopoietins interact with Tie2 in a structurally similar manner and pointed out that other still unidentified mechanisms were likely to be involved [26]. One of the proposed mechanisms involve the Tie1 receptor [27].

Contrary to Tie2, the Tie1 has been less well characterized. Tie1 is considered an orphan receptor and is mainly expressed at vascular bifurcations and branching points, with no yet identified in vivo ligand [28]. It is well known, however, that Tie1 has an important role in vascular development, since its inactivation causes late embryonic lethality and vasculature maturation failure [29,30]. Recent studies proposed that Tie1 forms a complex with Tie2 on the endothelial cell surface and acts as a Tie2 inhibitor [27]. Cells expressing both receptors are responsive to chemotactic signals and able to promote vessel branching and sprouting that is required for angiogenesis. On the other hand, Tie1 is absent in stable and quiescent mature vessels [27].

A mechanistic study indicated Tie1 as being responsible for angiopoientin’s differential function. In mature vessels, as Tie1 is absent, Tie2 can be activated by either Ang1 or Ang2, to promote vessel stability. On active angiogenesis sites, Tie1 and Tie2 form a complex and Ang2 fails to activate Tie2, allowing vessel branching to be promoted. On the other hand, Ang1 is able to dissociate Tie2 from the Tie1-Tie2 complex, activating Tie2 and thus enhancing vascular stability [27,31].

Human fetal adrenal (HFA) plays a critical role in fetal maturation and perinatal survival. HFA steroid hormones regulate intrauterine homeostasis and appropriate fetal organ systems maturation [32,33].

Contrary to the adult adrenal cortex that includes three distinct zones: glomerulosa, fasciculata and reticularis; the HFA is primarily composed of two single distinct zones: outer zone or definitive zone and inner zone or fetal zone [33,34]. The definitive zone comprises a narrow band of small cells that exhibit typical characteristics of cells in proliferative state. Definitive zone does not produce steroids until the third trimester. However, as gestation advances, definitive zone cells start to accumulate lipids and resemble steroidogenic active cells. The fetal zone is the largest adrenal cortex zone and consists of large cells that exhibit features characteristic of steroid-secreting cells [33,34,35,36,37]. In ultrastructural studies, a third zone in between definitive zone and fetal zone, named transitional zone, has been described. The transitional zone is composed by cells with intermediate characteristics, but capable to synthetize cortisol and so cells can be considered analogous to fasciculata layer cells of mature adrenal cortex [33,38,39,40,41].

Due to the HFA critical role in fetal maturation, the early and extensive vasculature development that occurs in this gland, is not only necessary but also particularly important. Angiogenesis is not only required for HFA growth and maturation, but it is also necessary for the influx of steroid precursors and trophic factors into the gland to enable mature steroids synthesis and secretion into circulation. Indeed, the fetal adrenal gland is one of the most highly vascularized organs in the human fetus [41].

Previous studies have reported that VEGF-A, FGF-2, Ang1, Ang2, and Tie2 are expressed in HFA since midgestation and to have a putative role in adrenal gland angiogenesis [34,42,43].

Ang2 expression in HFA is markedly higher when compared to the mature adrenal gland, whereas Ang1 and Tie2 expression seem to be similar in both fetal and adult adrenals. Thus, supporting higher angiogenesis activity and vascular instability in developing adrenal glands [42].

Ang2, FGF-2 and VEGF-A expression are mainly expressed in the gland periphery suggesting that the HFA periphery is the primary site of angiogenesis, in parallel to cell proliferation [42,43]. Further supporting this hypothesis, a dense network of irregular capillaries was also observed at the HFA periphery [44].

On the contrary, Ang1 is mainly expressed in the fetal zone, suggesting that the inner adrenal zone presents a greater vessel maturity. Tie2, was exclusively identified to be present in endothelial cells throughout the gland [42,43].

Adrenocorticotropic hormone (ACTH), the main regulator of HFA growth and function, also seems to be implicated in angiogenesis control. In vitro studies found that ACTH upregulates VEGF-A, FGF-2 and Ang2 in the HFA, therefore controlling angiogenesis while simultaneously exerting growth and secretion stimulatory actions [42,43,45,46].

The steroidogenic factor 1 has a critical role in adrenal development, steroidogenesis, and also in gonadal differentiation [47]. In addition, steroidogenic factor 1 also seems to be implicated in HFA angiogenesis regulation by direct interaction and activation of the Ang2 gene promoter. Furthermore, the authors demonstrated that steroidogenic factor 1 and Ang2 are strongly co-expressed in HFA periphery in early stages of development [48].

Overall, these findings support that the adrenal gland growth, steroidogenesis and blood vessel formation, are synchronized phenomena [42,43,45,46].

The adrenal gland is one of the most vascularized organs in adult mammalian organisms. Its developed intrinsic vasculature is required for an efficient secretion of steroid hormones into the systemic blood flow. The adrenal gland is supplied by three different arterial branches derived the abdominal aorta: inferior phrenic artery, middle adrenal artery and renal artery. The arterial blood enters in the adrenal gland and flows centripetally through the adrenal cortex into the adrenal medulla [49,50].

Previous studies have found that adrenocortical cells highly express VEGF-A and EG-VEGF—a VEGF specific of steroidogenic organs, both having been pointed out as important molecules for maintenance of the dense and fenestrated vasculature of the adrenal cortex. This expression also seems to be regulated by ACTH [51,52,53,54,55].

In addition, the vasculature of the adrenal cortex seems to be coordinated with the mass of the adrenal cortex, since it suffers fluctuations decreasing or increasing along regression or expansion of the adrenal cortex, respectively [52].