*1.4. VEGF Expression*

Several factors have been found to upregulate VEGF mRNA expression, including tumor necrosis factor (TNF-α), platelet-derived growth factor (PDGF), interleukins, angiopoietins, and erythropoietins [41–45]. Another molecule that regulates the expression of VEGF is nitric oxide, which contributes to the processes of permeabilization of blood vessels and in vasodilation stimulated by VEGF [29,46,47].

However, one of the main and more robust regulators of VEGF expression is hypoxia [48]. VEGF mRNA has a half-life of 30–45 min under normoxic conditions, whereas the mRNA half-life is prolonged in hypoxia [49,50] and cells increase the production of the hypoxia-induced transcription factor 1 (HIF-1), a heterodimer consisting of three subunits (HIF-1α, HIF-1β, and HIF-3) [51,52]. HIF-1α and HIF-1β are produced continuously, but HIF-1α is highly labile in the presence of oxygen, so it degrades under aerobic conditions [53]. When the cell is in a hypoxic environment, HIF-1α persists and translocates to the nucleus, where it associates with HIF-1β and forms the HIF-1α/HIF-1β complex. This complex binds to the hypoxia response element (HRE) [54,55], whose transcriptional activation requires the recruitment of the CREB-binding protein, which is a transcriptional coactivator. Thus, as the 2019 Nobel Laureates in Medicine Kaelin, Ratcliff, and Semenza described, through this mechanism, cells perceive and adapt to changes in oxygen levels, modifying both their metabolism and physiological functions [52,56]. Therefore, transactivation of HRE by the HIF-1α/HIF-1β complex stimulates the gene expression of erythropoietin, glucose transporters, glycolytic enzymes, and VEGF [57,58], among

others, the latter being in charge of promoting angiogenesis after binding to its specific receptors [5]. Little is known about the expression and function of HIF-3 [59].

#### **2. VEGF Receptors**

The biological activity of the VEGF family is mediated through binding to two classes of receptors: receptors with tyrosine kinase activity and receptors without tyrosine kinase activity. The first group consists of three structurally related receptors characterized by the presence of seven immunoglobulin-like domains in the extracellular region, a single transmembrane region, and an intracellular consensus tyrosine kinase sequence interrupted by a kinase insertion domain. These receptors are VEGR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4). On the other hand, the receptors without kinase activity are neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2), which are also receptors for semaphorins [15,20] (Figure 1).

**Figure 1.** VEGF receptors. The family of VEGF receptors includes three tyrosine kinase receptors (VEGFR-1, VEGFR-2, VEGFR-3) and two non-tyrosine kinase neuropilin receptors (NP-1, NP-2). The different members of the VEGF family bind to the different types of VEGF receptors, as illustrated. The main effect of VEGF-A as a neurotrophic factor is mediated by its binding to VEGFR-2 (Flk-1).

NRP-1 and 2 are expressed in different types of neurons [60,61] and play an essential role in regulating and developing the cardiovascular and nervous systems. Besides, they act as co-receptors for RTKs, presenting and improving VEGF binding to Flk-1 and promoting receptor phosphorylation and neurotrophic factor-mediated signal transduction [62,63].

Many studies indicate that both Flt-1 and Flk-1 activation could produce neuroprotection, but there are differences between the functions of both receptors [5,26,29,30]. Flk-1 predominates in neuronal and Schwann cells and is necessary for endothelial proliferation and migration, while Flt-1 is expressed mainly in vessels, astrocytes, and reactive microglia [29,64]. Besides, it has also been described that Flt-1 acts as a negative regulator for VEGF in endothelial cells, preventing its binding to Flk-1 [4] and that its functions and signaling properties may differ according to the stage of development of the animal, the cell type, and the binding ligand [65].

Unlike Flt-1, the Flk-1 receptor is an important survival promoter for endothelial and CNS cells, being the primary mediator of VEGF functions [4,66]. The main ligand of this Flk-1 receptor is the VEGF-A isoform, being the only one that triggers its autophosphorylation and final glycosylated form [4]. Therefore, the VEGF receptor that we will refer to throughout this review is Flk-1, which is expressed in motoneurons of the

human spinal cord [40], mouse [5], rat [38] and is reduced in some patients with amyotrophic lateral sclerosis (ALS) [40]. Flk-1 overexpression in spinal motoneurons of the ALS SOD1 mouse model (with mutations in the gene encoding the antioxidant copper/zinc superoxide dismutase) has been shown to delay both neurodegeneration and disease onset [67], being the primary mediator of the neuroprotective and anti-excitotoxic effects of VEGF on motoneurons [37,38,68]. All these functions are performed by activating the phosphatidylinositol-3 kinase-AKT (PI3-K/Akt) pathway, involved in the processes of cell growth, proliferation, cell survival, and intracellular traffic, among others, in addition to regulating the entry of glucose to the cell through an insulin signaling cascade [69]. Furthermore, VEGF binding to the Flk-1 receptor also exerts a protective effect by suppressing the activation of the mitogen-activated protein kinase p38 (p38MAPK), a determining factor in the cell death pathway [16,30,38,70].
