The Vascular Endothelial Growth Factor (VEGF) Family

The vascular endothelial growth factors (VEGFs) are potential targets for next-generation doping due to their blood vessel growth-inducing (angiogenic) effects. The VEGF family consists of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placenta growth factor (PlGF), each encoded by its own gene [32, 33]. Of these, VEGF-A through VEGF-D and PlGF are endogenous to humans (see Table 1). VEGF-A (often simply called VEGF in older literature) is the primary angiogenic factor [32], and its potential for gene doping has been previously recognized [34–38].

 | Interacting receptors | Biological effects | ReferencesFirst description(s)/recent review(s)
Angiogenesis | Vascular permeability | Lymphangiogenesis
VEGFR-1 | VEGFR-2 | VEGFR-3
VEGF-A | + | + |  | +++ | ++ | Conflicting reports1 | [53, 54] / [55, 56]
PlGF | + |  |  | Indirectly2 | – | Not determined | [57] / [58]
VEGF-B | + |  |  | Conflicting reports3 | – | – | [59] / [56]
VEGF-C |  | + | + | ( +)4 | + | +++ | [60, 61] / [62, 63]
VEGF-D |  | + | + | (+++)4 | + | ++ | [64–66] / [67, 68]
VEGF-E5 |  | + |  | + | ++ | Not determined | [69] / [56]
VEGF-F5 |  | + |  | + | +++ | Not determined | [70] / [71]

VEGF concentrations are highest during embryogenesis and fetal development, where, for example, VEGF-A is first essential for the survival of hemangioblasts, the common developmental ancestors of blood and endothelial cells [39–41], and subsequently for the growth and development of the cardiovascular system [42, 43]. The close relationship between blood and endothelial cells is maintained into adulthood through many shared cell surface receptors [44, 45].

VEGF-C is primarily known for its role in the development and growth of the lymphatic system [46–48] and in cancer metastasis via lymphatic dissemination [49–51]. In adults, VEGF-C regulates lymphangiogenesis during tissue regeneration, such as wound healing [52].

The other human VEGFs have more specialized roles. VEGF-B is prominently expressed during early embryonic development, acting in a species-specific manner in heart vascularization and as a general vascular survival factor [75, 76, 81, 82]. Unlike VEGF-C, which is primarily lymphangiogenic, VEGF-D can also be a potent angiogenic inducer, sometimes even outperforming VEGF-A [79]. In humans, it is believed to replace VEGF-A and thus allow tumors to resist drugs that target VEGF-A [83, 84].

PlGF, named for its high expression in the placenta [57], is vital for the endometrial cycle and fetal development. Outside the reproductive system, it indirectly boosts angiogenesis and vascular permeability by displacing VEGF-A from the inhibitory VEGFR-1, allowing VEGF-A to bind to the mitogenic receptor VEGFR-2 [74, 85, 86]. In mice, PlGF, VEGF-B, and VEGF-D contribute to, but are not essential for, angiogenesis, as studies have shown only minor changes in the cardiovascular system when each gene was deleted individually [87–94].

The Regulation of VEGF Expression

VEGF-A is regulated by a complex array of external factors at multiple levels, from gene transcription to mRNA translation [95–98]. In adults, VEGF-A levels are typically low in most organs, but are increased during wound healing [99, 100], muscle growth [101, 102], tissue repair [103], and the hair growth [104] and female reproductive cycles [105, 106], for example, but also during inflammation [107–109] (reviewed by [56]) and in pathologically oxygen-depleted tissues, such as tumors (reviewed by [110]). There is a rich literature about hypoxia-induced VEGF-A expression (reviewed by [111]). In contrast, the evidence for hypoxia-induced upregulation of other VEGF family members is either negative [112] or sparse, and often limited to pathological situations [113, 114]. Upregulation of VEGF-A expression is mediated by hypoxia-inducible factor-1 (HIF-1) [115, 116] (see Fig. 2). Under hypoxic conditions, HIF-1 degradation is suppressed, allowing it to move into the nucleus. There, it acts as a transcription factor interacting with hypoxia-responsive elements in hypoxia-inducible genes, including EPO and VEGFA [115, 117–120]. One goal of this hypoxia-induced change in gene expression is to improve oxygen transport to hypoxic tissue by increasing vascularization. Besides hypoxia, other factors and variables, such as growth factors, inflammatory cytokines, and an acidic pH, can increase HIF-1 levels and, therefore, VEGF-A expression [97].

Fig. 2: Induction of VEGF-A expression by HIF-1α. Prolyl hydroxylases limit the physiological activation of HIF-1α, the oxygen-sensitive subunit of the HIF-1 complex, and are central enzymes targeted by many interventions aimed at inducing hypoxia-responsive genes. Fe2+ in the active site of PHDs participates in the prolyl hydroxylation as a cofactor, being oxidized to Fe3+ (the same mechanism underlies the connective tissue phenotype in scurvy, where ascorbate deficiency prevents the reduction of Fe3+ to Fe2+ and thus the prolyl hydroxylation required for collagen maturation). Carbon monoxide is not only a toxic gas, but also an endogenously produced gasotransmitter. It is generated when heme oxygenases-1 and -2 degrade heme. Heme oxygenase-1 is strongly upregulated by hypoxia, and the generated CO further enhances the expression of hypoxia-response genes, such as VEGFA. Cytochrome c oxidase (Complex IV) is a heme-containing enzyme in the mitochondrial electron transport chain. Under hypoxia, reduced cytochrome c oxidase activity leads to ROS generation and HIF-1 stabilization [121]. ARNT Aryl hydrocarbon receptor nuclear translocator, HIF hypoxia-inducible factor, P proline, PHD/EGLN prolyl hydroxylase domain enzyme/egg-laying defective gene 9, ROS reactive oxygen species, Ub ubiquitin, VEGF vascular endothelial growth factor, VHL von Hippel-Lindau protein

The VEGF Receptors

VEGFs bind to VEGF receptors (VEGFRs), which are tyrosine kinase receptors [40]. Placental mammals such as humans and mice feature three VEGFRs (VEGFR-1, VEGFR-2, and VEGFR-3), while marsupial mammals feature one more (VEGFR-4, aka Kdr-like). The different receptors mediate distinct effects (Fig. 3). VEGFR-2 is the best-known of these, as it mediates most of the known responses to VEGF-A, including angiogenesis and vascular permeability [122–124]. VEGFR-1 is thought to limit angiogenesis by trapping VEGF-A and thus decreasing signaling via the angiogenic VEGFR-2 [125, 126]. VEGFR-1 also mediates some of the disease-related signaling of VEGFs [127]. VEGFR-3, on the other hand, mediates the effects of VEGF-C and VEGF-D on the lymphatic system, as these receptors are found mainly on lymphatic endothelial cells [128, 129]. All VEGFRs are primarily found on the surface of endothelial cells [130], but there are numerous exceptions, including immune cells [45, 129, 131], vascular smooth muscle cells [132, 133], and neurons [134–136].

Fig. 3: The VEGFs and VEGF receptors. Humans feature five endogenous VEGF growth factors, each of which interacts with a specific set of receptors, resulting in distinct biological effects. As elaborated in Sect. 4.6, VEGF-A and VEGF-D are the most promising targets for therapy and performance enhancement. Despite its ability to interact with VEGFR-2, in vivo application of VEGF-C is surprisingly specific for the lymphatic system, perhaps due to its unique activation by ADAMTS-3/14 [137, 138]. VEGF-D, on the other hand, can lose all of its affinity for VEGFR-3—and thus its lymphangiogenic potency—if activated by cathepsin D [139, 140]. VEGF vascular endothelial growth factor, VEGFR vascular endothelial growth factor receptor, PlGF placenta growth factor