VEGF Signaling Pathway

Introduction

Vascular endothelial growth factor (VEGF), a specific mitogen isolated from endothelial cells by researchers in the late 20th century, has been shown to have the ability to induce physiological and pathological angiogenesis. The VEGF family is a multifunctional cytokine that mainly includes six members, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PLGF). In many tumors, the level of expression is related to the degree of malignancy of the tumor.

Mammalian angiogenesis begins with the formation of an embryonic vascular network. Under the induction of hypoxia or inflammatory disease, pro-angiogenic factors such as VEGF, bFGF, and PDGF are produced in large quantities. It then binds to the corresponding receptor on the vascular endothelial cell membrane, which in turn triggers a downstream signaling cascade that promotes angiogenesis. Angiogenesis relies on the stimulation of extravascular pro-angiogenic factors. Under the action of extracellular matrix protease, the vascular basement membrane is first dissolved, thereby promoting the release of vascular endothelial cells to the extravascular vessels to form new blood vessels. Then, after remodeling and maturation, the new microvessels gradually integrate into the existing vascular system to form stable blood vessels. VEGF-mediated signal cascades are involved in each of the above-mentioned angiogenesis processes, directly controlling the occurrence and development of angiogenesis, and determining the results of angiogenesis to some extent.

VEGF receptor

VEGF has three receptors, VEGFR-1 (Flt-1), VEGFR-2 (KDR), and VEGFR-3 (Flt-4). They belong to the receptor tyrosine kinase superfamily membrane protein, which contains approximately 750 amino acid residues outside the cell membrane and consists of seven domains similar to IgG. It belongs to the tyrosine kinase domain in the membrane and is separated by a kinase consisting of 70 amino acids and the end is the C-terminus. The VEGFR domain and functional studies have shown that the second Ig domain outside the Flt-1 membrane is the major site of its binding to the ligand. The site where KDR binds to the ligand is mainly in the third Ig domain. VEGFR-1 binds to VEGF-A and VEGF-B in vivo and stimulates downstream signaling; VEGFR-2 binds primarily to VEGF-A and VEGF-E; VEGFR-3 binds to VEGF-C and VEGF-D; VEGF-C and VEGF-D can also bind to VEGFR-2 after proteolysis, but the binding efficiency is much lower than that of VEGR-3.

Figure 1 Schematic diagram illustrating the receptor-binding specificity of VEGF family members and the VEGFR-2 signalling pathways

Upstream signaling of VEGF

KLF4 is a pro-angiogenic factor and promotes proliferation, migration and tube formation by enhancing the VEGF signaling pathway in HRMEC. KLF4 interacts with KLF2 to maintain intact vascular endothelial and vascular integrity by up-regulating the expression of downstream eNOS, VEGFR2 and occludin genes.

Figure 2 Schematic representation of KLF4-mediated VEGF signaling pathway

Downstream signaling of VEGF

PI3K-Akt pathway

Numerous studies have shown that VEGF promotes endothelial cell proliferation mainly through its downstream PLCγ1, PI3K-AKT signaling pathway. VEGF-A/VEGFR2 signaling by PLCγ1 promotes vascular endothelial cell proliferation through Erk1/2 by promoting its agonistic kinase C and Raf-MEK signaling pathways. VEGF-mediated PLCγ1 signaling also promotes ATF6 and IRE1α present in the endoplasmic reticulum by up-regulating mTORC1 activity in endothelial cells. There are multiple tyrosine phosphorylation sites in VEGFR2, and Cbl is phosphorylated by sites 1054 and 1059. Phosphorylated Cbl inhibits tumor vascular endothelial cell proliferation by inhibiting PLCγ1 activation, thereby inhibiting angiogenesis.

VEGF-Akt is a classical signaling pathway that promotes endothelial cell proliferation and survival, inhibits apoptosis, and promotes angiogenesis. Studies have shown that VEGF inhibits apoptosis through the downstream PI3K-Akt signaling cascade through the BAD pathway, and promotes endothelial cell proliferation and angiogenesis via mTORC2 and FOXO1, respectively. The mTORC2 pathway is also important for PLCγ1 to promote endothelial cell proliferation. PLCγ1 finally up-regulates the expression of mTORC2 gene through proteins such as ATF6 in the endoplasmic reticulum and promotes endothelial cell proliferation together with Akt.

In brain microvascular endothelial cells, the phosphatidylinositol 3-kinases/Akt (PI3K/Akt) signal transduction pathway may be one of the important signaling pathways for direct regulation of VEGF gene expression. PI3K enters the cytosol to activate Akt phosphorylation, which promotes Akt entry into the nucleus to activate various anti-apoptotic or cytokines that promote tumor proliferation, which may act in a trans-activator of VEGF to promote VEGF expression and secretion. Studies have shown that inhibition of PI3K/Akt, mTOR and HIF-1α activity can inhibit the expression of VEGF. This suggests that VEGF expression is indeed regulated by the PI3K/Akt/mTOR/HIF-1α signaling pathway.

P38 pathway

P38 MAPK is an important pathway in the family of mitogen-activated protein kinase (MAPK) signaling pathways. P38 plays a key role in cell differentiation and apoptosis and has become a research hotspot in the field of signal transduction in recent years. For example, in human breast cancer MCF-7 cell line, 4-hydroxytamoxifen (4-OHT) has reduced sensitivity, which is caused by the circulation of p38 growth factor in VEGF/VEGFR2 and breast cancer cells. This suggests that VEGF expression can be regulated by the p38-mapk signaling pathway. In addition, MAPK pathway is known to be an important signaling system in the process of cerebral ischemic injury. P38 is a key protein of MAPK pathway, which is mainly involved in inflammatory reaction, cytokines, cell proliferation and differentiation, oxidative stress and other processes after cerebral ischemia. The study found that under a variety of cellular and stimulating conditions, p38 protein is an essential regulator of NOS2 expression, which in turn releases NO binding to ROS to activate MMP-9.

ERK1/2 pathway

Studies have shown that there are extracellular regulated protein kinases ERK1/2-Sp1-VEGF signaling pathway in cells. ERK1/2 kinase regulates the expression of VEGF and may be partially dependent on the activity of the transcription factor Sp1. Activation of oxidative stress → ERK1/2→AP1 signaling pathway is present in the retina of a diabetic rat model, and this pathway is associated with secretion of VEGF in vivo. By stimulating the diabetic environment in vitro and using Muller cells as the research object, it was confirmed that the oxidative stress→ERK1/2→AP1 signaling pathway is involved in the mechanism of secretion of VEGF. This suggests that VEGF expression can be regulated by the ERK1/2 signaling pathway, which in turn stimulates the release of NO from vascular endothelial cells. In summary, ERK1/2 is a key protein of the MAPK pathway, which regulates the expression of NOS and releases NO through the VEGF signaling pathway, and activates MMP-9 by binding ROS. It can be speculated that the ERK1/2 pathway can regulate the expression of NOS2, which in turn causes changes in the permeability of the blood-brain barrier.

Aquaporin

Studies have shown that aquaporins (AQP) are closely related to the function and integrity of the blood-brain barrier. Aquaporin 4 (AQP4) is a subclass of the water-protein-only family that is permeable to water. Up-regulation of AQP4 may be an important cause of cerebral edema. rhVEGF165 can increase the expression of AQP4 protein in astrocytes cultured in vitro, possibly increasing the expression of AQP4 protein by activating the ERK pathway.

ROS free radical

Free radicals can cause lipid peroxidation, protein oxidation or nitrification, and DNA damage in a variety of ways. The study found that VEGF expression is achieved through ROS-mediated PI3K and MAPK signaling pathways. Studies have shown that long-term incubation of vascular endothelial cells with high glucose medium can increase ROS levels, increase MMP-9 promoter activity, and increase protein expression and activity, and antioxidants can inhibit the elevated response of MMP-9. Reactive oxygen species can activate a variety of intracellular signaling pathways such as MAPK (ERK1/2, p38), PI3K/Akt, and others. For example, the process by which TGF-β1 stimulates vascular smooth muscle cells to express MMP-9 is involved in the ROS-dependent ERK-NF-κB signaling pathway. Moreover, its process of stimulating VSMC expression and secretion of MMP-9 is involved in the ROS-dependent MAPK/ERK signaling pathway.

VEGF and disease

Wang et al. showed that VEGFR plays an internal role in promoting angiogenesis. Jain et al. found that VEGFR-2 plays an important role in the development of early hemangioblasts, and knocking out this gene leads to the loss of embryonic hemangioblasts and hematopoietic stem cells. VEGFR-1 plays an important role in the assembly of hemangioblasts into hemangiomas, and the loss of this gene leads to angioangiogenesis. Recent studies have shown that VEGFR-3 directly affects the formation of microvascular branches and the composition of vascular networks under the regulation of Snail, and demonstrates that VEGFR-3, but not VEGFR-2, plays a major role in the longitudinal sprouting of retinal microvessels. In vivo, VEGF receptors usually do not act alone, and the VEGF receptors that play a major role in different cells and at different times are also in dynamic changes.

VEGF has a significant effect on the proliferation, migration, and chemotaxis of vascular endothelial cells in bone, lung, kidney, brain, tumor and other tissues. Under pathological conditions, hypoxia is the most important factor in promoting VEGF synthesis. VEGF not only provides the blood supply for tumor overgrowth, but also upregulates vascular endothelial VEGFR levels to regenerate blood vessels, and also mediates immune escape by dendritic cells. Zhou et al. showed that the expression level of VEGF-A in gastric cancer tissues was increased, and it was associated with increased vascular density, vascular invasion, lymph node metastasis, bone marrow micrometastasis and distant metastasis.

VEGF-A mainly binds to VEGFR-2 to promote tumor angiogenesis, while other VEGF members such as VEGF-C and VEGF-D can bind to corresponding receptors and participate in tumor lymphatic metastasis. VEGF-C produced by gastric cancer cells not only binds to VEGFR-3 on the vascular endothelium in a paracrine manner, induces angiogenesis, but also promotes the growth and migration of gastric cancer cells by autocrine binding to VEGFR-3.

References:

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