VEGF Pathway

Background
VEGF Pathway
   Ligands
   Receptors
VEGF – Signal Transduction
Factors Regulating VEGF and VEGFR Expression
VEGFR in Human Diseases

   Cancer
   Preeclampsia
   Age-related Macular Degeneration
   Amyotrophic Later Sclerosis
Challenges of VEGF/VEGFR Targeted Tumor Therapy
Conclusion

Background

Formation of new blood vessels, a process known as angiogenesis, is a fundamental biological process required for embryonic development, tissue repair, ovulation and menstruation. Angiogenesis involves sprouting of new blood vessels from the existing blood vessels through the proliferation, migration and invasion of endothelial cells. This process is tightly regulated by pro- and anti-angiogenic factors; dysregulation of which can lead to abnormal vasculature characteristic of multiple vascular diseases. There are several signaling mechanisms coordinated to form new blood vessel, including vascular endothelial growth factors (VEGFs)/VEGFRs, angiopoietin/Tie receptors1, Platelet-derived growth factors (PDGFs)/PDGFRs2 and EphirinB2/EphirinB43 . Among these, VEGF signaling has been often recognized as the key regulator of angiogenesis.

VEGF and VEGF Receptors

Vascular permeability factor (VPF) was initially known to show hyperpermeability of tumor blood vessels and involved in formation of tumor-associated ascites4. Later, studies revealed vascular endothelial growth factors (VEGF) as an angiogenic growth factor displaying high specificity for endothelial cells5. Subsequently, it was discovered that both VEGF and VPF are encoded by a single VEGF gene and are products of alternate splicing. Analysis of VEGF/VPF cDNA clones also revealed significant homology to platelet-derived growth factor (PDGF).

Ligands

There are five structurally related VEGF ligands: VEGFA, VEGFB, VEGFC, VEGFD and placenta growth factor (PIGF). All the VEGFs are homodimers with disulphide bonds. The native VEGF is heparin-binding homodimeric glycoprotein closely corresponds to the VEGF165 variant. VEGFA was shown to have five isoforms with 121, 145, 165, 189 and 206 amino acids as a result of alternate splicing6. VEGF ligands are secreted by various cell types and act in an autocrine and paracrine manner. Each VEGF ligand binds differently to the receptors and induces distinct biological responses7.

Receptors

VEGF ligands bind to three receptor tyrosine kinases, VEGFR1 (Flt-1), VEGFR2 (Flk-1, KDR) and VEGFR3 and to co-receptors like Neuropilin 1 (NRP1). While VEGF receptors have homology in kinase domains their signaling cascade differs significantly. Ligand binding induces receptor dimerization (homo/hetero dimers) and activation of the kinase domain. There are several substrates for the kinase domain; the protein-protein interactions are mediated through SH2 domain. The signaling molecules are either enzymes or are associated with enzymes and are regulated by the phosphorylation.

Phylogenetic analyses revealed that D-VEGFR/PVR, found in Drosophila melanogaster is the common ancestor of the vertebrate VEGF receptors. Both VEGFR and PDGFR in vertebrates were most likely obtained from the duplication/triplication of single D-VEGFR/PVR gene8,9.

VEGF – Signal Transduction

VEGF signaling involves the cascade of signals triggered when the ligand binds the VEGF receptors. The downstream signaling includes activation of phospholipase Cγ1, MAPK pathway via Ras/Raf1 activation and PI3K/Akt pathway. Phospholipase Cγ1 in turn regulates the concentration of intracellular Ca+2 ions and formation of endothelial nitric oxide synthase. The effect of all the cascades provides a balance of pro- and anti-angiogenic signals that maintain the vasculature and/or result in sprouting of new blood vessels, cell proliferation and cell migration (Figure 1). Table 1 indicates the biological effects of the signaling cascade triggered when the ligand bind to each of the VEGF receptors.

Table 1. Signaling Transduction by Each VEGF Receptor

Receptor VEGFR1 VEGFR2 VEGFR3 NRP1
Ligands VEGFA, VEGFB,PIGF VEGFA, VEGFC,VEGFD VEGFC,VEGFD VEGF165, PlGF
Cell Expression Endothelial, Dendritic, Monocytes/ Macrophages, Osteoblasts, Pericytes, Trophoblasts Endothelial, Neuronal, Retinal progenitors, hematopoietic stem cells,  Osteoblasts, Megakaryocytes Lymphatic endothelial cells, Monocytes/Macrophages Endothelial, Neuronal, Plasmacytoid Dendritic cells
Signal Transduction Activation of phospholipase C-γ1; regulation of mitogen activated protein kinase (MAPK) pathway10,11 Activation of PI3K/Akt pathway, BAD, FKHR1, Caspase1, Bcl-2, differentiation of mesenchymal stem cells Activation of ERK and PI3K/Akt pathways Activation of mitogen activated protein kinase (MAPK) pathway
Functions Inhibition of angiogenesis and recruitment of immune cells Angiogenesis; cell survival, proliferation, migration, vascular permeability and inhibition of apoptosis Lymphangiogenesis; development of vasculature; migration of endothelial cells Vascular development; apoptosis inhibition; migration

 

Figure 1. VEGF Pathway

Factors Regulating VEGF and VEGFR Expression

There are several factors regulating the expression of VEGF and VEGFR. They are broadly classified into external factors, transcription factors and oncogenes. External factors include the signals originating from tissue microenvironment. Hypoxia is one of such factor, which regulates the VEGF expression through Hypoxia Inducible Factor-1 alpha (HIF-1α) 12. Under hypoxic conditions HIF-1α dimerizes with HIF-1β, binds to the VEGF promoter inducing VEGF transcription. Growth factors and cytokines secreted in tissue microenvironment such as epidermal growth factor receptors (ErbB1 and ErbB2), insulin like growth factor-I receptor (IGF-IR)13, hepatocyte growth factors14, platelet-derived growth factors (PDGF)15 and cyclooxygenases (COX)16 regulate angiogenic factors and induce angiogenesis. Oncogenes and tumor suppressors such as like c-SRC (proto-oncogene)17, BCR-ABL18, Ras19, p5320 and PTEN21 also influence the expression of VEGF.

 

VEGFR in Human Diseases

Diseases that are associated with vascular abnormalities are related to anomalies in VEGF/VEGFR signaling axis. While anti-VEGF treatment in cancer treatment is popular it is also being employed to treat indications like preeclampsia, macular degeneration and amyotrophic lateral sclerosis.

Cancer

Although there are different pathways which contribute to angiogenic process, VEGF/VEGFRs axis is prominently considered for cancer therapy. Most of anti-angiogenic agents currently approved and under investigation are focused on VEGF pathway. One of the strategies employs neutralization VEGF activity through monoclonal antibodies22. Bevacizumab was first such monoclonal antibody approved for cancer treatment. It has been approved as treatment option for colorectal cancer, non-squamous cell lung carcinoma, renal cell carcinoma and glioblastoma. VEGF-Trap (Aflibercept) a decoy receptor for VEGFA and PIGF was developed for macular degeneration and later for colorectal cancer23. Sunitinib, Sorafenib and Pazopanib are some of the small molecules inhibitors against tyrosine kinase activity of VEGFRs approved for renal cell carcinoma. These inhibitors also act against several other kinases like FGF receptor, EGFR family members, PDGFRs and Flt3 and have broader applications in cancer therapy.

Preeclampsia

Preeclampsia affects 5-7% of pregnancies, causing hypertension and proteinuria that retards growth of the fetus. Soluble form of VEGFR1 (sVEGFR1) is over expressed in the serum of pregnant women with preeclampsia. Interestingly, the severity of the condition is correlated with the expression of VEGFR124. Furthermore, there is a correlation between the levels of sVEGFR1 in early stages of pregnancy to preeclampsia in later stages. Cancer patients who are given anti-VEGF drugs often show hypertension and proteinurea25, these drugs are simulating the function of sVEGFR1 by abrogating the VEGF signals, implicating therapeutic importance of sVEGFR1 in treating preeclampsia.

Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is one of the leading causes of blindness, caused by vascular changes in the choroid. VEGFA has been implicated as a key contributory factor in the pathophysiology of neovascular AMD since it is pivotal in the development of blood vessels and vascular permeability. With limited treatment options available, intravitreal injections of anti-VEGF drugs are being studied as first line treatment option for AMD26.

Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is characterized by degeneration of motor neurons in a cerebral cortex, brainstem and spinal cord. Apart from functioning as a key angiogenic factor, VEGF is an active mediator of neurogenesis, neuronal survival and neuronal migration27. VEGFB protects neuronal cells from degeneration and stimulates motor neuron survival28. Stimulation of VEGF signaling is being explored as a novel approach in the treatment of rare neurodegenerative diseases like ALS. 

Challenges of VEGF/VEGFR Targeted Tumor Therapy

The efficacy of antiangiogenic therapy for tumors is often met with the following challenges.

Intrinsic resistance: There are active pathways which generate angiogenic factors, which make VEGF signals signals. For instance, high levels of infiltration of inflammatory cells increase the levels of proangiogenic factors. While many patients may benefit initially by anti-VEGF therapy, it may be a transient benefit due to this phenomenon of intrinsic resistance to VEGF/VEGFR treatment strategies.

Acquired resistance: From both preclinical and clinical findings, it was known that tumor adapt rapidly by upregulating pathways that retain tumor growth. The secretion of proangiogenic factors by stromal cells, upregulation of compensatory proangiogenic pathways and recruitment of bone marrow-derived proangiogenic cells results in the tumor acquiring resistance.

Tumor microenvironment: Anti-VEGF treatment markedly decreases tumor vasculature. This however increases the intra-tumor hypoxia in patients with non-small cell lung cancer and breast cancer. Hypoxia stimulates the expression of factors like HIF-1alpha leading to increase in the number of cancer stem cells in the tumor niche. Hypoxia also increases metastatic ability and triggers treatment-resistant mechanisms.

Conclusion

The role of VEGF and VEGFRs is well recognized in angiogenesis and has been implicated in chronic disease conditions. In view of suppressing tumor growth, several small molecules and antibodies were designed. Although, they have shown good response in in-vitro studies, several tumors showed resistance to anti-VEGF therapy due to parallel angiogenesis signals from other growth factors. Considering the variability in pathological conditions across tumors and patients, universal treatment of anti-VEGF therapy is not the answer. Factors regulating tumor resistance and angiogenesis are to be extensively defined. Focus on VEGF/VEGFR axis is crucial to answer such challenges in tumor behavior and treatment.

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