1Jobu University, Director, Institute of Physiology and Medicine, Gunma 372-8588
2Tokyo Medical and Dental University, Department of Molecular Oncology, Tokyo 113-8519
3University of Tokyo, Tokyo 108-8639, Japan
Vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) system has been shown to play central roles not only in physiological angiogenesis, but also in pathological angiogenesis in diseases such as cancer. Based on these findings, a variety of anti-angiogenic drugs, including anti-VEGF antibodies and VEGFR/multi-receptor kinase inhibitors have been developed and approved for the clinical use. While the clinical efficacy of these drugs has been clearly demonstrated in cancer patients, they have not been shown to be effective in curing cancer, suggesting that further improvement in their design is necessary. Abnormal expression of an endogenous VEGF-inhibitor sFlt-1 has been shown to be involved in a variety of diseases, such as preeclampsia and aged macular degeneration. In addition, various factors modulating angiogenic processes have been recently isolated. Given this complexity then, extensive studies on the interrelationship between VEGF signals and other angiogenesis-regulatory systems will be important for developing future strategies to suppress diseases with an angiogenic component.
Vertebrates, including humans, have a closed circulatory system for supplying oxygen and nutrients to various tissues in the body, and for removing CO2 and waste materials from peripheral tissues (Risau, 1997). In addition to the blood circulatory system, vertebrates have a similar tubular system, the lymph vessel system, which is essential for the absorption and delivery of fluids, lipids and immune cells from peripheral tissues to lymph nodes and circulating blood.
The circulatory system plays a crucial role in the etiology of many diseases in humans. The hypothesis that suppressing tumor angiogenesis was a potentially novel anti-cancer strategy was first suggested by J. Folkman in 1970 (for review, Hanahan and Folkman, 1996), although the molecular basis of the regulation of angiogenesis was not clearly characterized prior the late 1980s.
Around 1990th, the genes encoding vascular endothelial growth factor (VEGF) and its receptor (VEGFR) were isolated and characterized. Based on extensive studies of these molecules, signals mediated by members of the VEGF and VEGFR families were shown to play central roles in angiogenesis and lymphangiogenesis (Leung
In parallel to the VEGF-VEGFR axis, other regulatory systems, including angiopoietin (Ang)-Tie, Delta-Notch and Ephrin-Eph, have been shown to play a role in angiogenesis (Suri
Based on the knowledge that VEGF signals are key players in tumor angiogenesis, a variety of antibodies and kinase inhibitors which suppress VEGF-VEGFR signaling have been developed that are now widely used in the treatment of cancer. The clinical efficacy of such therapeutics is limited however, and many future studies will focus on improving their performance in the clinic (Hurwitz
Recently, a soluble form of Flt-1/VEGFR-1, sFlt-1, has been shown to be associated with preeclampsia (PE) (Maynard
In 1989, two research groups independently isolated a cDNA, one encoding for vascular permeability factor (VPF) and another for VEGF, which proved to be identical, and encoded a single protein, now widely known as VEGF (for review, Dvorak, 2002; Ferrara, 2004). The human genome contains five genes encoding five distinct VEGF family members, namely VEGFA (also called VEGF), placenta growth factor (PlGF), VEGFB, VEGF-C, and VEGF-D (Fig. 1). Structurally, VEGF family proteins are homodimeric, with two subunits of about 120 to 200 amino acids in length. Given their overall structural resemblance to members of the platelet-derived growth factor (PDGF)/macrophage colony-stimulating factor (M-CSF)/stem cell factor (SCF) ligand family, the VEGF and PDGF families are considered to constitute a supergene family, the VEGFPDGF superfamily. With regard to interactions between VEGF ligands and their receptors, VEGF-A, which contains subtypes such as VEGF-121, -165 and -189, binds VEGFR-1/Flt-1 and VEGFR-2 (De Vries
In contrast to VEGF-A, PlGF and VEGF-B appear to have a relatively minor role in the regulation of angiogenesis, and have been shown to play a role in cardiac muscle function (Bellomo
In 1990, our group isolated a cDNA from human placenta that encoded a novel receptor-type tyrosine kinase containing seven extracellular immunoglobulin (Ig)-domains and a kinase insert sequence of approximately 70 amino acids in length. Based on its structural similarity to the Fms receptor, we named it Fms-like tyrosine kinase-1 (Flt-1) (Shibuya
VEGF-A binds to VEGFR-1/Flt-1 with high affinity (Kd=1–10 pM) and less strongly to VEGFR-2 (Kd=10–100 pM), although the tyrosine kinase (TK) activity of VEGFR-1/Flt-1 is about 10 fold weaker than VEGFR-2 (Keyt
Members of the PDGFR/Fms (M-CSF receptor)/Kit family are distantly related to VEGFRs, and are known to signal through PI3K activation domains with tyrosine (Y)-containing motifs such as Y-x-x-methionine (M) and Y-M-x-M, at the TK-insert domain. After autophosphorylation of PDGFR family members, the p85 subunit of PI3K binds the Y-x-x-M and YM-x-M motifs via SH2 domain, resulting in activation of downstream PI3K-Akt and Ras pathways and strong signaling for cell proliferation (Heldin and Westermark, 1999). Very interestingly however, VEGFRs, including VEGFR-2, lack Y-M-x-M and Y-x-x-M motifs in the TK-insert and other regions. In contrast, we found that VEGFR-2 Y1175, a major autophosphorylation site of this receptor, binds the SH2 domain of PLCγ, activating the PLCγ-C kinase-Raf-MEK-MAP kinase pathway to mediate endothelial cell proliferation (Takahashi
VEGFR-2 Y1175 has also been shown to be involved in von Willebrand factor release from endothelial cells (Xiong
While most TK family kinases engage the Ras activation pathway to stimulate cell proliferation, this pathway appears to be relatively unimportant in VEGFR-2 signaling. The dependence of VEGFR-2 on PLCγ-C kinase in signaling for the MAP kinase activation is therefore unique among TK family members.
The biological characteristics of VEGFR-1/Flt-1 are distinct from those of VEGFR-2. Fong
VEGFR-3 is highly expressed in lymphatic endothelial cells, and VEGF-C/D-VEGFR-3 signals have been shown to stimulate lymphangiogenesis and lymph-node metastasis in cancer. In addition, VEGFR-3 has been shown to play a role in tumor angiogenesis (Tammela
Recently a variety of angiogenesis-regulatory systems have been elucidated, including: the Ang-Tie receptor axis, which mediates stabilization or destabilization of blood vessels; the Ephrin-Eph receptor pathway, which plays a role in arterio-venous differentiation of vessels; the Delta-Notch pathway, which is involved in regulation of vascular morphology and tip cell-stalk cell communication; and the Netrins-UNC5R pathway, which plays a role in guidance during angiogenesis (Suri
In addition to these angiogenesis-regulatory systems, a variety of endogenous angio-suppressive factors have been identified. TSP-1, for example, has been shown to suppress angiogenesis by inducing apoptosis or cell cycle arrest of vascular endothelial cells via upregulation of p21 (Watnick
Future studies will be required to clarify which of these factors and signaling pathways are involved in suppressing tumor angiogenesis and which, if any, are suitable for development of therapeutics for clinical use.
Based on the evidence that VEGF-VEGFR signals play central roles in angiogenic processes in a variety of diseases such as cancer, various VEGF signal inhibitors, including anti-VEGF neutralizing antibodies and VEGFR kinase/multi kinase inhibitors, have been successfully developed and now widely used in the clinic (Kim
Given the potential of adverse clinical effects of small molecules kinase inhibitors, novel compounds are currently undergoing clinical trials to identify drugs with fewer side effects.
To date, there have been no successful results in the use of anti-VEGF signaling drugs in the treatment of pancreatic or gastric cancer.
While the clinical efficacy of anti-VEGFs has been demonstrated in solid tumors, it is not clear whether this benefit continues for long periods. OS curves in some phase III trials indicate that over time their efficacy decreases, and the potential for resistance or refractoriness exists in later periods. Refractoriness may be due to several possible mechanisms as follows: (1) other angiogenic factors such as HGF and FGF compensate for the loss of the VEGF proangigenic signal (Pàez-Ribes
Anti-VEGF signal inhibitors have been shown to be effective in suppressing symptoms of AMD. A recovery of visual acuity was demonstrated in response to intraocular injection of an anti-VEGF neutralizing antibody.
PE, which occurs in 5–7% of pregnancies, causes hypertension and proteinuria in the mother and growth retardation in the fetus, and Cesarean section is often required to save the fetus. sFlt-1 is present at high levels in the serum of PE patients, and the degree of sFlt-1 overexpression has been shown to correlate with the severity of PE (Koga
Since sFlt-1 is derived from the ligand-binding region of VEGFR-1/Flt-1, its major biochemical function is thought to be trapping of VEGF for suppression of VEGF signals (Tanaka
Furthermore, sFlt-1 is expressed in lens epithelial cells as well as pigment epithelial and photoreceptor cells in the eye, where it plays a role in maintaining avascularity in the cornea and in tissues outside of the retina (Ambati
sFlt-1 is also expressed in podocytes in the kidney, where it binds to the cell membrane lipid to maintain the physiological functions of podocytes and vascular endothelial cells in renal glomeruli (Jin
Another exciting advance in the VEGF-VEGFR field in past 12 years is the discovery of an intimate relationship between VEGF signaling and the neuronal system. Although VEGF signaling in neuronal tissues was thought to function directly upon blood vessels rather than neuronal cells, an extensive study clearly showed that this is not the case (Oosthuyse
Sensory nerve cells in the dorsal root ganglion of mice also express VEGFR-2, and VEGF signals are required to maintain the healthy condition of these cells (Verheyen
On the other hand, olfactory sensory neurons express VEGFR-1, and the VEGF-VEGFR-1 pathway has been shown to be important for physiological function of these neurons (Wittko
VEGF signaling plays a crucial role not only in cancer but also in a variety of other diseases, including neuronal degeneration and nephrotic syndrome. We anticipate that further studies on VEGF signaling and its modulators will herald a new era in which the severe diseases whose etiology is associated with abnormal VEGF signaling can be brought under control.