2012 Group 2 Project
|2012 Projects: Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | Group 9|
- 1 Vascular Endothelial Growth Factor (VEGF)
- 1.1 Introduction
- 1.2 History
- 1.3 Normal Function
- 1.4 Ligands and Receptors
- 1.5 Signaling Pathway
- 1.6 Abnormal Function
- 1.7 Research: Therapeutic Applications
- 1.8 External Links
- 1.9 Images
- 1.10 Glossary
- 1.11 References
Vascular Endothelial Growth Factor (VEGF)
Vascular endothelial growth factor (VEGF) is an important growth factor found in almost all cells of the body. When VEGF binds to a vascular endothelial growth factor receptor (VEGFR), the resulting signalling cascade induces angiogenesis and vasculogenesis as well as promoting the proliferation, migration, and survival of endothelial cells. All of these roles are particularly important during embryogenesis, skeletal growth, and reproduction.
VEGF has a number of subtypes- VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF, all of which are responsible for different processes. All of these subtypes share a common signaling pathway but utilise different receptors. Like other physiological regulators, over- or under-expression of VEGF can lead to dysfunction that gives rise to an array of diseases. Because each type of VEGF has a different function, the clinical manifestations vary depending on the VEGF affected. For the purpose of this page, we will be focusing on diseases where VEGF-A has been implicated, because it influences a larger number of processes than the other subtypes. Fortunately, many studies have demonstrated the successful treatment of various afflictions using VEGF-A and anti-VEGF-A therapies.
Here we will examine the function, signalling, and research history of the VEGF family, as well as addressing diseases, treatment and research associated with VEGF-A.
Discovery of tumor-secreted protein that potently increased microvascular permeability to plasma proteins. 
Senger and Dvorak were the first to discover a protein which increased vascular permeability in a number of vascular beds, including those of skin, subcutaneous tissue, peritoneum, pleura, mesentery, diaphragm, retina and skeletal muscle. This protein was named vascular permeability factor (VPF). 
Dvorak proposed that by secreting VPF, tumors induce angiogenesis. Dvorak's scientific research greatly contributes to our current understanding of tumor growth and its blood supply. He noted that tumors were like wounds in their secretion of VPF, which caused blood vessels to leak plasma fibrinogen and hence induced angiogenesis. However, unlike wounds which heal and stop secretion of VPF, malignant tumors would continue to grow and spread. 
VEGF-A was first cloned and isolated by Napoleone Ferrara and his colleagues at Genentech.  Subsequently, it was discovered that the DNA sequences of VEGF and VPF were identical.
Over the years, five VEGF-related genes have been identified (VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E). There are five characterized VEGF-A isoforms of 121, 145, 165, 189 and 206 amino acids in mammals. 
|2004||The antiangiogenic effects of bevacizumab led to its FDA approval for the treatment of metastatic colon cancer. |
|2006||Ranibizumab was approved by the US FDA for the treatment of patients with neovascular age-related macular degeneration. |
Vasculogenesis is one of the primary pathway in which new blood vessels are formed. It is more often related to the formation of the primary network of blood vessels during embryological development. Endothelial precursor cells, angioblasts, migrate to differentiate into vascular endothelial cells as a response to growth factors such as VEGF leading to the formation of new blood vessels .
Another process of blood vessel formation is through angiogenesis. Angiogenesisis the formation of blood vessels through the sprouting of capillaries from pre-existing blood vessels greatly influenced by the presence of VEGF-A. In humans with formed vasculature, angiogenesis is mainly a physiological response to trauma, injury and growth. Its formation is critical in injuries that can cause ischemia and is also responsible for the growth of tumours beyond the restrictions of their initial blood supply. Recently, it has been found that bone marrow stromal cells releases various cytokines that affect angiogenesis. Angiogenesis can be particularly helpful in the augmentation of the process in response to injuries for increased vasculature leading to better supply of nutrients to the affected area. On the other hand, angiogenesisis one of the contributing factors in the rapid growth of tumour cells. Several reviews showed that the up regulation of angiogenesiscan lead to cancer. Through the down regulation of this process, growth can be controlled and possibly even halted.
There are 5 main steps of angiogenesis:
- Vasodilation occurs as a physiological response to nitric oxide (NO) and increased vascular permeability stimulated by VEGF
- Endothelial cell migration at the site of injury
- Proliferation of endothelial cell
- Inhibition of endothelial cell proliferation and the formation of these cells into capillary tubes
- Recruitment of pericytes to form mature vessels
Recently formed blood vessels are usually leaky due the incomplete inter-endothelial junctions and because of the increased cellular permeability effect caused by VEGF.
Lymphangiogenesis is the formation of lymphatic vessels from pre-existing lymphatic vessels highly influenced by VEGF-C. The lymphatic system found all though out the body and is closely related to blood vessels as it serves as the drainage filtering out extracellular exudates. The lymphatic system is also associated with the body’s immune system. During inflammation, lymph flow is increased to accommodate the drainage of oedematous fluid in the tissue . Lymphatic vessels are leaky allowing the migration of leukocytes in areas of injury. In addition, lymph nodes are part of this system that is a major location of white blood cells (B and T-lymphocytes) and other macrophages. In spite of working in favour for the human body, lymphatic vessels can also increase cancer metastasis. Tumours found within a close proximity with lymphatic vessels increases the spread of cancer cells throughout the body.
Ligands and Receptors
Vascular Endothelial Growth Factor
VEGF is a homodimeric member of the cystine-knot growth factor family. A single gene encodes vascular endothelial growth factors, however, several isoforms of VEGF are present due to the altering splicing of the VEGF gene. The diagram below shows the different splicings locations resulting into VEGF-A, VEGF-B, VEGF-C, VEGF-D, and Placental Growth Factor (PGF). Here it can be seen how the removal of exons of a gene can result in differing proteins that will lead to varying biological processes and physiological changes.
Factors that induce/regulate VEGF production
a) Hypoxia and Trauma
All cells need oxygen to survive. However, in some cases, such as a decrease in inspired oxygen from places of high altitude, or pre-term birth, insufficient oxygen is supplied to tissues. Hypoxic cells, cells without adequate supply of oxygen, can stimulate the production of VEGF . When cells are lacking oxygen, it produces Hypoxia-Inducible Factor (HIF) a transcription factor that stimulates the release of VEGF. The free VEGF then binds to VEGFR on cell membranes of endothelial cells causing angiogenesis and vasculogenesis as a cellular response.
An oncogene is a gene that can latently cause cancer through induced production of VEGF leading to greater tumor vascularization. Oncogenes can be observed in high levels in tumour cells. Cell death or apoptosis is a normal process of a cell cycle, however, oncogenes suppresses this mechanism; hence, leading to further proliferation of mutated cells. Some of the manifestation of oncogenes is the error in protein structure that affects the level of enzyme activity and cell regulation. In cells with high levels of oncogenes, levels of kinase production are altered. Kinases are enzymes that add a phosphate group to different proteins which acts as an on and off switch as well as receptor kinases adding phosphate group to receptor proteins found in the surface of cell membrane to send out signal from the environment to the inside of the cell. Since VEGFRs are tyrosine kinase receptors, an alteration in the signaling pathway can cause cancer by switching on the receptor in spite having no signals from outside the cell. Evidences have shown that mutations in the p53 tumor suppressor up regulates the production of VEGF .
c) Other growth factors and cytokines
VEGF are also produced as a response to other growth factors eg. Platelet Derived Growth Factor (PDGF) and cytokines. PDGF supports endothelial cell survival and vascular maturation by the recruitment of pericytes and smooth muscle cells. Similarly, the enzyme Cyclooxygenase-2(COX-2) hastens the production of prostaglandins that induces inflammation therefore increasing the production of VEGF. This correlation has been found with the study of tumors.
Vascular Endothelial Growth Factor Receptors
VEGFR is a tyrosine kinase receptor found along the cell membrane. It has 7 immunoglobin-like structure found on its extracellular domain and a split tyrosine kinase found in the intracellular domain. There are 3 different VEGFR namely VEGFR-1, VEGFR-2, and VEGFR-3. VEGF ligands cause varying effects by binding to different VEGFR resulting into different physiological changes. VEGFR-1 and VEGFR-2 are linked to angiogenesis whilst VEGFR-3 is related to lymphangiogenesis. Effects of each ligand-receptor complex are shown on the table below:
VEGF binding to VEGFR-1 is shown to cause release of other factors. Researches showed that VEGF-1 stimulates the production of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), matrix metalloproteinase-9(MMP9) and other vascular bed-specific growth factors.
In addition, there were controversial findings stipulating that VEGFR-1 acts as a decoy to prevent VEGF from binding to VEGFR-2 and 3. Since VEGFR-1 is the only receptor which does not lead to mitogenesis, VEGF binding to VEGFR-1 competes with mitogenic action that VEGF bound to VEGFR-2 and 3 would result to.
VEGF binding with VEGFR-2 has 3 major effects, cellular survival, proliferation, and migration. Each is a result of different pathways yet acting together to induce angiogenesis, mitogenesis and increased permeability of blood vessels.
Endothelial cell survival is achieved through the PI3K/Akt pathway, migration through p38MAPK pathway and proliferation though the Raf/MEK/Erk pathway as shown on the diagram on the right.
Taking VEGFR-2 as an example. VEGF binds with the extracellular domain of the VEGFR-2 causing a receptor dimerization, which then activates the intracellular tyrosine kinase domain of the receptor and allowing autophosphorylation to occur. Autophosphorylation allows specific intracellular molecules to recognise and adhere to the VEGFR sending an extracellular signal into the cell.
The binding of VEGF to VEGFR-2 can lead to the activation of three pathways:
• P13K →Akt/PKB
• P38 MAPK
Phosphatidylinositol 3-kinase (PI3K) is a lipid-base protein that plays a crucial role in the PI3K pathway. The PI3K pathway is an extremely complex pathway comprised of several activators, inhibitors and affectors that is important for cellular signaling and cancer development.
This pathway is activated through the binding of VEGF to VEGFR causing receptor dimerisation. As a result, PI3K is recruited to the docking site(intracellular domain) of the phosphorylated VEGFR and becomes activated . PI3K can also be activated through a mediator protein called insulin substrate receptor-1 (IRS-1) that binds to the phosphotylated site of VEGFR. While bound to the VEGFR, IRS-1 acts as another binding and activating site for PI3K. The activated PI3K then moves and binds to a lipid membrane molecule, PIP2, converting it to active PIP3 through phosphorylation. PIP3 releases a signal that activates the signalling kinase, Akt. Reviews showed that activated Akt acts on mammalian target of rapamycin (mTOR) resulting the promotion of cell growth and propagation through protein synthesis .
Akt promotes a cascade of reaction starting from the activation of the protein Rheb that activates mTOR. mTOR then activates translation factor, S6K, which binds to the large subunit of a ribosome. The binding of S6K to the ribosome promotes the translation of mRNA to proteins in the cytoplasm. In addition, this process inhibits cell apoptosis by blocking Forkhead box (FoxO) activity. FoxO is a transcription factor characterised by its fork head DNA binding domain. Translocation of FoxO outside the nucleus inhibits the function of the said protein. FoxO initiates cellular apoptosis by up regulating genes needed for cell death.
To sum up, VEGF initiates the PI3K/Akt pathway through the phosphorylation of the VEGFR. This results in a cascade of intracellular signal promoting endothelial cell survival.This pathway has been highly associated with cancer and is linked to be responsible to some of resistance in cancer therapy. Errors in this pathway can be a result of mutations and augmentation of tyrosine kinases and defects on PI3K.
P38 mitogen-activated protein (MAP) kinase is a signalling molecule and a member of the ubiquitous MAP kinase that highly affects the VEGF-induced angiogenesis and vascular permeability by acting as a molecular switch that amplifies the pathway.
The initiation of the p38 MAPK pathway is mediated by 3 kinases, namely MAP kinase kinase, MAP kinase and MAP. MAP kinase 4 activates several MAPK kinase, MKK3, MKK4, and MKK6 that then phosphorylates p38 MAPK. Activation of p38 can lead to the activation of MK2 MK3, and Ets-1.
Activation of MK2 increases the production of TNF-α which is a major cytokine in inducing inflammation . Inflammation is a result of angiogenesis. On the other hand, p38 can activate MK1 that leads to a different physiological change. MK1 represses the activity of E47 through phosphorylation. E47 is a transcription factor associated with cellular differentiation. Ets-1 positively regulates the VEGF and VEGFR gene expression. Ets-1 activates the urokinase plasminogen activator (uPA), which leaves the zymogen plasminogen resulting in an active plasmin enzyme. Plasmin are used by the cell to break fibrin polymers that causes blood clots. Furthermore, Ets-1 regulates VEGF-induced matrix metalloproteinase-9 and 13.
Researches suggest that p38 MAPK is needed for the VEGF-mediated vascular permeability. On the contrary, inhibition of p38 MAPK would result in the reduced vascular permeability and angiogenesis.
The Mitogen-Activated Protein Kinase (MAPK)/Erk pathway is crucial in the control of gene expression, cellular growth, development and survival. MAPK is an inactive molecule that requires phosphorylation to be activated. The binding of VEGF to the VEGFR from the extracellular domain triggers this pathway. When the intracellular tyrosine kinase autophosphorylates signaling molecules, Growth Factor Receptor-Bound Protein 2 (Grb2) and Sos, attaches to the intracellular domain of the receptor . Ras-GTP complex is then recruited to the docking site (close to where Grb2 and Sos molecules are attached). This leads to the activation of the protein RAF and the enzyme Protein Kinase Suppressor of Ras (KSR) within the cell membrane .
The KSR enzyme has three distinct binding sites for 3 specific proteins. Upon activation of KSR, three protein molecules, RAF, MEK and Erk, drift towards their corresponding binding site found in the KSR molecule . This collective binding leads to the activation of Erk. Once activated, Erk migrates to the nucleus. Inside the nucleus, Erk induces several transcription factors that mediate gene expression that up regulates cellular survival and proliferation.
This signalling pathway is highly regulated by KSR that has three binding sites for the RAF, MEK and Erk molecules. Another mechanism that this pathway is regulated is through the inactivation of Ras complex shortly after its activation. Reviews have shown that regulation of this pathway is extremely important to avoid unwanted and unnecessary cellular signalling which often results to diseases like tumour proliferation that can lead to cancer.
Because VEGF-A plays such a crucial role in maintaining many key physiological processes, it's dysfunction can give rise to a vast array of diseases. The following examples are a small selection of diseases where VEGF-A has been implicated in the pathogenesis. The role of VEGF-A in these diseases is relatively well understood, and while there are many promising advancements in terms of treatment, extensive research is still required.
|Diabetic Retinopathy||A complication of diabetes affecting the retina. Possibly leads to complete blindness.
•Nonproliferative retinopathy-characteristic microangiopathy, edema, microaneurysms, haemorrhaging, exudates and venous dilations, in the retina.
•Proliferative retinopathy-haemorrhaging, capillary rupture, and retinal detachment are commonly observed
|VEGF-A is upregulated due to the hyperglycemia and oxidative stress that results from diabetes. This in turn causes the characteristic vascular leakage, retinal neovascularisation, and macular edema.  |
|Rheumatoid arthritis||Inflammatory disease of the synovial membrane. Most commonly impacts small joints in the hands and feet. According to the American College of Rheumatology, patients must exhibit at least four of the following criteria to be diagnosed-
• morning stiffness lasting at least 1 hour
• swelling in three or more joints
• swelling in hand joints
• symmetric joint swelling
• erosions or decalcification on x-ray of hand
• rheumatoid nodules
• abnormal serum rheumatoid factor
|Inflammation around the joint is maintained by extensive blood vessel proliferation. In rheumatoid arthritis, the protein CD147 upregulates VEGF-A, which in turn increases angiogenesis around the affected area. |
|Breast cancer||Malignant cancer cells form in the breast tissue. ||Inflammation around the joint is maintained by extensive blood vessel proliferation. In rheumatoid arthritis, the protein CD147 upregulates VEGF-A, which in turn increases angiogenesis around the affected area.
Koutras and colleagues (2010) made a very important point- “Tumors cannot grow beyond about 2 mm in diameter, in the absence of a vasculature providing oxygen and nutrients”. It is therefore no surprise that VEGF-A has been implicated in the proliferation of blood vessels that encourages tumor growth.
|Age-related macular degeneration||In developed countries, Age-related macular degeneration (AMD) is responsible for the most cases of vision-loss. 
Patients experience a loss of central and high resolution vision, which may eventually lead to irreversible blindness.
|The macula is located under the retina and is responsible for high resolution vision. VEGF-A induces the growth of abnormal blood vessels at the centre of the macula, which results in impaired function|
|Motor neuron disease||Initial degeneration of the upper and lower motor neurons leads to weakness of muscles in the abdnominal, bulbar, limb, and thoracic regions. Respiratory capacity is decreased due to impairment of the respiratory muscles. This deterioration often leads to death.||Studies have demonstrated that VEGF-A is essential for the survival of motor neuron cells. As a result, lowered levels of VEGF-A have been associated with motor neuron disease.|
Research: Therapeutic Applications
In recent years, antiangiogenesis has been at the core of alternate cancer therapy research. This includes a variety of methods that prevent tumor angiogenesis and/or that attack tumor blood vessels. Alternate cancer therapy is desirable as these approaches are relatively non-toxic and are thought to prevent tumor cell regrowth with long-term administration.
In particular, therapies have been designed to target VEGF-A, which is known to initiate and promote tumor vasculature in humans and animals. There have been recent successes in treating mouse cancers. Evidence shows that by neutralising antibodies against VEGF-A, and antibodies that block VEGF-A receptors, tumor growth can be significantly reduced. More recently, antibodies have been designed to selectively recognise VEGF-A forms found on tumor vessels, hence avoiding side effects that might result from inactivation of free VEGF-A. 
Research conducted by Ranpura et al (2011) suggested that a particular anti-angiogenesis drug called bevacizumab had caused fatal side effects in combination with particular types of chemotherapy. Some of these adverse effects included: wound dehiscence, bleeding, thromboembolic events, bowel perforation, and neutropenia.  Bevacizumab is a humanized monoclonal antibody that inhibits VEGF activity. In Ranpura et al's (2011) study, a total of 10217 patients who suffered from a range of advanced tumors were included in the analysis. Results indicated that in a meta analysis, the VEGF inhibitor in combination with chemotherapy or biological therapy, was associated with increased treatment-related mortality. 
|Indication||Treatment||Trial results on which the FDA based its approval |
|Metastatic colorectal cancer||In combination with IFL (irinotecan, fluorouracil, leucovorin)||Improved median overall survival (OS) by 4.7 months; also improved progression-free survival (PFS)|
|Metastatic colorectal cancer||In combination with FOLFOX4 (folinic acid [leucovorin], fluorouracil, oxaliplatin)||Improved OS by 2.2 months; also improved PFS|
|Metastatic kidney cancer, post-nephrectomy||In combination with interferon-alfa||Improved PFS|
Age Related Macular Degeneration (AMD)
AMD results from complex interactions of the eye, including local inflammation, which can lead to neovascularization (CNV). CNV can cause blindness if left untreated. Current treatment strategies include:
- Pegaptanib: was one of the first anti-angiogenic agent with proven efficacy in clinical trials for neovascular AMD.  Research showed significant reduction in moderate and severe vision loss. 
- Bevacizumab: as already mentioned above, it is a humanized monoclonal antibody that inhibits VEGF activity.
- Ranibizumab: an antibody fragment created to have a greater binding capacity in comparison to Bevacizumab. 
Current studies by Kovach et al (2011) confirm that the use of either bevacizumab or ranibizumab is the best treatment strategy for this moment in time. Clinical studies showed that either monthly or with individualised treatment plans with close follow up lead to the most improvement in AMD patients. 
Future Prospects for AMD
Regular invitreous injections of these drugs are not very cost effective. Therefore further research must be conducted to increase efficacy but also formulate cost-conscious treatment plans.
Additional Links: | Anti-VEGF Treatment Strategies for Wet age-related macular degeneration | Food and Drug Administration |
Another condition that can lead to the formation of abnormal neovascularisation is diabetic retinopathy. Proliferative diabetic retinopathy (PDR) is characterised by the uncontrolled formation of new vessels on the retinal surface. This spread can invade the vitreous cavity and lead to complications such as hemorrhage, fibrosis, and tractional retinal detachment. Although current techniques in treating this disease are quite successful, the increasing prevalence of type 2 diabetes raises future concerns. 
Current treatment of PDR involves laser therapy known as panretinal photocoagulation (PRP). Research shows that 50-60% of individuals experienced improvement in their vision and neovascularisation over a 3 month period using the laser treatment.  However, this type of therapy requires multiple administrations and can result in severe side effects such as pain, nyctalopia or loss of peripheral vision.
Current research is aimed at improving treatment and techniques to minimise consequent side effects and to maximise its efficiency. Tremolada et al (2012) reviews current research on proposed drug therapies such as Pegaptanib, Bevacizumab and Ranibizumab which are all VEGF inhibitors. In 2006, a group of researchers investigated the benefits and use of intravitreal injections in the treatment of PDR. In particular, this experiment focused on Bevacizumab which is an active inhibitor of all VEGF-A isoforms. Results showed that this method of treatment is proven to be effective and enhanced rapid regression of retinal and iris neovascularisation. 
Additional Links: | Current Therapies and Future Challenges |
External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.
This website is a great source of information - from current cancer research and funding to useful fact sheets.
This website directly tackles VEGF and VEGFR's role in the proliferation of cancer.
The FDA agency aims to protect and promote public health through regulations and supervision. This website provides information on new anti-VEGF approved drugs such as Bevacizumab.
- Angioblast: primordial mesenchymal cells that differentiates into embryonic blood vessels or vascular endothelium
- Angiogenesis: a biological process by which new capillaries are formed and it occurs in many physiological and pathological conditions.
- Chemotaxis: characteristic movement towards or away a chemical stimulus of an organism or cell as a response to a chemical gradient on its environment
- Cytokines: regulatory proteins produced by the immune system that acts as intracellular mediators in the modulation of immune response
- Dehiscence: splitting or opening of a surgical suture
- Dimerization: the formation of a compound consisting of two identical molecules
- Endothelial Cell: lining cell of a body cavity
- Exudate: fluid that has exuded out of a tissue or its capillaries as a result of injury or inflammation
- Hyperglycemia: condition of high glucose blood level
- In vivo: occurring or made to occur in side an organism in its natural setting
- Ischemia: restriction of blood supply to a tissue resulting to a lack of oxygen supply
- Lymphangiogenesis: biological process of lymphatic vessel formation from pre-existing lymphatic vessels
- Macrophages: activated monocytes circulating the body to protect against infection and foreign substances
- Mitogenesis: inducing mitosis in cells
- Neovascularisation: the formation of functional microvascular networks with red blood cell perfusion
- Neutropenia: condition of abnormally low neutrophil count making people with this disorder susceptible to infections
- Nyctalopia: impaired vision in dim light and in the dark, due to impaired function of the rods in the retina
- Pericytes: contractile cells found outside the basement membrane of precapillary arterioles
- Stromal Cells: connective tissue cells of the body
- Thromboembolic: the blocking of a blood vessel by a blood clot dislodged from its site of origin
- Vasculogenesis: biological process of creating new blood vessels during the early stage of development (embryological development)
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