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Metastasis.JPG

Vascular Endothelial Growth Factor (VEGF)

Introduction

Vascular endothelial growth factor plays a crucial role in angiogenesis and vasculogenesis[1] as well as the proliferation, migration, and survival of endothelial cells. All of it’s roles are particularly important during embryogenesis, skeletal growth, and reproduction. Like other physiological regulators, over- or under-expression of VEGF-A can lead to dysfunction that gives rise to an array of diseases[2]. 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 of VEGF-A, the way in which it works, it’s relevance to various diseases and treatments, as well as past, present, and future studies.

History

Date Brief description
Late 1970s

Discovery of tumor-secreted protein that potently increased microvascular permeability to plasma proteins. [3]

1983

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). [4]

1986

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. [5]

1989

VEGF-A was first cloned and isolated by Napoleone Ferrara and his colleagues at Genentech. [6] 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. [4]

1993

Ferrara reports that by inhibiting VEGF-induced angiogenesis with specific monoclonal antibodies, they were able to suppress the growth of a variety of tumours in vivo. [6]

2004 The antiangiogenic effects of bevacizumab led to its FDA approval for the treatment of metastatic colon cancer. [7]
2006 Ranibizumab was approved by the US FDA for the treatment of patients with neovascular age-related macular degeneration. [7]

Normal Function

Vascular Endothelial Growth Factors (VEGF) are growth factor ligands found in cells that initiates a cascade of signal resulting in vasculogenesis[8], angiogenesis[9] and lymphangiogenesis[10].

Vasculogenesis

Vasculogenesis is one of the primary pathway in which new blood vessels are formed[11][12]. 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 [13].

Angiogenesis

Another process of blood vessel formation is through angiogenesis. Angiogenesis is the formation of blood vessels through the sprouting of capillaries from pre-existing blood vessels greatly influenced by the presence of VEGF. 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 in 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[14]. Several researches have been linked to angiogenesis and its up regulating and down regulating mechanism[15][16]. This 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, angiogenesis is one of the contributing factors in the rapid growth of tumour cells[17]. Through the down regulation of this process, growth can be controlled and possibly even halted.

There are 5 main steps of angiogenesis:

Vasculogenesis angiogenesis.jpg

1) Vasodilation occurs as a physiological response to nitric oxide (NO) and increased vascular permeability stimulated by VEGF

2) Endothelial cell migration at the site of injury

3) Proliferation of endothelial cells

4) Inhibition of endothelial cell proliferation and the formation of these cells into capillary tubes

5) Recruitment of pericytes to form mature vessels[18]


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

Lymphangiogenesis is the formation of lymphatic vessels from pre-existing lymphatic vessels highly influenced by VEGF-C[19][20]. 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[21]. During inflammation, lymph flow is increased to accommodate the drainage of oedematous fluid in the tissue [22]. 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 cause the faster metastasis of cancer. Tumours found within a close proximity with lymphatic vessels increases the spread of cancer cells throughout the body[23].

Signalling Pathway

Vascular Endothelial Growth Factor

A single gene encodes vascular endothelial growth factors, however, several isoforms of VEGF are present due to the altering splicing of the VEGF gene[24]. 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.

Several factors causes the production of VEGF namely:

Hypoxia

All cells need oxygen to survive. However, in some cases not enough oxygen is supplied to tissues from various reasons, decrease in inspired oxygen from places of high altitude and preterm birth. 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.[25]

Oncogenes

An oncogene is a gene that can latently cause cancer. 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.

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 hastens the production of prostaglandins that induces inflammation therefore increasing the production of VEGF. This correlation has been found with the study of tumors.[26]

Vascular Endothelial Growth Factor Receptors

There are 3 different VEGFR namely VEGFR-1, VEGFR-2, and VEGFR-3. VEGF ligands causes 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 [27]. Effects of each ligand-receptor complex are shown on the table below:

VEGF Receptor Effect
VEGFR-1
  • Induces other factors
  • Acts as a decoy
  • Angiogenesis
VEGFR-2
  • Cellular survival
  • Angiogenesis
  • Vasculogenesis
  • Propagation
VEGFR-3
  • Lymphangiogenesis

VEGFR-1

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.


VEGFR-2

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. Shown on the diagram on the right.


VEGFR-3

One of the primary mediators of lymphangiogenesis is the VEGF/VEGFR-3 complex[28][27]

Abnormal Function

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.


Disease Description VEGF-A Relationship Image
Diabetic Retinopathy A complication of diabetes affecting the retina. Possibly leads to complete blindness.

Two types-

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 [29][30]. Diabetic retinopathy.JPG
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

[31]

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 [32].
A hand affected by rheumatoid arthritis Copyright Information
Breast cancer Malignant cancer cells form in the breast tissue [33]. 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[32].

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[34].

Excised human breast tissue, showing a stellate area of cancer 2cm in diameter. The lesion could be felt clinically as a hard mobile lump, not attached to skin or chest wall. The histology was that of a moderately well differentiated duct carcinoma Copyright Information
Age-related macular degeneration In developed countries, Age-related macular degeneration (AMD) is responsible for the most cases of vision-loss [35].

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[36][37]. Age related macular degeneration.jpg
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[38]. Studies have demonstrated that VEGF-A is essential for the survival of motor neuron cells[39]. As a result, lowered levels of VEGF-A have been associated with motor neuron disease [40]. Hawking.jpg

Research: Therapeutic Applications

(A) Fresh lung tissue was examined using laser scanning confocal microscopy to detect GFP-positive metastatic tumor cells (B)More metastatic lesions were visible in lungs (after 9 days) of mice injected with cells expressing VEGF compared with cells expressing GFP alone

Anti-VEGF Treatment

Cancer Therapy

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. [41]

Possible Implications

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. [42] 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. [43]

Indication Treatment Trial results on which the FDA based its approval [44]
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


Related Studies: | Schwannoma | Future Implications | Current Progress, hurdles and future prospects|


Ranibizumab is a recombinant humanized monoclonal antibody fragment. Bevacizumab is a recombinant humanized IgG antibody. Both bind to and inhibit all biologically active forms of VEGF-A and are derived from the same mouse monoclonal antibody. Ranibizumab has been genetically engineered to bind with higher affinity than bevacizumab.

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. [45] Research showed significant reduction in moderate and severe vision loss. [46]
  • 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. [7]

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. [7]

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 |

External Links

National Cancer Institute

This website is a great source of information - from current cancer research and funding to useful fact sheets.

Images

Glossary

  • 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:
  • 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:
  • 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
  • Neutropenia:
  • Stromal Cells: connective tissue cells of the body
  • Thromboembolic:
  • Vasculogenesis: biological process of creating new blood vessels during the early stage of development (embryological development)

References

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