Angiogenesis

Question 1

What are the three ways of making blood vessels?

Answer 1

1. Vasculogenesis – formation of new blood vessels from bone marrow progenitor cells
2. Angiogensis – formation of new blood vessels by sprouting from pre-existing vessels
3. Arteriogenesis – collateral growth of blood vessels that is dependent on shear stress and external factors like macrophages

Question 2

What is the main signal for angiogenesis?

Answer 2

Hypoxia

Question 3

What is the most important pro-angiogenic factor?

Answer 3

VEGF (Vascular Endothelial Growth Factor)

Question 4

Explain the mechanism by which hypoxia triggers angiogenesis

Answer 4

• HIF (hypoxia-inducible factor) is a transcription factor that is responsible for the expression of genes involved in angiogenesis In normoxic conditions,
• Under normoxic conditions, HIF is bound to von Hippel Lindau protein (tumour suppressor), which induces ubiquitination (binding of ubiquitin which) inhibits HIF from promoting angiogenesis
• In hypoxic conditions, HIF is not bound to von Hippel Lindau (or ubiquitin) so it can regulate transcription and express genes involved in angiogenesis e.g. VEGF

Question 5

How many members are there in the VEGF family? List them.

Answer 5

5 VEGF-A, B, C, D PIGF (placental growth factor)

Question 6

How many tyrosine kinase receptors are there for VEGF? List them.

Answer 6

3 VEGFR 1, 2 and 3

Question 7

How many coreceptors are there for VEGF? List them.

Answer 7

2 Neuropilin 1 (Nrp 1) and 2

Question 8

Which receptor is the major mediator in VEGF-dependent angiogenesis?

Answer 8

VEGFR2

Question 9

What pathway is crucial for the selection of tip cells?

Answer 9

Notch signalling

Question 10

Outline the process of Notch signalling in endothelial cells, in regards angiogenesis

Answer 10

1. The notch ligand (Delta-like ligand 4 or Dll4) on an endothelial tip cell binds the Notch receptor on an endothelial stalk cell. Note both the ligand and receptors have extracellular domains, and the receptor has an intracellular domain also
2. The intracellular NICD domain is cleaved
3. This then translocates to the nucleus and binds to the transcription factor RBP-J and regulates transcription essentially making the stalk cells grow while the tip cells are quiescent
4. This sets up this ‘escalator-like’ growth for tip cells to spread before they meet and fuse

Question 11

What is another name for the notch ligand?

Answer 11

Delta-like ligand (Dll4)

Question 12

What effect does VEGF have on notch signalling and vice versa?

Answer 12

• It increases expression of Dll4
• Dll4 then drives Notch signalling, which inhibits expression of VEGFR2 in the adjacent cell
• Dll4 expressing tip cells develop a motile, invasive and sprouting phenotype
• Adjacent stalk cells form the base of the emerging sprout and proliferate to support sprout elongation

Question 13

Describe the role that macrophages can play in angiogenesis

Answer 13

• Aid vessel anastomosis
• Macrophages help carve out the existing endothelia and basal lamina and tunnels through ECM at the site of sprouting - MMPs etc
• Help stabilise newly formed vessels by promoting tip cell fusion

Question 14

Apart from macrophages, which other cell type is recruited to help with the stabilisation of the newly formed vessel?

Answer 14

• Pericytes

Question 15

Which cell adhesion molecules are essential for vessel stabilisation and quiescence?

Answer 15

VE-Cadherin

Question 16

What roles does cadherin play and where is it found in cells? Then mention a unique role that a form of cadherin plays in endothelial cells (also naming this form of cadherin)

Answer 16

• Cadherin is an essential part of the adhesive junctions between cells that allow them to form continuous monolayers
• Adjacent cadherins bind homophilically forming adjacent cell junctions, and this is important also in signalling
• Cadherin plays a role in contact inhibition of proliferation
• VE-cadherin is important in endothelial cell quiescence and stabilisation

Question 17

What growth factor do pericytes produce that is important for stabilisation of new blood vessels?

Answer 17

Angiopoietin 1

Question 18

Which important signalling pathway modulates the activation and return to quiescence of endothelial cells?

Answer 18

Angiopoietin-Tie2 signalling pathway

Question 19

Describe the actions of angiopoietin 1

Answer 19

• Ang 1 promotes quiescence in the blood vessel
• Promotes stability
• Anti-inflammatory

Question 20

Describe the actions of angiopoietin 2

Answer 20

• Ang 2 is an antagonist against the action of angiopoietin-1
• It gets released when you need to form a new vessel (promotes angiogenesis) and promotes vessel destabilisation
• It is also anti-inflammatory

Question 21

What is the name given to the point at which a tumour begins to initiate angiogenic signals to generate new vasculature?

Answer 21

• Angiogenic switch

Question 22

Describe what is meant by the angiogenic switch and when and why this occurs?

Answer 22

• This is when the tumour can no longer survive on diffusion from host vasculature and begins to become hypoxic and this makes it generate angiogenic signals in order to develop vasculature around it to support its greater metabolic requirements

Question 23

What are some of the issues with tumour blood vessels?

Answer 23

• They are not properly formed because the signals are not physiological
• Vessels can be irregularly shaped, distended, tortuous
• Leaky and haemorrhagic etc - haemorrhage is common in tumours.

Question 24

What is the aim of anti-angiogenic therapy in cancer?

Answer 24

• You want to prevent too much angiogenesis which would supply the tumour but you want to balance this with not reducing drug delivery too much in doing so or causing hypoxia
• Normalise tumour blood vessels to reduce hypoxia
• Improve efficiency of drug delivery

Question 25

What are the consequences of being too aggressive with anti-angiogenic therapy?

Answer 25

* This can make the tumour blood supply inadequate for the delivery of drugs

Question 26

What is avastin?

Answer 26

* Anti-VEGF humanised mouse antibody * Also called bevacizumab

Question 27

What are the side effects of avastin?

Answer 27

* GI perforation * Hypertension * Proteinuria * Venous thrombosis * Haemorrhage

Question 28

What are the two main methods of unconventional resistance to VEGF blockade?

Answer 28

* Tumour adopts evasive strategy – adapts to bypass the angiogenic blockade * Intrinsic or pre-existing difference – a tumour may not have been particularly sensitive to VEGF in the first place

Question 29

What did anti-VEGF therapy start getting used for other than cancer and how is it useful to treat this? Start with the drug name that is used in this

Answer 29

* Lucentis * Age-related macular degeneration (AMD) * In AMD, there is abnormal growth of choroidal vessels which become leaky and thus oedematous, this oedema results in the visual impairment

Question 30

What slightly modified form of avastin became licensed by the FDA to treat AMD?

Answer 30

Lucentis (ranibizumab)

Question 31

Give 3 potential therapeutic uses for pro-angiogenic drugs

Answer 31

1. Ischaemic diseases (e.g. MI, peripheral ischaemic disease) - neovascularisation to reduce ischaemia 2. Vascular graft for coronary bypass surgery 3. Vascular networks for organ regeneration