7. Angiogenesis

Question 1

Define the term angiogenesis.

Answer 1

Angiogenesis is the formation of a new blood vessel from pre-existing blood vessels

Growth factors organized in a gradient on the wall

Blood vessels are the first organ systems that form in the embryo -­‐ without blood vessels, nothing else can form

• Physiological angiogenesis can occur in – embryonic development, wound healing and in the menstrual cycle.

Angiogenesis can be:

• Insufficient – e.g. baldness, MI.
• Involved in vascular malformations – e.g. Angiodysplasia (HHT & VWD).
• Excessive – e.g. Retinal disease, cancers, atherosclerosis.

Angiogenesis (describes “sprouting”) is just ONE method of creation of new blood vessels.

Others include VASCULOGENESIS (using bone marrow progenitor cells for development) and ARTERIOGENESIS (collateral growths of vessels to accommodate occlusions etc, is dependent on shear stress and external factors like macrophages.).

Question 2

Explain the basic mechanisms regulating angiogenesis.

Answer 2

Model of Sprouting Angiogenesis

To begin with, there is a need for new blood vessels (usually the result of hypoxia)

1. One cell gets hit by growth factor - Selection of sprouting ECs.
2. Sprout outgrowth and guidance.
3. Sprout fusion and lumen formation.
4. Perfusion and maturation.

To begin with, there is a need for new blood vessels (usually the result of hypoxia)

• Growth factors are released that activate endothelial cells in the pre-­‐existing capillaries (this happens in small vessels)
• The endothelial cells undergo a conformational change where they go from being part of a very organised monolayer, to sending out filopodia and begin to migrate towards the growth factors
• To allow the endothelial cell to do this, the cytoskeleton of the tip cell must be modified and it needs to control the interaction with neighbouring cells at cell-­‐ cell junctions
• The tip cells will keep on moving until they find another tip cell, with which they will fuse
• The tip cells themselves do not divide, they require their neighbouring cells to divide behind them to push the tip cells towards the growth factor
• Eventually, the tip cell will meet another tip cell and it will fuse and stabilise

NOTE: we know so much about angiogenesis by looking at angiogenesis in zebrafish embryos

Question 3

Summarise the main cellular and molecular pathways involved.

Answer 3

Hypoxia:

• Hypoxia is a common trigger for angiogenesis.

1. HIF – Hypoxia-Inducible Transcription Factor – controls gene regulation.
2. pVHL – protein Von Hippel-Lindau tumour suppressor gene – controls levels of HIF (at normal conditions)

Absence of oxygen:

• pVHL does NOT bind to HIF and HIF translocates to the nucleus and binds to HIF-region and induces translation of hypoxic factors- drive the expression of genes involved in angiogenesis

One of the targets of HIF is the expression of VEGF (vascular endothelial growth factor)

Presence of oxygen:

• pVHL adds a hydroxyproline group to HIF and HIF is degraded by a proteasome.

VEGF:

VEGF is, by far, the best known pro-­‐angiogenic growth factor

There are 5 members of the VEGF family:

• VEGF-­‐A
• VEGF-­‐B
• VEGF-­‐C
• VEGF-­‐D
• PIGF (placental growth factor)

There are 3 tyrosine kinase receptors for VEGF:

• VEGFR-­‐1
• VEGFR-­‐2
• VEGFR-­‐3

VEGFRs can dimerise with other forms of VEGFRs.

I.E. VEGFR-2 -> VEGFR-3.

There are 2 coreceptors for VEGF:

• Neuropilin-­‐1 (Nrp1)
• Neuropilin-­‐2 (Nrp2)

VEGFR-­‐2 is the major mediator of VEGF-­‐dependent angiogenesis -­‐ it activates signalling pathways that regulate endothelial cell migration, survival and proliferation

Question 4

Outline the steps of the angiogenetic process

Tip Cells selection & Canonical Notch Signalling:

Answer 4

(1) Tip Cells & Canonical Notch Signalling:

• In sprouting angiogenesis, specialised endothelial tip cells lead the outgrowth of blood-­‐vessel sprouts towards gradients of VEGF
• Once a tip cell has been selected, it seems to control the behaviour of the cells around it via cell-­‐cell communication
• There is a pathway called Notch that is crucial for the selection of tip cells

Canonical Notch Signalling:

1. Binding of the notch ligand to the notch receptor activates the receptor by cleaving the intracellular domain (NICD)
2. NICD then translocates to the nucleus where it binds to the transcription factor RBP-­‐J and regulates transcription
3. When a tip cell is chosen, it begins to express notch ligand which binds to the stalk cells’ notch receptors and tells them that ‘I am the tip cell, you are the stalk cells’
4. The stalk cells then begin to divide and push the tip cell towards the growth factor

NOTE: the notch ligand is also called Delta-­‐like ligand 4 (Dll4)

Selection of Tip cells:

1. Stable blood vessels – DII4 and Notch signalling maintain quiescence.
2. Unstable – VEGF activation increases expression of DII4.
3. DII4 drives Notch signalling in the adjacent cell which inhibits expression of VEGFR-2 (to prevent it from becoming tip)
4. DII4-expressing Tip cells acquire a motile, invasive and sprouting phenotype.
5. Stalk cells (adjacent cells) form the base of the sprout and proliferate to support the sprout elongation.

Question 5

(2) Outline sprout Outgrowth & Guidance:

Answer 5

(2) Sprout Outgrowth & Guidance:

Once the tip cell and stalk cells have been identified, the sprout needs to progress forwards

• Myeloid cells are recruited to support and guide the sprout (stimulated by Ang-II).
• The cells will interact with the ECM and there will be guidance systems in place

Macrophages also have an important role in vessel anastomosis (both physiological and pathological)

1. Macrophages have been shown to carve out tunnels in the ECM, thereby providing avenues for subsequent capillary infiltration
2. Tissue-­‐resident macrophages were shown to be associated with angiogenic tip cells during anastomosis

• So macrophages appear to help stabilise newly formed vessels (by promoting tip cell fusion)

Question 6

(3) Outline Stabilisation & Quiescence

Answer 6

Stabilisation & Quiescence:

• Once the tip cells have fused and the stalk cells are separating for form a patent tube, the new vessel needs to stabilise
• Stabilisation involves:

1. reforming the endothelial monolayer barrier
2. recruiting neural cells (pericytes)
3. switching off the active angiogenesis process

Mural Cells = generally refers to smooth muscle cells and pericytes

• Pericytes are important in the stabilisation of new blood vessels because they produce proteins such as Angiopoietin 1, that goes on to control junctional systems e.g. Notch system
• The angiopoietin/Tie-­‐2 system is specific to the endothelium

The angiopoietin-­‐Tie2 system is required to modulate the activation and return to quiescence of endothelial cells

• Tie2 is a receptor that can bind to Angiopoietin 1
• Angiopoietin 1, when it binds to Tie2, promotes quiescence in the vasculature
• Angiopoietin 2 is the one that gets released when you need to form new blood vessels or when you need to respond to inflammation or when the vasculature needs to be destabilised
• So Ang-­‐2 antagonises Ang-­‐1 signalling and has pro-­‐angiogenic effects

Question 7

Explain the role of angiogenesis in cancer.

Answer 7

• Tumours less than 1mm3 receive oxygen and nutrients by diffusion from the host.
• Tumours greater than 1mm3 require the “Angiogenic switch” so that the tumour can continue to grow. This is done once the tumour starts to secrete angiogenic factors.
• The “Switch” occurs discretely and at different steps in the tumour-progression pathway.

Tumour Blood Vessels

These are NOT properly formed because the signals are not physiological -­‐ there is an imbalance in the signals that are regulating angiogenesis

Tumour blood vessels display characteristics:

1. Irregular shape.
2. Dilated, torturous.
3. Not organised into arteries/veins/capillaries.
4. Leaky n haemorrhagic (overproduction of VEGF).
5. Perivascular cells loosely associated.
6. May recruit endothelial progenitor
7. cells from bone marrow.

Question 8

Targeting the VEGF pathway

Answer 8

• Anti-­‐angiogenic therapy can help normalise the tumour blood vessels
• However, if you go for very aggressive anti-­‐angiogenic therapy, you could end up damaging the ability to deliver other drugs to the tumour
• The aim now is to normalise tumour blood vessels to reduce hypoxia and improve the efficiency of drug delivery

Avastin/ Bevacizumab

• Used in combination with other drugs to treat cancer: Cervical, colorectal, glioblastoma, NSCLC, ovarian.

Avastin has relatively limited efficacy and it has many side effects:

1. GI perforation
2. Hypertension
3. Proteinuria
4. Venous thrombosis
5. Haemorrhage
6. Wound healing complications

• There is no overall survival advantage over chemotherapy alone
• No quality-­‐of-­‐life or survival advantage
• These side effects are because VEGF is essential for the homeostasis of the endothelium

Mechanisms of Resistance to Anti-VEGF.

1. VEGF inhibition leads to MORE hypoxia so more release of OTHER angiogenic factors or increases tumour invasiveness.
2. Tumour vessels may be less sensitive to VEGF inhibition due to tumour cells lining the vessels.
3. Tumour cells that recruit pericytes may be less responsive.
4. Tumour cell vasculogenic mimicry (VM) – the tumour cells remodel/organise themselves to resemble vessels which then once perfused by a single vessel allow adequate nutrient delivery to the whole tumour.

• Anti-angiogenic therapy then will not affect this VM.

Anti-angiogenic therapy sometimes facilitates effects that seem pro-tumour growth.

• Sustained anti-angiogenic therapy can lead to too much ischaemia and thus release of hypoxic factors that further induce angiogenesis.
• Vasculature may become refractory to treatment.
• Vasculature may be inadequate for further delivery of drugs/oxygen.

Aiming for a NORMALISED vasculature of the tumour:

• Reduces hypoxia so less hypoxic factors.

Question 9

Summarise the prospects for anti-angiogenic therapies

Answer 9

Anti-Angiogenic Therapy – AMD:

AMD – Age-related Macular Degeneration.

Abnormal growth of choroidal blood vessels à leaky vessels causing oedema à visual impairment.

AMD is the main cause of blindness.

Avastin (anti-VEGF AB) is an off-licence drug that can be used to combat AMD.

• Many patients become refractory to treatment >2 years.

Development of Anti-Angiogenic Drugs

• Problem – tumours are complex 3D structures that function in association with host vasculature and so is difficult to mimic on a 2D-cell line monolayer when creating anti-angiogenic drugs.
• “Tumour-on-a-chip” platform:
  
  • Small system that incorporates human cells on a 3D ECM supported by perfused human micro-vessels.