Angiogenesis

Question 1

When does angiogenesis occur?

Answer 1

It is essential during (early) development and organ growth, but in adults endothelial cell turnover is very low and angiogenesis normally occurs only in very few specific situations, e.g. reproduction and wound healing.

Ectopic angiogenesis is seen in several diseases, in which is it called neovascularisation, and can result in excessive supply of surrounding cells with nutrient/GFs.

Question 2

How do new blood vessels form?

Answer 2

New vessels sprout from existing ones. Each sprout is formed from one or a few endothelial cells.

When stimulated to by assorted signals, the vessels endothelial cells become motile and form a tip cell which produce long extensions called filopodia (cytoplasmic projections that extend beyond the leading edge of lamellipodia in migrating cells.) which guide the development of the new vessel towards the cells emitting the signal.

As the tip cell moves towards its angiogenic stimulus, other endothelial cells behind it form a stalk, these stalk cells start to hollow out and form a tube. When tip cells emerging from different blood vessels meet, they merge and blood can now begin to flow through the new vessel.

As the young vessel matures, the endothelial cells recruit pericytes which help to stabilise vessel structure.

Question 3

What is VEGF?

Answer 3

An angiogenesis inducing mitogen, chemoattractant and survival factor for endothelial cells.

VEGF is upregulated in many tumours and other neovascularising diseases such as AMD.

Question 4

Describe VEGF-axis knockouts.

Answer 4

Loss of one copy of the VEGF gene causes aberrant blood vessel formation and death in embryogenesis.

Disruption of genes for VEGF receptors VEGF-R2 or VEGF-R1 is embryonic lethal.

Question 5

What are VEGFRs?

Answer 5

Various VEGF family (VEGF-A, B etc) bind to different receptors, primarily VEGFR RTKs. There are three main types of VEGFR: VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1) and VEGFR3 (Flt-4).

VEGF-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well defined, although it is thought to modulate VEGFR-2 signaling perhaps through competition for VEGF.

VEGFR-3 mediates lymphangiogenesis in response to VEGF-C and VEGF-D.

Question 6

What receptors recognise VEGF?

Answer 6

Other than VEGFRs, VEGF can be recognised by neuropilis such as NRP1 which colocalise with the VEGFR to potentiate the signal.

VEGF binding to NRP1 and NRP2 has different effects - they have been shown to be more important than previously thought.

Neuropilins also act as neuronal adhesion molecules and form the ligand binding subunit of semaphorin receptors, which are involved in axonal guidance amongst other things.

Question 7

How was VEGF discovered?

Answer 7

• 1983: Senger et al partially purify a protein that induced vascular leakage, called Vascular Permeability Factor (VPF)
• 1989: Vascular Endothelial Growth Factor (VEGF) purified and cloned; secreted peptides

Question 8

What isoforms of VEGF are there?

Answer 8

VEGF A-D and PlGF (placental growth factor).

VEGF-A is the most important for general angiogenesis.

Question 9

What are the six ways by which blood vessels can form?

Answer 9

1. Sprouting angiogenesis
2. Vasculogenesis
3. Intussusception
4. Vessel co-option
5. Vascular mimicry
6. Tumour cell to EC differentiation

The latter three are important in tumour angiogenesis.

Question 10

What is sprouting angiogenesis?

Answer 10

Mechanistically the more important, this was described earlier. This relies on the tip cell forming and making filopodia that respond to VEGF gradients, bringing ECs with it that hollow to form a tube that recruits pericytes.

Question 11

What is vasculogenesis?

Answer 11

Endothelial progenitor cells (EPCs) differentiate directly into ECs to produce new tubes.

Question 12

What is intussusception?

Answer 12

When a blood vessel contains a smaller blood vessel that contains an even smaller blood vessel and then it’s just turtles all the way down.

Also refers to when there are a few endothelial cells that form an inner tube inside a vessel and then grow outwards to split the vessel into a fork.

Question 13

What is vessel co-option?

Answer 13

Vessel co-option (or vascular co-option) is a mechanism in which tumors obtain a blood supply by hijacking the existing vasculature and tumor cells migrate along the vessels of the host organ – this happens a lot in glioma.

Question 14

What is vascular mimicry?

Answer 14

Vasculogenic mimicry (VM) is a mechanism by which highly aggressive tumor cells can form vessel-like structures themselves, by virtue of their high plasticity.

Question 15

What is Tumour cell to EC differentiation?

Answer 15

Fairly self explanatory really. Could have come up with a less obvious name so’s not to ruin my cue card pattern. How thoughtless of them.

Here angiogenesis occurs by transdifferentiation of tumour cells into endothelial cells (Ricci-Vitiani et al, 2010).

Question 16

Okay apparently we’re doing sprouting angiogenesis mechanism again now in more detail, sorry to have wasted your time earlier.

Answer 16

I’m not okay with this.

Question 17

What are the three stages of sprouting angiogenesis?

Answer 17

Selection of tip cells
Stalk elongation and tip guidance
Sprout fusion, maturation, perfusion

Question 18

How are tip cells selected in sprouting angiogenesis?

Answer 18

This requires several local events beyond VEGF signalling. The membrane around the sprouting tip cell needs to be by matrix remodelling by MMPs. Junctions need to be loosened via VE-cadherin reduction. Delta-like ligand (DLL or notch) signalling inhibits tip cell phenotype in neighbouring cells by opposing the VEGFR2 signalling.

The tip cell then grows in the direction of the growth factor, MMP and other enzyme gradient using its filopodia. Pericyte detachment is required for the sprouting growth.

Question 19

How does stalk elongation and tip guidance occur in sprouting angiogenesis?

Answer 19

The MMPs release more GFs from the matrix, and the tip cell continues to migrate down the gradient. Its pathfinding is controlled by VEGFR1, Wnt, FGF, semaphorins and integrins amongst others.

Pericytes and myeloid cells are recruited to the ECs that trail the tip cell, the pericytes offering the srutural stability allowing for lumen formation.

Question 20

What occurs in sprout fusion, maturation and perfusion in sprouting angiogenesis?

Answer 20

Multiple sprouts from different directions fuse to link each sprout to the capillary network.

As they come together many signalling pathways, including notch and PDGF act to produce a mature vessel by rebuilding all the matrix and basement membrane components broken down to allow for its production, and maturation of the pericytes.

Question 21

How can angiogenesis actions in humans be classed?

Answer 21

Physiological (naturally occurring)
•Embryonic development and growth
•Reproduction -pregnancy
•Wound repair

Pathophysiological (involved in disease)
•Cancer
•Ocular neovascularisingdisease, egwet AMD
•Psoriasis
•Cardiovascular disease
•Diabetes

Question 22

What are the growth stages of a tumour?

Answer 22

1. Somatic mutation; ongenes/carcinogens
2. Small avascular tumour; lack of regulation
3. Tumour secretion of angiogenic factors (due to hypoxic stress) stimulates angiogenesis
4. Rapid tumour growth and metastasis

The transition to a vascular tumour is a point at which a cancer goes from bad to OH FUCK because it enables faster growth and metastasis.

Question 23

How does tumour vasculature differ from other vasculature?

Answer 23

Tumour capillaries are 10x more permable due to high levels of VEGF, which increase the EC proliferation rate from 3-13 to 47-2000 per day. Malformed vessels (dead ends, tangled knots, less order, rough and leaky endothelium) mean that tumour remains hypoxic, releasing more VEGF etc.

Damage to vessels here makes them more permeable to the cancer cells, increasing the likelihood of metastasis but and reduces the ability to deliver drugs to the tumour.

Question 24

What is the angiogenic switch?

Answer 24

The decision whether or not to begin angiogenesis, which is controlled by the fine balance of pro- and anti-angiogenic factors.

Both classes are important for vascular homeostasis.

Question 25

List some pro and anti-angiogenic factors

Answer 25

Pro-angio
VEGF, FGF, PDGF

Anti-angio
Angiostatin, Endostatin, Thrombospondin

Question 26

How does hypoxia affect the tumour phenotype?

Answer 26

• Hypoxia induces a metabolic switch from oxidative to glycolytic metabolism – the idea of the Warburg effect
• Hypoxic tumour cells are more resistant to radio- and chemotherapy
• Hypoxia selects for a more malignant phenotype, increases mutation rates, and increases expression of genes associated with angiogenesis (+ VEGF etc secretion) and invasion
• Hypoxia is associated with a more metastatic phenotype

Question 27

What does the hypoxic nature of tumour for allow in terms of treatment?

Answer 27

New hypoxia-selective therapies are being developed e.g. targeting of hypoxia-inducible factor 1 (HIF-1) transcription factor. Still in pre-clinical or early clinical development

Question 28

What is HIF1?

Answer 28

• HIF1-a is the master regulator of the hypoxic response, though there are other factors.
• Under hypoxia it escapes degradation, enabling hetero-dimerisation with HIF1-b (HIF-a’s partner) and subsequent binding to hypoxic response elements within the promotor regions of target genes e.g. VEGF, PDGF
• Positive regulator of tumour survival, progression and metastasis
• Clinical studies demonstrate that HIF-1 is associated with increased patient mortality

Question 29

How is HIF-1 regulated?

Answer 29

When oxygen levels are high the HIFa subunit is hydroxylated and cytoplasmically localised. The hydroxyl group allows fo rht ebinding of the Von Hippel-Lindau ubiquitin ligase, leading to proteasomal degradation.

In hypoxic conditions HIFa is not hydroxylated. In the nucleus it heterodimerises with HIFb and binds to hypoxia response elements to upregulate genes.

Question 30

What genes are upregulated by hypoxia elements?

Answer 30

HELP I’M STUCK IN A CUE CARD FACTORY

48. 0673° N, 12.8633° E
49. 7457° N, 0.3367° W

Question 31

What genes are upregulated by hypoxia elements?

Answer 31

pro-angiogenic factors such as VEGF, PDGF, FGF etc.

Question 32

How is HIF often dysregulated in cancer?

Answer 32

The VHL ubiquitin ligase which degrades HIFa is often mutated to be non-functional as this is a tumour supressor gene. This allows for continuous activation of the hypoxia response and thus increased angiogenesis.

Question 33

What advantages are there to targeting tumour-asociated vasculature rather than the tumour itself?

Answer 33

o Accessibility
o 1 capillary supports many tumour cells
o No drug resistance-more genetically normal
o Applicable to many solid tumours - all require vasculature so broad applications

Question 34

What kinds of treatments can target tumour-associated vasculature?

Answer 34

I) Vascular Disrupting Agents (VDAs)
II) Small molecule inhibitors
III) Antibodies

Question 35

What are vascular disrupting agents?

Answer 35

These are drugs which specifically target the cells comprising the tumour associated vasculature. The majority interfere with tubulin (with the same binding function as colchicine) activity to prevent proper microtubule function as, unlike established vasculature, the tortuous tumour capillaries are dependent upon the cytoskeleton for migration, proliferation, adhesion and cohesion and the structural support of connective tissue, pericytes and smooth muscle.

Thus targeting this makes the drugs, such as Combretastatin specific to tumour vasculature. Other VDAs target colchicine; there are various tactics.

Question 36

What is the disadvantage with VDAs?

Answer 36

They destroy all but the tumour rim, which continues to grow because the vasculature around the tumour rim is made up of more normalised cells.

This becomes a real problem as you get a dramatic response 24 hours later. You can see necrosis and the the loss of vasculature, but some of them remain viable and so can regrow.

However, combination therapy may be a way to overcome this.

Question 37

How can small molecule inhibitors be used to target tumour-associated vasculature? What are the problems with this?

Answer 37

These are used to inhibit the angiogenic pathways, inhibiting their signalling pathways. For examble, ZD6474 is used for NSCLC as a VEGFR2 inhibitor, binding and inhibiting the kinase domain.

However, this does present an issue with specificity due to the similarity between different kinase domains. Delivery and half-life in the plasma can also present issues.

Question 38

How can antibodies be used to target tumour-associated vasculature?

Answer 38

Antibodies such as avastin AKA bevacizumab (the first anti-angiogenic drug licensed by the FDA in 2004) are specific for VEGF, binding it and effectively sequestering it to prevent it from activating angiogenesis.

These are useful in combination with other chemotherapies.

Question 39

What is the disadvantage with using antibodies to target tumour-associated vasculature?

Answer 39

This is less specific to tumour-associated vasculature, so also reduces VEGF binding in other vasculature - a healthy component of endothelial stability stimulating NO and prostaglandin production.

Bevacizumab was withdarawn from use in breast cancer due to severe cardiovascular toxicity - increasing hypertension and thrombosis.

Too high a dose can lead to a reduction in the ability to deliver the chemotherapy used in combination with it.

Question 40

What have mouse models shown about avastin treatment?

Answer 40

It actually increased metastatic tumour growth. It is thought that the tumours develop resistance to the drug by inducing other pro-angiogenic factors, such as PDGF and FGF instead of VEGF allowing the vasculature to regrow.

By targeting all three simulataneously we may be able to produce the same anti-vasculature effect without the harsh side effects of VEGF targeting.