Chapter 10: Angiogenesis (Main)

Question 1

What is the definition of angiogenesis

Answer 1

Formation of capillary vessels from pre-existing blood vessels

Question 2

Which cells form the inner lining of all blood vessels?

Answer 2

Endothelial cells, they are the key players in angiogenesis

Question 3

Angiogenesis is a multistep endothelial cell process. What are the different steps (5)?

Answer 3

1. Quiescent/resting stage
2. Vessel dilation and pericyte detachment (= unstable blood vessel)
3. Degradation of matrix and basement membrane
4. Cellular migration and proliferation
5. Lumen formation, basement membrane formation and pericyte attachment. Fusion of blood vessel sprouts and further vessel maturation (=functional blood vessel)

Red = endothelial cell

Purple = basal membrane

Green = supporting cells / parasites

Question 4

What are the different reasons why we would need angiogenesis? (and also label these ‘good’ and ‘bad’)

Answer 4

• “Good” reasons:
  
  • Pregnancy, embryogenesis
  • Wound healing
  • Inflammation
• “Bad” reasons:
  
  • Cardiovascular disorders
  • Inflammatory diseases
  • Cancer

Question 5

Why is angiogenesis so important in cancer?

Answer 5

Because tumors (primary and metastases) require blood vessels to grow beyond 2-3mm^2 in size

Question 6

What is dormancy?

Answer 6

Dormancy is a stage in cancer progression where the cells cease dividing but survive in a quiescent state while waiting for appropriate environmental conditions to begin proliferation again.

Question 7

How is the balance between activators and inhibitors that dictate the angiogenic activity called?

Answer 7

The angiogenic balance/switch

Question 8

What is the most important angiogenesis activator?

Answer 8

Vascular Endothelial Growth Factor A (VEGF-A)

Question 9

Fill in: VEGF-A is frequently highly/poorly expressed by tumor cells

Answer 9

VEGF-A is frequently highly expressed by tumor cells

Question 10

Which combination of VEGFR will lead to vasculogenesis and angiogenesis?

Answer 10

VEGFR-2 (with VEGF-A)

Question 11

Which two types of signaling are activated upon activation of the VEGFR?

Answer 11

The AKT signaling and the RAS-RAF signaling

Question 12

What do the AKT and RAS-RAF signaling induce?

Answer 12

The AKT-signaling pathway inhibits apoptosis and activates vascular permeability. The RAS-RAF-signaling pathway activates migration and proliferation

Question 13

What three types of phenotypes can an endothelial cell take on due induction of VEGF?

Answer 13

The phalanx cells, tip cells and stalk cells

Question 14

What are the phenotypes/functions of the phalanx cells, tip cells and stalk cells?

Answer 14

• Phalanx cells: the resting/quiescent endothelical cell.
• Tip cells: the migratory endothelial cell at the forefront of the sprout.
• Stalk cells: the proliferative endothelial cells just behind the tip cell.

Question 15

Which pathway regulates the tip/stalk phenotype?

Answer 15

VEGF/DLL4/Notch signaling

Question 16

Explain the process of VEGF/DLL4/Notch signaling

Answer 16

Cells with high VEGFR-2 will turn into tip cells and increase expression of secretion of DLL4. The underlying cells have more Notch and more sensitive to DLL4. Activation of Notch will lead to activation of proliferation thereby the stalk cell. At the same time stalk cells will express more VEGFR-1 which makes them less sensitive to VEGF thereby preventing these cells to switch to a tip cell phenotype

Question 17

What drives VEGF expression?

Answer 17

Hypoxia (lack of oxygen). This can be due to a too high consumption or too low delivery of oxygen

Question 18

Which transcription factor is activated when there is hypoxia?

Answer 18

Hypoxia inducible factor-1 (HIF-1)

Note: this TF activates 100 genes, of which VEGF

Question 19

What is the opposite term (antonym) of hypoxia?

Answer 19

Normoxia (=normal levels of oxygen)

Question 20

Explain how in normoxic conditions, angiogenesis is inhibited

Answer 20

In presence of oxygen, propyl 4-hydroxylase will hydrolyse the HIF-1-alfa. This hydroxylated state can recruit VHL (amongst other proteins) which will ubiquitinate HIF-1-alfa. HIF-1-alfa will then be degraded, so no transcription of angiogenesis is induced.

Question 21

Explain how in hypoxic conditions, angiogenesis is induced

Answer 21

There are low levels of oxygen -> HIF-1-alfa is not hydroxylated -> no ubiquitination of HIF-1-alfa -> transcription of angiogenesis is induced

Question 22

Normally, if angiogenesis has occurred, there is more oxygen and thus a feedback loop exists. This is important so that there is not continuous angiogenesis. A tumor will evade this process. How?

Answer 22

Because of the ‘architecture’ and characteristics of the blood vessel network. It is immature, leaky, tortuous and has irregular flow. This creates continuous hypoxia, leading to distinct molecular characteristics

Question 23

There are five different therapeutic strategies for angiogenesis, but we will only be discussing cell activation and vessel targeting. What are the three therapeutics for cell activation?

(for those interested, the other three are ECM degradation, cell migration and cell proliferation)

Answer 23

VEGF scavengers, VEGF receptor antagonists and VEGF receptor blockers

Question 24

What do the VEGF scavengers, VEGF receptor antagonists and VEGF receptor blockers do?

Answer 24

• VEGF scavengers – so that it can no longer bind to the receptor
• VEGF receptor antagonist – block the VEGF binding site on the receptors
• VEGF receptor blockers – Tyrosin kinase inhibitors (TKIs); molecules that prevent activation of the kinase domain intracellular

Question 25

There are five different therapeutic strategies for angiogenesis, but we will only be discussing cell activation and vessel targeting. How does vessel targeting work?

Answer 25

Because tumor vessels are distinct from normal vessels, and have specific 'tumor endothelial cell markers', which can be targeted to destroy the existing tumor vasculature.

Question 26

Unfortunately, the benefit of the drugs are limited because resistance often occurs in the treated patients. Which two mechanisms are in play for this resistance?

Answer 26

1. Recruitment of bone-marrow derived cells 2. Vessel cooption and vasculogenic mimicry Definitions: **Vessel co-option**: Vessel co-option is a non-angiogenic process through which tumour cells utilize pre-existing tissue blood vessels to support tumour growth, survival and metastasis. **Vasculogenic mimcry**: Vasculogenic mimicry is the formation of microvascular channels by aggressive, metastatic and genetically deregulated tumour cells. This process differs from angiogenesis in that it occurs de novo without the presence of endothelial cells (tumour cells line tumour vessels effectively mimicking a true vascular endothelium) *(other factors that are in play here are growth factors redundancy, vessel stabilization by local stromal cells and increased invasiveness and metastasis, but you don't have to know that for the exam)*

Question 27

How is the recruitment of bone-marrow derived cells achieved (a form of **resistance** of angiostatic drugs)

Answer 27

**Recruitment of bone-marrow derived cells** * _Myeloid derived suppressor cells._ * Express angiogenic factors (that are not VEGF) * Induce angiogenic phenotype in immune cells * _Endothelial progenitor cells._ * Vasculogenesis

Question 28

How is the vessel cooption and vasculogenic mimicry achieved (a form of **insensitivity**/less sensitive of angiostatic drugs) **Vessel co-option**: Vessel co-option is a non-angiogenic process through which tumour cells utilize pre-existing tissue blood vessels to support tumour growth, survival and metastasis. **Vasculogenic mimcry**: Vasculogenic mimicry is the formation of microvascular channels by aggressive, metastatic and genetically deregulated tumour cells. This process differs from angiogenesis in that it occurs de novo without the presence of endothelial cells (tumour cells line tumour vessels effectively mimicking a true vascular endothelium)

Answer 28

_Vessel cooption_ * Migration and growth of tumor cells along pre-existing vasculature. _Vasculogenic mimicry_ * Tumor cell adaptation towards endothelial phenotype which allows formation of tumor cell lined vessels

Question 29

The total network of blood vessels is immense (about 84,000 km). By using **blood vessels markers**, tumor blood vessels can be found because these markers are different from 'regular' blood vessels. What are the _two_ blood vessel markers that are used regularly?

Answer 29

There are about 7 different kinds, of which the two you have to know are **Vimentin** and **Galectin-1**

Question 30

True/false: During angiogenesis, endothelial cells proliferate and migrate in the direction of an angiostimulatory signal.

Answer 30

True

Question 31

Put in the correct order: A. HIF1a is degraded. B. HIF1a is ubiquitinated. C. HIF1a is hydroxylated.

Answer 31

C-B-A HIF1a is hydroxylated -\> HIF1a is ubiquitinated -\> HIF1a is degraded.