Angiogenesis

Question 1

what are the physiological reasons for angiogenesis?

Answer 1

embryonic development
wound healing
menstrual cycle

Question 2

how can angiogenesis turn out to be in pathology?

Answer 2

o Insufficient – e.g. baldness, MI.

o Involved in vascular malformations – e.g. Angiodysplasia (HHT & VWD).

o Excessive – e.g. Retinal disease, cancers, atherosclerosis.

Question 3

what are the methods of creating new blood vessels and describe their action?

Answer 3

• vasculogenesis= use of bone marrow progenitor cells
• arteriogenesis= collateral growths to accommodate occlusions
• angiogenesis= sprouting dependent on endothelial cells

Question 4

what is the outline of sprouting angiogenesis?

Answer 4

1. Selection of sprouting ECs (tip cells and stalk cells)
2. Sprout outgrowth and guidance.
3. Sprout fusion and lumen formation.
4. Perfusion and maturation.

Question 5

name the initial trigger of angiogenesis

Answer 5

hypoxia

Question 6

name the regulator of gene expression to promote angiogenesis

Answer 6

Hypoxia-Inducible Transcription Factor (HIF)

leads to the expression of Hypoxia inducible Genes e.g. VEGF

Question 7

what controls the levels of HIF?

Answer 7

protein Von Hippel-Lindau

- tumour suppressor gene (pVHL)

Question 8

what happens with pVHL and HIF in the absence of oxygen

Answer 8

1) in hypoxia pVHL does not bind to HIF
2) HIF alpha and HIF beta associated to create a transcription factor
3) allows HIF TF to translocate to the nucleus and bind to the HIF-region

this induces the translation of hypoxic factors e.g. VEGF that enable angiogenesis

Question 9

what happens with pVHL and HIF in the presence of oxygen?

Answer 9

hypoxic trigger removed

1) so pVHL adds a hydroxyproline group to HIF (ubiquination)
2) HIF is degraded by a proteasome

no angiogenesis
pVHL is anti-angiogenesis

Question 10

what does VEGF stand for?

Answer 10

vascular endothelial growth factor

Question 11

what are the 5 members of the VEGF family?

Answer 11

VEGF-A/B/C/D and Placental GF (PlGF)

Question 12

what are the 3 receptors VEGF can bind to? what are the co-receptors they need?

Answer 12

VEGFR-1/2/3

these require the co-receptors Nrp1 and 2

Question 13

which of the VEGF receptor is the main mediator of VEGF-dependant angiogenesis?

Answer 13

VEGFR-2

is the major mediator of VEGF-dependant angiogenesis.

Question 14

what is the first step of angiogenesis?

Answer 14

the selection of the endothelial tip cell occurs which lead the outgrowth of vessels towards gradients of VEGF

Question 15

how is the tip cell selected?

Answer 15

the cell that receives the highest concentration of VEGF becomes the tip cell

by Canonical Notch Signalling between adjacent endothelial cells

Question 16

which cell would have the notch receptor?

Answer 16

the stalk cell (support cell)

adjacent to the tip cell with the delta/jagged ligand

Question 17

what happens to the notch receptor once it binds to the jagged ligand of the tip cell?

Answer 17

the intracellular domain of Notch called NICD translocates to the nucleus and binds to the TF RBP-J

Question 18

how is quiescence in the endothelial layer maintained when tip selection occurs?

Answer 18

stable state: the cells signal with DII4 and Notch to each other when VEGF comes to bind

Question 19

when does the induction sprouting occur?

Answer 19

in unstable state: VEGF activation increases the expression of DII4 (on tip cell)

this drives Notch signalling in the adjacent cell

Question 20

when the tip cell has been selected and starts to produce more DII4 (notch ligand) , what effect does this have?

Answer 20

this drives Notch signalling in the adjacent cell so it will reduce the expression of VEGFR-2 on the adjacent cell so VEGF can’t bind to it

the adjacent cells has now been given its role of stalk cell

Question 21

what is the role of the stalk cells?

Answer 21

while the tip cell is motile, invasive and sprouting the stalk cells adjacent form the base of the sprout and proliferate to support sprout elongation

Question 22

what cells are recruited to guide and support the growing sprout?

Answer 22

myeloid cells (stimulated by angiopoietin 2)

Question 23

which cells play a role in angiogenesis anastomosis? what do they do?

Answer 23

macrophages

carve out tunnels in the ECM for subsequent capillary infiltration and support the structure during anastomosis

Question 24

what facilities the stabilisation of the new vessels?

Answer 24

• a barrier formed made with tight junctions and adherins

- pericytes

Question 25

what molecules are associated with the barrier formation during stabilisation of the vessels?

Answer 25

VE-Cadherins and Angiopoietin 1

Question 26

what receptor does the angiopoietin-1 bind to?

Answer 26

TIE-2 receptors on the endothelial cells

Question 27

what are the effects of Angiopoietin 1 binding to TIE-2?

Answer 27

- stabilises the vessel - inhibits inflammatory gene expression no more angiogenesis required so reach stable state

Question 28

what are the roles of VE-cadherins?

Answer 28

o Constitutively expressed at junctions. o Controls contact inhibition of cell growth. o Promotes the survival of the EC.

Question 29

what cells allow the stabilisation of the vessels?

Answer 29

mural cells pericytes+ smooth muscles cells = mural cell using the Angiopoietin/TIE system

Question 30

what are the actions of Angiopoietin 2 on Tie-2?

Answer 30

they antagonise the receptor

Question 31

what are the effects of Angiopoietin-2 binding to Tie-2?

Answer 31

antagonises Angiopoietin-1 signalling therefore leads to vascular instability and VEGF-dependant angiogenesis

Question 32

in what pathologies is Angiopoietin-2 levels increased?

Answer 32

sepsis CKD congestive heart failure high levels destabilises the vascular system

Question 33

what size tumours are not dependent on angiogenesis for survival?

Answer 33

tumours less than 1mm^3 receive oxygen and nutrients by diffusion from the host

Question 34

what is required by tumours greater than 1 mm^3?

Answer 34

“Angiogenic switch” diffusion is insufficient for the tumor, so the tumour starts to produce angiogenic factors like VEGF so it can continue to grow

Question 35

what does the tumour do when it need vessels?

Answer 35

secretes angiogenic factors (e.g. VEGF, Angiopoietin 2) the "angiogenic switch" is driven by hypoxia

Question 36

what are the characteristics of new vasculature associated with a tumour?

Answer 36

- Irregular shape - Dilated, torturous. - Not organised into arteries/veins/capillaries - Leaky (overproduction of VEGF) - Perivascular cells loosely associated - May recruit endothelial progenitor cells from bone marrow.

Question 37

what are the 4 methods that can be used to target the VEGF pathway?

Answer 37

 Anti-VEGF antibodies – Avastin/Bevacizumab.  Soluble VEGF Receptors – VEGF-Trap.  Anti-VEGFR antibodies – IMC-1121b.  Small-molecule VEGFR inhibitors – Vatalanib, Sunitinib.

Question 38

what is avastatin? | what types of cancers is Avastin (with other drugs) used for?

Answer 38

anti VEGF antibodies ``` colorectal Cervical glioblastoma NSCLC ovarian ```

Question 39

what are the disadvantages of Avastin?

Answer 39

1) has many side effects: - GI perforation - hypertension - proteinuria - VTE - haemorrhage - wound healing complications. 2) a limited efficacy: No quality of life or survival advantage over chemo alone.

Question 40

how can sustained anti-angiogenic therapy actually have pro-tumour effect?

Answer 40

too much ischaemia leads to the release of hypoxic factors that further induce angiogenesis vasculature may be inadequate for further delivery of drugs/oxygen

Question 41

what is the aim for the vascular health in tumours?

Answer 41

normalised vasculature - reduce hypoxia to reduce hypoxic factors - increase efficacy of conventional therapies

Question 42

what are the mechanisms of resistance to anti-VEGF? [4]

Answer 42

1. VEGF inhibition -->more hypoxia--> 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)- de novo vessel synthesis independent of endothelial vessels to start

Question 43

what happens in tumour cell vasculogenic mimicry? what impact does this have on treatment?

Answer 43

the tumour cells remodel themselves to resemble vessels which then once perfused by a single vessel allow adequate nutrient delivery to the whole tumour. Anti-angiogenic therapy is useless against vasculogenic mimicry

Question 44

what happens in age-related macular degeneration (AMD) that leads to visual impairment?

Answer 44

Abnormal growth of choroidal blood vessels leads to leaky vessels causing oedema

Question 45

what can be used to treat AMD?

Answer 45

AMD is the main cause of blindness | can be treated by off-licence Avastin but can people become refractory to it

Question 46

what is the problem with creating anti-angiogenic drugs and what is a solution?

Answer 46

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 Solution: "Tumour-on-a-chip” platform: a small system that incorporates human cells on a 3D ECM supported by perfused human micro-vessels

Question 47

what will the selected tip cell now express? what is the purpose of it?

Answer 47

Delta Like Ligand 4 this will bind to the notch receptor on the adjacent cell to give it its stalk cell role

Question 48

what is the role of angiopoietin 1?

Answer 48

bind to Tie-2 to promote vessel stability and inhibit inflammatory gene expression

Question 49

what is the role of angiopoietin 2?

Answer 49

antagonise the action of Angipoietin 1 by binding to Tie 2 to promote vessel instability and therefore the need for VEGF dependent angiogenesis

Question 50

when is Angiopoietin 2 released?

Answer 50

- when new vessel formation is required - in response to inflammation - to destabilise vessels