3.4. Angiogenesis

Question 1

what is the aim of understanding angiogenesis?

Answer 1

• To know more about how cancer develops so that it can be prevented;
• To produce drugs which can be used to inhibit angiogenesis in cancer as well as in other pathological processes.

Question 2

Angiogenesis in adults

Answer 2

Pathological:1.wound healing

2.hypoxia/ischaemia

Question 3

what does VEGF stand for?

Answer 3

Vascular Endothelial Growth Factor

Question 4

where is Tie2 located?

Answer 4

it sits on the basal surface of the endothelial cell

Question 5

angiopoietin 1/Tie2

Answer 5

pericyte –> angiopoietin –> binds & joins Tie2 –> this binding results in endothelial cell in quoiescent state

Question 6

was it the function of angiopoietin 2?

Answer 6

under the influence of VEGF –> angiopoietin 2 is released from the endothelial cell –> inhibits angiopoeitin 1 from binding to Tie2 ==> changes in endothelial cell
* angiopoeitin 2 inhibits association of ANG1/Tie2 ==> cell is no longer quoiescent –> allows endothelial cell to become a tip cell –> moves away from capillary

Question 7

outline the process of angiogenesis

Answer 7

1. VEGF (Vascular Endothelial Growth Factor) is produced by cells under the influence of hypoxia or inflammatory mediators such as Fibroblast Growth Factor (FGF) and TGFβ. It is also stimulated by proliferative and anti-apoptotic signalling even in the presence of normal oxygen levels. This causes a concentration gradient of VEGF around nearby blood vessels.
2. The capillary which is going to start sprouting under the influence of this VEGF first has to become destabilised – the endothelial cells have to enter G1 and the surrounding pericytes have to lift off and move away. This occurs because of the release of Angiopoietin 2 from the endothelial cell which binds to a receptor called Tie 2. This antagonises the association of Angiopoietin 1 on pericytes with Tie 2 on the endothelial cells, which is responsible for keeping the pericytes in close association with the endothelial cells.
3. At the same time, VEGF makes the vessel far more permeable and the endothelium produces matrix metalloproteases which leak out into the perivascular extracellular matrix. These help to break down the basement membrane and ECM so that migration of the cells can take place. There is breakdown of the specific cadherin-catenin complexes making up the adherens junctions between the cells so that the cells can migrate independently.
4. One of the endothelial cells develops into a Tip cell. This cell has 2 special features.
   a) It has a very specific integrin (αvβ3) in its membrane which allows it to hook into the ECM so that it can migrate.
   b) This integrin also localises MMP-2, matrix metalloproteinase, so that the advancing tip can break down parts of the ECM to make migration possible.
5. The endothelial cells proliferate to form the stalk behind the tip cell under the influence of VEGF.
6. The endothelial cells of the stalk then form tubes instead of solid cords of cells and blood starts to flow into them.
7. When the tip cell makes contact with another sprout or capillary, it fuses and this causes signals which switch off proliferation. Also, there must be very specific recognition of sprouts coming from the arterial side and those from the venous side to make a functional loop. This is achieved by expression of a specific receptor- ligand pair – the arterial side expresses ephrinB2 and the venous side expresses the receptor EphB4.
8. At the same time there is proliferation of mesenchymal cells under the influence of PDGF (Platelet-derived growth factor) and these cells migrate along the vessel. These cells then differentiate into pericytes. Contact with pericytes switches off the production of metalloproteinases and allows the basement membrane and other ECM to be synthesised.
9. Once the blood flows into this area there is no further hypoxia and the production of VEGF ceases (in normal tissue). This allows the new vasculature to mature. Stabilisation of the vessels with basement membrane and recruitment of smooth muscle etc. is dependent on the association of Angiopoietin 1 from the pericyte and Tie2 on the endothelial cell.
10. Further refinement depends on flow through the vessels – superfluous vessels are “pruned” away depending on the oxygen concentration and the mechanical forces of the flowing blood lead to the alignment of the vessel within the tissue. Anti-angiogenic factors such as thrombospondin cause apoptosis of new and proliferating endothelial cells rather than the stable ones present in mature vessels.

Question 8

what controls VEGF production?

Answer 8

a transcription factor called HIF(hypoxia inducible factor)

Question 9

describe the relationship between VEGF & HIF

Answer 9

(refer to pg 16 of notes)

1. all cells produce HIF alpha
2. HIF alpha must bind to HIF beta to make a transcription factor
3. HIF alpha becomes hydroxylated in normal O2 because we don’t want VEGF because blood supply is enough

Question 10

what are alternative stabilisers of HIF-alpha?

Answer 10

• HIF-alpha is stabilised (i.e. prevented from being recognised by E3 ligase) by proliferative signalling and oncogene activation
  2. AKt & MAP kinase signalling can also stabilise HIF-alpha
• -> prevent E3 ligase targeting, ubiquitination & proteosome degradation –> drives blood vessel formation in cancer

Question 11

what are other factors capable of inducing angiogenesis?

Answer 11

1. other cytokines

2. growth factors such as TNF-α, TGF-βand PDGF

Question 12

what are natural inhibitors of angiogenesis?

Answer 12

1. thrombospondin
2. angiostatin
3. endostatin
4. tumstatin
5. TIMP
6. IL-4
7. interferons

Question 13

discuss the angiogenic switch

Answer 13

Hypothesis:
•Early cancers may produce VEGF but don’t show much angiogenesis
•The VEGF is initially bound by ECM surrounding the cells
•Inflammatory cells –mast cells and macrophages –recruited by the tumour produce MMP-9 which releases VEGF and also produce VEGF themselves
•Angiogenesis then proceeds rapidly
-Probably a number of other contributory factors e.g. Oncogene suppression of Thrombospondin

Question 14

Macroscopic Tumour vessel abnormalities:

Answer 14

Abnormalities in architecture and vessel density
•Overall the architecture is chaotic
•Variations in vessel diameter
•Tortuous, elongated vessels
•Collections of multiple small vessels and other variations in density
•Uneven blood flow within vessels, AV shunts

Question 15

microscopic tumour vessel abnormalities

Answer 15

• Decreased pericytes
• Poorly formed basement membrane
• Incomplete endothelial lining
• Incorporation of tumour cells in vessel wall
• Microvascular proliferation

Question 16

VEGF in cancer angiogenesis

Answer 16

Increased and continuous levels cause:
•Endothelial cell proliferation
•Leaky vessels because of effect on permeability via adherens junctions
•Failure of pericytes to adhere to endothelial cells
•Failure of appropriate vessel maturation
*Excessive in amount(both from cancer cells and macrophages and fibroblasts) and incorrectly distributedcausing abnormal concentration gradients
*Continuously produced and not switched off because of inflammation, tissue damage and oncogene-driven pathways

Question 17

function of Ang2 in cancer angiogenesis

Answer 17

Produced by EC in tumours destabilises the vessels

Question 18

what are the Consequences of abnormal tumour vessels

Answer 18

• Leakiness leads to increased interstitial pressure –effects on drug delivery, compression of vessels, concentration of blood and sluggish flow etc.
• Facilitates tumour cell entry into vessels
• Areas of hypoxia and ischaemia–selects for cells resistant to apoptosis, also for cells which can survive in a new hypoxic environment.
• Radiation and some chemotherapy depend on the presence of oxygen