Angiogenesis Flashcards
Define Angiogenesis, state the different types and the main methods.
Angiogenesis
- Angiogenesis is the formation of a new blood vessel from pre-existing blood vessels
- Angiogenesis can be:
- Insufficient – e.g. baldness, MI.
- Involved in vascular malformations – e.g. Angiodysplasia (HHT & VWD).
- Excessive – e.g. Retinal disease, cancers, atherosclerosis.
Vasculogenesis: During development, the contribution of bone marrow progenitor cells is very important in the early stages of blood vessel formation
Arteriogenesis - collateral growth that is dependent on shear stress and external factors like macrophages
- It is a cascade of events, start at the endothelial cells, Vascular Endothelia Growth Factor (released and binds to receptors) growth factors are organised in gradients to help the movement
What are some factors which activate and inhibit Angiogenesis?
When you form a new blood vessel, you need to destabilise the pre-existing blood vessel and then re-stabilize it
- There are activators and inhibitors of angiogenesis - a balance of these two groups regulates angiogenesis
- There are some proteins/regulators that are absolutely essential e.g. VEGF - a loss of one allele of VEGF is incompatible with life
What is the main trigger for Angiogenesis?
- The main trigger for angiogenesis is HYPOXIA
- There is a transcription factor called HIF (hypoxia-inducible transcription factor), which is important for the regulation of genes involved in angiogenesis, in the presence of Oxygen is inhibited
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 (involved in angiogenesis)
- One of the targets of HIF is the expression of VEGF (vascular endothelial growth factor)
Presence of oxygen:
- HIF is inhibited by Von Hippel-Lindau (a tumour suppressor gene)
- pVHL adds a hydroxyproline group to HIF and HIF is degraded by a proteasome.
- HIF does not drive the expression of angiogenesis genes
Define Vascular Endothelial Growth Factor (VEGF)
What are the different receptor types it can bind to?
Which receptor is most commonly used?
These receptors can form dimers which increase their complexity
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
- 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
Describe the selection of Tip and Stalk cell
What is the role of each?
How do they communicate?
In sprouting angiogenesis, specialised endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of VEGF
- Notch signalling pathway is used for tip selection between the adjacent endothelial cells at the angiogenic front.
Tip cell:
- Cell that receives and grows towards the heaviest dose of VEGF = Tip Cell.
- Once a tip cell has been selected, it seems to control the behaviour of the cells around it via cell-cell communication
Stalk cell:
- Tip cell expresses notch ligand ‘Delta-Like-Ligand 4 (DLL4)’ - binds to notch receptor in adjacent cells to say, “I am tip cell, you are Stalk Cell.”
Once tip and stalk cells identified, stalk cell proliferates and push tip cell up
Describe the use of the Canonical Notch Signalling Pathway
(what is the ligand, what is the receptor, what are their action?)
- NOTE: the notch signalling pathway is found in other tissues other than endothelia
Binding of the notch ligand to the notch receptor activates the receptor by cleaving the intracellular domain (NICD) and act as a transcription
- Tip cells has the ligand and the stalk cell has the receptor
- NICD then translocate to the nucleus where it binds to the transcription factor RBP-J and regulates transcription
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’
- 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)
Summarize the effect of the VEGF on angiogenesis
- In stable vessels DII4 and notch signalling maintain quiescence
- In an unstable vessel, VEGF activation increases the expression of DII4 (Delta-like ligand)
- DII4 drives expression of the notch signalling which inhibits the expression of VEGFR-2
- DII4-expressing Tip cells acquire a motile, invasive and sprouting phenotype.
- Stalk cells (adjacent cells) form the base of the sprout and proliferate to support the sprout elongation. (their role is to divide and push the cell forward)
Model of Sprouting Angiogenesis
- 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 (outgrowth)
- 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 (guidance)
- 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
Describe the process of Spout Outgrowth and Elongation, which cells take part in this?
Myeloid cells are recruited to support and guide the sprout, they are 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)
- Macrophages have been shown to carve out tunnels in the ECM, thereby providing avenues for subsequent capillary infiltration
- So macrophages appear to help stabilise newly formed vessels (by promoting tip cell fusion)
- Tissue-resident macrophages were shown to be associated with angiogenic tip cells during anastomosis
What is the main aspect of Stabilisation and Quiescence?
Stabilisation involves reforming the endothelial monolayer barrier and recruiting neural cells (pericytes) and switching off the active angiogenesis process
- Pericytes wrap around new vessel to stabilise.
- Mural cells = VSMCs and pericytes.
- Barrier formation is associated with – VE-Cadherins and Ang-I.
Too much VEGF and there is too much sprouting and not enough stabilisation.
State the process for the formation of Tight Junctions and Adherence Junctions
Which molecules are used and what is their function?
What is the pathway?
- The endothelial cells have junctions between each other that allows them to form a cohesive monolayer
- There are proteins on the membranes of both cells involved in the junction, that bind in a homophilic way
- Cadherin is an important protein that lines the junctions of endothelial cells
VE-Cadherin is essential for vessel stabilisation and quiescence
- The homophilic interaction between the cadherins on the endothelial cells
- mediates the adhesion between endothelial cells
- is important in intracellular signalling
- The cadherin interactions are also important in contact inhibition of cell growth
- Promotes survival of the EC
Describe the function of Mural cells in angiogenesis
- Mural Cells = generally refers to smooth muscle cells and pericytes, both of which are involved in the formation of normal vasculature and are responsive to VEGF
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
2 tip cells meet and fuse, stalk cells separate to form lumen. Pericytes wrap around new vessel to stabilise.
What is the main Signalling Pathways Controlling Stability?
The Angiopoietin-Tie2 Ligand-Receptor System
- Ang1- and Ang-2 are antagonistic ligands of the Tie receptor
- Tie2 is a receptor that can bind to Angiopoietin 1
- Angiopoietin 1, when it binds to Tie2, promotes stability in the vasculature, stabilizes the junction and inhibit inflammatory gene expression
- Angiopoietin 2: it promotes vascular instability and VEGF dependent angiogenesis. It 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, synergises with VEGEF
- So, Ang-2 antagonises Ang-1 signalling and has pro-angiogenic effects
Define Angiogenic Switch, when does it occur?
Angiogenic Switch - there’s a point at which the tumour gets to a certain size where diffusion is no longer sufficient, so some cells within the tumour become hypoxic and send angiogenic signals
- This can occur at different stages in the tumour-progression pathway, depending on the nature of the tumour and its environment
- Avastin the first drug that was used
Tumours <1 mm3 receive oxygen and nutrients by diffusion from host vasculature
When tumours grow larger than this, they require new vessel networks
Tumours secrete angiogenic factors that stimulate the formation of new blood vessels, facilitating its progressive growth
What is the characteristic of Tumour Blood Vessels?
There is an imbalance in the signals that are regulating angiogenesis
- So in tumours, haemorrhage is common
Tumour vessels are not properly formed:
- imbalance in regulation,
- irregular shapes
- loose associations with perivascular cells
- Leackage
∴ haemorrhage is common
What is the aim of the new drug treatments for the blood supply in a tumour?
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
- Less sensitive to VGEF inhibition
- The aim now is to normalise tumour blood vessels to reduce hypoxia and improve the efficiency of drug delivery
How can the tumour cells show resistance to VEGF pathway?
- VEGF inhibition leads to MORE hypoxia so more release of OTHER angiogenic factors or increases tumour invasiveness.
- Tumour vessels may be less sensitive to VEGF inhibition due to tumour cells lining the vessels.
- Tumour cells that recruit pericytes may be less responsive.
- 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.
