Angiogenesis: Health, disease and medicine Flashcards
What is the initial step of angiogenesis? how does the vasculature respond?
Following hypoxia, hypoxia genetic response leads to production and secretion of VEGF. This stimulates nearby vessel endothelial cells to become motile and form a tip cell.
What does a tip cell do following its formation?
Produces extending filopodia which guide the development of a new vessel toward the VEGF secreting cells
What forms behind the tip cell as it extends out toward the hypoxic cells>?
Stalk cells which begin to hollow out and form a tube.
What happens when tip cells from two different growing vessels meet?
They merge together, allowing blood to flow through the newly formed vessel.
What are recruited to the vessel walls following the formation of a new vessel?
Pericytes which stabilise the vessel structure. Recruited by endothelial-derived PDGF-BB
What is the organotypic co-culture model of angiogenesis?
Co-culture of human dermal fibroblasts with human endothelial cells. The interaction of these cells results in the formation of tubules with lumens that embed in naturally produced ECM and closely resemble capillaries formed during angiogenesis in vivo. It is dependent on angiogenic factors. The vessel is identified by CD31 or vWF staining. Branch points and tube length can be quantified.
What markers can be used for new vessel formation.
CD31 and vWF
How can the organic co-culture model of angiogenesis be used for drug development?
After establishing the production of new vessels through the addition of VEGF and staining with CD31/vWF. Drugs can be titrated in at differing concentrations to see if they have an anti-angiogenic effect
What is an antibody that has anti-angiogenic properties?
Avastin
What is the role of PLC-y in VEGF signalling?
PLC-y binds to phosporylated tyrosine residues on VEGF-A. PLC-y cleaves PIP2 to form DAG and IP3. IP3 stimulates the release of intracellular calcium stores which activate eNOS which generates NO. NO regulates vascular tone, angiogenesis and arterioprotection.
What are the different mechanisms of blood vessel formation?
1) Sprouting angiogenesis
2) Vasculogenesis - endothelial progenitors differentiate to form endothelial cells.
3) Intussuseption - one blood vessel inside another that splits to form two vessels.
4) Vessel co-option
5) Vascular mimicry
6) Tumour cell differentiates to an endothelial cell.
Which mechanisms of blood vessel formation tend to be seen in cancers?
1) Vessel co-option
2) Vascular mimicry - Tumour cells line tumour vessels by taking on properties of endothlial cells.
3) Tumour cell differentiation to an endothelial cell - Cancer stem-like cells differentiate into endothelial cells.
What cancer experiences vessel co-option and what is the effect of this?
Glioma - tumour cells co-opt pre-existing vessels so are able to spread and proliferate extremely quickly
During angiogenic sprouting, what breaks down the basement membrane?
increased activity of metallomatrixproteases
What signalling from a tip cell ensures that the cells forming behind it are stalk cells and not more tip cells.
Tip cell expresses DLL4 which interacts with notch on neighbouring cells to inhibit the tip cell phenotype.
Summarise angiogenic sprouting
1) VEGF activation of tip cells via VEGFR2
2) Suppression of tip cell phenotype in neighbouring cells via DLL4/Notch
3) Tip cell guidance along VEGF gradient
4) Stalk elongation
5) Lumen formation
6) Pericyte recruitment by endothelial derived PDGF-BB
How does the organisation of tumour-associated vasculature differ to normal tissue?
It is much more chaotic compared to the highly organised vasculature of normal tissue. This abnormality reduces the delivery and subsequent response of conventional therapies
How do endothelial monolayers differ between normal and tumour vessels?
Disorganised endothelium with gaps can be found in tumour vessels which permit seepage of plasma, producing a high interstitial pressure
How do pericytes associate with normal blood vessels compared to tumour vessels?
Very good coverage in normal vessels. In tumour vessels this is lost, becoming loosely attached. This reduces the stability of the vessel structure, contributing to disorganisation.
How does HIF get targeted in normoxic conditions?
1) Degradation - In normoxic conditions a proline residue on HIF-a is hydroxylated by HIF-PH. This tags it to be bound by VHL. This ubiquitinates HIF-a leading to its degredation at the proteosome.
2. Transcriptional deactivation: HIF-1a activity also regulated by factor inhibiting HIF (FIH) FIH hydroxylates HIF-1a at asparagine residues on its C terminus, blocking recruitment of coactivators rendering HIF-1a transcriptionally inactive.
How do vascular disrupting agents work?
Tubulin binding agent that targets established tumour vessels. It selectively inhibits tumour blood flow by blocking endothelial cell proliferation. This leads to necrosis of tumour cells.
What cells remain after treatment with combrestatin and why is this the case?
Combrestatin is a vascular disrupting agent. The tumour rim remains after treatment because the vasculature surrounding the tumour tends to be more normal so combrestatin doesn’t bind as well.
What is Vandetanib and how does it work?
A small molecule of the tyrosine kinase domain of VEGF2. Also has some effect on EGFR and VEGF3
What is Avastin?
An antibody to VEGF
Why was avastin removed from treatment for breast cancer.
Due to the severe cardiovascular toxicity
How does drug resistance to anti-VEGF develop?
Introduction of other pro-angiogenic factors such as PDGFs or FGFs. Vascular remodelling can then occur leading to resistance.
Summarise how angiogenesis is seen in tumour development.
Through hypoxia, or the action of oncogenes; tumour cells emit elevated levels of VEGF. Excessive VEGF leads to abnormal amounts of tip cell formation, creating malformed capillaries. These capillaries are more permeable than normal ones. Poorly assembled vessel walls may allow escaped tumour cells to enter the blood stream to metastesise.
The malformed capillaries may cause irregular blood flow, so tumour cells remain hypoxic and continue to secrete VEGF, perpetuating malformed vessel formation.
Why does Avastin cause strokes/ CVD?
VEGFR signalling induces PLCy activity which leads to NO production. NO keeps vascular tone under control so when it is disregulated by VEGF inhibitors thrombotic events are more likely to occur.
