Angiogenesis

What are mural cells?

• 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

ROle of VEGF in Notch signalling

• VEGF will activate endothelial cells in a capillary and increase expression of Dll4 • Dll4 then drives the Notch signalling, and inhibits the expression of VEGFR2 in the adjacent cell (so that it doesn't become the Tip cell) • By doing this, the cells on either side of the tip cell will then recognise their role as stalk cells that have to divide and push the tip cell forward

Two main modes of RESISTANCE to VEGF blockade

1. The tumour adopts an evasive strategy and adapts to bypass the specific angiogenic blockade 2. Intrinsic or pre-existing difference - the idea that a particular tumour in particular place in a particular person, was not very sensitive to VEGF anyway, so knocking out VEGF made little difference

Receptors for VEGF

1. VEGFR-1 2. VEGFR-2- major mediator of VEGF dependent angiogenesis. Activates signalling pathways that regulate endothelial cell migration, survival and proliferation 3. VEGFR-3

Broad stages of angiogenesis

1. selection of tip cell 2. stalk elongation and tip guidance 3. quiescent phalanx resolution

What is the aim of anti-angiogenic therapy and what happens if it's too aggressive?

Aim: to normalise tumour blood vessels in order to a) reduce hypoxia b) increase efficiency of drug delivery Aggressive therapy: - may damage ability to deliver other drugs to tumour

What is angiogenesis?

Angiogenesis is the formation of a new blood vessel from pre-existing blood vessels

What is the stimulus for angiogenesis?

Hypoxia

What stimulates VEGF expression?

Hypoxia inducing factor- drives expression of VEGF in hypoxia.

What is the best known pro-angiogenic growth factor?

VEGF

AMD

*main cause of blindness in the elderly*

Role of HIF in angiogenesis

- transcription factor - responsible for regulating genes involved in angiogenesis - usually bound by (and inhibited by) *Von Hippel-Lindau* (a TSF) in NORMOXIA -in hypoxic conditions, VHL is no longer bound to HIF-->HIF is mobilised and translcoated into nucleus-->drives expression of genes involved in angiogenesis -One of the targets is expression of VEGF (vascular endothelial growth factor)

Stabilisation and quiescence phase

-After fusion of neighbouring branches, lumen formation allows perfusion of the neovessel -the neovessel resumes quiescence by promoting a phalanx phenotype, re-establishing junctions, depositing basement membrane, maturation of pericytes and production of vascular maintenance signals. -Other factors promote transendothelial lipid transport. ____ • Once the tip cells have fused and the stalk cells are separating for form a patent tube, the new vessel needs to stabilise • Stabilisation involves reforming the endothelial monolayer barrier and recruiting neural cells (pericytes) and switching off the active angiogenesis process

What is avastin?

-Anti-VEGF Humanised MAb (mouse antibody) • This is also called Bevacizumab • Avastin has relatively limited efficacy and it has many side effects: ○ GI perforation ○ Hypertension ○ Proteinuria ○ Venous thrombosis ○ Haemorrhage ○ Wound healing complications • There is no overall survival advantage over chemotherapy alone • No quality-of-life or survival advantage • These side effects are because VEGF is essential for the homeostasis of the endothelium • There are TWO main modes of unconventional resistance to VEGF blockade: ○ The tumour adopts an evasive strategy and adapts to bypass the specific angiogenic blockade ○ Intrinsic or pre-existing difference - the idea that a particular tumour in particular place in a particular person, was not very sensitive to VEGF anyway, so knocking out VEGF made little difference

Does anti-angiogenic therapy work?

-In some cases benefits are transitory, and followed by a restoration of tumour growth and progression -In other cases there is no objective benefit _____ AVASTIN -so relatively limited efficacy -no overall survival advantage over chemotherapy alone -no quality of life advantage

How do pericytes stabilise newly formed blood vessels?

-They wrap around capillaries and send siganls to say you need to stabilise -Related to muscle cells

What is vasculogenic mimicry?

-Tumour cells undergo vascular mimicking. -They pretend to be endothelial cells. -Hook up with vascular system.

Therapeutic Angiogenesis for Coronary Artery Disease and Peripheral Artery Disease

-vascularisation is important in ischaemia -if you promote angiogenesis distal to occlusion you can bypass the occlusion

What transcription factor is important in controlling angiogenesis?

HIF: hypoxia inducing factor

Coreceptors for VEGF

Neuropilin-1 (Nrp1) Neuropilin-2 (Nrp2)

What pathway is crucial in selecting the tip cells?

Notch pathway

Angiogenic therapy in other diseases

Other fields are good targets for angiogenic therapy- both pro and anti, depending on the disease.

What is the angiogenic switch?

Point at which tumour becomes dependent on new vasculature

What is a major signalling pathway controlling stability of blood vessels?

The angiopoietin-Tie2 ligand-receptor system

Other ways of making new blood vessels

• During development, the contribution of bone marrow progenitor cells is very important in the early stages of blood vessel formation - this is vasculogenesis • Blood vessels are the first organ systems that form in the embryo - without blood vessels, nothing else can form • Angiogenesis is the most common way in which new blood vessels are formed e.g. in wound healing or the menstrual cycle • Arteriogenesis - collateral growth that is dependent on shear stress and external factors like macrophages

Role of tip cells in angiogenesis

• In sprouting angiogenesis, specialised endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of VEGF • Once a tip cell has been selected, it seems to control the behaviour of the cells around it via cell-cell communication • There is a pathway called Notch that is crucial for the selection of tip cells

Describe the notch signalling pathway

• 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) • NICD then translocates 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)

How does sprout outgrowth and elongation occur?

• Once the tip cell and stalk cells have been identified, the sprout needs to progress forwards • 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 • Tissue-resident macrophages were shown to be associated with angiogenic tip cells during anastomosis • So macrophages appear to help stabilise newly formed vessels (by promoting tip cell fusion) ______ -Tip cells navigate in response to guidance signals (such as semaphorins and ephrins) and adhere to the extracellular matrix (mediated by integrins) to migrate. -Stalk cells behind the tip cell proliferate, elongate and form a lumen, and sprouts fuse to establish a perfused neovessel. -Proliferating stalk cells attract pericytes and deposit basement membranes to become stabilized. -Recruited myeloid cells such as tumour-associated macrophages (TAMs) and TIE-2-expressing monocytes (TEMs) can produce pro-angiogenic factors or proteolytically liberate angiogenic growth factors from the ECM.

Describe the angiopoietin-Tie2 ligand-receptor system

• The angiopoietin-Tie2 system is required to modulate the activation and return to quiescence of endothelial cells • Tie2 is a receptor that can bind to Angiopoietin 1 • Angiopoietin 1, when it binds to Tie2, promotes quiescence in the vasculature • Angiopoietin 2 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 • So *Ang-2 antagonises Ang-1 signalling* and has *pro-angiogenic* effects • NOTE: angiogenesis can be studied in early post-natal mouse retina vasculature - the development of the mouse retina vasculature starts immediately after birth

Junction formation between endothelial cells

• 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 and is important in intracellular signalling • The cadherin interactions are also important in contact inhibition of cell growth • The cadherins also promote the survival of the endothelial cells • Pericytes are eventually recruited, which produce a load of proteins that are involved in stabilising the vessel

How are tumour blood vessels different from normal blood vessels and why?

• These are NOT properly formed because the signals are not physiological - there is an imbalance in the signals that are regulating angiogenesis • So in tumours, haemorrhage is common • Tumour blood vessels can be: ○ Irregularly shaped, dilated, tortuous ○ Not organised into definitive venules, arterioles and capillaries ○ Leaky and haemorrhagic, partly due to the *over-production of VEGF* ○ Perivascular cells often become loosely associated ○ Some endothelial cells may recruit endothelial progenitor cells from the bone marrow

Anti-VEGF therapy for age-related macular degeneration (AMD)- LUCENTIS

• Though Avastin wasn't designed for AMD, some clinicians tried it out and found that it was effective in AMD • The pharmaceutical company made the Avastin into a slightly modified form - Lucentis • A lot of studied have shown that Avastin works just as well as Lucentis and Avastin is much cheaper

The future of anti-angiogenic therapy

• Though the research into anti-angiogenic treatments didn't yield particularly good results in the field of cancer, it did massively benefit patients with *Age-related macular degeneration (AMD)* • There is research looking into the benefits of giving pro-angiogenic therapy following occlusion of an artery (e.g. in myocardial infarction or peripheral vascular disease) • There have been attempts to inject VEGF into the cardiac tissue soon after occlusion with the hope of stimulating neovascularisation that will reduce the damage due to ischaemia

Overview of steps in 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 • 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 • 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

How does size of tumour influence its nutrient supply?

• 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 • Newly vascularised tumour no longer relies solely on diffusion from host vasculature - this facilitates its progressive growth • The key point in tumour development with regards to neovasculature is the Angiogenic Switch

Angiogenesis as a balance

• When you form a new blood vessel, you need to destabilise the pre-existing blood vessel and then restabilise 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 • The loss of other, less important, regulators of angiogenesis could still mean that vasculature develops but it wont be quite normal

Side-effects of avastin?

○ GI perforation ○ Hypertension ○ Proteinuria ○ Venous thrombosis ○ Haemorrhage ○ Wound healing complications

Members of VEGF family

○ VEGF-A ○ VEGF-B ○ VEGF-C ○ VEGF-D ○ PIGF (placental growth factor)