Angiogenesis Flashcards
Define angiogenesis
Angiogenesis (literally meaning the creation of new blood vessels) may be defined as the “formation of neo-vessels from pre-existing blood vessels”.
Occurs in small blood vessels- where the endothelium is not surrounded by many layers-as to allow a new capillary to grow
Describe the physiological roles of angiogenesis
Embryonic Development
Menstrual cycle
Wound healing
Describe the roles of angiogenesis in pathology
Cancer Chronic inflammatory diseases Retinopathies Ischemic diseases Vascular malformations
Give a brief overview of the process of angiogenesis
Simulus (hypoxia) Angiogenic factor production (VEGF) Release of angiogenic factor Endothelial cell receptor binding- intracellular signalling EC activation- BM degradation EC proliferation Directional margining (gradient of angiogenic factor- directs the growth of the new vessel to where it is needed) ECM remodelling Tube formation Loop formation- a-v differentiation Vascular stabilisation
Describe how angiogenesis differs in different sites of the body
Blood vessels differ around the body (i.e blood vessels in the liver differ to those in the brain)
Microenvironments differ around the body
Therefore, the mechanisms of angiogenesis differ around the Body.
Describe the different types of angiogenesis
Vasculogenesis (bone marrow progenitor cell)- recruitment or mobilisation of vessels, formation of capillary plexus and formation of mature network
Angiogenesis
(sprouting) - main type that occurs in adults
Arteriogenesis
(collateral growth)- occurs to overcome partial occlusion of vessels- stimuli include shear stress, macrophage cytokines, matrix remodelling and SMC growth
Summarise the regulators of angiogenesis
A large number of molecules can influence Angiogenesis
Some molecules are essential (i.e. VEGF), other are required for modulation (i.e. VWF)- without VWF you will still have blood vessels, but they may be faulty
Many are best known for other functions (i.e. TNF-α, VWF)
Some have been reported to have both pro- and anti-angiogenic effects
Pathways may act in a tissue and stimulus-specific manner
What are the inhibitors of angiogenesis
Extracellular Matrix: Thrombospondin-1 Angiostatin Endostatin Soluble factors: sVEGF-R IL-10 IL-12 TNF-α Cell surface receptors: αvβ3
What are the activators of angiogenesis
Growth factors: VEGF family FGF family TGF β PDGF Soluble factors: IL-6 Factor XIII TNF-α Cell surface receptors: αvβ3
Describe how factors can have both pro- and ant-angiogenic effects
Depends on microenvironment, phase and whether it has been cleaved properly or not.
What else is important to consider in angiogenesis
The factors involved in the maturation and integrity of the newly formed vessels- these factors are critical for the functioning of the new blood vessels VE-Cadherin (Junctions) Angiopoietin/Tie2 Notch pathway ERG pathway Platelets
Give a brief outline for sprouting angiogenesis
tip/stalk cell selection; lateral inhibiton of neighbouring cells- stalk cells proliferate to push the tip up
tip cell navigation and stalk cell proliferation;
branching coordination;
stalk elongation, tip cell fusion, and lumen formation;
perfusion and vessel maturation (lumen extension and flow initiation).
What is the main signal for angiogenesis
Hypoxia
HIF: hypoxia-inducible transcription factor, controls regulation of gene expression by oxygen
What happens to HIF in the presence of oxygen
In normoxic conditions, o pVHL adds a hydroxyproline group to HIF and HIF is degraded by a proteasome.
Ubiquitin will also be attached to HIF-a
What happens to HIF in the absence of oxygen
Absence of oxygen:
o pVHL does NOT bind to HIF and HIF translocates to the nucleus and binds to HIF-region and induces translation of hypoxic factors (transcribed form hypoxia-inducible genes)
HIF-a binds to HIF-b on hypoxia inducible genes VEGF PDGF-B TGF-A EPO
What is pVHL
pVHL: Von Hippel–Lindau tumor suppressor gene, controls levels of HIF
What are the major vascular endothelial growth factors and their receptors
Family of 5 members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF)
Three tyrosine kinase receptors: VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3; and co-receptors neuropilin (Nrp1 and Nrp2)
Which receptor is the major mediator of VEGF-dependent angiogenesis
VEGFR-2 is the major mediator of VEGF-dependent angiogenesis, activating signalling pathways that regulate endothelial cell migration, survival, proliferation.
VEGF-A is the major GF
Summarise the structure of the VEGF-Receptors
Each contain a tyrosine kinase domain
Each contain a dimerisation or binding domain
VEGF-R3 has a lot of disulphide bonds in this region
Summarise the structure of the neuropilin receptors
VEGF binding domain (b1 and b2)
a1,a2 and c1
Essentially, what happens in sprouting angiogenesis
In sprouting angiogenesis, specialised endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of VEGF
What are the main drivers of sprouting angiogenesis
Astrocytes and macrophages respond to hypoxia and release angiogenic factors (VEGF, Ang 2, FGF, chemokines)
You then get tip cell formation- which is the cell that is selected to sprout (VEGFR-2, DLL4, JAGGED1, NRP1, Integrins, HIF-alpha, MT1-MMP, PGC-1a)
These tip cells instruct neighbouring cells to become stalk cells to support the sprouting
You also get:
Loosening junctions between endothelial cells (VE-cadherin)
Matrix remodelling (MMPs)
Pericyte detachment (ANG-2)
Permeability, vasodilation and extravasation (VEGF)
Why is it important not all the cells become tip cells
Otherwise all the cells would sprout and they would have no support and the process would not be coordinated.
So you need the neighbouring cells to become stalk cells
Tip cell selection is based on notch signalling between adjacent endothelial cells at the angiogenic front.
Summarise the canonical Notch signalling pathway
Notch ligand (delta/jagged ligand) binds to notch receptor This leads to ADAM gamma-secretase cleaving the intracellular domain of the notch receptor The intracellular domain of the notch receptor (NICD) translocates to the nucleus and binds to the transcription factor RBP-J, which will decrease the expression of VEGFR-2
Essentially, notch receptors and ligands are membrane-bound proteins that associate through their extracellular domains.
Outline the role of notch signalling in tip selection
In stable blood vessels, Dll4 and Notch signalling maintain quiescence
VEGF activation increases expression of Dll4
Dll4 drives Notch signalling, which inhibits expression of VEGFR2 in the adjacent cell
Dll4-expressing tip cells acquire a motile, invasive and sprouting phenotype
Adjacent cells (Stalk cells) form the base of the emerging sprout, proliferate to support sprout elongation.
Summarise sprout outgrowth and guidance
Tip-cell guidance and adhesion (integrins)
Myeloid cell recruitment (ANG 2, PIGF)
Liberation of angiogenic factors from ECM (VEGF, FGFs)
Stalk elongation (NOTCH, WNT, PIGFs, FGFs)
Pericyte recruitment (NOTCH, ANG-1)
Lumen formation (VE-cadherin, VEGF)
Describe the roles of macrophages in sprout outgrowth and guidance
Macrophages play a significant role in both physiological and pathological angiogenesis
Macrophages carve out tunnels in the extra cellular matrix (ECM), providing avenues for capillary infiltration
Tissue-resident macrophages can be associated with angiogenic tip cells during anastomosis- to support the new blood vessels formed
Summarise the role of platelets in angiogenesis
Contain pro- and anti-angiogenic factors- and are therefore described as modulators of angiogenesis.
Describe the role of platelets in physiological angiogenesis
Wound healing (growth factors and cytokines)
Vascular development and lymphangiogenesis (podoplanin, Cleo-2)
Describe the role of platelets in pathological angiogenesis
Atherosclerosis (P-selectin, VWF) Rheumatoid arthritis (PMPs) Diabetic retinopathy (VEGF, PDGF) Tumour development- angiogenesis, metastasis, and homeostasis (VEGF, G1ba)
Summarise the role of stabilisation and quiescence of the new blood vessels formed in angiogenesis
Barrier formation (VE-cadherin, ANG-1)
§ Too much VEGF and there is too much sprouting and not enough stabilisation.
§ Pericyte maturation facilitates the stabilisation of the vessel (ANG1,notch)
o Mural cells = VSMCs and pericytes.
§ Pericytes and mural cells use an Ang/Tie-2 system to stabilise the vessel
Basement membrane deposition (TIMPs) Transendothelial lipid support (VEGF-B) Vascular maintenance (VEGF, ANG-1) Phalanx cell (VE-cadherin, TIE-2)
Which type of cells are important in stabilising the new vessels formed by angiogenesis
Mural cells (pericytes) help to stabilise the neovessels These are closely related to smooth muscle cells
Summarise the types of junctions found between endothelial cells
Tight junctions (Claudins, occludins and lectins) Adherens junction (VE-cadherin)- this is particularly important in stabilising newly formed blood vessels.
Junctions are important in:
Controlling the flux of substances between tissues and blood- important in permeability
Controlling the influx of inflammatory cells into tissues- important in infection.
Describe VE-cadherin
Constitutively expressed at junctions
Homophilic interaction mediates adhesion between endothelial cells and intracellular signalling
Controls contact inhibition of cell growth
Promotes survival of EC
Summarise the angiopoietin-Tie2 signalling system
Ang-1 and Ang-2 are antagonistic ligands of the Tie2 receptor
Ang-1 binding to Tie2 promotes vessel stability and inhibits inflammatory gene expression
Ang-2 antagonises Ang-1 signalling, promotes vascular instability and VEGF-dependent angiogenesis
Describe the role of pericytes in promoting neovascular stability
ANG1 is constitutively released by pericytes
ANG1 binds to Tie 2 receptor expressed on vascular endothelial cells.
This inhibits Rho kinase- which promotes the stabilising of cell-cell junctions and thus blood vessel stabilisation
This blocks NFkB which has anti-inflammatory effects
Stimulates the PI3/Akt pathway which leads to cell survival
Inhibits leukocyte recruitment via adhesion molecules.
Where is ANG2 made
In endothelial cells
Weibel-Palade bodies release ANG 2 upon inflammatory stimulus.
In which diseases are plasma levels of ANG 2 raised
Ang-2 plasma levels raised in disease including:
congestive heart failure
Sepsis
Chronic Kidney Disease
Summarise sprouting angiogenesis
Initiation Selection Tip cell navigation Stalk elongation Fusion Perfusion and oxygenation Maturation and stabilisation Quiescence
What is Avastin
2004: Anti-VEGF humanised mAb: Avastin is FDA approved
for the treatment of advanced colorectal cancer.
Summarise tumour angiogenesis
Tumors less than 1 mm3 receive oxygen and nutrients by diffusion from host vasculature.
Larger tumors require new vessel network. Tumor secretes angiogenic factors that stimulate migration, proliferation, and neovessel formation by endothelial cells in adjacent established vessels (stimulated by hypoxia)
Newly vascularized tumor no longer relies solely on diffusion from host vasculature, facilitating progressive growth.
New blood vessels will be aberrant- tumour won’t be able to release all the angiogenic factors needed to create perfect vessels, may only release VEGF.
What are the benefits of angiogenesis for the tumour
Supply of nutrients
Removal of waste products
Potential to metastasise
What is the angiogenic switch
The angiogenic switch is a discrete step in tumour development that can occur at different stages in the tumour-progression pathway, depending on the nature of the tumour and its microenvironment
Essentially, it is when the tumour recruits its own blood supply by angiogenesis and no longer relies on diffusion.
Give a brief overview of tumour angiogenesis
Dormant
Perivascular detachment and vessel dilatation
Onset of angiogenic sprouting
Continuous sprouting; new vessel formation and maturation; recruitment of perivascular cells
Tumour vasculature
Describe the importance of targeting tumour angiogenesis in cancer treatments
Key mechanism for survival and proliferation of the tumour
Can recruit inflammation and raise interstitial pressure- which can inhibit the delivery of anti-cancer drugs.
Describe the characteristic of tumour blood vessels
irregularly shaped, dilated, tortuous
not organized into definitive venules, arterioles and capillaries
leaky and haemorrhagic, partly due to the overproduction of VEGF
perivascular cells often become loosely associated
some tumours may recruit endothelial progenitor cells from the bone marrow (controversial!)
Essentially:
occlusions, breaks, blind ends and arterial-venous shunts
What are some of the issues with tumour blood vessels
They are not properly formed because the signals are not physiological (won’t contain all of the mediators necessary)
Vessels can be irregularly shaped, distended, tortuous
Leaky and haemorrhagic etc.
Haemorrhage is common in tumours (which will recruit platelets)
They are tortuous and disorganised.
Describe the multicellular response that promotes tumour angiogenesis
Cancer-associated fibroblasts (CAFs) secrete extracellular matrix; pro-angiogenic growth factors, (VEGFA; FGF2; CXCL12; PDGFC)
Pericytes are loosely associated with with tumour-associated blood vessels (TABVs), and this favours chronic leakage in tumours. This is enhanced by angiopoietin 2 (ANGPT2)
Platelets release pro-angiogenic mediators and proteases that support the proliferation and activation of CAFs, such as PDGFB and TGFβ - platelets recruited as a result of haemorrhage
Describe the role of platelets in tumour angiogenesis
Link between cancer progression and thrombocytosis
Activated platelets are a source of:
pro-angiogenic factors: VEGFA, platelet-derived growth factors (PDGFs), FGF2
angiostatic molecules: thrombospondin 1, plasminogen activator inhibitor 1 (PAI1), endostatin
Tumours cause platelet activation, aggregation and degranulation
Disrupting platelet function does not obviously impair tumour angiogenesis, however the overall outcome of platelet activation in tumours appears to be pro-angiogenic
Too many side effects (increased bleeding) to justify use of anti-platelet drugs as part of cancer treatment.
What is a key therapeutic strategy to treat tumour angiogenesis
Anti-VEGF therapy
Summarise the different strategies to block VEGF signalling in tumour cells
use monoclonal antibodies (Bevacizumab)- to bind to VEGFA and prevent it from activating VEGF-Rs to stimulate angiogenesis
use decoy receptors (aflibercept)- to act as a decoy receptor for VEGFA, VEGFB and PIGF.
Use soluble VEGFR1 (sVEGFR1) to mop up VEGF
Use VEGF kinase inhibitors to disrupt intracellular signalling:
Preventing angiogenesis, lymphagoensis and proliferation of tumour cells.
Describe how VEGF inhibition by sVEGFR1 (Flt-1) reduced tumour growth
Cells stably transfected with control or sFlt-1 plasmid to promote Flt-1 (VEGFR1) expression
VEGFR1 binds to VEGF and “mops it up” preventing it from stimulating angiogenesis
Flt-1 expression reduces tumor growth in vivo, without affecting tumor cell growth in vitro: effect on vasculature
What is Avastin approved for
Avastin is FDA approved for the treatment of advanced colorectal cancer (i.e if it has metastasised)
Cervical cancer
Glioblastoma
Non-small cell lung cancer
Ovarian, Fallopian tube or primary peritoneal cancer
Describe the limited efficacy of Avastin as a cancer therapy
No overall survival advantage over chemo alone
No quality-of-life or survival advantage- VEGF is essential for survival of endothelial cells
In some cases benefits are transitory, followed by a restoration of tumour growth and progression
In other cases there is no objective benefit
State the side effects of Avastin
GI perforation Hypertension Proteinuria Venous thrombosis Haemorrage Wound healing complications
Describe the potential resitance mechanisms to anti-VEGF therapy in cancer cells
VEGF inhibition aggravates hypoxia increasing tumour’s production of other angiogenic factors or increases tumour invasiveness
Tumours vessels maybe less sensitive to VEGF inhibition due to vessel lining by tumour cells or endothelial cells derived from tumours
Tumour cells that recruit pericytes maybe less responsive to VEGF therapy
Essentially, tumour cells are different to normal cells! The mechanisms for angiogenesis differ in normal cells to cancer cells.
Describe vascular mimicry as a resistance mechanism used by tumours against anti-VEGF therapy
Tumor cell vasculogenic mimicry (VM), also known as vascular mimicry, describes the plasticity of aggressive cancer cells forming de novo vascular networks and is associated with the malignant phenotype and poor clinical outcome.
4. 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.
a. Anti-angiogenic therapy then will not affect this VM.
Describe the consequences of aggressive anti-angiogenic therapy
Anti-angiogenic therapy sometimes facilitates effects that seem pro-tumour growth.
o Sustained anti-angiogenic therapy can lead to too much ischaemia and thus release of hypoxic factors that further induce angiogenesis.
Can lead to loss of vessels
o Vasculature may become refractory to treatment.
o Vasculature may be inadequate for further delivery of drugs/oxygen.
What is the ultimate aim of anti-angiogenic therapy
Anti-angiogenic therapy which normalises vasculature
reduces hypoxia
Increase efficacy of
conventional therapies
To create a balance between pro- and anti-angiogenic factors- as to mimic normal vasculature
Summarise the future of anti-angiogenic therapies in cancer
Anti-angiogenic therapy in combination with other anti-cancer therapies
Resistance: combinatorial strategies involving angiogenesis inhibition & drugs targeting resistance mechanisms
Novel non-VEGF targets – novel molecular mechanism
How can we find novel cellular mechanisms
single cell RNASeq of tumor endothelium
Take cells from tumour- isolate them and sequence their RNA cell by cell to build up a profile of the cell types in the tumour to identify potential targets
May identify a different type of pericyte for example- which can be targeted by therapies.
Describe the role of angiogenesis in other diseases
Angiogenesis is important in many clinical diseases, not just cancer (e.g. heart and vascular disease, rheumatoid arthritis). Anti-angiogenic therapy in other diseases: Retina vascularization (diabetic retinopathy, wet AMD)
Summarise age-related macular degeneration
Abnormal growth of choroidal blood vessels
“Leaky” vessels cause oedema
Visual impairmen
Describe the use of Avastin in wet-AMD
AMD is the main cause of blindness
Avastin not FDA approved for AMD, but used off-label
Lucentis developed by Genentech from the parent molecule Avastin
June 2006: FDA approval for Lucentis for AMD
High efficacy of both treatments in maintaining or improving vision
Many patients become refractory to treatment >2 years
Ranibizumab (Lucentis), $2,023 per dose (up to 12 injections per year)
Bevacizumab (Avastin), $55 per dose.
What are the limitations of our cell culturing techniques we use for drug screenings
Tumours are complex three-dimensional (3D) structures with their own unique microenvironments
We lack good in vitro models - our understanding of tumour behaviour in a complex 3D environment is limited and drug screens are often misleading.
Studies are performed on cell lines growing as two-dimensional (2D) monolayers, which do not mimic the complex interplay between tumour cells and their extracellular environment
The phenotype of tumour cells when cultured in 2D vs 3D is different
Crucially, tumors receive nutrients and therapeutics through the vasculature, which is not included in any in vitro tumor models.
Describe how ‘tumour-on-a-chip’ can give a better representation of the tumour for studying drug design
Develp a microphysiological system that incorporates human cells in a 3D extracellular matrix (ECM), supported by perfused human microvessels
Improve drug screening- more accurate representation of what happens physiologically.
What is the distance between cells and blood
· Cells must be within 10 cells distance (100 micrometers) of a blood
What is the key difference between ANG1 and ANG2
Ang 1 promotes quiescence in the blood vessel
Ang 2 is an antagonist and gets released when you need to form a new vessel or when you need to respond to inflammation/vasculature needs to be destabilised
