angiogenesis Flashcards
where does angiogenesis occur
small bv - endothelium is not surrounded by lots of layers
summarise the cascade of events in angiogenesis *
startes becasue of hypoxia - triggers the release of angiogenic factors eg VEGF
VEGF binds to an extracellular receptor - causing intracellular signalling
this activates endothelial cells
extracellular actiation leads to BM degredation
leads to EC proliferation and directional margination
causes ECM remodelling, tube formation, loop formation (a-v differentiation)
vascular stabalisation
when does angiogenesis occur physiologically *
development in embryo - mediated by podoplanin and clec-2
menstrual cycle
wound healing - medialted by GF and cytokines
what pathology is angiogenesis involved in *
cancer
chronic inflammatory diseases
rheumatoid arthritis - mediated by RMPs
retinopathy - mediated by VEGF adn PDGF
ischemic diseases - bv grow at base of atherosclerotic plaque - mediated by p-selectin and vWF
vascular malformations
are all bv the same *
no - they have differnet angiogenic microenvironments
therefore angiogeneis is different in development to in wound healing
describe in vivo models of angiogenesis
in zebrafish blood vessels stained - can see the sprouting of new vessels
in mouse retina is vascularised after birth - we can stain for bv - green is a TF and is seen in every endothelial cells
describe the different types of vascular growth
vasculogenesis - in embryo - involves progenitors from the bone marrow
angiogenisis - sprouting - main form in adults
arteriogenesis - collateral growth when there is an occlusion
describe the regulators of angiogenesis *
large number of molecules can influence angiogenesis
VEGF is essentila
VWF - not essential but w/o it the vessel growth is not perfect
some molecules are pro- and anti-angiogenic depending in the microenvironment and the stage of the cascade, some molecules get cleaved
what are the inhibitors of angiogenesis *
Extracellular Matrix:
- Thrombospondin-1
- Angiostatin
- Endostatin
Soluble factors:
- sVEGF-R
- IL-10
- IL-12
- TNF-a
Cell surface receptors:
•avb3
what are the activators of angiogenesis *
Growth factors:
- VEGF family
- FGF family
- TGF b
- PDGF
Soluble factors:
- IL-6
- Factor XIII
- TNF-a
Cell surface receptors:
•avb3
why do we need molecules for maturation and stability of bv *
once we have made bv they are useful w/o stabalising them
need to be maintained because they are crucial for the function of tissues
factors that are involved in maturation and stability of bv *
VE-Cadherin (Junctions)
Angiopoietin/Tie2
Notch pathway
ERG pathway
Platelets
describe the mechanism of sprouting angiogenesis *
angiogenic factor stimulates endothelial activation
1 cell is hit be the factor - becomes the tip cell, surrounding cells become the stalk cells
the tip cell starts sprouting and stalk cells push them up
tip cell is involved in navigation and stalk cell in proliferation
the stalk cells elongate and tip cells fuse forming the lumen
then the vessel is perfused and matures
describe how hypoxia triggers apoptosis *
hypoxia-inducible TF controls regulation of gene expression by oxygen
Von Hippel–Lindau tumor suppressor gene, controls levels of HIF
in presence of oxygen - HIF-a binds to pVHL, pVHL takes HIF-a to protease so HIF-a is destroyed
in absence of oxygen ie hypoxia - pVHL leaves HIF-a - HIF-a binds to DNA dribing transcription of genes that promote angiogenesis eg VEGF
what is the family of VEGF *
Family of 5 members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF)
describe the VEGF receptors *
there are 3 TK receptors - VEGFR-1 VEGFR-2 and VEGFR-3
and co receptors neuropilin (Nrp1 and Nrp2) - have VEGF binding domains
the VEGF receptors can form dimers
what are the main VEGF(R) *
VEGFR-2 and VEGF-a are the main players of VEGF dependant angiogenesis, they activate signalling pathways that regulate endothelial cell preliferation, survival and migration
describe stage 1 of angiogenesis *
astrocyte or macrophage produces VEGF
VEGF binds to VEGFR on endothelial cells - these cells become the tip cells which drive the formation of new bv and tells the cells next to it to become stalk cells
the tip cells lead the bv to gradients of VEGF
tip cell selection is controlled by the NOTCH signalling pathway between adjacent endothelial cells at the angiogenic front
describe the canonical notch signalling pathway *
this pathway is essential for development and basic functioning
notch receptors and ligands are membrane bound proteins that associate through their extracellular domains
the ligand and receptors are on opposite cells
the ligand on the tip cell binds to the receptor on the stalk cell - the notch intracellular domain (NICD) of the receptor is cleaved and enters nucleus - binds to TF RBP-J - regulates proliferation and the behaviour of the cell
one of the big ligands is Dll4
describe how the selection of tip cells relates to VEGF and notch *
in stable bv Dll4 and notch signalling maintain quinescence
VEGF increases activation of Dll4 expression
Dll4 drives notch signalling which inhibits VEGFR2 expression in adjacent cell
DII4 expressing tip cells acquire motile, invasive and sprouting phenotype
stalk cells form the base of the emerging sprout, and proliferate to support elongation
describe stage 2 of angiogenesis - stalk elongation and tip guidance *
the tip cell migrates to the tissue
integrins are adhesion receptors and make the cells move and sense the ECM
myeloid cells are recruited - they support sprouting by carving a tunnel in matrix so endothelial cells dont have to work so hard - the macrophages wrap around the tip of the bv
platelets are full of regulators of angiogeneis - they release podoplanin, clec 2, GF and cytokines for physiological angiogenisis and p-selectin, vWF, PMPs, VEGF and PDGF for pathological angiogenesis
describe stage 3 of angiogenesis - stabalisation and quinescence *
need to form juntions - they are homophilic interactions ie by the same proteins on opposite cells - the proteins are TM proteins - have extracellular bit that binds and intracelluler bit that controls signalling
at adheren junctions adhesion is promoted by cadherin
at tight juction adhesion is mediated by claudins, occludin, members of the JAM family and ESAM
these junctions control permability, allow inflammatory cells to enter, and control contact inhibition - allow endothelial cells to grow in a single layer
the junctions promote the survival of
describe the role of pericytes *
they are mural cells - they help stabalise the neovessels
they dont cover the whole of the vasculature - they just wrap around
they produce molcules for stability ie angiopoitin
describe the angiopoitin-tie 2 ligand receptor system *
Tie2 is an endothelial receptor - Ang-1 and ang-2 bind to it
ang-1 binds in homeostatic bv to stabalise junction and promote survival - inhibits inflammatory gene expression
when bv is activated because of inflammation or during angiogenesis - ang-2 is released from weibel-palade bodies, transcription is increased; ang-2 synergises with VEGF
ang-2 antagonises ang-1 signalling and promotes vascular instability and VEGF-dependant angiogenesis
ang-2 plasma levels are increased in congestive HF, sepsis and CKD - high levels of ang-2 is not a good thing because destabalising systemically
it modulates angiogenesis but is not essential for initiation
describe angiogenesis in tumours *
tumors <1mm3 recieve oxygen and nutrients by diffusion from host vasculature
large tumours require a new vessel network
tumours secrete angiogenic factors eg VEGF driven by hypoxia that stimulate migration, proliferatioon and neovessel formation by endothelial cells in adjacent established vessels
tumours dont secrete all factors and stimulation of bv is not normal
newly vascularised tumour doesnt rely on diffusion from host vasculature, fascilitating progressive cell death
what is teh angiogenic switch *
a discrete step in tumour development taht can occur in differnet stages in tumour progression pathway - depending on the tumour and its microenv
tumour goes through stafes - dormant - perivascular detachment and vessel dilation - onset of angiogenic sprouting - continuous sprouting (new vessel formation and maturation, recruitment of perivascular cells) - tumour vasculature
describe tumour bv *
they are irregularly shaped, dilated and tortuous
not organised into definitive venules, arterioles and capillaries,
leaky and haemorrhagic - partly becasue of over production of VEGF
perivascular cells are loosely associated
some tumours may recruit endothelial progenorator cells from the marrow - controversial
there are occlusions
describe the promotion of tumour angiogenesis *
it is a multi-cellular response
cancer associated fibroblasts secrete ECM and factors that modulate angiogenesis (VEGFA, FGF2, CXCL12, PDGFC) - doesnt give off all the factors involved physiologically
pericytes are loosely associated with tumour associated bv (TABV) - this favours chronic leakage - enhanced by ang-2
tumour has haemorrhage because bv are not functioning properly - attracts platelets - platelets release proangiogenic mediators and proteases that support the proliferation and activation of cancer associated fibroblasts - PDGFB and TGFbeta
describe the role of platelets in tumour angiogenesis *
there is a link between cancer progression adn thrombocytosis
activated platelets are a source of pro-angiogenic factors - VEGFA, platelet derived GF (PDGFs), FGF2
also a source of angiostatic molecules - thromobospondin 1, plasmin activator inhibitor 1 (PAI1), endstatin
tumours cause plateley activation, aggregation and degranulation
disrupting platelets doesnt impair tumour angiogeneisis - but the overall action of platelets is pro-angiogenic
describe therapeutic strategies to inhibit VEGF signalling *
anti-VEGF Ab - molecules block VEGFR and then downstream signalling
aflibercept blocks VEGFA and VEGFB adn PIGF
bevacizumab blocks VEGFA
ramucizumab blocks VEGFR-2
can get VEGFR kinase inhibitors
evidence for VEGF inhibition by VEGFR1 (Flt-1)*
cells transfected with control or sFlt-1 plasmid to promote Flt-1 (VEGFR1 - soluble bit of receptor) expression
VEGFR1 binds to VEGF and mops it up - so VEGF cant bind to receptor on cells
Flt-1 acts in vivo to reduce tumour growth but not in vitro - indivating bv involvement
what is avastin *
FDA approved
it is anti-VEGF monoclonal Ab
describe avastin SE and limited efficacy *
no overall survival advantage over chemo alone
no QOL advantage
benefits are transient - followed by restoration of tumour growth and progression - tumour becomes resistant
SE are because VEGF is essential endo survival factor
SE:
- GI perforation
- Hypertension
- Proteinuria
- Venous thrombosis
- Haemorrage
- Wound healing complications
potential mechanisms of SE for anti-VEGF therapy in cancer *
VEGF inhibition aggravates hypoxia increasing tumour;s production of other angiogenic factors/increases tumours invasiveness
tumour vessels may be less sensitive to VEGF inhibition due to vessel lining by tumour cells or endo cells derived from tumours
tumour cells that recruit pericytes may be less responsiev to VEGF therapy
tumour cell vasculogenic mimicry - also known as vascular mimicry - describes plasticity of aggressive cancer cells - forming de novo vasculature - tumour cells pretend to be bv and form channels, vessels hook up to channels within tumour mass, so doesnt matter if block vegf
what are the anti-angiogenic strategies *
aim to normalise the vasculature
dont block all factors - otherwise make it hypoxic and also chemo will not be able to access the tumour
if you kept giving aggressive anti-angiogenic therapy - you would damage healthy vessels and create vasculature resistant to treatment and inadequate for delivering drugs and ox
what is the future for angiogenic therapy *
- 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
describe the process of finding novekl treatment mechanisms*
single cell RNASeq of tumour endo
this allows you to study all genes transcribed in cell by studying RNA
gives you a profile of expressuion of individual cells in body
eg look at tumour and normal endothelial cells and can see that the endothelial cells are differnet - know what genes are expressed in tumour and not in normal endo - help you find new molecular targets
describe anti-VEGF for age related macular degeneration
AMD is because of abnormal growth of choroidal bv - they are leaky vessels and cause oedema = visual impairment
avastin was used off label - lucentis was developed from avastin for treatment of AMD
High efficacy of both treatments in maintaining or improving vision
Many patients become refractory to treatment >2 years
what is the challenge in finding therapeutic strategies to inhibit ang *
tumours are complex 3d structures with own micro-env - lack in-vitro models
studies are performed on cell lines as 2d monolayes that dont mimic interplay between tumour and env
phenotype of tumour in 2 an 3d is different
crucially vascualture is not involved in vitro
describe tumour on a chip *
it is the development of new in vitro models for anti-angiogenic drug screens
model that is closer to the body microenv
have blood and all variety of cells
bv grow on microchip - put tumour cells on - show the growth of cells - use chemo and show that they work in this network
why would you use pro-angiogenic drugs *
to alloow drug delivery to the tumour - the chemo drugs need to reach a certain concentration to be effective - they would not be effective with anti-angiogenesis drugs
