7th March - Positive regulators of angiogenesis Flashcards
What is angiogenesis?
The sprouting of blood vessels from existing ones
What is vasculogenesis?
The de novo formation of blood vessels from endothelial cell precursors
What is a chick chorioallantoic membrane assay (CAM)?
- Implant a membrane/coverslip coated with compound of interest on a CAM of fertilised egg through a hole in the egg shell
- Incubate for 1-3 days
- Quantify vascularisation using image analysis or colourmetrics
What did Folkman state in 1971?
That tumours secrete an unknown factor to help increase its blood supply and if blocked the tumour will wither and die
Outline Folkmans 1966 experiment
Grew tumours in isolated perfused organs these were limited to 1-2mm3
Implanted these tumours into mice and the expanded rapidly to 1-2cm3 due to vascularisation
Outline Lien’s 1970 experiment
Resin Cast of metastasis in rabbit liver through injection into the blood supply showed that the tumours are:
- Avascular up to 1mm3
- Vascularised >1mm3
Outline Tannock’s 1970 experiment
[3H] Thymidine labelling index of tumour cells decreases with increasing distance from the nearest open capillary. [3H] thymidine labelling shows the extent of cell division
Outline Gimbrone’s 1972 experiment
Tumours suspended in the aqeous fluid of the anterior chamber of the eye remain viable, are avascular and limited to 1mm3
Once implanted into the cornea, neovascularisation is rapidly induces and the tumour reaches 16000x its original volume in 2 weeks
Outline Knighton’s 1970 experiment
CAM assay
Used [3H] thymidine labelling index of endothelial cells, which decreased with age. Tumours implanted in older embryos have a decreased growth rate which parallels endothelial proliferation rate
What are the positive regulators of angiogenesis?
VEGF Angiopoeitins Fibroblast GF Placental GF Angiogenin Hepatocyte GF Platelet derived endothelial cell GF Interleukin-8 Granulocyte colony-stimulating factor
What are the negative regulators of angiogenesis?
Angiostatin Thrombospondin Endostatin Metalloprotease inhibitors TGF-beta Interferon alpha Platelet factor 4 prolactin 16kDa fragment Tumstatin
What diseases can be caused by excessive angiogenesis?
Rheumatoid arthritis Blindness Cancer AIDs complications Psoriasis
What diseases can be caused by insufficient angiogenesis?
Stroke Heart Disease Ulcers Scleroolema Infertility
Briefly outline the process of angiogenesis in a tumour
Hypoxic conditions –> angiogenic factor production –> endothelial cell receptor binding –> endothelial cell activation –> endothelial cell proliferation –> directional migration –> ECM remodelling –> tube formation –> loop formation –> vascular stabilisation
What is the most potent isoform of VEGF?
165VEGF
Where do VEGF isoforms tend to differ?
In exon 6 and 7 - the heparin binding region
What is the function of VEGF?
It is angiogenic
It induces expression of serine proteases uPA and uPAR
It stimulates vascular permeability by loosening endothelial cells and causing plasma protein leakage and extracellular fibrin gels
It induces interstitial collagenasse in human umblical cord and vein endothelial cells (HUVECs)
It stimulates integrin expression promoting the migration of endothelial cells
- -collagen/lamin receptors: alpha1beta1, alpha2 beta1
- -vitronectin/fibronection receptors: alpha v beta 3, alphav beta 5
What are the VEGF receptors?
VEGFR1
VEGFR2
VEGFR3
Outline the transgenic models used to demonstrate that was used to determine the role of VEGF and it’s receptors
VEGF A -/- = Embryonic lethal, absence of vasculature
VEGF A -/+ = Embryonic lethal, severe vascular abnormalities, half dosage effect
Post-natal VEGF inactivation = Lethal in the first few weeks
VEGF B -/- = Normal fertile mice, hearts reduced in size –> VEGFB important in coronary vascularisation
VEGF X OE –> lymphatic hyperplasia
PlGF KO –> overtly normal, defect in adult vascular remoedelling
VEGFR-1 -/- = Embryonic letha, endothelial cells disorganised
VEGFR-2 -/- = Embryonic lethal, absence of endothelial cell precursors
VEGFR-1 Kinase domain deletion = Overtly normal with mild defect in monocyte migration –> VEGFR-1 kd not required
Outline Kim’s (1993) experiment to show VEGF was required for angiogenesis in tumours
Injected human rhabdomyosarcoma, glioblastoma multiforme or leiomyosarcoma cell lines into nude mice - 10^6 cells injected subcutaneously
Treated with anti-VEGF Ab –| Tumour growth in vivo, no effect on growth rate in vitro
Had greatest effect on the rhabdomyosarcoma as this is the most rapidly proliferating and therefore more angiogenesis dependent tumour
The Ab was injected intra peritaneally, twice a week
Explain the results of Millauer’s (1994) experiment to show that glioblastoma growth was inhibited by Flk-1 mutant
Dominant negative FLk-1 mutant –| glioblastoma growth
Emphasises the key role of VEGF/VEGFR in tumour growth
Produced MSV based genome with mutant lacking TM domain
Tumour volume was much lower without VEGFR
How does HIF-1 regulated VEGF?
It binds the hypoxic response element in VEGF-A promoter, up regulating VEGF transcription
HIF-1alpha also stabilises VEGF mRNA and IRES mediated translation maintains protein levels
How is HIF-1 activated?
Under hypoxic conditions Prolyl hydroxylase does not target HIF1alpha meaning that the VHL protein does not bind targeting it for proteasomal degradation
What is Von Hippel Lindau Disease?
A heriditary cancer syndrome which causes renal carcinomas, CNS hemangioblastomas
Results from pVHL inactivation leading to active HIF1alpha leading to increased VEGF
Outline the function of VEGF165b
In a corneal pocket assay it inhibits VEGF stimulated blood vessel growth
What TRKs are important for angiogenesis?
Angiopoeitins - Tie 1 and Tie 2
Outline a study that shows that Tie-1 is vital
Tie 1 -/- = embryonic lethal Impaired structural integrity of ECs Lack of supporting pericytes Oedema Haemozigging
What receptor does Ang-1 bind to?
Tie 2
Describe the phenotype of Ang-1 KO
Lack of pericyte recruitment during embryonic development
What receptor does Ang-2 bind to?
It inhibits Tie-2
What is the main function of Tie-2?
Key in capillary sprout formation
What is Tie-2?
Classic RTK which activates PI3K and FAK and with low potency the MAPK pathway
Describe the phenotype of Tie2 -/-
Embryonic lethal
Defect in development of endothelial cells in the heart
What is the role of Tie 2?
Dual functional
Ang-2 –> Tie 2
- -Pericyte loosening
- -Endothelial cell migration
- -Allows VEGF to bind to receptors and stimulate migration by loosening endothelial cell junctions
- –> Destabilisation
Ang-1 –> Tie 2
- -Recruitment of mesenchymal cells
- -Vessel stabilisation
- -At the end of angiogenesis process
- -Recruits Ang-1
- -Replaces GFs which attract the mesenchymal cells
- –> stabilisation
What is the evidence for the supporting role of alphavbeta3 in angiogenesis?
Not detectable in quiescent endothelial cells, but induced by angiogenic GFs
alphavbeta3 blocking Ab or peptide antagonists block blood vessel formation in vitro
alphavbeta3 block antibodies promote apoptosis of angiogenic vessels
alphavbeta3 binds MMP2
What is the evidence for alpha1beta1 and alpha2 beta1’s supporting role in angiogenesis?
Angiogenesis model system: human MEL2 tumour cell line engineered to OE VEGF
- -Subcutaneous injection in ECM gel in nude mice
- -IP injection of Ab to alpha 1 or alpha 2 integrin subunits –| angiogenic response
–> Collagen receptors are important in angiogenesis
