Lectures 29 and 30 - Angiogenesis Flashcards
Describe the basic structure of blood vessel.
- Vascular endothelial cells form in the inner surface of blood vessels
- Basal lamina is an extracellular matrix layer that the endothelial cells sit on
- Pericytes form a around endothelial cells and are contractile, due to the blood flowing through the vessels
When does the growth of vascular endothelial cells occur?
Growth is developmental, during embryogenesis
What are the two processes that generate new blood vessels?
- Vasculogenesis
- Angiogenesis
What is Vasculogenesis?
Differentiation of precursor angioblasts into endothelial cells (de novo formation of primitive vascular network)
- forms the primary network
What is Angiogenesis?
Formation or remodeling of blood vessels from exisitng blood vessels
- eg., wound healing, pre-existing blood vessels will be reforming around the injury
How does Vasculogenesis occur?
- Mesoderms differentiate into hemangioblasts
- Hemangioblasts begin to form tube like structures which will become the blood vessels
- The tube-like hemangioblasts form a primary capillary plexus (a network)
How do you detect and study Vasculogenesis in vitro?
- Plate embryonic stem cells on gelatin + LIF
- When LIF is withdrawn, embryoid bodies form
- To the plated strain, genetic modifications (gene overexpression and gene deletion) are done to the ES cells
- LIF is removed from the plate with the now modified ES cells
- Results in modified ES-derived embryoid bodies which you use to quantify the endothelial gene expression and analyse the vascular structure formation
- Stain cells with CD31, which is a marker for endothelial cells, to view the forming blood vessels
Vasculogenesis is highly restricted in adult tissues. During which four events does it occur in?
- During development, in the placenta, a whole new vascular system must be created
- In the the uterus, during the menstrual cycle
- During hair growth, the hair has to be supplied with nutrients and gas exchange
- During wound healing, has both angiogenesis and vasculogenesis traits
What is VEGF?
Vascular Endothelial Growth Factor
- regulates angiogenesis
What occurs to the cell when VEGF binds to the VEGF receptor?
- The VEGFR is located on the surface of endothelial cells
- When VEGF binds, a signal transduction cascade is adtivated
- VEGFR phosphorylates a tyrosine kinase, which activates it
- The tyrosine kinase then activates PI3K, p38MAPK and Raf
Why is it important that PI3K, p38MAPK and Raf become activated during the binding of VEGF?
- PI3K activates Akt/PKB which lead to vascular permeability and endothelial cell survival (need the cell to survive during remodeling)
- p38MAPK is responsible for endothelial cell migration (cells must go to the right place during remodeling)
- Raf activates MEK which activates Erk, which is required for cell proliferation (cells need to grow at the remodeling location)
What are the VEGF family members that are responsible for vasculogenesis and the ones responsible for tumour angiogenesis?
VEGF has different isoforms and binds with specificity to their cognate receptor
- VEGF-B is responsible for vasculogenesis
- VEGF-C and D are responsible for endothelial cell proliferation, migration and survival which could lead to tumour angiogensis
Why do you have to remodel the environment that the new vascularture is building off of?
- To allow the endothelial cells to lay down and fit/grow
- Involves a large amount of integrin remodeling
When VEGF-A binds to VEGFR-2, what occurs downstream?
- Raf and PI3K are activated
- Raf activates MEK which activates MAP kinase which results in cell division
- PI3K activates Akt which results in cell survival
- Permeability migration mobilization of precursor cells
What did Judah Foulkman discover?
- He came up with the idea that tumour angiogenesis was a central part of the tumour process
- When tumours are small, they can survive with just diffusion of nutrients
- But as they grow, diffusion is no longer sufficient to meet their nutritional needs
- A “switch” occurs (Tumour angiogenesis factor to initiate remodeling)
- This resulted in massive tumour cell survival and growth
What did Judah Foulkman hypothesis about tumour angiogenesis?
- Vascular limitation in tumour growth
- Angiogenic switch in tumours for nutrient replacement
- Tumours produce angiogenic factors that initiate remodeling (later discovered to be VEGF)
- Blocking tumour angiogenesis to inhibit tumour growth
How large can tumors grow before hypoxic conditions arise?
Tumors can grow avascular to a maximum size of 1-2 diameter before hypoxic conditions arise
How do hypoxic regions form in tumors?
- Tumor cells closest to the capillary is well-oxygenated
- As the tumor grows, hypoxic regions begin to form dur to diffusion not being enough to supply nutrients to the cells farther away from the capillary
- The cells would begin to undergo programmed cell death or become necrotic
Before angiogenesis can resupply nutrients, what occurs to the hypoxic tumors?
Their metabolism changes to adapt
- switches to glycolysis
- shuts down the entry of pyruvate into the TCA cycle
What is HIF1-alpha and how does it prevent pyruvate from entering the TCA cycel?
In hypoxic tumour cells, HIF1-alpha levels are high because it is a master sensor of oxygen
- A transcription factor
- TCA cycle is turned off because there is no oxygen so efforts are put into telling the cell to take up more glucose
- hypoxia inducible factor
- It inhibits PDK1 from assisting pyruvate form entering the mitochondria by activating pyruvate dehydrogenase which phosphorylates PDK1 and inhibits it
- Pyruvate is converted into lactate (a glucose transporter) by LDHA
During hypoxia, how are the new vessels different from normal vessels?
The new forming vessels may form temporary occlusions (a pinched of capillary), blind ends (dead end), shunts (bridges between vessels that reduce the flow) and are leaky (breaks in vessel walls)
- the new forming vessels are formed quickly to provide a nutrient source to survive
- pathological situation, not developmental
- An irregular vessel system
What does HIF-1 target?
It targets genes that support survival and stimulate angiogenesis (such as VEGF)
What happens to HIF-1 when there is sufficient oxygen in the environment?
HIF-1 is broken down by ubiquination
- Pyruvate dehydrogenase (PDH) adds an OH group onto the HIF-1 which results in it binding to VHL and getting a ubiquitin tag
What processes are activated by HIF-1 alpha binding to DNA?
- Metabolic reprogramming (GLUT-1)
- Angiogenesis (VEGF)
- Anti-apoptotic (IGF2)
- Stem cell properties (DLK1)
- Metastasis (COL5A1 - cells need to get to the location of the nutrients)
- Therapy resistance (ABCB1)
What is the angiogenic switch necessary for?
It is necessary to promote and recruit new host vasculature to support nutrient demands
- post angiogenic switch: blood vessels have infiltrated the tumor from the outside and within to provide the nutrients
How is remodeling initiated?
Tumors produce a wide variety of angiogenic factors that act on endothelial cells and initiate remodeling
- proangiogenic factors (involved in survival, movement, etc of the cell) outnumber endogenous inhibitors and breaks the tipping balance
What are the proangiogenic factors?
- VEGF
- FGF
- PDGF
- Ang
- IGF
- HGF
What are the five main steps in angiogenesis? Which factors are involved in each step?
- Signals (such as VEGF) are released which initiates activation
- Basement membrane degradation (MMPs involved)
- Endothelial cell proliferation and migration (VEGF involved)
- Tube formation, elongation and remodeling (integrins involved)
- Maturation (Ang1/2 and PDGF involved)
What is thought to be the role of ANG-1?
When it binds to the receptor TIE2, it is thought to increase circumferential proliferation (increase the circumference of the vessel)
How is angiogenisis regulated?
- It is a balance between “on” and “off”
- The balance between activators, such as VEGF and ANF-1, and inhibitors, such as IL-12
When tumor angiogenesis is blocked, what occurs to the cells?
Tumor angiogenesis was blocked to inhibit tumor growth
- Endothelial specific HIF-2 alpha was knocked down
- The result of this was the KO tumors were smaller, lighter, underwent more proliferation and underwent more apoptosis
What do drugs in development target in angiogenesis?
They either block the signal on the outside of the cell (at the receptor) or inside the cell (blocks the downstream kinase signaling angiogenesis)
Problems of getting the drugs into the cell and may lose specificity depending on where you target.
What does Bevacizumab inhibit?
It inhibits VEGF by binding it to prevent it from binding the VEGFR.
- Cannot undergo angiogenesis.
- An antibody that bind VEGF
- Doesn’t have to enter the cell to work
What does Sunitinib Sorafenib inhibit?
It inhibits VEGFR from activating PI3K. Cannot undergo angiogenesis.
What does Sorafenib inhibit?
It inhibits Raf to prevent the downstream activation of Mek and Erk. Endothelial cells are unable to proliferate.
How does tumor vasculature impact host immunity?
- As tumor cells become hypoxic, they can separate and migrate to other sites and metastasize
- Once they have metastasized and evaded, they release factors that inhibits the immune attack (includes TGF-beta)
- Encourages the production and activation of Tumour activating macrophages
How does a high dose of anti-angiogenic therapy work?
- The abnormal growth becomes pruned and a immunosuppresive tumor microenvironment develops
- The tumor inhibits T effector cells by itself and by increasing the production of M2-like TAMs, which also inhibit T effector cells
How does a low dose of anti-angiogenic therapy work?
- The abnormal growth becomes normalized and immunosupportive tumor microenvironment develops
- More M1-like TAMs are produced which results in the activation of T effector cells
