Angiogenesis

drugs for anti-angiogenesis?

*Iressa* blocks VEGF production *Avastin* neutralizes VEGF * Sutent* blocks VEGF receptor

examples of pro-angiogenic mediators?

*VEGF* -- most important/well-studied bFGF PDGF IL-8 HGF (hepatocyte growth factor) PIGF (placental growth factor)

name the 3 common anti-angiogenic drugs used clinically?

*avastin/bevacizumab* [single target] *sunitinib/sutent* [multiple targets] *sorafenib/nexavar* [multiple targets]

what are the 2 general strategies for anti-angiogenesis therapy?

1. reduce the activators - expression/production - bioavailability - signaling [most common target: VEGF] 2. increase the inhibitors - production - exogenous addition

Concept Review 3

Have to have switch turned on for angiogenesis to occur. Activity have to outweigh activity of anti-angiogenic mediators. Turned on through inflammation which causes an increase in activators because inflammatory cells are produin

can we target angiogenesis to treat cancer?

Judah Folkman, MD -Credited with the idea of anti-angiogenesis as a cancer therapy -Led research on regulation of angiogenesis and the identification of novel anti-angiogenic molecules

tumor progression?

This happens in the progression of different tumor types. Multi step process where benign tumor becomes malignant. As that progresses you see increase in angiogenesis. Off to on switch requires pro-angiogenic mediators to outweigh anti-angiogenic. switch in pics: OFF, ON, OFF, ON

Concept Review 4

Turn switch off to starve tumor. Starve it by targeting angiogenesis. Treat with inhibitors causes vessel regression Find ones that target multiple types

pro-angiogenic mediators?

a wide range of molecules are capable of stimulating angiogenesis

what are some of the characteristics of tumor vessels?

abnormal vessels! - vessels are *increased in number* compared to normal tissue - vessels have a disorganized distribution and are *tortuous* - vessels are *extremely permeable* (leaky) - coverage of basement membrane and pericytes around vessels is *abnormal* inject a dye into circulation. normal tissue it stays in blood vessels since they don't allow leakage, in a tumor, lots of blood vessels are leaky and you can see the dye leaking out basement membrane (ECM that surrounds them normally) doesn't completely cover bv. pericytes that wrap around bv are abnormal too!

how do new blood vessels form?

angiogenesis is the formation of blood vessels from existing blood vessels vasculogenesis is de novo blood vessel formation

what turns on the angiogenic switch?

*inflammation* -- delivery of pro-angiogenic mediators by inflammatory cells *hypoxia* -- sensing of low oxygen by HIF (hypoxia-inducible factor) and stimulation of pro-angiogenic mediator production Low oxygen levels: certain TFs like hypoxia inducible factors will be activated. That'll cause the transcription of pro-angiogenic mediators. Inflammation can also stimulate angiogenesis because inflammatory cells deliver pro-angiogenic mediators. Injury: see both of these things happening. Damaged vessels AND inflammation.

angiogenesis?

- ECs in quiescent blood vessels are activates - ECs proliferate, migrate, and differentiate to form new bv sprout - 2 adjacent sprouts fuse

blood vessels in cancer?

- blood vessels are needed for tumor growth and can be used to metastasize - blood vessels are part of the *tumor microenvironment*

vasculogenesis?

- endothelial progenitor cells (EPCs) are recruited from the bone marrow - EPCs incorporate into blood vessel - EPCs proliferate and differentiate into endothelial cells

role of neovascularization?

- provides a blood supply (oxygen/nutrients) to the tissue - required for *embryonic development, reproductive cycle, tissue repair* - contributes to multiple diseases, including *cancer*

problems with anti-angiogenesis therapy?

-High cost ($100,000/year for Avastin) -Modest overall survival advantage for patients -Side effects -Resistance

avastin/bevacizumab [single target]?

-humanized, function-blocking monoclonal antibody that binds VEGF -first VEGF inhibitor approved by FDA (2004) for colorectal cancer -used in combination with other chemotherapeutic drugs for metastatic colon cancer, metastatic renal cell carcinoma, non-small cell lung cancer, glioblastoma

intrinsic resistance?

-pre-existing non-responsiveness (tumors were always resistant) -absence of even a transient beneficial effect with therapy What kind of tumors do you think might not care if you inhibit angiogenesis? A lot of times you'll see this happen if you have a very highly vascularized tumor. So many blood vessels it doesn't care if you take some away! Or, if its hypovascular and its used to growing in a low oxygen environment.

sunitinib/sutent [multiple targets]?

-small molecule inhibitor of multiple receptor tyrosine kinases: VEGFR-1,-2,-3; PDGFR-α/β; c-KIT -approved for metastatic renal cell carcinoma, pancreatic neuroendocrine tumors, and gastrointestinal stromal tumors (GIST)

Sorafenib/Nexavar [multiple targets]?

-small molecule inhibitor of multiple receptor tyrosine kinases: VEGFR-1,-2,-3; PDGFR-β; c-KIT; raf-1 kinase -used to treat thyroid cancer, advanced renal cell carcinoma and unresectable hepatocellular carcinoma

evasive/acquired resistance?

-tumors respond for a limited period of time, then develop resistance to treatment -usually results from upregulation of alternative growth pathways Other type of resistance is evasive/acquired. In this situation, the tumors respond initially and end up regrowing and develop resistance. Usually this results from upregulation of alternative pathways. Normally a tumor that's growing will recruit new blood supply by angiogenesis that allows tumor to grow. Treat with VEGFi to shrink tumor. But because you have less bv's, the oxygen levels will be reduced! Hypoxia is one of the triggers for angiogenesis. That'll cause the production of pro-angiogenic mediators. Inhibit VEGF, a lot of other pro-angiogenic mediators will be produced. So, the tumor finds a way around the fact that you're blocking VEGF. Use combination therapies to inhibit VEGF and the alternative pathways!

side effects of anti-VEGF therapy?

Angiogenesis is uncommon in healthy adult tissues, but does occur during the female reproductive cycle, wound healing, and embryonic development... *wound healing complications* -- Increased percentage of patients with abnormal healing, wound dehiscence, etc. *abnormal fetal development* -- Even the loss of a single VEGF allele results in defective vasculature, impaired development, and embryonic lethality Wait until after the wound has healed. If a patient is about to go under surgery, wait for this treatment til AFTER so wound doesn't open back up.

resistance to anti-angiogenic drugs?

Anti-angiogenic drugs often fail to provide long-term clinical responses, which may be partially due to drug resistance

is VEGF direct or indirect?

DIRECT

what do VEGFR signaling pathways do?

Different VEGFR signaling pathways mediate endothelial cell survival, permeability, migration and proliferation Know VEGF binds to receptor which becomes phosphorylated and triggers downstream signaling that causes changes in endothelial cells.

steps of sprouting angiogenesis?

ECs line the lumen. light gray = ECs. tight barrier. outside of ECs is basement membrane, ECM that helps pack the bv. typically, just on the other side of the membrane you'll have pericytes or smooth muscle cells which help protect/stabilize the bv

...then?

Eventually if everything goes ok you have 2 adjacent sprouts that come together and form a mature vessel. Endothelial cells connected. Basement membrane. Pericytes being recruited! That's called *resolution*. Another thing that can happen to resolve this process is in the case that 2 new sprouts don't come together properly, typically you won't see blood perfusion and those vessels end up regressing, nonfunctional vessels regress.

concept of anti-angiogenic therapy?

Folkman: antiangiogenic drugs and anti-VEGF drugs will starve the tumor and cause regression = *Starvation hypothesis*

VEGF proteins and their receptors?

Lots of drugs target VEGF. Family of proteins that bind to multiple tyrosine kinase receptors. Lots of family members. When she says it she means *VEGF-A - primary one that's studied*. But there are others. All these proteins bind to different VEGF receptors and there are some co-receptors. But the *VEGFRs are tyrosine kinase receptors*. When the ligand binds to the receptor the receptor will be phosphorylated and that'll lead to downstream signaling.

Concept Review 1

Mediators cause loosening of ECs in quiescent vessel, proliferation and migration! Tip cell migrates out and other Ecs follow to form the new sprout. The process is resolved by 2 new sprouts binding each other and forming new stable connection which requires the endothelial cells to connect, basement membrane to be produced, and pericytes to be recruited. Or, if they don't connect properly, the new sprouts will regress and you'll be left with quiet vessels again.

are new vessels permanent?

NO - vessels are capable of regressing if the angiogenic stimulus is removed (removal of *TAF* = disappearance of capillaries)

what happens next in angiogenesis, after activation?

Once these endothelial cells in quiescent vessel become activated, you have loosening of junction between endothelial cells. Normally they're tightly opposed and have tight junction. Have to loosen up for cells to start migrating and forming new vessel. Need degradation of basement membrane. Tip cell is the first cell migrating out, the cells that follow are the stalk endothelial cells.

what happens during activation?

Phalanx EC in quiescent! Vessel surrounded by basement membrane and pericytes. Then an activation event occurs. Stimulus tells these blood vessels to make more! The major signals are low oxygen (hypoxia) or inflammation. And both of those things cause an increase in pro-angiogenic mediators! pro-angiogenic mediators: VEGF, Ang-2, FGF, chemokines

the angiogenic switch

There are also anti-angiogenic mediators. For angiogenesis to happen, the levels of activators of angiogenesis have to outweigh the inhibitors activity

advantages to using broad inhibitors?

broad inhibitors block multiple targets and multiple cell types ex: endothelial cells (VEGF) and pericytes (PDGFR) By targeting multiple pro-angiogenic mediators you can affect multiple cell types. These broad inhibitors block endothelial cells AND pericytes by blocking PDGFR signaling. What happens normally if you just treat with antiVEGF you see shrinkage of tumor. Take drug away. Vessels come back very quickly. Part of that's because some of the pericytes and basement membrane that line vessels can stick around. So here we have the blood vessels in green and pericytes stained by alpha-SMA in red. You can see when treat with just anti-VEGF some of the blood vessels don't go away but structured covered by pericytes. Tube with basement membrane and pericytes left there. Take antivegf drugs away see quick regrowth because the endothelial cells can repopulate those sleeves of old vessel.

tumor microenvironment?

everything besides tumor cells that support the growth. includes ECM, fibroblasts, inflammatory/immune cells, blood vessels that deliver oxygen/nutrients to help the tumor grow

blood vessel density correlation with tumor size/survival?

increase tumor volume, increase microvessels high microvessel density, higher disease probability

pro-angiogenic factors induce angiogenesis either...

indirect stimulate surrounding cells (cells that support angiogenesis) -- might have a mediator that recruits inf cells but doesn't affect endo cells directly. because it recruits inf cells that make pro-ang mediator, the ECs end up being stimulated and you get angiogenesis. another example of indirect is the GF like PDGF that affects the pericytes!

what did Folkman find regarding tumor growth?

previous observations established that angiogenesis occurs in tumors *tumor growth is angiogenesis-dependent* - angiogenesis ALWAYS coincides with tumor growth - tumors REQUIRE the recruitment of new blood vessels - avascular tumors are *dormant* and cannot grow more than ~2mm without new vessels to supply oxygen and nutrients - a *soluble factor 'TAF' (tumor angiogenesis factor)* is produced by tumor cells

capillaries?

small blood vessels that form a network within tissues to carry blood from arteries to veins; thin walls allow for efficient gas exchange

advantages to broad inhibitors again?

targeting VEGF and PDGFR should increase the effectiveness of anti-VEGF drugs We target VEGF and PDGFR. In theory can increase effectiveness of anti-VEGF. Pericytes normally help protect and stabilize. Treat with PDGFRi, they're detached and leave endo cells more sensitive to anti-VEGF therapy.

what is neovascularization?

the growth of new blood vessels angiogenesis is a TYPE of neovascularization! left: mouse skin, macroscopically new blood vessels grew in. right: CD31 stain endothelial cell marker

anti-angiogenic agents? NOT a comprehensive list

there are currently hundred of ongoing clinical trials testing anti-angiogenic agents She's showing this because it breaks it down into the type of drugs. VEGF blocking agents, small-molecules RTK inhibitors, and endogenous inhibitors!

inhibitors?

thrombospondin interferon alpha/beta angiostatin endostatin collagen IV fragments endostatin/angiostatin -- keep vasculature static, stop angiogenesis... interferon interferes with angiogenesis...

effectiveness of anti-angiogenic therapy?

treatment with VEGFR and PDGFR inhibitor in established tumors Its been shown to be a more effective strategy! Treat with an inhibitor that targets VEGF and PDGF signaling, see greater reduction in vessels and tumor size.

what are the receptors for many pro-angiogenic GFs?

tyrosine kinase, so most signaling inhibitors are RTKIs (ex: VEGF)