Tumor angiogenesis Flashcards
Overview: Tumor angiogenesis
1) Hypoxia
2) ECM degradation
3) Tip cell migration
4) tube formation
5) regulation of vessel size
6) pericyte recruitment
7) tumor angiogenesis -> cancer treatment?
Hypoxia
- Hypoxia is a physiological state characterized by decreased oxygen levels in organs and tissues
- Definition of hypoxia is an oxygen conc. of ≤2%, while concentrations of ≤0.02% oxygen are defined as severe hypoxia or anoxia
- Affects cellular functions and disrupts various biological processes including cell proliferation and differentiation, metabolism and pH homeostasis
- Solid tumors can grow to a size of approx. 1–2 mm3 before their metabolic demands are restricted due to the diffusion limit of oxygen and nutrients
Median oxygen concentrations as measured in selected mammalian tissues:
- ambient O2: 21%
- brain: 4.4%
- arterial blood: 13.2%
- lung: 5.6%
- kidneys: 9.5%
- uterus: 3-5%
- liver: 5.4%
- muscle: 3.8%
- venous blood: 5.3%
Spheroids
- Three-dimensional culture of cells can mimic tumor formation
- Investigating hypoxic conditions: Pimonidazole (green) binds to reduced thiol-containing proteins in hypoxic cells; cells are visualized with nucleic acid stain (blue)
Proliferation rate in spheroids:
* High in the outer area
* Low in the center
Apoptosis rate in spheroids:
* Low in the outer area
* High in the center
Hypoxia inducible factor (HIF)
- Hypoxia inducible factor (HIF) transcription factor
- Dimeric transcription factor composed of an oxygen-dependent subunit (α) and an oxygen-independent subunit (β)
- 3 subtypes: HIF-1 -> ubiquitously expressed and best investigated
bHLH – basic Helix-loop-helix domain required for dimerization
PAS – domain identified to be required for dimerization
ODDD – O2-dependent degradation domain
TAD – transactivation domain
NLS – nuclear localization signal
HIF regulation
Hypoxia-inducible factor alpha subunit -> Proline Hydroxylation -> + VHL leads to VHL conjugation -> ubiquitination -> targeting to proteasome -> proteasomal degradation -> peptides
Vascular endothelial growth factor (VEGF)
- HIf-1 activates the transcription of factors such as VEGF
- All members of the VEGF family (VEGF-A, VEGF-B, VEGF-C, VEGF-D, PGF) stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface
- Involved in vasculogenesis (de novo formation of the embryonic circulatory system) and angiogenesis (growth of blood vessels from pre-existing vasculature)
Vascular endothelial growth factor receptor
- Three main subtypes of VEGFR, numbered 1, 2 and 3
- Two VEGFR form a dimer to activate autophosphorylation of tyrosine residues on the cytoplasmic domain
- Consist of extracellular portion with 7 immunoglobulin-like (Ig) domains, a single trans- membrane spanning region and intracellular portion with a split tyrosine-kinase domain
- Structure of VEGFR: Ig-like VEGF binding domain, Tyrosine kinase domain
Tip cell formation
- Endothelial cells spearheading the vascular sprouts
- Characterized by position, long and dynamic filopodia and migratory behavior
- Respond to attractive and repulsive directional cues presented by the environment and thereby define the route in which the new sprout grows
- Required to create new connections between different sprouts to generate an interconnected and functional vascular network
Pericyte detachment
- Pericytes are multi-functional mural cells of the microcirculation that wrap around the endothelial cells ➜ embedded in the basement membrane
- regulation of blood flow, angiogenesis, structural stabilization, and vascular permeability
Pericyte-to-endothelial cell ratios in humans:
1:1 in CNS ➜ blood brain barrier
1:1 in the retina ➜ blood retina barrier
1:2.5 in the kidney ➜ glomerular filtration
1:10 in the liver ➜ stellate cells
1:10 in the lung
ECM degradation
- The extracellular matric (ECM) supports adhesion of cells and transmits signals through cell-surface adhesion receptors
- Matrix-Metalloproteinases (MMP) are calcium-dependent zinc containing endopeptidases degrading all kinds of extracellular matrix proteins
- Synthesized as inactive zymogens with a pro-peptide domain that must be removed before the enzyme is active
-> Extracellular activation by thiol modifying agents (e.g. by proteases) - Collagen: MMP1, 2, 8, 9, 13
- Laminin: MMP7, 10, 11, 14, 15, 16
- Elastin: MMP2, 7, 9, 12
- Fibronectin: MMP10, 11, 12, 14, 15, 16
- Proteoglycans: MMP3, 7l 10, 11, 12
Pericyte detachment
A.) Ischemic tissues (yellow) induce pro-angiogenic factors (VEGF and Ang2)
B.) Pro-angiogenic factor signaling destabilizes endothelial cell - pericyte interactions; promoting pericyte detachment
C.) Sprouts grow and penetrate hypoxic tissues; PDGF release activates and recruits pericytes
D.) Pericyte-derived Ang1 antagonizes the Ang2 receptor (Tie2) and serves as a stabilizing factor that strengthens pericyte-EC interactions
E.) Vessel maturation and lumen formation
Angioporetin/Tie signaling
- Angiopoietins (Ang) are a family of vascular growth factors and ligate to the endothelial cell tyrosine kinase receptor Tie2
- Angs can form dimers, trimers, tetramers and higher order multimers, but the Tie2 receptor can only be activated at the tetramer level or higher
Mechanism: - Ang1 acts paracrine; secreted by pericytes
- Ang1 binding to Tie-2 mainly on endothelial cells (but also on pericytes) promotes pericyte binding
- Ang2 is almost exclusively expressed by endothelial cells in Weibel-Palade bodies (WPB)
- Ang2 acts as dynamic autocrine modulator of Ang1/Tie2 signaling
- Ang2 acts paracrine on pericytes, thereby contributing to vascular destabilization through direct pericyte effects
Stalk cell formation
- Stalk cells follow the tip cells
- Produce fewer filopodia, are highly proliferative, establish adherent and tight junctions
- Ensure the stability of the new sprout, and form the nascent vascular lumen
- Endothelial tip cell: Filopodia (polarized), Migrates, Proteolytic, Limits tip cell number, recruits pericytes
- Endothelial stalk cells: proliferate, lumenogenesis
- Endothelial phalanx cells: quiescent, single cell layer, apical-basal polarization
Tip and stalk cell communication
How to select which cell is a tip and which is a stalk cell?
* Tip cells express VEGFR2/3 together with several co-receptors (neuropilin-1 (NRP1))
* Tip cell attraction by VEGF gradients
* Upon activation: tip cells express DLL4, that binds to Notch receptors on follower stalk cells
* In stalk cells: Notch is cleaved by γ-secretase, releasing Notch intracellular domain (NICD)
* NICD dampens VEGFR2/3 expression and increases VEGFR1 expression
* VEGFR1 is released by stalk cells and binds to VEGF molecules to block local VEGF signaling
* Jagged1 (Jag1) is expressed by stalk cells and negatively regulates Notch activity in tip cells
Platelet-derived growth factor
- Platelet-derived growth factor (PDGF) is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF-AB)
- PDGF-BB encourages maturation of the nascent vessels by activating and recruiting pericytes, which stabilize endothelial sprouts and prevent regression
- PDGF receptors are surface receptors, classified as a receptor tyrosine kinase
- PDGF-B expression is initially widespread and becomes concentrated to the sprouting tips
