Lecture 7 Flashcards
what happens when there is a lack in vascularization in regenerative medicine ?
gradient of malfunctoning : necrosis, and around it are alive cells but that didn’t differentiate into the required tissue
PAD : when is the critical stage ? current treatments ?
When there is pain at rest.
Bypass surgery and catheter based revascularization -> for many, that is not an option
what is the difference between arteriogenesis and angiogenesis ? Link between the two ?
1) arteriogenesis : formation of collateral arteries by enlargement of pre-existing small vessels -> induced by shear stress + monocytes
2) angiogenesis : expansion of capillary network responsible for metabolic exchanges -> induced by ischemia = lack of oxygen + nutrients
Angiogenesis induces arteriogenesis !
what is the factor that regulates the repsonse to hypoxia (lack of oxygen) ? Description
HIF-1.
It is constantly produced.
If normal O2 -> hydroxylation and degradation of HIF-1alpha.
If hypoxia -> HIF-1alpha gets stabilized -> goes to nucleus -> stimulates angiogenesis
3 steps of blood vessel growth (factors and cells involved)
1) induction : VEGF is the kick-off, sticks to ECM -> gradient that shows to the endothelial cells where to grow
2) Maturation : PDGF-BB produced by endo cells (also sticks to matrix) -> pericytes proliferation + migration
3) Stabilization : pericytes control proliferation of endothelium (signaling)
which VEGF type and receptor are we interested in ?
VEGF-A and receptor VEGFR2
VEGF-A isoforms : which are the most important, what is different between them, how do they bind to ECM and why it’s required (3 things)
189, 165, 121 : different exon recombination -> different length (nb of amino acids).
Positive and negative charges allow bonding to ECM -> amount of charge determines strength.
Required for gradient formation, modulation of receptor stimulation, synergistic signaling with integrins
what is the best degree of binding to ECM for normal vascular networks ?
Intermediate sticking (VEGF 165, or combo of short and long)
what are the different types of gene therapy vectors ?
Non-viral
Viral -> integrating (in genome) or non-integrating
what is an issue with VEGF (dose and duration) -> one way to solve it ?
Uncontrolled VEGF causes angiomas (aberrant vessels) but VEGF expression needs to last at least 4 weeks to achieve vessel stabiliztion.
Reducing the TOTAL dose (heterogeneous gene expression levels) still results in aberrant vessels.
Solution : microenvironmental VEGF levels (HOMOGENEOUS gene expression level) -> threshold between capillaries and angiomas. (70-100 ng/10^6 cells / day)
general question : 2 ways to control VEGF potency
1) control dose distribution
- genetic engineering
- factor decorated matrices
2) target downstream pathways that modulate the outcome of VEGF signaling
control VEGF expression by genetic engineering : how does it work ?
VEGF gene is linked to a cell-surface marker (CD8) through an internal ribosomal entry site (IRES). VEGF and CD8 are produced in same quantities : VEGF is released and CD8 binds to membrane -> can be detected with fluorescence
-> sorting cells based on CD8 expression
one problem with VEGF expressing mesenchymal cells for angiogenesis in bone grafts
Increase of angiogenesis but also increased osteoclast recruitement and bone resorption. So yes flow improved but less bone …
solution against bone resorption : decoration of fibrin matrices : how does it work ? result ?
No genetic modification this time.
We bind VEGF to TG (transglutaminase), which naturally cross-links to the fibrin matrix = enzymatically attached
-> increased vacularization and efficient bone formation
osteogenic grafts decorated with TG-VEGF : effects of VEGF dose ? Ideal dose ?
The dose controls speed and depth of vascular invasion.
Perfect dose = 0.1 microg/mL :
- proliferation up to core
- improved bone tissue formation
- prevents excessive osteoclast recruitement
- promotes MSC differentiation
