Flashcards in Angiogenesis: tissue engineering of capillary networks Deck (61):
what are the two main components of the vascular networks?
- cells and matrix (as with all tissues)
how can tissues be subdivided?
cell rich vs matrix mix
what is he vasculature system for?
Cells require nutrients, O2 and a system for the removal of metabolic waste products
what tissue doesn't have any vasculature?
where is cartilage found?
- at the point of joints- articular catalogue
what are the layers of cartilage?
- tangential layer, transitional layer, radial layer, calcified cartilage, bone
how does the oxygen supply change as you go through the layers?
as you move through the transitional layer, the tissue stats to require oxygen
why does cartilage need vasculature?
- low number of cells and they are quiescent
what is an example of a matrix risk tissue?
bone, tendon, muscle systems
are matrix rich tissues high in vasculature?
what is an example of cell wish organs?
- iver, kidney brain- require lots of vasculature
at what point in tissue growth is new blood vessel formation required?
- 100-200 microns
what is the rate of spontaneous blood vessel growth?
- several tenths of micrometers per day
what is the problem with the slow rate of vascular growth?
the disconnected tissue can necroses
where do enforthelial cells arise from?
- splanchnopleuric mesoderm
what is a vasculogenesis?
- formation of vessels from endothelial cells- a primitive network
what is angiogenesis?
- vascular nework is remodelled into more complex network
what is arteriogenesis?
- process of structural enlargement and remodelling of small arterioles into larger vessels
how can cells form a tube? (5)
- the first process that happens developmental is wrapping of a sheet- like in neural tube (this is used for larger vessels)
- budding- preexisitng tube gives rise to a bud of cells which gives rise to a tube
- cavitation: fused series of cells and lumen forms
- cord hollowing
- cell hollowing
how can intrinsic pre patterning and extrinsic responses to environmental parameters interact
- the intrinsic pre patterning can be influenced by the extrinsic responses to environmental parameters and this can work in the same way
what are endothelial cells?
Basic building blocks of endothelial sheets, capillaries, veins and arteries
- Promote stem cell development and organ formation
- Source of paracrine signalling
- Signals primarily used to promote endothelial development act directly on other cell types
- Reinforcing the complex interplay between vasculature and surrounding tissues.
- Earliest marker of angioblast precursors is Flk1 (VEGFR-2), which is the major receptor for VEGF-A.
what is the earliest marker of angioblast precursors?
flk1 (VEGFR-2) which is the major receptor for VEGF-A
how can angiogenesis occur in the adult?
- a tissue that is newly formed and requiring vasculature will be releasing angiogenic factors or an area of damage will be
- this will form a gradient
- cells within the lining of the vessel will respond to this and bud and start to move towards the angioblastic gradient
- the first cell that does this will be called the tip cell and this will be followed by the stalk cell
- the tip cell might divide allowing movement in two directions
- then process of cavitation and nd vacuoles will merge and lining up
then capillaries will mature
what are the 4 main component of angiogenesis?
- angiogenic growth factors
- cell matrix interactions
what are the main angiogenic growth factors?
VEGFs, FGFs, TGF-beta, BMPs, Notch, netrins, ephrins, semaphorins
what is the predominate ECM of most tissues in the body?
- collagen type I
how did they test how endothelial cells interact with collagen type I 3D gel?
- they had a 3D gel made from collagen type I
- the cultured endothelial cells on top of it
- they formed a sheet and they all travelled to the top of the cell
why did the think that the cells were moving to the top of the top of the sheet?
- polarity - they say signalling for par 3
- the cells naturally acquire apical basal polarity: at one time there is mate and one side there is medium
how did they measure the effects of cell-cell interactions?
they added bone marrow stromal cells to the culture - they found that instead of just going to the top, they were found all the way through
after seeing that the coculture resulted in cells all the way through, how did they try to make tubes?
- they added laminin and collagen IV which are basement membrane proteins
- they found semi tubular structures- end to end fusion of cells just by adding the matrix component
how did they measure the effects of different ECM components on tube formation in the ECM with endothelial cells?
-they then measured which combination were best: collagen I with collagen IV or collagen I with laminin and then they measured the average length of the cell to cell network, the average number of nuclei and the average area occupied
how did they investigate how laminin was causing the sheet?
- the targeted the receptor laminin- alpha 6 integrin y adding an antagonistic to the receptor and they reverse the tube formation. This showed that laminin is very important
how did they look at the effects of laminin on the uptake of growth factors on endothelial tube formation?
- they cultured endothelial cells (no somatic cells) only and with laminin they get sheets with collagen and without collagen.
- when they cultured the bone marrow stromal cells (stem cells) with collagen and laminin you get VEGF produced.
- when you use a co culture with collagen alone. You still get VEGF present in the medium
- but when you co-culture with collagen or laminin then the amount of VEGF decreases and his suggests that the cells are outpacing the growth factor.
- they then measured the activity of the VEGFR2 by looking for the expression of this in other collagen alone or collagen and laminin when there were no stream cells.
- they found that hey had 2% of cells expressing VEGF receptor 1 in collagen only and 4% expressing VEGF2. when you have collagen and laminin you se that 50% of cells are upregulating VEGFR2.
why is it that they use a cell source rather than adding the growth factors?
they use stromal bone marrows stem cells to supply a natural source of growth factors
how did they investigate the effects of cancer cells and a stromal body with ECs?
they developed a tumouroid model and then they had a tumour mass in collagen I and then a surrounding stroma which they populate with normal cells such as stromal cells. They always add lamininbecause they know this is important.
- they showed that they can grow a cancer which grows into the surrounding stroma and recruitment of vasclature and ingrowth of stromal cells into the dance
what is a cancer cell interaction with angiogenesis?
- how cancer cells form leaky vasculature- cancer cells spur out angiogenic growth factors in a random manner and this results in easy vasculature - diseased state of how vessels form.
how can you stop angiogenis from happening? for example in cancer?
- because tumours are good at forming poor vasculature- you want to normalise the vessel structure to allow drugs in to target the structure- cancer
- lower the release of angiogenic growth factors
- how cancer acquire resistance to anti angiogenic agents
- tumours can do vasculature mimicry
what do endothelial cells acres their origin with?
when can adult blood vessels proliferate?
- in response to physiological stimulus such as hypoxia for blood vessels and inflammation for lymph vessels
- angiogenesi is reactivated during wound healing and repair
name examples of diseases are linked to too much angiogenesis?
- inflammatory disorders
- autoimmune disease
what diseases are linked to too little angiogenesis?
ischaemic heart disease
how is angiogenesis linked to cancer?
- it allows the progression of tumours and metastasis
where re VECs progenitors found in adults?
- in the bone marrow
what have been used to generate Vascular endothelial cells and vascular smooth muscle cells?
HSCs and iPSCs
what is the problem with using HSCs or iPSCs to make VEC progenitors?
- there is yet to be a definitive endothelial cell marker
although not concrete, what marker has been used for VEC progenitors?
how can IPScs be used to help medicine in terms of breaking down the blood brain barrier, why would this be good?
As a specialized type of VECs, the brain microvascular endothelial cells (BMECs) compose the blood-brain barrier (BBB).This attribute also prevents the uptake of most neurotherapeutics and is com- monly compromised in neurological diseases, thereby driving great interest from pharmacology as well as disease modeling studies. However, because of the limited availability and fidelity of human BMECs, most BBB models demonstrated to date are either borrow- ing brain microvessels from primary animal sources or utilizing immortalized BMECs. Inevitably, they commonly fail to faithfully recapitulate the barrier properties of human BBB. Very re- cently, a positive breakthrough has been made. Lippmann and colleagues re- ported the derivation of human BMECs with BBB properties from hPSCs while co-differentiating with neural cells
how have VSMCs been produced?
using both HSCs and iPSCs
how can iPSCs be used to look at vascular diseases?
The successful establishment of disease-affected VECs and VSMCs may not only help to reveal cell type- specific manifestation, but also provide ideal platforms for cell type-specific high throughput drug screening in the future.
why os producing VECs and VSMCs fri iPSCs so difficult? how can this be improved?
- the process of differentiation is not hugely understood so the required factors to induce their differentiation is not known
-genome-wide gene expression and epigenetic analysis, such as RNA sequencing, DNA methylation and his- tone modification-associated sequenc- ing, and proteomics analysis may be needed to map and identify new VEC/ VSMC-specific markers and signaling nodes in hPSC-differentiated vascular derivatives
how can angiogenesis be used to treat cardiovascular disease?
Many cardio- vascular ailments, such as ischemic heart disease, stroke, and peripheral vascular disease, are caused by loss of vascular supply, which causes ir- reversible cell loss and organ failure. Successful treatment of these diseases through traditional or regenerative ap- proaches will require re-establishing normal vascular beds, both for proper nutrient exchange and for essential paracrine signals exchanged between vascular cells and organ stroma.
how was the paper which as successful in producing endothelial cells carried out?
- hECS were used and there were turned into ECs in three stages:
-In phase 1 (BMP4+ GSK3beta inhibitors), PSCs dif- ferentiate into mesodermal precursors, which commit to EPs in phase 2 (VEG-A+ DAPT a notch inhibitor), and then differentiate into mature endo- thelial cells (ECs) in phase 3.
what was the key finding with generating ECs from heck?
notch inhibition was needed in stage 2 (committment of mesoderm precursors to EPs) and then release of inhibition on stage 3
what are the main things that VEGF is needed for?
stimulating EC migration and proliferation , MMP production and matrix degradation
why are cells in co culture used to look at angiogenesis?
having a cell providing the support is more physiological
what three types of tube formation
ECs in collagen- laminin co-cultures with HBMSCs showed morphologies that could be mimicking cavitation, cord hollowing or cell hollowing.
what did the study looking at tube formation find in terms of VE-cahderin?
Qualitative evidence through immunostaining in this study showed that VE-cadherin surface localisation on day 7 was more prominent in our collagen-laminin co-cultures compared to collagen only. Higher VE-cadherin levels have previously been shown to correlate with higher VEGFR2 expression. VEGFR2 surface expression is stabilised as a result of the interaction with cadherins, decreasing receptor internalisation
what types of stem cells have been considered for ECs?
mesenchymal stem cells- A recent study demonstrated that MSCs are capable of differentiating toward vascular lineages both in vitro and in vivo
Has adding VEGF in vivo been good for promoting angiogenesis? what can be done instead?
- no , better to use combination:
how have mechanical stresses been implicated in angiogenesis?
Ye et al. demonstrated that shear stress, as well as vascular SMCs, promotes endothelial differentiation of EPCs via activation of Akt, which may provide new insight into vascular regeneration