C1-HC 3

Question 1

Regenerative medicine:

Answer 1

Regenerative medicine= the process of replacing or regenerating human cells, tissues or organs to restore or establish normal function.

Question 2

Anatomy of a mature blood vessel:

Answer 2

lumen
endothelial cells
elastica interna
medial layer
adventitial layer

Question 3

7 characteristics of the endothelial cells:

Answer 3

○ Mesodermal origin.
○ Selective barrier between blood and tissue.
○ Monolayer with tight junctions
○ Anti-coagulant surface (involved in keeping the blood moving) but also involved in haemostasis (blood clothing > the leak is quickly removed and plugged)
○ Role in vasodilation and vasoconstriction (by the factors they secrete, e.g. NO)
○ Mechanosensor (they sense the flow of blood across the surface)
○ Central player in angiogenesis (formation of new blood vessels from existing ones)

Question 4

Elastica interna:

Answer 4

Elastica interna: gives the blood vessel flexibility (ability to expand & contract)

Question 5

5 characteristics of medial layer:

Answer 5

○ Mesoderm origin.
○ consist of smooth muscle cells (more elongated cells, do not connect on all sides to other cells, gives strength and ability to expand & relax).
○ Vasodilation/contraction > regulation of blood pressure. Vascular remodelling (adaptation of blood vessels to change forces: larger of smaller when needed, haemodynamic forces)
○ Vascular wall pathologies (stenosis, atherosclerosis > produce extra cellular matrix causing problems e.g. Closure of the lumen)
○ Extracellular matrix production which gives structural stability of the blood vessel (by secreting extra cellular matrix and collagen).

Question 6

Adventitial layer:

Answer 6

Adventitial layer: protection of blood vessel and for rigidity. Usually consists of fibroblasts.

Question 7

3 different types of blood vessels & function:

Answer 7

Arteries: thicker wall with a lot of smooth muscle cells. From heart to capillary.

Capillary: smallest vessels, provides most of the nutrients & O2 to the tissues.

Vein: thinner wall & lumen is not round, but more collapsed (because of the thinner wall). From capillary to heart.

Question 8

4 types of blood vessel formation: name and method

Answer 8

Types of blood vessel formation:
1 Vasculogenesis: the process of forming a blood vessel network from zero.
2 Angiogenesis: Relies on a primary vascular network, and sprouts from the existing blood vessels.
3 Maturation: formation of larger blood vessels.
4 Arteriogenesis: Protective mechanism that has evolved to be able to cope with changing situations in blood supply.

Question 9

Epiblast:

Answer 9

Epiblast= the outermost layer of the embryo before it differentiates into ectoderm and mesoderm.

Question 10

How are blood vessels formed in the embryo?

Answer 10

Epiblast= the outermost layer (a) of the embryo before it differentiates into ectoderm and mesoderm. The extraembryonic ectoderm will form the placenta. The endoderm is on the outside in the beginning (b). Between the extraembryonic ectoderm and the epiblast, the primitive streak will appear (d). The endoderm cells migrate inward and there, differentiate into mesoderm cells (this process is called endothelial to mesenchymal transition). Mesoderm is the origin of the first endothelial and smooth muscle cells. Blood vessels are the first one to form, because they allow the embryo to grow.

Question 11

Vasculogenesis:

Answer 11

Vasculogenesis= the process of forming a blood vessel network from zero (in early vertebrate embryos).

The steps:

1. The angioblasts and the hematopoietic progenitors meet each other in the extra embryonic tissue where they form blood islands.
2. Hematopoietic prgenitors are in the middle and the angioblasts surround these blood islands.
3. The blood islands fuse together to form the first primitive vascular network.
4. This primary vascular network then develops further into larger blood vessels and capillaries > whole closed circulatory system.

Question 12

Angiogenesis:

Answer 12

Angiogenesis

- Relies on a primary vascular network, and sprouts form the existing blood vessels.
- Driven by metabolic demand of tissue, HIF (Hypoxia inducible factor) and VEGF (vascular growth factor) dependent. These factors are sensitive to O2 levels:

If you have a high oxygen level > HIF is continuously produced & broken down directly > so it cannot accumulate to higher levels in the cell.

Low oxygen level
HIF is produced, but not broken down
It accumulates
The chance that is goes to the nucleus is higher
It targets genes (e.g., VEGF)
Excreted VEGF is the trigger for local endothelial cells (with VEGF sensors) to break out of the membrane
These cells differentiate into different phenotypes (stock cells and tip cells)
Stock cells: provide the connection with your original blood vessel and keep a lumen.
Tip cells: the first cells on the tip and they are filled with VEGF receptors.
Capillary sprouts with tip cells leads the way to cells with the most VEGF (the most hypoxic tissues)
Blood and oxygen are provided to this tissue and they return to the normal state with low hypoxia inducible factors.

Result: all tissues are perfused optimally

Question 13

Maturation:

Answer 13

Maturation (or pruning)

- = formation of larger blood vessels. When a blood vessel is no longer needed, because there are plenty of blood vessels there, then it can also regress (sometimes smaller vessels go away, and only a few larger vessels remain).
- If a capillary is surrounded by smooth muscle cells (also  called pericytes) it's actually protected from regressing or being broken down.
- Endothelial cells secrete factors which carry the receptor for communication (e.g., PDGF beta, and TGF beta).

Question 14

Arteriogenesis:

Answer 14

• Protective mechanism that has evolved to be able to cope with changing situations in blood supply. Steps:
  a. Diseased blood vessel that causes the vessel to clog or to have a plaque formation
  b. Downstream tissue gets less blood and O2
  c. Not the same blood pressure
  d. Pressure gradient causes activation of pre-existing collateral arteries
  i. These are small blood vessels that connect one side to the other side, they don’t carry lots of blood
  e. Now blood is forced into these arteries (it follows the pressure gradient)
  f. Causes shear stress > activation of endothelial cells + perivascular macrophages quickly mature & grow to be able to withstand the increased pressure > typically you see lots of turns in the vessel (blood vessels grow quicker than what the surrounding tissue can accommodate).
  g. Blood flow is restored (the body has created its own bypass).

Question 15

2 types of hypothesis of how neovascularisation (blood vessel formation) occurred in adults:

Answer 15

• Asahara hypothesis: you have a population of stem cells in your bone marrow and they are recruited to the circulation. At the site were the new blood vessels are needed, they attach and migrate inwards. Then, they differentiate to form the building blocks of the new blood vessels. So, circulating cells would become the new blood vessels.
  
  • Paracrine hypothesis: bone marrow derived cells circulate and end up in the same place where new blood vessels are needed. They do not transdifferentiate into endothelial cells, but secrete factors that help other cells to either proliferate into normal differentiated endothelial cells or recruit additional progenitor cells that than cause new blood vessels to be formed.

Question 16

Now a days hypothesis for neovascularisaion:

Answer 16

• Now a days hypothesis: you have bone marrow derived cells (blue) and they contribute to the formation of new blood vessels, but will not transdifferentiate into any endothelial cells. The source for new endothelial cells comes from the existing material. There are some cells (ECFCs) that have a large replicative potential and they can also detach as all endothelial cells (circulating). They end up in the wound area and start dividing to provide new endothelial cells for new vessels. The ECFCs could also by themselves (without any prior structure) form a new capillary of new blood vessel together with the help of the pro angiogenic cells that provides growth factors for this process.

Question 17

Steps for finding info about EPCs

Answer 17

Experiments done to find more information about the different types of endothelial progenitor cells (EPC). Steps:

1. Use density gradient centrifugation to select specific cells from the blood.
2. Plate on a cell culture diss together with growth medium with a lot of endothelial growth factors (e.g., VEGF, IGF, and FGF).
3. Culture the cells (they get more characteristic).
4. Look for endothelial characteristics.

Question 18

3 types of EPC:

Answer 18

CFC-Hill
CAC
ECFC

Question 19

CFC-Hill:

Answer 19

CFC-Hill (early outgrowth endothelial progenitor cells):

	○ After 4-9 days in culture, you see colonies forming. 
	○ Star shaped.
	○ Now a days they are not studied anymore, because the colonies don't have EPC characteristics, but have myeloid and T-cell properties.

Question 20

CAC:

Answer 20

CAC (circulation angiogenic cell):

	○ No colony will appear. They have a small, elongated shape, simetimes still round. They resemble monocytes and don't proliferate. 
	○ You call these early outgrow cells.
	○ Put in 3D gel: they do not incorporate in tubule network. If you take blood from healthy people and from patient with cardiovascular disease/ diabetes, you find lower number of CAC in the sick patients. So, this disease impairs the formation of progenitor cells!

Question 21

ECFC:

Answer 21

ECFC (endothelial colony forming cells):

	○ After three weeks you see colonies appear. These colonies include cells that proliferate rapidly and also form this monolayer of cells that adhere to each other, much like endothelial cells. 
	○ Only the ECFC are able to form de novo (so without any prior structure) new blood vessels.
	○ You call these late outgrowth endothelial progenitor cells. 
	○ Put in 3D gel: they incorporate in tubule network.

Question 22

Interesting founding’s in the cluster dendrogram about:

1. CFU-Hill
2. CAC
3. ECFC

Answer 22

Interesting founding’s in the cluster dendrogram:

1. The CFU-hill cells don’t cluster with anything.
2. CACs are similar to monocytes.
3. ECFCs cluster with differentiated endothelial cells (HUVEC).

Question 23

Important for therapeutic possibilities with ECFC:

Answer 23

Therapeutic possibilities with ECFC: you can form a network by mixing MSCs and ECFCs in a collagen gel.

Conclusion I: the MSC donor is more important in determining the success of this network formation.

Conclusion II: ECFCs have vasculogenic capacity in vivo, when in combination with cells that support (e.g., MSCs).

Question 24

Hind limb ischemia mouse model:

Answer 24

Hind limb ischemia mouse model:

- Method: They ligate (afbinden) the femoral artery > one limb becomes ischemic (reduced blood supply resulting in a shortage of oxygen and nutrients). The mouse will use a technique to recover its blood flow (e.g., angiogenesis). You inject stem cells in the blood or in the muscles surrounding this ligated vessel and see if that can speed up the perfusion recovery. 
- Use Doppler laser (the same as what the police use to measure your speed) to image the perfusion. Top image: no perfusion in limb, but there is perfusion in tail and other limb. Bottom image: all perfused.

Question 25

What type of cells are MSCs?

Answer 25

A lot of cells can grow from MSCs. This shows that they are multipotent cells.

They can also stimulate blood vessel formation, but are not impaired by disease.

Question 26

Identification of MSCs:

Answer 26

Identification of MSCs:

- Look at a lot of markers. 3 primary markers that are used the most are CD105+, Cd73+, and CD90+. If they at leats express these markers, you can call them MSCs.
- Functional marker: MSCs adhere (stick) to tissue culture plastic.
- One way of really showing that MSCs are multipotent is that you expose them to different culture protocols (e.g., start from MSCs, then put them in a culture that leads to bone or cartilage formation or adipose tissue). 
- In vitro differentiation potential > the cells can be directed into different cell types.

Question 27

Sources for MSCs:

Answer 27

Sources for MSCs:

- Bone marrow (most common)
- Placental tissue
- Umbilical cord tissue (Wharton's Jelly is the sort of gelatinous material in the cord that gives you some protection and stability for the blood vessel) 
- Dental pulp - Adipose tissue

Question 28

which endothelial progenitor cell type is thought to become a structural part of newly formed blood vessels?

a. Colony-forming units (CFU) - Hill cells
b. Circulation angiogenic cells (CAC)
c. Endothelial colony-forming cells (ECFC)
d. All of the above

Answer 28

c. Endothelial colony-forming cells (ECFC)

Question 29

What is the difference between angiogenesis and vasculogenesis?

Answer 29

Angiogenesis: formation of new blood vessels (capillaries) from pre-existing vessels
Vasculogenesis: formation of new blood vessels de novo (without pre-existing vessels)