Angiogenesis is the growth of new blood vessels from pre-existing vessels. It is a normal process in growth and development as well as wound healing. It involves the breakdown of the basement membrane by proteolytic enzymes, migration and proliferation of endothelial cells stimulated by growth factors, and formation of new vessels. Key regulators of angiogenesis include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and angiopoietins. A balance between activators and inhibitors normally keeps angiogenesis in check, but it can be stimulated under conditions like hypoxia.

1. Angiogenesis Overview
Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince
College, Shimoga, Karnataka, India

2. • Angiogenesis is the physiological process
involving the growth of new blood
vessels from pre-existing vessels.
• vasculogenesis is the term used for
spontaneous blood-vessel formation,
• intussusception is the term for the formation
of new blood vessels by the splitting of
existing ones

3. • Angiogenesis is a normal and vital process in
growth and development, as well as in wound
healing and in granulation tissue, highly
regulated by angiogenic factors and
angiogenic inhibitors

4. angiogenesis occurs normally in the human body at specific times in
development and growth. For example, a developing child in a mother's womb
must create the vast network of arteries, veins, and capillaries that are found in
the human body. A process called vasculogenesis creates the primary network of
vascular endothelial cells that will become major blood vessels. Later on,
angiogenesis remodels this network into the small new blood vessels or
capillaries that complete the child's circulatory system.

5. Proliferation of new blood vessels also takes place in adults,
although it is a relatively infrequent event. In women,
angiogenesis is active a few days each month as new blood
vessels form in the lining of the uterus during the menstrual cycle.
Also, angiogenesis is necessary for the repair or regeneration of
tissue during wound healing.

6. The walls of blood vessels are formed by vascular endothelial
cells. These cells rarely divide, doing so only about once every
3 years on average. However, when the situation requires it,
angiogenesis can stimulate them to divide.

7. Angiogenesis is regulated by both activator and inhibitor molecules.
Normally, the inhibitors predominate, blocking growth. Should a need
for new blood vessels arise, angiogenesis activators increase in
number and inhibitors decrease. This prompts the growth and division
of vascular endothelial cells and, ultimately, the formation of new blood
vessels.

8. Angiogenic stimulators or factors
Stimulator Mechanism
FGF
Promotes proliferation & differentiation of
endothelial cells, smooth muscle cells, and
fibroblasts
VEGF Affects permeability
VEGFR and NRP-1 Integrate survival signals
Ang1 and Ang2 Stabilize vessels
PDGF (BB-homodimer) and PDGFR recruit smooth muscle cells
Chemical stimulation of angiogenesis is performed by
various angiogenic proteins, including several growth
factors like VEGF, FGF, Angiopoietins, MMPs, etc……
Contd….

9. Fibroblast growth factor
• Fibroblast growth factors, or FGFs, are a family of growth
factors involved in angiogenesis, wound healing, and
embryonic development.
• The FGFs are heparin-binding proteins and interactions with
cell-surface-associated heparan sulfate proteoglycans.
• FGFs are key players in the processes of proliferation and
differentiation of wide variety of cells and tissues.
• In humans, 22 members of the FGF family have been
identified, all of which are structurally related signaling
molecules.
• FGF1 is also known as acidic, and FGF2 is also known as basic
fibroblast growth factor, and plays a key role in angiogenesis
Contd….

10. FGF receptors and binding
The mammalian fibroblast growth factor receptor family has 4 members, FGFR1, FGFR2, FGFR3,
and FGFR4. The FGFRs consist of three extracellular immunoglobulin-type domains (D1-D3), a
single-span trans-membrane domain and an intracellular split tyrosine kinase domain.
Between receptor protein Domains D1 and D2 is a short span of acidic residues called the "acid
box.“ A short stretch of acidic amino acids located between the D1 and D2 domains has auto-
inhibitory functions.
This 'acid box' motif interacts with the heparan sulfate binding site to prevent receptor
activation in the absence of FGFs.
One important function of FGF1 and FGF2 is the promotion of endothelial cell proliferation and
the physical organization of endothelial cells into tube-like structures. They thus
promote angiogenesis, the growth of new blood vessels from the pre-existing vasculature. FGF1
and FGF2 are more potent angiogenic factors than vascular endothelial growth factor (VEGF)
or platelet-derived growth factor (PDGF)

11. Vascular endothelial growth factor
• Vascular endothelial growth factor (VEGF) is a signal protein
produced by cells that stimulates vasculogenesis and angiogenesis.
It is part of the system that restores the oxygen supply to tissues
when blood circulation is inadequate. Serum concentration of VEGF
is high in bronchial asthma and low in diabetes mellitus. VEGF's
normal function is to create new blood vessels during embryonic
development, new blood vessels after injury, muscle following
exercise, and new vessels (collateral circulation) to bypass blocked
vessels
• When VEGF is overexpressed, it can contribute to disease. Solid
cancers cannot grow beyond a limited size without an adequate
blood supply; cancers that can express VEGF are able to grow and
metastasize. Overexpression of VEGF can cause vascular disease in
the retina of the eye and other parts of the body.
Contd….

12. VEGF and types
Type Function
VEGF-A
•Angiogenesis
• ↑ Migration of endothelial cells
• ↑ mitosis of endothelial cells
• ↑ Methane monooxygenase activity
• ↑ αvβ3 activity
• creation of blood vessel lumen
• creates fenestrations
•Chemotactic for macrophages and granulocyte
s
•Vasodilation (indirectly by NO release)
VEGF-B
Embryonic angiogenesis (myocardial tissue,
specifically)[
VEGF-C Lymphangiogenesis
VEGF-D
Needed for the development of lymphatic
vasculature surrounding lung bronchioles
PGF
Important for Vasculogenesis, Also needed for
angiogenesis during ischemia, inflammation,
wound healing, and cancer.
A number of VEGF-related proteins have also been discovered encoded by
viruses (VEGF-E) and in the venom of some snakes (VEGF-F). (no proper
reference)

13. VEGF, receptors and production
• All members of the VEGF family stimulate cellular responses by binding to tyrosine
kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become
activated through transphosphorylation, although to different sites, times and extents.
• The VEGF receptors have an extracellular portion consisting of 7 immunoglobulin-like
domains, a single transmembrane spanning region, and an intracellular portion containing a
split tyrosine-kinase domain.
• VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1). VEGFR-2 appears to mediate
almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well-
defined, although it is thought to modulate VEGFR-2 signaling.
• Another function of VEGFR-1 may be to act as a dummy/decoy receptor, sequestering VEGF
from VEGFR-2 binding (this appears to be particularly important during vasculogenesis in the
embryo). VEGF-C and VEGF-D, but not VEGF-A, are ligands for a third receptor (VEGFR-3),
which mediates lymphangiogenesis.
• VEGFproduction can be induced in cells that are not receiving enough oxygen. When a cell
is deficient in oxygen, it produces HIF,hypoxia-inducible factor, a transcription factor. HIF
stimulates the release of VEGF, among other functions (including modulation of
erythropoeisis). Circulating VEGF, then binds to VEGF Receptors on endothelial cells,
triggering a Tyrosine Kinase Pathway leading to angiogenesis.
• HIF1 alpha and HIF1 beta are constantly being produced but HIF1 alpha is highly O2 labile,
so, in aerobic conditions, it is degraded. When the cell becomes hypoxic, HIF1 alpha persists
and the HIF1alpha/beta complex stimulates VEGF release.

14. Basement membrane breakdown: proteolytic enzymes
Interaction between the uPA and
MMP systems
To initiate the formation of new
capillaries, endothelial cells of the
existing blood vessels must
degrade the underlying basement
membrane and invade into the
stroma of the neighbouring tissue .
These processes of endothelial cell
invasion and migration require the
cooperative activity of
the urokinase-plasminogen
activator (uPA) and the matrix
metalloproteinases (MMPs)
systems.
Process of angiogenesis

15. Endothelial cell migration and proliferation: angiogenic factors
Following proteolytic degradation of the ECM, "leader" endothelial cells start to migrate
through the degraded matrix. They are followed by proliferating endothelial cells, which
are stimulated by a variety of growth factors, some of which are released from the
degraded ECM. Other ECM products, such as peptide fragments of fibrin) also stimulate
the angiogenic process. Therefore, a local collapse of the ECM results in an increased
extracellular concentration of soluble mediators of endothelial cell migration and
proliferation.

16. Cell-cell and cell-matrix interactions: adhesion
molecules
The processes of cell invasion, migration and proliferation do not only depend on angiogenic
enzymes, growth factors and their receptors, but are also mediated by cell adhesion molecules .
To initiate the angiogenic process, endothelial cells have to dissociate from neighbouring cells
before they can invade the underlying tissue. During invasion and migration, the interaction of
the endothelial cells with the ECM is mediated by integrins. Also, the final phases of the
angiogenic process, including the construction of capillary loops and the determination of the
polarity of the endothelial cells, which is required for lumen formation, involve cell-cell contact
and cell-ECM interactions (18).
Cell adhesion molecules can be classified into different families depending on their biochemical
and structural characteristics. These families include the cadherins and the integrins. Members
of each family are implicated in neovascularization.
Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are
expressed on quiescent endothelium, but are upregulated after stimulation with TNF-a, IL-1 or
interferon-g (IFN-g).

17. Capillary formation and vessel maturation
After proteolytic degradation of the basement membrane and endothelial cell migration,
the newly-forming capillaries synthesize a new basement membrane.
During this process extracellular proteolysis must be locally inhibited to permit the
deposition and assembly of ECM components. Once a capillary sprout is formed,
degradation of the newly formed ECM again occurs at the tip of the sprout, which then
allows further invasion. Thus, capillary formation results from alternate cycles of activation
and inhibition of extracellular proteolysis. The endothelial cells also form branches, which
connect with other branches to form capillary loops. In order to form a lumen, the polarity
of the endothelial cells, luminal versus abluminal, has to be established by cell adhesion
molecules. Further stabilization of the new capillaries requires the recruitment
of pericytes and smooth muscle cells, which is regulated by PDGF.
Finally, when sufficient neovascularization has occurred, angiogenic factors are
downregulated or the local concentration of inhibitors increases. As a result, the
endothelial cells become quiescent and the vessels remain or regress if no longer needed.
Thus, angiogenesis requires many interactions that must be tightly regulated in a spatially
and temporally manner.

18. The angiogenic process, as currently understood, can be summarized as
follows:
• A cell activated by a lack of oxygen releases angiogenic molecules that
attract inflammatory and endothelial cells and promote their
proliferation.
• During their migration, inflammatory cells also secrete molecules that
intensify the angiogenic stimuli.
• The endothelial cells that form the blood vessels respond to the
angiogenic call by differentiating and by secreting matrix
metalloproteases (MMP), which digest the blood-vessel walls to enable
them to escape and migrate toward the site of the angiogenic stimuli.
• Several protein fragments produced by the digestion of the blood-vessel
walls intensify the proliferative and migratory activity of endothelial
cells, which then form a capillary tube by altering the arrangement of
their adherence-membrane proteins.
• Finally, the capillaries emanating from the arterioles and the venules will
join, thus resulting in a continuous blood flow.