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L11 Angiogenesis
Angiogenesis is the formation of vessels from pre-existing vessels
Functions of endothelial cells
 Barrier function
 Blood clotting
 Inflammation
 Vasoconstriction and vasodilation
 Angiogenesis
Roles of angiogenesis
 During development
 Physiological processes
o Wound healing
o Menstrual cycle
o Adaptation to increased muscle activity
 Pathological processes
o Cancer
o Inflammatory
o Diabetic retinopathy
Vessel formation during development I – Vaculogenesis
 Vasculogenesis is the formation of blood vessels from mesodermal derived angioblasts
 Endothelial progenitors (angioblasts) differentiate from mesodermal cells
 Angioblasts coalesce to form first embryonic vessels – dorsal aorta and cardinal vein
 Angioblasts from blood islands which fuse and remodel to form a primitive capillary plexus (no hierarchy of
arteries and veins in size)
Vessel formation during development II - Angiogenesis
 Angiogeneic remodelling of dorsal aorta, cardinal vein and vascular plexi give rise to arteries, veins and
capillaries
 Angiogeneic sprouting of new vessels
 Pericytes are recruited to stabilise capillaries
 Lymphatic endothelial cells sprout from veins and form the lymphatic system via lymphangiogenesis
HYPOXIA DRIVES ANGIOGENESIS
As distance from a vessel increases, there is:
 Increased acidity
 Increased lactic acid
 Decreased ATP and glucose can’t do anerobic resp
 Decreased O2
HIF-1 is a transcription factor (Hypoxic inducible factor) a master regulator of O2 homeostasis
 Drives production of VEGF and Angiopoietin 2
 HIF-1 is a transcription factor consisting of two subunits HIF-1a and HIF-1B
 Degradation of the HIF-1a subunit is regulated in response to oxygen levels
In high O2 HIF-1a is degraded
 Proline hydroxylase enzyme hydroxylates proline residues on HIF-1a
 Is at dynamic equilibrium in these conditions if favours the hydroxylated state
 This is recognised and bound by a ubiquitin ligase which catalyses the poly-ubiquitinates of HIF-1a
 Poly-ubiquitinate HIF-1a is degraded by the proteasome

In low O2 HIF-1a is stabilised and induces expression of proangiogenic factors
 Not enough O2 to drive the proline hydroxylation of HIF-1a
 HIF-1a associates with HIF-1B and induces transcription of genes needed for adaptation to hypoxic conditions
 A key target of HIF-1 is VEGF
The functions of VEGF in endothelial cells
 VEGF-A is the most potent of the VEGF family of growth factors at driving angiogenesis
 VEGFR signals via activation of VEGFR-2, a receptor tyrosine kinase
 Dimerisation of VEGFR-2 activates signalling pathways which affects
o Proliferation
o Survival
o Migration
o Vascular permeability – via modulation of junctional proteins
The angiogenic switch
 There are a number of pro and anti-angiogenic factors
 Angiogenesis occurs when the effects of angiogenic activators are greater than those of the inhibitors
Modes of angiogenesis
 New vessels can form by sprouting or splitting (intussusception)
 Intussusception involves the formation of a pillar which elongates
splitting the vessel in two
o Rapid increase in capillarity
o Requires minimal endothelial proliferation
Sprouting angiogenesis
 Loosening of adherens junctions by VEGF regulates VE-cadherin
 Endothelial cells secrete proteases to remodel pre-existing interstitial matrix
 Selection of tip cell to guide the newly forming sprout
 Filipodia actin driven extensions, sense the extracellular environment
 Angiogenic factors (e
...
VEGF, FGF)
 Pericyte detachment mediated by angiopoietin 2 (Ang2)
 Increased permeability permits extravasation of plasma proteins (such as fibrinogen and fibronectin) to deposit a
provisional matrix layer
...
g
...

 Stalk cells behind the tip cell proliferate and extend the sprout, VEGFR-2 is downregulated in stalk cells
 The sprouts from adjacent vessels grow towards one another
 Stabilisation of the newly formed vessel
o Signalling between endothelial cells and pericytes maintain quiescence of the newly formed vessel,
Angiopoietin 1 (Ang1)
o Formation of tight junctions and barrier function
o Basement membrane deposition
o Pericyte maturation (PDGF, Ang1)
Specifying tip and stalk cell fate
 Notch and delta signalling
 VEGF signals  one cell will have higher Delta levels, the other will have higher notch
 Tip cells have high Delta-4, stalk cells have higher notch
 Stalk cells less responsive to VEGF so that tip stays the tip

Exercise and angiogenesis in skeletal muscle
 Exercise and the contractile activity of muscle influences capillary growth
 Mechanical stretch and shear stress (flow) stimulate angiogenesis
 VEGF plays an essential role in driving capillary growth
 Shear stress primarily induces longitudinal splitting and this requires eNOS
signalling
 Tissue stretch induces angiogenic sprouting
 Passive stretch also stimulates VEGF  sprouting
Pathological Angiogenesis
 Angiogenesis contributes to the pathology of inflammatory diseases and the
growth and metastasis of solid tumours
 Tumour growth and hyperplasia in inflammatory disease increases the distance of cells from vessels and hypoxia
drives VEGF production
 Chronic hypoxic giving rise to high levels of VEGF and inflammatory cytokines create a pro-angiogenic
environment
 Vessel formation is abnormal leading to leaky and poorly perfused vessels and persistent hypoxia
 The leaky vessels facilitate leukocyte extravasation in inflammatory disease
 In tumours/inflammation the vessels aren’t very organised because there is constant VEGF
...

Angiogenisis in Rheumatoid Arthritis
Modulating angiogenesis – inhibiting VEFG signalling
 Monoclonal antibody to VEGF
o Bevacizumab
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g
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Assessing angiogenic sprouting – the spheroid assay
Assessing angiogenic sprouting – the co-culture assay
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Fibroblast produce extracellular matrix and secrete VEGF
Co-culture fibroblasts with endothelial cells, then image tubule formation
Can add VEGFR-2 inhibitors to see difference

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