Angiogenesis Overview

Vijay Avin BR, Molecular Biomedicine Laboratory, Sahyadri Sceince College, Shimoga, Karnataka, India

• 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

• 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

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.

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.

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.

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.

Angiogenic stimulators or factors

Stimulator Mechanism

FGFPromotes 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….

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….

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)

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….

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 granulocytes•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

PGFImportant 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)

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.

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

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.

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). 

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.

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.