Blood vessels are required due to the distance-limitation of diffusive transport. Their role is to supply the body with oxygen and nutrients, and to remove carbon dioxide and waste products; blood vessel growth is fundamental to life. Blood vessel formation is seen physiologically in many aspects of life; growth, ovulation, menstruation, placenta formation and wound healing are some typical examples. Growth of new blood vessels is also seen in pathologies such as tumour formation, Psoriasis, and vascular malformations (Folkman J, 2001). Indeed some pathologies are also associated with a reduction in vascular growth for example delayed ulcer healing, emphysema and pulmonary fibrosis. New blood vessel growth can occur by Vasculogenesis which is defined as De novo formation of vessels from progenitor cells, or Angiogenesis which is the sprouting of new capillaries from existing capillaries. VEGF is widely reported as a long process of remarkable scientific discoveries. It was the late 1800s when Rudolf Virchow first observed that tumours were highly vascularised (David H, 1988); this finding was not elaborated until several decades later. Warren Lewis the John Hopkins educated scientist described variations in blood vessel structure in different types of tumour in rats (Lewis WH, 1927). By the mid 1940s vast amounts of research were undertaken in the field with a pivotal paper published by Glenn Algire's group at the National Cancer Institute. Algire transplanted tumour tissue and normal tissue into mice and quantitatively observed blood vessel growth, with concurrent tumour size comparisons. These studies highlighted the importance of tumour cells in vessel growth, and the requirement of blood vessel growth to fuel increases in tumour size. The idea of a diffusible factor derived from an implanted tumour which led to vessel proliferation was presented in 1968. Tumours were transplanted into hamster cheeks with a filter separating the tumour from the host. Vessel proliferation still occurred despite the presence of a filter, hence giving rise to the idea of tumour-released growth factors (Greenblaat M. et al. 1968 & Ehrmann R. L. et al. 1968). In 1971 Judah Folkman attempted to isolate this diffusible factor from animal and human tumours which he named 'tumour angiogenesis factor' - a factor which is mitogenic to capillary endothelial cells. Folkman suggested the blockade of 'tumour angiogenesis factor' could arrest blood vessel proliferation in tumours which could be of great therapeutic importance with respect to cancer (Folkman J, 1971). In 1983 Donald Senger and his colleagues came across a protein which increased vascular permeability in guinea pigs, suggesting its role in tumour-associated ascites. The protein was called tumour vascular permeability factor (VPF) but was not isolated and sequenced; at this point in time Senger and his team were unaware of the mitogenic activity tumour vascular permeability factor has on capillary endothelial cells (Senger DR, 1983). Napoleone Ferrara isolated and purified a factor which is mitogenic to endothelial cells hence the logical name of Vascular Endothelial Growth Factor (VEGF) was given to the protein (Ferrara N, 1989). Subsequent investigations through sequencing revealed that VEGF and VPF are the same protein (Keck P. J. et al, 1989). Molecular cloning studies of VEGF show that there are many isoforms of the enzyme which is derived from variations in RNA transcript splicing. There is a low sequence homology in the VEGF family of proteins; different isoforms have different levels of angiogenic potency. VEGF B is reported not to affect vessel permeability and angiogenesis. The role of VEGF B is maintenance of pathological blood vessels (Zhang F. et al, 2009). VEGF C and D are important in the development of lymphatics (Feng Y. et al, 2010). Placental like Growth Factor is considered under the VEGF family due to its homology. PIGF is only expressed during pregnancy and pathologically (Selvaraj S.K. et al, 2003) VEGF165 is a 165 amino acid splice variant of VEGF A; it is the dominant isoform of greatest importance with respect to endothelial cell mitogenic action and angiogenesis (Houck K. A. et al, 1991). Figure 1 compares the VEGF family in detail. There are three different types of VEGF receptors, VEGFR-1, VEGFR-2, and VEGFR-3. VEGR-1 and VEGFR-2 are of particular importance with respect to binding VEGF A, and angiogenesis. VEGF receptors have seven Immunoglobulin like domains extracellularly, and an intracellular kinase domain. VEGFR-1 is the fms-like tyrosine kinase receptor (flt1) which binds VEGF A, VEGF B, and PIGF (de Vries C. et al, 1992). VEGFR-1 is responsible for vessel morphogenesis and organisation. This was shown by gene knockout studies where VEGFR-1 knockout mice had disorganised vasculature which lead to their subsequent death (Fong G.H. et al, 1995). VEGFR-1 is present in vascular endothelial cells, macrophages and monocytes (Cross M. J. et al, 2003).VEGFR-1 is also involved in monocyte migration (Barleon B. et al, 1996). VEGFR-2 is a Kinase Domain insert Receptor (KDR) which binds to VEGF A, C and D (Terman B. I. et al, 1992). VEGFR-2 is crucial in blood vessel formation shown by gene knockout studies where VEGFR-2 knockout mice had deficient blood vessel formation which was lethal (Shalaby F. et al, 1995). VEGFR-2 is present in vascular endothelial cells and haematopoietic stem cells (Cross M. J. et al, 2003). The importance of VEGF A is demonstrated in experiments by Carmeliet and his team. In heterozygous VEGF A-deficient (VEGF A+/-) embryos blood vessel formation is not ceased, but abnormal. In homozygous VEGF A-deficient (VEGF A-/-) embryos blood vessel formation was even more impaired resulting in mid-gestational death. The conclusions drawn from this study were that VEGF A dose-dependently regulates vessel development. (Carmeliet P. et al, 1996) Angiogenesis is a dynamic process involving the permeabilisation of the vascular endothelium allowing breakage of the vessel then subsequent sprouting of new vessels from the existing vessel. The majority of angiogenesis occurs in the microvasculature. There are a variety of promoters and inhibitors of angiogenesis present in blood vessels. The most protent promoters of angiogenesis include VEGF, Angiopoietins and Fibroblast growth Factors (FGFs). The most potent inhibitors of angiogenesis include Angiopoietins and Angiostatin (Smith A, 2010). Vessel growth is also regulated by numerous factors such as metabolic stimuli (hypoglycaemia, low pH), mechanical stimuli (shear stress), and oncogene upregulation (Kerbel R. S. et al, 1998). One of the most important stimuli for vessel growth is hypoxia. Vessel hypoxia can occur during increased oxygen consumption, blood vessel occlusion or when there is an increased tissue mass which prompts compensatory vessel growth. Hypoxia Inducible Factors (HIFs) are turned over in normoxia by hydroxylation by prolyl hydroxylases (PHDs) which then undergo Ubiquitin proteosomal degradation (Kaelin W. G. et al, 2008). In the absence of oxygen this hydroxylation fails to occur thus there is a build up of HIF which affects the Hypoxic response element leading to altered gene expression of VEGF (Dor Y. et al, 2001). When promoters of angiogenesis overwhelm inhibitors a phenomenon called the 'angiogenic switch' occurs. The 'angiogenic switch' causes activation of quiescent vasculature into an angiogenic state. It is important to note the existence of VEGF gradients which are established by the affinity of binding of the VEGF to the matrix. Once the local endothelium is activated into an angiogenic state it begins to secrete Matrix Metalloproteinase-9 (MMP-9). MMP-9 increases the availability of VEGF A to take part in angiogenesis in addition to decomposition of the extracellular matrix and basement membrane (Bergers G. et al, 2000). This allows release of the endothelial cells from the extra cellular matrix. Binding of VEGF A to the tyrosine kinase receptors VEGFR-1 and VEGFR-2 causes them to dimerise and activate (Blechman J. M. et al, 1995). The binding of VEGFA to the VEGFR-2 initiates increases in vascular permeability. Increases in vascular permeability are important to allow Fibroblast growth factor to pass into adjacent tissue to promote sprouting. It also allows plasma proteins to penetrate the blood vessel and conditionally form a matrix where angiogenesis will occur. Fibroblast growth factors (FGF) are other well known promoter of angiogenesis. FGF 2 is the most important isoform which passes into tissues near the existing vessel to aid VEGF A in sprouting (Parsons-Wingerter P. et al, 2000). Sprouting is a phenomenon where growth of the new vessel occurs by branching off from the existing vessel. Tip cells are highly specialised endothelial cells with long filipodia extensions. They lack a lumen and are enriched with VEGFR2 receptors. Tip cells also express platelet derived growth factor Î² (PDGFB) which is important in pericyte recruitment during vessel stabilisation later on in the angiogenic process. Endothelial cells differentiate into Tip cells at the point of highest VEGFA concentration; this is mediated by delta-like ligand 4/Notch signalling (Phng L. K. et al, 2009). Tip navigation is initiated as by ligand binding dimerisation of the VEGFR2 receptors. Tip cells also produce proteases to digest the basement membrane allowing mobility into surrounding tissues. The high density of VEGFR2 receptors on Tip cells allows migration in the direction of the VEGFA gradient. Tip cells produce further VEGF which promotes migration. Tip cells do not proliferate, but they are at the leading edge of the new vessel (Gerhardt H. et al, 2003). Ligand-induced dimerisation of VEGFR-2 also promotes mitogenesis and migration of endothelial cells. The most commonly reported molecular transduction is through autophosphorylation at tyrosine residue 1175. This activates Phospholipase CÏ’ which produces Diacyl glycerol, leading to Protein Kinase C activation which eventually leads to activation of a cascade of Mitogen-activated protein (MAP) kinases. This activates DNA synthesis of endothelial cells promoting proliferation. Another effect is through autophosphorylation of the tyrosine residue 1214 of the VEGFR-2 receptor. Through a cascade of phosphorylations this leads to p38 MAP kinase activation which leads to endothelial cell migration. Fibronectin is an important component as it has high affinity for VEGFR2. VEGF A binds to fibronectin at the HEP2 site; in the absence of fibronectin there is delayed endothelial cell migration(Takahashi T. et al, 2001). Figure 2 depicts this receptor activation and the subsequent signal transduction pathways involved. Stalk elongation occurs behind the non proliferating tip cells through the receptor activation described, causing endothelial cell proliferation and migration. Tip-cell fusion is a process where two sprouting vessels meet. The tip cells of each respective sprout fuse by forming cell-cell junctions to form a patent blood vessel as seen in figure 3. The Tip cells differentiate back into normal endothelial cells and cease to migrate further. However there are many stages of vessel maturation that are required for the new vessel to become fully functional (Carmeliet P. et al, 2009). Vessel maturation encompasses a change from the developing vessel into a quiescent fully mature blood vessel. The new vessel undergoes perfusion which brings to it oxygen and nutrients. Hyperpermeability of the vessel is a feature which allows fibrin rich granulation tissue to be laid down surrounding the new vessel aiding vessel maturation (Levick J. R, 2010). Oxygen supply to the local area allows PHDs to continue phosphorylating HIFs to their normoxic levels thus reducing pro-angiogenic signals. Pericytes, vascular smooth muscle and extracellular matrix cells begin to be incorporated into the new vessel (Carmeliet P. et al, 2009). It has been shown that platelet derived growth factor Î² (PDGFB) is essential in pericyte recruitment. PDGFB is a functional homodimer with homology to the VEGF family. The PDGF receptor (PDGFR) is similar to the VEGFR in the sense that it is a tyrosine kinase receptor but it has five immunoglobulin like domains extracellularly as opposed to the seven of VEGFR. As described before tip cells produce PDGFB which creates a concentration gradient during sprouting. Pericytes contain the PDGFR which allows migration and incorporation into the newly formed blood vessel (Karamyasheva A. F, 2008). Pericytes are stimulated to divide, migrate and incorporate into the newly formed blood vessel wall by PDGFB (Bjarnegard M. et al, 2004). In PDGFB deficient mice embryos microvasculature pericyte migration and differentiation to new blood vessels failed to occur, leading to microaneurysms. This experiment highlights the role of PDGFB in blood vessel wall formation (Lindahl P. et al, 1997). Pericytes arrest growth and migration of newly formed vessel endothelial cells; this is enhanced by TGF- Î² activation (Betsholtz C. et al, 2005). Once the mural progenitor cells have differentiated and formed smooth muscle cells or pericytes the vessel wall is complete and the vessel is deemed to be quiescent. The determinant of artery or vein structure of a vessel is determined by the expression of protein ephrin B (Levick J. R, 2010). In tumour angiogenesis there is disruption of mural cell incorporation into the new vessel wall. This leads to disorganised, 'leaky' vessels prone to haemorrhage which can further potentiate metastasis (Dor Y. et al, 2002). Angiopoietins are also responsible for vessel stability; there is four isoforms with the two of main interest being Ang1 and Ang2. Ang1 and Ang2 are ligands to the tyrosine kinase receptor Tie2. Ang1 phosphorylates and activates the receptor whereas Ang2 binds but does not activate the receptor (therefore it is essentially a reversible inhibitor). Ang1/Tie2 knockout studies in mice embryos have shown that death occurs due to poor development of the basement membrane in newly formed vessels and detachment of pericytes. This demonstrates the pivitol role that angiopoetin1 and its receptor have on pericyte-endothelium attatchment (Von T. D. Et al, 2006). When in excess, Ang2 the competitive inhibitor reverses the function of Ang1 by deactivating Tie2 receptors. This has been demonstrated in studies by injection of Ang2 into mice retinae resulting in pericyte dissociation from blood vessels (Hammes H. P. et al, 2004). Angiopoetin is also an important angiogenesis factor in reducing new blood vessel permeability in the final stages of stabilisation, reducing oedema and haemorrhage of new blood vessels (Uemura A. et al, 2002). Vessel stabilisation is a complex process encompassing oxygenation, vessel wall formation and tightening of the vessel wall mediated by Ang1, PDGFB and TGF-Î². Once this process is complete the newly formed vessel goes into a quiescent stage and is fully functional. Vasculogenesis is another method of new blood vessel formation. As defined previously it is the De novo formation of blood vessels from progenitor cells. Figure 4 represents a schematic diagram of Vasculogenesis. Originally it was believed that Vasculogenesis only occurred during embryo development. Vasculogenesis is driven by the embryological environment,by molecules such as VEGF, Angiopoietin and Fibronectin. The first stage of Vasculogenesis is mesoderm formation which requires a myriad of growth factors such as FGF and TGF-Î² (Cornell R. A. et al, 1994). Blood island formation is the next stage where there is a core of haemopoietic progenitor cells surrounded by angioblasts, endothelial cell precursors (Coffin J. D. et al, 1991). Many individual blood islands come together and the angioblasts differentiate into fully formed endothelial cells predominantly driven by FGF and VEGF (Cox C. M. et al, 2000). The lumen becomes patent to form a primary capillary plexus. This structure is further developed into a developed blood vessel and undergoes angiogenesis to make intricate pathways of blood vessels in embryogenesis (Risau W. et al, 1995). It is now believed that bone marrow-derived cells also assist in blood vessel repair and formation in adults and not just embryos (Crosby J. R. et al, 2000). Crosby and his team quantified the amount of circulating haematopoietic endothelial cell progenitors contributing to new vessel formation. Haematopoietic chimeras were prepared and marked by a DNA marker. Granulation tissue formation was stimulated by sponge implant and left for four weeks. The sponges were then removed and the DNA marker tested for. The percentage of endothelial cells which were haematopoietic derived in the granulation tissue was between 0.2-1.4%. The proportion of haematopoietic derived cells is small but changes the outlook on the traditional view that Vasculogenesis can only occur during embryogenesis (Crosby J. R. et al, 2000). Experiments have also found that Vasculogenesis occurs in adults following vascular trauma such as ischaemia. Takahashi T. et al showed this by introducing bone marrow derived endothelial progenitor cells (EPCs) which expressed endothelial specific Tie2/lacZ in mice and rabbits (see figure 5). Mice with hindlimb ischaemia had significantly more EPCs incorporated in corneal neovascularisation than sham-operated mice (a). Similarly rabbits with granulocyte macrophage-colony stimulating factor (GM-CSF, an exogenous cytokine therapy) had significantly more EPCs incorporated in corneal neovascularisation than control rabbits. Both these results are statistically significant with a p value of less than 0.01. This study shows how vascular injury can lead to mobilisation of endothelial progenitor cells and incorporation into neovascularisation (Takhashi T. et al, 1999). Vasculogenesis which was once thought to be a process only related to embryogenesis is now known to be incorrect. Postnatal Vasculogenesis has been shown to occur in situations of blood vessel repair and formation. In conclusion the growth of new blood vessels is a complex and tortuous process. Neovascularisation can occur through Angiogenesis or Vasculogenesis. Decades of research have been dedicated to this topic in the pursuit of scientific knowledge and cancer therapy. The early findings of Glenn Algire and his team about the requirement of vessel growth in tumour development is central to cancer therapeutics. Aspects of cancer therapy aim to inhibit one or more of the processes in blood vessel formation. In light of resistance to novel therapies for cancer emerging rapidly (for example VEGF inhibition) there is still growing need for research in the field of growth of new blood vessels.
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Figure 1: Table comparing the VEGF family (Smith A, 2010)
Figure 2: VEGF receptors and signal transduction pathways (Kowanetz M. et al, 2006)
Figure 3: Stages of Angiogenesis (Carmeliet P. et al, 2009)
Figure 4: A schematic representation of Vasculogenesis (Risau W. et al, 1995)
Figure 5: EPC incorporation into corneal neovascularisation (Takhashi T. et al, 1999)