Steps and Switches in Angiogenesis
The development of new blood vessels is determined by a fine balance of promoting and inhibiting influences, controlling a so-called "angiogenic switch" which must be flipped by an imbalance between the two. Activators and inhibitors of angiogenesis can act upon any step of the process. Anti-angiogenic inhibitors such as Thrombospodin, Endostatin, and Angiostatin seem to inhibit angiogenesis by interfering with proliferation and migration of endothelial cells, and may promote apoptosis (6). Pro-angiogenic factors include uPA and plasminogen, which are involved in basement membrane degradation (recall that uPA converts plasminogen, a zymogen, into its active form, plasmin), as well as a host of growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), and perhaps most importantly VEGF (6). Levels of these different factors regulate whether vessels are in a quiescent or angiogenic state, and changes in the balance mediate the angiogenic switch. In cancer, this switch has become dysregulated, resulting the proliferation and invasion of blood vessels into tumours and surrounding tissue.
Mechanism of angiogenesis
Angiogenesis, being quite complex, requires several key steps to occur. In order to form new capillaries, endothelial cells must first break down the basement membrane surrounding the blood vessel (1). This is accomplished through the release of proteolytic enzymes. Plasmin is one such key proteolytic enzyme, and is responsible for the breakdown of basement membrane proteins such as laminin, fibrin, fibronectin and the protein component of proteoglycans, all of which are important structural components of the extra cellular matrix (1). This break down appears over active in tumour vasculature, seeing as blood vessels supplying tumours have severely reduced basement membranes (1). In addition, plasmin may activate many different matrix mettaloproteinases (MMPs). Each of these MMPs possesses specific substrates (1). However, collectively these proteins bind and degrade a broad range of ECM proteins including collagen, elastin, gelatin, laminin, and proteoglycans among others (1). Plasmin is the active form of a zymogen called plasminogen, which is cleaved to form plasmin by urokinase-plasminogen activator (uPA), a serine protease (1). Expression of uPA is up-regulated by vascular endothelial growth factor (VEGF), a very important factor in angiogenesis, and one which is commonly over-expressed in tumours.
Once the basement membrane has been degraded, epithelial cells begin to proliferate and migrate. Cells called "Tip" cells begin to migrate toward the angiogenic stimulus, followed by a column of proliferating "stalk" cells (2). Migration of tip cells is stimulated by chemotaxis, which involves three major promoters: VEGF, bFGF, and angiopoetins (3). These factors act through receptors on the surface of "tip" cells, and induce dynamic actin remodelling, which drives cell motility (3). Three actin based cellular structures are mainly responsilbe for motility: filopodia (A), lammelipodia (B), and stress fibers (C) (3).
Filopodia are parallel bundles of actin which extend out as projections from tip cells and serve as "feelers", sensing motile stimuli (1). Lammelipodia are cortical actin networks that form flat protrusions, conferring a swimming-type of motility, and are found at the leading edge of migrating cells (1). Stress fibers are parallel arrangements of actin and myosin found anchored to focal adhesion sites, providing traction for the rear of the cell towards the leading edge (1). These migrating tip cells are followed by a stalk of proliferating cells, which are stimulated by a variety of growth factors, including VEGF, some of which are released by the degraded ECM (1)
Once tip and stalk cells have reached their chemotaxic target, it is vitally important that this new growth of cells be able to carry blood. In order to do this, the stalk of cells must re-form itself into a hollow, tubular structure (4). This process, in which a column of cells undergoes a transformation to create an interior lumen, is called tubulogenesis (4). Tubulogenesis is presently not well understood, however two main models have emerged as the most supported theories. The first, referred to as cell hollowing, involves the collection and fusion of vesicles or vacuoles in the center of the stalk of cells to form a lumen. These vesicles are generated through pinocytosis, and carry markers which will identify the new apical end once the lumen is formed, allowing polarization (4).
The other model of tubulogenesis is called cord hollowing. The cord-hollowing model notes that in tubulogenesis, the tip cells migrate out, followed by the proliferation within the column (4). Therefore, the cells in this structure lose their apico-basal polarity, and the cord thickens to become two to three cells wide (as opposed to the single cell wide column in the cell hollowing model) (4). Repolarization is initiated by external cues, with cell surfaces exposed to the extracellular environment taking on basal polarity, and those surfaces in the column interior becoming apical (4). As in the cell-hollowing model, the interior lumen is formed by the fusion of apical vesicles.
Once a newly formed capillary has entered its target tissue, it switches from a proliferative phase to a stabilization and maturation phase. It is thought that pericytes play a major role in this process (5). Pericytes are recruited to new vessels, through differentiation of mesenchymal precursors or migration from nearby sites, and contribute to the suppression of endothelial proliferation and migration (5). This has the effect of fixing the new vessel in a beneficial position, guiding or stabilizing the development of the new vessel bed (5). Pericytes also contribute to the re-establishment of the basement membrane around the new vessel. This is theorized to be achieved through the interaction of endothelial cells and pericytes, which promotes expression of basement membrane proteins such as laminin and fibronectin (5). Tumour blood vessels have been seen with little to no pericytes, which helps explain the severe reduction in their basement membranes, overactive proliferation, and strange disorganized positioning (5). The absence of pericytes in tumor vasculature also results in abnormal vessels which are more porous or "leaky". This abnormal vasuclature can have the effect of reducing the effectiveness of certain chemotherapies, making "normalization" of the vasculature important in treatment, as you will see in later sections.
The prevention and regulation of vascularization is maintained by several proteins, but of recent interest is the involvement of the guardian of the genome - p53 (7). P53 exerts its negative effects on angiogenesis, and thereby preventing tumor growth and metastasis, via three mechanisms (7). First, it disrupts hypoxia sensing mechanisms that promote angiogenesis by physically binding and inhibiting the powerful proangiogenic protein, hypoxia inducible factor (HIF) (7). Second, it decreases proangiogenic factor production by either binding and inhibiting the transcription factors that activate their genes, or by binding to their core promoters to prevent transcription (7). Examples of proangiogenic factors heavily regulated by p53 include VEGF, bFGF, and COX-2 (7). Third, p53 increases the production of proteins that inhibit angiogenesis by binding to its target antiangiogenic genes' promoter via a p53 response element, leading to upregulation (7). Examples of such upregulated antiangiogenic targets include TSP-1, BAI-1, EPHA2, and antiangiogenic collagens (7).
1. Liekens, S., De Clercq, E., & Neyts, J. (2001). Angiogenesis: regulators and clinical applications. Biochemical Pharmacology 61(3), 253-270.
2. M. Slevin (ed.). (2011). Therapeutic Angiogenesis for Vascular Diseases, DOI 10.1007/978-90-481-9495-7_4
3. Lamalice, L., Le Boeuf, F., & Huot, J. (2007). Endothelial Cell Migration During Angiogenesis. Circulation Research 100, 782-794.
4. Tung, J., Tattersall, I., & Kitajewski, J. (2012) Tips, Stalks, Tubes: Notch-Mediated Cell Fate Determination and Mechanisms of Tubulogenesis during Angiogenesis. Cold Spring Harbor Perspectives in Medicine, 2(2): a006601.
5. Ribatti, D., Nico, B., & Crivellato, E. (2011). The role of Pericytes in Angiogenesis. Int. J. Dev. Biol. 55, 261-268.
6. Bergers, B., & Benjamin, L. (2003). Tumorigenesis and the Angiogenic Switch. Nature Reviews Cancer 3(6), 401-410.
7. Teodoro, J. G., Evans, S. K., & Green, M. R. (2007). Inhibition of tumor angiogenesis by p53: a new role for the guardian of the genome. Journal of Molecular Medicine 85(11), 1175-1186.