Angiogenesis

Key features of angiogenesis Tumour growth is angiogenesis-dependent Microvascular endothelial cells (ECs) are genetically-stable ECs release angiogenic factors that stimulate proliferation

Composition of nascent and mature blood vessel walls Nascent vessels consist of a tube of ECs, which mature into specialized capillaries, arteries and veins. (b) Capillaries consist of ECs surrounded by basement membrane and a sparse layer of pericytes embedded within the EC basement membrane. Capillary endothelial layer can be continuous (muscle), fenestrated (kidney/ endocrine glands) or discontinuous (liver sinusoids). The endothelia of the blood-brain barrier or blood-retina barrier are further specialized to include tight junctions, and are thus impermeable to various molecules. (c) Arterioles and venules have an increased coverage of mural cells compared with capillaries. a) Nascent vessels consist of a tube of ECs. These mature into the specialized structures of capillaries, arteries and veins. (b) Capillaries, the most abundant vessels in our body, consist of ECs surrounded by basement membrane and a sparse layer of pericytes embedded within the EC basement membrane. Because of their wall structure and large surface-area-to-volume ratio, these vessels form the main site of exchange of nutrients between blood and tissue. Depending upon the organ or tissue, the capillary endothelial layer is continuous (as in muscle), fenestrated (as in kidney or endocrine glands) or discontinuous (as in liver sinusoids). The endothelia of the blood-brain barrier or blood-retina barrier are further specialized to include tight junctions, and are thus impermeable to various molecules. (c) Arterioles and venules have an increased coverage of mural cells compared with capillaries. Precapillary arterioles are completely invested with vascular SMCs that form their own basement membrane and are circumferentially arranged, closely packed and tightly associated with the endothelium. Extravasation of macromolecules and cells from the blood stream typically occurs from postcapillary venules

Steps in network formation and maturation during embryonic (physiological) angiogenesis Figure 2. Steps in network formation and maturation during embryonic (physiological) angiogenesis (a) and tumor (pathological) angiogenesis (b). (a) The nascent vascular network forms from an initial cell plexus by processes of vasculogenesis or angiogenesis. This is regulated by cell-cell and cell-matrix signaling molecules (Box 2) and mechanical forces, as is further growth and expansion of the network (by proliferating and migrating cells) alongside its normal remodeling by cell death (apoptosis). Ordered patterns of growth, organization and specialization (including the investment of vascular channels by mural cells) produce mature networks of arteries, capillaries and veins—networks that are structurally and functionally stable and appropriate to organ and location. (b) ECs and mural cells derived from circulating precursor cells or from the host vasculature form networks that are structurally and functionally abnormal. Continual remodeling by inappropriate patterns of growth and regression (cell apoptosis and necrosis) contribute to the instability of these networks.

Key differences in tumour vasculature Different flow characteristics or blood volume Microvasculature permeability Increased fractional volume of extravascular, extracellular space

Steps in network formation and maturation during tumour angiogenesis Figure 2. Steps in network formation and maturation during embryonic (physiological) angiogenesis (a) and tumor (pathological) angiogenesis (b). (b) ECs and mural cells derived from circulating precursor cells or from the host vasculature form networks that are structurally and functionally abnormal. Continual remodeling by inappropriate patterns of growth and regression (cell apoptosis and necrosis) contribute to the instability of these networks.

Cellular mechanisms of tumour angiogenesis (1) host vascular network expands by budding of endothelial sprouts or formation of bridges (angiogenesis); (2) tumour vessels remodel and expand by the insertion of interstitial tissue columns into the lumen of pre-existing vessels (intussusception); and (3) endothelial cell precursors (angioblasts) home from the bone marrow or peripheral blood into tumours and contribute to the endothelial lining of tumour vessels (vasculogenesis) (4) Lymphatic vessels around tumours drain the interstitial fluid and provide a gateway for metastasizing tumour cells. 1 3 2 2 1 3 In the adult, the majority of angiogenesis is associated with wound healing, the reproductive tract, and inflammation. At the cellular level, this process occurs by several different mechanisms, including a phenomenon termed sprouting (9), which is a major method by which tumors recruit the preexisting vasculature. Sprouting is stimulated in response to local molecular cues, such as hypoxia- or inflammation-induced VEGF-A production. Thought to be a stepwise process, sprouting begins with local increases in vascular permeability, followed by basement membrane and ECM degradation. Subsequently, endothelial cells sometimes called tip cells send out projections and initiate migration along newly deposited ECM tracts. Finally, lumen-containing vessels are formed and integrated into the circulation. Recent evidence using real-time imaging techniques in zebrafish demonstrates that the latter steps involve, at least in some vessels, endothelial pinocytosis followed by coalescence of intracellular vacuoles 4 4

Cellular angiogenesis-overview Tumour cells release pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which diffuse into nearby tissues and bind to receptors on the endothelial cells of pre-existing blood vessels, leading to their activation. Such interactions between endothelial cells and tumour cells lead to the secretion and activation of various proteolytic enzymes, such as matrix metalloproteinases (MMPs), which degrade the basement membrane and the extracellular matrix. Degradation allows activated endothelial cells — which are stimulated to proliferate by growth factors — to migrate towards the tumour. Integrin molecules, such as v 3-integrin, help to pull the sprouting new blood vessel forward. The endothelial cells deposit a new basement membrane and secrete growth factors, such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel. PDGFR, PDGF receptor; VEGFR, VEGF receptor. Nature Reviews Drug Discovery 1, 415-426 (2002)

Cellular angiogenesis-overview Balance between inhibitory factors and angiogenic factors Inhibitory – endostatin, angiostatin, thrombospondin Angiogenic - VEGF, bFGF, PDGF Tumour cells release pro-angiogenic factors which activate receptors (VEGFR) also stimulates secretion and activation of MMPs which degrade the basement membrane This allows activated endothelial cells (ECs) to migrate towards tumour, helped by integrins ECs deposit a new basement membrane and secrete growth factors such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel. Tumour cells release pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which diffuse into nearby tissues and bind to receptors on the endothelial cells of pre-existing blood vessels, leading to their activation. Such interactions between endothelial cells and tumour cells lead to the secretion and activation of various proteolytic enzymes, such as matrix metalloproteinases (MMPs), which degrade the basement membrane and the extracellular matrix. Degradation allows activated endothelial cells — which are stimulated to proliferate by growth factors — to migrate towards the tumour. Integrin molecules, such as v 3-integrin, help to pull the sprouting new blood vessel forward. The endothelial cells deposit a new basement membrane and secrete growth factors, such as platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel. PDGFR, PDGF receptor; VEGFR, VEGF receptor. VEGF – Vascular Endothelial Growth Factor bFGF - basic Fibroblast Growth Factor MMPs – Matrix MetalloProteinases PDGF – Platelet-Derived Growth Factor

Metastasis

Invasion & Metastasis 1 4 3 2 5 6 Red: E-Cadherins Green: Integrins

Integrins – the ‘velcro’ of the cell The cell moves by "ruffling" it's membrane. This is done by a series of actin fibers, whose function is controlled by the integrins. These fibers cause the cell membrane to move in certain directions, and the integrins attach to the matrix as this happens, pulling the cell along a micrometer at a time

Invasion & Metastasis Red: E-Cadherins Green: Integrins

Epithelial-mesenchymal transition (EMT) necessary for invasiveness

Then why do secondary tumours histopathologically resemble primary tumours? EMT induced by stromal signals EMT may be reversible depending on the stromal signals e.g. TGF-b, TNF-a, EGF, HGF, IGF-1.

Stromal signals that trigger EMT

Cell invasiveness controlled by Matrix Metalloproteinases (MMPs) MMPs secreted by stromal cells Can be PM-bound or soluble enzymes MMP activation can be indirect E.g. via urokinase plasminogen activator (uPA)

Cell motility regulated by RhoGTPases Binary switches like Ras 3 sub families; Rho, Rac and cdc42 Lysophosphatidic acid Overexpressing Rac Serum-starved GEF

Metastatic cells travel via lymphatic systems

Colonisation depends on a variety of factors metastatic tropisms (Paget’s ‘seed & soil’theory)

Colonisation depends on complex interactions between metastasising cells and their microenvironments E.g Osteolytic metastasis initiated by breast cancer

Breast cancer initiated osteolytic metastasis Bone growth versus loss

Reading Chapter 13 and 14 : Biology of Cancer by R Weinberg AND /OR Chapter 12: Cancer Biology by RJB King Angiogenesis in cancer and other diseases by P Carmeliet & RK. Jain Nature vol 407 14 september 2000 pp 249