Método

Angiogenesis may be defined as the formation of new blood vessels and vasculature. This is a complex process requiring the synchronised activity of various cell and vascular components; endothelial cells division and later migration, with vascular basement membrane and surrounding extracellular matrix degradation (Folkman, 2002). In adults, angiogenesis is involved in many normal physiological processes including wound repair, ischemia and embryogenesis (Ching et al. 2010). Its role in cancer pathogenesis has been well defined. Both tumour growth and metastasis are dependent on angiogenesis and lymphangiogenesis. Indeed, angiogenesis is

characterised as one of the hallmarks of cancer (Hanahan and Weinberg, 2011) where quiescent endothelial cells display a proliferative phenotype in response to pro

Hypoxia is a characteristic state in solid tumours (Voupel and Mayer, 2007). This arises from oxygen deprivation in the tumour microenvironment that initiates various

processes associated with tumour angiogenesis (Ching et al.2010). Other stimulus includes mechanical stress and hypoglycaemia (Ferrara, 2004). The’ angiogenic switch’ whereby tumours progress exponentially to propagate past their primary location from unbalanced equilibrium of pro and anti angiogenic factors has been observed in both human tumour and experimental models (Folkman, 2002). These various studies have led to the characterisation of a plethora of angiogenic factors involved in tumour angiogenesis. In particular, VEGFA, a potent pro angiogenic factor (Ferrara 2009).

Under normal physiological processes, the angiogenic network is tightly controlled. Conversely, in tumour angiogenesis perturbation of this controlled network is typical due to an upregulated state arising from the shift ‘switch’ between pro and anti

angiogenic factors (Folkman, 2002). Hypoxia is the most well studied stimuli for tumour angiogenesis. This hypoxic micro-environment stimulates the angiogenic network resulting in sprouting angiogenesis (sprouting of blood vessels) from adjacent tissues into the tumour (Folkman, 2002). Growth of the tumour at this stage around 2 mm in diameter is typical. Angiogenesis pathology may be defined by a change in endothelial cell phenotype to that of invasive and proliferative with atypical tumour vasculature morphology arising from structural abnormalities of venules, capillaries and arterioles. The typically tight vasculature becomes leaky due to the loose EC monolayer.

The angiogenic signalling network involved in tumour pathogenesis is both complex and dynamic. It comprises a plethora of pro and angiogenic signals including

angiopoietins, endothelial and oxygen sensors, growth factors and integrins, in addition to various others described in detail in (Ferrara, 2004). Numerous studies in vivo and in

vitro have uncovered numerous key pro angiogenic factors involved in this process;

VEGF, TGFβ, IL-8, angiogenin and growth factors: endothelial, placental, hepatocyte and epidermal, probably the most significant (Nishida et al.2006). In particular, the VEGF family (A-D), (Ribatti, 2008). Each has varying angiogenic roles differing in their receptor binding specificity dependant on function and tissue. VEGF C and D are typically involved in lymphanogenesis, whilst the role of VEGFB is less defined (Nishida

et al.2006). VEGFA however is a well characterised pro angiogenic stimulator inducing various independent angiogenic cascades. This heparin binding glycoprotein

constitutes six isoforms as a consequence of alternative mRNA splicing (Stalmans et al.2002). Figure 4.1.7 depicts a simplified view of hypoxia induced VEGFA angiogenic pathways.

VEGFA expression is highly regulated by the HIF family members, particularly HIF-1a (Ribatti et al.1999). However, hypoxia induced VEGF expression may also be via HIF independent mechanisms (Mizukami et al.2007). EGF, TGFβ cytokines along with p53 are also pivotal in inducing VEGF expression by several different mechanisms (Dvorak

etal.1999). VEGFA is the most studied member and its increased expression is correlated with poor patient prognosis. VEGFA intra-tumoral expression has been observed in various tumours including breast, lung, ovarian and bladder (Ferrara, 2004). VEGFA mechanism of action is exerted via interaction with tyrosine kinase receptors; VEGFR-1/2, resulting in perturbed effects to vascular ECs and an aggressive tumour phenotype. This is characterised by extracellular matrix degradation, increased vascular permeability and enhancement of EC survival via opposing apoptosis (Ferrara, 2004), thereby allowing tumour formation and progression. These various different, yet overlapping independent angiogenic pathways are challenging. Indeed, numerous anti angiogenic therapies targeting VEGF have low efficacy with limited clinical success. Some has even shown to promote cancer progression by increased invasion and

metastasis (Ribatti, 2010). This inefficacy is probably as a result of such complexity and resistance. Even systematic approaches have been used to dissect angiogenic network dynamics for effective single and multi-combined angiogenic therapies (Montanez et al.2011).

Given this, elucidating the role of pro and anti angiogenic factors, identifying effective markers and targeting these pro-proliferative processes is pivotal to implementation of successful anti-cancer therapy.

Figure 4.1.7 A simplified view of the hypoxia induced angiogenic pathway via VEGF

VEGFA activation via VEGFR induces several pathways to induce angiogenesis via cell

proliferation, survival migration and vascular permeability. Signalling via RAS/MEK.ERK induces expression and proliferation. Signalling via PLC activates PKC which results in increased

intracellular Ca+ inducing vascular permeability by Nitric oxide (NO) production and also prostaglandin (Not shown). FAK (focal adhesion kinase) signalling results in cell migration. VEGFA induction of p38 results in MAP2/3 and hsp27 activation and subsequent cell migration.

p38 further induces p53 with p21 activation, however hsp27 inhibits p21 affecting cell cycle arrest. The p13K pathway ensures cell survival via PI3K activated conversion of PIP2 to PIP3. This results in cellular membrane translocation and activation of AKT which also induces endothelial NO synthase further affecting vascular permeability, however in parallel inhibits caspase 9 and BAD (Pro apoptotic genes) thus maintaining cell survival. Other angiogenic inducers; αv integrins, (transmembrane receptors which bind EM proteins promote sprouting), cytokines such as IL6, and growth factors including FGF2 and PDGFRB.