Inhibiting Angiogenesis

(The Angiogenesis Foundation, 2013)

Naturally occurring negative inducers angiostatin, endostatin, interferon-alpha, interferon-beta, and PEX (chemopexin domain of matrix metalloproteinase II) (Jansen et al, 2004).

Angiogstatin binds many proteins, binding angiomotin, endothelial surface ATP synthase and integrin C annexin-II, c-MET receptor, No2-protoglyclans, and CD26 (O'Reilly et al, 1994). This binding capability inhibits endothelial migration, proliferation, and apoptosis (Sharma et al, 2004). Treatment using angiostatin showed: decreased vascularity, and reduced VEGF mRNA expression (Choudhury et al, 2010).

The second naturally occurring negative inducer, Endostatin targets integrin α_vβ₃ (Yokoyama et al, 2004) inhibiting endothelial cell proliferation and migration, inducing apoptosis in proliferating endothelial cells (Fuetal et al, 2009). Through its association with integrin α_vβ₃ and the proteolytic plasminogen activator system, the adhesion of endothelial cells to the extracellular matrix is affected (Rabbani et al, 2001).

IFN-α and IFN-β exert anti-angiogenic properties by suppressing bFGF expression and decreasing endothelial cell migration (Greenberg et al, 2005). IFN-α and IFN-β presented anti-glioblatoma activity by a mechanism involving central tumor necrosis followed by neovascularization or blood vessel death (Choudhury et al, 2010).

The final negative inducer, PEX, presents significant antimitotic, anti-invasive, and anti-angiogenic properties against GBM (Bello et al, 2001). Originally MMP-2 and integrin α_vβ₃ binding promotes endothelial cell invasion. PEX binds to this integrin, inhibiting the binding of MMP-2, decreasing endothelial cell proliferation (Choudhury et al, 2010).

Alongside naturally occurring negative inducers, clinical trials have used physiological factors to inhibit certain factors in angiogenesis.

Figure 2: Physiological Factors for the Inhibition of Angiogenic Receptors (Choudhury et al b, 2010)

Physiological Factors for the Inhibition of Angiogenic Receptors
Target of the Inhibitor	Inhibitors
EGFR	Gefitinib Erlotinib OSI-774 ZD1839 Tyrophostin AG1478 Lapantinib (EGFR, ErbB-2 inhibitor) AEE788 (EGFR, VEGFR inhibitor) ZD6474 (EGFR, VEGFR inhibitor) EKB569 (erbB1, erBb2 inhibitor) Cetuzimab (anti-EGFR monoclonal antibody)
VEGFR	Valatanib (PTK787) (PDGFR, VEGFR inhibitor) Sorafenib (VEGFR, PDGFR, Raf kinase inhibitor) AZD2171 (VEGFR2 inhibitor) ZD674 (VEGFR, EGFR inhibitor) SU5416 (VEGFR2 selective inhibitor) SU6668 (VEGFR2, PDGFR, FGF inhibitor) CEP 7055 SNS-032 (VEGF, CDK2,7,9 inhibitor) AA481 (VEGFR2 and Raf inhibitor) Pazopanib
PDGFR	Imatinib mesylate PTK787 (PDGFR, VEGFR inhibitor) SU011248 (PDGFR, VEGFR, c-KIT, FLT3 inhibitor) Sorafenib (PDGFRβ, VEGFR1, VEGFR2 inhibitor)
PI3K/Akt Pathway	LY294002 Perifosine PI-103
Integrin	Cliengitide (αvβ3 + αvβ5 inhibitor)

Angiopoietins in Glioblastoma Angiogenesis

(Medizinische Hochshule Hannover, 2013

 

 

Angiopoietins are a family of growth factors that works with VEGF promoting angiogenesis and tumor formation (Brunckhorst et al, 2010). Angiopoietin 1-2 bind to Tie-2 (tyrosine kinase). Angiopoietin 1, secreted from pericytes, plays a role in maturation and stabilization of the tumor vasculature (Choudhury et al, 2010).                 

Tumor cells cause Tie-2 receptor phosphorylation, which stabilizes VEGF-A (Hawighorst et al, 2002) inducing vasculature, endothelial cell sprouting, migration, and survival (Sako et al, 2008).                                 

Angiopoietin-2 expressed by endothelial cells (Hegen et al, 2004) has destabilizing ion effects on Ang-1/Tie-2 facilitating neovasculation and vessel sprouting in the presence of VEGF-A (Asahara et al, 1998). In the absence of VEGF, vessel regression and endothelial cell apoptosis is induced (Choudhury et al, 2010). 

The role that TGF-β and TGFR play in Angiogenesis

(National Academy of Science, 2013)

 

Like EGFR, TGF-β and TGFR are growth factors and mitogens for endothelial cells (Held-Feindt et al, 2003) expressed on GBM (Choudhury et al, 2010). TGF-β receptors I and II are trans membrane serine/threonine receptor kinases mediating intracellular signaling (Grznil et al, 2011).

TGF-β functions by regulating and inducing the expression of other growth factors: bFGF, PDGF, PDGFR, EGFR (Van Meeteren et al, 2011). It also potentiates the secretion of EGF in GBM as well as up regulating α2 or βintegrin subunit, contributing to endothelial cell motility (Choudhury et al, 2010). ­­­­

The Role that TGF-α, EGF, EGFR play in Glioblastoma Multiforme

(Hooper, 2013)

The epidermal growth factor, stimulating VEGF expression in GBM (Poulaki et al, 2003), and transforming growth factor beta are both RTKs involved in the RAS pathway (Serban et al, 2008). Different forms of the epidermal growth factor are created via mutation, alternative splicing, deletion, genetic rearrangement, and translational alterations (Talasila et al, 2012).

(Shi, 2013)

EGFRvIII and ΔEGFR are over expressed in 60% of all GBMs (Johnson et al, 2012). Both EGFRvIII and ΔEGFR lack a regulatory portion of the extracellular binding site, and function via phosphorylation (Hwang et al, 2012).

(Janku et al, 2010)

 

TGF-α and EGF are mitogens (Rutten et al, 1993) for endothelial cells, promoting cell proliferation, survival, and GBM angiogenesis (Kaur et al, 2005).

The Role that FGF and FGFR play in Glioblastoma Multifrome

FGF-1 and FGF-2 are mitogens and chemo attractants that both play an important role in angiogenesis (Friesel et al, 1995). Their biological activity is mediated by RKS’s receptor tyrosine kinases, modulating endothelial cell activity and regulating VEGF expression in tumor cells (Choudhury et al, 2010).

Figure 7: uPA leads to degradation of the extracellular matrix (Universita Degli Studi di Brescia b, 2013)

FGF-2 up regulates and induces the presence of uPA and collagenase (Sahni et al, 2004) in endothelial cells as well as potentiating the presence of VEGF on GBM in a dose dependent manner (D'Orazio et al, 1997). uPA is important because it converts inactive zymogen plasminogen to active proteolytic enzyme plasmin (Ribatti et al, 1999). This in turn degrades the extracellular matrix components: fibronectin and laminin allowing endothelial cell migration (Lamalice et al, 2013).   

 

The Role that PDGF and PDGFR play in GBM Angiogenesis

(Nature Reviews, 2002)

The platelet derived growth factor (PDGF) targets endothelial cells, vascular smooth muscle cells, osteoblasts, glia, and neurons resulting in proliferation, resistance to apoptosis, tumor growth, and angiogenesis (Nobuo et al, 2006).

The isoforms of PDGF are activated by dimerization (Fredrikkson et al, 2004). Dimerized ligands bind to tyrosine trans receptor subunits inducing dimerization (Roskoski, 2007). Dimerization leads to trans-auto phosphorylation by intracellular tyrosine domains which activate signal transduction pathways promoting downstream gene transcription activity (Choudhury et al, 2010).

PDGFB and PDGF-β are overexpressed in hyperplastic tumor endothelial cells (Hermansson et al, 1988). The expression of these proteins leads to increased transcription and secretion of VEGF by endothelial cells via the PI3K pathway (Karar et al, 2011).

The Role that VEGF and the VEGFR play in Glioblastoma Angiogenesis

Figure 1: Function of VEGF Overview (Qiagen Company, 2013)

Angiogenesis begins when tumor cells release proangiogenic factors (Bao et al, 2006) such as the vascular endothelial growth factor (VEGF) (Black et al, 2005). VEGF is one of the most potent factors playing a role in the formation of angiogenesis (Choudhury et al, 2010).

 VEGF functions by diffusing into nearby tissues and binding to receptors on the endothelial cells of the preexisting blood vessels, activating them (Black et al, 2005). The interaction between VEGF and the endothelial cells stimulates proliferation, migration, survival, and increases blood vessel leakiness as well as plasma protein extravasation (Choudhury et al, 2010). VEGF also plays a role in the production of nitric oxide (NO) by stimulation of endothelial nitric oxide synthase further activating the angiogenic cascade (Cooke et al, 2013) and stimulating the migration of monocytes and neutrophil (Choudhury et al, 2010).

There are six VEGF homologues that have been identified (Cho et al, 2003). VEGF-A is the major promoter of angiogenesis because of it’s high potency and specific responses to endothelial cells (Nagy et al, 2007). The expression of VEGF-A is facilitated by hypoxia-inducible factor 1 (HIF-1) and a transcriptional regulator (containing α and β subunits) (Choudhury et al, 2010). 

The presence of VEGF is dependent upon the binding of HIF-1 (Harada et al, 2007). In hypoxic conditions, HIF-1 binds increasing the transcription of VEGF-A, therefore increasing VEGF-A protein secretion (Choudhury et al, 2010). In turn the expression of VEGF-1 is increased, which results in angiogenesis (Bussolati et al, 2006). In hypoxic conditions, TSP-1 is down regulated, which is unfortunate because TSP-1 inhibits endothelial cell division and tube formation (Taraboletti et al, 2010).

Figure 2: Hypoxia Condition Overview (Case Western Reserve University, 2013)

In ischemia, a condition where the glucose and oxygen intake is depleted, (Fraum et al, 2011) intracellular signaling via the MAPK pathway increases resulting in endothelial cell proliferation (Seger et al, 1995).

     

Figure 3: Activation of PKB/AKT and its targets (Scheid et al, 2001)

The MAPK pathway functions through VEGF-A (Doanes et al, 1999). This pathway begins with the phosphorylation of PI3K, activating Pkb/Akt, (Hemmings et al, 2012) inducing endothelial cell survival (Shiojima et al, 2002). VEGFR-2 complexes are also activated reducing p53, p21, and BAX expression, resulting in an increase of BCL-2 expression (Brakenhielm, 2007) promoting further survival in endothelial cells (Choudhury et al, 2010).  

When the expression of VEGFR-1 and VEGFR-2 is increased, tumor angiogenesis is initiated (Vrendenburgh et al, 2007) via integrin interactions with VEGFR-2 (Choudhury et al, 2010).  Both VEGFR-1 and VEGFR-2 are important receptors involved in signaling with VEGF-A (Olsson et al, 2006). VEGF-A, released by tumors, signals to VEGFR-1 for support of the vascular system (Roskoski, 2007).

The microenvironment is also involved in angiogenesis because of VEGFR-1 expression. Not only is VEGFR-1 expressed on endothelial cells, but also, macrophages, pericytes, vascular smooth endothelial muscle, progenitor cells, EPC’s (bone marrow endothelial cells), and hematopoietic cells (Hall, 2006).

Involving the microenvironment strengthens angiogenesis because VEGFR-1 is the receptor for the placental P1GF (placental growth factor), VEGF-A, (Carmeliet et al, 2001) and VEGF-B (Hoeben et al, 2013). Upon the binding of VEGF-B tumor progression and metastasis is promoted via proteolytic enzymes (Bambace et al, 2011).

VEGFR-3, a receptor for VEGF-C and VEGF-D, (Lawrence et al, 2003) is also up regulated. Because it is a key regulator of angiogenesis, binding to VEGFR-3 promotes angiogenesis and metastasis in the lymphatic system (Choudhury et al, 2005).

             Figure 4: VEGFR system overview (Universita Degli Studi di Brescia a, 2013)

To summarize, VEGFR-1 is in charge of drawing other cells to the system (Nam et al, 2004), VEGFR-2 is essential for endothelial cell contribution to angiogenesis (Black et al, 2006), and VEGFR-2 aids in the metastasis of the lymphatic system (Choudhury et al, 2010).