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).

Angiogenesis, a Major Risk Factor of All Cancers

 

(Dominique, 2013)

Because Glioblastoma multiforme is rapidly growing; it needs more oxygen and nutrients than what is supplied by the current blood that nourishes normal tissue (Black et al, 2005).  Glioblastoma gets this additional supply of oxygen and nutrients via angiogenesis (Genentech BioOncology, 2013). Not only does Angiogenesis supply additional nutrients, but it also plays an important role in the metastases of tumors and the enlargement of the tumor (Black et al, 2005).                 

Angiogenesis can be simply defined as “the formation of blood vessels from pre-existing blood vessels” (Choudhury et al, 2010). These blood vessels play a vital part in reproduction, development, and repair of cancer cells (Black et al, 2005).                        

Angiogenesis originates from angioblasts of extraembryonic mesoderm (Polin et al, 2011). Once the embryo has formed a primary vascular plexus called vasculogenesis, more blood vessels are modeled via sprouting and non-sprouting angiogenesis (Choudhury et al, 2010). Adults can form new blood vessels under pathologic conditions such as wound healing, ophthalmologic disorders, and tumors (Black et al, 2005).                                                     

Even though, angiogenesis has been known for over a 100 years now, the mechanism at which it functions is unclear. (Italiano et al, 2008) In 1971, Judah Folkman proposed the interesting hypothesis that, “tumor growth is angiogenesis dependent and that endothelial cells may be switched from a resting state to a rapid growth phase by diffusible chemical signal from tumor cells” (Pollack et al, 2008). Today evidence supports angiogenesis to being essential for tumor growth and propagation in glioblastoma multiforme (Black et al, 2005).

Tumor angiogenesis parallels developmental angiogenesis, except for that fact that tumor angiogenesis continues uncontrollably ceasing to stop (Hillen et al, 2007). The tumor vasculature consists of vessels from a preexisting network, as well as vessels resulting as an angiogenic response to cancer cells (Murat et al, 2009).                                                                  

 In order for Angiogenesis to occur, there is a complex interplay between tumor cells, endothelial cells, and several angiogenic factors that promote endothelial cell migration, (Lamalice et al, 2013) proliferation, vascularization, and capillary formation (Black et al, 2005). Angiogenesis is facilitated by growth factors, adhesion molecules, and matrix-degrading enzymes (Ingber et al, 1989).

Figure 2 Factors Influencing Angiogenesis (Koontongkaew, 2013)

Facilitation via cytokines (regulatory proteins) (Lee et al, 2013) occurs if there is an overexpression of angiogenic factors through hypoxia or mutations (Choudhury et al, 2010).

Angiogenesis begins with the breaking down of the basement membrane of the vessel, (Rundhaug, 2003) and continues with the migration of the endothelial cells towards a stimulus. Endothelial cells start proliferating and trail behind leading cells invading the stroma (Choudhury et al, 2010). Lumen begins to form in the endothelial sprout, and branches and loops develop to allow blood flow. Pericytes will than provide support around immature vessels (Black et al, 2005).

Once the endothelial cells are activated, they release proteolytic enzymes such as matrix metalloproteinase (MMPs), (Page-McCaw et al, 2007) which degrade the extracellular matrix and the basement membrane, allowing activated cells to migrate towards the tumor (Birkedal-Hansen et al, 1993). Integrin molecules play a role in pulling the sprouting new blood vessels forward (Mizejewsk et al, 1999). The endothelial cells deposit a new basement membrane and secrete growth factors such as the platelet derived growth factor (PDGF), attracting supporting cells to stabilize the new vessel (Sato et al, 1993).

Another mechanism of formation can be by the insertion of interstitial tissue column into to the lumen of existing vessels (Davis et al, 2012). Despite the mechanism of formation, the vessels lose the normal anatomic structural arrangements and can be leaky and fragile leading to hemorrhaging (Xia et al, 2004).

Glioblastoma Multiforme, the Brain Cancer Killer, Statistics and Current Therapies

Glioblastoma multiforme (GBM) is one of 150 recorded brain tumors (Bellenir, 2007), accounting for 78% of 18,500 cases of all malignant central nervous system cancers (Choudhury et al, 2005). Unfortunately, it is the most invasive, malignant, highly vascularized, infiltrative, and lethal glial tumor (Choudhury et al, 2010) with a poor prognosis. (Bellenir, 2007) The World Health Organization categorizes glioblastoma to be a high-grade (IV) fast growing astrocytoma containing dead tumor cells, developing in the cerebrum, especially in the frontal and temporal lobes. (Bellenir, 2007) The median age of diagnosis for glioblastoma is 50 to 70 and it is more prevalent in men than women.

 

 

Glioblastoma multiforme is characterized by differentiated cells with multinuclei in the presence of nuclear atypia, mitoses, microvascular proliferation, and pseduopalisading necrosis (Chodhurey et al, 2005).

 

Glioblastoma multiforme has two mechanisms of development. It may develop de novo (primary development), or it can originate from low-grade astrocytomas (secondary development). Primary tumors can be classified as glial or non-glial functioning as either benign or malignant. (Bellenir, 2007) An adult diagnosed with a primary tumor will only live for six months. The primary tumor over expresses the epidermal growth factor, mutation in the phosphatase and tension homolog on the chromosome, and deletions of the cyclin-dependent kinase inhibitor 2A.  (Chodhurey et al, 2005)

 

Unlike primary tumors, secondary tumors are more common in children. The secondary tumors will contain mutation in the tumor protein 53 (TP53) and over expresses the platelet derived growth factor (PDGFR).

 

Current therapies for glioblastoma multiforme include surgery, radiation, and chemotherapy. Decisions as to what treatments are used are based on the specific condition of the patient. (Bellenir et al, 2007)

 

In surgery, the neurosurgeon’s challenge is to remove as much of the brain tumor as possible. Common procedures include craniotomy and sterotactic biopsy. However, even if 90% of the tumor is removed a patient will only live for 9-15 months. Because glioblastoma is a high-grade glioma, it forms tentacle like structures that invade surrounding tissue, making surgery a less likely option. (Bellenir et al, 2007)

 

If surgery is not an option, radiation, the use of high-energy x-ray to kill cancer cells, can be used. The main types of radiation include standard external beam radiotherapy, proton beam treatment, and sterotactic radiosurgery. (Bellenir et al, 2007)

 

The final treatment, chemotherapy only positively effects 20 percent of all patients. Chemotherapy functions by causing cell damage that is better repaired by normal tissue than tumor tissue. Resistance occurs if the tumor tissue survives, and it is unable to respond to the drug. Resistance may also occur, if the drug is unable to pass through the blood brain barrier. (Bellenir et al, 2007)