Targeting Angiogenesis in Cancer Treatments: Where do we Stand?

Since early seventies of the twentieth century, through seminal work of Judah Folkman, angiogenesis, the process of new blood vessel sprouting from the existing vasculature, was recognized as a necessary part of wound healing, development of placenta, tissue growth and regeneration as well as cancer progression. This process is induced by low tissue oxygenation and it is a crucial prerequisite for rapid tissue growth, providing proper oxygen supply and removal of toxic metabolites. Suppression of angiogenesis as a way of slowing down tumor progression continues to be one of the most important areas of cancer research. The angiogenic process is relatively complex and it is regulated by numerous proand anti-angiogenic factors. Intensive research in the last twenty years resulted in identification of more than 300 angiogenesis inhibitors, a trend that is expected to continue. Unfortunately, most of these treatments have demonstrated unacceptable toxicities or failed to show activity in clinical studies. Although not yet completely understood, the complex process of tumor angiogenesis involves highly regulated orchestration of multiple activating and inhibiting factors. Vascular endothelial growth factor (VEGF) and its cognate receptors appear to play a central role in angiogenesis activation. Thus, initial efforts to develop anti-angiogenic treatments focused largely on inhibiting VEGF action. Such approaches, however, often lead to transient responses due to multiple pathways able to compensate for a single pathway inhibited. Accordingly, more recent treatments have focused on simultaneous inhibition of multiple signaling pathways. This review concentrates on identifying those antiangiogenic treatments that made to the clinic by receiving approval by USA Food and Drug Administration (FDA) as treatments for cancer. Regardless of observed problems, it is an imperative that research in angiogenesis regulation continues. Consequently, pharmacological manipulation of angiogenesis may yet to introduce truly new pharmacological therapies into the field of cancer therapy, the field that was rather dormant in the last several decades. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page. _______________________________________________________________________________________


INTRODUCTION
Formation of functional vasculature is the crucial prerequisite for growth and most of physiological processes in mammalian tissues. Proper supply with oxygen and nutrients, together with efficient removal of unwanted metabolites and CO2 are necessary for growth of the healthy tissues as well as tumors. It is estimated that growth of tissue to the sizes above 2-3 mm 3 requires proper blood supply (1). During embryonic development blood vessels are created de novo from endothelial cell precursors (angioblasts) through the process called vasculogenesis (2). Vasculature formation during development is not the end of vascular network growth. Vascular tissue formation is highly dynamic process continuing well after cessation of development. Adjustment of vascular network coverage is continuously occurring throughout adult life and it is progressing by new blood vessel outgrowth from pre-existing capillaries in the process of angiogenesis (3). Such growth of new vasculature is usually quite limited since apart from growing tumors, relatively few adult tissues require ongoing angiogenesis. These include female reproductive organs, organs that are actively growing or tissue regenerating following injury (4). Transition from quiescent vascular state to active angiogenesis is termed "angiogenic switch". While vasculogenesis and angiogenesis have been considered to be distinct processes, they share many similarities. Thus, some evidence suggests that endothelial progenitors may contribute to vessel growth both in embryo and in ischemic and malignant adult tissues (5,6). ________________________________________ However, the percentage of endothelial precursors incorporated into newly formed blood vessels during angiogenesis is generally low, supporting the concept that adult tissues and tumor neovascularization occurs primarily via the process of existing vessel sprouting (5).
In early tumor development, cancer cells are oxygenated through simple diffusion in a phase defined as "avascular state". Tumors of small size can remain dormant for many months after reaching a steady state between proliferation and apoptosis (7). The demand for oxygen and nutrients varies widely among tumors and it is often dependent upon tumor type, its stage of growth and its location. These physiological requirements could also change over the course of tumor progression (8).
Tumor cells characteristically show a reduced oxygen dependency compared to normal cells, and are thought to sustain their metabolism under relatively anaerobic conditions by increasing glycolysis to maintain ATP production (so called Warburg effect) (9). However, despite their increased tolerance to hypoxia, tumors remain strictly dependent on adequate oxygen supply for their exponential growth. After initial acute oxygen deprivation and as growth progresses and the lack of oxygen becomes chronic. Induction of specific genes expression allows tumors to overcome hypoxia by increasing their blood supply through angiogenesis. This neovascularization signals the change from relatively dormant state to a tumor capable of rapid growth and metastasis. It is characterized by a set of physiological events termed the "angiogenic switch" which is provoked by a change in balance of pro-and anti-angiogenic molecules (4). In addition to physiological stimuli, factors influencing the angiogenic switch include genetic mutations, metabolic and mechanical stress, inflammation and activation of oncogenes and/or inactivation of tumor-suppressor genes (10,11). Factors affecting the onset of the angiogenic switch may be tumor and tissue-specific and may also change during tumor progression (12,13). The switch can occur at different stages of tumor growth, again depending on the type of the tumor and the surrounding tissue. Angiogenesis will continue as long as the tumor grows and demand for oxygen and nutrients by the hypoxic and necrotic areas of the tumor exists. Furthermore, angiogenesis is a crucial process that impacts not only malignant tumor growth, but also increases tumor metastasis (13).
This review aim is to summarize current status of angiogenesis suppressive therapies through identifying those treatments that made to the clinic by receiving approval by USA Food and Drug Administration (FDA) as treatments for cancer. Also, limitations of such therapy are outlined, as well as some new directions. Regardless of significant complications encountered in application of such therapies, angiogenesis suppression is still an important new addition to the classical cancer treatments that did not include any new pharmacological options in the past several decades.

Angiogenesis phases
The process of angiogenesis progresses through several distinct stages (Diagram 1). It begins with perivascular detachment and vessel dilation (Diagram 1B) in the vicinity of the tissue affected by hypoxia, followed by disassembly of existing blood vessel into individual activated endothelial cells and pericytes (14). Pericytes are cells embedded within the basement membrane of small blood vessels. Their major function appears to be reinforcing and stabilization of vascular structure to achieve proper microvascular blood flow. Activated pericytes and endothelial cells participate in digestion of basal lamina by releasing proteases (Diagram 1C). Interaction between endothelial cells and extracellular matrix appears to be crucial for proper endothelial angiogenic activation (15). Angiogenic process continues through division and migration of endothelial cells and pericytes toward the hypoxic tumor (Diagram 1D), followed by assembly and differentiation of endothelial cells into the new blood vessel (Diagram 1E). The process is finalized by establishment of new vascular connection between primary vessel and tumor. This is achieved by maturation and renewed recruitment of pericites to the newly formed blood vessel (Diagram 1F). Successful completion of each of these steps is required for the final formation of the functional new blood vessels.

Regulation of angiogenesis
Major signal for initiation of angiogenesis is tissue or tumour hypoxia and subsequent decrease in oxidative metabolism and ATP production (16). Intracellular induction of hypoxia-inducible factors (HIFs) appear to be crucial for transcriptional induction of genes that mediate subsequent angiogenic steps. HIFs are constantly expressed and degraded under normoxia, but stabilized under hypoxia (17).

Diagram 1: Stages of tumor angiogenesis
Specifically, HIF-2α has the major role in vascular remodelling through angiogenesis (17). As a consequence of HIFs induction in tissues affected by hypoxia, numerous humoral factors are released from affected tissue to initiate functional changes in neighbouring vasculature leading to angiogenesis (18).
Hypoxia-inducible factor-1α (HIF-1α)induced expression and release of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) during tissue and tumor hypoxia plays the most important part in activation of vascular endothelial cells and initiation of angiogenesis process (19,20).
In order to respond to these signals, vascular endothelial cells express corresponding receptors. VEGF is not a single factor but a relatively large family of growth factors. VEGF-A is the prototypical member of a family that includes placental growth factor (PLGF), VEGF-B, VEGF-C, and VEGF-D (also known as c-Fos-induced growth factor, FIGF) (21). In that family, VEGF-A appears to be the main regulator of angiogenesis. VEGF-A is not a single protein, but a group of three major isoforms resulting from alternative splicing of mRNA from a single, 8-exon VEGFA gene. Such process is producing three isoforms (VEGFA120, VEGFA164, and VEGFA188) all of them physiologically active. VEGF-A family binds to two tyrosine-kinase receptors: VEGF receptor 1 (VEGFR-1 also known as flt-1) and VEGF receptor 2 (VEGFR-2, also called KDR/Flk-1), which are expressed almost exclusively in endothelial cells (22,23). VEGF-A has been shown to be the necessary factor in angiogenesis induction. It stimulates endothelial cell mitogenesis, cell migration and expression of genes that regulate angiogenic switch (24).
Similarly to VEGF, physiological effects of fibroblast growth factors (FGFs) are mediated by four structurally related receptor tyrosine kinases, denoted FGFR-1, -2, -3 and -4, which display broad expression patterns. FGFs are important angiogenic growth factors, and basic FGF (bFGF or FGF2) is recognized as a potent inducer of angiogenesis (25,26).
In addition to growth factors, interaction of endothelial cells with extracellular matrix appears to be crucial for proper regulation of angiogenesis. Major constituents of extracellular matrix are structural proteins such as laminin, entactin, collagen and heparan sulfate proteoglycans (27). Cell adhesion to such complex substrate is essential for the expression of numerous endothelial genes and differentiation of endothelial cells during angiogenesis. Importance of matrix-endothelial integrin interaction in angiogenesis has been well-documented (28).
Considering that angiogenesis is heavily regulated sequential set of events and that its progression is dependent on successful completion of each of the proceeding steps, it is not surprising that there are many endogenous factors that could stop or slow down the whole process (29 and Table  1). Consequently, blocking of excessive pathological angiogenesis could be theoretically achieved by either increasing activity of endogenous inhibitors or blocking the action of angiogenesis activators. Endogenous inhibitors of angiogenesis include interferons (α, β and γ) which act by decreasing expression of angiogenesisactivators (30) such as bFGF or induce apoptosis of endothelial cells (31); Interleukin 12 promotes apoptosis of endothelial cells (32) whereas interleukin-10 down-regulates synthesis of VEGF and matrix metalloproteinase 9 (MMP-9) (33); Tissue inhibitor of matrix metalloproteinase (TIMP), inhibits degradation and remodelling of extracellular matrix which is necessary for efficient endothelial cells migration and proliferation (34,35); Angiostatin, a 38 KDa internal fragment of plasminogen, down-regulates VEGF expression and suppresses endothelial proliferation and migration (36)(37)(38); Endostatin, a 20 KDa fragment of type XVIII collagen blocks action of VEGF (39); Pigment epithelium-derived factor (PEDF) inhibits endothelial migration and proliferation (40); Vasostatin, a 180 amino acid calreticulin fragment, inhibits endothelial cell proliferation (41); Kringle 5 fragment of human plasminogen induces apoptotic death of endothelial cells (42); Thrombospondin-1 is a glycoprotein that influences cellular phenotype and the structure of the extracellular matrix. It inhibits angiogenesis through direct effects on endothelial migration and survival and through indirect effects on growth factor mobilization (43); Chondromodulin-I (ChM-I), a 25-kDa glycoprotein in cartilage matrix secreted from chondrocytes into the extracellular matrix inhibits endothelial migration (44).
Unfortunately, there are no clinical treatments or drugs developed based on those endogenous inhibitors of angiogenesis apart from interferon-α. Potential reasons for such outcomes could reside in the fact that peptide drug treatments are extremely difficult to design and implement. Furthermore patient treatment with interferons and cytokines has been plagued with significant systemic adverse effects and unacceptable high toxicity (45). Table 1. Endogenous inhibitors of angiogenesis Consequently, design of new treatments aimed at blocking angiogenesis through suppressing VEGF action are currently the main objective in clinical anti-angiogenesis research (46). Currently available anti-angiogenic drugs have shown to result in only limited clinical benefits. Findings in early clinical studies suggest that decrease in dynamic of cancer progression could be achieved, but overall patient survival is in most cases unaffected. Furthermore, when tested in early stage cancers, there seems to be no benefit of angiogenesis inhibitors, possibly due to the low angiogenic potential of tumors at this stage (47). As the discovery of numerous new molecules with angiogenesis suppressive action grows, many new potential treatments have been proposed. The following examples are just some of the new research directions that deviate from the standard approaches outlined above.
A newly-developed vaccination treatment consisting of inducing an immune response against the tumor vasculature combines the benefits of immunotherapy and angiogenesis suppression and may overcome the limitations of current antiangiogenic drugs (48). Such therapy relies on the fact that active angiogenesis is usually absent in adults, thus minimizing potential adverse effects. Most studies have focused on VEGF as a target for active immunization in animal models, although attempts were also made to use glutaraldehydefixed endothelial cells as antigens in three malignant brain tumor patients and showed partial or complete tumor responses (49). Further studies in that direction are certainly needed to fully assess validity of such therapy.
Another example is the use of small interfering RNA (siRNA) to inhibit specific molecular targets involved in activation of angiogenesis. SiRNA, sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length designed specifically to bind to and cause degradation of mRNA of specific gene targets. For example, siRNA suppressing expression of survivin was tested for its effects on angiogenesis in vitro. Survivin is a member of the inhibitor of apoptosis family. It inhibits caspase activation, thereby leading to suppression of apoptosis or programmed cell death. Blocking of survivin expression is found to have effects in cancer and endothelial cells, causing inhibited proliferation and apoptosis (50).
Furthermore, many agents from Chinese herbal medicines have shown powerful anti-angiogenic activities against tumor in in vitro and animal studies (51).
Regardless of these exciting new findings of new anti-angiogenesis factors, none of those treatments ever successfully passed clinical trials and their clinical potential is yet to be tested.
While cancer therapy aimed at suppressing angiogenesis in solid tumors is relatively easy to conceptualize, role of that therapy in blood cancers (various types of leukemia) is much more difficult to understand. Since all white blood lineage cells are produced in bone marrow, higher requirement for oxygen in bone marrow during increased production of blast cells in leukemic conditions could be the connection to solid tumor angiogenesis. Pronounced vascularization in pathologically increased function of bone marrow during leukemia is well documented. In these conditions, as in augmented requirement for new vasculature in growing solid tumors, bone marrow angiogenesis appears to be a prerequisite for increased production of blast cells in leukemia. Despite bone marrow high degree of vascularization, endosteal surface of cortical bone appears to be among the most hypoxic areas in the body (52). Consequently, numerous clinical trials are underway to determine efficacy of antiangiogenic treatments in various hematological malignancies (53). There is emerging evidence that progression of multiple myeloma, acute leukemia and myeloproliferative neoplasms depends on new blood vessel formation. Several agents aimed at blocking angiogenesis, like monoclonal antibodies, receptor tyrosine kinase inhibitors, immunomodulatory drugs have been entered in clinical trials or have been approved for treatment of these cancers. Multiple myeloma was the first hematological malignancy in which increased angiogenesis rate was detected (54) and it was shown that in this condition extent of bone marrow angiogenesis represents a good prognostic predictor since it is positively correlated with severity of the blood-born malignancies (55). Thus, it was demonstrated that bone marrow of acute lymphoblastic leukemia patients had increased blood vessel content compared to normal counterparts. Moreover, urine and peripheral blood samples from acute lymphoblastic leukemia patients contained elevated levels of proangiogenic growth factors, namely bFGF and VEGF, which correlated with the increase of bone marrow angiogenesis (56). In support of the role of pro-angiogenic factors in development of blood cancers, significantly increased bone marrow microvascular density and VEGF expression was found in patients with myeloproliferative neoplasms (57). Similarly, increased VEGF production and VEGF receptor expression was detected in three leukemic cell lines (HL-60, HEL, and K562) and primary leukemia samples. Injection of neutralizing monoclonal antibody specific to human VEGFR-2 into mice with transplanted human leukemia cells inhibited their proliferation of and significantly prolonged animal survival (58). It appears that regardless of leukemia type, increased microvascular density in bone marrow is always an indicator of disease severity. Similar observations were reported in bone marrow of patients with multiple myeloma, acute lymphoblastic leukemia, acute myeloid leukemia, myelodysplastic and myeloproliferative syndrome (59). Thus, a positive correlation between micro vessel score expression of VEGF and bFGF RNA in bone marrow blast cells in acute myeloid leukemia patients' was detected (60). Such changes correlate with extent of bone marrow hypoxia. Thus, elevated expression of VEGF correlated with Hif-1α levels in B cell chronic lymphocytic leukemia (61). Increased response to proangiogenic factors is not restricted just to bone marrow vasculature, since the ability of leukemia cells to directly respond to angiogenic factors through expression of VEGF and VEGFRs have also been reported (62,63).
These findings, taken together, suggest that anti-angiogenic therapy could become a part of clinical protocols for many leukaemia types and it is reasonable to expect new developments in that direction (64,65). In addition, changes in expression of angiogenesis-related markers could become important prognostic tool in assessing disease severity and progression (66,67).

Current clinical application of anti-angiogenic treatment
The anti-angiogenic therapy currently being approved for clinical use include: (1) direct anti-VEGF treatments to include anti-VEGF, anti-VEGFR antibodies and VEGF "traps"; (2) immunomodulatory drugs with antiangiogenic properties and (3) receptor tyrosine kinase inhibitors, targeting VEGF receptors and their signalling (Diagram 2). Apart from those therapies, a large number of potential anti-angiogenic therapies are currently at the research stage (68). The major problem associated with therapy aimed to suppress angiogenesis and ultimately block tumour growth and development is transient improvement in patient condition but not overall survival compared to chemotherapy alone. Future research is expected to optimize the antiangiogenic therapy that would be successfully combined with traditional treatments.
The following drug classes of angiogenesis inhibitors are currently approved by the U.S. Food and Drug Administration (FDA) to treat cancerous conditions. Typically, these drugs are given together with main types of cancer treatment, such as chemotherapy. In the U.S., there are currently fifteen approved anti-cancer therapies with recognized antiangiogenic properties (USA Angiogenesis Foundation (http://www.angio.org/) all falling into the six categories based on their mechanism of action. Diagram 2 summarizes mode of action of the currently approved drugs.

Endothelium-specific drugs
Cumulative information about mechanism of action of these drugs is given in Diagram 2.

1.
Anti-VEGF and anti-VGFR antibodies Bevacizumab (Avastin) is a monoclonal antibody against VEGFA (69). It blocks the interaction of VEGF to its receptors (Flt-1 and KDR) on the surface of endothelial cells. It is approved for treatment of colorectal, kidney and lung cancer. As an antibody, it is given intravenously. After a series of clinical trials in 2004, Avastin was approved by the FDA, becoming the first commercially available anti-angiogenesis drug. FDA approval of Avastin for breast cancer treatment was later revoked in 2011 due to lack of effectiveness and required safety, although bevacizumab still remains on the market as approved treatment for certain types of colon, lung, kidney and brain cancers (70). Ramucirumab (Cyramza) is a monoclonal antibody directed against VEGFR-2 and it is approved for treatment of advanced stomach cancers and gastroesophageal junction adenocarcinoma (71). As an antibody, it is applied intravenously.
Drugs affecting endothelium as well as other tissues 1.
mTOR inhibitors Everolimus (Afinitor) is a derivative of sirolimus (also known as rapamycin, a macrolide produced by the bacterium Streptomyces hygroscopicus) an inhibitor of mammalian target of rapamycin (mTOR), also known as FK506-binding protein and 12-rapamycin-associated protein 1 (FRAP1). It is a serine/threonine protein kinase that regulates cell growth, cell proliferation, motility, survival, protein synthesis, autophagy, and gene transcription (73). It belongs to the phosphatidylinositol 3-kinase-related kinase protein family. Sirolimus has immunosuppressant functions in humans and is used to prevent rejection after organ transplantation and suppress activation of T and B cells by inhibiting production of interleukin-2. It is taken orally. As a general immunosuppressant and action relatively nonspecific to angiogenesis, everolimus is shown to possess expected adverse effects characteristic of immunosuppressant drugs (74). Temsirolimus (Torisel) acts through its active metabolite sirolimus, binds to an intracellular protein (FKBP-12), form a protein-drug complex inhibiting the activity of mTOR that controls cell division (75).

2.
Interferon-α Interferon-alpha (IFN-α) has anti-angiogenic properties in addition to its immune-stimulatory and anti-proliferative effects. IFN-α has been shown to inhibit angiogenesis through downregulation of bFGF expression in murine cancer models (76,77). In clinical testing, administration of bevacizumab with IFN-α led to a clinical response in 24% of patients with stage IV melanoma and stabilization of disease in another 20% of patients (78).

3.
Small molecule receptor tyrosine kinase inhibitors There are currently seven receptor tyrosine kinase inhibitors approved for cancer treatment. Although both VEGFR and bFGFR are tyrosine kinase receptors and are inhibited by those drugs, many other growth factor receptors are of the same kind, making the treatment with receptor tyrosine kinase inhibitors relatively non-specific to angiogenesis.
Axitinib (Inlyta) is an approved option to treat kidney cancer (79). Phase III data validated sunitinib, pazopanib and sorafenib as the bestsupported drugs in first line therapy for treatment of metastatic renal cell carcinoma, whereas second-line treatment possibilities include axitinib, everolimus and sorafenib (80).
Cabozantinib (Cometriq) a small molecule inhibitor of the tyrosine kinases c-Met and VEGFR2 is currently approved for use in medullary thyroid cancer treatment (81).
Pazopanib (Votrient) is approved for treatment of kidney cancer and advanced soft tissue sarcoma. It is an orally-active drug and multi-target receptor tyrosine kinase inhibitor that blocks tumor growth and inhibits angiogenesis (82). Its main targets are c-KIT, FGFR, PDGFR and VEGFR.
Regorafenib (Stivarga) is an oral multi-kinase inhibitor which targets angiogenic, stromal and oncogenic receptor tyrosine kinases. It shows antiangiogenic activity due to its dual VEGFR2-TIE2 tyrosine kinase inhibition (83).
Sorafenib (Nexavar) is an inhibitor of receptor tyrosine kinases (VEGFR and PDGFR) and Raf kinases (84). Platelet-derived growth factor receptors (PDGFRs) are cell membrane tyrosine kinase receptors for members of the plateletderived growth factor (PDGF) family. PDGFRs regulates cellular proliferation, differentiation and growth in many tissues. Sorafenib has broad mechanism of action, including suppression of angiogenesis. It is taken orally (85).
Sunitinib (Sutent) previously known as SU11248 is a small-molecule oral drug and multitargeting receptor tyrosine kinase inhibitor of intracellular signaling. Targets include all receptors for platelet-derived growth factor (PDGFRs) and vascular endothelial growth factor (VEGFRs), which play a role in both tumor angiogenesis and tumor cell proliferation (86). The simultaneous inhibition of these targets reduces tumor vascularization and triggers cancer cell apoptosis (87).
Vandetanib (Caprelsa) is used for the treatment of medullary thyroid cancer. It acts as a kinase inhibitor of a number of cell receptors, mainly the VEGFR, epidermal growth factor receptor (EGFR) and the RET-tyrosine kinase family (88).

4.
Immune-suppressive and antiinflammatory drugs Lenalidomide (Revlimid) is used to treat multiple myeloma. It is an orally active drug with immunomodulatory, anti-angiogenic, and direct apoptotic properties causing cancer cell apoptosis (89). While recent preclinical and clinical studies put forward a dual mechanism of its action involving both direct anti-tumor activity as well as immunomodulation, it is presently unclear which mechanism(s) are responsible for its effectiveness.
Thalidomide (Thalomid) is an immunomodulatory drug. Its precise mechanism of action is currently unknown. Possible mechanisms include tissue anti-angiogenic and oxidative stressinducing effects (90). It also inhibits TNF-α, IL-6, IL-10 and IL-12 production, modulates the production of IFN-γ and enhances the production of IL-2, IL-4 and IL-5 by immune cells (91). It is highly teratogenic oral drug.
Current limitations of angiogenesis-suppressing cancer therapy Existing preclinical and clinical investigations clearly indicate transient efficacy of current antiangiogenic drugs where tumorigenesis resumes after withdrawal of therapy. Consequently, the impact of current angiogenesis suppressing therapies on overall patient survival has been somewhat disappointing primarily due to development of drug resistance (92)(93)(94)(95)(96)(97)(98). One of the potential reasons for those observations might be related to the discovery of numerous compensatory vascular mechanisms existing in tumors. Those mechanisms allow tumors to become relatively resistant to angiogenesis-suppression therapy (92). In addition, new vascular remodeling processes have been identified that are relatively angiogenesis-independent (93). These include vessel co-option (growth without angiogenesis through "recruiting" of pre-existing vessels (94), intussusception (occurring by internal division of the pre-existing capillary network without sprouting) (95) and vascular mimicry (where cancer cells form vessel-like structures able to transport blood) (96). It was estimated that up to 30% of tumors possess ability to use these alternative ways to insure adequate blood supply (97). Taking into account growing number of anticancer agents directed toward suppressing vascular growth, identification of factors causing observed drug resistance is of great importance (98).
The second hurdle of anti-angiogenic therapy is its toxicity predominantly related to drug targets. It is important to emphasize that such toxic effects are usually milder compared to the classic cytostatic therapy. Considering that bevacizumab is one of the first angiogenesis suppressive therapies approved for inclusion into cancer treatment protocols, it is not surprising that it is the most investigated drug compared to the rest of currently available options. Consequently adverse drug effects of those treatments are reported the most. Thus, both bevacizumab and VEGFR kinase inhibitors have been shown to cause hypertension and these toxicities correlate with the response (99). In the study using FOLFIRI protocol (folinic acid, 5-fluorouracil and irinotecan) combined with bevacizumab as a first-line treatment for colorectal cancer, grade 3/4 toxicities were reported (grade 3 is classified as severe and grade 4 as life-threatening) to include neutropenia (16.1%), diarrhea (11.3%) and nausea-vomiting (1.6%) (100). Based on those findings, authors concluded that such treatment has good safety profile compared to existing chemotherapy protocols and could be used in clinical setting. Furthermore, current experience with angiogenesis inhibitors in clinical trials indicates that observed short-term toxicities are mostly manageable (101). In addition, reported adverse effects do not happen with every drug or with every patient. Rarely, those effects could be more serious, to include serious bleeding, heart attacks, heart failure or thrombosis (102). Another rare side effects include bowel mucosal damage induced by chemotherapy that may be exacerbated by treatment with bevacizumab, resulting in extensive necrosis (103). Regardless of the observed side effects, most of therapies aimed at suppression of tumor angiogenesis exhibited acceptable safety profile, particularly when compared to standard cytotoxic protocols (104).

CONCLUSIONS
From its discovery, more than forty years ago, angiogenesis is considered one of the most promising new targets in pharmacological treatment of various types of cancer. Taking into account that growth and metastatic potential of any cancer are very dependent on proper tumor vascularization, the possibility to suppress angiogenesis in cancer treatment generated significant excitement in the field.
Today, after accumulated knowledge about angiogenesis and its regulation we have all realized that the process itself appears to be much more complex than previously thought. Consequently, although angiogenesis remains an important target for new treatments, current expectations regarding success of such therapy are somewhat lowered. Currently, combinatory anti-angiogenesis treatments are investigated in many clinical studies and that direction (at least in short term) is expected to continue (reviewed in [105][106][107]. Regardless of observed complications, angiogenesis suppression is still an extremely important addition to the cancer treatment, which did not include any new pharmacological options for the past several decades.