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 Table of Contents  
REVIEW ARTICLE
Year : 2015  |  Volume : 5  |  Issue : 3  |  Page : 121-130

The basic molecular biology of angiogenesis and its implication in anticancer therapeutics


Department of Surgery, Late Shri Baliram Kashyap Memorial Government Medical College, Jagdalpur, Chattisgarh, India

Date of Web Publication19-Oct-2015

Correspondence Address:
Dr. Sujan Narayan Agrawal
Department of Surgery, Late Shri Baliram Kashyap Memorial Government Medical College, Jagdalpur - 494 001, Chattisgarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-9596.167472

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  Abstract 

Angiogenesis is a physiological process through which new blood vessels are formed from the existing blood vessels. It is a multistep process, involving proliferation of activated endothelial cells (ECs) and migration of ECs to target organs. At target organs, the ECs get organized into capillary tubes and synthesize new basement membrane. These capillary tubes then get canalized to form a vascular lumen. The process of Angiogenesis plays an important role in growth and progression of cancer. The control of tumor angiogenesis depends upon the net balance of angiogenic factors (activators) and antiangiogenic factors (inhibitors), which are secreted by both tumor cells and host cells. The key signaling angiogenic factors include vascular endothelial growth factor (VEGF), Notch, angiopoietin, ephrins, transforming growth factor beta, and platelet derived growth factors (PDGFs). The potential therapeutic implication of tumor angiogenesis was coined by Folkman in 1971. The knowledge of tumor angiogenesis can be utilized in the development of novel strategies of anticancer therapy by targeting tumor vessels instead of tumor cells. It works best when combined with cancer chemotherapy. However, there are many hurdles to the road of success. The long-term survival and overall improvement in advanced cancer are still limited, and the final outcome may be different from what is obtained in experimental animals. In this article, an effort is made to address the process of angiogenesis, the basic molecular biology of key signaling pathways and the concept, present status and challenges in cancer therapeutics as far as angiogenesis is concerned.

Keywords: Angiogenesis, angiopoietins, Eph/Ephrins, neuropilins, Notch, platelet derived growth factor, semaphorins, transforming growth factor beta, vascular endothelial growth factor


How to cite this article:
Agrawal SN. The basic molecular biology of angiogenesis and its implication in anticancer therapeutics. Arch Int Surg 2015;5:121-30

How to cite this URL:
Agrawal SN. The basic molecular biology of angiogenesis and its implication in anticancer therapeutics. Arch Int Surg [serial online] 2015 [cited 2024 Mar 28];5:121-30. Available from: https://www.archintsurg.org/text.asp?2015/5/3/121/167472


  Introduction Top


Angiogenesis is the formation of new blood vessels from the endothelium of existing blood vessels. It differs from vasculogenesis that denotes differentiation of endothelial precursor cells from the mesoderm and their coalescence into tubes to form a primary vascular plexus. Angiogenesis is a fundamental process that is responsible for tumor growth, progression and metastasis. Tumors cannot grow beyond 1-2 mm in size unless they are adequately vascularized. This is because of the fact that, cancer cells also require delivery of oxygen, nutrients and removal of waste products for their survival and growth. Presumably the 1-2 mm range represents the maximum, across which oxygen, nutrients and waste products diffuse from blood vessels. [1]

In 1945, Algire and Chalkey were the first to conclude that growth of the solid tumor is closely connected to the development of an intrinsic vascular network. [2]

Angiogenesis is a finely regulated play between pro and antiangiogenic molecules. In the last decade, the use of homologous recombination, to abate, gene functions in the experimental animal have provided a major breakthrough in our understanding of angiogenesis. Key signaling pathways to vascular morphogenesis includes vascular endothelial growth factor (VEGF), Notch signaling pathways, angiopoietins (ANGPTs), ephrins, transforming growth factor beta (TGF-β), and platelet derived growth factor (PDGF). The antiangiogenic factors include angiostatin, endostatin, interferon, platelet factor 4, thrombospondin, prolactin 16 kD fragment and tissue inhibitors of metalloproteinase-1, 2, and 3.

Although the molecular mechanism of angiogenesis is relatively well characterized, the successive interaction and relationship between these growth factors during the angiogenic cascade is less understood. This could explain in part, the results of antiangiogenic therapy in human cancers that do not give as good results, as expected, in comparison with those, reported in experimental models.


  The Angiogenic Switch Top


Tumor angiogenesis is influenced by the angiogenic switch, which is a balance between pro and antiangiogenic factors [Table 1]. It is not a simple up-regulation of angiogenic activities but a result of the net balance between positive and negative regulators. When pro-angiogenic factors overcome the effect of angiostatic molecules, the tumor acquires an angiogenic phenotype that leads to the formation of new blood vessels. The acquisition of the angiogenic phenotype is considered to be a key step in early tumor progression that allows the tumor to transform from a microscopic lesion to a rapidly expanding mass with metastatic potential. Oncogene derived protein expression, as well as a number of cellular stress factors such as hypoxia, low pH, nutrient deprivation or inducers of reactive oxygen species, are important stimuli of angiogenic signaling. The main pro- and anti-angiogenic factors are tabulated in [Table 1].
Table 1: Pro- and anti-angiogenic factors


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The process of angiogenesis

The formation of the embryonic vasculature is initiated by the appearance of blood islands from progenitor cells called hemangioblasts, in the visceral yolk sac. The hemangioblast differentiates into either hemopoietic or endothelial cells (ECs). The hemopoietic cells are located within the channel space, and ECs are lying on the edges. This first phase of vessel formation is called vasculogenesis. The second phase is angiogenesis where the new blood vessels are formed from the preexisting blood vessels. The interconnections in these primitive vessels result in the formation of the primary vascular plexus, which undergoes a process of remodeling, selective fusion, and regression. The circulatory system is the first functioning organ system to develop, in the vertebrate embryo.

Early vascular morphogenesis

The primitive vascular plexus consists of differentiated ECs that initiate their function as a barrier between blood and tissues. The vascular plexus has uniform tubes and are not differentiated into a vascular hierarchy. This formation of the vascular plexus is a transient phenomenon. Vascular plexus expands by vascular sprouting (angiogenesis). During angiogenesis, ECs migrate and proliferate, forming tubules containing the cell to cell tight junction. The leading cells are called "tip cells." The tip cells respond to VEGF signaling by sending cellular processes (filopodia) and sensing its environment.

Recruitment and differentiation of smooth muscle cells, fibroblast and pericytes is also initiated at this stage. Recruitment events are highly regulated and coordinated by a large number of molecules. The main group of these molecules is VEGF and Notch signaling pathways. Thus, a blueprint of vascular system is formed. It is matured by vascular patterning and remodeling, which occurs simultaneously.

Vascular patterning

The vascular patterning is characteristic in every organism suggesting that there is specific genetic programming, which controls these important processes. These processes are extensively studied and documented in Zebrafish embryo during experiments.

The tip cells in the sprout exhibit numerous active filopodia, which extends and retract in intermittent fashion like the movement of Amoeba. The directionality is guided by the combined set of attractive and repulsive cues, which in turn, determines the selection of branching sites, the directionality and the fusion. VEGF and its isoform VEGF 188 are specifically responsible for stimulation of sprouts and guidance cue. [3]

Neuropilin is a receptor that promotes guidance of axonal sprout in the nervous system. It is also found to bind with isoform of VEGF and help in patterning of the vasculature. Plexins are another important signaling molecule in vascular patterning. Experimentally it is documented that disruption of Plexin-D1 signaling and its ligand semaphorin 3E removes the propulsive signals necessary for correct guidance during vascular sprouting events.

Vascular remodeling

This is a process in which the specific vessels fuse to form large vascular channels and others regress, ultimately giving rise to the typical hierarchical character. The process of vascular remodeling is influenced by various signaling pathways and signaling molecules such as Tie2, ANGPT-1, and Notch1 etc. The extracellular matrix molecule plays the most important role in vascular stabilization and remodeling. Signals from matrix molecules are conveyed through integrins such as alpha-v, beta-3, etc.

Arterial and venous network

The endothelial tubes in a primitive vascular matrix are not differentiated in arteries and vein. In the mature vascular matrix, they should be differentiated in arterial and venous network with interconnection as a capillary bed in the target organ. It is seen that the arteries and vein differ in their arrangement of the extracellular matrix, smooth muscle cells and other supportive cells which give rise to different mechanical and physiological properties characteristic of arteries and vein.

There is genetic predetermination to drive the fate of endothelial tubes into arteries or vein. It is observed that transmembrane ligand ephrin-B2 and its tyrosine kinase receptors Eph B4 are differently expressed in arterial and venous endothelium. Not only these but the Notch signaling pathways and the expression of neuropilin-1 and neuropilin-2 play an important role in arterial and venous differentiation. Along with the genetic predetermination local hemodynamic forces also play a role such as blood flow, blood pressure, etc. It has been experimentally demonstrated that after transplanting the arterial endothelium into vein and vice-versa, the transplanted endothelium adopts the specific molecular programs of the host vessels, suggesting that, the local cues modulate vessel specificity. [4]

The signaling pathways for angiogenesis

The most important ligand/receptor families essential for vascular morphogenesis are VEGF/VEGF receptor (VEGFR), Notch-DSL, ANGPT-Tie-2, and Ephrin-B2-Eph-B4. Other equally important are TGF-β and PDGF, extracellular matrix protein and their receptors, adhesion molecules and other growth factors. ECs activated by VEGF produce matrix metalloproteinase (MMPs). The extracellular matrix is broken down by MMPs. These MMPs fill the space between cells and is made of protein and polysaccharides. This matrix permits the migration of ECs. The ECs begin to divide as they migrate into the surrounding tissues. Soon they organize into hollow tubes that evolve gradually into a mature network of blood vessels with the help of an adhesion factor, such as integrins α or β. Newly formed blood vessels need to stabilize or mature. Angiotensin-1, -2 and their receptor Tie-2 can stabilize and govern vascular growth.


  Molecular Biology of Signaling Proteins Top


Vascular endothelial growth factor

VEGF is a subfamily of PDGF family. They are important signaling proteins involved both in vasculogenesis and angiogenesis. The representative of the VEGF family is VEGF-A, (labeled as VEGF) and VEGF-B, C, and E. They act on their respective receptors and cause proliferation of blood vessels, while VEGF-C and VEGF-D are involved in lymphangiogenesis.

There is multiple isoform of VEGF-A that results from alternative splicing of messenger RNA (mRNA) from a single 8-exon VEGF-A gene. They are classified into two groups that are referred to according to their terminal exon (exon 8) splice site, that is, the proximal splice site (denoted as VEGFxxx) or distal splice site (denoted as VEGFxxxb). In addition, alternate splicing of exon 6 and exon 7 alters their heparin-binding affinity and amino acid number (in human, VEGF 121, VEGF 121 b, VEGF 145, VEGF 165, VEGF 165 b, VEGF 183 , VEGF 189 , VEGF 206 , etc.). These domains have important functional consequences for the VEGF splice variants, as the terminal (exon 8) splice site determines whether the proteins are proangiogenic (proximal splice site: Expressed during angiogenesis) or the anti-angiogenic (distal splice site: Expressed in normal tissues). Also, inclusion or exclusion of exon 6 and exon 7 mediates interaction with heparin sulfate proteoglycans and neuropilin co-receptors on the cell surface, enhancing their ability to bind and activate the VEGFRs.

VEGF-B exists as two protein isoforms, VEGF-B167 and VEGF-B186, resulting from different spliced mRNA and binds specifically to VEGFR-1. However, VEGF-B forms a heterodimer with VEGF-A, which may alter its interaction with its biological receptors and modify its normal physiological functions. VEGF-C has a mature form that consists of a VEGF homology domain, which contains receptor binding sites and is almost 30% identical in the amino-acid sequence of VEGF165. The expression of VEGF-C appears to be restricted to early development and certain pathological settings such as tumor angiogenesis and lymphangiogenesis.

VEGF-D is known as c-FOS-induced growth factor, and the mature form has 61% identical amino acid sequence with VEGF-C and both these growth factors bind to the same receptors on human ECs, that is, VEGF-2 and VEGF-3. The binding of VEGF-C and VEGF-D to the receptor

VEGFR-3 regulates lymphangiogenesis as well as angiogenesis in mid-stage of embryogenesis. VEGF-E is encoded by parapox virus or Orf virus. The interaction of VEGF-E with its receptor seems to promote EC growth. VEGF-2 appears to mediate almost all of the known cellular responses to VEGF. The function of VEGFR-1 is less well defined, although it is thought to mediate VEGFR-2 signaling. [5] VEGFR-2 is exclusively expressed in the ECs and appears to play a pivotal role in EC differentiation and vasculogenesis. VEGF-A production can be induced in cells that are not receiving enough oxygen. Oxygen deficient cells produce hypoxia inducible factor, which in turn stimulates the release of VEGF-A. Circulating VEGF-A then binds to VEGFRs on ECs triggering a tyrosine kinase pathway leading to angiogenesis.

All members of VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (VEGFRs) on the cell surface causing them to dimerize and become activated through trans-phosphorylation. VEGF binds to VEGFR-1/Flt-1 and VEGFR-2/Flk-1/DKR, and VEGFR-3/Flt-4. [6]

VEGF-receptor tyrosine kinase (RTKs) are single pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the gamma-phosphate of ATP to a tyrosine residue in protein substrates. Activation of VEGF-RTKs occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins.

Neuropilins and semaphorins

Neuropilins are transmembrane receptors best understood for their interaction with semaphorins. They also bind to VEGF isoforms 164 and 188. Eight classes of semaphorins have been identified, and most of these appear specific to the neural system. Class three semaphorins bind to neuropilin receptors, also expressed by ECs, in addition to neurons. There are six members of class-3 semaphorins (sema A-F). Sema-3A activates neuropilin-1 and sema-3F activates neuropilin-2. Class-3 semaphorines play a role in both physiological and pathological angiogenesis. [7]

Both the antiangiogenic sema-3A and sema-3F are expressed by ECs suggesting an autocrine function, whereas sema-3C is thought to be a proangiogenic factor. There may be potential to exploit therapeutically sema-3A, sema-3F and sema-3B as an antiangiogenic and antitumor agent.

Angiopoietins

It belongs to the family of vascular growth factor and plays a vital role in embryonic and postnatal angiogenesis. There are four identified ANGPTs, viz. ANGPT1, ANGPT2, ANGPT3, and ANGPT4. Also there are a number of proteins that are closely related to ANGPTs, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, and ANGPTL7. ANGPTs are responsible for assembling and disassembling the endothelial lining of blood vessels. [8] ANGPTs are critical for vessel maturation, adhesion, migration and survival. Structurally ANGPTs have an N-terminal super clustering domain, a central coiled domain, a linker region and a C-terminal fibrinogen-related domain responsible for binding between the ligand and receptors. [9]

The ANGPTs works through two signaling pathways Tie-1 and Tie-2. These pathways mediate the cell signals by inducing phosphorylation of specific tyrosine; this in turn initiates the binding and activation of downstream intracellular enzymes. There is a collective interaction of ANGPTs, RTK, VEGF and their receptors through Tie-1 and Tie-2 signaling pathways. Tie-2/Ang-1 signaling activates β1-integrin and N-cadherine in LSK-Tie-2+ cells and promotes hematopoietic stem cells (HSC) interaction with extracellular matrix and its cellular components. This signaling contributes to the maintenance of the long-term repopulating ability of HSC and the protection of HSC compartment from various cellular stresses.

ANGPT proteins 1, 2, 3, and 4 are all ligands for Tie-2 receptors. Tie-1 heterodimerizes with Tie-2 to enhance and modulate the signal transduction of Tie-2 for vascular development and maturation. Tyrosine kinase receptors are typically expressed on vascular ECs and specific macrophages for immunoresponse. ANGPT-1 and tyrosine kinase signaling are essential for regulating blood vessel development and stability of mature vessels. [10]

The expression of ANGPT-2 in the absence of VEGF leads to cell death and vascular regression. Increased levels of ANGPT-2 promote tumor angiogenesis, metastasis and inflammation. Ang-2 works as an antagonist to Ang-1 and promotes vessel regression if VEGF is not present. Targeting Ang-2 could be an effective strategy in therapeutics of malignancies and inflammation.

The notch signaling pathway

It is a cell signaling system present in most multicellular organisms. There are four different notch receptors labeled as Notch1, Notch2, Notch3, and Notch4. The notch receptors are single pass transmembrane receptor protein. It consists of a large extracellular portion, a single transmembrane pass and a small intracellular region. [11]

Ligand protein binding to extracellular domain induces proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to modify gene expression.

The receptor is normally triggered in a direct cell to cell contact, in which the transmembrane protein of the cell in direct contact forms the ligands that bind the notch receptors. The notch binding allows the group of cells to organize themselves, in such a manner that if one cell expresses a given trait, this may be switched off in neighboring cells by intracellular notch signals. In this manner, groups of cells influence each other to make large structures. This lateral inhibition mechanism, are key to notch signaling. Lin-12 and Notch mediate binary cell fate decisions and lateral cell inhibition involves feedback mechanisms to amplify initial differences. [12]

The notch extracellular domain is composed primarily of small cystic knot motifs called EGF-like repeats. Notch1 for example, has such 36 repeats. Each EGF-like repeats is composed of approximately 40 amino acids, and its structure is defined largely by six conserved cysteine residues that form three conserved disulfide bonds. Each EGF-like repeat can be modified by O-linked glycans of specific sites. [13]

An O-glucose sugar can be added between first and the second conserved cysteine. These sugars are added by an O-glucosyltransferase 1 and GDP-fucose protein O-fucosyltransferase 1 (POFUT1), respectively. The addition of the O-fucose by POFUT1, is absolutely necessary for notch functioning and without the enzymes to add O-fucose all notch protein fail to function properly.

Once the notch extracellular domain interacts with a ligand, an ADAM-family metalloprotease called ADAM10 cleaves the notch protein just outside the membrane. This releases the extracellular portion of the notch, which continues to interact with the ligand. The ligand plus the notch extracellular domain is then endocytosed by the ligand-expressing cells. After this first cleavage, an enzyme called ϒ-secretase cleaves the remaining part of the notch protein just inside the inner leaflet of the cell membrane of the notch expressing cells. This releases the intracellular domain of the notch protein, which then moves to the nucleus, where it can regulate gene expression by activating the transcription factor CBF/Suppressor of Hairless/LAG-1 (CSL).

ECs use notch signaling pathways, to co-ordinate cellular behavior, during the blood vessel sprouting that occurs in angiogenesis. Activation of notch takes place primarily in the "connector" cells and cells that line patent stable blood vessels. Notch signaling may be needed to control the sprouting pattern of blood vessels during angiogenesis. In a patent blood vessel, when cells are exposed to VEGF signaling, only a restricted number of them initiate the angiogenic process. VEGF is able to induce DLL4 expression. In turn, DLL4 expressing cells down regulate VEGFRs in neighboring cells through activation of the notch, thereby preventing their migration into developing sprout. Likewise during the sprouting process itself the migratory behavior of the connector cells must be limited to retain a patent connection to the original blood vessel.

Eph receptor signaling and ephrins

The Eph receptors are the largest of the RTK family. They transduce signals from cell exterior to the interior through ligand-induced activation of their kinase domain. They generally mediate contact dependent, cell to cell communication by interacting with the surface associated ligands (The ephrins) on neighboring cells. The complex of Eph receptor-Ephrin emanates bidirectional signals that affect both receptor and ephrin expressing cells. The Eph receptors have a multidomain extracellular region that includes the ephrin ligand binding domain, a single transmembrane segment and a cytoplasmic region that contains the kinase domain. There are nine Eph receptors in the human genome that bind to five ephrin-A ligands. There are five Eph receptors that bind to three ephrin-B ligands. [14]

Both ephrin classes include a conserved Eph-receptor binding domain, which is connected to the plasma membrane by a linker segment whose length is affected by alternate splicing. The ephrin-As are attached to the cell surface by a glycosylphosphatidylinositol anchor. The ephrin-Bs contains a transmembrane segment and a short cytoplasmic region. The Eph receptor family is greatly expanded and includes almost a fourth of 58 human RTKs. They are highly expressed in brain and most of the other tissues.

Eph receptor and ephrins engage in multiple activities. They mediate contact dependent communication between cells of the same or different types to control cell morphology, adhesion, movement, proliferation, survival and differentiation. [15]

The forward signaling of the Eph receptor is typical of prototypical RTK mode of signaling, which is triggered by ligand binding and involves activation of the kinase domain. Binding between Eph receptors and Ephrins on the juxtaposed cell surface leads to oligomerization through not only Eph receptor-Ephrin interface but also receptor-receptor cis interface located in multiple domains. The proximity of clustered Eph receptor molecules leads to trans-phosphorylation. Phosphorylation of two conserved tyrosine in the transmembrane domain, relieves inhibitory intramolecular interaction with the kinase domain, enabling efficient kinase activity. [16]

Besides the forward signaling, the Eph receptors can also stimulate reverse signaling in the ephrin expressing cells. Recruitment of signaling protein containing PDZ domains to the ephrin-B carboxyl terminus is crucial for reverse signaling. Ephrin-B interacts with PDZ domain protein and promotes angiogenesis and lymphangiogenesis, by enabling VEGFR endocytosis and regulates axon guidance and synaptic plasticity. [17]

Fallowing ligand-dependent activation, RTKs are typically internalized by endocytosis and can continue to signal from intracellular compartment until they are inactivated by dephosphorylation and degradation of traffic back to the cell surface.

The signaling of Eph RTK family is unique in many ways and differs from other RTK families. The peculiar character includes membrane bound nature of ephrins, the bidirectional mode of Eph receptor-Ephrin signaling, the ability of the ephrins not only to stimulate but also to attenuate Eph receptor signaling, and the ability of the Eph receptors to signal without ephrin involvement or even independent of kinase activity are a few example.

The transforming growth factor-beta signalling

The TGF-beta is a member of a large superfamily that includes bone morphogenic protein, activins, inhibits and Mullerian inhibitory substance. Three members of TGF-beta has been identified, that is, TGF-beta 1, 2, 3. The growth factors are secreted in latent form, and its activation is dependent on either proteolytic processing or binding to thrombospondin-1. Signal transduction by TGF-beta requires a series of serine/threonine receptors, Smad proteins, and Smad transcription factors that convey these signals to specific genes. [18]

Genetic inactivation studies have clearly shown TGF-beta signaling is important for differentiation of ECs. TGF-beta signaling is mediated by its Type 1 and Type 2 serine/threonine kinase receptors.

Type 1 receptors are again of two types that is:

  1. Activin receptor-like kinase-1 (ALK-1).
  2. Activin receptor-like kinase-5 (ALK-5).


Type 2 receptor has only one variant called TGF-beta receptor-II (TGF-β-R-II). By activation of Smad 1/5, it has been shown that ALK-1 is responsible for the stimulation of EC proliferation and migration. ALK-5 activation occurs through Smad 2/3 and results in inhibition of cell proliferation and migration. [19]

Ultimately the net expression of these two receptors and their respective target Smads dictates the end result of TGF-beta response in ECs. [20] Mutation in ALK-1 or its accessory receptor endoglin has been linked to hereditary hemorrhagic telangiactesis.

Platelet-derived growth factor

PDGF is a 30 kDa dimer composed of an A and/or B chain, which are encoded by separate genes and regulated independently. Two additional genes were identified encoding PDGF-C and PDGF-D polypeptides. Each chain is encoded by an individual gene located on chromosomes 7, 22, 4 and 11 respectively. So, PDGF is a heparin binding family of polypeptide growth factor denoted A, B, C, and D. All four PDGF chains contain growth factor domain of approximately 100 amino acids that is, also found in VEGF family. Until now, five dimeric compositions have been identified, that is, PDGF-AA, BB, AB, CC and DD. [21]

PDGF targets mesoderm derived cells like fibroblast, pericytes smooth muscle cells, glial cells, etc. The receptor of PDGF are two class-3 RTK, that is, PDGF receptor α (PDGFRα) and PDGFRβ. Binding of the ligand leads to autophosphorylation of the receptors on tyrosine residue that induces activation of several signaling molecules. The individual PDGF chain has different affinity for two receptors. PDGFRα has a high affinity for PDGF-A, PDGF-B and PDGF-C whereas PDGFRβ has a high affinity for PDGF-B and PDGF-D. Ligand binding to the receptor induces receptor dimerization, which leads to activation of the intrinsic tyrosine kinase domain and subsequent recruitment of SH-2 domain containing signaling proteins. Finally, activation of these pathways leads to cellular responses like proliferation and migration. [21]

PDGF signals through two cell surface tyrosine kinase receptors, PDGFRα and PDGFRβ. It induces angiogenesis by up-regulating VEGF production and modulating the proliferation and recruitment of perivascular cells. [22]

VEGF-A enhances endothelial PDGF-B expression, whereas fibroblast growth factor-2 (FGF-2) enhances perivascular PDGFR-β expression. Another function of PDGF is to regulate transcriptional activities. In this process, PDGF-B, upregulates the transcriptional factor E26 transformation specific sequence-1 (Ets-1). PDGF plays an important role in wound healing, stimulating cell proliferation, migration and angiogenesis. This role is related to some specific molecules of extracellular matrix such as collagens or heparin. The PDGF-C is widely expressed in muscle tissues and also in active angiogenic tissues such as placenta, ovary, some embryonic tissues and tumors. PDGF-C is thought to activate PDGFRαα heterodimers. PDGF and PDGFR play a crucial role in the normal development of various organs such as lung, intestine, kidney, skin, and testis. All members of PDGF family display potent angiogenic activity and thus PDGF-B/PDGFRβ axis is very valuable for angiogenesis. PDGF-B is produced by developing and quiescent ECs and PDGFRβ is expressed by perivascular cells and ECs. Recruitment of pericytes is completely dependent on PDGF-B/PDGFRβ signaling. The biggest importance of PDGF-B, during development, is to promote perivascular cell recruitment during angiogenesis. In the absence of PDGF-B the number of perivascular cells in the small blood vessels is reduced, and their rate of proliferation is slower. The crucial role of the PDGF-B is the maintenance of vascular stability. Increased PDGFRβ activity is associated with overexpression of VEGF-A and VEGFR-2, which results in increased sprouting, pericytes coating and vessel formation.


  The Therapeutics Top


The concept

The conventional approach to the treatment of cancerous growth consists of surgery, chemotherapy and radiotherapy. Basically they all target removal or destruction of a cancerous mass. The concept of targeting the tumor angiogenesis was first introduced by Folkman. [23]

The tumor depends on angiogenesis for its growth and dissemination, so blocking this process reduces tumor growth and dissemination. The antiangiogenic therapy can be introduced by several modalities as follows:

  1. To block the angiogenic factor and/or receptor, e.g., blocking VEGF/VEGFR is the most studied and commonly targeted molecule, due to its central role in tumorogenesis. This approach is found useful in gastric carcinoma, colonic cancer, pancreatic and hepatocellular carcinoma etc.
  2. When only one factor is blocked the tumor growth may shift to other angiogenic factor, so blocking multiple pathways is more effective than inhibition of single factor. The blockade of tyrosine kinase is one such approach. VEGF, basic FGF, platelet derived EC growth factor use this pathway for action to complete. Blocking the tyrosine kinase pathway has got several advantages over a single protein/receptor blockage. This concept is tested and developed in practical studies and also in phase I/II clinical trials.
  3. Another approach is to use drugs that directly inhibit proliferation and/or survival of ECs. Thalidomide is one such drug which inhibits EC proliferation, although the exact mechanism is not yet understood.
  4. It is known that during angiogenesis there is degradation of extracellular matrix and basement membrane. Thus it facilitates progression of angiogenesis. The inhibitor of MMP-2 and MMP-9 has shown to reduce tumor vascularity and metastasis in human colon cancer xenograft implanted in mice. [24]
  5. Yet another approach is to inhibit vascular cellular adhesion molecules. [25]


The present scenario

The most targeted protein is VEGF. The clinical benefit of VEGFR inhibition is due to:

  1. They prevent the tumor vessel expansion by blocking its branching power.
  2. The anti-VEGF drugs induce regression of preexisting tumor vessels.
  3. They sensitize the ECs to the effect of chemotherapy and irradiation by depriving them the special benefit of VEGF.
  4. They normalize the abnormal blood vessels by pruning immature, pericytes deprived blood vessels. However, this mechanism may be transient since tumor vascularization may escape VEGF blockade and develop an alternative pathway for angiogenesis.


The actual therapeutics

The agents which interrupt the critical cell signaling pathways involved in tumor angiogenesis and growth can be grouped into three main categories [Table 2] as follows:
Table 2: The actual therapeutics


Click here to view


  1. Monoclonal antibodies, they are directed against specific proangiogenic growth factors and/or their receptors.
  2. Small molecule tyrosine kinase inhibitors of multiple proangiogenic growth factor receptors.
  3. Inhibitors of mammalian target of rapamycin.
  4. Miscellaneous agents.


There are other antiangiogenic agents that inhibit angiogenesis, but the exact mechanism is not completely understood. Finally in the field of dermatology, there are several agents used in the treatment of skin tumours [Table 2].

The pitfalls and challenges

  1. The long-term survival and overall benefit in advanced disease are limited and same may acquire resistance and become refractory to anti-VEGF therapy. The mechanism of developing resistance is yet to be evaluated.
  2. Monotherapy with VEGFR kinase inhibitor induces benefit in some and is ineffective in others or evolves side effects when combined with chemotherapy.
  3. Some tumors become nonresponsive during treatment when hypoxia up regulates rescue angiogenic molecules such as placental growth factor, FGFs, interleukin-8 etc.
  4. Poor vascularization as in pancreatic cancers or mature tumor capillaries as in hepatocellular carcinoma may reduce sensitivity to VEGFR inhibitor treatment.
  5. Depriving the tumor of its blood supply may divert and select hypoxia resistant clones of tumor cells. [26]


Trying to block the VEGF/VEGFR molecule, looks like a secured option theoretically, but the tumor cells are more clever and they circumvent the blockade by several strategies like VEGF independent vascular growth, in which cells can survive in hypoxic environment. They may also opt to disseminate by lymphatics.

The future

  1. The mechanism of antiangiogenic therapy is to deprive tumor, its blood supply and to starve them of oxygen and nutrients. VEGF is one of the most studied and promising targets. Other strategies are being developed to achieve the same goal with a different pathway.
  2. It is a known fact that VEGF (receptor) inhibitors work more efficiently on the vessels that are devoid of pericytes. So, VEGF inhibitors may be combined with PDGFRβ inhibitors, where the later reduces tumor progression by facilitating pericytes detachment and vessel becomes more immature and vulnerable to regression.
  3. Understanding the molecular basis of angiogenesis has progressed much, but the optimal dosing, duration and modalities of combination therapy is yet to be decided.
  4. After resection of the primary tumor, the usefulness of VEGF (receptor) inhibitors is yet to be evaluated.
  5. Predictive and prognostic biomarkers are lacking which can be studied in any given tumor, stage, and treatment.
  6. It is not yet clear whether available vessel pruning therapy has got an edge over sustained vessel normalization, to suppress metastasis and enhance the chemotherapeutic effect.
  7. Whether it is useful and/or how much it is useful in nonsolid malignancies like Leukemia is not known.
  8. The safety in children and pregnant women is not known.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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