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The process of angiogenesis has a number of regulatory molecules factors, but over the last two decades vascular endothelial growth factor (VEGF) has emerged as one of the major angiogenic regulators1. Not only has VEGF transpired as an essential regulator in angiogenesis, but also it has been established to have a vital role during developmental, physiological, and pathological conditions. VEGF consists of four different isoforms, generated through alternative splicing, represented as VEGF121, VEGF165, VEGF189, and VEGF2062.

Moreover, VEGF165 is the predominate form of VEGF and has the ability to freely diffusible or become sequestered within the extracellular matrix; whereas the other isoforms bioactivity and availability are represented by length3. VEGF belongs to a gene family consisting of VEGF-A, placenta growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D. Each specific protein has different binding affinities and signaling responses to the tyrosine kinase receptors, which include the following: vascular endothelial growth factor 1 (VEGFR-1), VEGFR-2, and VEGFR-3.

The importance of VEGFs and VEGFRs in physiological conditions has proven to be vital in the regulation, binding, and expression of each. Demonstrated through gene targeting knockout studies in mice, the elimination of VEGF activity proved to be lethal, as the consequence in these deficient mice did not advance past embryonic stages4. Furthermore, the dependence of VEGF and its expression prove to be necessary in embryonic stages of angiogenesis for early stages, as well as postnatal development, and the maturation and formation of bone5. When VEGF is inhibited, the ability of blood vessels to infiltrate and induce correct directional growth and cartilage cause invasion was eliminated due to the lack of VEGF mRNA expression by hypertrophic chondrocytes.

VEGF has been found to be produce at excess levels during pathological conditions. The regulation of VEGF expression can be stimulated by many factors including hypoxia, exposure to additional growth factors, and cell differentiation. These characteristics of VEGF mRNA expression has identified VEGF as a hypoxic-inducible angiogenic factor6. Change in cell differentiation, and the transition of growth in oncogenes, also increases VEGF protein secretion and stability. As a result, the up regulation of VEGF makes for a major candidate in tumor and atherosclerosis plaque formation; as well as influential responses in other disorders.

The potential of tumor growth relies on new blood vessels, and VEGF is a key mediator that can stimulate such a response. Thus, the ability to target VEGF through inhibition actions has been a topic of great interest in the perspective aspects in therapeutics. Not only has the goal of elimination to treat cancers been investigated, but the ability to induce VEGF could also have possible benefits in ischemic conditions. To date, VEGF inhibitors have shown promise in many animal models and clinical studies in humans have shown an onward progress.



A. Vascular Endothelial Growth Factor-A

The structural component of the human VEGF gene (VEGF-A) is characterized by eight exons allowing for the production of various protein coding regions2. The process of alternative splicing of VEGF mRNA results in the generation of four different isoforms: VEGF121, VEGF165, VEGF189, and VEGF206, which correspond to the length of amino acids sequence2,7. Undergoing cleavage of a 26-amino acid hydrophobic signal sequence, and comprised of identical amino-terminal residues, the following proteins of VEGF are generated7. Both VEGF165 and VEGF121 are absent of exon 6 residues, while VEGF121 is void of exon 7 residues8. VEGF189 and VEGF206 both contain the full sequence of VEGF165, with the addition of 24 amino acids, and VEGF206 has an additional 17 amino acids encoded due to varying selection of the 5'-splice donor site2,7. The different exons of all VEGF mRNA splice variants are displayed in Fig 1.

The different forms of VEGF have been associated with a genetic regulatory mechanism for their bioavailability and bioactivity3,9 depending on the resulting amino acid length through generated gene splicing of VEGF2,7. The most abundant form of VEGF is VEGF1653 which is expressed as a 45 kDa homodimer of 23 kDa momomers and has a heparin-binding biological characteristic10. VEGF121, spliced of the 44 amino acids found relative to VEGF165 in the carboxyl terminus, is an acidic protein with no binding affinity for heparin, allowing the shortest isoform to be freely soluble3. While VEGF121 is freely diffusible, the longer isoforms VEGF189 and VEGF206 are basic proteins that have a high binding affinity for heparin. In correspondence with the isoform length, there is a greater affinity for allowing the sequestering of these isoforms in the extracellular matrix to heparan sulfate proteoglycans3,9. The predominate form, VEGF165, has an intermediary characteristic since 50-70% of this form is bound to the extracellular matrix and plasma membrane proteins and the remainder can be secreted3,9.

In concordance with the findings from the studies, the availability of VEGF proteins in soluble forms to endothelial cells can be induced by two methods: 1) the generation of smaller and diffusible proteins such as VEGF121 and VEGF165 or 2) the release of longer VEGFs by protease activity of plasmin, through the plasminogen activation cascade, allowing the cleavage of bound isoforms in the extracellular matrix3,9.

In order to understand the importance of the roles of the bioavailability of each isoform and their structural characteristics, Keyt et al. 11 examined the role of plasmin cleavage. The cleavage of the protease plasmin produced two fragments consisting of an amino terminal and a carboxyl terminal, which respectively include a homodimeric protein receptor binding determinant and a polypeptide binding heparin. The findings from this study showed that the carboxyl terminal domain has a significant role in the reduction of the bioactivity response on the mitogenic activity of VEGF on endothelial cells, as the proteolysis or alternative splicing of this terminal removed the heparin binding domain11. The different isoforms and their biological characteristics proved that VEGF165 is the most optimal and effective signal for endothelial cells8,11.

To further investigate the importance of the VEGF heparin-binding domain isoforms, Carmeliet et al.12 produced mice that solely express the VEGF120 isoform through the removal of exons 6 and 7 that encode for VEGF164 and VEGF188 (VEGF in mice are shorter by one amino acid compared to human VEGF). In the absence of the larger expressed isoforms of VEGF164 and VEGF188, impaired cardiac performance and myocardial angiogenesis ensued, myocardial perfusion was reduced, and fatal ischemic cadiomyopathy resulted. Of the 230 neonates in this study, the expressions of only the shortest isoform lead to mortality of about half of the mice within a few hours of birth and the remaining population died before postnatal day 1412. Consequently, the different isoforms that are expressed can be considered to have different biological roles by binding specific receptors.

B. VEGF-Related Molecules

Placenta Growth Factor (PlGF)

VEGF is part of a protein family consisting of VEGF-A, placenta growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D. Compared with VEGF165, the encoded genes display similar homology sequences. These characteristics strengthen the idea that VEGF and the genes discussed below are working together in the regulation of angiogenesis and related vessel development14. Specifically, PlGF shares a 53% sequence homology with VEGF and was one of the first VEGR-related molecules to be identified15. Primarily expressed in the placenta, heart, and the lungs, two isoforms are generated through gene splicing: PlGF131 and PlGF15216,17. Ischemia, inflammation, and wound healing in PlGF deficient mice was impaired during angiogenesis and collateral growth under these conditions18. In contrast, mice over expressing PlGF had elevated development in vessels and vascular permeability19.


Highly expressed in skeletal muscle, brown fat, and the myrocardium, VEGF-B can be found as two different isoforms: VEGF-B167 and VEGF-B18620,21. VEGF-B isoforms are characterized as membrane bound (VEGF-B181) and freely diffusible (VEGF-B167) proteins, with the predominate form being VEGF-B16720. Inhibition of VEGF-B via gene targeting knock out in mice suggests the potential of coronary arteriogenesis since recovery was shunted in ischemic induced events. The development of smaller hearts were also a characteristics of limited VEGF-B expression22. In addition, after 7 days of induced ischemia in mice, an elevated expression of VEGF-B is demonstrated leading to neovascularization23.



A. Hypoxia

Gene expressions of VEGF, especially during oxygen tension levels, have shown to be regulated in both in vitro and in vivo studies. In a hypoxic induced state, VEGF messenger RNA levels are significantly up regulated, with VEGF functioning as a hypoxic-inducible angiogenic factor6,24,25. Studies conducted on human retinal pigment epithelium24 and human vascular smooth muscle cells25 both identified and characterized the potent stimulus of VEGF gene expression as correlated with hypoxic conditions and to be the major endothelial mitogen. Furthermore, Shweiki et al.6 used glioblastoma multiforme, a rapidly growing tumor undergoing necrosis, to examine the levels of expression of VEGF mRNA in oxygen deprived conditions. They found high levels of VEGF gene expression within these tumors, suggesting that hypoxia conditions are a key characteristic for the regulation of vascular permeability.

VEGF gene expression has also been shown to have neuronal developmental activities. As retinal vasculature undergoes formation under the regulation of oxygen, there is an increase of VEGF secretion during hypoxic conditions26. In addition to hypoxia in neuronal development and vascular permeability in endothelial cells, ischemic conditions have also shown positive up regulation in the revascularization process in a study done by Banai et al.27. Occluding the left anterior descending coronary artery in pig myocardium, ischemia induced an increase in VEGF RNA expression.

Adding support to the regulation of genes in response to hypoxia, researchers where able to show similarities between the regulation of VEGF and erythropoietin gene expression28. Goldberg et al.28 were able to lend support to the notion that the mechanisms that sense hypoxia within the expression of these genes are conserved at a molecular level. The sequence of the 5' VEGF transcription start site is homologous to the human erythropoietin hypoxia-responsive enhancer28. The transcriptional regulation similarities revealed a 28 base pair sequence located at the 5' promoter of the human VEGF gene which meditates hypoxia-inducible transcription and protein binding29,30. This 5' promoter site proved to be homologous to the hypoxia-inducible factor 1 (HIF-1) binding site found at the 3' erythropoietin enhancer29,31. HIF-1 was discovered to have homologous sequences and similarities with VEGF and erythropoietin, and Forsythe et al.32 provided additional support of HIF-1 serving as a key activation mediator of gene transcription of VEGF. These results demonstrated that cells that lacked the HIF-1 binding site did not induced VEGF gene expression during hypoxic conditions32.

Several other studies have further investigated transcriptional control in the up regulation response of VEGF due to low oxygen tension. Hypoxic induction of the VEGF gene was up regulated with the Levels of adenosine33 and c-Src34 were found to up regulate the hypoxic induction of the VEGF gene. During hypoxia, adenosine levels continue to rise and more adenosine binds to its receptors triggering an increase in cAMP. In correspondence to the rise of cAMP levels through the activation of the protein kinase A pathway, an increase of VEGF mRNA expression is observed33. Not only was there an increase of VEGF expression through hypoxia and adenosine levels, activation of c-Src was shown to also up regulate VEGF expression through kinase activity34. The same study by Mukhopadhyay et al.34 confirmed a down regulation of VEGF in hypoxia due to a c-Src negative mutation.

In relation to the regulation and expression during the transcriptional level of VEGF in a hypoxic condition, researchers have also tested the importance of post-transcriptional regulation35-37. The transcription of the VEGF gene cannot, by itself, be solely responsible for up regulation of VEGF mRNA levels and this has proven to be true by the increase of mRNA stability that ensues during hypoxia. Results from Levy el al.35 revealed an enhanced half-life of VEGF mRNA during hypoxia from 43 ± 6 to 106 ± 9 minutes. Also, comparing both hypoxic and normoxic levels, the stability of mRNA was further increased by hypoxia-induced proteins, which bound to the 3'-untranslated region29. These results indicate that mediated expression of VEGF during hypoxia relies on both pre and post-transcriptional regulation.

B. Cytokines and Growth Factors

Exposure to several different types of growth factors has been linked with regulation of the release of VEGF. The up regulation and release of VEGF proteins have been shown to correlate with the following cytokines: Transforming growth factor b and a (TGF)38-40, epidermal growth factor (EGF) , and keratinocyte growth factor (KGF)38. Frank et al.38 demonstrated that, during the wound healing process, an increase in VEGF mRNA expression occurred through the treatment of cultured keratinocytes to EGF, TGF-b, and KGF. Likewise, TGF-b was also shown to have a positive stimulatory response in fibroblastic and epithelial cells in terms of increasing the transcriptional expression of VEGF mRNA and the release of the protein40. Furthermore, Detmar et al.39 found an up regulation in the three isoforms of VEGF (121, 165, and 189 amino acids) by keratinocytes when introduced to TGF-a and EGF.

Inflammatory cytokines have also caused induction of VEGF gene expression through the study of interluekin-1b (IL-1b)41, prostaglandin E and interleukin-1a (IL-1a)42, as well asinterluekin-6 (IL-6)43. The increase of VEGF expression was studied in isolated rat aortic smooth muscle cells through dose and time dependent exposure of IL-1b in order to acquire a better understanding of the mechanism that controls atherosclerosis42. The activity of IL-1b enhanced the mRNA levels of transcription through lengthening the half-life stability and increasing the rate of transcription. Not only were transcriptional levels amplified, levels of VEGF protein were also increased 3.3 fold, leading to the conclusion that VEGF and IL-1b play significant roles in atherosclerosis lesions41.

Furthermore, Ben-Av et al.42demonstrated up regulated expression of VEGF through exposure of synovial fibroblasts from rheumatoid arthritis cell to inflammatory mediators of prostaglandin E and IL-1a. In response to the likelihood that VEGF is, in response, also regulated in inflammatory angiogenesis, other researchers conducted studies to examine the correlation of IL-6 and VEGF. Not only did VEGF mRNA expression increase with treatment of IL-6 over time, but, through the deletion and mutations, the VEGF 5'-untranslated regions were found to be important in the promoter activity and up regulation for the expression of VEGF43.

Interestingly, insulin-like growth factor 1 (IFG-I), a mitogen that evokes many cancers, has been shown to produce up regulation of VEGF mRNA and protein in colon carcinoma cells. The treatment of IGF-I increased the rate of transcription of VEGF mRNA 5-fold over 4 hours of exposure, and compared to control cells, improved the stability of VEGF mRNA half-life to 2 hours44. Goldman et al.45 also examined the expression of VEGF in glioblastoma multiforme, a human malignant brain tumor, which stimulated the induction and release of VEGF in conjunction with epithelial growth factor.

C. Cell Transformations and Oncogenes

The ability of cells to transform and undergo developmental changes, either during physiological or pathological conditions, has elevated VEGF gene expressions. Under normal conditions, Claffey et al.46 examined the role of VEGF gene expression during cell differentiation in rodent tissues and models. Studying the development and differentiation of adipocyte and myogenic cells, the expression of VEGF mRNA levels increased as both cells transitioned in their cell lines. The ability of cells to develop and differentiate under conditions of elevated expression of VEGF mRNA required specific signal transduction pathways. Further investigation during these studies concluded that the up regulatory response of VEGF could be linked to protein kinase C and cAMP-dependent protein kinase pathways through the use of second messenger effectors46.

In addition to the regulation of VEGF expression, transformations of cells can also be related to pathological conditions. The transformation of key regulators and cells linked to these conditions has significant roles in regulation of VEGF gene induction47-49.

During tumor development in humans, alteration and mutation of the p53 tumor suppressor gene is commonly observed. The transformation of this tumor regulator has been linked to elevated levels of VEGF expression in relations to the increased activation of protein kinase C47. Similarly, the alteration and mutational expression of Ras oncogenes has also been a significant genetic change resulting in the occurrence of human cancers48.

With the up regulation of VEGF gene expression required in the growth of tumors, Rak et al.48 where able to establish an increase of VEGF mRNA and protein secretion through mutant Ras oncogene expression in both human and rat tissues. To further prove the importance of mutant Ras and the regulatory expression of VEGF in tumor angiogenesis, investigators blocked the mutant gene through a protein farnesyltransferase inhibitor and found marked decreases in the induction of VEGF gene expression48. Equally important, studies conducted by Mazure et al.49 on the expression of VEGF were further elevated during both oncogneic transformation of Ras and hypoxia.

The von Hippel-Lindau (VHL) tumor suppressor gene has also shed new insight on the transcriptional mechanisms of VEGF50,51. Mutations in the VHL gene have lead to the increase of carcinomas, including renal cancers52, and in result, these highly vascular tumors have shown a high production of VEGF expression53. The advances and findings by Simeister et al.50 helped show that the mediator of tumor growth due to the lack of VHL protein was, in itself, VEGF. Using human renal carcinoma cells that either lacked the wild type, or expressing an inactive mutant form of the VHL gene, Siemeister el al.50 showed an increased induction of VEGF expression. To illustrate the correlation of the VHL tumor repressor gene and the loss of regulation corresponding to VEGF at the mRNA and protein levels, the treatment with the wild type VHL gene reversed the induction of VEGF expression.

The importance of HIF-1 as being a key activation mediator of gene transcription of VEGF32 was further investigated by Iliopoulos et al.51 in relation to the regulatory mechanisms of the VHL tumor suppressor protein. To study the regulatory role and function of the VHL protein, their model consisted of the comparison of a mutant and wild type VHL protein. Encoding for the expression of mRNAs for hypoxia-inducible genes consisting of VEGF, platelet-derived growth factor and glucose transporter GLUT1 genes were observed in both normoxic and hypoxic conditions. mRNAs for all the hypoxic-induced genes were expressed in both conditions, whereas in the treatment in which wild type VHL was transected and introduced into the cells, the expression of the genes was reverted51. Those that contained the wild type VHL protein showed regulation within those hypoxia-induced genes, especially VEGF deregulation, indicating that the VHL protein has characteristic regulatory mechanisms in tumor angiogenesis51. The cellular pathway has many alterations that can change the regulation of gene expression, and most importantly, can be the cause of elevating both the VEGF mRNA and the protein level14.


The VEGF Receptors

A. VEGF Binding Site Distributions and Characteristics

The characteristics of VEGF binding sites where revealed in a study conducted by Vaisman et al.54. Through the use of bovine endothelial cells, researchers were able to discover the presence of two VEGF binding sites that contained a relative high affinity. The binding of VEGF to endothelial cells was found to be highly specific with each receptor having a binding dissociation of 10 pM and 1pM and a cell density of 40,000 and 3,000 receptors per cell, respectively54.

VEGF binding receptors lead researchers to study the cross-linking activities of the different isoforms of VEGF expressed and found the existence of lower affinity binding sites55. The cross-linking revealed the binding selectively of the lower affinity receptors of tumor and endothelial cells selected the predominate form, VEGF16555. Moreover, the study conducted by Jakeman et al.56 supported the role and importance of VEGF as the major factor in binding vascular endothelium. Using tissue sections from adult rats, and studying the ligand binding capabilities of VEGF, researchers were able to demonstrate the high affinity of VEGF on both microvessels and larger vascular endothelium in all tissues and organs that were studied56. Thus, the role of VEGF, and the importance of the binding receptors, was further supported in the growth and maintenance of vascular endothelial cells56.

The two receptors that bind VEGF isoforms have been identified as VEGFR-1 and VEGFR-2; both classified as receptor tyrosine kinases (RTKs)57,58. Both VEGFR-1, known as Flt-1 (fms-like tyrosine kinase), and VEGFR-2, classified as a KDR/Flk-1 (kinase domain region/fetal liver kinase 1), show binding affinities for VEGF. The two VEGF domain receptors are similar in structure. They are composed of seven extracellular immunoglobulin-like domains, a single transmembrane region, and an intracellular tyrosine kinase domain sequence that is interrupted by a kinase-insert domain59.

Although these two receptors are the major binding factors for VEGF, another receptor, VEGF-3, has been identified as an additional factor of the family of RTKs60. Also known as Flt-4 and containing the same amount of immunoglobulin-like domains in the extracellular domain, this receptor does not bind VEGF; however, VEGF-3 has a binding affinity for VEGF-C and VEFG-D 61,62. Not only have RTKs been identified as receptors for varying VEGF ligands, recent studies have shown a group of co-receptors, the neurophilins, that interact with VEGF 55,63. A schematic illustration of all binding interactions of VEGF and VEGFR are indicated


One of the key characteristics of VEGFR-1 is, not only does it bind VEGF, but it also has binding affinities for PlGF and VEGF-B64,65. Using endothelial cells, Park et al.64 were able to show the high affinity of VEGFR-1 to PlGF, whereas no binding was recognized with VEGFR-2. Olosfsson et al.65 demonstrated the selective binding characteristics VEGF-B has to VEGFR-1. Through the HIF-1 mechanisms, which also proves as an enhancer site for VEGF, the expression of VEGFR-1 also shows characteristics of the same inducible factor since hypoxic conditions have shown to cause an up regulation expression for this receptor66.

Although VEGFR-1 has been shown to have binding characteristics with different growth factors, the mitogenic response is very weak through the binding of VEGF67. As a result of VEGF only inducing a weak tyrosine kinase response, the VEGFR-1 was presumed to contain a negative regulatory characteristic64. By binding VEGF, and thus reducing the ability to bind to other receptors, the over all mitogenic activity that could occur on endothelial cells would diminish. Studies investigating the binding properties of VEGFR-1 have also found a correlation with both PlGF and VEGF18 and the ability to heterodimerize with VERGF-268 have amplifying effects on the mitogenic responses.

Although studies have shown the limited signaling responses through the binding of VEGFR-1, others have demonstrated the importance of this receptor through developmental events and different cell types14. Fong et al.69,70 established the importance of the VEGFR-1 through gene targeting by the deletion of the exon encoding for the signaling peptide. Mice targeting in the deletion of this gene died within 8 days of gastrulation due in part to the development of disorganized vascular channels, blood islands, and elevated endothelial growth and angioblasts69,70.

To further investigate the role of VEGFR-1 in embryonic development and the importance of the tyrosine kinase, Hiratsuka et al.71 deleted the kinase domain without altering the ligand binding. As a result, the ability of normal vessels developed and the mice were able to survive; however, monocyte migration was reduced71. They conducted that the tyrosine kinase domain is significant in monocyte migration as the recruitment promotes angiogenesis and arteriogenesis72.

The importance of the VEGFR-1 signaling pathway has also been shown by Hattori et al.68 through the stimulation of PlGF. Binding of PlGF with VEGFR1 was able to restored early and late hematopoiesis with the recruitment and survival of endothelial and hematopoietic progenitor stem cells. Although many studies have been conducted, the true function and signaling methods of this receptor is still unknown and being investigated.


With a similar structural formation in relation to VEGFR-1, VEGFR-2 has been shown to have larger biological function and signal activity14. Through studies conducted, VEGFR-2 has proven to have major enhancing effects on mitogenic, angiogenic, and permeability factors. In contrast to gene targeting studies on VEGFR-1 conducted in mice that still developed endothelial cells69,70, studies of deficient VEGFR-2 binding failed to develop both organized blood vessels and yolk sac blood islands4. Thus, the lack of VEGFR-2 binding capabilities within these mice proved to have a significant aspect in the role of developmental angogenesis and hematopoiesis, as fatality ensued within 9 days in utero4.

The binding of VEGF, VEGF-C, and VEGF-D allow the phosphorylation of many tyrosine residues leading to a variety of different cell signaling pathways in endothelial cells. The tyrosine phosphorylation characteristics of this receptor have been shown to have roles in the proliferation, survival, migration and permeability factors13.

The ability to induce proliferation through the binding of VEGF has been linked to the ability to initiate the Raf-Mek-Erk pathway, ultimately increasing gene transcription73. A schematic of all the signaling pathways are demonstrated in figure 3. The ability to stimulate this pathway relies on the activation of Raf through protein kinase C activation73. Activating the binding receptor of VEGFR-2 also signals the phosphoinositide 3-kinase (PI3K)74. The resulting pathway further acts on Akt/Protein kinase B (PKB) and Rac intracellular molecules, resulting in the inhibition of B-cell lymphoma 2 (Bcl-2)-associated death promoter homologue (BAD) and Capsase 9. The following inhibition of BAD and Capsase 9 allows cell survival of endothelial cells, preventing apoptosis cell signaling74. Not only does the Akt/PKB pathway regulate cell survival, but it also has the capability of increasing nitric oxide (NO) production through triggering endothelial nitric oxide synthase (eNos)75,76. Thus, the ability to increase NO production permits an increase in vascular permeability and migration of endothelial cells.

Other signaling pathways that have been investigated through the action of VEGFR-2 are the p38 mitogen-activated protein kinase (MAPK)77 and Src kinase activities78. Yet the exact interactions of these pathways are unknown.

Furthermore, studies have demonstrated a relation between VEGFR-2 and adhesions of endothelial cells through complex formations involving vascular endothelial cadherin (VE-cadherin)79. The importance of this role was demonstrated by Carmeliet et al.79 with regard to signaling endothelial cell survival, as the absence of the VE-cadherin gene resulted in cell apoptosis and unregulated Akt/PKB pathways.


VEGFR-3 is a high affinity receptor for VEGF-C and VEGF-D61,62, and unlike VEGFR-1 and VEGFR-2, consists of six immunoglobulin-like domains cleaved within the fifth loop and held together by disulfide bridges60. Like the signaling activities involved in VEGFR-2, VEGFR-3 is involved in promoting the proliferation, migration, and survival in endothelial cells that pertain to the lymphatic's80.The function roles and characteristics are activated and stimulated through the cell signaling pathways previously mentioned: MAPK signaling cascade, PI3K, and Akt/PKB80.

The presence of VEGFR-3 has important roles in embryogenesis and adult stages81. During the early developmental stages of embryogenesis, this receptor can be found on all endothelial cells; however, in the adult, VEGFR-3 is only expressed and found in lymphatic endothelial cells. The significance of the receptor was made known by Dumont et al.82 through gene targeting VEGFR-3 expression in mouse embryos. Lacking the inability to express this receptor, mice die within 9 and half days due to defective vessel remodeling. This defect leads to the disorganization of larger vessels and fluid accumulation, ultimately causing cardiovascular death82. The importance of VEGFR-3 in the induction of lymphatic vessel development was shown through the blocking of the receptor. Through this method, Makinen et al.83 showed an occurrence of an obstruction in the lymphatic system through an increase in fluid retention and reduction in lymphatic vessels. In support of the signaling importance and receptor binding, a congenital hereditary lymphedema has shown to been linked to mutations in the activation of VEGFR-3 phosphorylation84. Furthermore, inhibiting the binding of VEGFR-3 suppressed tumor lymphangiogenesis and lymph node metastasis85.

In contrast, the over expression of VEGFR-3 was also demonstrated to be related to the growth of tumors. A study conducted by Skobe et al.86 revealed an over expression of VEGF-C is correlated with an increase of lymphangiogenesis resulting in breast cancer. Not only was the increase of VEGF-C binding capabilities able to increase lymphatic tumors, but VEGF-D has also been linked to the spread of tumors by the lymphatic system stimulating tumor angiogenesis and growth87.

D. Neuropilins

The detection of other VEGF binding sites were observed upon tumor and endothelial cells that showed an expression on cell surfaces which were distinct for specific VEGF isoforms55. The ability to distinguish the binding affinity was conducted through the different binding characteristics of VEGF165 and VEGF121. As a result, no binding of the VEGF121 isoform was seen, suggesting that exon 7 found in VEGF165 is needed to bind this receptor55. An investigation conducted by Soker et al.63 indicated this receptor mediates neuronal patterning and axonal guidance by proving it to be analogous to human neuropilin-1 (NP-1).

Identification of this new receptor lead to the interest of the mediated roles and signaling induced during the binding of VEGF. The expression of both VEGFR-2 and neuropiln-1 (NP-1) in cells indicated NP-1 functions as a co-receptor, elevating the binding interaction with VEGF165 and VEGFR-2. The binding of VEGF165 increased the mitogenic signaling and chemotaxis through the expression of both receptors87. In addition, both the over expression and the gene targeting of NP-1 was illustrated88. The over expression of NP-1 in mouse embryonic stem cells resulted in abundant cardiac and vessel defects, including dilated and increase in blood vessels, excess capillaries, and abnormal hearts89. In contrast, NP-1 plays a significant role in embryonic vessel formation as seen in a study conducted by Kawasaki et al.88. Using NP-1 mutant mouse embryos, the lack of this receptor produced malformations in the development of neural and yolk sac vascularization, blood vessels and cardiovascular functions88. Either the up regulation or down regulation of this receptor seems to have important signaling regulations on the potency of the mitogenic response to VEGF.



A. Embryonic, early, and postnatal development

VEGF has many essential roles in physiological development pertaining to the progress and growth of new blood vessels. The occurrence of angiogenesis is a key mediator in the development of proper embryonic and postnatal events. The cause and effect of VEGF in embryonic angiogenesis and vasculogenesis were shown through gene alteration studies90,91. Inactivation of the VEGF gene enabled the comparison of homozygous VEGF-deficient (VEGF-/-), heterozygous VEGF-deficient (VEGF+/-), and wild type mice. Each altered gene expression group showed signs of inhibited VEGF secretion: VEGF-/- cells showed no secretion, whereas VEGF+/- was able to express half of the normal amount91. Evaluating both altered gene expressions and vascular development; VEGF-/- displayed a larger decrease in the overall branching patterns and the number of blood vessels91. The vascular development was greatly inhibited, as well as formation of large vessels, the lumens, and interconnections90. VEGF+/- mice also showed many organelle abnormalities, which included underdeveloped cranial, branchial, and forebrain regions. Not only where those areas altered, the atrium and ventricle showed changes as well as a reduction in wall thickness of the ventricle91. Furthermore, defects in the nervous tissue and the placenta were also observed91. The developmental importance of VEGF is apparent: not only is it needed to induce vascular advancement, but without it an increase in apoptotic events also ensues. Accordingly, the lack of the VEGF allele, either containing only one single mutation or both, leads to a decrease in the maturation of embryonic development and death, which occurred within 11 days of these studies91.

Gerber et al.92 further indicated the importance of VEGF in the development of mice during neonatal stages. During the first and eighth day of postnatal old mice, mice were administered with mFlt(1-3)-IgG; a VEGF inhibitor. The results suggest that impairment of VEGF, starting administration at day one (Fig. 4A) or day eight (Fig 4B), shunted the overall growth in mass of mice compared to the controls. In addition, all the organs during the blocking of VEGF had signs of impaired and reduced size of 5 and 14 day old mice (Fig 4C, D). The treatment of this inhibition of VEGF also proved lethal during postnatal stages, similar to those seen previously in embryonic development. The most significant defects and malformations in VEGF deficiency were seen in the liver, kidney, and heart (Fig 4E, F).

Kitamoto et al.93 further supported the necessity of VEGF for proper development through blocking endogenous VEGF activity during the kidney developmental process of mice. Researchers treated mice with an anti-VEGF neutralizing antibody to test the possible deficiencies that result in the inhibition of VEGF and found an important role between VEGF and the crucial need in the proper development of the glomerulus and nephrons. Furthermore, the regulation of VEGF-A in the renal system, and the expression of podocytes, is important in pre and postnatal stages as demonstrated by Eremina et al.94. Heterozygous and homozygous deletion of VEGF-A in glomeruli, as well as the over expression, were conducted in mice. The mice of heterozygous strains showed signs of renal disease within 2.5 weeks, homozygous mice resulted in death during birth, and over expression of VEGF lead to renal failure 94. In conclusion, from the studies that were conducted, VEGF expression is necessary for physiological development during both embryonic and postnatal stages.

B. Bone Formation

The role of VEGF and angiogenesis is vital for the embryonic and early postnatal development, as is the maturation and formation of bone. Gerber et al.5 injected 24 day postnatal mice with mFlt(1-3)-IgG once a day to inhibit VEGF protein over 14 days. After treatment, mice were allowed to recover. Inhibiting VEGF, femur length was reduced and the hypertrophic chondrocyte zone of the growth plate showed large expansions (Fig 5A, B). The hypertrophic chondrocyte zone, compared to the controls, showed a 300-600% thickness in the mice that were injected with mFlt(1-3)-IgG. The resulting expansion of the growth plate was caused by the inhibition of resorption of the hypertrophic chondrocytes; even though chondrocyte proliferation, differentiation, and maturation were normal under treatment. The recovery stages indicated that the inhibition of VEGF resulted in the suppression of blood vessel invasion, since withdrawal of the inhibitor resumed rapid blood vessel growth, resorption of the chondrocytes, and normalized the size of the growth plate. Thus, for the necessary blood vessels to infiltrate and induce correct directional growth and cartilage invasion, VEGF mRNA must originate and be expressed from the hypertrophic chondrocytes5.

In addition to VEGF on bone development, Ryan et al.95 performed inhibition studies using the monoclonal antibody, recombinant humanized anti-vascular endothelial growth factor (rhuMAbVEGF). Administered to adult cynomolgus monkeys, rhuMAbVEGF was given twice during weekly intervals at a dose of 50 mg/kg. Further supporting the VEGF dependence for bone growth, treatment of this antiangiogneic agent resulted in increased hypertrophied chondrocytes and inhibition vascular invasion of the growth plate.

In contrast to the embryonic and post-natal development, the inhibition of VEGF in mice and primates during juvenile and adult stages did not have significant abnormalities seen in other studies conducted during early developmental stages90-94. The juvenile and adult stages of development can be concluded to no longer have a vital need for VEGF to survive, but rather require VEGF to maintain angiogenesis5,92.



A. Tumorogenesis

The formation of new blood vessels is a vital process for growth and developmental, but increase in vascular formation has been associated with tumor growth and formation. To date, the exact mechanisms by which angiogenesis mediates tumor growth are not fully known; however, angiogenic factors have been a primary focus. Most studies in the literature suggest that the effects of VEGF expression by tumor or non-malignant cells are a potent factor in the association of vascular proliferation for tumor growth. The up regulation of VEGF mRNA expression in many human tumors studies have demonstrated an elevated increase compared to controlled tissues8.

The expression of VEGF mRNA and vascular development in tumor cells were examined by Mattern et al96. These researchers used 91 previously untreated human patients with epidermoid lung carcinoma to study the relationship of VEGF, known to increase tumor cell proliferation and tumor vessels. Tissue samples were analyzed through VEGF antibodies, von Willebrand factor (factor VIII), and proliferating cell nuclear antigen (PCNA) in order to detect VEGF expression, blood vessels, and proliferating cells, respectively. Comparing between VEGF positive and VEGF negative tumor cells, there was a correlation in the expression of VEGF mRNA with the increase of vessel density, supporting the role of VEGF and angiogenesis in tumor cells (Fig. 6). Associations between VEGF expression and PCNA also showed a positive link, indicating that the ability of tumor cells to proliferate relies on the up regulated expression of VEGF

In addition, Brown et al.97 also demonstrated a correlation between VEGF mRNA expression and tumor development and growth, by the fixation and staining of 24 tissue specimens of various breast carcinomas. Through hybridization and VEGF RNA probe labeling of the collected tissues, there was a strong expression of VEGF mRNA. Not only was the expression of mRNA found to be up regulated in this study, vessels of the surrounding endothelial cells closest to the malignant tumor cells showed an increase in VEGF mRNA receptor. Yoshiji et al.98 added to the findings by conducting a similar study in 18 women with malignant breast tissues. Conducting northern blot analysis of VEGF, VEGFR-1, basic fibroblast growth factor, and transforming growth factors in human breast cancers, VEGF up regulation showed the greatest gene expression (Fig 8). In contrast to the elevated expression of VEGF receptors, VEGFR-1 showed no sign of increase within the tissues studied. However, this supports the evidence and idea in the literature that VEGFR-2 is the critical receptor for VEGF mitogenic responses. As a result, studies have been conducted in which both receptor mRNAs for VEGFR-1 and VEGFR-2 have been up regulated in tumor samples97,99,100.

The significance of up regulation in VEGF mRNA expression in carcinomas has further been defined in human tissue samples including the gastrointestinal tract 100, kidney and bladder101, brain99, and the central nervous system97. These results all suggest that the increase in VEGF mRNA and the receptors has important implications for the growth and vascular development of tumors in comparison with normal tissues.

The inhibition of VEGF has provided additional support for the relationship between VEGF and tumor growth. Kim et al102 studied the primary role of VEGF through the inhibitor result of VEGF-induced angiogenesis by the use of specific monoclonal antibodies. In vivo expression of VEGF mRNA was studied in nude mice through subcutaneous injection of the following human tumor cells lines: A673 rhabdomyosaroma, G55 gliblastoma multiforme, and SK-LMS-1 leiomyosarcoma. Administration of the anti-VEGF antibody (A6.4.1) at different doses, as well as an anti-body control (5B6), was given to tumor-infected mice twice a week, over a four-week period (Fig. 9A,B). Anti-VEGF antibody showed an inhibitory response to both cell lines in comparison to the control antibody tumor size. Greater inhibitory results were witness in the A673 tumor line since there is a greater dependence on angiogenesis, whereas SK-LMS-1 had no growth over weeks. Treatment of the tumor cell line mice also decreased both vascular density in areas and tumor weight compared with controls. A decrease of 96%, 80%, and 70% in weight for A673, G36, SK-LMS-1 cell lines were demonstrated, respectively (Fig. 9C). In support of inhibition of tumor development with anti-VEGF antibodies, and the requirement for VEGF in tumor growth, other studies of different human tumor cell lines were demonstrated including A431, a human epidermoid carcinoma, and HT-1080, a human fibrosarcoma cell line{{; 193 Melnyk,O. 1996; 194 Asano,M. 1995; }}.

In addition to the tumor growth development factor of VEGF, a study by Yuan et al.103 shows that endogenous VEGF is also involved with the increase in vascular permeability of tumors. Moreover, the constant expression of VEGF has also been demonstrated to maintain vascular proliferation and integrity.

The experiment performed by Yuan at al.103 was conducted on severe combined immunodeficient (SCID) mice with the implantation of different human tumor cell lines consisting of: LS174t, a human colon adenocarcinoma, U87, a human gioblastoma, and P-MEL, a human melanoma. Tumor cell lines were transplanted into two different locations: the cranium and dorsal skinfold chamber. Treatment of anti-body VEGF antibody (A4.6.1) or a bolus were giving to the to the infected mice containing the vascularized tumors over a period of 7 to 23 days post-tumor implant. Four different groups were made up of between 3 or 7 mice and vascular permeability and morphology were performed in a time dependent fashion of 6 hours to 11 days post treatment. Calculations were conducted through rhodamine labeled BSA injections for vascular permeability, whereas vessel length, diameter, and density were conducted by image analysis.

Results were similar to that of Kim et al.102. Treatment of anti-VEGF antibody resulted in a time dependent reduction of the tumors implanted; in addition, the permeability factor was reduced in comparison to the controls (Table 1, 2). Not only was vascular permeability reduced upon the treatment of anti-VEGF antibody, but the vascular integrity was reduced in both size and distortion (Fig. 10,11). Taken together, the results implicate the growth of the tumor was slowed by reducing the infiltration of new blood vessels, morphology, and permeability by blocking VEGF over time103.

B. Atherosclerosis

Various inflammatory responses have shown an increase in the expression of VEGF39,42. Elevated expression of VEGF, and its pivotal role of inducing neovascularizaion, has been related to the pathological condition of plaque progression in the vascular system, known as atherosclerosis104. The localization of VEGF in atherosclerotic lesions has been further supported through immunohistochemical techniques of 38 human coronary artery segments105. Specimens of human coronary atherosclerosis consisted of different stages of severity, and compared to normal arterial segments, showed positive expression of VEGF and its receptors.

The early stages of atherosclerosis lesions of the ‘fatty streak' increase macrophage and T lymphocytes resulting in the accumulation of lipoproteins and ultimately producing lipid filled foam cells106. These events and associated factors leading to the formation of the atherosclerotic plaque have been correlated with VEGF induction, further enhancing plaque progression.

Human monocytes have demonstrated the ability to express VEGFR-1, and in response to VEGF, the chemotatic action of monocytes is activated107,108. To provide a correlation with VEGF and monoctye activation in the enhancement of atherosclerotic plaque progression, Celletti et al.109 used apolipoprotein E/apolipoprotein B100 deficient mice treated with recombinant human VEGF165 (rhVEGF) or albumin as a control. Placed on a 0.25% cholesterol diet, time points of 1, 2, and 3 weeks were conducted in both treated and control groups and comparison of endothelial precursor cells, macrophages, and plaque formations were evaluated.

Significant increases of macrophages in both bone marrow and peripheral blood treated groups are seen when VEGF is administered. Versus albumin, all time points recorded in the treated group showed increase in levels of macrophages (Table 3). Treatment with rhVEGF, compared to the controls, also had considerable elevations in plaque formation in the following measurements: cross sectional area (Fig. 12A), circumference (Fig. 12B), and maximal thickness (Fig. 12C) over the experimented time. Total plaque areas for VEGF treated mice under histological sections were 14,909 ± 2,534 mm2 in the first week, 230,935 ± 4,612 mm2 in the second week, and 64, 328 ± 23,254 mm2 during the third week. In contrast, control mice treated with albumin had 2,228 ± 1,385 mm2, 5,136 ± 4,612 mm2, and 64,238 ± 23, 254 mm2 in weeks 1, 2 and 3, respectively. Increases in circumference and maximal thickness were also demonstrated in comparison with treated and control groups, as well as less uniform plaque build up shown in thoracic aorta histological slides.

Endothelial and macrophage density where also demonstrated among treated and control grouped in cholesterol deficient mice. To evaluate endothelial cross sectional density staining with either factor VIII or CD3 antigen was applied and subjected to immmunohistochemistry. All treated mice showed a large difference in the cross-sectional density over three weeks compared to control specimens. However, in spite of plaque growth and increase of vascular density, macrophage infiltration was dampened until week three in treated groups.

Along with testing on the deficient mice, the experiment was also repeated in cholesterol fed rabbits following the same protocol to show the concluded results were not species specific. Mean plaque area, circumferential plaque, and plaque thickness all showed a significant increase compared to controls 109. Taken together, these results indicated that VEGF is able to induce macrophage mobilization within the vascular system and increase both the formation and progression of atherosclerosis. The increase due to VEGF expression and stimulation to elevate atherosclerotic plaque formation may have a detrimental role on plaque stability.

Proving additional insight for VEGF in promoting atherosclerosis through the increase of macrophages during inflammatory responses and invoking addition angiogenesis, the inhibition of plaque neovascularization was examined. Moulton et al110 inhibited angiogenesis in apolipoprotein E deficit mice on high cholesterol diet over 75 days through the administration of angiostatin. Atherosclerosis lesions were significantly reduced in treated mice when compared to controlled samples. In addition to the reduction of vascular lesions, both the capillary plaque density and frequency seen in association with angiogenesis were reduced. Along with the inhibition of angiogenesis, inflammatory responsive cells included macrophages and leukocytes were diminished.

In concurrence with the pervious study by Celletti109, and further applied by Moulton,110 angiogenesis can be considered as a positive feedback cycle. By the induction of macrophages through inflammatory responses, this stimulation future enhances not only VEGF, but accumulates additional inflammatory response factors to further mediate the progression of angiogenesis and ultimately arteriosclerosis plaques.

Once inflammatory cells have recruited to the sites of lesions, the progression of atherosclerosis plaques are deposited with lipoproteins and develop foam cells. The relationship with the progression of lipoproteins with VEGF and macrophages has been investigated. Salomonsson et al.111 were able to prove a correlation with the secretion and expression of VEGF by oxidized low-density lipoprotein (OxLDL) in vitro.

Using human monocyte-derived macrophages (hMDMs), the expression of VEGF was measured in response to the exposure to OxLDL. Varying the concentration of OxLDL to hMDMs over 24 or 48 hour, researchers were able to show an elevated expression of VEGF mRNA in both time frame exposures (Fig. 13 A,B). Maximum stimulation of mRNA was seen at OxLDL concentration doses of 25 mg mL-1. OxLDL was further able to increase VEGF mRNA protein secretion within equivalent time periods.

To address the correlation in the increase of VEGF expression and protein secretion by lipoprotein, the stability of VEGF mRNA was addressed through mRNA decay assay studies. The treatment of VEGF mRNA macrophages to OxLDL resulted in an elevated half-life beyond 120 minutes. In contrast to control hMDMS, which only produced a half-life of roughly 45 minutes, the treated group tripled in half-life decay, concluding that lipoproteins and macrophages can increase VEGF mRNA stability and expression.

The cell singalling pathways of macrophages induced by OxLDL and VEGF were also investigated to support kinase activation in the role of VEGF expression. Cells were introduced to anisomycin, a protein kindase activator, and stimulation with OxLDL proved to enhance the p38 MAPK signaling pathways. In contrast, an inhibitor against p38 MAPK expression showed no reduction of the VEGF expression by OxLDL 111.

Taken together, VEGF expression is regulated by the p38 MAPK signaling and increased through the treatment of OxLDL. This is in support to the results obtained by Rousseau77, where binding of VEGF to specific receptors induces the p38 MAPK signaling pathways. The concluding findings were able to show an increase of VEGF mRNA and expression by low-density lipoproteins through the activation of known signaling pathways. Although these findings were conducted in in vitro studies, it would add more value if the same increase of VEGF could be demonstrated in vivo.

C. Other pathological conditions

The biological characteristics and activities of VEGF have placed this angiogenic factor at the forefront in progressing pathological conditions. Easily diffusible and induced by hypoxic conditions, elevated levels of VEGF have been found in diabetic retinopathy112, age-related macular degeneration visual loss 113, rheumatoid arthritis 114, and psoriatic skin115 and bullous disorders116. The key pathological condition in all of these disorders is the elevated induction of neovasclarization. As a result, detection and induction of VEGF, a potent mediator during the growth of new vessels, is specific for this process in these syndromes. With a vast range of diseases, the ability to target VEGF expression could play an important role in the diagnosis and treatment of patients undergoing such conditions.



The current knowledge and immense research in the role, regulation, expression, and signaling mechanisms leading to both physiological and pathological conditions of the vascular system by VEGF still calls for supplementary investigation of the potential mechanisms for this angiogenic factor. By the study and discovery of related angiogenic growth factors, and in comparison of studies throughout the scientific community and literature, VEGF is a key regulator in physiological angiogenesis. VEGF production and expression in many endothelial tissues is vital for development, but has also shown to be a mediator in pathological angiogenesis. As a potent growth factor for specific tissues, VEGF stands to be a potentially important regulator in growth and differentiation of multiple components of the cardiovascular system.

Through the use of gene targeting knockout mice, both heterozygous and homozygous mutations proved to have significant and detrimental consequences. The mutations that resulted in the inactivation of gene expression, and also blocking of both VEGF receptors, VEGFR-1 and VEGFR-2, showed substantial changes in angiogenesis and vasculogenesis, and ultimately progressing to a fatal state. The ability of gene knockout studies has potential in examining and demonstrating signaling pathways in the development and differentiation during specific signaling transduction pathways during VEGF expression of both endothelial and vascular cells.

Expression of VEGF has the potential to be up regulated in many circumstances. Hypoxia has been proven to be a strong inducer for increases of VEGF expression as demonstrated in tumors and necrosis conditions. The potential to target these regulations and control their influences, could also prove to dampen pathological conditions. Not only will this prove to be beneficial, but also other regulations of VEGF that increase expression can be targeted to stabilize excessive growth as additional cytokines, cell transformations, and oncogenes elevate VEGF expression as well.

Futhermore, the use of developed inhibitors, recombinant humanized antivascular endothelial growth factor (rhuMAbVEGF) and mFlt(1-3)-IgG in preventing the vasculogenic and angiogenic mechanisms, researchers have identified VEGF as a critical role in the developmental process. These results solidified the aspect of VEFG expression and regulation is necessary for embryonic, early, and postnatal growth. Important in development, recombinant inhibitors have potential therapeutic values in targeting cancer and atherosclerosis plaque progressions.

The implications during pathological angiogenesis, and the excessive neovacularization, have shown to be essentially linked to VEGF. Thus, future directions of therapeutic interest have focused on the inhibition of such roles and production of VEGF inhibitors to target pathological conditions are forth going. Currently, clinical studies and trials are being conducted addressing such pathological conditions through the used of VEGF inhibitors targeting a range of lung, renal, and colorectal carcinomas. In order to fully understand the consequences on inhibiting VEGF in humans, additional studies over time need to be conducted to fully elucidate all pathways and potentials the role VEGF inflicts in vascular pathological conditions.

In contrast to inhibiting VEGF for therapeutic implications, the induction of VEGF can also prove to be beneficial in specific conditions. In ischemic induced animals models, the regulation of VEGF expression has shown to be positively regulated in these circumstances. Thus, the ability to promote angiogenesis during the elevation of VEGF in ischemic conditions could prove favorable in the development of collateral vessel growth. This possibility could have clinical and experiment research in inducting recombinant VEGF to promote angiogenesis.

Although it has been accepted that VEGF is a major mediator of angiogenesis in physiological and pathological conditions, there is still much to be concluded in future studies about the possible roles in the influence of later stages of development. Consequently, pathological roles and regulation of VEGF expression are still conducted to target the onset of carcinomas while also finding beneficial outcomes of its induction. This progress has shown potential and hopes of fully elucidating the mechanism, progress, and signaling of vascular endothelial growth factor that will prove valuable in hopes of improving development and therapeutic implications.



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