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Vascular endothelial growth factor is a signalling protein involved in the Angiogenesis (formation of the new blood vessels from the pre existing blood vessels) and Vasculogenesis (new blood vessels formation where there are no pre existing blood vessels) Vascular endothelial growth factor receptors are mainly divided in to
VEGFR-3 is one of the VEGFR receptors essential for cardio vascular development during the embryogenesis. A strong protein expression of the vascular endothelial growth factor-3 was found on the tumour endothelium of the glioblastomas.VEGF-3 is also known as Flt-4. VEGFR-3 is found in the lymphatic endothelial cells in adults and in the human hereditary lymphedema, the mutant VEGFR-3 alleles have been implicated.(1).VEGFR-3 is one of the member of the tyrosine kinase family that binds to VEGF-C and VEGF-D, but does not binds to VEGF-A. According to the biology of the VEGF its family consists of five glycoproteins known as VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor [PIGF] (2). In this we discuss about.
The Biology/ Biochemical properties of VEGFR-3.
Role of VEGFR-3 in Normal cells.
Role of VEGFR-3 in Cancer cells.
Validation of VEGFR-3.
Expression of VEGFR-3 in Normal and cancer cells.
Anti Cancer therapies of VEGFR-3
Biological/Biochemical properties of VEGFR3.
Structure of VEGFR-3.
Structure of the VEGFR-3 receptor protein
The human gene VEGFR-3 consists of 31 exons in which 13th exon is largest and 362bp in length. VEGFR-3 proximal promoter contains stretches of the sequences homologus with the mouse VEGFR-3. VEGFR-3 proximal promoter is a TATA-less. The basis of VEGFR-3 specific endothelial lineage restricted expression can be under stand by the VEGFR-3 gene and its regulatory 5 flanking region. The molecular genetic analysis of the lymphatic system and its disorders can be known by the sequence, organisation and variation in the VEGFR-3 region. In human gene 31 exons are present in these 30a and 30b are alternatively spliced in to mRNA produces the polypeptide isoforms differing by the 65 amino acid residues. The kinase insert sequence interrupts the transmembrane region [TM] and tyrosine kinase domains [TK I and TK II]. VEGFR-3 exons encodes I-VII Ig-homology domains (1).
Exon-intron structure of the Human VEGFR-3 and the comparison of Human and mouse VEGFR-3 promoters.
Role of VEGFR-3 in normal cells:
Vascular endothelial growth factor is mostly found in the endothelium of lymphatic vessels such as the thoracic duct and also expressed in many organs including endocrine glands, kidney glomeruli , splenic and hepatic senusoids and bone marrow. VEGF-C and VEGF-D ligands of VEGFR-3 is mostly found in vascular smooth muscle cells. In addition to the vascular smooth muscle cells, intence cytoplasmic staining of VEGF-C is found in neuro endocrine glands such as the prolactin secreting cells of the anterior pituitary and alpha cells of islets of langerhans, while VEGF-D was found in the dispersed neuro endocrine cells and in the adrenal cortex. The ligands of VEGFR-3 has paracrine function.It has a role in the release of peptide from the secretory granules of the certain neuro endocrine cells to the surrounding capillaries(2).
Role of VEGFR-3 in cancer:
VEGFC a specific ligand activates VEGFR-3 and promotes the cancer progression. VEGFR-3 axis is not only expressed by the Lymphatic endothelial cells but also expressed by the number of human tumour cells. VEGFR-3 plays an important role in the lukaemic proliferation of the cell, survival and resistance to the chemotherapy. Cancer cell mobility and the invasion capabilities promoting the cancer cell metastasis in several types of solid tumours is enhanced by the activation of the VEGFC / VEGFR-3. Metastasis is facilitated by increase in lymphogenesis [formation of the lymphatic vessels] around the tumours by the activation of VEGFC / VEGFR-3 axis in lymphatic endothelial cells. VEGFC a specific ligand of VEGFR-3 characterised as a lymphangiogenic and angiogenic growth factors. VEGFR-3 plays a crucial role in determining the potential for a lymphangiogenic response. Recent studies have concluded that VEGFR-3 is expressed in different types of human malignancies and has been proposed as a marker for the lymphatic endothelial cells. VEGFR-3 axis has different types of biological effects on the cancer cells that cause tumour progression. The intra tumoral lymphangiogenesis and also the amount of the peri tumoral activity has been increased by the over expression of the VEGFR-C a specific ligand of VEGFR-3.VEGFR-C has a role in the growth of the malignant mesothelioma cells. Recent studies has also revealed that VEGFR-3 axis has increased the cancer cell motility and invasiveness and it also provides an evidence that it contributes to the promotion of the cancer cell metastasis in different types of cancers like lung adenocarcinoma, breast cancer, cervical prostate cancer and colorectal cancer. In invasion and metastasis the role of VEGFR-3 has shown high levels of expression, which has correlated closely with the clinical metastasis and patient survival that was revealed by examining the tumour tissues of different types of cancers (3).
Validation of VEGFR-3:
In the drug discovery validation of a target is an important step, as many drugs are the inhibitors which blocks the action of the target protein. Validation process is important in order to know the role of the target in the disease before the drugs are sought (4). The VEGFR-3 shows prognostic value in endometrial carcinoma and this can be predicted by the target validation. By this study we can know the presence of VEGF-D and VEGFR-3 in normal endometrial, CAH's and endometrial carcinoma. The materials required for this study are population and tissues. Here 71 patients with endometrial carcinoma from whom formalin-fixed, paraffin- embedded blocks which were induced in this study are available from these patients. By tracing cell the patients at different stages and by reevaluation for histological type and are graded by the gynaecological pathologists. The specimens were taken from 14 CAH's and 16 women with normal endometrium.
Here the goat polyclonal anti VEGF-D [ catalogue number AF 286] and anti VEGFR-3 [catalogue number AF 349] antibodies were used at a concentration 5 and 1µgm/ml now the tissue sections were passed through xylene and alcohols and are placed in the citrate buffer with PH 6.0 and then heated at 800W for 5 minutes for antigen retrival. The tissue were then treated with H20 2 in methanol for ten minutes to know the activity of peroxidase in the tissue and these tissues were incubated over night in the presence of primary antibody at 4oc. Now the slides were washed in PBS containing the 0.1% tween20 [PBS/ tween]. This is done before the application of the secondary antibody. Now the tissue was incubated with the secondary antibody for one hour before it has been washed for 15 minutes with 3 changes of PBS/ tween. Avidine-biotinylated-horse radish peroxidase complex is used during the incubation of the tissues for 30 minutes. And therefore the reactivity was visualised. This is done by using 0.02% of 3,3-diaminobenzidinetetrahydrochloride as a chromogene in tris-Hcl buffer at PH 7.6 containing 0.03% H202. Later the tissues were couter stained using Mayer's Hemalum. The negative control studies were done in the presence of normal goat serum for each antibody in which it is observed that there is no significant staining in the negative control sections. Formalin-fixed, paraffin-embedded sections which were taken from the human placenta were stained for VEGF-D and VEGFR-3 by the same above staining procedure. In both the tumour cells and adjacent stromal cells VEGF-D was stained. The staining was estimated by the percentage of the tumour cells in which the intensity of the staining is more or less than that of the positive control cells. The staining levels are of four groups absent [0% of cells] weak staining [10-15% of cells] moderate staining[50% of cells] and strong staining. Tumours are observed as positive for VEGF-D when they shows the moderate staining or strong staining (5). In tumour cells and adjacent endothelial cells was stained by VEGFR-3 and the staining on the tumour cells was done by estimating the percentage of tumour cells in which the intensity of staining is more or less than that of the positive control cells. The endothelial cells which are stained for VEGFR-3 shows positive when 5% are strongly immuno reactive as positive control cells. It is known that in the lymphatic and vascular endothelia neighbouring tumour nests VEGFR-3 expression was detected. Further stastical analysis was done to know the significance of the relation between the expression of VEGF-D and VEGFR-3 by X2 - test and Fishers exact probability test.
Immunohistochemical staining of endometrial carcinoma with anti-VEGF-D antibody and anti-VEGFR-3 antibody.
Endometrial carcinoma showing the positive staining for the VEGF-D on both the stromal and carcinoma cells.
Serial section of the A as negative control using the non immune goat serum in place of the VEGF-D antibody.
Endometrial carcinoma showing positive staining for VEGFR-3 on both carcinoma cells and endothelial cells. The arrows indicates the endothelial cells.
Endometrial carcinoma showing the positive staining for VEGFR-3 on the endothelial cells and the scale bars in A&B are in 12.5µm and the scale bars in C and D are 25µm.
The results shows that in normal endometrium and adjacent stromal cells immuno staining of VEGF-D and VEGFR-3 shows very weak staining as well as 90% of CAH's fails to stain for VEGF-D and VEGFR-3. How ever in carcinoma cells VEGFD is seen in the cytoplasm of the tumour cells in positive cases and shows moderate or strong staining at the stages 1/2 and 3/4 stage. It is observed that 24 carcinomas are positive for VEGF-D out of 71 carcinomas in stromal components adjacent to the carcinoma nests. Where as in the carcinoma cells and the endothelial cells adjacent to carcinoma nests VEGFR-3 was expressed. This VEGFR-3 staining was observed only in the endothelial cells in the stromal components. Therefore from the 71 carcinomas 33 of them showed positive result for the VEGFR-3in endothelial cells adjacent to carcinoma nests. From the above results it is predicted that myometrial invasion and lymph node metastasis are identified by the presence of VEGF-D and VEGFR-3 in endometrial carcinoma and these can be a promising targets for new therapeutic strategies in endometrial carcinoma (6).
Expression of VEGFR-3 in normal and cancer cells:
Rile Li and Mamoun Younes studied the expression of VEGFR-3 in prostate carcinoma. In these study tissues from 640 patients were taken and tissues arrays of the normal prostate, benign prostate hyperplasia [BPH] and prostate carcinoma [PCA] were built by using micro array technology. Now the tissues were stained with polyclonal antibody to VEGFR-3 using standard immune peroxidase method. Later the immuno reactivity was known by semi quantitation scoring system in order to know the intensity and percentage. From the above study the results showed that VEGFR-3 is expressed in normal prostate, benign prostate hyperglasia, and prostate carcinoma. But shows an up regulation in the prostate carcinoma. It is also found that VEGFR-3 correlated with lymph node metastasis. Therefore the expression of the high levels of VEGFR-3 increases the risk of the biochemical recurrence in prostate cancer patients as well as it plays a crucial role in facilitating the lymphatic spread of prostate carcinoma and tumour progression (7). VEGFR-3 is mostly expressed in the lymphatic endothelial cells in adults. VEGFR-3 and its ligands VEGF-C, VEGF-D expression has been detected in many human tumours like gastric carcinoma, colorectal carcinoma, lung and breast carcinoma, malignant melanoma (8).
Anti cancer therapies of VEGFR-3:
VEGFR-3 is expressed mostly in lymph node metastasis. The drug rapamycin shows significant decrease in the number and the area of the lymphatic vessels in the primary tumours which was demonstrated by the in vivo metastatic experiment. Therefore rapamycin has the anti lymph angiogenetic effect and causes the inhibition of the lymphatic metastasis (9). Phosphorylation of the VEGFR-3 is blocked by the Axitinib with average Ic 50s of 0.2 and 0.1 to 0.3 nM. BAY 73-4506 [regorafenib] also inhlbits the activity of the VEGFR-3. Bevacizumab is an VEGF blocking antibody these antibody reduces the lymph node metastasis and breast cancer which can be done by inhibiting the activity of VEGFR-3 (10).
The advantage of the targeted therapy is that the cancer cells shows their dependence for the growth and survival on the oncogene product. The cancer cells are more sensitive then the normal cells for the treatment in the discovery of the anticancer drugs there is an involvement in the micro RNAs in the initiation and progression of cancer can develop new targets for anticancer treatment.