VEGF (Vascular endothelial growth factors )

Presentedby –
BhawnaKushawah
Ph.DBiotech,IISem
B-1531/15
DUVASU
mathura

Vascular Endothelial Growth Factor
Originally described as endothelial cell-specific mitogen (Abraham and
Schilling, 1989);
Now as VEGF and also known as vascular permeability factor (VPF).
VEGF is a sub-family of growth factors, to be specific,
the platelet-derived growth factor family of cystine-knot growth factors.
Native VEGF is a basic, heparin-binding, homodimeric glycoprotein of
45 kDa (Ferrara, 1992).
VEGF .... ????????

Tumor cells , macrophages, platelets, keratinocytes, and renal
mesangial cells etc .
Important signalling protein .
Mainly involved in angiogenesis and vasculogenesis .
VEGF plays a role in normal physiological functions such as
bone formation, hematopoiesis, wound healing, and
development. (Tischer and Vaisman 1990).

VEGF - First identified in guinea pigs, hamsters, and mice
(Senger et al. in 1983)
Purified and cloned (Ferrara and Henzel , 1989)
Tischer et al. (1991) – discovered alternative splicing of
VEGF
Crystal structure of VEGF- first at 2.5 Å resolution and later
at 1.9 Å Discovered by Christinger and De Vos (1996-1997)
Historical perspectives

Classification of VEGF
Crystal structure of Vammin, a VEGF-F
from a snake venom
Mammals – Five classes
VEGF-A
VEGF-B
VEGF-C
VEGF-D
PGF
Viruses - VEGF-E
Snake venom - VEGF-F

Consist of 121, 165, 189 and 206 peptides
in humans.
Up regulate -nitric oxide production.
Main isoforms- 121 &165.
Found in chromosome 6 in human and 11
in rats.
VEGF – A (Senger et al., 1983)
Oloffson et al., 2000

Angiogenesis
↑ Migration of endothelial cells (Bates and Harper, 2003)
↑ Mitosis of endothelial cells
↑ Activity of Matrix metalloproteinases (MMPs)
(Garrido-Urbani et al. 2008).
↑ αvβ3 integrin activity (Sato 2001).

 Age-related macular degeneration (AMD)
 Proliferative diabetic retinopathy
 Diabetic macular edema
 Rubeosis iridis associated with retinoblastoma
 Central and branch retinal vein occlusion
 Von Hippel-Lindau syndrome
 Ocular melanomas and retinoblastomas
VEGF-A
Levels increased in many ocular neo-vascular diseases
Pe'er et al. Ophthalmology 1997; 104: 1251-1258; Pe'er et al. Ophthalmology
1998; 105: 412-416; Harris. Oncologist 2000; 5 Suppl 1: 32-36; Stitt et al. J
Pathol 1998; 186: 306-312

Consists of 188 amino acids.
Present on chromosome 6 in humans (Olofsson, 1996).
Regulates Embryonic angiogenesis
(Creates new blood vessels during embryonic development)
Promotes fatty acid uptake in endothelial cells, which is critical in organs
with high metabolic stress such as the heart (Hagberg et al. 2010).
Contributes to formation of muscular tissue in the heart;
Protects neurons in the cerebral cortex and retina during a stroke.
VEGF-B

Isoforms of VEGF –B : VEGF 186 & VEGF 167.
Highest levels in brain, heart, kidney and testes in adult mice,
Lower levels in liver, lungs and spleen (Lagercrantz et al.,
1997).
VEGF B-deficient animals survive, but display reduced heart
size and impaired recovery after cardiac ischemia (Bellomo et
al. 2000; Aase et al. 2001).
Functional mechanism ???? (Tammela et al., 2005).

Encoded by the VEGFC gene in humans and located on
chromosome 4q34 (Paavonen et al.1995).
Regulates Lymphangiogenesis- Creates new lymph vessels and plays a role in
vascular endothelial cell growth, production, and transition (Alitalo et al., 2002).
Important for neural development, and blood pressure regulation.
Closest structural and functional relative of VEGF-D.
VEGFC/VEGFR3 are critical regulators of lymph endothelial function. Loss-of-
function VEGFR3 mutants in humans cause lymphedema (Maes et al., 2002)
VEGF-C

The VEGF-D gene contains seven exons and is found on the X chromosome
(Gera Neufeld et al., 1999).
Function - Development of lymphatic vasculature surrounding lung
bronchioles.
Its expression correlates with lymph node metastasis in colorectal , lung and
ovarian carcinomas .
VEGF-D

VEGF-D serum levels are significantly elevated in patients
of angiosarcoma (Katsuoka , 2004).
VEGF-D signalling represents a potential target for anti-
cancer and antimetastasis therapy.
►Adult colon, heart, lungs, skeletal muscle, and small intestine
contain high levels of VEGF-D .
►Ovary, pancreas, prostate, spleen, and testes contain low levels
(Bhaskari et al ., 2014).

The Orf virus, a parapox virus infecting sheep, goats, and sometimes
humans, is known to induce angiogenesis at sites of infection on the skin
(Lyttle et al., 1994).
Degree of identity -
25% to human VEGF-A ,
and 19% to human PDGF.
VEGF-E tightly binds to and activates VEGFR-2, but not other VEGFRs
(VEGFR-1, VEGFR-3) nor PDGFR2.
VEGF -E

VEGF-F is a weak angiogenic protein present in snake venom
(T. flavoviridis, habu) (Takahashi et al.1994).
This protein, named T. Flavoviridis svVEGF, binds tightly to VEGFR-1 and weakly
to VEGFR-2 .
Biological function is not for increasing the vascular permeability within
the snake body itself but rather promotes vascular permeability in local
tissues of targeted animals.
Vascular permeability activity is linked to circulation of real toxin into the
target animals and promotion of their efficacy.
Mechanism .....??????

Receptors & Mechanism of action of VEGF
VEGF is a diametric glycoprotein and acts via tyrosine kinase receptors 1
,2 3, and NRP 1 & 2 receptors; predominantly located on endothelial cells
(Devries et al., 1992)
All members of VEGF family stimulate cellular responses by binding to
tyrosine kinase receptors on cell surface, causing them to dimerize and
become activated through transphosphorylation.
Neuropilin-1 and Neuropilin-2 co-receptors, which bind selectively
VEGF165 that encode VEGF specific tyrosine-kinase receptors.

VEGF = ONLY mitogenic for
endothelial cells
FGF = mitogenic for many cell types.
PDGF=platelets derived growth
factors.
Angiopoietin GFs=Angi1 and Angi2,
MMPs
Class-3 semaphorins
Physiological significance of VEGFs
Angiogenesis

Blood vessel formation –various ways

Ovarian folliculogenesis
Follicular growth, ovulation, corpus luteum development, and ovarian
steroidogenesis.
VEGF expression is enhanced at the mid-proliferative and mid-secretory
stages when E2 and P4 levels are high, respectively (Sugino et al., 2003).
Follicular development and E2 biosynthesis are suspended by short-term
administration of anti-VEGF antibody at the late follicular stage in monkeys
(Zimmermann et al., 2005).

Human endometrium strongly expresses VEGF during
menstruation, when both E2 and P4 levels are low.
VEGF expression can also be up-regulated by hypoxia, which
occurs as a result of tissue ischemia during menstrual shedding
(Sharkey et al., 2000).
Ovarian VEGF expression is gonadotropin-dependent (Sugino
et al., 2000).
Follicular VEGF expression is increased after the
administration of hCG in primates (Hazzard et al., 1999).

VEGF stimulates expression of proteases such as PA and collagenase in
endothelial cells (Olofsson et al., 1998).
VEGF and flt-1 have been localized in GCs especially in pre-ovulatory
follicles (Otani, 2000), but whether the increased PA expression in GCs is
mediated by VEGF or not is an open question????
VEGF/VPF may increase vascular permeability that contributes to the
physiologic process of antrum formation. Mechanism of development of
the antrum is not fully understood, further research is needed to address
the question of whether the VEGF/VEGFRs are involved in the regulation of
follicular antrum formation.

Loss of ability of cell to synthesize VEGF results in angiogenic
diseases (Pierce and Stone, 1996)
VEGF-A implicated with poor prognosis in breast cancer
Although VEGF-A has been correlated with poor survival, its exact
mechanism of action in progression of tumors remains unclear.
Over expression of VEGF A -an early step in metastasis, a step that
is involved in "angiogenic" switch.
Role of VEGF in Pathophysiology

Angiogenesis is as essential for the growing tumor as it is for a developing
organ such as the corpus luteum, since delivery of blood-borne nutrients to
tumor cells is essential for their survival (Folkman, 1971).
VEGF over expression in tumors is associated with increased angiogenesis,
proliferation and metastasis.
VEGF secreted from neoplastic cells influences nearby endothelial cells
(Ferrara and Dvorak, 1997).
VEGF in Tumor angiogenesis

VEGF-related tumour angiogenesis can be induced by production of VEGF-
like viruses proteins.
AIDS virus protein, HIV-1/Tat, can bind and activate VEGFR-2 (Albini et al.,
1996).
This probably represents a mechanism by which AIDS virus enhances the
angiogenic stimuli provided by Kaposi sarcoma .
Herpes virus-encoded proteins, contribute to development of Kaposi
sarcoma in AIDS patients (Ganem et al., 1997).
Tumor growth requires formation of new blood vessels, and agents inhibiting
angiogenesis might be an effective antitumor strategy .

In retinopathy, premature babies are placed in hyper-oxygenated incubators because their
lungs are not yet fully developed.
In premature babies, there is decreased production of VEGF during development of
retina , thus newly formed blood vessels regress and halt the orderly process of retinal
angiogenesis during retina development.
When the babies are moved out of the incubators, all the cells in the retina suddenly
experience extreme oxygen and produce simultaneously large amounts of VEGF.
Overproduction of VEGF results in rampant angiogenesis, which is not as finely
choreographed as the angiogenesis that takes place during normal retinal development.
Deregulated growth of blood vessels causes blindness in babies that have been subjected to
such treatment .
Angiogenic diseases of retina

Isoforms - VEGFxxx and VEGFxxxb .
Role of VEGF-A is important in DR.
Mechanism(s) of DR still largely obscure (Kusari J,2010).
Diabetic Retinopathy (DR)

Polycystic ovary syndrome (PCOS)
Hyperplasia and hyper-vascularity of ovarian theca interna and stroma are prominent
features of PCOS, a leading cause of infertility.
Endocrine gland VEGF (EG-VEGF), also known as prokineticin-1, is a promising
angiogenic factor that is expressed at earlier stages of follicular development.
25-38 % or approximately two fold elevation in serum levels of VEGF in PCOS
patients compared to controls (Agrawal et al., 1998)
There may be Positive and strong correlation between EG-VEGF and hyperplasia
and angiogenesis in PCOS.
EG-VEGF might be of even greater pathophysiological importance in acyclical
angiogenesis occurring during chronic an ovulation (Napoleone et al., 2003)

OHSS - a life threatening complication occurring most commonly in
stimulated ovarian cycles after treatment with gonadotropins.
Characterized by enlargement of ovary and increased vascular permeability
leading to extra- vasation of protein-rich fluid.
Evidence that hCG up-regulates VEGF expression of ovarian GCs in patients
with OHSS but not in those without (Wang et al., 2002).
Ovarian hyper-stimulation syndrome (OHSS)

VEGF A induces expression of anti-apoptotic proteins BCL-2 and A1
(Gerber et al., 1998).
VEGF-A induces activation of RAS/RAF/ERK/MAPK pathway through
VEGFR-2 (Meadows and Zachary, 2001). But exact mechanism of VEGFR2-
induced RAS activation is not clear.
Over-expression of VEGF - contributes to disease.
Solid cancers cannot grow beyond a limited size without adequate blood
supply; Cancers expressing VEGF are able to grow and metastasize (Hattori
et al.2000).
Current research

High VEGF secretion is necessary for growth of leukemia cells,
VEGF inhibition causes apoptosis (Broggini et al., 2003)
Serum VEGF concentrations remain elevated up to 20 weeks of pregnancy
and that concentrations are positively correlated with placental volume at mid-
pregnancy and with placental and fetal weight at delivery. (Williams et al,.
2005)
VEGF-A, development of the kidney, in maintenance of the glomerular
capillary structure and filtration barrier, and in the renal repair process after
injury. (Eremina V,2005)

Knockout of VEGF is lethal
Ferrara & Alitalo, NatureMed. 2000
Loss of a single VEGF allele in
mice leads to vascular
deformities and embryonic
death between Days 11 and 12
(Ferrara & Alitalo , 2000).
Formation of blood vessels was
abnormal, but not abolished in
VEGF-deficient (VEGF+/−)
embryos .

The first, a monoclonal antibody named bevacizumab, was approved in
2004. Approximately 10-15% patients benefit from bevacizumab therapy, an
anti-VEGF drug; however, biomarkers for its efficacy are not yet known.
VEGFR antagonists for example (lenvatinib, motesanib) are under
investigation for treating various cancers.
Pazopanib - renal cell carcinoma in 2009.
Regorafenib -colorectal cancer in Sept 2012.
Inhibition of angiogenesis - further growth is not possible because of
inadequate supply of blood , oxygen and other nutrients.
Anti-Angiogenic Therapy

Inflammatory molecules (TNF-α, IL-1β, IL-6), macrophage-migration inhibitory
factor, IL-17, IL-18, prostaglandins, nitric oxide, and transforming growth factor
(TGF)-β promote VEGF production by synovial fibroblasts (Kim et al.2008).
VEGFR2-induced migration has also been shown to involve signalling via
heterotrimeric G-proteins and activation of PLCb3 (Bhattacharya et al., 2009).
Many VEGF SNPs have been reported showing association with DR, such as
rs833061 (Awata, 2002)
rs3025039 (Uthra , 2008)
rs699947 (Chun ,2010),

NRP1 has been shown to be essential in VEGFA-induced 3D biology, such
as vessel sprouting and branching (Kawamura et al., 2008).
VEGF C-634G, C-2578A,C+936T polymorphisms are associated with
increased lung cancer risk in Asian population (Liang et al., 2012).
VEGF-A is also released in rheumatoid arthritis in response to TNF-α,
increases endothelial permeability and swelling and also stimulates
angiogenesis (Hae-Rim Kim et al., 2015).

There are five members of the human VEGF family of growth factors, and
several of these possess multiple isoforms.
VEGF is a central regulator of angiogenesis and vasculogenesis.
Diseases like cancer, glomerular endothelial integrity and blood pressure
homeostasis in adults are characterized by abnormal angiogenesis.
Modulation of VEGF function may contribute to successful therapeutic treatment
of several diseases.
VEGF signal transduction is not sufficiently understood. Therefore, VEGF signal
transduction study seems an area of intensive investigation in near future.
Research of VEGF will enable us to better understand the physiological and
pathophysiological role and target it as new drug target.
Conclusion

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Acad. Sci. USA 86, 7311–7315.
Agrawal R, Sladkevicius P, Engmann L, Conway GS, Payne NN, Bekis J, Tan SL, Campbell S,
Jacobs HS: Serum vascular endothelialgrowth factor concentrations and ovarian stromal blood
flow are increased in women with polycystic ovaries. Hum Reprod 1998, 13:651–655
Alitalo, K. Korpelainen, E. I., (1998) and Signaling angiogenesis and lymphangiogenesis. Curr.
Opin. Cell Biol. 10, 159–164
Albini, A., Soldi, R., Giunciuglio, D., Giraudo, E., Benelli, R.,Primo, L., Noonan, D., Salio, M.,
Camussi, G., Rockl, W., andBussolino, F. (1996) The angiogenesis induced by HIV-1 Tat protein
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Broggini M, Marchini SV, Galliera E, Borsotti P, Taraboletti G, Erba E, Sironi M, Jimeno J,
Faircloth GT, Giavazzi R and D’Incalci M. Aplidine, a new anticancer agent of marine origin,
inhibits vascular endothelial growth factor (VEGF) secretion and blocks VEGF-VEGFR-1 (flt-1)
autocrine loop in human leukemia cells MOLT-4. Leukemia 2003; 17: 52-59.
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