1. ANGIOGENESIS
DR. RAJKUMAR , R M.D.
III YR POST GRADUATE
DEPT. OF MEDICAL ONCOLOGY
2. Historical Highlights of the
Anti-Angiogenesis Field
• 1787 - British surgeon Dr. John Hunter first uses the term
'angiogenesis' (new blood vessel growth) to describe
blood vessels growing in the reindeer antler
• 1971 - Surgeon Dr. Judah Folkman hypothesizes that tumor growth is
dependent upon angiogenesis. His theory, published in
the New England Journal of Medicine, and is initially
regarded as heresy by leading physician and scientists.
• 1975 - The first angiogenesis inhibitor is discovered in cartilage by Dr.
Henry Brem and Dr. Judah Folkman.
• 1984 - The first angiogenic factor (basic fibroblast growth factor,
bFGF) is purified by Yuen Shing and Michael Klagsbrun at
Harvard Medical School.
• 1989 - One of the most important angiogenic factors, vascular
endothelial growth factor (VEGF), is discovered by Dr.
Napoleone Ferrara and by Dr. Jean Plouet. It turns out
to be identical to a molecule called Vascular
Permeability Factor (VPF) discovered in 1983 by Dr.
Harold Dvorak.
2
3. Historical Highlights of the
Anti-Angiogenesis Field
• 1997 - Dr. Michael O'Reilly publishes research finding in the journal
Nature showing complete regression of cancerous tumors
following repeated cycles of anti-angiogenic therapy
using angiostatin and endostatin
• 1999 - Massive wave of anti-angiogenic drugs in clinical trials: 46 anti-
angiogenic drugs for cancer patients; 5 drugs for macular
degeneration; 1 drug for diabetic retinopathy; 4 drugs
for psoriasis.
• 1999 - Dr. Richard Klausner, Director of the U.S. National Cancer
Institute designates the development of anti-angiogenic
therapies for cancer as a national priority.
• 2003 - The monoclonal antibody drug Avastin (Bevacizumab) becomes
the first anti-angiogenic drug shown in large-scale
clinical trials inhibiting tumor blood vessel growth can
prolong survival in cancer patients.
3
4. JUDAH FOLKMAN:
Father of Angiogenesis
First person to observe
angiogenesis as
having pathological
Implications in cancer in 1971
Born in Cleveland in 1933
Began his surgical residency
at the Massachusetts General
Hospital and served as chief
resident in surgery from 1964-1965
5. Folkman Facts
While serving as a lieutenant in the U.S. Navy from
1960-1962, Folkman and a colleague first reported the
use of silicone rubber implantable polymers for the
sustained release of drugs
Formed basis of development of Norplant
6. INTRODUCTION
Angiogenesis : A fundamental biological
process
Regulated by a fine balance
Deranged in various diseases
Historically, implicated in few diseases
In recent years, it has been increasingly
evident that excessive, insufficient or
abnormal angiogenesis contributes to the
pathogenesis of many more disorders. 6
7. DEFINITION
The formation of new blood vessels out of pre-existing
capillaries.
INVOLVES : Sprouting
Splitting
Remodeling of the existing vessels
WHY IT IS IMPORTANT?
Supply of oxygen and nutrients
7
Removal of waste products
8. VASCULOGENESIS : the generation
of
blood vessels from hemangioblasts
(endothelial cell precursors).
ANGIOGENESIS VASCULOGENESIS
New blood vessels mainly New endothelial cells
emerge from pre-existing differentiate from stem cells.
ones. Seen during embryonic
Can be seen in adult life development( for primary
also. vasculature).
Physiologic stimuli during Vasculogenesis is absent even
wound healing and the in presence of physiologic
reproductive cycle in stimuli. 8
women lead to
angiogenesis.
9. Definitions
Vasculogenesis Formation of new vessels from EC
precursors (angioblasts)
Angiogenesis Formation of new vessels from pre-
existing BV by sprouting
Arteriogenesis Subsequent stabilisation and
maturation
Collateralisation Enlarging existing vessels as bridges
between networks
(Myogenesis)
10. What Is Tumor Angiogenesis?
Small localized tumor Tumor that can grow and spread
Angiogenesis
Blood vessel
Signaling
molecule
16. Without Angiogenesis,
Tumor Growth Stops
Infuse nutrient solution
Isolated organ
(e.g., thyroid gland)
Injected cancer cells
stop growing as
mass reaches
1–2 mm in diameter
17. With Angiogenesis,
Tumor Growth Proceeds
Tumor
suspended
in anterior
chamber Tumor
growing
on the iris
Tumor
Cornea growing
Tumor size
on the iris
Tumor
suspended
in anterior
Iris chamber
Lens
2 4 6 8 10
Days
18. What Prompts Angiogenesis?
Chamber
Cancer cell Signaling molecule
Place chamber beneath an animal's skin
Angiogenesis
21. Vascular Endothelial Growth
Factor
• Glycoproteins consisting of A-, B-, C-, D-, E- forms and
Placenta Growth Factor (PLGF)
• Within the six subtypes multiple isoforms exist
• Loss of even a single VEGF-A allele results in embryonic
lethality due to cardiac complications
26. Platelet-derived growth factor
• The platelet-derived growth factor (PDGF)
regulates
the recruitment of pericytes and
smooth muscle cells
required for further stabilization of the new capillaries
26
27. Fibroblast growth factor
• Fibroblast growth factor (FGF) family are also
potent inducers of angiogenesis. The effects of
FGFs are mediated via high-affinity tyrosine
kinase receptors.
• Cellular responses mediated by FGFs include
cell migration
proliferation
differentiation
27
28. The Angiogenesis Signaling Cascade
Cancer cell
VEGF (or bFGF)
Receptor protein Endothelial
Relay cell surface
proteins
Genes are
activated in
cell nucleus
Proteins stimulate
new endothelial
cell growth
29. Endothelial Cell Activation
Secretes
MMPs that Activated
digest endothelial
surrounding cell
matrix
Matrix
Cell migrates
and divides
31. Angiogenesis Inhibitors
• Other angiogenesis inhibitors have been found in
nature - in green tea, soy products, fungi,
mushrooms, Chinese cabbage, tree bark, shark
tissues, snake venom, red wine, and many other
substances.
• Still other angiogenesis inhibitors have been
manufactured synthetically in the laboratory.
• Some FDA-approved medicines have also been "re-
discovered" to have anti-angiogenic properties.
31
32. ENDOSTATIN
• It was first discovered in
1995 in Dr. Folkman’s
lab
• Phase I clinical studies
began at M.D. Anderson
November 1999
• A naturally-occurring 20-
kDa C-terminal fragment
derived from type XVIII
collagen.
• Interfere with the pro-
angiogenic action of
growth factors such as
basic fibroblast growth
factor (bFGF/FGF-2) and
vascular endothelial
growth factor (VEGF)
32
33. ANGIOSTATIN
• Naturally occurring protein
found in several animal
species, including humans.
• It is an endogenous
angiogenesis inhibitor
• Angiostatin is produced by
autoproteolytic cleavage
of plasminogen,
• Can be cleaved from
plasminogen by different
metalloproteinases
(MMPs), elastase, prostata-
specific antigen (PSA), 13
KD serine protease, or
24KD endopeptidase.
33
34. ANGIOSTATIN
• It is a 57 kDa fragment of a
larger protein, Plasmin
(itself a fragment of
plasminogen)
• Encloses three to five
contiguous Kringle
modules.
• Each Kringle module
contains two small beta
sheets and three disulfide
bonds.
• Considerable uncertainty
on its mechanism of
action, but it seems to
involve the inhibition of
endothelial cell migration,
proliferation and induction
of apoptosis.
34
35. Angiogenesis Inhibitors and
Primary Tumors
Tumor
size
in
mice
0 40 80 120 160 200 240
Days
Start Start
Stop Stop
Endostatin Treatment
36. Angiogenesis Inhibitors and Metastasis
Inject
cancer cells
Let initial tumor
grow for
several weeks
Remove
initial tumor
Allow time for
metastases to appear
Angiostatin No
injections treatment
Few metastases Many metastases
37. Angiogenesis and Tumor Dormancy
Angiostatin inhibits
Large primary tumor
Tiny dormant tumor masses
38. Cancer in Angiogenesis-Deficient Mice
Normal mouse Angiogenesis-deficient
mutant mouse
Inject breast
cancer cells
Cancer No cancer
39. Angiogenesis Inhibitors in the
Treatment of Human Cancer
Cancer
cell
VEGF (or bFGF)
Receptor
protein
Endothelial Angiogenesis
cell Inhibitors
MMPs
Matrix
40. Drugs That Inhibit Angiogenesis Directly
Cancer
cell
Endostatin
VEGF EMD121974
(or bFGF) TNP-470
Squalamine
Receptor
protein Apoptosis
Endothelial
cell
Combretastatin A4
MMPs Matrix
Integrin Drug
molecule
Integrin interacts with drugs to destroy
proliferating endothelial cells
41. Old Drug With a New Use
Cancer
cell
VEGF (or bFGF)
Receptor
protein
Endothelial
cell Thalidomide
MMPs
Matrix
42. Drugs That Block the
Angiogenesis Signaling Cascade
Cancer
cell Interferon-alpha
VEGF (or bFGF)
Receptor
protein
Anti-VEGF
antibody
SU5416
SU6668
Endothelial PTK787/ZK 22584
cell
MMPs No
endothelial
cell growth
Matrix
43. Drugs That Block
Extracellular Matrix Breakdown
Cancer
cell
VEGF (or bFGF)
Receptor
protein Marimistat
AG3340
COL-3
Neovastat
Endothelial BMS-275291
cell
No
MMPs endothelial
cell migration
Matrix
44. Potential Mechanism of Efficacy
Folkman Hypothesis – Glioblastomas are
angiogenesis- dependent – Growth advantage
Jain Hypothesis – Normalization of vessels →
Reduction of hypoxia, interstitial pressure, and
increased drug delivery
Stem Cell Hypothesis – Glioma stem cells
promote angiogenesis via VEGF – Vascular niche
protects stem cells (Bao et al., Cancer Res, 2006; 66:7843-8)
45. NORMAL BODY BLOOD
VESSEL FORMATION
• Stages
A: Vasculogenesis
B: Angiogenic remodeling
C: Stabilization and maturation
D: Destabilization
E: Regression
F: Sprouting
46. STAGE A: VASCULOGENESIS
• Undifferentiated
vascular bedding
during embryonic
development
• Vascular
Endothelial Growth
Factor (VEGF) triggers this process
47. STAGE B: ANGIOGENESIS
• Pruning of primitive tubular network to
form blood vessels
• Vascular Endothelial Growth Factor
(VEGF) is required
48. STAGE C: STABILIZATION
AND MATURATION
Endothelial cells integrate
tightly with supporting cells
such as smooth muscle cells
and pericytes
Cell walls mature
49. STAGE D: DESTABILIZATION
• Angiogenic sprouting into
previously avascular tissue occurs
• Distinct angiogenesis
from previous type
• Only possible if pre-existing
vessels are first destabilized
50. TUMOR ANGIOGENIC
DEPENDENCY
• Tumor- undesired growth of cells
• Once a tumor grows beyond 100-200 μM
in size, the development of new
vasculature becomes essential to maintain
adequate tumor oxygenation and
sustained tumor growth
51. Structure of vessels and capillaries
Small artery: Monocellular layer of endothelial cells
Capillary: endothelial cell,
basal lamina, pericytes
52. Angiogenesis:
Sprouting of cells from mature endothelial cells of the vessel wall
(secretion of proteases, resolution of
Basal lamina, migration towards
Chemotactic gradient, proliferation,
Tube formation)
VEGF is factor largely specific for
endothelial cells,
bFGF can also induce,
not specific for EC)
Mouse cornea:
wounding induces
angiogenesis,
chemotactic
response to
angiogenic factors
54. Hypoxia - HIF - VEGF
every cell must be within 50 to 100 m of a capillary
HIF: hypoxia inducible factor
VEGF: vascular endothelial growth factor
55. Von Hippel-Lindau Tumor Suppressor, HIF and VEGF
VEGF-gene:
Regulated by HIF,
HIF is continously produced,
ubiquitinylated,
degraded in proteasome,
therefore low concentration;
Ubiquitinylation dependent on
Hippel-Lindau tumor
suppressor
(part of an E3 ubiquitin-ligase
complex)
HIF1 is modified by a
prolyl hydroxylase,
then better interaction with
vHL protein, high turnover;
Hydroxylase is regulated by O2
56.
57.
58. ROLE OF VEGF
• VEGF production is under control of :
hypoxia inducible factor (HIF)
• VEGF receptor expression is up-regulated under :
hypoxic or ischemic conditions.
So, early involvement of VEGF in this process.
• VEGF is a major player in angiogenesis initiation
because: i) it induces vasodilatation
via endothelial NO production
ii)it increases endothelial cell 58
permeability
59. So it cause:
1. vasodilatation
2. increased vascular permeability
3. can induce the expression of proteases and
receptors important in cellular invasion and
tissue remodeling
4. prevent endothelial cell apoptosis
But angiogenesis is not completely dependent
on VEGF production. Recently shown by :
Hansen-Algenstaedt et al.
59
60. VASCULOGENESIS
Formation of vessels by
differentiation of cells from
angioblasts in the yolk sac
of the embryo:
Is differentiation and proliferation
of endothelial cells
in a non-vascularized tissue
Leads to formation of a primitive
tubular network
Has to undergo angiogenic
remodeling to stable vascular
system
62. TUMOR ANGIOGENESIS
Three major steps
(A) Initiation of the angiogenic response,
(B) Endothelial cell(EC) migration,
proliferation and tube formation,
(C) Finally the maturation of the 62
neovasculature.
63. Proteases
matrix metalloproteases plasminogen activator(PA) /
(MMPs) plasmin system
PAs activate the plasminogen
degrade different into plasmin, which degrades
protein types several components of
extracellular matrix (ECM)
• Both PAs and MMPs are secreted together with their inhibitors: PAI
&TIMP
• It ensures a stringent control of local proteolytic activity.
63
64. TUMOR ANGIOGENESIS
So, the extracellular matrix is
degraded
An increased concentration of
various
growth factors
64
So, EC(„leader EC‟) migration
and
65. (b) Endothelial cell migration,
proliferation, and tube formation
• The „leader EC‟ starts migrating and
proliferating
• More EC starts to migrate through the
degraded matrix
• So, forms small sprouts.
• After the initial period of migration, rapid EC
proliferation begins, thus increasing the rate of
sprout elongation.
65
• These processes are also mediated by cell
adhesion molecules(CAM).
66. cell adhesion molecules(CAM)
• Integrin, cadherin, vascular cell adhesion molecule-
1, P-selectin and E-selectin are implicated in
angiogenesis.
• Integrin αvβ3 plays a critical role in angiogenesis.
• It is expressed at high levels in : tumor vasculature
and wound-healing tissues , but at extremely low
levels in normal blood vessels.
66
67. (C) Maturation of the
neovasculature
• THE FINAL PHASE
• Establishment of polarity of the endothelial cells :
by CAM
Finally, when sufficient neovascularization has
occurred, the angiogenic factors are down
regulated
or
the local concentration of the inhibitors increases.
“A FINELY BALANCED EQUILIBRIUM”
As a result, the endothelial cells become
quiescent.
67
68. Cellular mechanisms of tumour angiogenesis
(1) host vascular network
1 expands by budding of
endothelial sprouts or
3
formation of bridges
(angiogenesis);
(2) tumour vessels remodel
2 2 1
and expand by the insertion
of interstitial tissue columns
into the lumen of pre-
existing vessels
(intussusception); and
3
(3) endothelial cell precursors
(angioblasts) home from the
bone marrow or peripheral
blood into tumours and
contribute to the endothelial
lining of tumour vessels
(vasculogenesis)
4 Lymphatic vessels around
(4)
tumours drain the interstitial
4
fluid and provide a gateway
for metastasizing tumour
cells.
70. Steps in network formation and maturation
during tumour angiogenesis
71. Key differences in tumour vasculature
Different flow
characteristics or
blood volume
Microvasculature
permeability
Increased fractional
volume of
extravascular,
extracellular space
77. CHALLENGES OF ANGIOGENIC
THERAPY
• VEGF forms leaky and tortuous
vessels
• Adverse Effects of increased levels of
angiogenic factors such as triggering
of dormant tumors and acceleration of
atherosclerosis.
77
78. development of angiogenesis
inhibitors
Usually follows any the following :
1. inhibition of tumor cell synthesis of
angiogenic proteins
2. the neutralization of angiogenic proteins
by antibodies or traps
3. inhibition of endothelial cell binding to
angiogenic proteins 78
4. direct induction of endothelial cell
80. ANTIANGIOGENIC THERAPY
• A large number of agents that target
angiogenesis are in clinical development.
They can be broadly classified as :
I. agents that have been developed
primarily for their antiangiogenic activity
II. those that have been developed or
used for other biologic effects but also
have anti-angiogenic activity e.g.
80
celecoxib, rosiglitazone, zolendronic acid,
interferon alpha, everolimus,vorinostat
81. HOW TO MAKE THESE AGENT
MORE ATTRACTIVE FOR USE
• One possible approach to improve the
therapeutic efficacy and selective toxicity
of anticancer drugs is by targeting
anticancer drugs through
I. monoclonal antibodies (MAbs) or
II. peptide ligands that bind to molecules
that are over expressed on the plasma
membrane of cancer cells or tumor-
associated endothelial cells.
81
82. DRUGS THAT BLOCK THE ANGIOGENESIS
SIGNALING CASCADE
Anti-VEGF antibodies that block the VEGF receptor from
binding growth factor. Bevacizumab, is the first of these anti-
VEGF antibodies.
Interferon-alpha, is a naturally occurring protein that inhibits the
production of bFGF and VEGF, preventing these growth factors
from starting the signaling cascade
82
83. DRUGS THAT INHIBIT ANGIOGENESIS
DIRECTLY
Endostatin, the naturally occurring protein known
to inhibit tumor growth in animals.
Combretastatin A4, causes growing endothelial
cells to commit suicide (apoptosis).
83
85. DRUGS WITH OTHER MECHANISMS OF
ACTION
Involves mechanisms that are either nonspecific or
are not clearly understood.
A drug called CAI, exerts its effects by inhibiting the
influx of calcium ions into cells.
While this inhibition of calcium uptake suppresses
the growth of endothelial cells, such a general
mechanism may affect many other cellular
processes.
85
86. Current Angiogenic Inhibitors in
Clinical Use and Clinical Trials
Bevacizumab (Avastin™)
Sunitinib (Sutent™)
Sorafenib (Nexavar™)
Cederanib (Recentin™ - AZD- 2171)
Cilengitide
VEGF-Trap
Many others in development
87. “AVASTIN BEVACIZUMAB- REACH BEYOND
CONVENTION”
Recombinant, humanized monoclonal antibody that
binds to all isoforms of VEGF-A such that KDR
signaling is inhibited
Developed by Genentech BioOncology
Not a chemotherapy drug: “Targeted Therapy”
88. BEVACIZUMAB CONTINUED
FDA-approved for first and second-line treatment of colorectal
and rectum cancer in combination with oxaliplatin, leucovorin
and fluorouracil (FOLFOX4) in 2004
Approved for first-line treatment of Non-Small Cell Lung
Cancer in combination with Carboplatin and Paclitaxel
Previously investigated in combination with Fluorouracil in
phase II and III trials in a wide variety of tumors
Study results initially presented at the 2003 Annual Meeting of
the American Society of Clinical Oncology (ASCO)
89. EFFICACY
Adding Bevacizumab to chemotherapy results in
increased median Progression Free Survival by
33%
Median survival was 15.1 and 18.3 months in the
Leucovorin (IFL)/placebo, and 5-
FU/LV/Bevacizumab trial groups respectively
Overall Response Rate and duration of response
were also increased in the Bevacizumab-containing
group
90. DOSAGE
Colorectal and rectum cancer
AVASTIN in combination with intravenous 5-
FU-based chemotherapy
- 5 mg/kg or 10 mg/kg every 14 days
AVASTIN in combination with bolus-IFL
- 5 mg/kg
AVASTIN in combination with FOLFOX4
- 10 mg/kg
Non-Squamous, Non-Small Cell Lung Cancer
15 mg/kg, as an IV infusion every 3 weeks
91. BENEFITS
Non chemotherapeutic- biological agent that is less
invasive to the body than chemotherapeutic agents
Half life of 20 days- good drug retention
92. CONCERNS
Since Bevacizumab is expected to inhibit new
angiogenic growth, concerns have been raised
regarding postoperative wound-healing and
bleeding complications in patients who undergo
surgery within 1 to 2 months of Bevacizumab
therapy
93. BOXED WARNINGS AND ADDITIONAL
IMPORTANT SAFETY INFORMATION
Gastrointestinal (GI) perforation
Wound healing complication
Hemorrhage
Neutropenia
94. FUTURE DIRECTIONS-VEGF-TRAP
Composite decoy receptor based on VEGFR-1 and
VEGFR-2 fused to a human Fc segment of IgG1
that binds VEGF
Decreases free VEGF to bind to receptors and
prevent vessel growth
FDA approved for macular degeneration
95. Bevacizumab- Efficacy in Clinical Trials –
Metastatic Colorectal Cancer
From Ferrara N, Nat Rev Drug Discovery, 2004; Hurwitz et al, NEJM, 2004
97. Patient 2
before and after (2 mos apart)
Courtesy Dr. Sajeel Chowdhary, Moffitt Cancer Center
98. Response Rates
6-month PFS of 43% and median PFS of 24 weeks
compares favorably to historical controls (Wong et al., J. Clin.
Oncol., 1999) of 15% and 9 weeks, using 8 previous
chemotherapy regimens
Overall 1-year survival of 37% compares favorably
to historical control of 21% (Wong et al., 1999)
Temozolomide, in combination with other agents
(e.g., irinotecan, erlotinib, etoposide) produced modest
improvements in R.R. or O.S., but not as dramatic
as bevacizumab + irinotecan
100. CONCLUSION
The study of angiogenesis is making a profound
impact on the biological and medical world.
The hope of being able to build new, functional, and
durable blood vessels in ischemic tissues, or
conversely, to prevent their further growth in
malignant and inflamed tissues is becoming more
realistic every day.
100
101. However, efforts to therapeutically stimulate new
blood vessels have significantly lagged behind
those to inhibit angiogenesis.
Better understanding of the underlying process will
enable the scientist to develop new drugs and
therapies that will significantly enhance our ability to
treat intractable diseases, such as, cancer,
diabetes, and heart disease.
101
102. Modulation of angiogenesis may have
an impact on diseases in the
twenty-first century
similar to that which the discovery of
antibiotics
had in the twentieth century….
102
