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LYMPHATIC RESEARCH AND BIOLOGY
Volume 6, Number 3–4, 2008
© Mary Ann Liebert, Inc.
DOI: 10.1089/lrb.2008.1016
Judah Folkman’s Contribution to the
Inhibition of Angiogenesis
Diane R. Bielenberg
1,3
and Patricia A. D’Amore
1,2,3
203
Introduction
J
UDAH
F
OLKMAN
is the undisputed founder of the field of
angiogenesis research. His early observations, along with
Dr. Michael Gimbrone, then a Harvard Medical student, that
tumors separated from a vascular source could not grow
1
propelled the concept of anti-angiogenesis therapy. In addi-
tion to postulating the angiogenesis-dependence of solid tu-
mors, Dr. Folkman predicted the existence of angiogenesis
factors when he commented:
some diffusible message is released from tumor to
nearby endothelial cells’ these cells are then switched
from a previously resting, non-regenerative state to a
rapidly dividing group of regenerating cells capable of
forming new capillary sprout that can grow at a rate of
1 mm per day.
2
In his very early work, he and Dr. David Kessler (who
would later become the Commissioner of the Food and Drug
Administration) described the association of mast cells with
growing vessels.
3
Early efforts were aimed at isolating the
elusive “tumor angiogenesis factor,” leading to the isolation
and purification of the fibroblast growth factors, the first
known angiogenic factors, by members of the Folkman
group.
4
,
5
In addition to studies aimed at identifying angiogenic fac-
tors and inhibitors and investigations to unravel the cellular
and molecular basis of the process of angiogenesis, Dr. Folk-
man and his colleagues made major contributions to the de-
velopment of reagents and assays that helped to drive the
field. These include the isolation and culture of microvascu-
lar endothelial cells,
6
the development of the chick chorioal-
lantoic membrane assay,
7
corneal pocket assay,
8
and de-
scription of polymer systems for the slow release of
macromolecules,
9
all of which were central to the subsequent
discoveries and success in the field.
While other researchers, many of them associated with
Dr. Folkman, focused on elucidating the steps of angiogen-
esis, Dr. Folkman was largely occupied with isolating and
identifying molecules that might be used clinically to block
tumor growth. Very early in his career, he recognized the
complexity of the angiogenic process, particularly as it re-
lates to tumor vascularization (Fig. 1) and he applied this
knowledge to his search to understand the angiogenic pro-
cess so as to develop more rational inhibitors. As we de-
scribe below, these molecules ranged from re-inventing new
uses for drugs that had been described for other purposes
(e.g., interferon and thalidomide) to the isolation of mole-
cules from unexpected sources such as fungi to the discov-
ery of endogenous inhibitors of angiogenesis (e.g., angio-
statin and endostatin).
Interferon
The Folkman group published the first angiogenesis in-
hibitor describing the inhibition of endothelial cell motility
with interferon (see timeline, Fig. 2) in Science.
10
Dr. Bruce
Zetter describes how this discovery came about:
In 1980, I learned the phagokinetic motility assay from
Gunter Albrecht Buehler at Cold Spring Harbor. He
used it to show that cells moved in opposite directions
after dividing but it wasn’t a quantitative assay. I had
the idea to quantify it by measuring the size of the tracks
made by moving cells. To make this possible, Dr. Folk-
man bought the lab a primitive image analyzer made by
Bosch and Lomb that had 16 kb of memory and cost
$100,000—an enormous amount at the time. I was able
to quantify the cell motility and then showed that tu-
mor factors stimulated endothelial cell motility and sub-
mitted a paper to Nature on that finding.
Danielle Brouty-Boyé arrived as a visiting scientist
from France. She had worked with Ion Gresser whose
lab had some of the purest samples of interferon avail-
able at the time. Because interferon seemed able to in-
hibit many cellular processes, we wanted to investigate
whether it had any anti-endothelial cell properties and
applied it to the quantitative endothelial cell motility as-
say. The result was a striking inhibition of endothelial
cell motility, implying a potential anti-angiogenic activ-
ity.
Although, he ran the lab and provided much of the
funding for the experiments, Dr. Folkman refused to
1
Department of Surgery, Children’s Hospital, Boston, MA
2
Schepens Eye Research Institute, Boston, MA
3
Harvard Medical School, Boston, MA
have his name added to the paper because he felt he
hadn’t contributed directly to the work, and he wanted
to make sure that the findings furthered our careers. We
submitted the interferon paper to Science. Because pub-
lication of the Nature paper was delayed, the two papers
came out in the same week, causing quite a stir. Dr. Folk-
man was, of course, the first to suggest that interferon
might be used to suppress vessel growth in heman-
giomas.
Interferon (IFN) is an endogenous protein first named
for its ability to interfere with virus replication. IFNand
IFNboth bind to the same receptor, the IFN/recep-
tor. Dr. Bruce Zetter who was part of the Folkman group
found that IFN(leukocyte interferon) could inhibit the
proliferation and migration of bovine capillary endothelial
cells and aortic endothelial cells in a reversible manner.
11
They suggested that IFNwas cytostatic rather than cyto-
toxic, as the cells could recover if IFN was removed. It was
later established that IFN’s mechanism of action was based
on both direct effects on cytostasis and motility as well as
indirect effects on tumor cells by inducing the down-reg-
ulation of bFGF and MMPs. IFNwas shown to have a U-
shaped dosing curve–in other words its anti-angiogenic ef-
fects are not seen at maximum tolerated doses but rather
at lower doses.
12
As a pediatric surgeon Dr. Folkman often saw patients at
Children’s Hospital Boston with life-threatening heman-
giomas. Infantile hemangiomas are benign tumors composed
of blood vessels that rapidly develop in the first months of
life and then slowly involute over the next 10 years. These
tumors often occur in the head and neck region and occa-
sionally can become very serious if they endanger an airway
or obstruct the vision of the child. For corticosteroid-resis-
tant hemangiomas, there were no other treatment options
available at that time. Drs. Ezekowitz, Mulliken, and Folk-
man tried IFNtherapy in these patients since it was an ap-
proved drug. They reported their findings in the New Eng-
land Journal of Medicine in 1992.
13
Using daily administration
of IFN, they saw accelerated regression of these heman-
giomas after an average of eight months of treatment. IFN
therapy is still being used today with new papers just re-
cently published from Mexico
14
and Greece.
15
TNP-470
During the routine culture of bovine capillary endothelial
cells, Dr. Don Ingber (at that time in the Folkman Lab) no-
ticed a fungal growth in the dish that produced a local gra-
dient of endothelial cell rounding, while cells a few diame-
ters away were spread normally. He and Dr. Folkman
hypothesized that this effect might be due to a soluble fac-
tor secreted by the fungus that caused endothelial rounding
and rendered them unable to proliferate. The group identi-
fied the fungus and purified the factor from large-scale con-
ditioned media cultures containing the fungus. The factor
was identified as fumagillin, an antibiotic used to treat amoe-
biasis in humans. Purified fumagillin potently inhibited en-
dothelial cell proliferation and angiogenesis in the chorioal-
lantoic membrane (CAM) assay.
16
The application of
fumagillin as an inhibitor was hindered by the fact that it
caused severe weight loss in animals. Synthetic analogs were
produced that resembled fumagillin in structure. An analog
of fumagillin called AGM-1470 showed anti-angiogenic ac-
tivity in tumor studies. Takeda Chemical Industries (Osaka,
Japan) licensed this analog and it became known as TNP-
470.
BIELENBERG AND D’AMORE204
FIG. 1. Possible targets for anti-angiogenesis.(From Dr. Judah Folkman)
Dr. Don Ingber reflects on his memories from this time:
The development of the potent angiogenesis inhibitor,
TNP-470, resulted from my serendipitous discovery of
a fungal contaminant in one of my capillary cell cultures
when I was a postdoc with Judah Folkman in the mid
1980s. When we first submitted a manuscript describ-
ing this discovery to Science, it was rejected with little
consideration. Dr. Folkman responded by handing me
a book that contained a figure showing a copy of the re-
jection letter that Alexander Fleming had received from
the equally high impact journal Nature when Fleming
submitted his paper describing the discovery of peni-
cillin in 1929. The editors told Fleming that his findings
were “more appropriate for a specialty journal,” and his
world-transforming discovery was eventually pub-
lished in the British Journal of Experimental Pathology. It
turns out that we were more fortunate, and we eventu-
ally published our findings in Nature in 1990.
17
How-
ever, Dr. Folkman taught me that there are no experts
of the future, and that the sting of these attacks should
only serve to awaken the lion that lies asleep inside all
of us—knowledge that I have found invaluable in my
own career.
In clinical trials, TNP-470 showed evidence of antitumor
activity when used as a single agent, but many patients ex-
perienced neurotoxicity. Consistent with Dr. Folkman’s per-
sistent nature, he and Dr. Ronit Satchi-Fainaro further con-
jugated TNP-470 to an HPMA polymer to increase its tumor
retention and prevent it from crossing the blood-brain bar-
rier.
18
HPMA-TNP-470 was renamed caplostatin and dra-
matically inhibited tumor angiogenesis and decreased vas-
cular permeability.
19
Statins
As a surgeon, Dr. Folkman had noticed that the removal
of certain tumors (both in humans and in mice) led to the
rapid growth of distant metastases. After reading Noel
Bouck’s article in Cell in 1989 about the balance of positive
and negative regulators of angiogenesis within a tumor,
20
Dr. Folkman hypothesized that the tumors themselves may
be making an angiogenesis inhibitor.
21
He rationalized that
this inhibitor must be present in the circulation to inhibit the
metastases. Dr. Michel O’Reilly set out to try to purify just
such a molecule.
Dr. Michael O’Reilly and colleagues found that the serum
and urine from tumor-bearing (Lewis lung carcinoma) mice
could inhibit endothelial cell proliferation.
22
They purified
18 liters of mouse urine from tumor-bearing mice and found
that the anti-angiogenic activity co-purified with a 38-kDa
fragment of plasminogen. This protein, which they named
angiostatin, accumulated in the serum and urine of tumor-
bearing mice but disappeared from serum and urine after
the removal of the tumor. When the primary tumors were
surgically removed, the metastases in the lungs grew
rapidly, but when angiostatin was administered to mice af-
ter surgery, metastases in the lungs remained small dormant
lesions.
Several years later, O’Reilly and Folkman published an-
other novel inhibitor in Cell called endostatin using a simi-
lar strategy of isolating the inhibitor from a tumor source.
23
Endostatin, a 20 kDa internal fragment of the C-terminal re-
gion of collagen XVIII, was purified from the conditioned
media of cultured hemangioendothelioma cells called
EOMA. E. coli-derived endostatin was able to dramatically
inhibit several different tumor types. To date it has been used
to inhibit more than 65 different tumor types in preclinical
trials. Many groups are currently using peptides containing
the active regions of endostatin.
24
In September 2005, endo-
statin (called Endostar) was approved by the state in China
for the treatment of non-small-cell lung cancer.
21
In 2006, Dr.
Folkman described endostatin as one of the most broad spec-
trum and least toxic anti-angiogenesis inhibitors. Even more
important than the finding of endostatin may be the concept
that inhibitors exist as fragments of matrix molecules as this
approach has led to the discovery of numerous other inhib-
itors such as tumstatin, arresten, and canstatin.
25,26
Thalidomide
Thalidomide was developed by a German pharmaceutical
company and sold to pregnant women in the late 1950s and
early 1960s as an antiemetic to combat morning sickness and
as a sleep aid. The drug caused serious side-effects to the fe-
tus, and children were born with severe malformities. When
Dr. Robert D’Amato joined the Folkman laboratory in the
early 1990s, he reasoned that drugs that had previously un-
known anti-angiogenic effects would affect physiological an-
giogenesis during menstruation or pregnancy. He recalled
the terrible history of thalidomide and hypothesized that the
mechanism of its action may be through the inhibition of an-
giogenesis. Thinking back to this time Dr. D’Amato com-
mented, “Dr. Folkman was very open-minded to different
approaches to find novel angiogenesis inhibitors.”
D’Amato and Folkman demonstrated that thalidomide in-
hibited bFGF-induced angiogenesis in the rabbit corneal mi-
cropocket assay and published in 1994 in PNAS a paper en-
titled, “Thalidomide is an Inhibitor of Angiogenesis.”
27
Soon
after, clinical trials were initiated using thalidomide against
age-related macular degeneration and brain tumors.
FOLKMAN AND THE INHIBITION OF ANGIOGENESIS 205
FIG. 2. Timeline of the identification and characterization of anti-angiogenic agents.
The following year in 1995, Beth Wolmer contacted Dr.
Folkman after reading about his theories of anti-angiogene-
sis. Her husband, Ira Wolmer, a cardiologist, had been di-
agnosed with multiple myeloma and treatments had failed.
Dr. Folkman suggested thalidomide. Though Wolmer’s on-
cologist Dr. Bart Barlogie was reluctant at first, he was able
to obtain permission to try thalidomide. Unfortunately, Dr.
Wolmer did not survive, but a second patient had a near
complete remission. Dr. Barlogie went on to complete a
larger clinical trial in which one third of the patients had a
positive response to thalidomide.
28
On May 26, 2006, the U.S.
Food and Drug Administration granted accelerated approval
for thalidomide (Thalomid, Celgene Corporation) in combi-
nation with dexamethasone for the treatment of newly di-
agnosed multiple myeloma patients.
Anti-Angiogenic Chemotherapy
Chemotherapy has been used for decades, and the dosing
has historically been at maximum tolerated doses. Since
these doses are toxic to normal dividing cells as well as tu-
mor cells, they require treatments to be separated in time
(usually by two to three weeks) to permit the recovery of the
normal cells such as bone marrow cells. In 2000, Dr. Timo-
thy Browder and Dr. Folkman challenged this concept. They
hypothesized that tumor endothelial cells would also regrow
during these treatment-free periods, thereby reducing the ef-
ficacy of the chemotherapy. Based on this thinking, they ex-
amined whether lower doses of chemotherapy given on a
continuous schedule, termed anti-angiogenic chemotherapy,
would be beneficial. Cyclophosphamide given at lower
doses and more frequently regressed even drug-resistant tu-
mors by inducing endothelial cell apoptosis.
29
TNP-470 used
in combination with anti-angiogenic chemotherapy could
eradicate aggressive drug-resistant tumors in 32 of 38 mice.
Similar results were also reported by Dr. Robert Kerbel’s lab-
oratory in a dose scheduling called metronomic chemother-
apy.
30,31
Dr. Mark Kieran in collaboration with Dr. Folkman trans-
lated this concept to the clinic. In a feasibility trial, they stud-
ied 20 children with brain tumors that were not operable and
were refractory to radiotherapy and chemotherapy. Patients
received daily oral thalidomide and celecoxib (Celebrex,
Pfizer) in combination with daily oral low-dose cyclophos-
phamide alternated every three weeks with daily oral low-
dose etoposide. Twenty-five percent of the patients were pro-
gression free after 2.5 years.
32
Conclusions
Dr. Folkman’s efforts to discover and characterize inhibi-
tors of angiogenesis spanned four decades and included the
description of more than 13 molecules that could block the
growth of new vessels (Fig.2). This heroic effort was termi-
nated abruptly with the unexpected and untimely death of
Dr. Folkman on January 24, 2008. He did, however, live to
see the FDA-approval of anti-angiogenesis therapies that
were being used as adjuvants with chemotherapy for a va-
riety of cancers as well as for the treatment of the “wet” form
of age-related macular degeneration. There are currently
scores of pharmaceutical and biotechnology companies purs-
ing a variety of anti-angiogenic approaches. Moreover, hun-
dreds of basic science laboratories around the world con-
tinue to push the field of angiogenesis forward, and repre-
sent the living legacy of Dr. Folkman’s vision and genius.
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Address reprint requests to:
Dr. Diane Bielenberg
Childrens Hospital
Vascular Biology
300 Longwood Ave
Boston, Massachusetts 02115
E-mail: diane.bielenberg@childrens.harvard.edu
FOLKMAN AND THE INHIBITION OF ANGIOGENESIS 207
