Sprouting angiogenesis was the first
identified form of angiogenesis. It occurs in several well-characterized
stages. First, biological signals known as angiogenic growth factors
activate receptors present on endothelial cells present in pre-existing
venular blood vessels. Second, the activated endothelial cells begin to
release enzymes called proteases that degrade the basement membrane in
order to allow endothelial cells to escape from the original (parent)
vessel walls. The endothelial cells then proliferate into the
surrounding matrix and form solid sprouts connecting neighboring
vessels. As sprouts extend toward the source of the angiogenic stimulus,
endothelial cells migrate in tandem, using adhesion molecules, the
equivalent of cellular grappling hooks, called integrins. These sprouts
then form loops to become a full-fledged vessel lumen as cells migrate
to the site of angiogenesis. Sprouting occurs at a rate of several
millimeters per day, and enables new vessels to grow across gaps in the
vasculature. It is markedly different from splitting angiogenesis,
however, because it forms entirely new vessels as opposed to splitting
existing vessels.
Types of angiogenesis
Sprouting angiogenesis
Intussusceptive angiogenesis
Intussusception, also known as
splitting angiogenesis, was first observed in neonatal rats. In this
type of vessel formation, the capillary wall extends into the lumen to
split a single vessel in two. There are four phases of intussusceptive
angiogenesis. First, the two opposing capillary walls establish a zone
of contact. Second, the endothelial cell junctions are reorganized and
the vessel bilayer is perforated to allow growth factors and cells to
penetrate into the lumen. Third, a core is formed between the two new
vessels at the zone of contact that is filled with pericytes and
myofibroblasts. These cells begin laying collagen fibers into the core
to provide an extracellular matrix for growth of the vessel lumen.
Finally, the core is fleshed out with no alterations to the basic
structure. Intussusception is important because it is a reorganization
of existing cells. It allows a vast increase in the number of
capillaries without a corresponding increase in the number of
endothelial cells. This is especially important in embryonic development
as there are not enough resources to create a rich microvasculature
with new cells every time a new vessel develops.
Modern Terminology of Angiogenesis
Besides the differentiation between
“Sprouting angiogenesis” and “Intussusceptive angiogenesis” there exists
the today more common differentiation between the following types of
angiogenesis:
Vasculogenesis
– Formation of vascular structures from circulating or tissue-resident
endothelial stem cells (angioblasts), which proliferate into de-novo
endothelial cells. This form particularly relates to the embryonal
development of the vascular system.
Angiogenesis –
Formation of thin-walled endothelium-lined structures with/without
muscular smooth muscle wall and pericytes (fibrocytes). This form plays
an important role during the adult life span, also as "repair mechanism"
of damaged tissues.
Arteriogenesis – Formation of medium-sized blood vessels possessing tunica media plus adventitia.
Because it turned out that even this
differentiation is not a sharp one, today quite often the term
“Angiogenesis” is used summarizing all different types and modifications
of arterial vessel growth.
Therapeutic angiogenesis
Therapeutic angiogenesis is the
application of specific compounds which may inhibit or induce the
creation of new blood vessels in the body in order to combat disease.
The presence of blood vessels where there should be none may affect the
mechanical properties of a tissue, increasing the likelihood of failure.
The absence of blood vessels in a repairing or otherwise metabolically
active tissue may retard repair or some other function. Several diseases
(eg. ischemic chronic wounds) are the result of failure or insufficient
blood vessel formation and may be treated by a local expansion of blood
vessels, thus bringing new nutrients to the site, facilitating repair.
Other diseases, such as age-related macular degeneration, may be created
by a local expansion of blood vessels, interfering with normal
physiological processes.
The modern clinical application of the
principle “angiogenesis” can be divided into two main areas: 1.
Anti-angiogenic therapies (historically, research started with); 2.
Pro-angiogenic therapies. Whereas anti-angiogenic therapies are trying
to fight cancer and malignancies(because tumors, in general, are
nutrition- and oxygen-dependent, thus being in need of adequate blood
supply), the pro-angiogenic therapies are becoming more and more
important in the search of new treatment options for cardiovascular
diseases (the number one cause of death in the Western world). One of
the world-wide first applications of usage of pro-angiogenic methods in
humans was a German trial using fibroblast growth factor 1 (FGF-1) for
the treatment of coronary artery disease. Today, clinical research is
ongoing in various clinical trials to promote therapeutic angiogenesis
for a variety of atherosclerotic diseases, like coronary heart disease,
peripheral arterial disease, wound healing disorders, etc.
Also, regarding the “mode of action”,
pro-angiogenic methods can be differentiated into three main categories:
1. Gene-therapy; 2. Protein-therapy (using angiogenic growth factors
like FGF-1 or vascular endothelial growth factor, VEGF); 3. Cell-based
therapies.
There are still serious, unsolved
problems related to gene therapy including: 1. Difficulty integrating
the therapeutic DNA (gene) into the genome of target cells; 2. Risk of
an undesired immune response; 3 Potential toxicity, immunogenicity,
inflammatory responses and oncogenesis related to the viral vectors; and
4. The most commonly occurring disorders in humans such as heart
disease, high blood pressure, diabetes, Alzheimer’s disease are most
likely caused by the combined effects of variations in many genes, and
thus injecting a single gene will not be beneficial in these diseases.
In contrast, pro-angiogenic protein therapy uses well defined, precisely
structured proteins, with previously defined optimal doses of the
individual protein for disease states, and with well-known biological
effects. On the other hand, an obstacle of protein therapy is the mode
of delivery: oral, intravenous, intra-arterial, or intramuscular routes
of the protein’s administration are not always as effective as desired;
the therapeutic protein can be metabolized or cleared before it can
enter the target tissue. Cell-based pro-angiogenic therapies are still
in an early stage of research – with many open questions regarding best
cell types and dosages to use.
FGF
The fibroblast growth factor (FGF)
family with its prototype members FGF-1 (acidic FGF) and FGF-2 (basic
FGF) consists to date of at least 22 known members. Most are 16-18 kDa
single chain peptides and display high affinity to heparin and heparan
sulfate. In general, FGFs stimulate a variety of cellular functions by
binding to cell surface FGF-receptors in the presence of heparin
proteoglycans. The FGF-receptor family is comprised of seven members and
all the receptor proteins are single chain receptor tyrosine kinases
that become activated through autophosphorylation induced by a mechanism
of FGF mediated receptor dimerization. Receptor activation gives rise
to a signal transduction cascade that leads to gene activation and
diverse biological responses, including cell differentiation,
proliferation, and matrix dissolution – thus initiating a process of
mitogenic activity critical for the growth of endothelial cells,
fibroblasts, and smooth muscle cells. FGF-1, unique among all 22 members
of the FGF family, can bind to all seven FGF-receptor subtypes, making
it the broadest acting member of the FGF family, and a potent mitogen
for the diverse cell types needed to mount an angiogenic response in
damaged (hypoxic) tissues, where up regulation of FGF-receptors occurs.
FGF-1 stimulates the proliferation and differentiation of all cell types
necessary for building an arterial vessel, including endothelial cells
and smooth muscle cells; this fact distinguishes FGF-1 from other
pro-angiogenic growth factors, such as vascular endothelial growth
factor (VEGF) which primarily drives the formation of new capillaries.
Until now (2007), three human clinical
trials have been successfully completed with FGF-1 in which the
angiogenic protein was injected directly into the damaged heart muscle.
Also, one additional human FGF-1 trial has been completed to promote
wound healing in diabetics with chronic wounds.
Besides FGF-1, one of the most
important functions of also fibroblast growth factor-2 (FGF-2 or bFGF)
is the promotion of endothelial cell proliferation and the physical
organization of endothelial cells into tube-like structures, thus
promoting angiogenesis. FGF-2 is a more potent angiogenic factor than
VEGF or PDGF (platelet-derived growth factor), however, less potent than
FGF-1. As well as stimulating blood vessel growth, aFGF (FGF-1) and
bFGF (FGF-2) are important players in wound healing. They stimulate the
proliferation of fibroblasts and endothelial cells that give rise to
angiogenesis and developing granulation tissue, both increase blood
supply and fill up a wound space/cavity early in the wound healing
process.
VEGF
VEGF (Vascular Endothelial Growth
Factor) has been demonstrated to be a major contributor to angiogenesis,
increasing the number of capillaries in a given network. Initial in
vitro studies demonstrated that bovine capillary endothelial cells will
proliferate and show signs of tube structures upon stimulation by VEGF
and bFGF, although the results were more pronounced with VEGF.
Upregulation of VEGF is a major component of the physiological response
to exercise and its role in angiogenesis is suspected to be a possible
treatment in vascular injuries. In vitro studies clearly demonstrate
that VEGF is a potent stimulator of angiogenesis because in the presence
of this growth factor plated endothelial cells will proliferate and
migrate, eventually forming tube structures resembling capillaries. VEGF
causes a massive signaling cascade in endothelial cells. Binding to
VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade
that stimulates the production of factors that variously stimulate
vessel permeability (eNOS, producting NO), proliferation/survival
(bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into
mature blood vessels. Mechanically, VEGF is upregulated with muscle
contractions as a result of increased blood flow to affected areas. The
increased flow also causes a large increase in the mRNA production of
VEGF receptors 1 and 2. The increase in receptor production means that
muscle contractions could cause upregulation of the signaling cascade
relating to angiogenesis. As part of the angiogenic signaling cascade,
NO is widely considered to be a major contributor to the angiogenic
response because inhibition of NO significantly reduces the effects of
angiogenic growth factors. However, inhibition of NO during exercise
does not inhibit angiogenesis indicating that there are other factors
involved in the angiogenic response.
Angiopoietins
The angiopoietins, Ang1 and Ang2, are
required for the formation of mature blood vessels, as demonstrated by
mouse knock out studies . Ang1 and Ang2 are protein growth factors which
act by binding their receptors, Tie-1 and Tie-2; while this is somewhat
controversial, it seems that cell signals are transmitted mostly by
Tie-2; though some papers show physiologic signaling via Tie-1 as well.
These receptors are tyrosine kinases. Thus, they can initiate cell
signaling when ligand binding causes a dimerization that initiates
phosphorylation on key tyrosines.
MMP
Another major contributor to
angiogenesis is matrix metalloproteinase (MMP). MMPs help degrade the
proteins that keep the vessel walls solid. This proteolysis allows the
endothelial cells to escape into the interstitial matrix as seen in
sprouting angiogenesis. Inhibition of MMPs prevents the formation of new
capillaries. These enzymes are highly regulated during the vessel
formation process because destruction of the extracellular matrix would
decrease the integrity of the microvasculature.
Applications of Angiogenesis
Tumor angiogenesis
Cancer cells are cells that have lost
their ability to divide in a controlled fashion. A tumor consists of a
population of rapidly dividing and growing cancer cells. Mutations
rapidly accrue within the population. These mutations (variation) allow
the cancer cells (or sub-populations of cancer cells within a tumor) to
develop drug resistance and escape therapy. Tumors cannot grow beyond a
certain size, generally 1-2 mm³, due to a lack of oxygen and other
essential nutrients.
Tumors induce blood vessel growth
(angiogenesis) by secreting various growth factors (e.g. Vascular
Endothelial Growth Factor or VEGF). Growth factors, such as bFGF and
VEGF can induce capillary growth into the tumor, which some researchers
suspect supply required nutrients -- allowing for tumor expansion. On 18
July 2007 it was discovered that cancerous cells stop producing the
anti-VEGF enzyme PKG. In normal cells (but not in cancerous ones), PKG
apparently limits beta-catenin which solicits angiogenesis.Other
clinicians believe that angiogenesis really serves as a waste pathway,
taking away the biological end products put out by rapidly dividing
cancer cells. In either case, angiogenesis is a necessary and required
step for transition from a small harmless cluster of cells, often said
to be about the size of the metal ball at the end of a ball-point pen,
to a large tumor. Angiogenesis is also required for the spread of a
tumor, or metastasis. Single cancer cells can break away from an
established solid tumor, enter the blood vessel, and be carried to a
distant site, where they can implant and begin the growth of a secondary
tumor. Evidence now suggests that the blood vessel in a given solid
tumor may in fact be mosaic vessels, comprised of endothelial cells and
tumor cells. This mosaicity allows for substantial shedding of tumor
cells into the vasculature. The subsequent growth of such metastases
will also require a supply of nutrients and oxygen or a waste disposal
pathway.
Endothelial cells have long been
considered genetically more stable than cancer cells. This genomic
stability confers an advantage to targeting endothelial cells using
antiangiogenic therapy, compared to chemotherapy directed at cancer
cells, which rapidly mutate and acquire 'drug resistance' to treatment.
For this reason, endothelial cells are thought to be an ideal target for
therapies directed against them. Recent studies by Klagsbrun, et al.
have shown, however, that endothelial cells growing within tumors do
carry genetic abnormalities. Thus, tumor vessels have the theoretical
potential for developing acquired resistance to drugs. This is a new
area of angiogenesis research being actively pursued.
Angiogenesis research is a cutting
edge field in cancer research, and recent evidence also suggests that
traditional therapies, such as radiation therapy, may actually work in
part by targeting the genomically stable endothelial cell compartment,
rather than the genomically unstable tumor cell compartment. New blood
vessel formation is a relatively fragile process, subject to disruptive
interference at several levels. In short, the therapy is the selection
agent which is being used to kill a cell compartment. Tumor cells evolve
resistance rapidly due to rapid generation time (days) and genomic
instability (variation), whereas endothelial cells are a good target
because of a long generation time (months) and genomic stability (low
variation).
This is an example of selection in
action at the cellular level, using a selection pressure to target and
differentiate between varying populations of cells. The end result is
the extinction of one species or population of cells (endothelial
cells), followed by the collapse of the ecosystem (the tumor) due either
to nutrient deprivation or self-pollution from the destruction of
necessary waste pathways.
Angiogenesis-based tumour therapy
relies on natural and synthetic angiogenesis inhibitors like
angiostatin, endostatin and tumstatin. These are proteins that mainly
originate as specific fragments pre-existing structural proteins like
collagen or plasminogen.
Recently, the 1st FDA-approved therapy
targeted at angiogenesis in cancer came on the market in the US. This
is a monoclonal antibody directed against an isoform of VEGF. The
commercial name of this antibody is Avastin, and the therapy has been
approved for use in colorectal cancer in combination with established
chemotherapy.
Angiogenesis for cardiovascular disease
Angiogenesis represents an excellent
therapeutic target for the treatment of cardiovascular disease. It is a
potent, physiological process that underlies the natural manner in which
our bodies respond to a diminution of blood supply to vital organs,
namely the production of new collateral vessels to overcome the ischemic
insult. Perhaps the greatest reason for these trials’ failure to
achieve success is the high occurrence of the “placebo effect” in
studies employing treadmill exercise test readout. Thus, even though a
majority of the treated patients in these trials experience relief of
such clinical symptoms such as chest pain (angina), and generally
performed better on most efficacy readouts, there were enough
“responders” in the blinded placebo groups to render the trial
inconclusive. In addition to the placebo effect, more recent animal
studies have also highlighted various factors that may inhibit an
angiogenesis response including certain drugs, smoking, and
hypercholesterolemia.
Although shown to be relatively safe
therapies, not one angiogenic therapeutic has yet made it through the
gauntlet of clinical testing required for drug approval. By capitalizing
on the large database of what did and did not work in previous clinical
trials, results from more recent studies with redesigned clinical
protocols give renewed hope that angiogenesis therapy will be a
treatment choice for sufferers of cardiovascular disease resulting from
occluded and/or stenotic vessels.
Early clinical studies with
protein-based therapeutics largely focused on the intravenous or
intracoronary administration of a particular growth factor to stimulate
angiogenesis in the affected tissue or organ. Most of these trials did
not achieve statistically significant improvements in their clinical
endpoints. This ultimately led to an abandonment of this approach and a
widespread belief in the field that protein therapy, especially with a
single agent, was not a viable option to treat ischemic cardiovascular
disease. However, the failure of gene- or cell-based therapy to deliver,
as of yet, a suitable treatment choice for diseases resulting from poor
blood flow, has led to a resurgence of interest in returning to
protein-based therapy to stimulate angiogenesis. Lessons learned from
earlier protein-based studies, which indicated that intravenous or
intracoronary delivery of the protein was not efficacious, have led to
completed and ongoing clinical trials in which the angiogenic protein is
injected directly into the beating ischemic heart.
Such localized administration of the
potent angiogenic growth factor, human FGF-1, has recently given
promising results in clinical trials in no-option heart patients.
Angiogenesis was documented by angiographically visible “blushing”, and
functional exercise tests were also performed on a subset of patients.
The attractiveness of protein therapy is that large amounts of the
therapeutic agent can be injected into the ischemic area of interest, to
pharmacologically start the process of blood vessel growth and
collateral arteries’ formation. In addition, from pharmacokinetic data
collected from the recent FGF-1 studies in the human heart, it appears
that FGF-1, once it exits the heart is cleared in less than three hours
from the circulation. This would presumably prevent FGF-1 from
stimulating unwanted angiogenesis in other tissues of the bodies where
it could potentially cause harm, such as the retina and in the kidneys.
No serious adverse events have yet to be noted in any of the completed
or ongoing clinical trials in which the FGF-1 protein is utilized as the
therapeutic agent tom stimulate angiogenesis
