A Systems Biology Approach to Hypoxia Induced Angiogenesis
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A Systems Biology Approach to
Hypoxia-Induced Angiogenesis
Mark Ormiston, MASc. candidate, Dept. of Chemical Engineering and Applied Chemistry and IBBME

Abstract— Angiogenesis can be regulated by hypoxia to
coordinate vascular oxygen supply with metabolic demand. This
process is mediated through the activity of hypoxia-induced
transcription factors (HIF). HIFs regulate the expression of
genes encoding several angiogenic inducers in a manner that is
directly related to cellular oxygen concentration. This paper
reviews the function of several key angiogenic inducers in the
process of vascular formation, the role of HIFs in the regulation
of angiogenesis, and the potential for applying a systems biology
approach to this complex regulatory system.
Index Terms— Angiogenesis, hypoxia-inducible transcription
factors (HIF), systems biology, therapeutic applications
I. INTRODUCTION
O
XYGEN is an essential metabolic requirement for most
higher eukaryotes. In order to deliver sufficient oxygen
to meet their metabolic demands, multicellular organisms have
evolved elaborate oxygen transport systems. In humans, the
lungs, heart, red blood cells and an intricate network of blood
vessels make up the system that delivers oxygen to all cells of
the body. The vascular network of this system is formed de
novo during embryo development through the process of
vasculogenesis [1]. Following development, new blood
vessels are generated from the endothelial cells of pre-existing
vessels by a process known as angiogenesis [2].
Angiogenesis is a tightly regulated process that requires the
coordinated interaction of several cell types, growth factors,
cell surface receptors, adhesion molecules, and components of
the extracellular matrix (ECM) [3]. While rare in healthy
adults, angiogenesis can be up-regulated under exceptional
conditions, including when a low oxygen concentration, or
hypoxia, requires increased blood flow to a particular region
of the body. Under these circumstances, vascular remodeling
is governed by a hypoxia response system that regulates
angiogenesis in a manner that is sensitive to cellular oxygen
concentration. This system functions through the activity of
specific DNA binding proteins known as hypoxia inducible
transcription factors (HIF) [4]. HIFs have been found to act as
a “master switch” for angiogenesis, up-regulating the
expression of several genes that encode proven angiogenic
inducers [5].
Over the last decade, researchers have developed a thorough
understanding of how growth factors, mechanical forces, cell
types, and signaling mechanisms interact to achieve vascular
remodeling. While the genomic and proteomic data obtained
from these studies is critical to our current understanding of
angiogenesis, a complete understanding of this complex
process requires the systemic integration of this knowledge.
The investigation of hypoxia-induced angiogenesis using a
systems biology approach would shed new light on the
interactions that govern new vessel formation. This systemlevel understanding could aid in the development of
therapeutic applications of angiogenesis.
Applications of therapeutic angiogenesis that are currently
being investigated include the controlled delivery of
angiogenic growth factors to treat disease or vascularize tissue
engineering constructs [2].
The inhibition of specific
angiogenic factors is also being investigated with the goal of
treating cancers that use the up-regulation of angiogenesis to
facilitate their growth and metastasis [3].
For these
applications to achieve success, a more complete
understanding of the angiogenesis regulatory system is needed.
II. ANGIOGENESIS
The process of angiogenesis can be broken down into six
basic steps: (1) vasodilatation of the existing vessels, reducing
the contact between adjacent endothelial cells; (2) secretion
and activation of a wide range of proteases by “stimulated”
endothelial cells, and the subsequent degradation of the
vessel’s basement membrane; (3) migration and proliferation
of endothelial cells to form a leading edge of the new
capillary; (4) generation of the capillary lumen and formation
of a tube-like structure; (5) basement membrane synthesis; and
(6) recruitment of pericytes and vascular smooth muscle cells
to form mature vessels [2].
The formation of an orderly network of mature, functional
vessels requires the regulation of angiogenic inducers and
inhibitors with precise spatial and temporal organization.
Studies conducted using in vitro and in vivo experimental
models of angiogenesis have identified a number of growth
factors that are vital to the process of vessel formation and
remodeling. These angiogenic growth factors can be divided
into three classes, depending on the method by which they
regulate vessel formation. Some of the most notable growth
factors in angiogenesis research will be discussed in detail.
The first class of molecules stimulate angiogenesis in the
most direct fashion, acting specifically on endothelial cells.
This group includes vascular endothelial growth factor
A Systems Biology Approach to Hypoxia Induced Angiogenesis
(VEGF) and the angiopoietins (Ang1 and Ang2). VEGF is
secreted by many cell types. In vitro, it has been proven to
stimulate ECM degradation [6], as well as the proliferation,
migration, and tube formation of endothelial cells [7]. It also
induces the expression of a number of proteases by endothelial
cells. These proteases are essential for the degradation of the
ECM and basement membrane [6]. In vivo, VEGF has been
found to mediate microvessel permeability, which is an
important step in the initiation of angiogenesis [8].
Angiopoietins have been found to play a complementary role
to VEGF, regulating new vessel stabilization and maturation
[9].
The second class of angiogenic inducers, which includes
members of the fibroblast growth factor (FGF) family of
cytokines, stimulates angiogenesis by activating a broad range
of target cells [3]. FGFs accumulate in the ECM of tissues,
and can be released during the degradation of the ECM
surrounding stimulated vessels. The most notable of the FGFs,
basic fibroblast growth factor (bFGF), can induce the
expression of proteases by endothelial cells [10], as well as
endothelial cell migration and proliferation [11]. bFGF is also
produced by endothelial cells to recruit pericytes and smooth
muscle cells [12].
The third group of angiogenic inducers play more indirect
roles in vascular remodeling. This group includes platelet
derived growth factor (PDGF) and transforming growth factor
beta (TGF-). When secreted by endothelial cells, PDGF
serves as a mitogen for smooth muscle cells [13] and increases
their expression of VEGF [14]. Endothelial cell secreted
PDGF has also been linked to the recruitment of pericytes
[12]. TGF- induces angiogenesis by up-regulating the
expression of PDGF in endothelial cells, as well as bFGF [11],
and VEGF in smooth muscle cells [15].
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regulation [5].
Hypoxia
inducible
transcription
factors
are
-heterodimers. Both the HIF- and HIF- subunits have
several isoforms encoded by distinct genetic loci [5]. While
HIF- subunits are constitutive nuclear proteins that can also
dimerize with transcription factors other than HIF-, HIF-
subunits are apparently dedicated to hypoxia response [17].
Of the three murine HIF- isoforms that have been identified,
HIF-1 and HIF-2 are the most closely related. These
isoforms are both able to up-regulate hypoxia responsive
transcriptional activity through the binding of HREs [18].
In oxygenated cells, HIF- proteins are inactivated by
two independent mechanisms: rapid proteolytic destruction
and inhibition of transcriptional activity [5]. These regulatory
mechanisms, which prevent the up-regulation of angiogenic
genes under normal oxygen conditions, are controlled through
the action of two types of HIF hydroxylases. Figure 1 outlines
the function of both HIF hydroxylase types. The first set of
these enzymes, the HIF prolyl hydroxylases, can hydroxylize
HIF- proteins at evolutionarily conserved prolyl residues
(two prolyl residues, Pro402 and Pro564, in the human
orthologue of HIF-1). Hydroxylization of these prolyl
residues increases the affinity of HIF- for proteasomal
destruction via the von Hippel-Lindau (VHL) E3 ubiquitin
ligase complex [19]. Once hydroxylized, either of the two
prolyl sites can interact independently with VHL E3, resulting
in rapid degradation [20]. The second set of HIF hydroxylases
are HIF asparaginyl hydroxylases. These enzymes, termed
III. REGULATION OF ANGIOGENESIS BY HYPOXIA
The role of hypoxia as a regulator of angiogenesis is
illustrated by the large number of genes involved in
angiogenesis that are responsive to hypoxia in tissue culture.
Examples include genes for angiogenic growth factors such as
VEGF, angiopoietins, and FGFs, as well as the genes encoding
their various receptors [5]. For a substantial number of these
genes, the hypoxic response can be linked directly to HIF
activity. HIFs regulate the transcription of hypoxia-inducible
genes through the binding of hypoxia response elements
(HRE) in their promoter and enhancer regions [16]. HREs
have been identified in genes associated with a variety of
cellular and systemic responses to hypoxia, including
erythropoiesis, vasodilatation, glycolysis, and angiogenesis
[16]. HREs have been isolated on the promoter regions of
genes encoding VEGF, the VEGF receptor Flt-1, nitric oxide
synthases (associated with vasodilatation), and some proteases
[5]. The expression of genes for angiogenic factors such as
angiopoietins, FGFs, and PDGF may also be indirectly
affected by HIFs through secondary cascades of gene
Fig. 1.
Dual regulation of HIF- subunits by HIF
hydroxylases [5].
A Systems Biology Approach to Hypoxia Induced Angiogenesis
FIH (factor inhibiting HIF), inhibit HIF transcriptional activity
through the -hydroxylation of an asparaginyl residue in the Cterminal activation domain of HIF- (Asn803 in human HIF1). Hydroxylation of this residue blocks the interaction of
HIF- with the transcriptional activator p300 [21].
The function of both groups of HIF hydroxylases can be
directly correlated to cellular O2 concentration. Both enzymes
hydroxylize amino acids through the cleavage of molecular
oxygen [22]. Under hypoxic conditions, the absence of
molecular oxygen results in decreased HIF hydroxylase
activity and the increased expression of genes encoding
angiogenic inducers. As illustrated in figure 2, this system
couples angiogenic regulation to metabolic oxygen demand
through a pathway that links O2 availability, HIF hydroxylase
activity, HIF-dependent transcription and angiogenic growth
factor expression [5]. It is important to note that hypoxia also
influences transcriptional pathways other than the HIF
pathway [5]. The interaction of these pathways with HIF and
HIF hydroxylases would greatly increase the complexity of the
pathway outlined in figure 2. The interaction of other
pathways with elements of the HIF pathway is the subject of
active research.
The pathway outlined in figure 2 could be modeled using a
systems biology approach. Recent advances in computational
biology and systems biology have provided tools capable of
organizing the vast amounts of genomic and proteomic data
associated with the various elements of this regulatory system.
This data could be used to create a model of the regulatory
control pathways that link different elements of the system in
order to study the dynamic interactions associated with
hypoxia-induced angiogenesis [23]. This type of research
would be useful in the development of new therapeutic
angiogenesis strategies that capitalize on HIF as a “master
switch” for angiogenic gene expression.
IV. THERAPEUTIC APPLICATIONS OF ANGIOGENESIS
Advances in our understanding of vascular remodeling have
prompted attempts by researchers to manipulate angiogenesis
for therapeutic applications. These applications include the
targeted delivery of angiogenic growth factors such as VEGF
or bFGF to treat disease states like ischemia [24] and coronary
artery disease [25]. Controlled growth factor delivery has also
been investigated as a means of vascularizing tissue
engineering constructs [26]. Early applications, based on the
delivery of a single growth factor (often VEGF or bFGF) to
stimulate vessel remodeling achieved minimal success. The
newly formed vessels were often leaky, immature and
disorganized [27]. Many applications using VEGF or bFGF
delivery exclusively also resulted in edema from nonfunctional vessel formation [27]. These results are not
surprising considering the therapies were attempting to use a
single growth factor to regulate a process that requires the
regulated interaction of several angiogenic inducers and
inhibitors.
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Fig. 1. Pathway linking angiogenesis to oxygen availability
through the regulation of HIF [5].
Recently, investigations of pro-angiogenic applications have
adopted more system-based strategies.
Modern tissue
engineering scaffolds are designed to induce functional vessel
formation through the sequential delivery of complementary
growth factors such as VEGF and Ang1 [27,28]. These
scaffolds produce more mature, organized vessels that are
surrounded by pericytes [28].
Researchers have also designed successful therapy strategies
that make use of the system that governs hypoxia-induced
angiogenesis. A more stable version of HIF-1 can be created
through the deletion of one of the prolyl residues that is
sensitive to hydroxylation by prolyl hydroxylases. The
transgenic expression of this molecule in the skin of mice
resulted in marked activation of HIF transcriptional targets and
a significant increase in blood vessel growth. The vessels
appeared functional, and showed no signs of the edema often
associated with VEGF therapy [29]. Successful strategies
have also been designed that induce angiogenesis through the
inhibition of HIF hydroxylases, as well as the blocking of the
VHL E3 complex [5].
Our increased understanding of angiogenesis can also be
applied to the inhibition of angiogenic factors associated with
tumor growth and metastasis. Tumors often up-regulate
angiogenic growth factors in order to grow beyond the size
limits imposed by oxygen diffusion [3]. Among these factors,
VEGF, bFGF, and the angiopoietins play a predominant role
[30]. Several anti-angiogenic cancer therapies are currently in
clinical trials. These therapies are based on strategies that (1)
interfere with angiogenic ligands, their receptors or
downstream signaling; (2) up-regulate or deliver endogenous
inhibitors; or (3) directly target tumor vasculature [30]. A
major problem with these therapies is that they often only
target one angiogenic inducer. As tumors grow, they produce
A Systems Biology Approach to Hypoxia Induced Angiogenesis
an increasing assortment of angiogenic growth factors. This
means that if the therapy only works to block one factors (eg.
VEGF), tumors may switch to another factor (eg. bFGF) and
continue growing [30].
In concordance with their physiological role, HIFs have also
been identified as important factors in tumor angiogenesis.
Up-regulation of HIF is observed in many common cancers.
This up-regulation can have several causes, including hypoxia
in the region of the tumor, stimulation by particular growth
factors, and tumor suppressor mutations. The most notable of
these tumor suppressor mutations occurs in the VHL complex,
which has been identified as a key component of the HIF
regulatory system [5].
The identification of HIF as a key player in tumor
angiogenesis has also made it an important anticancer target.
There is little doubt that an improved system-level
understanding the HIF regulatory system will be helpful in the
development of new therapies that target HIF activity the for
treatment of cancer.
V. CONCLUSION
The importance of a systems biology approach to the study
of hypoxia-induced angiogenesis is illustrated by the wealth of
new therapies that are being developed using elements of the
regulation HIF pathway. As the interactions of angiogenic
system elements become more clearly defined, and
representative models are created to test new theories, the
importance of a system-level understanding of angiogenesis
will become even more evident.
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A Systems Biology Approach to Hypoxia Induced Angiogenesis
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