Hypoxia and Matrix Manipulation for Vascular Engineering

Abstract

The substantial majority of cell types are known to be capable of sensing changes in O2 tension and in the extracellular matrix (ECM), resulting in various responses depending on the cell type and other factors in the microenvironment, such as cell-cell interactions. A growing body of evidence suggests that hypoxia greatly influences angiogenesis and vasculogenesis through the transcription of numerous genes, including vascular endothelial growth factor (VEGF), the major regulatory protein of these processes. At the same time, the spatial and temporal distribution of ECM components affects ECM properties and growth factor (GF) availability, which, in turn, regulates vascular development. This chapter will discuss how hypoxia and the ECM influence vascular morphogenesis. It seeks to provide a better understanding of vascular development by considering recent research and emerging technologies focused on controlling O2 tension and manipulating ECM properties. We will first focus on the influences of O2 tension and ECM composition on neovascularization. Then we present strategies for manipulating the microenvironment using both synthetic and naturally derived biomaterials. Control over O2 in three-dimensional (3D) microenvironments is thoroughly highlighted, along with the currently available O2 measurement techniques and mathematical models that are necessary to monitor O2 gradients in 3D microenvironments. Finally, we discuss the state-of-the-art technology in microfluidics and smart biomaterials to provide insight into future directions of these exciting research areas.

Full text

73© Springer Nature Switzerland AG 2018
S. Gerecht (ed.), Biophysical Regulation of Vascular Differentiation and
Assembly, Biological and Medical Physics, Biomedical Engineering,
https://doi.org/10.1007/978-3-319-99319-5_4
Chapter 4
Hypoxia andMatrix Manipulation
forVascular Engineering
MichaelR.Blatchley, HasanE.Abaci, DonnyHanjaya-Putra,
andSharonGerecht
4.1 Introduction
The permanency of multicellular organisms on Earth is stringently reliant on the
ability of cells to respond and adapt to their surrounding milieu. Cells respond to a
complex array of biological, biophysical, and biochemical cues to develop and
regenerate functional tissues. Alterations to these properties can catastrophically
result in impaired development or healing, as well as tumor development and
M. R. Blatchley
Department of Biomedical Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical
Sciences–Oncology Center and Institute for NanoBioTechnology,
Baltimore, MD, USA
H. E. Abaci
Department of Dermatology, Columbia University, New York, NY, USA
D. Hanjaya-Putra
Department of Aerospace and Mechanical Engineering, Notre Dame, IN, USA
S. Gerecht (*)
Department of Biomedical Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical
Sciences–Oncology Center and Institute for NanoBioTechnology,
Baltimore, MD, USA
Department of Materials Science and Engineering, Johns Hopkins University,
Baltimore, MD, USA
Department of Oncology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
e-mail: gerecht@jhu.edu

74
metastasis. At the most basic level, two of the most critical components of the cel-
lular microenvironment are oxygen (O2) and the composition of the extracellular
matrix (ECM).
According to the most recent ndings, O2 reached sufcient levels (estimated to
be 0.2–2% O2) for aerobic organisms to be able to survive between 2.45 and 2.2
billion years ago in the oceans [19, 154] and between 540 and 600 million years
ago in Earth’s atmosphere [72, 154, 194]. Ever since, O2 has been a highly avail-
able potential source of energy for multicellular organisms to commence, survive,
and multiply. Multicellular organisms require specialized systems to enable suf-
cient amounts of O2 to reach their cells. For instance, insects regulate the transport
of O2 into their tissues with a special respiratory system consisting of spiracles and
trachea. Around their tissues, they retain the relatively low O2 levels (1.4mmHg)
thought to be equivalent to the atmospheric O2 concentrations at the time of their
evolution [161, 162]. In vertebrates, O2 is carried by proteins in the blood, particu-
larly by hemoglobin, and is transported to tissues through endothelial cells (ECs).
The cells throughout the body are highly dependent on the dynamics of O2. In
humans, O2 concentrations vary between 1 and 10% in tissues (other than the
lungs) and between 5 and 13% in blood vessels [126, 155]. Therefore, the ability
for cells to sense and respond to O2 is critical, and O2 acts as a signaling molecule
for cells, regulating their metabolism, survival, cell-cell interactions, migration,
and differentiation.
Besides O2 availability, the transition from unicellular to multicellular organisms
requires that cells be connected together in a way that allows them to interact with
each other as parts of the same system. This interconnectedness could happen either
by having junctions at the cell peripheries or by having connecting cement between
the cells. Many multicellular organisms connect their cells in both ways: they use
cellular junctions to allow direct signaling between cells, and they use the ECM to
regulate the transport of molecules (e.g., O2, glucose, and signaling proteins)
between the cells by remodeling the components of the ECM.Thus, both cell-cell
and cell-ECM interactions are signicant for determining the fate of cells in tissues.
Transmembrane proteins known as integrins are responsible for signaling from the
ECM to the cell. Therefore, cells have different responses with respect to the com-
position and structure of the surrounding ECM.In particular, vascular morphogen-
esis is regulated by endothelial cell (EC) interactions with the ECM through
integrins and is highly dependent on the ECM context [49].
In the eld of vascular engineering, the effects of both O2 tension and the ECM
on blood vessel formation continue to be extensively investigated. Blood vessel
formation essentially occurs by angiogenesis or vasculogenesis. Angiogenesis is
the formation of blood vessels from pre-existing vasculature, orchestrated with the
proliferation, migration, and assembly of ECs, as well as the remodeling of the
ECM [127, 213]. Most of the ECs comprising the blood vessel walls are in a state
of quiescence in physiological conditions. A stimulus is required for ECs to switch
from their resting state to their navigating state, where they are activated to pro-
duce angiogenesis-promoting proteins [70]. Angiogenesis occurs in several situa-
tions, such as wound healing, arthritis, cardiovascular ischemia, and solid tumor
M. R. Blatchley et al.

75
growth [43, 199]. In all of these situations, the tissue or vasculature is deprived of
oxygen, leading to hypoxic conditions that promote angiogenesis. For vessel
sprouting, the ECM surrounding the vasculature needs to be degraded so that ECs
can easily navigate into the tissue and proliferate. Hypoxia is known to promote
the production of ECM-degrading enzymes via secretion from activated ECs [21,
61, 65]. Thus, EC sprouting is more favorable toward hypoxic regions in the ECM
through which the secretion of enzymes is upregulated by hypoxia, whereas the
invasion of vessels into the ECM is not favored in the direction of sufciently oxy-
genated regions.
An oxygen gradient emerges in early development, which guides cellular dif-
ferentiation and morphogenesis [141]. While the O2 uptake of early embryonic
cells relies on the simple diffusion of oxygen, hypoxia starts to be observed in
different regions as the embryo expands [5, 141]. The initial vascularization, vas-
culogenesis, starts with the differentiation of angioblasts (embryonic progenitors
of ECs), which surround hemopoietic cells to form blood islands. Blood islands
ultimately fuse as angioblasts differentiate into endothelial cells to form the pri-
mary capillary plexus and then undergo further tubulogenesis and vascular net-
work formation throughout the yolk sac [156, 185, 230]. This process of
vasculogenesis has been suggested to occur in hypoxic conditions [141, 156].
Hypoxia also stimulates microvascularization and the capillary network to form
around the developing organs. Vasculogenesis in adult organs has been demon-
strated to originate from endothelial progenitor cells (EPCs) circulating in the
blood [225]. The migration of EPCs and their recruitment to the appropriate sites
to induce the formation of new blood vessels depends on complex cell signaling.
Circulating EPCs home to hypoxic regions along both O2 and growth factor gra-
dients, in particular gradients of stromal-derived factor 1 (SDF-1) [33, 51].
Investigations of tumor growth and wound healing have revealed that hypoxia
occurs in both situations, inducing EPCs to migrate from the circulating blood
through the ECM.Hypoxia also plays a role in the recruitment of EPCs by pro-
moting receptor expression on the tissue that recognizes EPCs [242], as well as
on the EPCs themselves [35], which is followed by their differentiation into
mature ECs [32]. Moreover, vascular endothelial growth factor (VEGF), a key
regulatory protein known to induce vasculogenesis and angiogenesis, was found
to be upregulated in hypoxia [159]. These processes take place in the milieu of the
ECM, which is mostly composed of bronectin during early development [49,
148]. In adult tissue, on the other hand, collagen becomes abundant and controls
the cellular fate.
The formation of new blood vessels through angiogenesis or vasculogenesis
depends on the dynamic effects and interplay between the ECM and oxygen ten-
sion. A thorough understanding of the mechanisms involving the ECM and O2 dur-
ing angiogenesis and vasculogenesis is essential for the fundamental understanding
that can be harnessed for developing vascular engineering applications. Indeed, the
effects of these two factors on vascular cells are being investigated extensively. The
invitro vascularization of primary vascular cells has been studied using many dif-
ferent biomaterials [10, 24, 88] as three-dimensional (3D) matrix components, and
4 Hypoxia andMatrix Manipulation forVascular Engineering

76
they were shown to inuence various aspects of angiogenesis and vasculogenesis.
Similarly, a considerable amount of work has focused on the effects of hypoxia-
inducible factors (HIF1α, HIF2α, HIF3α) on the regulation of genes that induce
vasculature network formation [68, 159, 241]. In addition, some researchers have
also investigated the effects of hypoxia and the ECM context together [170, 177].
Success in engineering blood vessels from primary vascular cells or stem cells relies
on understanding the inuence of all critical parameters and controlling them in
targeted directions.
The main focus of this chapter is a review and discussion of how the cells in the
body respond to variations in oxygen tension and ECM components, leading to new
vasculature formation.
4.2 Concepts intheRegulation oftheVasculature byOxygen
andtheECM
4.2.1 The Inuence ofOxygen Tension onVascularization
Variations in oxygen concentrations at every stage of embryogenesis and in differ-
ent regions of adult tissues lead to diverse vascular responses, depending on the cell
type and microenvironment. Many cell types respond differently, but also collec-
tively, to the changes in O2 equilibrium through specialized sensing mechanisms
and effectors in order to maintain homeostasis. In this section, we will rst discuss
the formation and location of poorly oxygenated regions in the body, as well as the
mechanisms that cells utilize to sense changes in oxygen levels. Then, we will then
focus on several responses of pluripotent and vascular cells to low O2 tensions in
terms of gene regulation, differentiation, oxygen consumption, and cell survival.
4.2.1.1 The InVivo Consequences ofOxygen Gradients
Oxygen Availability intheBody
In vertebrates, O2 transport to the tissues relies on three main processes: the oxy-
genation of the blood in the alveoli in the lungs, the convectional transport of
oxygen in the blood along the veins, and the diffusion of oxygen across the vessel
walls followed by penetration of O2 to the deeper tissues. There are three distinct
resistances to the mass transfer of the O2 molecule, which result in O2 gradients
throughout the body.
O2 deprivation has been observed early in the development of mouse embryos
[141]. Additionally, polarographic oxygen measurements in the human placenta
have shown that O2 levels are 1.3–3.5% in the rst 8–10weeks and reach between
7.2 and 9.5% in weeks 12–13 of pregnancy [188, 202]. Oxygen levels measured in
M. R. Blatchley et al.

77
the gestational sac revealed even lower O2 levels in earlier stages of embryogene-
sis, where O2 is only transported by simple diffusion [115]. Diffusion, as opposed
to convection, transports nutrients between cells very slowly. Before vasculogene-
sis begins, the maximum diameter that a spherical embryo can reach without hav-
ing any anoxic cells was calculated to be 2mm [29]. This value varies with the
embryo’s geometry and, most importantly, with the O2 consumption of the animal
cells. The results of invivo imaging of various animal embryos show that the maxi-
mum diameter remains below 1mm, which agrees with the theoretically estimated
value [29, 230].
Vasculogenesis is crucial to facilitate cell proliferation and for the embryo to
grow larger. In mouse embryos, vasculogenesis commences after day 7, with the
differentiation of the mesoderm into angioblasts, which then assemble to form a
simple circulatory system consisting of a heart, dorsal aorta, and yolk sac by day 8
[56, 107]. Afterward, spatial increases are observed in O2 levels throughout the
course of embryonic development [141]. These profound spatiotemporal O2 level
changes in the embryo can be accepted as evidence for vascular formation during
embryogenesis. The large existing vasculature then sprouts and proliferates to sup-
ply O2 and nutrients to cells located in poorly oxygenated regions. Hypoxia, consid-
ered the most critical factor controlling the angiogenesis process, works via
numerous protein-signaling pathways. The mechanism determining the directional-
ity of angiogenesis and the complex networking of endothelial capillaries around
the tissues is manipulated by several other parameters, including hemodynamic
forces and cytokines; this mechanism will be discussed later in the chapter [150].
Once embryonic development is complete and sufcient concentrations of O2 and
nutrients are supplied to the tissues, the oxygen gradient still persists in some tissues,
providing several benets to specic cell types. In adults, O2 distribution ranges
from 1 to 13in normal tissues. Although the formation of blood vessels and capillary
networking is complete, some tissues still lack vasculature, such as the bone marrow
niche [91, 132, 175]. In such tissues, diffusion is the controlling mechanism for
nutrient transport, thus resulting in a wide range of O2 distributions from the internal
hypoxic region to the external regions, which remains at physiological O2.
The discovery of circulating EPCs in blood vessels revealed that neovasculariza-
tion in adults is directed not only by angiogenesis but also by the vasculogenesis
process, which depends on the renewal, mobility, recruitment, and differentiation of
EPCs [12, 13, 96, 219]. The bone marrow (BM) provides a host microenvironment
for a variety of cells, including hematopoietic stem cells (HSCs), mesenchymal
stem cells (MSCs), and EPCs. The development of EPCs occurs in the BM, which
has a unique structure that allows severe hypoxic regions to exist. Although the BM
is inaccessible for noninvasive oxygen measurements, both simulation studies and
qualitative measurements have demonstrated the existence of hypoxic regions.
Several theoretical models have been developed in order to simulate the distribution
of oxygen throughout the BM [41, 132, 133]. Chow et al. used homogeneous
Kroghian models to estimate oxygen levels in the BM [41]. Their simulations sug-
gested that both HSCs and EPCs are exposed to low O2 tensions in the BM.There
are various BM architectural organizations possible depending on parameters such
4 Hypoxia andMatrix Manipulation forVascular Engineering

78
as the spatial arrangement of vasculature and the distribution of many different cell
types populating the BM.Therefore, in the absence of supporting evidence from
invivo quantitative measurements, model predictions must be used to assess the
effects of different parameters on the O2 tension distribution in the BM.The model
described by Kumar etal. considered three possible vessel arrangements to simulate
oxygen level variations under various conditions [132]. They suggested that hypoxic,
and even anoxic, regions could be found in the BM, assuming that the cells’ oxygen
consumption is constant and that the density of arterioles in the BM is low.
On the other hand, qualitative observations in the study by Parmar etal. demon-
strated that HSCs are distributed according to oxygen availability in the BM [175].
Staining with pimonidazole and sectioning revealed the oxygen gradient throughout
the BM, showing that HSCs more likely reside at the lower end of the gradient.
These results are in agreement with other invitro studies suggesting that hypoxia
supports the maintenance of stemness [45, 64, 69]. Moreover, BM transplantation
studies have shown that BM-derived EPCs enhance neovascularization and the for-
mation of arteries [231, 235]. The renewal of EPCs in the BM depends on the dif-
ferentiation dynamics of HSCs, which are regulated by the microenvironments (i.e.,
the niches) they reside in. Osteoblasts, bone cell progenitors, bind to each other and
to HSCs via adhesion molecules to form the osteoblastic niche that is located far
from the sinusoidal arteries. Researchers have discovered the existence of another
type of niche within the BM, the vascular niche, which is located closer to the sinu-
soidal arteries than the osteoblastic niche. The differences in physicochemical fac-
tors within the various niches play fundamental roles in controlling the dynamics of
HSC migration and differentiation. Since the vascular niche’s close proximity to
arteries means that it is richer in O2 than the osteoblastic niche, Heissnig’s group
hypothesized that HSCs are in a quiescent state in the osteoblastic niche’s severe
hypoxic conditions [92]. When vasculogenesis is necessary in neighboring tissues,
specic cell signaling stimulates the migration of HSCs from the osteoblastic niche
to the more oxygenated vascular niche, where HSCs can switch from their quiescent
state to a proliferative state. The proliferation and differentiation of HSCs reconsti-
tute the EPC pool in the vascular niche before they enter the circulation.
Wound healing, another situation where tissue hypoxia is prevalent, consists of a
series of events that includes new vasculature formation, which is regulated by vary-
ing O2 levels. Platelets interfere with microcirculation in the wounded tissue, fol-
lowed by the release of coagulation factors to reinforce the clotting process.
Histamine and bradykinin, secreted by mast cells, also inuence the microcirculation
by enhancing vascular permeability and arteriolar vasodilation, thereby increasing
the blood ow rate [11, 102]. Recruitment of leukocytes and macrophages into the
damaged tissue is followed by their activation in response to several growth factors
(GFs) and integrins. High rates of O2 consumption in activated macrophages, along
with perturbation of the microcirculation, lead to a further decrease in O2 levels and
result in hypoxia [204], which leads to the accumulation of HIF1α at the wound site
[246]. Albina etal. [9] showed that the HIF1α mRNA of inammatory cells peaks
about 6h after injury. On the other hand, HIF1α protein levels could be detected
between 1 and 5days after wounding. More recently, Zhang etal. [246] demon-
M. R. Blatchley et al.

79
strated that, during the burn wound-healing process, the accumulation of HIF1α
increases the number of circulating angiogenic cells, as well as smooth muscle actin-
positive cells, in the wounded tissue. These hypoxic conditions—either directly or
indirectly through the accumulation of HIF—stimulate angiogenesis during wound
healing.
Ischemic tissues, including those affected by myocardial infarction or peripheral
artery occlusion (e.g., limb ischemia), have also been a hotbed for study of the
effects of low O2 on cellular recruitment and tissue regeneration. In particular, lack
of O2 delivery to these diseased tissues results in HIF stabilization and subsequent
upregulation of recruitment chemokines, perhaps most importantly SDF-1, as well
as transmembrane proteins integrin β2 and ICAM-1 that facilitate adhesion of cir-
culating cells to the damaged endothelium [33, 35, 51, 242]. Importantly, hypoxic
conditions also facilitate ECM remodeling through upregulation of proteases, such
as cathepsins and matrix metalloproteinases [4, 106, 226]. These factors, in concert
with HIF-induced production of other pro-angiogenic factors, such as VEGF, lead
to robust formation of neovasculature.
Oxygen-Sensing Mechanisms ofVascular Cells
Most cell types in the body respond to variations in O2 tensions [233]. Gene expres-
sion, viability, metabolism, and the oxygen uptake rate of the cells change with
alterations in O2 levels, in order to maintain homeostasis. When cells experience a
change in extracellular O2 levels, they adapt to the new conditions, which may occur
rapidly. Hence, O2 sensing in cells is expected to be controlled by well-organized,
highly sensitive mechanisms.
Several mechanisms have been proposed in the literature to account for O2 sens-
ing in cells. Although their sensitivities may differ from one another, more than one
such mechanism can coexist in a cell, resulting in various cellular responses. Within
the cell, the O2 molecule mainly engages in two distinct processes: it is involved
directly in biosynthesis reactions; or it participates in metabolic processes, such as
the electron transport chain occurring in mitochondria. Any change in the concen-
tration of O2 extensively perturbs these processes and, following a sequence of
events, may have a number of different effects on the cell. Therefore, O2 sensors in
cells can be mainly categorized as mitochondria-related sensors (bioenergetic) and
biosynthesis-related sensors (biosynthetic)—although they can be linked to each
other in some cases, making the distinction not completely clear [233].
Among the several effectors of O2-sensing mechanisms, HIFs are the most essen-
tial in terms of the diversity of their inuences. The family of HIFα subunits (HIF1α,
HIF2α, and HIF3α) has been shown to be responsible for regulating expression of a
large number of genes, including those coding for key regulatory proteins of angio-
genesis and vasculogenesis. Although HIFα is expressed at every oxygen tension, it
is rapidly ubiquitinated in normoxic conditions, resulting in its degradation. Thus,
the amount of intracellular HIFα protein depends on the balance between its expres-
sion and degradation. In conditions of low O2 availability, all HIFα proteins heterodi-
4 Hypoxia andMatrix Manipulation forVascular Engineering

80
merize with HIFβ (ARNT) and form a transcriptional complex which regulates the
transcription of numerous genes [159]. Stabilization of HIFα in the cell is controlled
by two main O2 sensing proteins, prolyl hydroxylase domain (PHD) and factor-
inhibiting HIFα (FIH), which belong to the previously mentioned biosynthetic sen-
sors category. Three isoforms of PHDs are present in all mammals [28]. Specic
proline residues on the oxygen-dependent domain of HIFα are hydroxylated by
PHDs at separate hydroxylation sites, leading to HIFα degradation. The activity of
PHDs in the cytoplasm is controlled by various O2-dependent molecular events and,
directly, by the concentration of the O2 molecule [68]. All three PHDs remain par-
tially active in normoxia. PHD activity is expected to be very sensitive to small
changes in cytoplasmic O2 levels since Km, the Michaelis-Menten parameter for the
activation of PHDs, is approximately 230–250μM, which is much higher than physi-
ological oxygen concentration (approximately 60μM) [98]. Besides, mitochondria
are also involved in the PHD activation process through their consumption of O2,
regulation of reactive oxygen species (ROS), and production of nitric oxide (NO).
While the stabilization of HIFα depends on PHD activity, the expression of HIFα is
controlled by FIHs. Therefore, when O2 levels are lowered, both the stabilization and
transactivation of HIFα increase, resulting in several angiogenic responses that will
be discussed in the following section.
NO and ROS not only contribute to the HIFα stabilization process, but they also
have several direct effects on vascular cells and blood vessels. A number of studies
have shown that NO induces angiogenesis, hyperpermeability, and vasodilation
[73]. Moreover, NO also perturbs EC respiration through the inhibition of cyto-
chrome c oxidase, which causes lower mitochondrial O2 consumption [118].
Mitochondrial ROS are also increased as a consequence of electron transport chain
inhibition, which then contributes to the deactivation of PHDs via oxidizing cofac-
tor Fe (II) and helps to stabilize HIFα. ROS production, in respect to hypoxia, is
proportional to the concentrations of intracellular O2 and electron donors. Under
hypoxia, the amount of O2 required to form superoxides is decreased, whereas the
concentration of the electron donors increases as a consequence of the reduction in
the proximal electron transport chain. Therefore, ROS production can change in
both manners, depending on the variations in these molecules’ concentrations [233].
Ushia-Fukari etal. [227] showed that ROS inuence the expression of surface adhe-
sion molecules of ECs and stimulate EC proliferation and vessel permeability.
Moreover, the hypoxia-induced decrease in ROS production leads to the inhibition
of K+ channels of pulmonary artery smooth muscle cells (SMCs), whereas an
increase in ROS production leads to intracellular Ca+ release from ryanodine-
sensitive stores [233]. Another molecular path found between mitochondrial energy
generation and K+ channel inhibition occurs through AMP kinases. The energy of
the cell is generated by the conversion of ADP to one molecule of ATP and
AMP.Hence, AMP kinase becomes highly dependent on the ADP/ATP ratio, which
is very sensitive to changes in cytoplasmic O2 concentrations. AMP kinases were
shown to inhibit K+ channels through the regulation of Ca+ release in pulmonary
arterial SMCs and also to induce cellular survival in tumor cells when exposed to
severe hypoxia [63, 171].
M. R. Blatchley et al.

81
Moreover, heme oxygenases (HOs) and NADPH oxidases (NOXs) play impor-
tant roles in the biosynthetic oxygen sensing of cells. NOX-2, one of the three iso-
forms of NOX, is used for superoxide production from molecular O2. Hypoxic
conditions can cause a decrease in NOX-2-derived ROS concentrations, due to the
low Km values (18μM) of NOX-2; this helps Ca+ release in pulmonary artery SMCs
[239]. However, some studies also suggest that hypoxia increases NOX-2 activity,
therefore causing the generation of a greater amount of ROS [233]. On the other
hand, Ca+-activated K+ channels in glomus cells were shown to be related to the
activity of HO-2, an isoform of HO which can convert heme to CO, biliverdin, and
Fe(II) using O2 and NADPH [237].
The effectiveness of an oxygen sensor can be determined by evaluating (a) its
sensitivity to small changes in intracellular O2 levels and (b) the subsequent diver-
sity of triggered cellular responses. Taking this considerations into account, PHDs
and FIHs appear to be the most critical oxygen sensors responsible for controlling
HIF activity [94]. Deactivation of these two sensors leads to HIFα stabilization,
initiating the regulation of hundreds of different genes. Using O2 as a controlling
parameter to engineer vascular tissues demands a clear understanding of the bio-
chemical events that follow changes in O2 tension, as well as the net response of the
cells and how O2 affects their collective behaviors.
4.2.1.2 Cellular Responses toDifferent Oxygen Concentrations
Metabolism andOxygen Uptake Rate
Several studies have observed that the O2 consumption of cells depends on O2 avail-
ability [1, 26, 171, 210]. We have recently shown that the O2 uptake rates (OURs) of
EPCs and human umbilical vein endothelial cells (HUVECs) are similar, but not
identical, to each other and that both decrease when O2 availability is lowered
(Fig.4.1a) [1]. Many mechanisms have been proposed to explain the relationship
between mitochondrial O2 consumption and variations in O2 levels. HIF1α was
found to be responsible for inducing the enzymes required for glycolysis [171]. It
also plays a role in activating pyruvate dehydrogenase kinase-1, which reduces the
Fig. 4.1 O2 tension regulates vascular cell responses. Comparison of EPCs and HUVECs at three
different O2 tensions in terms of (a) oxygen uptake rate (OUR) and (b, c) gene regulation [1]
4 Hypoxia andMatrix Manipulation forVascular Engineering

82
amount of pyruvate that ows into the TCA cycle and therefore decreases aerobic
respiration in mitochondria [171]. In addition, the increases in both the transcription
and expression of glucose transporter protein 1 (GLUT-1) were shown to be HIF1α-
dependent in hypoxic conditions [7]. In other words, deactivation of PHDs and FIHs
at low O2 levels leads to the stabilization of HIFα, which then reduces O2 aerobic
respiration by inducing pyruvate degradation while, at the same time, promoting
glycolysis by increasing the expression of glucose transporter proteins. Another
proposed mechanism involves the inhibition of cytochrome oxidase by NO, which
is known to be regulated by shear stress and O2 tension. NO inuences mitochon-
drial respiration by the competitive inhibition of cytochrome oxidase with O2 and by
inhibiting electron transfer between cytochrome b and c, therefore increasing ROS
production [26].
The effects of blood ow and O2 tension are crucially important for the ECs
comprising the vessel walls, since these conditions can be perturbed in many patho-
physiological situations in the body. Some studies have shown mitochondrial respi-
ration of ECs to be lower than other cell types, suggesting that most of the O2
consumption is non-mitochondrial [78, 223]. Helmlinger etal. demonstrated that
ECs consume O2 during capillary formation, whereas they also preserve and expand
the capillary structures, even under severe hypoxia (about 0.6% O2), by upregulat-
ing VEGF expression [93]. It is not surprising that ECs possess a special type of
metabolism—aerobic glycolysis in their resting state (physiological conditions) and
anaerobic glycolysis in their navigating state (hypoxic conditions)—since O2 is
transported through ECs to other tissues, and thus they must possess the ability to
survive and commence angiogenesis under hypoxic conditions [70].
Moreover, when ECs are exposed to excess glucose, their ATP generation shifts
to glycolysis, and lactate levels, increased as a by-product of glycolysis, contribute
to the inactivation of PHDs and, therefore, the stabilization of HIFα [240]. Where
blood ow is perturbed, such as in ischemia and wound healing, both NO and O2
levels are changed in blood vessels, and all of the metabolic variations discussed
become more important.
Transcription ofAngiogenic Genes
Manalo etal. showed in their study of ECs that 245 genes are upregulated and 325
genes are downregulated at least 1.5-fold in response to hypoxia and HIF1α. These
genes are responsible for the expression of collagens, GFs, receptors, and transcrip-
tion factors, all of which are signicant for the processes of angiogenesis and vas-
culogenesis. This wide range of hypoxia-related transcription factors also indirectly
affects HIF1α. The genes directly regulated by HIF1α include VEGF-A, VEGFR-1,
Flt1-1, and erythropoietin (EPO). Examples of indirectly regulated genes include
broblast growth factor (FGF), placental growth factor (PLGF), platelet-derived
growth factor (PDGF), angiopoietins (ANG-1 and ANG-2), and Tie-2, the receptor
of ANGs [68]. Although VEGF is the major GF that stimulates blood vessel forma-
tion, when it alone was transgenically overexpressed in mice, defective blood
M. R. Blatchley et al.

83
vessels formed, which then led to tissue edema and inammation [184]. On the
other hand, overexpressing both VEGF and ANG-1, which is important for main-
taining vascular integrity, has been shown to induce hypervascularity without
imperfections in mice [218]. ANG-2 is responsible for EC apoptosis and vascular
regression in the absence of VEGF, whereas, when combined with VEGF expres-
sion, it enhances angiogenic responses by destabilizing the blood vessels [100,
101]. More recently, ANG-4 was shown to function similarly to ANG-1 and to
induce angiogenesis by binding the ANG receptor TIE-2, which is also upregulated
by HIF1α [241]. We have recently shown that VEGF and ANG-2 genes are upregu-
lated in hypoxic (1% O2) cultures of EPCs and HUVECs [1], and the fold differ-
ences in upregulation levels of VEGF and ANG-2in EPCs were shown to vary
during the 3-day exposure period (Fig. 4.1b), where no signicant change was
observed for HUVECs (Fig. 4.1c). How hypoxia affects the regulation of these
angiogenic genes depends on the cell type; for instance, VEGF is upregulated in
ECs, SMCs, cardiac broblasts, and myocardiocytes, whereas ANG-2 is induced
only in ECs [159]. Therefore, from a tissue engineering perspective, co-culturing of
different cell types under controlled hypoxic conditions should be considered, since
a combination of hypoxia- induced angiogenic proteins is required to obtain vascu-
lar formation without excessive permeability.
Cell Death andSurvival
Hypoxia inuences the proliferation and viability of many cell types [1, 69, 172,
245]. The wide spectrum of HIF1α-dependent genes also includes proapoptotic and
prosurvival genes. BH3-only proapoptotic genes, a subfamily of BCL-2 that
includes BNIP3, BNIP3L, NOXA, RTP801, and HGTP-P, are directly activated by
HIF1α [234]. Although these genes play important roles in cellular apoptosis, a
growing body of evidence suggests that hypoxia mediates cellular survival in many
cell types [160, 172, 245]. Programmed cell death is, of course, a very critical step
for cells and is most likely taken only after all possible survival mechanisms have
been exhausted. One of these mechanisms, autophagy, is a cellular catabolic pro-
cess where cytoplasmic organelles are degraded to provide ATP generation in nutri-
ent deprivation. Hypoxia was found to induce mitochondrial autophagy via both
HIF1α- dependent and HIF1α-independent pathways [172, 245]. Small interfering
RNA silencing of BNIP3 and BNIP3L together suppresses autophagy to a greater
extent than silencing only one of them at a time [20]. Zhang etal. have shown that
mitochondrial autophagy is induced by HIF1α-dependent upregulation of BNIP3
incorporated into the constitutive expression of BECLIN-1 and ATG-5 [245]. On
the other hand, the neuron-derived orphan receptor (NOR-1), which is overex-
pressed in ECs exposed to hypoxia, mediates cellular survival as a downstream
effector of HIF1α signaling [160]. CD105, one of the EC markers also shown to
play a role in cellular survival, is signicantly upregulated under hypoxia [146]. In
vivo studies of rats subjected to hypoxia also found the induction of mitochondrial
autophagy by overexpression of BNIP3 [17]. In addition, Papandreou etal. propose
4 Hypoxia andMatrix Manipulation forVascular Engineering

84
that hypoxia induces autophagy in tumor cells through AMP kinase, which is acti-
vated by hypoxia independently of HIF1α, as discussed previously in the O2 sensing
section [172].
4.2.1.3 Cell Pluripotency andDifferentiation
Vasculogenesis takes place in low O2 environments, such as the early development
of the embryo, EPC regeneration in the BM, or EPC attachment and differentiation
into mature ECs at neovascularization sites. All of these processes rely on pluripo-
tent/unipotent cells differentiating into the endothelium, where O2 tension is a cru-
cial parameter regulating their differentiation characteristics. As already discussed,
EPC regeneration in the BM depends on cellular dynamics between the osteoblas-
tic niche (low O2) and vascular niche (high O2); HSCs are quiescent in the osteo-
blastic niche and differentiate into EPCs in the vascular niche before joining the
circulation [111]. Therefore, it is important to understand the effect of O2 tension
on the differentiation of cells into EPCs/ECs as a primary step of vasculogenesis.
Hypoxia enhances human embryonic stem cell (hESC) pluripotency via the upreg-
ulation of Oct-4, NANOG, and SOX-2, which are pluripotent markers [45, 64, 69,
116]. HIF2α is responsible for the overexpression of Oct-4, SOX-2, and NANOG,
while HIF3α also plays a role in the process by inducing HIF2α transcription [45,
69]. Prasad etal. demonstrated that hypoxic conditions (5% O2) prevent the spon-
taneous differentiation of hESCs, whereas the inhibition of Notch activation
revoked this effect, suggesting that hypoxia-induced pluripotency occurs via Notch
signaling [182]. On the other hand, the efciency of the process of reprogramming
mouse and human somatic cells into induced pluripotent stem cells (iPSC) was
shown to be improved in 5% O2 cultures, compared to atmospheric O2 cultures
[243]. In contrast, other studies have demonstrated that hypoxia induces the expres-
sion of early cardiac genes in spontaneously differentiating embryoid bodies (EBs)
[125, 168]. In a more recent study, Lopez etal. showed that EPCs/ECs can be
obtained from hESCs more efciently when cultured in 5% O2, compared to previ-
ous methods that induce EB formation in atmospheric O2 [181]. Additionally, sim-
ply priming EBs in hypoxic conditions mediated suppression of pluripotent marker
Oct-4 and upregulated VEGF [140]. When hPSCs are differentiated toward an
endothelial lineage, hypoxia has also been shown to enhance EC differentiation
through changes in the early stages (mesodermal specication) of EC lineage com-
mitment. Interestingly this affect, which was dependent on low O2 tension, was
driven by production of ROS [135]. More in-depth investigations have uncovered
the specic role of NADPH oxidase 2 (Nox2)-produced ROS in upregulating Notch
signaling to facilitate differentiation toward arterial endothelial cells [119]. Another
group showed a biphasic regulation of EC fate specication via a HIF1α-mediated
pathway in mESCs. Hypoxia led to upregulation of the transcription factor Etv2in
the early stages of differentiation, which resulted in development of endothelial
progenitor cells. Continued exposure to hypoxia led to HIF1α-induced upregula-
tion of Notch1 signaling and formation of functional arterial endothelial cells, with
M. R. Blatchley et al.

85
the capacity to contribute to revascularization of ischemic tissues [224]. Similarly,
HIF1α also induces the differentiation of peripheral blood mononuclear cells into
EPCs, and hypoxia stimulates the further differentiation of EPCs into mature ECs
[8, 117].
All of these ndings highlight the signicance of O2 tension as a critical param-
eter to control vascular differentiation of pluripotent or multipotent cells. Although
some of these studies suggest contrary hypotheses, the importance of O2 consider-
ations in cell culture environments cannot be overstated, as O2 tension can be
manipulated to prevent spontaneous differentiation of pluripotent cells and to
enhance the efciency of the differentiation into EPCs and mature ECs.
4.2.2 Vascular Responses toECM
In the human body, vascular cells are surrounded by diverse components of the
ECM, the unique spatial and temporal distribution of which affects GF availability
and matrix properties which, in turn, regulate vasculogenesis and angiogenesis. Just
like oxygen tension, which varies throughout vascular development, ECM compo-
nents are also uniquely distributed; for example, hyaluronic acid (HA; also known
as hyaluronan) levels were found to be highest during embryogenesis and to be
replaced by bronectin and then collagen, which remains abundant throughout
adulthood. In this section we will discuss ECM distribution and its effects on vascu-
lar development and maintenance. Then, we will discuss various ECM components
that affect vascular morphogenesis. Lastly, we will describe strategies for manipu-
lating the ECM using synthetic biomaterials and emerging technology.
4.2.2.1 Types ofECM Found Participating inVascularization
The ECM surrounding blood vessels contributes signicantly to their diverse func-
tions and complexity. This ECM diversity encompasses different vascular develop-
ment periods (i.e., embryonic versus adult) and specialized vessels at various
locations in the body (i.e., capillary, arteriole, and venule) or tissues in the body
(i.e., heart, kidney, lung, etc.). During early vascular development, the ECM pro-
vides informational cues to the vascular cells, thus regulating their differentiation,
proliferation, and migration. Fibronectin and HA, which are major components of
the embryonic ECM, have been shown to be vital regulators for vascularization dur-
ing embryogenesis [222]. Fibronectin, a unique glycoprotein, contains cell adhesion
and heparin-binding sites that synergistically modulate the activity of VEGF to
enhance angiogenesis [236]. Various lineage studies have found developmental
abnormalities in embryonic hearts and vessels in bronectin-null mice, suggesting
its crucial role in mediating EC interactions [14, 71]. The levels of hyaluronan, a
nonsulfated linear polysaccharide, are greatest during embryogenesis and then
decrease at the onset of differentiation [221], where it plays a crucial role in
4 Hypoxia andMatrix Manipulation forVascular Engineering

86
regulating vascular development [18]. Hyaluronan and its receptor, CD44, have
been shown to be essential in the formation and remodeling of blood vessels [18, 30,
62]. We have previously reported that a completely synthetic HA hydrogel can
maintain the self-renewal and pluripotency of hESCs [79, 86, 87]. Interestingly,
when VEGF is introduced into the culture media, this unique HA microenvironment
can direct the differentiation of hESCs into vascular cells, as indicated by positive
staining for α-smooth muscle actin and an early stage of the endothelial cell marker,
CD34 (Fig.4.2a). More recent studies have employed higher throughput methods to
explore the effects of ECM composition on EC fate. As the ECM of the developing
Fig. 4.2 Matrix composition and orientation affect vasculogenesis. (a) Hyaluronic acid microen-
vironment for vasculogenesis. Human ESC colonies were cultured in conditioned medium for
1week, followed by the replacement of medium containing 50ng/ml VEGF165. Left: Cell sprout-
ing was observed after 48h of culture in medium containing VEGF (indicated by arrowheads).
Middle and right: After 1week of differentiation, sprouting elongating cells were mainly positive
for alpha-smooth muscle actin (a-SMA) (middle), while some were positive for the early-stage
endothelial marker CD34 (right). Scale bars—left, 100μm; middle and right, 25μm. Printed with
permission [79]. (b) Nanotopography induces the formation of supercellular band structures in
long-term EPC culture. EPCs cultured on at substrates began forming conuent layers of cells
after 6days of culture. In contrast, EPCs cultured on nanotopography began to form supercellular
band structures aligned in the direction of the features (as indicated by the arrow) after 6days of
culture. These morphological differences are evident through staining of PECAM-1 and
VE-CAD.Scale bars are 50μm. Printed with permission [23]. (c) Organized capillary tube forma-
tion in vitro. Capillary-like structures (CLSs) were induced by the addition of Matrigel after
6days. EPCs cultured on at substrates (upper left) formed low-density unorganized structures,
while EPCs cultured on nanotopographic substrates (upper right) formed extensive networks of
organized structures with (lower panel) longer average tube lengths than EPCs cultured on at
substrates (*** p<0.001). The direction of the linear nanotopographic features is indicated by the
arrow. Scale bars are 200μm. Printed with permission [23]
M. R. Blatchley et al.

87
embryo consists of multiple components, culturing ECs on combinatorial ECM
arrays revealed optimal conditions for EC survival, in response to low O2 and low
nutrient availability [104], as well as enhancements in EC fate which were regulated
by ECM composition, at least partially through upregulation of integrin β3 and its
associated signaling pathway [105].
In contrast, the adult ECM consists mostly of a laminin-rich basement mem-
brane, which maintains the integrity of the mature endothelium, and interstitial col-
lagen I, which promotes capillary morphogenesis [50]. Although collagen I is
present during development, its role becomes increasingly important in postnatal
angiogenesis, after its reactive groups have been cross-linked to further stabilize the
interstitial matrix [186]. EC integrins, which interact with collagens and brin, are
key receptors in EC activation, proliferation, and tubular morphogenesis. The
collagen- I-mediated activation of Src and Rho and the suppression of PKA promote
the formation of prominent actin stress bers, which mediate EC retraction and
capillary morphogenesis. Moreover, the activation of Src also disrupts VE-cadherin
from cell junction and cell-cell contact which, in turn, facilitates multicellular reor-
ganization. Conversely, basement membrane laminin-1 is responsible for maintain-
ing the mature endothelium. During the proliferative stage of morphogenesis, the
laminin-rich basal lamina is degraded, exposing the tips of sprouting ECs to the
underlying interstitial collagens and activating signaling pathways that drive cyto-
skeletal reorganization and vascular morphogenesis. This sharp difference in how
ECM components affect capillary morphogenesis is responsible for controlling the
delicate balance between vascular sprouting and maturation.
Once nascent vessels are formed, ECM components regulate their maturation
and specialization into capillaries, arteries, and veins. Capillaries, the most abun-
dant vessels in our body, consist of ECs surrounded by pericytes and basement
membrane. Exchanges of nutrients and oxygen occur through diffusion between
blood and tissue in these regions, due to the capillary’s thin wall structure and large
surface-area-to-volume-ratio. Maturation of the vessel wall involves the recruitment
of mural cells, development of the surrounding matrix, and organ-specic special-
ization [113]. ECM distribution in various tissues dictates the specialization of these
capillaries to support the functions of specic organs. The capillary endothelial
layer is continuous in most tissues (e.g., muscle), while it is fenestrated in exocrine
and endocrine glands (e.g., kidney and pancreas). Moreover, the enlarged sinusoidal
capillaries of the liver, spleen, and BM are discontinuous, allowing increased
exchange of hormones and metabolites between the blood and the surrounding tis-
sues. In contrast, where the excess exchange of molecules is not desirable, such as
at the blood-brain barrier and the blood-retina barrier, the interendothelial connec-
tion is further reinforced with tight junctions, such as occludin and ZO-1 [238].
Compared with capillaries, arterioles and venules have an increased coverage of
mural cells and ECM components. Arterioles are completely surrounded with vas-
cular SMCs that form a closely packed basement membrane. The walls of larger
vessels are composed of three layers: the tunica intima, the tunica media, and the
tunica adventitia. The EC layer of blood vessels is anchored to a basement mem-
brane, which is the major component of the tunica intima [57]. The basement mem-
4 Hypoxia andMatrix Manipulation forVascular Engineering

88
brane contains network-organizing proteins, such as collagen IV, collagen XVIII,
laminin, nidogen, entactin, and the proteoglycan perlecan. The tunica media con-
tains vascular SMCs (v-SMCs) and elastic tissue composed of elastin, brillins,
bulins, emilins, and microbril-associated proteins. The tunica adventitia contains
broblasts and elastic laminae and has its own blood supply, known as the vasa
vasorum [57]. SMCs and elastic laminae contribute to the vessel tone and regulate
vessel diameter and blood ow. This generic blood vessel architecture is modied
with various ECM components to fulll their individual tasks. Arteries, which func-
tion to deliver oxygenated blood, usually have a thick tunica media with numerous
concentric layers of v-SMCs, whereas veins have a thick tunica adventitia layer
enriched in ECM components with elastic properties, such as elastin and brillin.
As described, the composition of the ECM is inherently dynamic throughout
development as well as vascular regeneration, positing the importance of remodel-
ing and deposition of new ECM as these processes progress. Additionally, stability
of mature vessels requires a different ECM composition than developing or regen-
erating vasculature. Several studies have highlighted these changes in ECM deposi-
tion and have identied regulators of these important mechanisms. Much of the
work to date has established the role of perivascular cells, including pericytes and
smooth muscle cells, in ECM production [232]. Crucially, ECs also produce ECM
as blood vessels form. Of particular interest, endothelial progenitor populations and
mature ECs produce ECM differently; EPCs produce collagen IV, bronectin, and
laminin, while mature ECs have limited ECM production in standard cell culture
conditions. However, when subjected to hypoxic conditions, mature ECs adopt an
ECM secretome similar to the pro-regenerative EPCs, wherein they secrete collagen
IV, bronectin, and laminin at low O2 (1%). At moderate hypoxia (5% O2), both cell
types produce collagen I [134]. When developing engineered vasculature, these fac-
tors are critical to consider to obtain mature, long-lasting blood vessels, as ECM
composition is an important parameter governing vascular stability.
4.2.2.2 Properties oftheECM that Affect Vascular Morphogenesis
Recent decades have vastly expanded our understanding of how ECM properties
affect vascular assembly, primarily due to newly available, well-dened invitro
models. The most common models are cultures of ECs in gels made of different
ECM components, such as collagen, brin, bronectin, and Matrigel. These ECM
components contain instructive physical and chemical cues that direct vascular mor-
phogenesis, which involves several steps: (1) proteolytic degradation of basement
membrane proteins by both soluble and membrane-bound matrix metalloprotein-
ases (MMPs); (2) cell activation, proliferation, and migration; (3) vacuole and
lumen assembly into a tube with tight junctions at cell-cell contacts; (4) branching
and sprouting; (5) synthesis of basement membrane proteins to support the forma-
tion of capillary tube networks; and (6) tube maturation and stabilization by peri-
cytes. These complex processes require a delicate balance between various
immobilized and soluble GFs, as well as endothelial and perivascular cell
M. R. Blatchley et al.

89
interactions. Gels made from ECM components, engineered to have properties
resembling those of native tissues, have been widely explored as a tool to study the
molecular regulation underlying vascular development [49] and as a scaffold to
transplant vascular progenitor cells [15, 46, 164]. However, their manipulation for
vascular tissue engineering has been narrowly limited by their inherent chemical
and physical properties. Therefore, a great need exists to chemically modify these
ECM components [40, 130] or to utilize biomaterials to form scaffolds from hydro-
gels, which are xeno-free and instructive for vascular tissue engineering [152].
Hydrogels are cross-linked polymer networks which can store a large amount of
uid and which have biophysical properties similar to many soft tissues [138].
Hydrogels can be engineered from natural biomaterials (including ECM compo-
nents), articial protein polymers, self-assembling peptides, and synthetic polymers
to form scaffolds which mimic the native ECM.For example, dextran and chitosan,
natural biomaterials with similar structures, do not possess any inherent cross-link-
ing ability [214, 215]. However, a simple chemical modication, such as introduc-
ing double bonds into the repeating unit, allows the cross-linking of these
polysaccharides to form hydrogels. Alginate is another natural material which can
be physically cross-linked by adding cations (e.g., Ca2+ or Mg2+) [80]. Another
approach utilizes a purely synthetic polymer, like polyethylene glycol (PEG) or
poly-[lactic-co- glycolic acid] (PLGA), whose physical and chemical properties can
be easily manipulated. A simple modication can turn PEG, a cell-resistant mate-
rial, into an instructive scaffold designed to promote vascularization [58, 59, 166,
176]. Furthermore, the synthetic material of choice must be biodegradable and bio-
compatible, and such physical properties as pore size, degradation kinetics, and
matrix mechanical properties must be easily tunable to favor vascular morphogen-
esis. Bioactive molecules—like GFs, cell adhesion motifs such as arginine-glycine-
aspartic acid (RGD), and MMP-sensitive peptides—must be presented with correct
spatial and temporal distributions within the synthetic biomaterials. Next, we will
discuss several strategies for manipulating the chemical and physical properties of
synthetic biomaterials.
Cell Adhesion Regulates Neovascularization
In order to support vascular cells and instruct them to undergo vascular morphogen-
esis, synthetic biomaterials must rst be able to provide cell adhesion. Instead of
incorporating ECM components to make such materials bioactive, certain synthetic
peptides important for vascular morphogenesis can be incorporated into these inert
synthetic materials. The most common template is the integrin-binding domain of
bronectin, RGD [178], and the laminin-derived peptide IKVAV [203]. The rst
crucial step in vascular morphogenesis occurs when vascular cells utilize integrin
receptors to sense their surrounding microenvironments. Integrins are transmem-
brane receptors which not only maintain cell adhesion to ECM but also control cell
proliferation, migration, differentiation, and cytoskeletal organization. Since blood
vessels must be able to assemble in diverse tissue environments (e.g., adult versus
4 Hypoxia andMatrix Manipulation forVascular Engineering

90
embryo and muscle versus kidney), which have different distributions of ECM com-
ponents (as discussed in the previous section), it is evident that both β1 and αv integ-
rins can support vascular morphogenesis. For example, αvβ3 and α2β1 integrins
associate with vascular morphogenesis in collagen-rich ECM, like adult tissue,
while α5β1 and α6β1 integrins involve bronectin- and brin-rich ECM, like in embry-
onic tissue and healing wounds [50]. The binding of integrins onto RGD triggers
several downstream signaling events mediated by Rho GTPase, particularly Rac1
and Cdc42 [49]. Extensive work by Davis and his colleagues revealed the molecular
mechanism that regulates this EC morphogenesis in brin and collagen gels (an
excellent review of their work can be found in Chap. 20 of this book). This mecha-
nism has also been observed and controlled in synthetic (HA-based) hydrogels [89].
To further substantiate the role of cell-ECM interactions, particularly those
mediated by integrin engagement, several groups have identied the importance of
integrin specicity in vascular regeneration. In tumor vessels, αvβ3 is preferentially
expressed, leading to formation of new, albeit disorganized, leaky vasculature [53].
In order to establish organized, mature neovessels, engagement of α3/α5β1, rather
than αvβ3, was necessary [147]. While RGD peptides facilitate cell adhesion in syn-
thetic matrices, it is important to consider the non-specic integrin engagement
potential of these peptides, which may inuence vascular regeneration.
The number of RGD adhesion sites and the method of their presentation to the
vascular cells are also crucial in affecting cell migration [82] and vascular morpho-
genesis [110]. Using an invitro angiogenesis model, Folkman and Ingber were able
to show that, when cultured on a moderate coating density that only partially resisted
cell traction forces, ECs could retract and differentiate into branching capillary net-
works [67, 110]. High ECM density was saturated with RGD adhesion peptide,
which allowed the ECs to spread and proliferate, while low ECM density resulted in
rounded and apoptotic cells. Interestingly, in medium ECM density, with the appro-
priate RGD adhesion peptide, ECs collectively retracted and differentiated into
branching capillary networks with hollow tubular structures. It is evident that the
ECs exerted mechanical forces on the surrounding ECM to create a pathway for
migration and branching in forming vascular structures [48]. Hence, both the quan-
tity of RGD peptide and the method of presentation within the engineered synthetic
biomaterials determine the initial morphogenetic events in angiogenesis.
Scaffold Degradation Regulates Vascular Morphogenesis
Scaffolds made from ECM components, like collagen and brin gels, contain pro-
teolytic degradable sequences which can be degraded by the MMPs and other pro-
teases (e.g., cathepsins) secreted by vascular cells. This cell-mediated degradation
controls both structural integrity and temporal mechanical properties, which dictate
the presentation of chemical and mechanical cues at various stages of angiogenesis.
However, the degradation kinetics of these ECM-based scaffolds is determined by
their inherent cross-linking density which, in turn, limits their manipulation for vas-
cular tissue engineering. In contrast, synthetic biomaterials can be engineered to
M. R. Blatchley et al.

91
have degradation proles ranging from days to months, in order to suit the specic
needs of the engineered vascularized tissue constructs [215]. The polymer backbone
can be cross-linked using a nondegradable cross-linker that provides structural
integrity and/or a degradable cross-linker that allows directed cell migration and
vascular morphogenesis. Hydrolytic degradation by the body uid can break down
the ester bonds within the polymer backbone, allowing tissue inltration over time
[214, 215]. MMP-sensitive peptides can also be used to cross-link hydrogels, allow-
ing cell-mediated degradation, leading to a rapid response of vascular growth.
Overall, by adjusting the percentages of nondegradable and degradable cross-link-
ers, scaffold degradation can be tuned to allow cellular inltration, lumen forma-
tion, and ECM synthesis and distribution.
In order for the intracellular vacuoles to coalesce into a lumen, ECs require adhe-
sive ligands for traction [152] and utilize membrane-type-1 MMPs (MT1-MMPs) to
create physical spaces which facilitate the directed migration of cells to align with
neighboring cells [48, 192, 212]. Therefore, ECs can only invade this synthetic scaf-
fold if the minimal pore size is larger than the cell diameter (e.g., a soft self-
assembling peptide) [201] or if the scaffold bears an MMP-degradable sequence
[153]. The Hubbell research group has pioneered this approach by incorporating an
MMP-degradable sequence as a cross-linker into PEG scaffolds to promote vascular
healing and therapeutic angiogenesis [196, 249]. When grafted invivo, ECs were
able to invade, remodel, and vascularize this MMP-sensitive scaffold [248, 249].
Using concepts from this work, synthetic (HA-based) biomaterials utilized spatial
control of degradation through photopatterning to organize vascular morphogenesis
(Fig. 4.3) [90]. Hence, incorporating MMP-degradable peptides is essential for
directing vascular morphogenesis in 3D synthetic biomaterials.
Physical Orientation oftheECM
The native ECM provides an instructive template for ECs and perivascular cells to
orient, interact, and organize into tubular structures. Studies have demonstrated that
a stable vasculature could be achieved by co-transplantation of ECs and perivascu-
lar cells, such as MSCs or SMCs [15, 16, 128, 142, 164]. Recent studies showed that
engineering a stable vascularized tissue construct requires the triculture of ECs,
broblasts, and tissue-specic cells, such as cardiac or skeletal muscle cells [31,
142]. Perivascular cells, such as broblasts, stabilize the developing vascular tube
through physical support, by differentiating into v-SMCs and wrapping around the
nascent tube [114, 229], and chemical support, by secreting Ang-1, PDGF-BB, and
tissue inhibitor of metalloproteinase-3 (TIMP-3) [95, 97]. These perivascular cells
are also responsible for laying down ECM components in early embryogenesis and
continue to do so throughout adulthood. Many studies using broblast-derived
matrices have further revealed the 3D complexity of these ECM networks [206–
208]. A study by Soucy and Romer showed that broblast-derived matrix alone is
sufcient to induce HUVECs to undergo vascular morphogenesis, independent of
any angiogenic factors. Further analysis of protein colocalization suggested that
4 Hypoxia andMatrix Manipulation forVascular Engineering

Fig. 4.3 Spatial control of vascular morphogenesis in synthetic hydrogels. (a, b) Uniform −UV
(permit cell-mediated degradation) and +UV (inhibit cell-mediated degradation) hyaluronic acid
(HA) hydrogels are grafted onto the CAM membrane. (a) LM imaging and (b) confocal analysis
of the boxed regions in a shows CAM vessels penetrating into the −UV but not into the +UV
hydrogels. CAM vessels are stained with Fluorescein-conjugated Lens culinaris lectin. Scale bars
in a, 20mm, and b, 100 μm. H = hydrogels. Dotted white lines indicate the boundaries of the
hydrogels. (c) ECFCs are seeded on top of a uniform −UV (c) and 100μm stripe photopatterned
(d) HA hydrogels for an angiogenesis assay. After 3days in culture, ECFCs invade and sprout into
the 3D hydrogels (c). When photopatterned HA hydrogels are used, invasion and sprouting are
observed only within the −UV regions and not within the inhibitory +UV region (d). Conuent
monolayer of ECFCs sprouts and invades the −UV region (i) and further branches along the −UV
regions (ii). The +UV regions are labeled using MeRho (red); ECFCs are stained with uorescein-
conjugated UEA-I lectin (green) and DAPI (blue). Scale bars are 100 μm. Reproduced and re-
formatted with permission [90]

93
bronectin with a distinct structure and organization was uniquely distributed
among other secreted matrix components, such as collagen, tenascin C, versican,
and decorin. Cell matrix adhesions and MT1-MMP activities were reported to ori-
ent and localize within this brous bronectin, which is indicative of integrin-
mediated vascular morphogenesis [190]. In fact, ECs initiate neovascularization by
unfolding soluble bronectin and depositing a pericellular network of brils that
serve as a structural scaffolding on a mechanically ideal substratum for vessel
development [247]. We have studied how such bronectin organization inuences
endothelial tube formation by patterning bronectin on cell culture surfaces to opti-
mize vasculogenic potential and understand how microstructure inuences vascular
tube formation [54]. Alignment of other important ECM proteins, such as collagen,
has also been shown to guide vascular regeneration by enhancing EC organization
and migration [136]. Interestingly, similar effects in vascular organization are
observed when tensile forces are incurred upon vascular brin-based constructs,
where vascular network alignment was induced by application of force. Aligned
microvasculature was shown to enhance vascular integration upon implantation in
abdominal muscle [191]. This last study suggests a potential mechanism for organi-
zation of ECM components to guide vascular network organization through force-
induced remodeling. It is likely such a mechanism is coupled with ECM degradation
and secretion of new ECM components to establish a microenvironment amenable
to the formation of new blood vessels.
The unique orientation, organization, and nanotopography of brous bronectin
represent features that can be integrated into synthetic scaffolds. Synthetic poly-
mers, like PLGA and polycaprolactone (PCL), can be electrospun to produce vari-
ous ber sizes with micro- to nanoscale features that resemble brous bronectin.
We previously showed that surface nanotopography enhanced the formation of
capillary-like structures (CLSs) invitro [23]. Growing EPCs on grooves that were
600 nm wide reduced their proliferation and enhanced their migration without
changing the expression of EC markers. Moreover, after 6 days of culture, the EPCs
organized into superstructures along the nanogrooves, in signicant contrast to the
EPCs grown on planar surfaces (Fig.4.2b). The addition of Matrigel further induced
the formation of CLSs, with enhanced alignment, organization, and tube length
compared to a at surface (Fig.4.2c). This underscores the increasingly important
role of nanotopography in guiding and orienting vascular assembly. When inte-
grated into the tissue-engineered construct—for instance, using lamentous scaf-
fold geometry [75] and micropatterning [55, 108, 165]—the orientation and
structure of the engineered vasculature can be controlled.
Regulating Matrix Mechanics
It has become increasingly evident that the biomechanical properties of the ECM,
such as matrix orientation and mechanics, profoundly inuence the control of vas-
cular morphogenesis. Due to their versatility with respect to mechanical properties
(e.g., cross-linking density, pore sizes, and topography), synthetic biomaterials have
4 Hypoxia andMatrix Manipulation forVascular Engineering

94
powerful features that can be exploited to further direct vascularization. Changes in
ECM mechanics can lead to changes in GF availability [40, 110], drive capillary
morphogenesis [109], and stimulate angiogenesis invivo [122]. By altering matrix
adhesive characteristics and mechanics, Ingber and Folkman illustrated how bFGF-
stimulated ECs can be switched between growth and differentiation during angio-
genesis [110]. Recently, biomechanical cues from the ECM and signals from GF
receptors have been implicated in regulating the balance of activity between TFII-I
and GATA2 transcription factors, which govern the expression of VEGFR2 to insti-
gate angiogenesis [158]. Matrix stiffness regulates not only the cell’s response to
soluble GFs but also cell morphogenesis during angiogenic sprouting. Primarily
due to MMP activity, the tip of a new capillary sprout becomes thinner, locally
degrading the basement membrane proteins. This region, with its high rate of ECM
turnover and thin basement membrane, becomes more compliant and stretches more
than the neighboring tissue. Consequently, the decrease in matrix stiffness changes
the balance of forces across the cell integrin receptors, increases cell tension, and
results in cytoskeletal arrangement to form branching patterns that are characteristic
of all growing vascular networks [109].
The pioneering work by Deroanne etal. showed that a decrease of matrix stiff-
ness increased capillary branching and the elongation of tubes. A reduced tension
between ECs and ECM, accompanied by a profound remodeling of the actin-FAP
complex, is sufcient to trigger an intracellular signaling cascade leading to tubulo-
genesis [52]. This observation has been further conrmed in collagen gels [52, 200],
brin gels [211], self-assembling peptides, and HA-gelatin hydrogels.
Although ECM-based gels, such as collagen, brin, and Matrigel, have been
widely used in angiogenesis assays, their inherent physical properties have limited
their usage when studying the effects of matrix mechanics on angiogenesis. The
stiffness of ECM-based gels can be increased either by increasing their concentra-
tion, which also alters their ligand and bril density [189], or by altering the cross-
linking of ECM proteins in a narrow range using a microbial transglutaminase
[244]. Therefore, examining the effects of matrix stiffness alone on angiogenesis
requires the use of synthetic hydrogels, the stiffness of which can be easily adjusted
over a wide range of moduli without altering other chemical properties. Unlike nat-
urally available ECM-based gels, the elasticity of which is limited to their inherent
cross-linking density, synthetic HA hydrogels can be used to study a physiologi-
cally relevant range of matrix elasticity [88]. When the cross-linking density of the
HA-gelatin hydrogels was further reduced, the matrix elasticity became relatively
compliant, resulting in an increase of capillary branching, elongated tubes, and
enlarged lumen structures [88]. On a relatively compliant matrix, EPCs can produce
fewer MMPs than a stiffer matrix would require and still degrade, exert mechanical
tension on, and contract the matrix to enable vascular morphogenesis. On the other
hand, EPCs must produce more MMPs on a stiffer matrix, to overcome the extra
mechanical barriers; even then, this local decrease in substrate stiffness cannot sup-
port vascular morphogenesis (Fig.4.4). This model also explains the rapid appear-
ance of large functional vessels in granulation tissue, as a response to the
wound-healing mechanism [122].
M. R. Blatchley et al.

95
In addition to the effects of matrix stiffness on postnatal vascular regeneration,
matrix stiffness has been probed as an important regulator of stem cell fate.
Beginning with the pioneering work of Engler etal. [60], studies examining the
effects of substrate stiffness and mechanical signaling transduction pathways on
stem cell fate have proven instrumental in enhancing our collective knowledge of
differentiation schema. To this end, our group has shown that substrate stiffness can
govern EC fate through alterations in mesodermal precursors. Similar to the
enhancements in EC fate observed upon culture in low O2 environments (Fig.4.5a–
d) [135], compliant substrates enhance mesodermal differentiation, which results in
robust EC differentiation (Fig.4.5e–i) [205].
A recent illuminating study identied stress relaxation as an important, yet
understudied, regulator of mechanical signal transduction. Specically, in
alginate- based hydrogels with the same matrix stiffness and pore size, altering stress
relaxation modulated MSC cell fate [34]. While studies of the effect of stress relax-
ation on EC fate and vascular morphogenesis have not been published, stress relax-
ation is an important parameter to bear in mind for biomaterial design, particularly
because covalently cross-linked hydrogels do not exhibit stress relaxation behavior
similar to that of the native ECM.
Fig. 4.4 Mechanoregulation of vascularization. (a) EPCs were seeded on rigid, rm, and yielding
substrates for 12h, supplemented with 1ng/ml (low) VEGF (upper panel) and formed CLSs when
supplemented with 50ng/ml (high) VEGF (lower panel), as demonstrated by uorescence micros-
copy of F-actin (green) and nuclei (blue). (b) Real-time RT-PCR revealed a signicantly increased
expression of (1) MT1-MMP, (2) MMP-1, and (3) MMP-2in response to 50ng/ml VEGF (high)
concentration for EPCs cultured on the rigid, rm, and yielding substrates, respectively. As the
matrix substrate was reduced, EPCs cultured in medium supplemented with 50ng/ml (high) VEGF
showed a decrease in expression of these MMPs. (c) Metamorph analysis of CLSs revealed a sig-
nicant increase of mean tube length and mean tube area, as substrate stiffness decreased. Confocal
analysis of nuclei (blue), VE-CAD (red), and lectin (green) further revealed that branching and
hollow tubular structures formed on the yielding substrate. Signicance levels were set at *p<0.05,
**p<0.01, and ***p<0.001. Scale bars (a) 100μm and (c) 20μm. Printed with permission [88]
4 Hypoxia andMatrix Manipulation forVascular Engineering

Fig. 4.5 Low O2 and compliant substrates enhance induction of mesodermal precursor popula-
tions, thereby improving EC fate specication. (a) Schematic of manipulated O2 environments stud-
ied during differentiation. (b) RT-PCR analysis of VEcad and CD31 expression of EVCs
differentiated under the four studied oxygen conditions. Comparison of secondary and primed 5%
O2 conditions demonstrated by (c) light microscopy images (arrows indicate elongated cell bundles;
arrowheads indicate cobblestone area-forming cells; scale bar is 100 μm) and (d) ow cytometry for
VEcad expression. Isotype control in gray. *p<0.05; **p<0.01; ***p<0.001. (e) Schematic of stiff-
ness-primed mesoderm induction followed by EC differentiation on E~3GPa substrates. a-MEM,
a-minimum essential medium; FBS, fetal bovine serum; EGM, endothelial growth medium. (f)
Gene expression of mesodermal markers for cells differentiated on soft 3-kPa substrates and stiff
1.7-MPa substrates, normalized to expression from E~3GPa surfaces. Color key is presented in
log10 scale. (g) Bright-eld images of cobblestone endothelial colonies (white arrows) on day 12
EVCs. (h) Day 12 EVC ow cytometry plots of VECad expression in red, with corresponding
HUVEC VECad expression in green. Black font, VECad+ cells; green font, highly expressing
VECad+ cells. Data are presented as means ± SEM. (i) Representative immunouorescence images
of VECad expression on day 12 EVCs: low-magnication (top) and high- magnication (bottom)
images are shown (green, VECad; red, phalloidin; blue, nuclei). Reproduced and re-formatted with
permission [135, 205]

97
These studies underline the importance of engineering a tissue construct with a
matrix stiffness amenable to promote invivo vascularization. However, investigat-
ing how matrix stiffness may affect invivo vascularization remains challenging due
to the complexity of the system, which involves matrix remodeling, host capillary
ingrowth, as well as anastomosis of the vascular construct and contributions from
other cell types. For example, invivo vascular ingrowth into Matrigel scaffolds was
found to be optimal at intermediate matrix stiffness, in sharp contrast to the observed
invitro ingrowth [158]. Elegant work by Yoder’s research group also found that
increasing the collagen concentration yielded stiffer scaffolds, which in turn pro-
moted host capillary ingrowth invivo. Compared to stiffer scaffolds, softer scaf-
folds might have experienced excessive invivo remodeling and failed to retain the
vascular constructs. Moreover, invitro angiogenesis studies have found that ECM-
based gels produce a much narrower range of stiffness [46, 158] than synthetic
hydrogels [88, 158]. Future investigations are needed to evaluate vascularization by
both the host capillary and the engineered vascular construct over a wider range of
physiologically relevant matrix elasticities. Despite the differences in scaffold com-
position (ECM-based gels versus synthetic hydrogels), culture conditions (in vitro
versus invivo), assay type (2D versus 3D), and ranges of matrix stiffness, all of
these studies highlight the relevance of engineering scaffolds with mechanical elas-
ticity suited to the specic needs of tissue vascularization.
4.2.3 The Effects ofOxygen Availability andtheECM
In this section, we will consider O2 tension and the ECM as two interdependent fac-
tors determining the efciency of vasculature formation. We will review currently
available O2 measurement techniques and challenges, along with the mathematical
modeling approaches used to overcome some of these challenges in describing O2
gradients in 3D environments. Then, we will discuss cellular adaptations and
responses to O2 availability in 3D ECM constructs and the possible outcomes of
variations in O2 distribution in 3D cultures of vascular cells.
4.2.3.1 Varying Oxygen Tensions intheECM ofTissue andMatrix
Scaffolds: Measuring andModeling
Oxygen Measurement Techniques andChallenges
Manipulation of oxygen, in order to direct pluripotent or vascular cells to form
blood vessels, requires knowing the precise O2 tension that the cells are exposed to
under varying conditions. Many different O2 measurement techniques have been
used invitro and invivo. The accuracy of these measurements is fundamental to
condently describe the cellular responses under various O2 availabilities, as well as
to controlling the O2 tension in order to direct angiogenesis and vasculogenesis. An
4 Hypoxia andMatrix Manipulation forVascular Engineering

98
O2 measurement method needs several properties to be considered superior, includ-
ing accuracy, sensitivity, repeatability, rapidity, and noninvasiveness. Although
some methods are used more commonly in a broader range of applications, no “gold
standard” exists for all applications, since the method chosen usually depends on
the purpose of the measurement. In vivo O2 measurement methods can be divided
into two main categories: (1) direct measurements, where the concentration or the
partial pressure of O2 is directly measured, and (2) indirect measurements, where
levels of O2-indicative molecules (e.g., hemoglobin, cytochrome) are detected and
correlated to relative O2 concentrations.
The most common direct measurements are electrodes, phosphorescent probes,
electron paramagnetic resonance (EPR) oximetry, and nuclear magnetic resonance
(NMR). Some of the indirect measurement methods involve monitoring of hemo-
globin/myoglobin, mitochondrial cytochromes, and NADH/FADH [209].
Springett’s paper thoroughly reviews the benets and limitations of the most recent
methods [209].
In addition, invitro studies have applied these currently available methods to
monitor O2 levels quantitatively, such as by measuring O2 tensions at the cellular
level in 2D monolayer cell cultures or O2 gradients in 3D gels or scaffolds. Two
major methods used to measure O2 levels during invitro cultures are polarographic
and uorescence quenching techniques. The latter has been shown to surpass the
polarographic technique, which consumes O2 during the measurements [197]. When
an implemented measurement technique, like the polarographic technique, con-
sumes O2, it more likely generates even greater inaccuracies and leads to incorrect
conclusions in low O2 environments, as occurred in studies investigating the effect
of hypoxia in 3D scaffolds [36, 120, 145]. Fluorescence quenching technology is
available both for invasive applications, using an electrode probe with a very thin
(approximately 5μm) tip, and for noninvasive applications, using a sensor patch
composed of a ruthenium-based metal complex that can be excited by an external
uorescent light source.
Modeling Oxygen Transport inTissues
The limitations of these measurement techniques, caused mostly by the difculties
in measuring spatial O2 concentrations in tissues or scaffolds, raise a need for pre-
dictive mathematical models. Transport of O2 invivo is controlled by several param-
eters, including blood ow rate, degree of vascularization in the tissue, physiological
distance of the cells from the microvasculature, and, depending on cell type, the
cells’ rate of O2 consumption. These factors affect O2 distribution in the tissue, and
some can also have an impact on O2 transport in 3D invitro cultures of pluripotent
or vascular cells. Additional factors that in vitro studies should consider are the
geometry of the scaffold, the available surface area for O2 transport from the envi-
ronment to the system, and controlled dissolved O2 levels in the culture media.
In general, fundamental mathematical models estimating O2 distribution in 3D
constructs can be classied into: (1) static models, where O2 is only transported via
diffusion, and (2) dynamic models, where convectional transport of O2 is also incor-
M. R. Blatchley et al.

99
porated using perfusion systems, such as microuidic devices, or microcirculation
in the tissues.
Static Models
In tissues cultivated under static conditions within 3D scaffolds, using different
types of biomaterials, spatial O2 concentration can be dened with a one- dimensional
(1D), unsteady-state species continuity equation:
∂
∂
=∂
∂
−
Co
t
Do Co
z
R
2
2
2
2
2 (4.1)
where Co2 is the spatial O2 concentration in the scaffold changing with time (t) and
axial position (z), DO2 is the diffusion coefcient of O2 in the scaffold material, and
R is the oxygen consumption rate of cells. This form of the transport equation has
been used in many studies attempting to predict the O2 gradients in 3D scaffolds
[27, 76, 137]. The equation implies that O2 changes both with time and depth, while
being consumed by the cells as it diffuses from the environment into the scaffold.
Boundary conditions, which are critical for O2 distribution, depend on the O2 equi-
librium between the environment (media/air) and the boundaries of the scaffold.
Therefore, for a 3D scaffold with a depth of L and open boundaries from both sides,
the boundary conditions can be given as:
At andzzLC SP
OO
= = =
0
22
,.
(4.2)
Thus, the solubility (S) of O2 in the scaffold material is one of the determining
parameters of O2 distribution. Although the diffusion coefcient can also be consid-
ered a critical factor in relatively stiff scaffolds of the sort usually used for cartilage
and cardiomyocyte tissues [27], it has been shown to be less signicant for the natu-
ral hydrogel scaffolds commonly used for vascular tissues, such as collagen and
HA.For instance, the diffusion coefcient of O2 in collagen gels was found to be
99% of that in water [83]. Therefore, modeling studies usually assumed that it has
the same O2 diffusion coefcient as water or cell media (3.3×10−5 cm2/s at 37°C)
[157]. The consumption rate of O2 (R) given in Eq. (4.1) is a function of both CO2
and ρcell and is governed by the Michaelis-Menten equation, which states that the O2
uptake rate of each cell increases with O2 availability, reaching a maximum at a
point, Vmax:
RVC
KC
O
mO
=+
ρ
cell
ma
x2
2
(4.3)
where Km is the O2 concentration at which the O2 uptake rate is half of its maximum
value and ρcell is the cell density as a function of time and position. Different groups
have reported the Vmax and Km parameters of many vascular cell types at various cell
seeding densities [78, 163]. For example, the Vmax and Km of HUVECs, at a density
4 Hypoxia andMatrix Manipulation forVascular Engineering

100
of 1×106 cells/ml, are found to be 22.05±1.92 (pmols−110−6 cells) and 0.55±0.02
(μM), respectively [78]. It should be noted that these parameters are estimated as to
mitochondrial consumption of O2. However, as already discussed, ECs also con-
sume O2 for ROS production, and the theoretical models should also take this addi-
tional O2 consumption into account by, for example, including a linear correlation
in the O2 consumption rate equation (Eq. (4.3)). Besides, all estimations of the Vmax
and Km parameters for the O2 consumption of different cell types are carried out in
2D cultures. The literature currently lacks studies investigating whether or not,
depending on the composition of the extracellular matrix, encapsulating cells in 3D
gels changes their consumption of O2.
Vascular cells proliferate, die, migrate, and assemble during 3D cultivation,
which affects their spatial and temporal density and, therefore, O2 distribution.
Models developed for 3D cultures of cardiomyocytes take into account the cellular
proliferation and changes in the dimensions of the cells during nutrient transport in
scaffolds [76, 77]. However, we need more detailed models, which consider how
capillary formation affects O2 transport, to achieve more reliable estimations of spa-
tial O2 concentration. Tube formation and the networking of ECs in 3D gels have
been simulated by more complicated numerical models [47, 139], although the
effects of O2 concentrations on tube formation dynamics still need to be
incorporated.
Dynamic andInVivo Models
The models used for static cultures in 3D scaffolds can also be used to describe O2
distributions invivo when combined with a uid perfusion model that considers the
convectional O2 transfer to the tissues. The velocity prole of a uid in capillaries
or in an engineered microchannel system can be calculated using the simplied
Navier-Stokes equation with cylindrical coordinates given for a laminar, one-
dimensional, steady-state, and fully developed ow of an incompressible uid:
dP
dz r
d
dr
rdV
dr
z
=









µ
1
(4.4)
where P is the total pressure in the uid changing in an axial direction, μ is the vis-
cosity of the uid, and Vz is the axial velocity of the uid changing in a radial direc-
tion. After estimating the blood velocity prole, the species continuity equation,
which involves both diffusional and convectional transfers of O2, can be used to
obtain the O2 distribution inside the capillary or microchannel:
VC
z
D
rr
rC
r
C
r
z
O
O
OO
∂
∂=∂
∂
∂
∂




+∂
∂





2
2
2
2
2
1
Blood
(4.5)
The technical difculties of making quantitative O2 measurements in BM have
led many researchers to develop mathematical models to describe BM O2 distribu-
M. R. Blatchley et al.

101
tion [41, 131, 133]. Additional parameters that need to be considered invivo are the
vascularization of the tissue and the transport of O2 via hemoglobin proteins, mak-
ing the concentration of hemoglobin another essential factor for determining the
oxygenation of the tissue. Studies take these additional factors into account using
the following equation:
DCVzCN
O
j
O
j
zO
j
O2
2
22
2
∇


=∂
∂+



ϕ
(4.6)
The superscript j denotes each sinusoid/arteriole around the BM. N is the O2 car-
rying capacity of the blood and is the concentration of O2 bound to hemoglobin,
which depends on the plasma O2 concentrations [180]. Finally, spatial and temporal
CO2 in tissue can be estimated in a similar manner to the invitro models, using Eq.
(4.1) incorporated with the continuity of uxes assumption at the ECM-vessel inter-
face as a boundary condition.
4.2.3.2 Targeted Cellular Responses toO2 Availability inMatrix
Hydrogels
Engineering vascular tissues in a 3D ECM is well-orchestrated process combining
proliferation, apoptosis, migration, activation, and assembly of vascular or precur-
sor cells inside the construct. As discussed in the previous section, the composition
of the biomaterial used to encapsulate the cells is critical for cellular fate and vessel
formation. In addition to the effects of the chemical and physical properties of the
ECM material on blood vessel formation, temporal and spatial levels of O2 and
other nutrients are also crucial for various targeted cellular responses. A number of
studies have investigated the effects of matrix content and stiffness on angiogenesis/
vasculogenesis [109], and many others have proposed using different types of bio-
synthetic materials to develop more precise blood vessels [10, 152]. However, only
a few studies have highlighted how O2 gradients occurring in the matrix contribute
to the angiogenic process [93, 170, 177]. The availability of O2 and other nutrients
decreases at the center of the gel compared to the periphery, especially in engi-
neered vascular tissues, which require a high cell seeding density for sufcient vas-
cular tissue generation or repair. Hence, cells that reside along various layers of the
matrix respond differently to the nonuniform distribution of O2 and nutrients. For
primary vascular cells to form blood vessels, they require survival, activation, and
the induction of angiogenesis by GFs, cell signaling, and regression. All of these
responses, necessary for blood vessel formation, are controlled by ECM properties,
as well as by O2 availability. Therefore, the inuences of both the ECM and dis-
solved O2 distribution should be considered simultaneously.
Cell assembly and tube formation in the ECM require a sufcient cell density.
Deprived of O2 and nutrients, vascular cells can undergo apoptosis or necrosis [21].
These two cellular death mechanisms should be distinguished; apoptosis contrib-
utes to the process of angiogenesis at any O2 tension, whereas necrosis usually
4 Hypoxia andMatrix Manipulation forVascular Engineering

102
results in the collapsing and deformation of tubes [195]. The critical issues to con-
sider to prevent cellular necrosis during 3D vascular cell cultures are the permeabil-
ity of the ECM material to O2 and glucose, the cell seeding density, and the thickness
of the gel. Thus, cell seeding density is constrained by an upper limit, above which
the cells undergo necrosis due to nutrient deprivation, and a lower limit, below
which the cells cannot assemble sufciently to form tube-like structures. Both limits
depend on the equilibrium O2 levels in the environment.
Cells may also die as a result of apoptosis after their encapsulation in the gel.
Interestingly, some groups have demonstrated that programmed cellular death is
necessary for angiogenesis/vasculogenesis [195]. Segura etal., having studied tube
formation of ECs in both 2D Matrigel and 3D collagen, concluded that a consider-
able number of cells undergo apoptosis at the initial stages of cultivation and that,
once angiogenesis is induced and tube formation has started, no further apoptosis
occurs throughout the process. Inhibition of proapoptotic proteins has been shown
to correlate with defective tube formations, suggesting that apoptosis is important
for avoiding imperfections during blood vessel growth. Hypoxia, as already dis-
cussed, induces angiogenic responses and also regulates proapoptotic gene expres-
sions. Thus, spatial variations in O2 levels may alter the apoptotic responses in the
gel and therefore regulate vascular tube morphogenesis.
MMPs are promoted by integrin-ligand interactions between cells and the ECM,
leading to the degradation of the ECM and facilitating the migration of the cells
[81]. It is hypothesized that ECM fragmentation, orchestrated by the secretion of
MMPs, can mediate caspase activity through the rebinding of ECM protein frag-
ments to unligated integrins, namely, death receptors [38]. Therefore, the survival of
ECs depends on the balance between cell survival promoters, such as FAK, Src, and
Raf, and cellular apoptosis promoters, such as caspase 8 and caspase 3. Hypoxia
may again play a critical role here, affecting both sides of the equilibrium, by upreg-
ulating MMPs and VEGF at the same time [21, 93]. Hypoxia, accompanied by
nonuniform distribution of O2 throughout the gel, can result in spatial differences of
cellular viability, which may subsequently disrupt vascular networking.
Overall, blood vessel growth requires remodeling of the ECM, which is based on
two distinct mechanisms: (1) degradation of the ECM by secreted proteases, and (2)
production of new ECM to support the invading vasculature. Many studies have
shown that hypoxia can regulate the degradation, maintenance, and synthesis of the
ECM [61, 179]. ECM degradation is important for cellular migration into and blood
vessel invasion of tissue. MMPs, as mentioned above, are a major family of protein-
ases that participate in the degradation of the ECM during angiogenesis. In particular,
MMP-2 and MMP-9, both members of the gelatinase subgroup of MMPs, have been
shown to contribute to the process of angiogenesis [85]. MMP-2 secreted by the cells
is activated through membrane MT1-MMPs where the activation can be avoided in
the presence of tissue inhibitor of MMP-2 (TIMP-2) at high levels [99]. Furthermore,
hypoxia was shown to inuence the expression of MMP-2, as well as of MT1-MMP
and TIMP-2, in ECs [21]. Lahat’s group demonstrated the upregulation of MMP-2
expression in hypoxic (0.3% O2) cultures of HUVECs, whereas MT1-MMP and
TIMP-2 are downregulated, enhancing migration and tube formation [21].
M. R. Blatchley et al.

103
ECM degradation is accompanied by ECM production and the secretion of cells.
Once the quiescent state of the ECs composing the blood vessel walls is perturbed
and angiogenesis is induced, ECs start to proliferate and invade the neighboring
ECM by using proteinases. At the same time, they start to remodel the existing ECM
by synthesizing new ECM.In healing wounds, ECs produce transitional ECM pro-
teins, including brinogen and bronectin, and temporarily deposit them in the
ECM in order to provide available ligands during vessel growth [38]. Moreover,
ECs also produce such matricellular proteins as tenascin C and SPARC in the ECM
to mediate angiogenesis [38]. Clearly, the new ECM synthesis of cells is crucial for
angiogenesis, and hypoxia, through HIF1α, has been shown to regulate the expres-
sions of many different types of ECM proteins [167]. For example, many in vivo
and invitro studies have shown that hypoxia enhanced the synthesis of collagen, the
most abundant protein in mammalian tissues [22, 103, 217].
Moreover, proliferation of the cells during angiogenesis/vasculogenesis in 3D
scaffolds is regulated by basic broblast growth factor (bFGF) and VEGF, which are
known to be hypoxia-dependent proteins [177]. In most studies of the vasculariza-
tion of 3D scaffolds, both GFs are broadly used as soluble factors that supplement
cell growth medium to induce proliferation and migration [127, 193]. In addition,
Shen etal. demonstrated that immobilization of VEGF into a 3D collagen scaffold
promotes EC viability, proliferation, and vascularization [198]. VEGF has been
shown to promote blood vessel formation, not only by inducing cellular prolifera-
tion and migration but also by directly regulating elongation and capillary network-
ing in 3D ECM constructs deprived of O2 and nutrients [93]. Helminger etal. used
a sandwich system to seed HUVECs inside a collagen gel [93]. The transfer of O2
and nutrients was accomplished with only simple diffusion through the edges of the
collagen, so that O2 and nutrient levels decreased toward the center. They found that,
in a short time period (about nine hours), VEGF intensity increased in the interior
regions deprived of O2, which correlates well with cell elongation and branching.
VEGF promoted capillary networking independently of proliferation, highlighting
the role of autocrine VEGF in the reorganization of vascular networks in hypoxic
regions of solid tumors. Another study focusing on quantitative measurements of O2
gradients in 3D collagen also showed that increased VEGF concentrations corre-
lated well to decreasing O2 levels throughout the 3D constructs during a ten day
period of cultivation [36].
A few studies considering the induction of angiogenesis by hypoxia in 3D scaf-
folds have emphasized that lowering the O2 tension in 3D gels improved cellular
branching and tube formation of ECs [170, 177]. As made abundantly clear through-
out this chapter, both O2 and matrix mechanics act as potent upstream regulators of
a variety of signaling pathways that contribute signicantly to the regulation of
vascular differentiation as well as morphogenesis. To better understand these
processes, engineers and biologists together have built an impressive library of
materials and assays to examine an array of signaling cascades guiding the forma-
tion of blood vessels. There are several interesting platforms that can be used to
study the effect of hypoxia and O2 gradients on cellular function, including hydro-
gels (Fig.4.6) [143, 144, 173, 187] and microfabrication or microuidic devices [2,
4 Hypoxia andMatrix Manipulation forVascular Engineering

104
3, 6, 37, 74, 169]. Stiffness gradients can also be controlled to study ranges of
viscoelasticity and their effect on cell fate, migration, and other parameters [84].
Finally, O2 and matrix mechanics can be independently controlled in the same sys-
tem using gelatin- and dextran-based hydrogels. These polymers can be both enzy-
matically cross-linked by an O2-consuming reaction to create hypoxic conditions
and cross- linked by a secondary non-O2-consuming reaction, to create a stiffer
microenvironment without appreciably affecting microenvironmental O2 [25].
A growing body of publications have both investigated the inuence of the ECM
composition on angiogenesis/vasculogenesis and suggested the crucial roles of
hypoxia in blood vessel formation. In addition, the evidence discussed above is suf-
cient to suggest that the ECM composition and O2 tension are coupled factors that
need to be taken into account concurrently when developing and repairing vascular
tissues in 3D microenvironments.
Fig. 4.6 O2 controllable hydrogels can be used to study the effects of hypoxic gradients both
invitro and invivo. (a) Synthesis of Gtn–FA, which can form a hydrogel network via a laccase-
mediated cross-linking reaction with O2 consumption. (b) The Gtn–FA precursor solution can be
mixed with either cells or tissues to provide articial hypoxic microenvironments with O2 gradi-
ents (see inserted computer simulation of O2 tension and gradients). The precursor solution can
also be directly injected into the animal as a hypoxia-inducible acellular matrix that induces tem-
poral hypoxia and an O2 gradient in the body (see inserted computer simulation of oxygen tension
and gradients). Reproduced and re-formatted with permission [144]
M. R. Blatchley et al.

105
4.3 Future Directions
Understanding the simultaneous effects of the ECM and O2 tension on the processes
of angiogenesis/vasculogenesis will enable researchers to control these two factors
and thereby manipulate cellular responses in desired directions. Recent develop-
ments in many different elds of research, such as smart biomaterials and microu-
idics, have made it possible to design and construct novel invitro microenvironments
for cells. Smart biomaterials have been developed that can dynamically respond to
external stimuli, such as light [79], pH [174], temperature [216], and cytokines
[124]; these materials can truly mimic the complexity of a native ECM environ-
ment. The ability to control the physical and chemical properties of the gels at dif-
ferent spaces and times will provide better control over different stages of
angiogenesis. Light-sensitive hydrogels can be used to create biomaterials with dis-
tinct cross-linking densities to promote and inhibit cell spreading and migration
[121], which in turn can be used to pattern complex vascular networks. Since vas-
cular morphogenesis is sensitive to tissue stiffness [52], orientation [23], and polar-
ity [42, 149], researchers could also induce vascular assembly into a tube by creating
elasticity, GFs, adhesion peptide, and oxygen gradients along the 3D scaffold [144,
151, 228]. The development of photodegradable hydrogels, as well as the control of
cell-mediated degradation in synthetic hydrogels, whose mechanical and chemical
properties are controllable during the timescale of cellular development [123], has
enabled control of vascular assembly [89, 90]. On the other hand, creating smart
biomaterials that can shrink, swell, or degrade in response to oxygen tension would
also be desirable to prevent the formation of anoxic regions inside the gels. More
precise temporal control of O2 gradients inside the constructs could also be bene-
cial to explain various phenomena taking place in the body, such as EPC regenera-
tion in the BM and embryonic development, where the O2 gradient plays a critical
role in differentiation and migration dynamics.
Figure 4.7 illustrates two proposed approaches for controlling oxygen distribu-
tion and ECM properties. One proposal for regulating O2 gradients inside the gel
would be to incorporate microuidic technology [66, 129, 163, 220]. Although this
approach provides better O2 control over 3D microenvironments, the problem of
spatial variations in O2 levels throughout the gel, due to the cells’ O2 consumption,
must still be addressed. Advancements in microuidic technology could enable spa-
tial O2 control over 3D microenvironments; for instance, the gel could be prepared
around a microtube, which would supply O2 by ushing growth media containing a
desired amount of O2 (Fig.4.7a). Hence, different O2 gradients could be generated
via the manipulation of O2 concentrations in the outside environment and inside the
microtube.
Another method for controlling and improving O2 transport in the gel would be to
microencapsulate O2 carrier liquids, such as peruorocarbons (PFCs). Due to their
high capacity to dissolve O2, PFCs have been used as a blood replacement to improve
O2 delivery to tissues [112, 183]. Based on the high oxygen-carrying capacity of
PFCs, Radisic etal. [183] developed a PFC-perfused system to supply sufcient
4 Hypoxia andMatrix Manipulation forVascular Engineering

106
levels of oxygen to 3D cardiomyocyte cultures. A study by Chin etal. [39] made a
similar attempt, developing hydrogel-PFC composite scaffolds to improve oxygen-
ation throughout the gel. In a similar manner, taking advantage of the high O2 solu-
bility of PFCs, controlled release of O2 in 3D microenvironments could be improved
via microencapsulation of PFCs (Fig.4.7b). Polymeric microspheres loaded with O2
have also been shown to be effective in enhancing cell viability in anoxic microenvi-
ronments [44].
The continued development of novel biomaterial technologies, which enable the
study of human cells in highly biomimetic settings invitro, will continue to guide
discovery of cell behavior and vascular morphogenesis. These powerful systems,
coupled with continued innovation throughout all areas of biotechnology, including
gene editing and stem cell technology, have positioned the eld of vascular tissue
engineering in a fascinating arena, where new therapeutic targets and more robust
vascularized constructs continue to be discovered and translated to the clinic.
Combining expertise in biology, materials science, engineering, and medicine will
continue to inform our understanding of the complex cell-cell and cell-matrix
interactions that drive tissue formation and regeneration.
Acknowledgments We would like to acknowledge funding from various agencies that supported
our studies throughout the years, primarily the American Heart Association, the Maryland Stem
Cell Research Fund, the National Science Foundation, and the National Institutes of Health.
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