Histol Histopathol (2017) 32: 1239-1279

Histology and
http://www.hh.um.es Histopathology
From Cell Biology to Tissue Engineering

Review

Morphofunctional basis of the different types

of angiogenesis and formation of postnatal
angiogenesis-related secondary structures
L. Díaz-Flores1, R. Gutiérrez1, M.P. García-Suárez2, F.J. Sáez3,
E. Gutiérrez1, F. Valladares1,6, J.L. Carrasco1, L. Díaz-Flores Jr4 and J.F. Madrid5
1Department of Basic Medical Sciences, Faculty of Medicine, University of La Laguna, 2Department of Pathology, Hospiten®
Hospitals, Tenerife, 3Department of Cell Biology and Histology, UFI11/44, School of Medicine and Dentistry, University of the Basque
Country, UPV/EHU, Leioa, 4Department of Physical Medicine and Pharmacology, Faculty of Medicine, University of La Laguna,
Tenerife and 5Department of Cell Biology and Histology, School of Medicine, Campus of International Excellence “Campus Mare
Nostrum”, IMIB-Arrixaca, University of Murcia, Murcia and 6CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III,
Madrid, Spain

Summary. We review the morpho-functional basis of
the different types of angiogenesis and report our
observations, including the formation of angiogenesis-
related secondary structures. First of all, we consider the
following issues: a) conceptual differences between
angiogenesis and vasculogenesis, b) incidence of
angiogenesis in pre- and postnatal life, c) regions of
vascular tree with angiogenic capacity, d) cells
(endothelial cells, pericytes, CD34+ adventitial stromal
cells of the microvasculature and inflammatory cells)
and extracellular matrix components involved in
angiogenesis, e) events associated with angiogenesis, f)
different types of angiogenesis, including sprouting and
intussusceptive angiogenesis, and other angiogenic or
vascularization forms arising from endothelial precursor
cells (postnatal vasculogenesis), vasculogenesis
mimicry, vessel co-option and piecemeal angiogenesis.
Subsequently, we consider the specific morpho-
functional characteristics of each type of angiogenesis.
In sprouting angiogenesis, we grouped the events in
three phases: a) activation phase, which includes
vasodilation and increased permeability, EC, pericyte
and CD34+ adventitial stromal cell activation, and
recruitment and activation of inflammatory cells, b)
Offprint requests to: Lucio Díaz-Flores, Departamento de Ciencias
sprouting phase, encompassing EC migration (concept
and characteristics of endothelial tip cells, tip cell
selection, lateral inhibition, localized filopodia
formation, basal lamina degradation and extracellular
changes facilitating EC migration), EC proliferation
(concept of endothelial stalk cells), pericyte
mobilization, proliferation, recruitment and changes in
CD34+ adventitial stromal cells and inflammatory cells,
tubulogenesis, formation of a new basal lamina, and
vascular anastomosis with capillary loop formation, and
c) vascular remodelling and stabilization phase (concept
of phalanx cells). Subsequently, the concept, incidence,
events and mechanisms are considered in the other forms
of angiogenesis. Finally, we contribute the formation of
postnatal angiogenesis-related secondary structures: a)
intravascular structures through piecemeal angiogenesis,
including intravascular papillae in vessel tumours and
pseudotumours (intravascular papillary endothelial
hyperplasia, vascular transformation of the sinus in
lymph nodes, papillary intralymphatic angioendo-
thelioma or Dabska tumour, retiform hemangio-
endothelioma, hemangiosarcoma and lymphangio-
sarcoma), vascular septa in hemorrhoidal veins and
intravascular projections in some tumours; b) arterial
intimal thickening; c) intravascular tumours and
pseudotumours (e.g. intravenous pyogenic granulomas
and intravascular myopericytoma); d) vascular

glomeruloid proliferations; and e) pseudopalisading

Médicas Básicas, Facultad de Medicina, Universidad de La Laguna,

necrosis in glioblastoma multiform.

Tenerife, Spain. e-mail: kayto54@gmail.com
DOI: 10.14670/HH-11-923
1240
Angiogenesis and related secondary structures
Key words: Stromal cells, Fibroblasts, Fibrocytes,
Telocytes, Sprouting Angiogenesis, Pericytes,
Endothelial cells, Intussusceptive Angiogenesis,
Vasculogenesis, Piecemeal Angiogenesis
1. Definition
Angiogenesis is a process characterized by the
formation of new vessels from an established
microvasculature and is therefore responsible for
vascular network expansion and remodelling (Folkman,
2003).
Conventionally, when the term angiogenesis is not
specified, it generally refers to blood vessels (blood
vessel angiogenesis). In lymphatic vessels, the more
precise term of lymphangiogenesis is used.
2. Conceptual differences between angiogenesis and
vasculogenesis
Vasculogenesis and angiogenesis are two principle
types of mechanisms that intervene in vessel
development and growth. Classically, the concept of
vasculogenesis was restricted to the embryonic
development of certain vessels, such as the dorsal aortae
and the posterior cardinal veins (Sabin, 1920), and is
defined as capillary development (primary capillary
plexus) from differentiating endothelial cells (ECs) in
situ (Risau and Flamme, 1995; Auerbach et al., 1997; Jin
and Patterson, 2009). Currently, the concept of
vasculogenesis is broader and is considered as occurring
in pre- and postnatal life, with neo-vessel formation
from endothelial progenitor cells (EPCs). In the embryo,
vasculogenesis takes place in blood islands, which
originate from the splachnopleuric mesoderm. Thus,
after segregation from the mesoderm, some
mesenchymal cells transform into nests of isolated cell
cords of hemangioblasts, which are the precursors of
both ECs and blood cells. The peripheral cells of the
angioblastic masses differentiate into ECs, while the
blood precursor cells are found in the centre of the blood
islands, where a lumen develops (Axnick and Lammert,
2012). The newly formed lumens soon coalesce (Risau
and Flamme, 1995). Finally, smooth muscle cells and
pericytes are aggregated from undifferentiated
mesenchyme.
In the current concept of vasculogenesis, new blood
vessels may develop from circulating and/or bone
marrow-derived CD34+ EPCs (C-EPCs and BM-EPCs,
respectively) (Ashara et al., 1997; Gehling et al., 2000;
Asahara and Isner, 2002; Grant et al., 2002; Pelosi et al.,
2002), from CD34+, CD110+ cord blood cells (Aoki et
al., 2004), and from stem cells in a vasculogenic zone
(between vessel adventitia and the media layer) (Zengin
et al, 2006). Therefore, the recruitment, integration,
migration and maturation of circulating EPCs with
vascular development is considered to be a type of
angiogenesis (see section Postnatal angiogenesis with
endothelial precursor cell participation. Postnatal
vasculogenesis by EPCs).
3. Incidence of angiogenesis in pre- and postnatal
life
Angiogenesis is indispensable in embryonic and
foetal development, as well as in a large number of
normal and pathological processes during postnatal life.
Therefore, the area involved in the study of angiogenesis
is far-reaching, covering numerous fields and many
disciplines.
During embryonic and foetal development, new
vessel formation or neovascularization from pre-existing
microvasculature is an important event and has been
studied at morphologic (Fig. 1A) and molecular levels
(for review: Betz et al., 2016).
Angiogenesis is generally a quiescent process in the
adult organism. Nevertheless, it may occur rapidly in
several normal circumstances. For example, the need for
additional vasculature is imposed on the cyclic evolution
of transient structures in the female reproductive system
(for review, see Rizov et al., 2017), including the
sequential maturation of ovarian pre-ovulatory follicles
(Kim et al., 2017) and subsequent development of the
corpora lutea (Berisha et al., 2016; Woad and Robinson,
2016), cyclic extensions and repair of the endometrium
(Zhang et al., 2016), decidual transformation,
implantation and placentation (Okada et al., 2014; Chen
et al., 2017), and mammary gland changes during
pregnancy, lactation and involution (Andres and Djonov,
2010; VanKlompenberg et al., 2016).
Furthermore, angiogenesis is an important
component of many pathological processes, such as
chronic inflammation, regeneration, wound healing,
organization of thrombi, collateral circulation
development and neoplasias. Moreover, angiogenesis
occurs in severa1 conditions in which the term
‘angiogenic disease’ has been proposed, since an
abnormality of capillary growth is their principal
pathological feature (Folkman, 1989). Angiogenic
diseases include hemangiomas, psoriasis (Heidenreich et
al., 2009; Capkin et al., 2017), scleroderma (Mulligan-
Kehoe and Simons, 2008; Manetti et al., 2016), arthritis
(Szekanecz et al., 2010; Elshabrawy et al., 2015),
atherosclerotic plaque (Camaré et al., 2017), diabetic
retinopathy (Capitão and Soares, 2016), neovascular
glaucoma (Kim et al., 2015) and ischaemic diseases. For
example, the newly formed capillaries invade the joint
and cartilage in arthritis, and the vitreous in diabetes,
with secondary effects such as cartilage destruction and
bleeding, respectively. The role of angiogenesis in
neoplasias is of great interest, especially in the
progressive growth and metastases of solid tumours
(Folkman, 1985), and has resulted in numerous
descriptive studies on angiogenesis, as well as on the
possible mechanisms involved in this process (for
review: Hillen and Griffioen, 2007; Makrilia et al., 2009;
Krishna Priya et al., 2016; Ronca et al., 2017).
 1241
Angiogenesis and related secondary structures

Fig. 1. Angiogenesis in pre- (A) and postnatal (B and C) life. A. An embryonic sprouting vessel (arrow) originating from a vasculogenic vessel (vv). B. A
sprouting vessel (arrow) originating from a postcapillary venule (pv) in the adipose tissue. C. A sprouting vessel (arrow) originating from the rat femoral
vein (fv) induced by peri-venous administration of PGE2 and glycerol. D. Size of the rat femoral artery (black asterisk) and vein (white asterisk) (scale in
mm). Scale bars: A, 20 µm; B, 15 µm; C, 10 µm.
1242
Angiogenesis and related secondary structures
4. Regions of vascular tree with angiogenic capacity
(mainly venous side of circulation)
The regions with angiogenic capacity are the most
ubiquitous and have a common characteristic: the
presence, within or near, of an active preexisting
microvasculature, where angiogenesis begins as a result
of neighbouring angiogenic stimuli. Thus, the mother
vessel microvasculature forms part of a general
angiogenic-inflammatory-repair system in which the
vessels not only intervene in new capillary formation but
also in the recruitment of inflammatory cells and in the
contribution of matrix-forming cells (fibroblasts-
myofibroblasts, osteoblasts, chondroblasts), and
contractile and adipose cells. Interestingly, sprouting
angiogenesis mainly occurs in the venous side of the
circulation, above all in the postcapillary venules (Fig.
1B) (Ausprunk and Folkman, 1977; Díaz-Flores et al.,
1992, 1994a) with a negative pressure, an important fact
in initial stages of this process, in which sprouts are
immature (Díaz-Flores et al., 1992; 1994a) (see below).
Although some authors restrict the origin of newly
formed vessels, pointing out that they only arise from the
microvessels of the venous side of the circulation,
vessels of greater calibre, such as the rat femoral vein,
with a discontinuous internal elastic lamina and smooth
muscle cells in their media layer, are also capable of
contributing to angiogenesis (Fig. 1C,D) (Diaz-Flores et
al., 1994a,b, 2017; Madrid et al., 1998).
In avascular tissues (e.g. cartilage and cornea) and in
fibrin deposits (e.g. wounds), vessels can only originate
from neighbouring vascularized tissues (Díaz-Flores et
al., 2012a). In this case, the intensity of vascular
penetration into an avascular tissue depends on the
properties of the latter (e.g. inhibitory action of
antiangiogenic substances, as occurs in cartilage).
5. Cells and extracellular matrix involved in
angiogenesis
Cells involved in angiogenesis include those of the
vessel wall, circulating precursor cells, inflammatory
cells and cancer cells. The cells in the vessel wall are
ECs (intimal layer), pericytes/smooth muscle cells
(mural cells in the media layer) and CD34+ fibroblasts/
stromal cells (adventitial or external layer) (Fig. 2A-C).
The participation of ECs and pericytes/smooth muscle
cells in angiogenesis has received great attention in the
literature (for review: Diaz-Flores et al., 2009a; Ribatti
et al., 2011); not so the CD34+ adventitial
fibroblasts/stromal cells. Here we consider the
coordination and interactions between pericytes and
ECs, and the role of CD34+ adventitial stromal cells.
The coordination and interactions between pericytes
and ECs may be as follows: a) direct contact, which
includes interdigitations (peg and socket) (Fig. 2D), gap
junctions, and adherent and occluding plaques, leading
to intercellular communications, passage of molecules
and transmission of mechanical forces (Tillet et al.,
2005; von Tell et al., 2006; Díaz-Flores et al., 2011a), b)
release of vesicles (Fig. 2D), such as multivesicular
bodies, shed vesicles and exosomes, which contain
several molecules including microRNA (Dellett et al.,
2017; Todorova et al., 2017), and c) paracrine regulators,
including vascular endothelial growth factor A (VGFA),
sphingosine-1-phosphate, angiopoietin 1 and 2,
transforming growth factor beta (TGF beta),
semaphoring 3-A and several cytokines (Caporali et al.,
2017). These cells have the ability to modulate cellular
interactions during angiogenesis and vessel stabilization
(Diaz-Flores et al., 1991; 2009a).
The major fibroblastic cell components of adventitial
vessel walls are CD34+, CD31-, CD146- and CD45-,
and are considered MSC progenitors (Corselli et al.,
2012; Lin and Lue, 2013; Díaz-Flores et al., 2014;
2015a,b, 2016a,b). These cells have received numerous
names, including fibroblasts, fibrocytes, dendrocytes,
keratocytes, telocytes and stromal, dendritic, adventitial,
supraadventitial, perivascular, paravascular and
delimiting cells (for review: CD34+ stromal cells/
fibroblasts/fibrocytes/telocytes, Díaz-Flores et al., 2014).
Some authors point out that the microcirculation
contains no CD34+ adventitial stromal cells. However,
these cells extend continuously through most of the
vascular system. Therefore, a very thin rudimentary
adventitia is found in several microvessels (Fig. 2A),
except for some small vessels in certain locations.
Examples of microvessels with CD34+ adventitial
stromal cells are those of the reticular dermis and
submucosa, muscular propria, and serosa of the
intestinal wall and gallbladder (Fig. 2A-C). Examples of
microvessels without CD34+ fibroblasts are those of the
papillary dermis, and the intestinal and gallbladder
mucosa (Fig. 2E) (Díaz-Flores et al., 2014). The
presence of these ‘delimiting fibroblasts’ around small
vessels is easily demonstrated by electron microscopy
(Fig. 2C). In the adventitia of large vessels, CD34+
fibroblasts/stromal cells appear in several layers, which
decrease in number as vessel size reduces and, when
present in the capillaries, are arranged in a single layer.
Although CD34 is also intensely expressed in ECs, both
types of cells are easily distinguished by this different
response to CD31 (anti-CD31 stains ECs but not CD34+
adventitial stromal cells), and by their characteristics and
respective locations in the vessel wall. Pericytes and
SMCs are positive for anti-αSMA and negative for anti-
CD34. Since CD34 is expressed in stromal cells of the
adventitia and in ECs of the tunica intima, the CD34-
stained vessels show a double-ring appearance (stained
endothelium and adventitia), owing to two concentric
circles sandwiching the unstained media layer (smooth
muscle or pericytic layer) (Fig. 2A) (Lin et al, 2010;
Maumus et al., 2011; Díaz-Flores et al., 2014). A similar
image is obtained with double stained (CD34/brown and
αSMA/red) vessels, although with red staining in the
media layer (Fig. 2B). Some CD34+ fibroblasts/stromal
cells in the vascular wall may extend their processes into
 1243
Angiogenesis and related secondary structures

Fig. 2. Vessel wall

cells involved in
angiogenesis.
A. CD34 expression in
a microvessel with a
double ring
appearance (CD34
stained endothelial -
ec- and stromal
adventitial cells -sc-
sandwiching the
unstained mural cells
of the media layer).
B. A double stained
vessel with anti-CD34
(staining ECs -ec-and
stromal adventitial
cells -sc - brown) and
anti-αSMA (staining
mural cells -mc- red).
Note the double ring
appearance of CD34
stained cells
sandwiching αSMA
mural cells of the
media layer.
C. Electron
photomicrograph
showing projections of
adventitial stromal
cells (sc), a pericyte
(p) and endothelial
cells (ec).
D. Interdigitations (peg
and socket) (arrow)
between the process
of a pericyte (p) and
an endothelial cell
(ec). A multivesicular
body in formation is
also observed in this
interdigitation. E. In
the mucosae of the
bladder, double
staining with anti-
CD34 (brown) and
anti-αSMA (red) show
capillaries devoid of
CD34+ stromal cells.
Only one vessel
presents αSMA+
perivascular mural
cells (asterisk). Scale
bars: A, 10 µm; B, 15
µm; C, 2 µm; D, 1 µm;
E, 35 µm.
1244
Angiogenesis and related secondary structures
surrounding tissues and vice versa. Frequently, these
extensions associate with other tissue cells (native or
recruited).
The other cells involved in angiogenesis will be
considered in other sections, thus circulating precursor
cells in “Postnatal angiogenesis with endothelial
precursor cell participation”, the inflammatory cells in
“Sprouting angiogenesis” and the cancer cells in
“Vasculogenic mimicry”.
The pre-existing or incorporated components in the
extracellular matrix comprise collagens (including
collagen I, III, IV, V, VIII, XV, XVIII – mainly collagen
IV), elastin, laminins, perlecan, hyaluronan, fibronectin,
entactin/nidogen, TsP, SPARC, and plasminogen. In the
basement membrane of quiescent microvasculature there
are several proteins, including laminins, type IV
collagens, perlecan, nidogens, SPARC, fibulins,
trombospondins, and collagen types VIII, XV and
XVIII, (for review: Seano and Primo, 2015; Randles et
al., 2017). During angiogenesis, these molecules may
undergo local modifications (matricryptic sites of Davis)
(Davis et al., 2000) by the action of proteases, such as
MMPs, and their extracellular matrix fragments
originate matrikines (Bellón et al., 2004), which are pro-
or anti-angiogenic factors (see below).
6. Events associated with angiogenesis
Angiogenesis is a complex biological process, which
may be associated with coagulation, inflammation,
immunity, tissue repair and remodelling. Despite the
continuous and orderly nature of the process, these
findings occur in several overlapping phases, according
to predominant mechanisms. Therefore, several
phenomena, such as vascular dilation and increased
vascular permeability, coagulation, activation of
numerous cell types, including vascular and
inflammatory cells, as well as modifications of the
extracellular matrix, take place during new-vessel
formation.
7. Types of angiogenesis
Several types of angiogenesis have been described,
including sprouting and intussusceptive angiogenesis,
and other forms of vascularization by means of
circulating endothelial precursor cells (EPCs) (postnatal
vasculogenesis), vasculogenic mimicry and vascular co-
option. Following, we describe these types of
angiogenesis, and we also consider a recently reported
form: piecemeal angiogenesis, which intervenes in the
formation of angiogenesis-related secondary structures.
7.1. Sprouting angiogenesis
7.1.1. Definition
Sprouting angiogenesis is the process by which
vessel sprouts grow out of a pre-existing vasculature.
7.1.2. Events of new blood vessel formation in
sprouting angiogenesis
Sprouting angiogenesis is a complex multistep
process, in which the following overlapping events have
been described: a) activation of vascular cells, b) EC
migration, c) proteolytic destruction of extracellular
matrix, including basement membrane degradation, d)
EC proliferation, e) pericyte mobilization, proliferation
and recruitment, f) vascular lumen formation, g) changes
in extracellular matrix with development of a new
basement membrane, and h) vascular maturation and
remodelling, with capillary network formation, eventual
organization of larger microvessels, and involution of
most newly formed vessels. Vasodilation and an
inflammatory response may precede and accompany the
process. Furthermore, during angiogenesis and vascular
involution, other phenomena may occur in the
interstitium, mainly the participation of precursor
stromal cells, which may originate matrix-forming cells
(fibroblast-myofibroblasts, osteoblasts, chondroblasts),
contractile cells (SMCs, myofibroblasts and myointimal
cells) and adipose cells.
In this review, we group these events in three phases:
Activation phase, Sprouting phase and Stabilization
phase.
7.1.3. Activation phase
Vascular dilation, increased vascular permeability,
activation of vascular cells and diapedesis of leukocytes
form part of the activation phase. Below, we outline
these findings, which precede and accompany sprouting
angiogenesis.
7.1.3.1. Vasodilation and increased permeability
Dilation and increased permeability of the blood
venules and capillaries occur rapidly in the initial phase
of angiogenesis (Fig. 3A), in which the opening of
interendothelial cell junctions is observed (Fig. 3B).
Indeed, in response to proangiogenic factors, such as
VEGF (see below), capillaries and postcapillary venules
show vasodilation and increased vascular permeability.
For example, in corneal vascularization induced by
silver nitrate, dilation takes place within an hour
(McCracken et al., 1979) and intensifies progressively.
The changes in vascular permeability during
angiogenesis were studied using the intravenous
injection of different markers, such as colloidal carbon
or Monastral blue to label leaky vessels (Fig. 3C)
(Garbett and Gibbins, 1987; Diaz-Flores et al., 1992).
Increased vascular permeability is also demonstrated
by the presence of extravascular fibrin and dilated
lymphatics. Extravascular fibrin deposits, which are
found during inflammation, wound healing (Madri and
Pratt, 1988) and in the periphery of tumours (Nagy et al.,
1989), are important during angiogenesis. Thus,
neovascularization is induced by fibrin gels in vitro and
 1245
Angiogenesis and related secondary structures

Fig. 3. Phenomena in the activation phase of angiogenesis. A. A dilated and congestive capillary between oedematous spaces. Note perivascular
activated CD34+ adventitial stromal cells (arrows). B. Ultrastructural image of the opening of the interendothelial junctions (arrow). C. Demonstration of
vascular permeability during angiogenesis. Under electron microscopy, Monastral Blue particles (asterisk) are observed between endothelial cells (ec)
and pericytes (p) in a leaky vessel. D. A dilated vessel showing early migration of leukocytes (L) between an opened interendothelial cell junction
(arrow). Note an activated CD34+ adventitial stromal cell (sc) with a nucleus increased in size and with a prominent nucleolus. E. Portion of an
activated mast cell with degranulation between a process of a stromal cell (sc) and pericytes (p). Endothelial cell: ec). Scale bars: A, 25 µm; B, D, E, 1
µm; C, 2 µm.
1246
Angiogenesis and related secondary structures
in vivo (Dvorak et al., 1987). Also, it has been
demonstrated that cultured ECs synthesize fibronectin
and that growing capillaries produce fibronectin in situ
(Clark et al., 1982a).
7.1.3.2. Activation of ECs
In pre-existing vasculature (parent vessels), the
earliest morphological changes in the normally quiescent
ECs consist of hypertrophy with bulging in the vascular
lumen, nuclear enlargement, nucleolar prominence,
dispersal of the ribosomes into their free form, and an
increase in the number of organelles and projections
formation (Ausprunk and Folkman 1977; McCracken et
al., 1979; Díaz-Flores et al., 1992, 1994a). The ECs
projecting towards the interstitium have been described
as filopodia (Kurz et al., 1996; Ruhrberg et al., 2002).
Increased endothelial DNA-synthesis occurs at the onset
of angiogenesis (Burger et al., 1983; Díaz-Flores et al.,
1992) and an increased proliferative index is
demonstrated with Ki67. In addition, modifications of
the tight junctions (organized by claudins, occludins and
junctional adhesion molecules) and adherens junctions
(organized by catenins and cadherins), as well as
extracellular and intracellular associations play an
important role in EC activation (Dejana et al., 2008;
Dejana and Orsenigo, 2013; Dejana and Lampugnani,
2014). All these phenomena facilitate the recruitment of
inflammatory cells (Sprague and Khalil, 2009) and lead
to local deposition of serum proteins, originating a
fibrin-rich provisional matrix (Mehta and Malik,
2006).
Tissue hypoxia activates ECs and angiogenesis-
promoting signals, mainly VEGF, through stimulation of
VEGFR2. VEGF acts not only in this EC activation, but
also in other phases of vessel sprouting (see below).
7.1.3.3. Activation of pericytes
During the initial phase of angiogenesis, activated
pericytes in pre-existing vasculature bulge, shorten their
processes and increase their somatic volume, adopting
an angiogenic phenotype (Díaz-Flores et al., 1992,
1994a). Their nuclei display some folding and the
nucleoli become prominent. The cytoplasm acquires
numerous ribosomes, either singly or in aggregates, and
multiple profiles of rough endoplasmic reticulum are
observed. Micropinocytic vesicles are widely distributed
along the cell membrane. Moreover, pericytes may show
increased DNA synthesis, and a sudden and intense
pericyte proliferation occurs (Diaz-Flores et al., 1992).
Proliferation markers, such as Ki-67, also demonstrate a
higher proliferative index in pericytes. In the parent
vessels, the pericyte basement membrane is disrupted
and fragmented, sometimes away from the pericyte cell
surface. Many pericytes project into the perivascular
space and appear detached from the vessel walls (Díaz-
Flores et al., 1992).
VEGF is a negative regulator of pericyte function
and vessel maturation, and facilitates the loss of pericyte
coverage, leading to vessel destabilization (Greenberg et
al., 2008). In addition, VEGF induces overexpression of
Ang2 in ECs, which also facilitates pericyte dissociation,
and vessel destabilization and regression (Zhang et al.,
2003; Cao et al., 2007). Pericyte dissociation may also
be facilitated by migrating macrophages.
7.1.3.4. Activation of CD34+ adventitial stromal
cells
In this phase, CD34+ adventitial stromal cells
separate from the vascular wall, increase in size and
appear as large, plump, stellate or ovoid cells in
oedematous spaces (Fig. 3A). The nuclei also increase in
size and show one or two prominent nucleoli (Fig. 3D)
(Díaz-Flores et al., 2014, 2015a,b, 2016a,c). These cells
secrete VEGF, bFGF, PDGF-α and IL-8, which
contribute to the induction of an angiogenic phenotype
in ECs, and promote angiogenesis in vivo and in vitro
(Hartlapp et al., 2001; Miranville et al., 2004) (e.g.
adipose derived stromal cells support postnatal
neovascularization - Miranville et al., 2004).
7.1.3.5. Recruitment and activation of inflammatory
cells
Inflammation may play an important role in
angiogenesis, with vascular and leukocyte
communication, and inflammation-induced angiogenesis
(for review, Szade et al., 2015; Kreuger and Phillipson,
2016). Intravascular accumulation of platelets and
polymorphonuclear leukocytes (PMNs) occurs within a
few hours, with early diapedesis of leukocytes outside
the vessel lumen. Thus, before and during vascular
sprouting, inflammatory cells are observed adhering to
the endothelium of the parent vessels, as well as passing
through the endothelial junctions and the pericyte-
endothelial space. Between 1 and 6 h after angiogenic
stimuli, the PMNs predominate. Thereafter, the number
of monocytes/macrophages increases, while the number
of PMNs decreases dramatically. Frequently, the
monocytes/macrophages, either individually or in small
clusters of two or three, simultaneously appear trapped
between the ECs and the pericytes, within the basement
membrane (Diaz-Flores et al., 2009b).
VEGF, CSF-1 (colony stimulating factor 1), PIGF
(placental growth factor) and chemokines participate in
leukocyte recruitment. For example, in monocytes,
CXC, CC, C and CX3C chemokines act through
transmembrane G protein-coupled receptors, mainly
CCL2 (C-C chemokine ligand 2)/CCR2 (Ding et al.,
2012). The new vessels may arise following the
margination and diapedesis of the leukocytes, which do
not seem to be essential for the initiation and
continuation of angiogenesis. For example, corneal
vascularization has been produced in the absence of
leukocytes in rats and rabbits (Sholley et al., 1978).
Nevertheless, leukocytes may have a facilitatory or
augmentative role in vascularization, by the secretion of
cytokines, chemokines, growth factors and matrix-

Angiogenesis and related secondary structures
degrading enzymes (see inflammatory cells in sprouting
phase).
Activated mast cells (Fig. 3E) play an important role
in angiogenesis by producing and releasing pro-
angiogenic factors (for review: Maltby et al., 2009;
Ribatti and Crivellato, 2012a; Cimpean et al., 2017).
Thus, mast cells may release VEGF stored in their
granules or in a degranulation-independent form,
enhancing EP2 receptor by PGE2 (Boesiger et al., 1998;
Abdel-Majid and Marshall, 2004). Other angiogenic
mediators stored in their granules, such as histamine,
heparin, IL-8, TNF-alpha, TGF-beta, NGF and FGF-2
may be released by degranulation (Kanbe et al., 2000).
Finally, mast cell granules contain tryptase and chymase,
which participate in the degradation of extracellular
matrix components. In particular, tryptase stimulates the
proliferation of endothelial stalk cells and facilitates
tubulogenesis, activation of MMPs, and VEGF and
FGF-2 release (Blair et al., 1997).
7.1.4. Sprouting phase
This phase includes EC migration, extracellular
matrix changes with basement membrane degradation
facilitating EC migration, EC proliferation, pericyte and
CD34+ stromal cell mobilization, proliferation,
differentiation and recruitment, inflammatory cell
participation, tubulogenesis (vascular lumen formation),
formation of a new basement membrane and sprouting
connection. During the process in which the ECs
protrude and the vascular basement membrane is
degraded, scant microscopic bleeding may occur.
7.1.4.1. EC migration
Traditionally, it was considered that blood vessels
grew through EC movement (His, 1868). This concept of
EC migration is maintained as an important step during
angiogenesis (Ausprunk and Folkman, 1977). Most
researchers agree that these changes precede endothelial
replication, and that migration and mitoses are
independent phenomena (Sholley et al., 1977a,b; Wall et
al., 1978). Therefore, angiogenic stimuli may operate
through chemotaxis, and EC mitosis may be a secondary
event (Sholley et al., 1977a,b).
The current concept of endothelial tip, stalk and
phalanx cells is important to understand these findings
(Gerhardt et al., 2003; Ribatti and Crivellato, 2012b)
(see below). In this phase, endothelial tip cells are
predominantly involved in EC migration, whereas stalk
cells are involved in proliferation and have the capacity
to acquire a lumen-forming cell phenotype. Tip cells
were previously known as migrating, leader and
projecting ECs. The term filopodia was also applied
(Kurz et al,, 1996; Ruhrberg et al, 2002).
7.1.4.1.1. Concept and characteristics of endothelial tip
cells. Currently, the term tip cells is used for specialized
cells at the tips of a wide variety of developing
structures, predominantly of tube or branched formations
1247
(Gerhardt et al., 2003; Weavers and Skaer, 2014). In
angiogenic sprouts, endothelial tip cells are therefore
leader migrating (invading) cells (Fig. 4A,B) that sense
the environment, and precede and guide endothelial stalk
cells. In the initial phase of neo-vascularization, tip cells
degrade cell-cell junctions (VE-cadherin and tight
junction protein-1), and contribute to extracellular
matrix proteolysis (Arroyo and Iruela-Arispe, 2010) and
degradation of the vascular basement membrane of the
parent vessel. Subsequently, they protrude through the
vessel wall and begin to migrate in a polarized form into
the interstitial space towards the angiogenic stimulus.
Tip cells show an EC transient modified phenotype
(De Smet et al., 2009), with numerous free
polyribosomes and abundant intermediate filaments (Fig.
4A), as well as filopodia that sense attractant or repellent
signals and guide polarized migration (Ruhrberg et al.,
2002; Gerhardt et al., 2003; Lu et al., 2004; De Smet et
al., 2009; Geudens and Gerhardt, 2011). They proliferate
minimally and express the tyrosine-kinase receptor
VEGFR2, VEGFR3/FIT-4, Delta-like 4 (DLL4), PDGF
beta, EC specific molecule I ECM-I1), homolog b
(netrin receptor UNC5b) and angiomotin. Notch-
signalling activity is low (Gerhardt et al., 2003; Claxton
and Fruttiger, 2004; Lu et al., 2004; Siekmann and
Lawson, 2007; Suchting et al., 2007; Hellstrom et al.,
2007a; Tammela et al., 2008; Zheng et al., 2014a). In
early migration, ECs show extracellular-matrix-
degrading podosomes on their surface. These specialized
cell-matrix contacts contain adhesive (e.g. stalin,
vinculin, paxilin) and proteolytic (e.g. MT1-MMP)
molecules, surrounding a core rich in actin (Moreau et
al., 2006; Osiak et al., 2005). Individual podosomes and
podosome rosettes are induced by VEGF-A, which up-
regulates integrin α6β1 (Primo et al., 2010; Seano et al.,
2014), and participate in the degradation of the basement
membrane and in the control of blood vessel branching
(Seano et al., 2014).
Attractant signals, mainly VEGF-A, and repulsive
signals, mainly semaphoring 3A, guide sprout migration
(see below). The chemotactic behaviour of ECs at the
tips of growing vessels is facilitated by the secretion of
plasminogen activator and collagenases (Moscatelli et
al., 1981). EC migration, in response to the extracellular
matrix, depends on the integrin family of cell adhesion
receptors (Leavesley et al., 1993). Indeed, EC
attachment, spreading and migration are mediated by
integrins alfa2B1 and alfa4B3 (Leavesley et al., 1993).
EC migration on collagen and vitronectin is mediated by
alfa4B3 and occurs in a calcium-dependent mechanism,
while collagen recognition by alfa2B1 promotes EC
migration in the absence of calcium (Leavesley et al.,
1993).
Important findings during EC migration are tip cell
selection with lateral inhibition, localized filopodia
formation and sprouting guidance.
7.1.4.1.2. Tip cell selection and lateral inhibition. Not all
activated ECs acquire the tip cell phenotype, since the
ECs that participate in migration are selected by a
1248
Angiogenesis and related secondary structures

Fig. 4. EC migration in sprouting phase. A. An endothelial tip cell (tc), devoid of basement membrane, is observed in an angiogenic vessel. Note
numerous free polyribosomes and abundant filaments. In the insert, an image under light microscopy of a migrating CD34-stained EC. B. Migrating
CD34+ ECs with projections in their tips (arrows). C. Double stained microscopic section with anti-CD34 (staining endothelial cells - brown) and anti-
αSMA (staining pericytes - red). Note the absence of pericytes around the migrating and neighbouring ECs (arrow). Scale bars: A, 1 µm; B, C, 14 µm.

Angiogenesis and related secondary structures
process in which EC stimulated migration and lateral
inhibition occur. VEGF, Dll4 (delta-like 4)/Notch
signalling and the ligand Jagged I participate in this
process of tip cell phenotype specification and lateral
inhibition with stalk cell phenotype acquisition
(Hellström et al., 2007b; Leslie et al., 2007; Lobov et al.,
2007; Siekmann and Lawson, 2007; Suchting et al.,
2007; Bentley et al., 2008; Benedito et al., 2009; De
Smet et al., 2009; Potente et al., 2011). VEGF activates
VEGFR2 (De Smet et al., 2009) and is an important
inducer of tip cell selection and formation. Thus, only
ECs exposed to the highest VEGF levels become tip
cells (VEGF activation of VEGFR2 predominates in this
mechanism). VEGFR2 signalling induces Dll4
expression, which binds to NOTCH receptor on adjacent
ECs and activates NOTCH signalling. The latter inhibits
VEGFR2 and VEGFR3 in these adjacent ECs,
preventing tip cell formation and promoting a stalk cell
phenotype in immediately neighbouring ECs (Williams
et al., 2006a; Hellstrom et al., 2007b; Leslie et al., 2007;
Lobov et al., 2007; Suchting et al., 2007). The ligand
Jagged I antagonizes Dll4 and controls the tip cell
number (Benedito et al., 2009). Determination of stalk
cell phenotype is also facilitated by bone morphogenetic
protein signalling pathway (Moya et al., 2012). Taking
into consideration the above, the mechanism that
facilitates the selection of the tip cells may be a small
imbalance in local VEGF concentration. A time-based
formulation of EC behaviour (temporal adaptation) has
been hypothesized for the cell decisions (Bentley and
Chakravartula, 2017). Therefore, this competitive
cellular mechanism limits the number of sprouts
(excessive branching) (Hellström et al., 2007b; Lobov et
al., 2007; Suchting et al., 2007; Jakobsson et al., 2010;
Geudens and Gerhardt, 2011) and facilitates sprout
guidance and the change of tip cells for other cells
within the stalk region (relative levels of VEGFR1 and
VEGFR2) (see below). FGF might also control
endothelial tip cell migration (Vitorino and Meyer,
2008). Moreover, several inflammatory signals that act
in vascular permeability also participate in EC activation
and in extracellular matrix proteolysis by endothelial tip
cells. These factors include TNF alpha, CCL2/MPC-1,
bradiquinin, nitric oxide, PGE2 and sphingosine-1-
phosphate (SIP) (Langlois et al., 2004; Galvez et al.,
2005; Lee et al., 2006; Petrovic et al., 2007; Alfranca et
al., 2008; Sainson et al., 2008). Likewise, the action of
some inflammatory cells (e.g. monocyte/macrophage
MPPs) induces tip cell phenotype, guides tip cells and
cooperates with neovascularization (Anghelina et al.,
2006; Chung et al., 2009).
7.1.4.1.3. Localized filopodia formation in tip cells.
Filopodia are slender, polarized, highly dynamic plasma-
membrane protrusions (microspikes) (Fig. 4B), which
contain actin filaments, establish adhesions with
components of the extracellular matrix, sense their
environment (like antennae) and participate in cell
migration.
1249
Remodelling of the cytoskeleton, mainly down-
regulation of myosin II contraction, determined by
extracellular matrix properties, facilitates filopodia
formation (Gerhardt et al., 2003; Fisher et al., 2009;
Myers et al., 2011). Heparin-binding VEGF isoforms,
ephrin B2 and a small GTPasa of the Rho family
(CDC42) mediate internalization of VEGFR2, and
participate in the directionality and extension of
filopodia, as well in actin remodelling (Ruhrberg et al.,
2002; Etienne-Manneville, 2004; Sawamiphak et al.,
2010; Fantin et al., 2015). The non-tyrosine kinase
transmembrane protein, neuropilin 1(NRP1) is essential
for extracellular matrix-induced CDC42 activation
(Fantin et al., 2015). Pericytes may or may not be
present near the tip cells (Fig. 4C).
7.1.4.1.4. Basement membrane degradation and
extracellular changes facilitating EC migration. During
angiogenesis, local proteolysis of the basement
membrane of the parent vessels is observed, which is a
necessary step for EC migration (Ausprunk and
Folkman, 1977). Complete disintegration occurs on the
side closest to the angiogenic stimulus, coinciding with
those areas in which the ECs start to grow outwards
(Fig. 4A), while subtle alterations appear around the
whole circumference of the parent vessel. Therefore, EC
sprouts have no basement membrane, but a
homogeneous provisional substratum with altered
proteoglycans (Clark et al., 1982b). Proteases act in this
process (Moscatelli and Rifkin, 1988; Ghajar et al.,
2008; van Hinsbergh and Koolwijk, 2008), and matrix
metalloproteinases (MMPs) and ADAM family proteins
are the main proteases of the metzincin superfamily that
control capillary morphogenesis. MMPs involved in
these functions include membrane-type matrix
metalloproteinases (MT1-MMP, MT2-MMP/MMP-15,
MT3-MMP), and MMP-2, MMP-3, MMP-7, MMP-9
and MMP-13. Among these MMPs, the MT1-MMP is
the main pericellular fibrinolisin and collagenase
involved in EC sprouting (Hiraoka et al., 1998; Zhou et
al., 2000; Yana et al., 2007), and crosstalk between neo-
vessels and mural cells directs the site-specific
expression of MT1-MMP to endothelial tip cells (Yana
et al., 2007). The extracellular-matrix-degrading
podosomes (see above) participate in the degradation of
the basement membrane (Primo et al., 2010; Seano et al.,
2014), since individual podosomes and podosome
rosettes, induced by VEGF-A, which up-regulate
integrin α6β1, contain proteolytic molecules, mainly
MT1-MMP (Primo et al., 2010; Seano et al., 2014).
Likewise, the components of the microvascular
extracellular matrix, such as laminins and collagens
undergo dramatic changes (Nicosia and Madri, 1987).
The peptides released by partial proteolysis of
extracellular matrix macromolecules (matrikines, see
above) (Maquart et al., 2004) may have pro- and/or anti-
angiogenic activity, promote protease and cytokine
production, and/or facilitate neutrophil migration and
monocyte recruitment (Bellon et al., 2004; Adair-Kirk
1250
Angiogenesis and related secondary structures
and Senior, 2008; Monboisse et al., 2014).
7.1.4.2. EC proliferation
Mature ECs, normally in a resting state, have an
extremely slow turnover rate (2 months or more)
(Folkman and Cotran, 1976; Chen et al., 1995).
Therefore, angiogenesis is generally a quiescent process
in the healthy adult organism. Nevertheless, ECs can
quickly convert to a proliferative state during
angiogenesis in several processes, such as endothelium
repopulation in organ transplants, repair of large vessel
defects and thrombi rechannelling (Cavallo et al., 1973;
Folkman, 1984). However, EC proliferation is not
absolutely essential, since angiogenesis has been shown
to take place even in the absence of EC replication
(Sholley et al., 1984). Thus, initial sprouts may progress
without cell division, although proliferation is required
for sustained sprouting (Ausprunk and Folkman, 1977).
As mentioned above, endothelial DNA synthesis occurs
in parent vessels before sprouting and, according to
some authors, as early as 6 to 8 hours after an angiogenic
stimulus is applied (Cavallo et al., 1973). EC mitoses
appear in both the parent and newly formed vessels.
Classically, it was pointed out that mitoses occur at the
tip of the sprouts (Clark and Clark, 1939, Hadfield,
1951), but it is now accepted that when capillary sprout
budding begins, endothelial proliferation takes place in
cells that follow the endothelial tip cells. In other words,
the zone of replication is closer to the parent vessel.
7.1.4.2.1. Concept of endothelial stalk cells. Cells with
capacity to proliferate, and to form tubes and branches
are currently named endothelial stalk cells (see above)
(Gerhardt and Betsholtz, 2005). Thus, these cells
proliferate facilitating sprout extension, establish
intercellular junctions, develop basement membrane and
are coated by pericytes (Fig. 5A-C) (Eiken and Adams,
2010; Geudens and Gerhardt, 2011; Wacker and
Gerhardt, 2011).
Stalk cell proliferation depends on VEGF
concentration, while tip cell migration depends on a
VEGF gradient (Ruhrberg et al., 2002; Gerhardt et al.,
2003). VEGF induces filopodia formation and the
expression of Dll4 in tip cells, and bridge formation with
filopodia of other tip cells during vessel sprout
connection (Bentley et al., 2009). The ability of
angiogenic stimuli to induce replication in confluent ECs
is associated with disruption of cell-cell contacts
(Bavisotto et al., 1990). Likewise, the replicative state
and its ability to respond to endogenous mitogens may
depend on cytoskeletal organization, such as microtubule
destabilization or changes in the cell shape (Liaw and
Schwartz, 1993). Finally, collagen in the interstitium
may influence EC proliferation (Madri and Stenn, 1982;
Schor et al., 1983).
The orientation of stalk cell divisions is important
and is also regulated by VEGF gradient (Zeng et al.,
2007). During sprout extension in absence of blood flow,
the plane of division is perpendicular to the axis of the
vessel sprout (Zeng et al., 2007). Conversely, in perfused
vessels, through CD42 activation, VEGFR2, PECAM1
and VE-cadherin sense the blood flow, and the plane of
EC division is determined by the shear-stress direction
(Tzima et al 2003, 2005; Gomes et al., 2005).
Therefore, when an entire endothelial tip cell
migrates into the interstitium, it is followed by another
stalk cell, and EC sprouts are formed in the perivascular
stroma. Endothelial stalk cells align with others to create
either a solid sprout or one with an intercellular slit-like
lumina. The proliferation, elongation and rearrangement
of endothelial stalk cells progressively lengthen the
sprout (the sprout extension and tubulogenesis being
interlinked processes) (Aydogan et al., 2015). The
presence of abundant contractile intermediate filaments
in the endothelium of the sprouts might be important for
the extension and migration of these sprouts (Sauteur et
al., 2014).
7.1.4.3. Pericyte mobilization, proliferation and
recruitment in the sprouting phase of angiogenesis
Pericyte mobilization, proliferation and association
with EC sprouts occur during the sprouting phase of
angiogenesis (Rhodin and Fujita, 1989; Schlingemann et
al., 1990, 1991, 1996; Díaz-Flores et al., 1991, 1992,
1994a; Nehls et al., 1992; Wesseling et al., 1995;
Morikawa et al., 2002; Gerhardt and Betsholtz, 2003;
McDonald and Choyke, 2003). In addition, pericyte
recruitment and EC investment include alignment,
contacts and phenotype changes. Although EC sprouts
may initially form without pericyte involvement
(representing a plasticity window, allowing ECs to
remodel), pericytes are also among the first cells to
invade newly vascularized tissues (Díaz-Flores et al.,
1991; Nehls et al., 1992; Reynolds et al., 2000). Most of
the authors are of the opinion that the investment of
sprouts by pericytes occurs around trailing trunk ECs
(stalk cells). Therefore, endothelial tube formation is
followed by investment of pericytes, which use EC
sprouts as migration clues. In the association of pericytes
with the endothelium, cytoplasmic processes of pericytes
and ECs have been observed penetrating each other (peg
and socket) (Fig. 2D) (Diaz-Flores et al., 2011a).
Pericytes are recruited in the vascular sprouts (Fig.
5A-C) in response to growth factors, such as PDGF and
TGF beta, and angiopoietin 1 and sphingosine 1
phosphate (Nicosia and Villaschi, 1995; Abramsson et
al., 2002; Hashizume and Ushiki, 2002; Bergers and
Song, 2005; Hoffmann et al., 2005). Nitric oxide also
mediates pericyte recruitment (Kashiwagi et al., 2005).
This association between pericytes and ECs is key to
vascular maturation during remodelling and to vascular
patterning, diameter regulation, vessel stabilization and
endothelial cell survival (Hellström et al., 2001;
Gerhardt and Betsholtz, 2003; Betsholtz et al., 2005;
Gerhardt and Semb 2008). Although the mere presence
of pericytes appears not to protect vessels from
undergoing aggression after inhibition of VGFG
(Morikawa et al., 2002; Inai et al., 2004), pericyte
 1251
Angiogenesis and related secondary structures

Fig. 5. Endothelial
stalk cells in
sprouting phase of
angiogenesis.
A, B. Vascular
sprouts (arrows) are
observed in double
stained sections with
anti-CD34 (staining
endothelial stalk cells
- brown) and anti-
αSMA (staining
pericytes - red). Note
in A that sprouts
originate from one
side of the parent
vessels, probably
leading to angiogenic
stimulation.
C. Ultrastructural
image of endothelial
stalk cells (ec)
around a narrow or
virtual lumen (L).
Interendothelial cell
junctions (arrows)
and a mitotic
endothelial stalk cell
are observed. Also
note developing
basement
membrane, a
perivascular cell (p)
and projections of
perivascular cells.
Observe the contacts
between the
perivascular cell and
endothelial stalk
cells. Insert 1: Detail
of the interendothelial
cell junctions
(arrows). Insert 2:
Pericytes (stained in
red with anti-αSMA)
and endothelial stalk
cells (stained in
brown with anti-
CD34) under light
microscopy. Scale
bars: A, 20 µm; B, 30
µm; C, 2 µm.
1252
Angiogenesis and related secondary structures
coverage intervenes favourably in vessel protection
during development (Chan-Ling et al., 2004) and
prevents vessel regression (Benjamin et al., 1998, 1999;
Enge et al., 2002). Pericyte-endothelial maturation
appears to be more important than simple pericyte
coverage (Hoffmann et al., 2005), and TGF beta 1
participates in this process of pericyte differentiation
from precursor cells (Darland and D’Amore, 2001;
Darland et al., 2003). Recently, it has been demonstrated
that nerve injury-induced protein, Ninjurin (Ninj1), is a
factor to regulate angiogenesis through the function of
pericytes (Ninj 1 negatively regulates the formation of
neovessels) (Matsuki et al., 2015).
Alternatively, pericytes may locate at the growing
front of the endothelial sprouts, participating in
angiogenesis by determining the location of sprout
formation and by guiding newly formed vessels (Nehls
et al., 1992; Tsuzuki and Sasa, 1994; Ozerdem et al.,
2001; Morikawa et al., 2002; Ozerdem and Stallcup,
2004). Therefore, pericytes could be the first to extend
beyond sprouting ECs, promoting EC survival and
guiding EC migration (Amselgruber et al., 1999;
Morikawa et al., 2002; Darland et al., 2003; Kale et al.,
2005). Moreover, pericytes and macrophages can invade
tissues in the absence of ECs and can form tubes
(Moldovan et al., 2000; Anghelina et al., 2002, 2004;
2006; Ozerdem and Stallcup, 2004), enabling the
posterior penetration of ECs. In this case, pericytes and
macrophages act as a scaffold along which ECs migrate
during sprouting.
One question is the source of pericytes in the newly
formed vessels, which may originate from pre-existing
pericytes or other precursor cells, including bone
marrow-derived precursor cells and CD34+ stromal cells
(Nakayasu, 1988; Rhodin and Fujita, 1989; Diaz-Flores
et al., 1991; Nehls et al., 1992; Rajantie et al., 2004;
Lamagna and Bergers, 2006; Bexell et al., 2009).
Among other functions attributed to pericytes is a
role in angiogenesis regulation. The absence of pericytes
in EC sprouts is believed to stimulate EC mitosis, while
their presence in older regions of the growing capillaries
may inhibit EC proliferation and migration (Ordlidge
and D'Amore, 1987; Sato and Rifkin, 1989).
7.1.4.4. Perivascular CD34+ stromal cells in the
sprouting phase of angiogenesis
Migration, proliferation and differentiation into other
cell types may occur in the activated and dissociated
perivascular CD34+ stromal cells during sprouting
angiogenesis. The migration of these cells from their
pre-existing vascular niches takes place in a modified
extracellular matrix in which oedematous spaces and a
provisional matrix with fibrin and fibronectin are
present. During migration, these cells show shortening
of their processes, present mitoses (Fig. 6A) with a high
proliferative index (demonstrated in double staining with
Ki-67 and CD34) (Fig. 6B) (Díaz-Flores et al., 2015a,b)
and acquire transitional cell forms in several processes.
An example of transitional cell forms occurs in
granulation tissue formation, in which there is a loss of
CD34 expression and a gain of αSMA expression in
successive steps, including irregular cell labelling
intensity for anti-CD34, co-localization of CD34 and
actin, and finally loss of CD34 expression and gain of
αSMA (Fig. 6C). Moreover, there are simultaneous
changes in ultrastructural morphology of the cells, which
show characteristics of fibroblasts, protomyofibroblasts
and myofibroblasts, with a progressive increase in
intracytoplasmic microfilament bundles (first pre-
stressing with β and γ actin in protomyofibroblasts, and
later with typical αSMA+ stress filaments in
myofibroblasts) (Díaz-Flores et al., 2015a,b). Likewise,
there is an increase in organelles involved in protein
synthesis, with remodelling of the extracellular matrix.
These activated and migrating CD34+ stromal cells
show PDGFR α and β, and secrete vascular endothelial
growth factors (VEGF), epidermal growth factor (EGF),
nitric oxide synthase (iNOS), macrophage inflammatory
protein 1α (MIP-1 α), MIP-2 and cytokines, IL-2, IL-6,
IL-10 and Il-13 (Suciu et al., 2010; Chen et al., 2013;
Zheng et al., 2014b,c; Albulescu et al., 2015).
7.1.4.5. Inflammatory cells in sprouting phase
Diapedesis of leukocytes (neutrophils, macrophages
and lymphocytes) precedes and accompanies
angiogenesis during repair. The transmigration of these
cells through blood vessel walls is not passive, since
ECs, pericytes, CD34+ SFCs and migrating cells interact
by means of a cascade of signalling events, in different
regulatory mechanisms. Thus, neutrophils are a source
of VEGF-A and Interleukin-8 (Li et al., 2003; Ohki et
al., 2005; Schruefer et al., 2006), macrophages for
Interleukin-1, TNF α, PDGF-AB, TGF α and β, FGF β,
VEGF, EGF, prostaglandin, reactive oxygen species and
several pro-angiogenic chemokines via regulation of the
chemokine receptor CXCR4 by hypoxia (Polverini et al.,
1977; DiPietro and Polverini, 1993; Schioppa et al 2003;
Moldovan and Moldovan, 2005). T-lymphocytes
produce interferon gamma, Interleukins and TNF-α (Du
et al., 2012; Dunk et al., 2012). Platelets contribute with
PDGF-AB, TGF β, epidermal growth factor (EGF)/
TNF-α, tromboxane and insulin-like growth factor.
Moreover, platelet derived phospholipids and
microparticles play an important role as regulators of
angiogenesis (Walsh et al., 2015).
During the initial phase of angiogenesis, the
migrating monocytes/macrophages may also contribute
to the dissociation and detachment of the activated
pericytes and CD34+ SFCs in parent vessels before
vascular sprouts (Díaz-Flores et al., 2009b, 2011b,
2014). Indeed, we have described morphologic
associations between monocytes/macrophages and
resident cells (pericytes and CD34+ SFCs) during this
stage. These associations continue when the perivascular
cells are detached from the vessel wall (Fig. 6D), and
when they acquire transitional cell forms with
myofibroblasts, suggesting interactive cooperation in
migration, differentiation and functional activity (like a
 1253
Angiogenesis and related secondary structures

Fig. 6. Perivascular CD34+ stromal cell and macrophage behaviour, and tubulogenesis in sprouting phase of angiogenesis. A. One CD34+
perivascular stromal cell in mitosis (arrow) around a microvessel (asterisk). B. Double staining with anti-ki67 and anti-CD34 reveals ki-67 expression in
nuclei of CD34+ perivascular stromal cells (arrows). C. Double staining with anti-CD34 (brown) and anti-αSMA (red) shows stromal cells expressing
either CD34 or αSMA. D. Double staining with anti-CD68 (staining macrophages – red) and anti-CD34 (staining stromal cells – brown) reveal
association between macrophages and stromal cells. E. Ultrastructural image of vascular lumen formation (tubulogenesis) (asterisks) between
endothelial stalk cells (ec). Pericyte: p. Scale bars: A, 10 µm; B, 15 µm; C, 25 µm; D, 20 µm; E, 1 µm.
1254
Angiogenesis and related secondary structures
tug-macrophage towing a ship-perivascular cell-
myofibroblast) (Díaz-Flores et al., 2009b).
7.1.4.6. Tubulogenesis (vascular lumen formation)
In the formation of the lumen during sprouting
angiogenesis, ECs form tubular channels (Fig. 6E)
capable of carrying blood. This process requires a
complex mechanism and the participation of numerous
molecules (for review: Iruela-Arispe and Davis, 2009).
Current and classical studies, and according to the five
potential morphogenetic processes described for
different lumen and tubule formation (Lubarsky and
Krasnow, 2003), two mechanisms have been considered
in vascular sprout lumenogenesis: cell hollowing or
intracellular vacuolization and budding or cord
hollowing. In the cell hollowing or intracellular
vacuolization, contiguous endothelial stalk cells
originate exocytotic vacuoles/vesicles, which
interconnect and lead to a luminal space (Folkman and
Haudenschild, 1980; Furusato et al., 1984). In the
budding or cord hollowing mechanism, initial
intercellular channelling occurs by curvature of the
trailing trunk ECs (stalk ECs) (Wakui, 1988; Jin et al.,
2005; Axnick and Lammert, 2012; Yu et al., 2015). In
general, it is accepted that the lumen of the capillary
sprout is formed between the adjacent endothelial
processes, which bud off the wall of the parent vessels
and conserve their cellular polarity (Wakui, 1988). Thus,
the capillary sprout lumen may be an elongation of the
parent vessel lumen (Wakui et al., 1988). Other authors
are of the opinion that the new vessel lumen appears first
in the sprout, merging later with the mother vessel
(Folkman, 1984).
An important finding, which has received little
attention in vascular tubulogenesis in vivo, is the origin
of sprouts from the venous side of circulation with
negative blood pressure (see above). In our view,
although ECs have cell-cell interactions in early
lumenogenesis, they are devoid of sufficient support.
Therefore, low physical forces facilitate structural
preservation of the developing endothelial tubes. When
tip cells join to form capillary loops and contact with the
arterial side of circulation, the gradual impact of positive
blood pressure occurs in the newly formed vessels and
contributes to remodelling them (Díaz-Flores et al.,
2009b).
7.1.4.7. Changes in extracellular matrix. Formation
of a new basement membrane
Initially, active ECs secrete matrix-degrading
enzymes, such as plasminogen activator and collagenase,
causing fragmentation of the basement membrane (see
above). Thus, in the initial stages of the developing
microvessels, fibronectin is a predominant component of
the provisional matrix, making up a delicate fibrillary
network. Fibrils of type IV and V collagen, patchy
amorphous deposits of laminin and rare to absent fibrils
of type I and III collagen have also been described in
these initial stages (Nicosia and Madri, 1987).
Progressively, the deposits of fibronectin decrease,
becoming discontinuous, while laminin and type IV
collagen increase, accumulating and forming a
continuous feltwork in the subendothelial space. In the
late stages of angiogenesis, increased amounts of types I
and III collagen in the perivascular space are observed.
In some conditions, greatly thickened and/or
multilayered basement membrane is present in mature
vessels, probably by repeated episodes of EC death and
regrowth (Díaz-Flores et al., 1994a). Pericyte and
endothelial cells participate in the formation of mature
basement membrane. Thus, pericytes stimulate vascular
basal membrane formation by specific induction of
fibronectin, nidogen-1, perlecan and laminin isoforms
(Stratman et al., 2009). Likewise, connective tissue
growth factor (CCN2) is essential for basement
membrane formation and pericyte adhesion (Hall-Glenn
et al., 2012).
7.1.4.8. Vascular anastomosis and capillary loop
formation
Vascular anastomosis occurs as a result of the
formation of stable contacts between ECs of two
angiogenic sprouts and/or between sprouts and
functional blood vessels (Fig. 7A). The connection
between sprouts is initiated by contacting tip cell
filopodia and is followed by the formation of adherent
junctions, in which cDh5/VE-cadherin plays an
important role (Lenard et al., 2013). When the fusion is
with a functional vessel, tip cell filopodia in angiogenic
sprout and ECs of the target vessels form adhesive sites
(apical membrane initiation sites) (Betz et al., 2016).
Macrophages may act as bridging cells between tip cells
for vascular anastomosis down-stream of VEGF-
mediated tip cell induction (Maden et al., 2009).
When vascular anastomosis occurs, capillary loops
are formed. Other capillary sprouts then appear from
these loops to form a plexus. It has been suggested that
pericytic processes, which seem to bridge the gap
between the leading edges of opposing endothelial
sprouts, may serve as guiding structures for loop
formation (Nehls et al., 1992).
Vessels originating from the venous side of the
circulation form vascular networks, which connect then
to the arterial side and become functional. Subsequently,
remodelling of circulation occurs to adapt to tissue needs
(see below).
7.1.4.2. Phase of vascular remodelling and
stabilization
We have considered three main phases in
angiogenesis for a better understanding of this process,
although the phenomena in each phase overlap with the
next. Thus, most vascular remodelling and stabilization
phenomena initiate in the sprouting phase (see above).
Therefore, in this section, we address only the most
outstanding phenomena in this final phase of
 1255
Angiogenesis and related secondary structures

Fig. 7. Sprouting angiogenesis: vascular anastomosis, remodelling and stabilization. A. Double staining showing CD34+ endothelial cells (brown)
anastomosing to (arrow) microvessels (v) with periendothelial αSMA+ mural cells (red). B, C. Ultrastructural images in which regressing vessels with
EC degenerative phenomena (B) (arrow) and with intravascular accumulations of platelets (C) are observed. D. Orderly arrangement of phalanx
endothelial cells (ec), which show a sessile morphology. Mural cells: mc. Scale bars: A, 15 µm; B-D, 2 µm.
1256
Angiogenesis and related secondary structures
angiogenesis.
7.1.4.2.1. Vascular remodelling. During angiogenesis, a
dense network of neovessels is generally created and is
followed by a process of remodelling, in which many
vessels undergo involution and branching remodelling.
In this mechanism, pruning of excessive blood vessels is
essential. Two different types of pruning have been
described, depending on whether vessels are lumenized
or not (Kochhan et al., 2013; Franco et al., 2015; Lenard
et al., 2015; Betz et al, 2016). When vessels are
lumenized, pruning involves cell-self fusion (Lenard et
al., 2015) and formation of a unicellular tube, in which
the transcellular lumen collapses (vessel constriction and
occlusion), followed by apical membrane retraction and
reduction in cell-cell contacts, with EC apoptosis and/or
migration. This process is similar to intussusceptive
angiogenesis (see below), in which intravascular pillars
may form in vessels originated by sprouting
angiogenesis (Djonov et al., 2001; Makanya et al., 2005;
Hlushchuk et al., 2011). In non-lumenized vessel
pruning, cell rearrangements occur with formation of a
unicellular bridge, followed by apical compartment
separation, a reduction in cell-cell contacts, EC
apoptosis and/or migration and detachment of the vessel.
In both types of pruning, residual ECs may migrate and
return to neighbouring vessels by a reverse sprouting
mechanism (Chen et al., 2012; Udan et al., 2013).
Elevated levels of oxygen, low VEGF levels,
angiopoietin II signals and vessel flow changes play an
important role in vascular pruning (Claxton and
Fruttiger, 2005; Lange et al., 2009; Hlushchuk et al.,
2011). Indeed, pruning involves EC apoptosis (Fig. 7B)
(high oxygen level—Lange et al., 2009—and
macrophage through WNT7b—Lobov et al., 2005—
induced apoptosis), endothelial migration-dependent
regression (apoptosis-independent vascular regression)
(Dimmeler and Zeiher, 2000; Wietecha et al., 2013) and
‘reverse intussusception’ (intussusceptive vascular
pruning) (Hlushchuk et al., 2011) (for review: Ricard
and Simons, 2015). Intraluminal flow changes (flow
level, orientation and periodicity) also regulate cellular
rearrangement during blood vessel pruning and
branching remodelling, in which the axial polarity
between the Golgi and nucleus of ECs determines
polarized EC migration (Le Noble et al., 2004; Lee et al,
2011; Chen et al., 2012; Tirziu et al., 2012; Kochhan et
al., 2013; Udan et al., 2013; García and Larina, 2014;
Lenard et al., 2015; Franco et al., 2015; Ricard and
Simons, 2015) Thus, it has been proposed that EC
migration and incorporation into high-flow segments
depend on Notch pathway signalling (Phng et al., 2009)
and low blood flow, resulting in a smaller total vessel
area and an increase in blood flow. Alternatively, EC
apoptosis and intussusceptive regression are triggered by
low VEGF levels, resulting in lower blood flow and a
smaller total vessel area (Ricard and Simons, 2015;
Lenard et al., 2015; Franco et al., 2015).
Involutive vessels may also show marked
intravascular accumulation of platelets (platelet thrombi)
(Fig. 7C). We have hypothesized that this accumulation
of factor-releasing platelets during the massive
regression of neocapillaries may convert highly
vascularized zones (as occurs in granulation tissue) into
a ‘paracrine transitional organ’, facilitating fibroblast/
myofibroblast proliferation (Díaz-Flores et al., 2009b).
Subsequently, homogenized platelets, endothelial cell
debris and basement membrane residues are observed in
the regions affected by vascular remodelling. This
massive regression of capillaries originates capillary-free
zones and marked reduction in overall vascular density
(Benjamin et al., 1998).
7.1.4.2.2. Vascular maturation and stabilization.
Maturation and stabilization of persistent vessels
involves maturation regulating molecules, recruitment of
pericytes (see above), extracellular matrix formation,
and vessel type (arteries, arterioles, capillaries, venules
and veins) and organ-specific differentiation. Maturation
regulating molecules include angiopoietins and PDGFs
(Potente et al., 2011). Pericytes act in vessel stabilization
by the release of stabilizing factors including
angiopoietin-1 and TMP3 (Suri et al., 1996).
Angiopoietin-1 induces DLL4 expression through TIE2-
receptor. DLL4 activates Notch signalling which down-
regulates VEGRF2, thereby preventing further sprouting
and inducing the expression of NRARP, which enhances
WNT signalling, increasing tight junction stabilization.
In addition, pericytes stimulate vascular basal membrane
formation (Stratman et al., 2009) (see above). Likewise,
pericytes prevent regression by releasing inhibitors of
metalloproteinases (e.g. tissue inhibitor of
metalloproteinase 3). However, there are apparently
conflicting data in the role of pericytes in vessel
stabilization (von Tell et al., 2006). Although pericyte
coating may prevent vessel regression (Benjamin et al.,
1998), pericytes have been observed in immature,
mature and involutive microvessels (Díaz-Flores et al.,
1991; Inai et al., 2004). Moreover, pericyte-like cells
persist in some involutive vessels presenting EC
apoptosis.
Concept of phalanx cells
During vessel maturation and stabilization, ECs may
adopt a thickly apposed, orderly cobblestone-like
appearance, acquiring a more sessile cell fate (Fig. 7D) and
acquiescent phenotype, as well as the capacity to sense and
regulate perfusion, and to inhibit metastasis in tumours (De
Bock et al., 2009; Mazzone et al., 2009). In this stage,
these cells are known as endothelial phalanx cells, since
their alignment and characteristics resemble those of
ancient Greek soldiers forming military phalanxes.
7.2. Intussusceptive angiogenesis
7.2.1. Definition and nomenclature
Intussusceptive angiogenesis is the process by which
pre-existing vessels split or remodel through the

Angiogenesis and related secondary structures
formation of transluminal tissue pillars, leading to
expansion (intussusceptive microvascular growth),
arborisation (intussusceptive arborisation) or branching
remodelling (intussusceptive branching remodelling) of
the pre-existing vasculature (Caduff et al., 1986; Burri
and Tarek, 1990; Patan et al., 1996, 2001b; Augustin,
2001; Djonov et al., 2003; Burri et al., 2004; Makanaya
et al., 2009; Paku et al., 2011).
Other terms used to describe intussusceptive
angiogenesis are intussusception/ intussuceptional
angiogenesis (Caduff et al., 1986), non-sprouting
angiogenesis, splitting angiogenesis, inverse sprouting
angiogenesis (Paku et al., 2011), intraluminal or internal
splitting angiogenesis (Zhou et al., 1998; Williams et al.,
2006b) and longitudinal splitting or longitudinal division
angiogenesis (Egginton, 2009). In addition, and
depending on the final results of the process, the
aforementioned terms, intussusceptive microvascular
growth, intussusceptive arborisation and intussusceptive
branching, are also specifically used for the three main
forms of intussusceptive angiogenesis.
7.2.2. Incidence
Intussusceptive angiogenesis may occur in
developing tissues and in several pathologic processes,
including tumours. Thus, intussusceptive angiogenesis
has been described during development of lung, kidney,
ovary, retina, bone and skeletal muscle, among other
tissues and organs (Macchiarelli et al., 2006; Andres and
Djonov, 2010; De Spiegelaere et al., 2010, 2012), and
experimentally in hypoxic and inflammatory processes
(Konerding et al., 2010), as well as in liver cirrhosis
(Van Steenkiste et al., 2010). Several tumours also show
intussusceptive angiogenesis, including colon, renal and
mammary carcinomas, gliomas and lymphomas (Patan
et al., 1996; Djonov et al., 2001; Nico et al., 2010;
Ribatti and Djonov, 2012).
7.2.3. Events in intussusceptive angiogenesis
The events in intussusceptive angiogenesis mainly
refer to pillar formation, in which four successive phases
have been described (Burri and Tarek, 1990; Patan et al.,
1997; Burri and Djonov, 2002; Djonov et al., 2000,
2003; Egginton et al., 2009; Styp-Rekowska et al., 2011;
Paku et al., 2011): 1) intraluminal endothelial pillar
formation (trans-capillary inter-endothelial bridge) from
endothelial cells of the opposite sites of a vessel wall
(contact between opposite capillary walls) (Fig. 8A), 2)
reorganization of the junctions between the pillar
endothelial cells, which increase in size, flatten and
adopt a bilayer arrangement with a central virtual core,
3) central core perforation and invasion by pericyte and
myofibroblast extensions with extracellular matrix
formation, including collagen fibres (Fig. 8B), and 4)
increased size of the pillar, fusion with other pillars and
splitting of the vessel lumen. Throughout this process,
vessel permeability and the basement membrane remain
1257
unmodified. Intussusceptive angiogenesis phenomena
may occur together with sprouting angiogenesis,
predominantly in the later stages of growth and
remodelling (Makanya et al., 2005; Macchiarelli et al.,
2006).
Paku et al. (2011) have followed the mechanisms for
pillar formation during tumour-induced intussusceptive
angiogenesis, observing suction and subsequent
transport of perivascular collagen bundles into the vessel
lumen. For this mechanism, the authors coined the term
“inverse sprouting”.
Taking into account the three main forms of
intussusceptive angiogenesis (intussusceptive
microvascular growth, intussusceptive arborisation and
intussusceptive branching remodelling), in
intussusceptive microvascular growth, expansion of the
microvasculature occurs through the development of
numerous pillars, which fuse and split the pre-existing
vessels (Makanya et al., 2009; Mentzer and Konerding,
2014). In intussusceptive arborisation, series of pillars
participate in vascular branch angles (Ackermann et al.,
2014). Finally, in intussusceptive branching remodelling,
two forms may optimize the vascularization: branching
pattern or branching pruning (see above). Changes in
blood flow and in metabolic needs occur in all types of
intussusceptive angiogenesis (Djonov et al., 2003).
Experimentally, we demonstrated that perivenous
application of PGE2 and glycerol originates intense
vascularization of the wall of the rat femoral vein from
its intimal endothelial cells (Díaz-Flores et al., 1994b;
2011a; 2017). In these conditions, the intraparietal
microvessels interconnect, encircle and separate
components of the vein wall, giving rise to intravenous
projections, in which the EC cover is formed from
outgrowing ECs and the core by the incarcerated
components of the vein wall, including pericyte-like
cells (Díaz-Flores et al., 1994b, 2011a, 2017). The
results resemble the pillars/folds formed by
intussusceptive angiogenesis in the vein of the ovarian
pedicle after ovariectomy or tumour implantation (Patan,
2000, 2001a,b).
7.2.4. Mechanisms
Several molecules and blood flow participate in the
regulation of intussusceptive angiogenesis. Variable
expression of VEGF and VEGF receptors (VEGFR1 and
VEGFR2) may condition sprouting or intussusceptive
angiogenesis (Makanya et al., 2005; De Spiegelaere et
al., 2010; Hlushchuk et al., 2011; Gianni-Barrera et al.,
2013). Hypoxia-inducible factor 2 alpha, angiopoietin 1
and 2, FGF (fibroblast growth factor 2), and platelet-
derived growth factor beta (PDGF beta) also act in
intussusceptive angiogenesis regulation (Patan, 1998;
Augustin et al., 2001; Kurz et al., 2003; Makanya et al.,
2005, 2007; Taylor et al., 2010). Blood flow changes
(e.g. shear stress and/or cyclic stretch) play an important
role in vascular plexus remodelling and differentiation of
venules and arterioles (Djonov et al., 2002; Le Noble et
1258
Angiogenesis and related secondary structures

Fig. 8. Intussusceptive, mimicry and co-option forms of vascularization. A. In double staining with anti-CD34 (staining ECs: brown) and anti-αSMA
(staining pericytes: red), an endothelial pillar (transcapillary interendothelial bridge) is observed (arrow). B, insert. ultrastructural images of cross-
sectioned pillars (arrows), covered by endothelial cells -ec-, and with central core formation (presence of collagen fibres -co). C. channels containing
red cells and lined with melanotic cells and some melanophages (vascular mimicry) in a melanoma. D. observe connection between endothelial-lined
vasculature (elv) and a channel lined with melanotic cells (clm), and with red cells in its lumen. E. melanotic cells forming cuff around vessels (arrows)
are observed in a melanoma (co-option form of vascularization). Scale bars: A, C-E, 25 µm; B, 1 µm.

Angiogenesis and related secondary structures
al., 2005; Filipovic et al., 2009; Tsuda et al., 2009; Lee
et al., 2010, 2011; Styp-Rekowska et al., 2011).
7.3. Postnatal angiogenesis with endothelial
precursor cell participation (postnatal
vasculogenesis by EC precursors – EPCs)
7.3.1. Concept and incidence
Angiogenesis with participation of endothelial
precursor cells (EPCs) can be defined as the process by
which these precursor cells enter the circulation from
their niches (bone marrow, vessel wall ECs, cord blood),
incorporate into the pre-existing vasculature, and
differentiate into mature ECs, contributing to the newly
formed vasculature.
The contribution of EPCs in angiogenesis is difficult
to establish, and the intensity of their participation is
controversial, mainly in tumours (Kim et al., 2003;
Larrivee et al., 2005).
7.3.2. Characteristics of EPCs
As the name suggests, EPCs are precursor cells
capable of differentiating into mature ECs and may act
in vasculogenesis and angiogenesis (pre- and postnatal
angiogenesis, also considered postnatal vasculogenesis).
In successive works, the characteristics of EPCs were
established mainly by the expression of specific markers
(Asahara et al., 1997; Shi et al., 1998; Gehling et al.,
2000; Peichev et al., 2000). Moreover, as these cells
mature, they lose some markers and acquire others.
Thus, expression in the cells of CD34+, AC133+ and
VEGFR2+ has been considered representative of a
highly proliferative EPC population, whereas CD34+,
AC133-, VEGFR2+ cells represent more mature
circulatory EPCs (Asahara et al., 1997, Shi et al., 1998;
Gehling et al., 2000; Peichev et al., 2000). In more
mature EPCs, other markers may be observed, including
CD31, CD144 (VE-cadherin), CD62E (E-selectin) and
CD184 (chemokine receptor CXCR-4).
7.3.3. Events and mechanisms in angiogenesis
with EPC participation
The principal events in angiogenesis in which EPCs
participate are as follows: a) EPC release from the bone
marrow niche, and incorporation into the blood
(circulating EPCs), b) recruitment and integration within
angiogenic vasculature (incorporation into the vascular
endothelium of EPCs) and c) differentiation into mature
ECs.
7.3.3.1. Release of EPCs and incorporation into the
blood
This phase includes detachment of c-kit positive
cells from their niches, movement of EPCs to the
vascular zone and incorporation in the circulation
(Heissig et al., 2002). In general, several factors,
1259
including VEGF, participate in the release and
mobilization of EPCs from their niches (Asahara et al.,
1999). Factors other than the VEGF involved in release
and mobilization of EPCs include SDF-1 (stromal
derived-1) through CXR-4, FGF, osteopontin, PLGF
(placental growth factor), angiopoietin-1 and
macrophage colony stimulating-factor (Takahashi et al.,
1999; Moore et al., 2001). Moreover, VEGF and SDF-1
promote MMP9/endothelial nitric oxidase synthase-
mediated release of membrane-bound kid ligand
(transformation to a soluble form).
7.3.3.2. Recruitment and integration of EPCs within
angiogenic vasculature
When EPCs reach sites of injury, they participate in
the restoration of vascular integrity and angiogenesis.
Thus, it has been demonstrated that EPCs can integrate
into blood vessels of neovascularized ischemic limbs in
different experimental models (Asahara et al 1999;
Kalka et al., 2000). HMGB1 (high-mobility group box
1) activates integrin-dependent homing of EPCs
(Chavakis et al., 2007), and P-selectin and E-selectin
also act in the adhesion of EPCs (Vajkoczy et al., 2003).
Using a parabiosis model, EPC location was
demonstrated at sites of intussusceptive angiogenesis
(Chamoto et al., 2013).
7.3.3.3. Differentiation of EPCs into mature EPCs
When EPCs in blood vessels lose their progenitor
characteristics, they acquire those of mature ECs,
including expression of EC markers, such as endothelial
nitric oxide synthase, von Willebrand factor and VE-
cadherin (Lin et al., 2000). VEGF participates in this
differentiation of EPCs (Gehling et al., 2000).
7.4. Vasculogenic mimicry (vascular mimicry)
7.4.1. Concept
Vasculogenic mimicry or vascular mimicry is the
generation of perfusable, non-endothelial-lined tube-like
or channel structures, which are generally formed by: a)
highly plastic migratory/invasive tumour cells that
upregulate endothelial-selective markers, and b)
remodelled PAS+ extracellular matrix on the inner wall
of the tube-like structures (Maniotis et al., 1999; Paulis
et al., 2010). The network formed by these structures is
connected with or adjacent to blood vessels, transporting
blood or fluid from connecting or adjacent leaky vessels,
respectively (Maniotis et al., 1999; Paulis et al., 2010).
Therefore, the networks of channels originated by
vascular mimicry may form part of the complex
vasculature in certain tumours, in which all the types of
angiogenesis described here participate (sprouting and
intussusceptive angiogenesis, postnatal vasculogenesis
by precursor ECs, vessel co-option and vascular
mimicry) (Döme et al., 2007; Hillen and Griffioen, 2007;
1260
Angiogenesis and related secondary structures
Carmeliet and Jain, 2011). This complex vasculature
explains the difficulty of disrupting tumour growth using
anti-angiogenic therapy (Dey et al., 2015), since the loss
of one type of angiogenesis may be compensated by
other types (e.g. resistance to anti-VEGF therapy in
glioblastoma multiform - Soda et al., 2013).
7.4.2. Incidence
Vascular mimicry formed by tumour cells has been
described in several types of aggressive tumours.
Likewise, non-endothelial or tumoral cells may also
participate in vascular mimicry, including fetal cells of
the trophoblastic lineage in the hemochorial maternal
vascular spaces (Rai and Cross, 2014) and macrophages
(Barnett et al., 2016) (see other forms of vascular
mimicry). Most vascular mimicry studies have been
carried out on highly invasive melanoma (Fig. 8C,D), in
vivo and in vitro. In addition to melanoma, vascular
mimicry has been observed in several neoplastic
processes, such as carcinomas of the lung, breast, ovary,
skin, kidney, prostate, liver and bladder, gliomas (above
all in glioblastomas), and sarcomas, including synovial
sarcoma, alveolar rhabdomyosarcoma, osteosarcoma,
mesothelioma and Ewing tumour (Maniotis et al., 1999;
Paulis et al., 2010).
7.4.3. Events, and characteristics of tumour cells
and extracellular matrix forming vascular channels
The most important findings in vascular channels are
those related to tumour cells (Fig. 8C) and extracellular
matrix. Tumour cells involved in vascular mimicry
exhibit high plasticity and down-regulation of the
original phenotype markers, showing some
characteristics of embryonic progenitors and ECs
(Bittner et al., 2000; Seftor et al., 2002; Zhang et al.,
2007; Paulis et al., 2010). Moreover, these tumour cells
have anti-coagulant properties and act in matrix
remodelling, providing a perfusion pathway. Thus, in
melanoma, the tumour cells that form vascular channels
show down-regulation of genes related to a melanocytic
phenotype (e.g. tyrosinase and melan A) (Demou and
Hendrix, 2008), while expression of endothelium-
associated genes occurs (e.g. CD34, CD105, neurophilin
1, E-selectin, VE-cadherin and tyrosine-kinase receptor
1). Extracellular matrix in the networks of tubular
structures shows PAS positivity and presence of collagen
IV and VI, laminin, heparan-sulphate proteoglycans and
other components.
7.4.4. Mechanisms
Signalling pathways in vascular mimicry are highly
complex (for revision: Seftor et al., 2012). Briefly, hypoxia
participates in tumour cell plasticity, contributing to the
phenotype switch of tumour cells (Mihic-Probst et al.,
2012). VE-cadherin and EphA2, P13K (phosphoinositide 3
– kinase) and FAK (focal adhesion kinase) play an
important role in vascular mimicry (Hendrix et al., 2001;
Hess et al., 2001, 2003, 2006a,b).
7.4.5. Functionality
Vascular mimicry acts as a complementary perfusion
pathway of the tumours, facilitates metastasis (Hendrix et
al., 2003) and is important for the evaluation of clinical
tumour outcome (poor clinical outcome) (Cao et al., 2013).
As mentioned above, perfusion in vascular mimicry may
occur through interconnecting channel structures and
endothelial-lined vasculature (Fig. 8D) or through the
transportation of fluid from adjacent leaking vessels.
7.4.6. Other forms of vascular mimicry by non-
tumour cells
In both tumours and angiogenesis in in vivo models,
it has been demonstrated that macrophages may form
primitive non-endothelial, functionally connected
channels (Barnett et al., 2016). Hypoxia inducible
factors play an important role in the formation of these
channels (Barnett et al., 2016).
7.5. Vessel co-option
7.5.1. Concept
Vessel co-option is the process by which tumour
cells grow using pre-existing vessels (tumour cells co-
opt host vessels). Therefore, this process is not a true
angiogenic mechanism, since the neoplasic cells grow
over and along vessels (tumour cells parasitize on the
pre-existing vasculature).
7.5.2. Incidence
Tumours in well-vascularized organs, such as the
lung and brain, can be a substrate for this type of growth
(Wesseling et al., 1994; Pezzella et al., 1997), mainly
during initial tumour phases. This process has been
described in glioblastoma, melanoma, Kaposi sarcoma,
and in a model of neuroblastoma, non-small cell
carcinoma, Lewis lung carcinoma and murine ovarian
cancer (Pezzella et al., 1997; Holash et al., 1999; Döme
et al., 2002; Kim et al., 2002; Zhang et al., 2003). A
similar mechanism has been suggested for
myofibroblasts during wound healing (Kilarski et al.,
2009), although this finding may be the expression of
differentiating precursor adventitial cells (Díaz-Flores et
al., 2014). We have observed vessel co-option in
melanoma implanted in mice, particularly following
prior radiation of the tumour bed before implantation,
and in human glioblastoma multiforme. In the latter, the
growing tumour cells emit numerous processes around
the pre-existing vessels, which follow an undulating
route. Subsequently, vessel regression is observed. In
this case, cell projections form what is described as
palisade necrosis (see below).

Angiogenesis and related secondary structures
7.5.3. Events and mechanisms in vessel co-option
Tumours with vessel co-option show viable tumour
cells forming cuffs around the co-opted vessels (Fig.
8E). In some of these tumours, perivascular cuffs appear
between necrotic areas. An important finding is the
involution of the pre-existing co-opted vessels (Holash
et al., 1999; Kim et al., 2002; Zhang et al., 2003), in
which loss of pericytes and smooth muscle cells occurs
with regression of the ECs. The balance of angiopoietin
II and VEGF is involved in the regression of the co-
opted vessels. Angiopoietin II and VEGF expression
facilitate vessel sprouting, while angiopoietin II
expression in the absence or marked decrease of VEGF
induces vessel regression (Holash et al., 1999; Kim et
al., 2002). These regressive phenomena have been
considered as a host defence mechanism (Hillen and
Griffioen, 2007) and may lead to the angiogenic
formation of secondary structures (see below).
7.6. Piecemeal angiogenesis
7.6.1. Concept
Piecemeal angiogenesis is a mechanism by which a
variable number of intravascular papillary structures
form in tumours, pseudo-tumours, reactive processes,
and malformations of the blood and lymphatic vessels,
and in the intravascular incorporation of different tissue
components, including neoplasic cells. Through this type
of angiogenic mechanism, ECs grow from a pre-existing
vessel, and encircle and split up portions of the wall of
the vessel itself, perivascular tissues and/or fibrin
components, giving rise to the intravascular papillae
partially connected to the vessel wall (Díaz-Flores et al.,
2016d, 2017). In these intravascular papillae, ECs form
the cover and encircled components the core.
This piecemeal angiogenic mechanism has also been
demonstrated experimentally and is of interest to explain
several forms of angiogenic-related intravascular
structures. In this section, we consider the experimental
studies, while the angiogenic-related structures
originated by this process will be outlined in the section
“Participation of angiogenesis in formation of secondary
structures during postnatal life”.
7.6.2. Events and mechanisms in experimental
piecemeal angiogenesis
Experimentally, a large number of vessel sprouts and
intravascular papillae have been demonstrated in the rat
femoral vein wall after PGE2 and glycerol injection into
the soft tissue surrounding the vein (Díaz-Flores et al.,
1994b, 2011a, 2017). Vascular sprouts emerge from
exuberant EC growth in the intima of the vein, forming
conspicuous microvascular networks in the media layer
and papillae with the following sequence: a) activation
of vein ECs, b) migration of intimal ECs through gaps in
the discontinuous internal elastic lamina, originating
numerous vascular sprouts in the media layer, where
1261
they are surrounded by modified SMCs, which adopt
pericytic characteristics, c) interconnection between
vascular sprouts (anastomosing channels) in the vein
wall forming microvessel networks, in which their slit-
like or more apparent lumens connect to the vein lumen,
d) presence of papillary structures (Fig. 9A), whose
endothelial cover is formed from one side of the pairs of
migrating ECs in the innermost networks of the vein
wall, and whose cores are formed from stromal wall
components of the original vessel (extracellular matrix,
modified SMCs and/or other microvessels).
Taking into account the above, the anastomosing
vascular channels that penetrate the vessel wall and
perivascular structures remain connected to the vessel
lumen, form networks encircling components of the vein
wall or perivascular structures and give rise to
apparently free papillae. Therefore, in the papillae, the
EC cover is formed of ECs from these anastomosing
vascular channels, and the core is formed by the
incarcerated components of the vein wall and
perivascular structures. These components may vary
depending on the depth of microvessel penetration in the
vessel wall (penetration is scant when papillae with little
stromal support are formed). Proliferating endothelial
cells may even fold onto themselves, originating tuft-like
structures, or onto thrombotic fibrin components. This
pathogenic mechanism of vascular network and papillae
formation combines the sprouting and intussusceptive
mode of angiogenesis (Díaz-Flores et al., 1994b; Patan,
2000; Patan et al., 2001a; Burri et al., 2004; Ackermann
et al., 2014) with development of loops in the vein wall
(which may occur on the venous side in vessels of all
sizes).
8. Participation of angiogenesis in formation of
secondary structures during postnatal life
Angiogenesis may participate in the formation of
postnatal angiogenesis-related secondary structures,
including: a) intravascular structures through piecemeal
angiogenesis, such as intravascular papillae in vessel
tumours and pseudotumours, vascular septa in
hemorrhoidal veins and intravascular projections in
some tumours, b) arterial intimal thickening, c)
intravascular tumours and pseudotumours, d) vascular
glomeruloid proliferations, and e) pseudo-palisading
necrosis. In this review we do not include the
participation of angiogenesis in postnatal formation of
granulation tissue (a provisional tissue during repair) and
mature mesenchymal tissues (connective, bone, cartilage
and adipose), nor the widely studied pericyte and CD34+
stromal cell plasticity.
8.1. Angiogenesis in the formation of intravascular
structures through piecemeal angiogenesis
A piecemeal form of angiogenesis (see above) plays
an important role in the formation of several
intravascular structures, including intravascular papillae
in vessel tumours and pseudotumours, vascular septa in
1262
Angiogenesis and related secondary structures

Fig. 9. Piecemeal angiogenesis in experimental conditions and in the formation of angiogenesis-related secondary structures (papillae) in vessel tumours
and pseudotumours. A. semithin section showing intravascular papillae (arrows) after PGE2 and glycerol administration around the rat femoral vein. B.
intravascular papillary endothelial hyperplasia. Numerous papillae are observed in a section stained with anti-CD34 (staining ECs: brown) and anti-αSMA
(staining mural cells: red). C. numerous papillae are also observed in a cavernous hemangioma. Note the wall of a vessel with endothelial (brown) and
mural (red) cells (arrow), as well as the papillae in the lumen. D-F. Papillae in a cavernous lymphangioma. Sections stained with podoplanin show a linear
arrangement of papillae, some of which are joined by ECs (F, arrow). Scale bars: A, 25 µm; B, C, 15 µm; D, 35 µm; E, F, 7 µm.

Angiogenesis and related secondary structures
hemorrhoidal veins and intravascular projections of
some tumours (e.g. in minimally invasive follicular
carcinoma of thyroid).
8.1.1. Intravascular papillae in vessel tumours
and pseudotumours as a piecemeal form of
angiogenesis
A piecemeal form of angiogenesis that originates
intravascular papillae occurs in several types of vessel
tumours and pseudotumours, and has been described in
experimental conditions (Díaz-Flores et al., 1994b,
2011a, 2016d, 2017). By this procedure, growing ECs
encircle and separate components of the vessel wall,
perivascular tissue and/or fibrin, giving rise to
intravascular structures, partially connected to the
vessel wall (Díaz-Flores et al., 1994; 2011a, 2016d,
2017).
Examples of vascular pseudotumours, and benign
and malignant vascular tumours with intravascular
papillae include intravascular papillary endothelial
hyperplasia (IPEH), vascular transformation of the
sinuses in lymph nodes, papillary intralymphatic
angioendothelioma (PILA/Dabska tumour), retiform
hemangioendothelioma, hemangiosarcoma and
lymphangiosarcoma (Clearkin and Enzinger, 1976; Kuo
et al., 1976; Hashimoto et al., 1983; Sanz-Trelles et al.,
1997; Bhatia et al, 2006; Neves et al., 2011; Li et al.,
2013; Mota et al., 2013; Kugler et al., 2016; Liu et al.,
2015; Emberger et al., 2009; Kuo et al., 2015; Diaz-
Flores et al., 2016d). In IPEH (Diaz-Flores et al., 2016d)
and in other lesions, papillae, which may be numerous
(myriad of papillae), show a cover formed by ECs and a
connective or fibrinous core (Fig. 9B-F), as occurs in
experimental conditions (perivenous administration of
PGE2 and glycerol) (Díaz-Flores et al., 1994b, 2011a,
2017) (see above). However, there are differences in the
number of papillae, as well as variations in papillary
size, distribution, arrangement, EC cover and core
components (Fig. 9B-F). The resulting papillae are
similar to folds and pillars described in the ovarian
pedicle after ovariectomy or tumour implantation (Patan,
2001a,b), and in florid intussusceptive-like
microvascular disangiogenesis of bronchopulmonary
dysplasia (De Paepe et al., 2017).
Therefore, these intravascular projections in vessel
tumours and pseudotumours (papillae for pathologists),
and in intussusceptive angiogenesis (folds and pillars),
present a similar structure, supporting the hypothesis of
a piecemeal angiogenic mechanism in papillary
formation in the experimental and pathological
conditions, with association of sprouting and
intussusceptive types of angiogenesis (Díaz-Flores et al.,
2016d, 2017).
8.1.2. Compartmentalization of hemorrhoidal
veins
Closely associated with intravascular papillae is the
1263
compartmentalization of hemorrhoidal veins. In the
latter, successive series of secondary papillae may be
arranged in linear fashion, forming several septa with
compartmentalization of the vein lumen (Fig. 10A,B)
(unpublished observations).
8.1.3. Piecemeal angiogenesis in vascular
invasion/pseudoinvasion.
In some tumours, angioinvasion/pseudo-
angioinvasion may be based on a piecemeal mechanism
of angiogenesis. For example, in minimally invasive
follicular thyroid carcinoma (angioinvasive encapsulated
carcinoma), the invasion of small to medium vessels
within or immediately adjacent to the tumour capsule
may also be related to the formation of intravascular
structures. Indeed, ECs grow out of the vessel walls and
encircle tumour glands following a piecemeal
mechanism (Fig. 10C) (see above). In these conditions,
the glands remain in the core of the newly formed
peculiar papillae and therefore isolated from the
circulation. This mechanism may explain how vascular
invasion evolves with infrequent metastasis and requires
further studies.
8.2. Arterial intimal thickening
In several forms of intimal thickening (e.g. arterial
segments isolated between ligatures), an intense
intraarterial neovascularization originates from the vasa-
vasorum, predominantly in the early phase of intimal
thickening development (Díaz-Flores and Domínguez,
1985). We have demonstrated this penetration of
microvessels through the arterial wall by injecting
contrast, and subsequent tissue clearance and
observations in HE stained sections (Fig. 11A,B).
Indeed, in occluded arterial segments, the following
occurs: a) early capillary ingrowth through the adventitia
and tunica media, b) development of numerous
microvessels in the occluded arterial lumen, originating
a granulation-like tissue, c) coalescence of the lumen of
microvessels, forming an axial arterial neolumen, d)
involution of most microvessels and persistence of
interstitial cells, especially those with a pericytic aspect,
which acquire characteristics of intimal cells, and e)
presence of intimal thickening and a patent lumen in the
occluded segment connected to some perforating vessels
(Díaz-Flores and Dominguez et al., 1985, Díaz-Flores et
al., 1990).
8.3. Formation of intravascular tumours and
pseudotumours
Intravascular tumours and pseudotumours may be
related to angiogenesis. For example, in intravenous
pyogenic granuloma and intravascular myopericytoma.
In intravenous pyogenic granuloma, numerous
microvessels with prominent ECs and pericytes form the
intravascular lesion (Fig. 11C,D). In myopericytoma, as
1264
Angiogenesis and related secondary structures

Fig. 10. Piecemeal angiogenesis in compartmentalization of hemorrhoidal veins and in vascular invasion/pseudoinvasion. A, B. images of hemorrhoidal
veins in which successive series of secondary papillae, arranged in a linear fashion (arrow) (A), form several septa (arrows) with compartmentalization of
the vein lumen (B). C. minimally invasive follicular thyroid carcinoma. Detail of an intravascular tumour gland (asterisk), encircled by CD34+ endothelium,
as occurs in papillary structures. Note a gland (double asterisk) partially encircled by CD34+ endothelial cells, which originate from the vessel wall. A and
B: double staining with anti-CD34 (brown) and anti-αSMA (red). C: staining with CD34 (brown). Scale bars: A, 15 µm; B, 40 µm; C, 12 µm.
 1265
Angiogenesis and related secondary structures

Fig. 11. Angiogenesis

in intimal thickening
formation and in
intravascular tumours.
A, B. in early phases
of intimal thickening
development in
occluded arteries,
microvessel ingrowth
in the arterial lumen
from the vasa vasorum
is demonstrated by
injecting contrast after
tissue clearance (A)
and in an HE stained
section (B). Observe in
B microvessels
crossing the artery wall
(aw), reaching the
intimal thickening (it).
C, D. intravenous
pyogenic granuloma
with numerous
microvessels similar to
those of granulation
tissue. Double staining
with anti-CD34
(staining ECs - brown)
and anti- αSMA
(staining mural
cells/pericytes - red).
Scale bars: A, 0.5 mm;
B, 40 µm; C, 0.1 mm;
D, 35 µm.
1266
Angiogenesis and related secondary structures
occurs with arterial intimal thickening, intravascular
neovascularization plays an important role, and these
lesions are therefore related (Diaz-Flores et al., 2011c,
2012b). This hypothesis is supported by the following:
a) frequent participation of intimal thickening as a
myopericytoma component, b) phenotypic similarities
between the myoid cells (myopericytes) of the tumour
and the neointimal (myointimal) cells of the concomitant
intimal thickening, and c) possibility of a common cell
origin for both lesions and of an angiogenic pattern in
their initial stages (Díaz-Flores et al., 2011c, 2012b).
8.4. Glomeruloid vascular proliferations
8.4.1. Definition
Glomeruloid vascular proliferations or vascular
glomeruloid bodies are structures that resemble the renal
glomeruli and are formed by closely packed
anastomosing capillaries, which show irregular and
tortuous narrow lumina, prominent ECs and pericytes.
8.4.2. Incidence
Vascular glomeruloid bodies have been described in
vascular tumours (frequently associated with POEMS
syndrome – Forman et al., 2007; Yuri et al., 2008;
Gonzalez-Guerra et al., 2009) and malformations of the
skin, in glioblastoma multiforme (Fig. 12A,B) (Rojiani
and Dorovini-Zis, 1996), and in other tumours, including
breast, lung, endometrium, prostate and meninge
tumours (Ohtani 1992; Bläker et al., 1999; Straume et
al., 2002; Goffin et al., 2003; Kandemir et al., 2014), as
well as in experimental conditions (e.g. after
VPF/VEGF-164 gene delivery) (Sundberg et al., 2001).
8.4.3. Characteristics
In glomeruloid bodies, florid microvascular
proliferation, with packed anastomosing capillaries,
shows prominent ECs, which express CD31 and CD34
(Fig. 12A). These ECs present weak VEGF reactivity
and a Ki-67 proliferation index above 10% (Kandemir et
al., 2014). In addition, the pericyte population, with
αSMA expression, is relatively important in vessel walls
of glomeruloid structures (Fig. 12B).
8.4.4. Mechanisms and sequence of formation and
prognostic importance
Experimentally, it has been demonstrated that VPF-
VEGF-164 is sufficient for induction of glomeruloid
bodies. Likewise, VPF-VEGF-164 is also necessary for
maintaining these vascular bodies (Sundberg et al.,
2001). The sequence of glomeruloid body formation is
as follows: a) vessel dilation and focal accumulation in
the vessel EC layer of plump primitive cells, which show
EC markers, increased expression of VGFR-2 and lack
of pericyte markers; b) the cells in the accumulations
proliferate and extend toward the vessel lumen and the
extravascular tissue; c) incorporation of proliferating
pericytes, which intermingle with the ECs; d) formation
of a multilayered basement membrane between ECs and
pericytes; e) partial occlusion of the mother vessel lumen
and formation of small channels in the developing
glomeruloid structures and f) development of
microvessels with erythrocytes containing lumens,
originating characteristic glomeruloid bodies. In
glioblastoma multiforme, we have observed (non-
published observations) that several glomeruloid
microvascular structures may appear along a pre-existing
vessel, adopting an arciform distribution. The presence
of glomeruloid microvascular proliferation indicates an
aggressive angiogenic phenotype in human cancers and
is therefore an unfavourable biological marker (Straume
et al., 2002). However, in some conditions, glomeruloid
structures have been considered as reactive vascular
proliferations of undetermined biological importance
(Kandemir et al., 2014).
8.5. Angiogenesis in the formation of pseudo-
palisading necrosis
Two important structures in glioblastomas are
related to angiogenesis: pseudopalisading necrosis and
glomeruloid vascular structures described above.
In glioblastoma, two main types of necrosis can be
observed: pseudopalisading necrosis and large necrosis.
Pseudopalisading necrosis in glioblastoma is formed by
a garland-like arrangement of tumour cells (rim of cells)
around a central degenerative region (Fig. 12C). The
resulting pseudopalisades can be long and undulating or
small and rosette-like. This pseudopalisading
configuration of astrocytic cells is essentially unique for
glioblastoma. The hypothesis of a mixed population of
neoplasic and inflammatory cells to explain the
pseudopalisades is not sustainable, since practically all
cells in this location are neoplasic astrocytic cells.
Currently, several authors are of the opinion that
pseudopalisades could represent an astrocytic cell
population that rapidly migrates away from a
dysfunctional vasculature (from a hypoxic zone) (Brat et
al., 2004; Rong et al., 2006). In pseudopalisades, we
have observed that neoplasic astrocytic cells emit
numerous processes toward an involutive vessel in the
axis of the degenerative central zone. These processes
are positive for anti-gliofibrillar acid protein (Fig. 12D
and E) and therefore form most of the degenerative
central zone, in which debris of CD34+ ECs of the
involutive vessel are also observed (Fig. 12F). Loss of
pericytes or vascular smooth muscle cells occurs in these
vessels. Normally, astrocyte processes envelop synapses,
make contact with nodes of Ranvier, form gap junctions
between distal processes of other astrocytes and
establish interactions with blood vessels, where they
form astrocytic endfeet. Our results (obtained in
conjunction with Spreafico MA†, nonpublished
observations) agree with the concept that
pseudopalisades are formed by a mechanism in which
neoplasic astrocytes grow over and along a vessel, which
 1267
Angiogenesis and related secondary structures

Fig. 12. Angiogenesis in glomeruloid vascular proliferations and in pseudopalisading necrosis formation. A, B. Glomeruloid bodies with florid
microvascular proliferation, showing prominent CD34+ ECs (A) and an abundant pericyte population (B). C-F. pseudopalisading necrosis in
glioblastoma formed by a garland-like arrangement of tumour cells (rim of cells) around a central degenerative zone (cz), observed in HE (C), anti-
protein gliofibrillar acid (D, E) and in anti-CD34 stained sections. Note that the central degenerative region is formed by the gliofibrillar acid + projections
of the neoplasic astrocytic cells (D, E), which converge in a central vessel (E, arrow). F. Degenerative phenomena (residual expression of CD34)
(arrow) in a central vessel of the pseudopalisading necrosis. Scale bars: A, 20 µm; B, 15 µm; C-E, 50 µm; F, 40 µm.
1268
Angiogenesis and related secondary structures
shows involutive phenomena. Therefore, our
interpretation of the findings is that mummified or
partially disintegrated processes of the astrocytes form
the degenerative central zone around vessels (Fig.
12D,E). This concept may also justify the absence of
these specific pseudopalisades in tumours other than
glioblastomas. The explanation of the involution of the
central vessel may be similar to that in vessel co-option
(see above).
References
Abdel-Majid R.M. and Marshall J.S. (2004). Prostaglandin E2 induces
degranulation-independent production of vascular endothelial growth
factor by human mast cells. J. Immunol. 172, 1227-1236.
Abramsson A., Berlin O., Papayan H., Paulin D., Shani M. and Betsholtz
C. (2002). Analysis of mural cell recruitment to tumor vessels.
Circulation 105, 112-117.
Ackermann M., Houdek J.P., Gibney B.C., Ysasi A., Wagner W., Belle
J., Schittny J.C., Enzmann F., Tsuda A., Mentzer S.J. and
Konerding M.A. (2014). Sprouting and intussusceptive angiogenesis
in postpneumonectomy lung growth: mechanisms of alveolar
neovascularization. Angiogenesis 17, 541-551.
Adair-Kirk T.L. and Senior R.M. (2008). Fragments of extracellular
matrix as mediators of inflammation. Int. J. Biochem. Cell Biol. 40,
1101-1110.
Albulescu R., Tanase C., Codrici E., Popescu D.I., Cretoiu S.M. and
Popescu L.M. (2015). The secretome of myocardial telocytes
modulates the activity of cardiac stem cells. J. Cell Mol. Med. 19,
1783-1794.
Alfranca A., López-Oliva J.M., Genís L., López-Maderuelo D., Mirones
I., Salvado D., Quesada A.J., Arroyo A.G. and Redondo J.M. (2008).
PGE2 induces angiogenesis via MT1-MMP-mediated activation of
the TGFbeta/Alk5 signaling pathway. Blood 112, 1120-1128.
Amselgruber W.M., Schäfer M. and Sinowatz F. (1999). Angiogenesis in
the bovine corpus luteum: an immunocytochemical and
ultrastructural study. Anat. Histol. Embryol. 28, 157-166.
Andres A.C. and Djonov V. (2010). The mammary gland vasculature
revisited. J. Mammary Gland Biol. Neoplasia 15, 319-328.
Anghelina M., Schmeisser A., Krishnan P., Moldovan L., Strasser R.H.
and Moldovan N.I. (2002). Migration of monocytes/macrophages in
vitro and in vivo is accompanied by MMP12-dependent tunnel
formation and by neovascularization. Cold Spring Harb Symp. Quant
Biol. 67, 209-215.
Anghelina M., Krishnan P., Moldovan L. and Moldovan N.I. (2004).
Monocytes and macrophages form branched cell columns in
matrigel: implications for a role in neovascularization. Stem Cells
Dev. 13, 665-676.
Anghelina M., Krishnan P., Moldovan L. and Moldovan N.I. (2006).
Monocytes/macrophages cooperate with progenitor cells during
neovascularization and tissue repair: conversion of cell columns into
fibrovascular bundles. Am. J. Pathol. 168, 529-541.
Aoki M., Yasutake M. and Murohara T. (2004). Derivation of functional
endotelial progenitor cells from human umbilical cord blood
mononuclear cells isolated by a novel cell filtration device. Stem
Cells 22, 994-1002.
Arroyo A.G. and Iruela-Arispe M.L. (2010). Extracellular matrix,
inflammation, and the angiogenic response. Cardiovasc. Res. 86,
226-235.
Asahara T. and Isner J.M. (2002). Endothelial progenitor cells for
vascular regeneration. J. Hematother. Stem Cell Res. 11, 171-178.
Asahara T., Murohara T., Sullivan A., Silver M., van der Zee R., Li T.,
Witzenbichler B., Schatteman G. and Isner J.M. (1997). Isolation of
putative progenitor endothelial cells for angiogenesis. Science 275,
964-967.
Asahara T., Takahashi T., Masuda H., Kalka C., Chen D., Iwaguro H.,
Inai Y., Silver M. and Isner J.M. (1999). VEGF contributes to
postnatal neovascularization by mobilizing bone marrow-derived
endothelial progenitor cells. EMBO J. 18, 3964-3972.
Auerbach R., Gilligan B., Lu L.S. and Wang S.J. (1997). Cell
interactions in the mouse yolk sac: vasculogenesis and
hematopoiesis. J. Cell Physiol. 173, 202-2055.
Augustin H.G. (2001). Tubes, branches, and pillars: the many ways of
forming a new vasculature. Circ. Res. 89, 645-647.
Ausprunk D.H. and Folkman J. (1977). Migration and proliferation of
endothelial cells in preformed and newly formed blood vessels
during tumor angiogenesis. Microvasc. Res. 14, 53-65.
Axnick J. and Lammert E. (2012). Vascular lumen formation. Curr. Opin.
Hematol. 19, 192-198.
Aydogan V., Lenard A., Denes A.S., Sauteur L., Belting H.G. and
Affolter M. (2015). Endothelial cell division in angiogenic sprouts of
differing cellular architecture. Biol. Open 4, 1259-1269.
Barnett F.H., Rosenfeld M., Wood M., Kiosses W.B., Usui Y., Marchetti
V., Aguilar E. and Friedlander M. (2016). Macrophages form
functional vascular mimicry channels in vivo. Sci. Rep. 6, 36659.
Bavisotto L.M., Schwartz S.M. and Heimark R.L. (1990). Modulation of
Ca-dependent intercellular adhesion in bovine aortic and human
umbilical vein endothelial cells by heparin-binding growth factors. J.
Cell. Physiol. 143, 39-51
Bellon G., Martiny L. and Robinet A. (2004). Matrix metalloproteinases
and matrikines in angiogenesis. Crit. Rev. Oncol. Hematol. 49, 203-
220.
Benedito R., Roca C., Sorensen I., Adams S., Gossler A., Fruttiger M.
and Adams R.H. (2009). The notch ligands Dll4 and Jagged1 have
opposing effects on angiogenesis. Cell 137, 1124-1135.
Benjamin L.E., Hemo I. and Keshet E. (1998). A plasticity window for
blood vessel remodelling is defined by pericyte coverage of the
preformed endothelial network and is regulated by PDGF-B and
VEGF. Development 125, 1591-1598.
Benjamin L.E., Golijanin D., Itin A., Pode D. and Keshet E. (1999).
Selective ablation of immature blood vessels in established human
tumors follows vascular endothelial growth factor withdrawal. J. Clin.
Invest. 103, 159-165.
Bentley K. and Chakravartula S. (2017). The temporal basis of
angiogenesis. Philos. Trans. R Soc. Lond. B Biol. Sci. 372,
20150522.
Bentley K., Gerhardt H. and Bates P.A. (2008). Agent-based simulation
of notch mediated tip cell selection in angiogenic sprout initialisation.
J. Theor. Biol. 250, 25-36.
Bentley K., Mariggi G., Gerhardt H. and Bates P.A. (2009). Tipping the
balance: robustness of tip cell selection, migration and fusion in
angiogenesis. PLoS Comput Biol. 5, e1000549.
Bergers G. and Song S. (2005). The role of pericytes in blood-vessel
formation and maintenance. Neuro Oncol. 7, 452-464.
Berisha B., Schams D., Rodler D. and Pfaffl M.W. (2016). Angiogenesis
in the ovary - The most important regulatory event for follicle and
corpus luteum development and function in cow - An overview. Anat.
Histol. Embryol. 45, 124-130.

Angiogenesis and related secondary structures
Betsholtz C., Lindblom P. and Gerhardt H. (2005). Role of pericytes in
vascular morphogenesis. EXS 94, 115-125.
Betz C., Lenard A., Belting H.G. and Affolter M. (2016). Cell behaviors
and dynamics during angiogenesis. Development 143, 2249-2260.
Bexell D., Gunnarsson S., Tormin A., Darabi A., Gisselsson D., Roybon
L., Scheding S. and Bengzon J. (2009). Bone marrow multipotent
mesenchymal stroma cells act as pericyte-like migratory vehicles in
experimental gliomas. Mol. Ther. 17, 183-190.
Bhatia A., Nada R., Kumar Y. and Menon P. (2006). Dabska tumor
(endovascular papillary angioendothelioma) of testis: a case report
with brief review of literature. Diagn. Pathol. 1, 12.
Bittner M., Meltzer P., Chen Y., Jiang Y., Seftor E., Hendrix M.,
Radmacher M., Simon R., Yakhini Z., Ben-Dor A., Sampas N.,
Dougherty E., Wang E., Marincola F., Gooden C., Lueders J.,
Glatfelter A., Pollock P., Carpten J., Gillanders E., Leja D., Dietrich
K., Beaudry C., Berens M., Alberts D. and Sondak V. (2000).
Molecular classification of cutaneous malignant melanoma by gene
expression profiling. Nature 406, 536-540.
Blair R.J., Meng H., Marchese M.J., Ren S., Schwartz L.B., Tonnesen
M.G. and Gruber B.L. (1997). Human mast cells stimulate vascular
tube formation. Tryptase is a novel, potent angiogenic factor. J. Clin.
Invest. 99, 2691-2700.
Bläker H., Dragoje S., Laissue J.A. and Otto H.F. (1999). Pericardial
involvement by thymomas. Entirely intrapericardial thymoma and a
pericardial metastasis of thymoma with glomeruloid vascular
proliferations. Pathol. Oncol. Res. 5, 160-163.
Boesiger J., Tsai M., Maurer M., Yamaguchi M., Brown L.F., Claffey
K.P., Dvorak H.F. and Galli S.J. (1998). Mast cells can secrete
vascular permeability factor/vascular endothelial cell growth factor
and exhibit enhanced release after immunoglobulin E-dependent
upregulation of fc epsilon receptor I expression. J. Exp. Med. 188,
1135-1145.
Brat D.J., Castellano-Sanchez A.A., Hunter S.B., Pecot M., Cohen C.,
Hammond E.H., Devi S.N., Kaur B. and Van Meir E.G. (2004).
Pseudopalisades in glioblastoma are hypoxic, express extracellular
matrix proteases, and are formed by an actively migrating cell
population. Cancer Res. 64, 920-927.
Burger P.C., Chandler D.B. and Klintworth G.K. (1983). Corneal
neovascularization as studied by scanning electron microscopy of
vascular casts. Lab. Invest. 48, 169-180.
Burri P.H. and Djonov V. (2002). Intussusceptive angiogenesis--the
alternative to capillary sprouting. Mol. Aspects Med. 23, S1-27.
Burri P.H. and Tarek M.R. (1990). A novel mechanism of capillary
growth in the rat pulmonary microcirculation. Anat. Rec. 228, 35-45.
Burri P.H., Hlushchuk R. and Djonov V. (2004). Intussusceptive
angiogenesis: its emergence, its characteristics, and its significance.
Dev. Dyn. 231, 474-488.
Caduff J.H., Fischer L.C. and Burri P.H. (1986). Scanning electron
microscope study of the developing microvasculature in the
postnatal rat lung. Anat. Rec. 216, 154-164.
Camaré C., Pucelle M., Nègre-Salvayre A. and Salvayre R. (2017).
Angiogenesis in the atherosclerotic plaque. Redox Biol. 12, 18-34.
Cao Y., Sonveaux P., Liu S., Zhao Y., Mi J., Clary B.M., Li C.Y., Kontos
C.D. and Dewhirst M.W. (2007). Systemic overexpression of
angiopoietin-2 promotes tumor microvessel regression and inhibits
angiogenesis and tumor growth. Cancer Res. 67, 3835-3844.
Cao Z., Bao M., Miele L., Sarkar F.H., Wang Z. and Zhou Q. (2013).
Tumour vasculogenic mimicry is associated with poor prognosis of
human cancer patients: a systemic review and meta-analysis. Eur. J.
1269
Cancer 49, 3914-3923.
Capitão M. and Soares R. (2016). Angiogenesis and inflammation
crosstalk in diabetic retinopathy. J. Cell Biochem. 117, 2443-2453.
Capkin A.A., Demir S., Mentese A., Bulut Ç. and Ayar A. (2017). Can
signal peptide-CUB-EGFdomain-containing protein (SCUBE) levels
be a marker of angiogenesis in patients with psoriasis? Arch.
Dermatol. Res. 309, 203-207.
Caporali A., Martello A., Miscianinov V., Maselli D., Vono R. and Spinetti
G. (2017). Contribution of pericyte paracrine regulation of the
endothelium to angiogenesis. Pharmacol. Ther. 171, 56-64.
Carmeliet P. and Jain R.K. (2011). Molecular mechanisms and clinical
applications of angiogenesis. Nature 473, 298-307.
Cavallo T., Sade R., Folkman J. and Cotran R.S. (1973). Ultrastructural
autoradiographic studies of the early vasoproliferative response in
tumor angiogenesis. Am. J. Pathol. 70, 345-362.
Chamoto K., Gibney B.C., Ackermann M., Lee G.S., Konerding M.A.,
Tsuda A. and Mentzer S.J. (2013). Alveolar epithelial dynamics in
postpneumonectomy lung growth. Anat. Rec. (Hoboken). 296, 495-
503.
Chan-Ling T., Page M.P., Gardiner T., Baxter L., Rosinova E. and
Hughes S. (2004). Desmin ensheathment ratio as an indicator of
vessel stability: evidence in normal development and in retinopathy
of prematurity. Am. J. Pathol. 165, 1301-1313.
Chavakis E., Hain A., Vinci M., Carmona G., Bianchi M.E., Vajkoczy P.,
Zeiher A.M., Chavakis T. and Dimmeler S. (2007). High-mobility
group box 1 activates integrin-dependent homing of endothelial
progenitor cells. Circ. Res. 100, 204-212.
Chen Y.L., Jan K.M., Lin H.S. and Chien S. (1995). Ultrastructural
studies on macromolecular permeability in relation to endothelial cell
turnover. Atherosclerosis 118, 89-104.
Chen P.Y., Qin L., Barnes C., Charisse K., Yi T., Zhang X., Ali R.,
Medina P.P., Yu J., Slack F.J., Anderson D.G., Kotelianski V., Wang
F., Tellides G. and Simons M. (2012). FGF regulates TGF-β
signaling and endothelial-to-mesenchymal transition via control of
let-7 miRNA expression. Cell Rep. 2, 1684-1696.
Chen X., Zheng Y., Manole C.G., Wang X. and Wang Q. (2013).
Telocytes in human oesophagus. J. Cell Mol. Med. 17, 1506-1512.
Chen X., Man G.C.W., Liu Y., Wu F., Huang J., Li T.C. and Wang C.C.
(2017). Physiological and pathological angiogenesis in endometrium
at the time of embryo implantation. Am. J. Reprod. Immunol. 78,
e12693.
Chung E.S., Chauhan S.K., Jin Y., Nakao S., Hafezi-Moghadam A., van
Rooijen N., Zhang Q., Chen L. and Dana R. (2009). Contribution of
macrophages to angiogenesis induced by vascular endothelial
growth factor receptor-3-specific ligands. Am. J. Pathol. 175, 1984-
1992.
Cimpean A.M., Tamma R., Ruggieri S., Nico B., Toma A. and Ribatti D.
(2017). Mast cells in breast cancer angiogenesis. Crit. Rev. Oncol.
Hematol. 115, 23-26.
Clark R.A. and Clark E.L. (1939). Microscopic observations on the
growth of blood capillaries in the living mammal. J. Anat. 64, 251-
301.
Clark R.A., DellaPelle P., Manseau E., Lanigan J.M., Dvorak H.F. and
Colvin R.B. (1982a). Blood vessel fibronectin increases in
conjunction with endothelial cell proliferation and capillary ingrowth
during wound healing. J. Invest. Dermatol. 79, 269-276.
Clark R.A., Quinn J.H., Winn H.J., Lanigan J.M., Dellepella P. and
Colvin R.B. (1982b). Fibronectin is produced by blood vessels in
response to injury. J. Exp. Med. 156, 646-651.
1270
Angiogenesis and related secondary structures
Claxton S. and Fruttiger M. (2004). Periodic Delta-like 4 expression in
developing retinal arteries. Gene Expr. Patterns 5, 123-127.
Claxton S. and Fruttiger M. (2005). Oxygen modifies artery
differentiation and network morphogenesis in the retinal vasculature.
Dev. Dyn. 233, 822-828.
Clearkin K.P. and Enzinger F.M. (1976). Intravascular papillary
endothelial hyperplasia. Arch. Pathol. Lab. Med. 100, 441-444.
Corselli M., Chen C.W., Sun B., Yap S., Rubin J.P. and Péault B.
(2012). The tunica adventitia of human arteries and veins as a
source of mesenchymal stem cells. Stem Cells Dev. 21, 1299-1308.
Darland D.C. and D'Amore P.A. (2001). TGF beta is required for the
formation of capillary-like structures in three-dimensional cocultures
of 10T1/2 and endothelial cells. Angiogenesis 4, 11-20.
Darland D.C., Massingham L.J., Smith S.R., Piek E., Saint-Geniez M.
and D'Amore P.A. (2003). Pericyte production of cell-associated
VEGF is differentiation-dependent and is associated with endothelial
survival. Dev. Biol. 264, 275-288.
Davis G.E., Bayless K.J., Davis M.J. and Meininger G.A. (2000).
Regulation of tissue injury responses by the exposure of matricryptic
sites within extracellular matrix molecules. Am. J. Pathol. 156, 1489-
1498.
De Bock K., De Smet F., Leite De Oliveira R., Anthonis K. and Carmeliet
P. (2009). Endothelial oxygen sensors regulate tumor vessel
abnormalization by instructing phalanx endothelial cells. J. Mol.
Med. (Berl) 87, 561-569.
De Paepe M.E., Benny M.K., Priolo L., Luks F.I. and Shapiro S. (2017).
Florid intussusceptive-like microvascular dysangiogenesis in a
preterm lung. Pediatr. Dev. Pathol. (in press).
De Smet F., Segura I., De Bock K., Hohensinner P.J. and Carmeliet P.
(2009). Mechanisms of vessel branching: filopodia on endothelial tip
cells lead the way. Arterioscler. Thromb. Vasc. Biol. 29, 639-649.
De Spiegelaere W., Cornillie P., Casteleyn C., Burvenich C. and Van
den Broeck W. (2010). Detection of hypoxia inducible factors and
angiogenic growth factors during foetal endochondral and
intramembranous ossification. Anat. Histol. Embryol. 39, 376-384.
De Spiegelaere W., Casteleyn C., Van den Broeck W., Plendl J.,
Bahramsoltani M., Simoens P., Djonov V. and Cornillie P. (2012).
Intussusceptive angiogenesis: a biologically relevant form of
angiogenesis. J. Vasc. Res. 49, 390-404.
Dejana E. and Lampugnani M.G. (2014). Differential adhesion drives
angiogenesis. Nat. Cell Biol. 16, 305-306.
Dejana E. and Orsenigo F. (2013). Endothelial adherens junctions at a
glance. J. Cell Sci. 126, 2545-2549.
Dejana E., Orsenigo F. and Lampugnani M.G. (2008). The role of
adherens junctions and VE-cadherin in the control of vascular
permeability. J. Cell Sci. 121, 2115-2122.
Dellett M., Brown E.D., Guduric-Fuchs J., O'Connor A., Stitt A.W.,
Medina R.J. and Simpson D.A. (2017). MicroRNA-containing
extracellular vesicles released from endothelial colony-forming cells
modulate angiogenesis during ischaemic retinopathy. J. Cell Mol.
Med. (in press).
Demou Z.N. and Hendrix M.J. (2008). Microgenomics profile the
endogenous angiogenic phenotype in subpopulations of aggressive
melanoma. J. Cell Biochem. 105, 562-73.
Dey N., De P. and Brian L.J. (2015). Evading anti-angiogenic therapy:
resistance to anti-angiogenic therapy in solid tumors. Am. J. Transl.
Res. 7, 1675-1698.
Diaz-Flores L. and Dominguez C. (1985). Relation between arterial
intimal thickening and the vasa-vasorum. Virchows Arch. A Pathol.
Anat. Histopathol. 406, 165-177.
Diaz-Flores L., Valladares F., Gutierrez R. and Varela H. (1990). The
role of the pericytes of the adventitial microcirculation in the arterial
intimal thickening. Histol. Histopathol. 5, 145-153.
Diaz-Flores L., Gutierrez R., Varela H., Rancel N. and Valladares F.
(1991). Microvascular pericytes: a review of their morphological and
functional characteristics. Histol. Histopathol. 6, 269-286.
Diaz-Flores L., Gutierrez R. and Varela H. (1992). Behavior of
postcapillary venule pericytes during postnatal angiogenesis. J.
Morphol. 213, 33-45.
Díaz-Flores L., Gutiérrez R. and Varela H. (1994a). Angiogenesis: an
update. Histol. Histopathol. 9, 807-843.
Diaz-Flores L., Gutierrez R., Valladares F., Varela H. and Perez M.
(1994b). Intense vascular sprouting from rat femoral vein induced
bypmstaglandins El and E2. Anat. Rec. 238, 66-76.
Díaz-Flores L., Gutiérrez R., Madrid J.F., Varela H., Valladares F.,
Acosta E., Martín-Vasallo P. and Díaz-Flores L. Jr (2009a).
Pericytes. Morphofunction, interactions and pathology in a quiescent
and activated mesenchymal cell niche. Histol. Histopathol. 24, 909-
969.
Diaz-Flores L. Jr, Gutierrez R., Madrid J.F., Varela H., Valladares F. and
Diaz-Flores L. (2009b). Adult stem cells and repair through
granulation tissue. Front Biosci (Landmark Ed). 14, 1433-1470.
Díaz-Flores L. Jr, Gutiérrez R., Madrid J.F., Sáez F.J., Valladares F.,
Villar J. and Díaz-Flores L. (2011a). Peg-and-socket junctions
between smooth muscle cells and endothelial cells in femoral veins
are stimulated to angiogenesis by prostaglandin E₂ and glycerols.
Histol. Histopathol. 26, 623-630.
Díaz-Flores L. Jr, Gutiérrez R., Madrid J.F., Álvarez-Argüelles H.,
Valladares F. and Díaz-Flores L. (2011b). Adult stem and transit-
amplifying cells in repair through granulation tissue: Role of the
pericytic vasculature niche. In: Stem Cell, regenerative Medicine.
Chapter 6. Singh S.R. (ed.). Nova Science Publishers.
Díaz-Flores L., Gutiérrez R., García M.P., Alvarez-Argüelles H., Díaz-
Flores L. Jr and Madrid J.F. (2011c). Myopericytoma and arterial
intimal thickening: the relationship between myopericytes and
myointimal cells. J. Cutan. Pathol. 38, 857-864.
Diaz-Flores L. Jr, Gutierrez R., Madrid J.F., Acosta E., Avila J., Diaz-
Flores L. and Martin-Vasallo P. (2012a). Cell sources for cartilage
repair; contribution of the mesenchymal perivascular niche. Front
Biosci (Schol Ed). 4, 1275-1294.
Díaz-Flores L., Gutiérrez R., García M.P., Díaz-Flores L. Jr, Valladares
F. and Madrid J.F. (2012b). Ultrastructure of myopericytoma: a
continuum of transitional phenotypes of myopericytes. Ultrastruct.
Pathol. 36, 189-194.
Díaz-Flores L., Gutiérrez R., García M.P., Sáez F.J., Díaz-Flores L. Jr,
Valladares F. and Madrid J.F. (2014). CD34+ stromal
cells/fibroblasts/fibrocytes/telocytes as a tissue reserve and a
principal source of mesenchymal cells. Location, morphology,
function and role in pathology. Histol. Histopathol. 29, 831-870.
Díaz-Flores L., Gutiérrez R., Lizartza K., Goméz M.G., García M.del P.,
Sáez F.J., Díaz-Flores L. Jr and Madrid J.F. (2015a). Behavior of in
situ human native adipose tissue CD34+ stromal/progenitor cells
during different stages of repair. Tissue-resident CD34+ stromal
cells as a source of myofibroblasts. Anat. Rec (Hoboken) 298, 917-
930.
Díaz-Flores L, Gutiérrez R., García M.P., González M., Sáez F.J.,
Aparicio F., Díaz-Flores L. Jr and Madrid J.F. (2015b). Human
resident CD34+ stromal cells/telocytes have progenitor capacity and

Angiogenesis and related secondary structures
are a source of αSMA+ cells during repair. Histol. Histopathol 30,
615-627.
Díaz-Flores L., Gutiérrez R., García-Suárez M.P., González M., Díaz-
Flores L. and Madrid J.F. (2016a). Telocytes as a source of
progenitor cells in regeneration and repair through granulation
tissue. Curr. Stem Cell Res. Ther. 11, 395-403.
Díaz-Flores L., Gutiérrez R., Díaz-Flores L. Jr, Goméz M.G., Sáez F.J.
and Madrid J.F. (2016b). Behaviour of telocytes during
physiopathological activation. Semin. Cell Dev. Biol. 55, 50-61.
Díaz-Flores L., Gutiérrez R., González-Gómez M., Díaz-Flores L. Jr,
Valladares F., Rancel N., Sáez F.J. and Madrid J.F. (2016c).
Telocyte behaviour during inflammation, repair and tumour stroma
formation. Adv. Exp. Med. Biol. 13, 177-191.
Díaz-Flores L., Gutiérrez R., Madrid J.F., García-Suárez M.P.,
González-Álvarez M.P., Díaz-Flores L. Jr and Sáez F.J. (2016d).
Intravascular Papillary Endothelial Hyperplasia (IPEH). Evidence
supporting a piecemeal mode of angiogenesis from vein
endothelium, with vein wall neovascularization and papillary
formation. Histol. Histopathol. 31, 1271-1279.
Díaz-Flores L., Gutiérrez R., García M.P., Sáez F.J., Díaz-Flores L. Jr.
and Madrid J.F. (2017). Piecemeal mechanism combining sprouting
and intussusceptive angiogenesis in intravenous papillary formation
induced by PGE2 and glycerol. Anat. Rec. (Hoboken) (in press).
Dimmeler S. and Zeiher A.M. (2000). Endothelial cell apoptosis in
angiogenesis and vessel regression. Circ. Res. 87, 434-439.
Ding Y., Song N. and Luo Y. (2012). Role of bone marrow-derived cells
in angiogenesis: focus on macrophages and pericytes. Cancer
Microenviron. 5, 225-236.
DiPietro L.A. and Polverini P.J. (1993). Angiogenic macrophages
produce the angiogenic inhibitor thrombospondin 1. Am. J. Pathol.
143, 678-684.
Djonov V., Schmid M., Tschanz S.A. and Burri P.H. (2000).
Intussusceptive angiogenesis: its role in embryonic vascular network
formation. Circ. Res. 86, 286-292.
Djonov V., Andres A.C. and Ziemiecki A. (2001). Vascular remodelling
during the normal and malignant life cycle of the mammary gland.
Microsc. Res. Tech. 52, 182-189.
Djonov V.G., Kurz H. and Burri P.H. (2002). Optimality in the developing
vascular system: branching remodeling by means of intussusception
as an efficient adaptation mechanism. Dev. Dyn. 224, 391-402.
Djonov V., Baum O. and Burri P.H. (2003). Vascular remodeling by
intussusceptive angiogenesis. Cell Tissue Res. 314, 107-117.
Döme B., Paku S., Somlai B and Tímár J. (2002). Vascularization of
cutaneous melanoma involves vessel co-option and has clinical
significance. J. Pathol. 197, 355-362.
Döme B., Hendrix M.J., Paku S., Tóvári J. and Tímár J. (2007).
Alternative vascularization mechanisms in cancer: Pathology and
therapeutic implications. Am. J. Pathol. 170; 1-15.
Du J.W., Xu K.Y., Fang L.Y. and Qi X.L. (2012). Interleukin-17,
produced by lymphocytes, promotes tumor growth and angiogenesis
in a mouse model of breast cancer. Mol. Med. Rep. 6, 1099-1102.
Dunk C., Smith S., Hazan A., Whittle W. and Jones R.L. (2008).
Promotion of angiogenesis by human endometrial lymphocytes.
Immunol. Invest. 37, 583-610.
Dvorak H.F., Hawey V.S., Estrella P., Brown L.F., McDonach J. and
Dvorak A.M. (1987). Fibrin containing gels induce angiogenesis.
lmplications for tumor stroma generation and wound healing. Lab.
Invest. 57, 673-686.
Egginton S. (2009). Invited review: activity-induced angiogenesis.
1271
Pflugers Arch. 457, 963-977.
Eilken H.M. and Adams R.H. (2010). Dynamics of endothelial cell
behavior in sprouting angiogenesis. Curr. Opin. Cell Biol. 22, 617-
625.
Elshabrawy H.A., Chen Z., Volin M.V., Ravella S., Virupannavar S. and
Shahrara S. (2015). The pathogenic role of angiogenesis in
rheumatoid arthritis. Angiogenesis 18, 433-448.
Emberger M., Laimer M., Steiner H. and Zelger B. (2009). Retiform
hemangioendothelioma: presentation of a case expressing D2-40. J.
Cutan. Pathol. 36, 987-990.
Enge M., Bjarnegård M., Gerhardt H., Gustafsson E., Kalén M., Asker
N., Hammes H.P., Shani M., Fässler R. and Betsholtz C. (2002).
Endothelium-specific platelet-derived growth factor-B ablation
mimics diabetic retinopathy. EMBO J. 21, 4307-4316.
Etienne-Manneville S. (2004). Cdc42--the centre of polarity. J. Cell Sci.
117, 1291-1300.
Fantin A., Lampropoulou A., Gestri G., Raimondi C., Senatore V.,
Zachary I. and Ruhrberg C. (2015). NRP1 Regulates CDC42
Activation to promote filopodia formation in endothelial tip cells. Cell
Rep. 11, 1577-1590.
Filipovic N., Tsuda A., Lee G.S., Miele L.F., Lin M., Konerding M.A. and
Mentzer S.J. (2009). Computational flow dynamics in a geometric
model of intussusceptive angiogenesis. Microvasc. Res. 78, 286-
293.
Fischer R.S., Gardel M., Ma X., Adelstein R.S. and Waterman C.M.
(2009). Local cortical tension by myosin II guides 3D endothelial cell
branching. Curr. Biol. 19, 260-265.
Folkman J. (1984). What is the role of endothelial cells in angiogenesis.
Lab. Invest. 51, 601-602.
Folkman J. (1985). Tumor angiogenesis. Adv. Cancer Res. 43, 175-203.
Folkman J. (1989). Sucessful treatment of an angiogenic disease. N.
Engl. J. Med. 320, 1211-1212.
Folkman J. (2003). Fundamental concepts of the angiogenic process.
Curr. Mol. Med. 3, 643-651
Folkman J. and Cotran R. (1976). Relation of vascular proliferation to
tumor growth. Int. Rev. Exp. Pathol. 16, 207-248.
Folkman J. and Haudenschild C.C. (1980). Angiogenesis in vitro. Nature
288, 551-556.
Forman S.B., Tyler W.B., Ferringer T.C. and Elston D.M. (2007).
Glomeruloid hemangiomas without POEMS syndrome: series of
three cases. J. Cutan. Pathol. 34, 956-957.
Franco C.A., Jones M.L., Bernabeu M.O., Geudens I., Mathivet T., Rosa
A., Lopes F.M., Lima A.P., Ragab A., Collins R.T., Phng L.K.,
Coveney P.V. and Gerhardt H. (2015). Dynamic endothelial cell
rearrangements drive developmental vessel regression. PLoS Biol.
13, e1002125.
Furusato M., Fukunaga M, Kikuchi Y., Yokota S., Joh K., Aizawa S. and
lshikawa E. (1984). Two- and three dimensional ultrastructural
observation of angiogenesis in juvenile hemangioma. Virchows
Arch. (Cell Pathol.). 46, 229-237.
Gálvez B.G., Genís L., Matías-Román S., Oblander S.A., Tryggvason
K., Apte S.S. and Arroyo A.G. (2005). Membrane type 1-matrix
metalloproteinase is regulated by chemokines monocyte-
chemoattractant protein-1/ccl2 and interleukin-8/CXCL8 in
endothelial cells during angiogenesis. J. Biol. Chem. 280, 1292-
1298.
Garbett P.K. and Gibbins J.R. (1987). Experimental neovascularization
in vivo: lhe early changes in a stable adult vasculature responding
toangiogenic stimulation by a syngeneic neoplasm. Br. J. Exp.
1272
Angiogenesis and related secondary structures
Pathol. 68, 625-635.
Garcia M.D. and Larina I.V. (2014). Vascular development and
hemodynamic force in the mouse yolk sac. Front. Physiol. 5, 308.
Gehling U.M., Ergün S., Schumacher U., Wagener C., Pantel K., Otte
M., Schuch G., Schafhausen P., Mende T., Kilic N., Kluge K.,
Schäfer B., Hossfeld D.K. and Fiedler W. (2000). In vitro
differentiation of endothelial cells from AC133-positive progenitor
cells. Blood 95, 3106-3112.
Gerhardt H. and Betsholtz C. (2003). Endothelial-pericyte interactions in
angiogenesis. Cell Tissue Res. 314, 15-23.
Gerhardt H. and Betsholtz C. (2005). How do endothelial cells
orientate? EXS 94, 3-15.
Gerhardt H. and Semb H. (2008). Pericytes: gatekeepers in tumour cell
metastasis? J. Mol. Med. (Berl). 86, 135-144.
Gerhardt H., Golding M., Fruttiger M., Ruhrberg C., Lundkvist A.,
Abramsson A., Jeltsch M., Mitchell C., Alitalo K., Shima D. and
Betsholtz C. (2003). VEGF guides angiogenic sprouting utilizing
endothelial tip cell filopodia. J. Cell Biol. 161, 1163-1177.
Geudens I. and Gerhardt H. (2011). Coordinating cell behaviour during
blood vessel formation. Development 138, 4569-4583.
Ghajar C.M., George S.C. and Putnam A.J. (2008). Matrix
metalloproteinase control of capillary morphogenesis. Crit. Rev.
Eukaryot. Gene Expr. 18, 251-278.
Gianni-Barrera R., Trani M., Fontanellaz C., Heberer M., Djonov V.,
Hlushchuk R. and Banfi A. (2013). VEGF over-expression in skeletal
muscle induces angiogenesis by intussusception rather than
sprouting. Angiogenesis 16, 123-136.
Goffin J.R., Straume O., Chappuis P.O., Brunet J.S., Bégin L.R., Hamel
N., Wong N., Akslen L.A. and Foulkes W.D. (2003). Glomeruloid
microvascular proliferation is associated with p53 expression,
germline BRCA1 mutations and an adverse outcome following
breast cancer. Br. J. Cancer 89, 1031-1034.
Gomes E.R., Jani S. and Gundersen G.G. (2005). Nuclear movement
regulated by Cdc42, MRCK,myosin, and actin flow establishes
MTOC polarization in migrating cells. Cell 121, 451-463.
González-Guerra E., Haro M.R., Fariña M.C., Martín L., Manzarbeitia L.
and Requena L. (2009). Glomeruloid haemangioma is not always
associated with POEMS syndrome. Clin. Exp. Dermatol. 34, 800-
803.
Grant M.B., May W.S., Caballero S., Brown G.A., Guthrie S.M., Mames
R.N., Byrne B.J., Vaught T., Spoerri P.E., Peck A.B. and Scott E.W.
(2002). Adult hematopoietic stem cells provide functional
hemangioblast activity during retinal neovascularization. Nat. Med.
8, 607-612.
Greenberg J.I., Shields D.J., Barillas S.G., Acevedo L.M., Murphy E.,
Huang J., Scheppke L., Stockmann C., Johnson R.S., Angle N. and
Cheresh D.A. (2008). A role for VEGF as a negative regulator of
pericyte function and vessel maturation. Nature 456, 809-813.
Hadfield G. (1951). Granulation tissue. Ann. Roy. Coll. Surg. Eng.
9,397-407.
Hall-Glenn F., De Young R.A., Huang B.L., van Handel B., Hofmann
J.J., Chen T.T., Choi A., Ong J.R., Benya P.D., Mikkola H., Iruela-
Arispe M.L. and Lyons K.M. (2012). CCN2/connective tissue growth
factor is essential for pericyte adhesion and endothelial basement
membrane formation during angiogenesis. PLoS One 7, e30562.
Hartlapp I., Abe R., Saeed R.W., Peng T., Voelter W., Bucala R. and
Metz C.N. (2001). Fibrocytes induce an angiogenic phenotype in
cultured endothelial cells and promote angiogenesis in vivo. FASEB
J. 15, 2215-2224.
Hashimoto H., Daimaru Y. and Enjoji M. (1983). Intravascular papillary
endothelial hyperplasia. A clinicopathologic study of 91 cases. Am.
J. Dermatopathol. 5, 539-546.
Hashizume H. and Ushiki T. (2002). Three-dimensional cytoarchitecture
of angiogenic blood vessels in a gelatin sheet implanted in the rat
skeletal muscular layers. Arch. Histol. Cytol. 65, 347-357.
Heidenreich R., Röcken M. and Ghoreschi K. (2009). Angiogenesis
drives psoriasis pathogenesis. Int. J. Exp. Pathol. 90, 232-248.
Heissig B., Hattori K., Dias S., Friedrich M., Ferris B., Hackett N.R.,
Crystal R.G., Besmer P., Lyden D., Moore M.A., Werb Z. and Rafii
S. (2002). Recruitment of stem and progenitor cells from the bone
marrow niche requires MMP-9 mediated release of kit-ligand. Cell
109, 25-37.
Hellström M., Gerhardt H., Kalén M., Li X., Eriksson U., Wolburg H. and
Betsholtz C. (2001). Lack of pericytes leads to endothelial
hyperplasia and abnormal vascular morphogenesis. J. Cell Biol.
153, 543-553.
Hellström M., Phng L.K. and Gerhardt H. (2007a). VEGF and Notch
signaling: the yin and yang of angiogenic sprouting. Cell Adh. Migr.
1, 133-136.
Hellström M., Phng L.K., Hofmann J.J., Wallgard E., Coultas L.,
Lindblom P., Alva J., Nilsson A.K., Karlsson L., Gaiano N., Yoon K.,
Rossant J., Iruela-Arispe M.L., Kalén M., Gerhardt H. and Betsholtz
C. (2007b). Dll4 signalling through Notch1 regulates formation of tip
cells during angiogenesis. Nature 445, 776-780.
Hendrix M.J., Seftor E.A., Meltzer P.S., Gardner L.M., Hess A.R.,
Kirschmann D.A., Schatteman G.C. and Seftor R.E. (2001).
Expression and functional significance of VE-cadherin in aggressive
human melanoma cells: role in vasculogenic mimicry. Proc. Natl.
Acad. Sci. USA 98, 8018-8023.
Hendrix M.J., Seftor E.A., Hess A.R. and Seftor R.E. (2003).
Vasculogenic mimicry and tumour-cell plasticity: lessons from
melanoma. Nat. Rev. Cancer 3, 411-421.
Hess A.R. and Hendrix M.J. (2006). Focal adhesion kinase signaling
and the aggressive melanoma phenotype. Cell Cycle. 5, 478-480.
Hess A.R., Seftor E.A., Gardner L.M., Carles-Kinch K., Schneider G.B.,
Seftor R.E., Kinch M.S. and Hendrix M.J. (2001). Molecular
regulation of tumor cell vasculogenic mimicry by tyrosine
phosphorylation: role of epithelial cell kinase (Eck/EphA2). Cancer
Res. 61, 3250-3255.
Hess A.R., Seftor E.A., Seftor R.E. and Hendrix M.J. (2003).
Phosphoinositide 3-kinase regulates membrane Type 1-matrix
metalloproteinase (MMP) and MMP-2 activity during melanoma cell
vasculogenic mimicry. Cancer Res. 63, 4757-4762.
Hess A.R., Seftor E.A., Gruman L.M., Kinch M.S., Seftor R.E. and
Hendrix M.J. (2006). VE-cadherin regulates EphA2 in aggressive
melanoma cells through a novel signaling pathway: implications for
vasculogenic mimicry. Cancer Biol. Ther. 5, 228-233.
Hillen F. and Griffioen A.W. (2007). Tumour vascularization: sprouting
angiogenesis and beyond. Cancer Metastasis Rev. 26, 489-502.
Hiraoka N., Allen E., Apel I.J., Gyetko M.R. and Weiss S.J. (1998).
Matrix metalloproteinases regulate neovascularization by acting as
pericellular fibrinolysins. Cell 95, 365-377.
His W. (1868). Untersuchungen uber die erste Anlage
desWirbelthierleibes. F.C.W. Vogel. Leipzig.
Hlushchuk R., Ehrbar M., Reichmuth P., Heinimann N., Styp-Rekowska
B., Escher R., Baum O., Lienemann P., Makanya A., Keshet E. and
Djonov V. (2011). Decrease in VEGF expression induces
intussusceptive vascular pruning. Arterioscler. Thromb. Vasc. Biol.

Angiogenesis and related secondary structures
31, 2836-2844.
Hoffmann J., Feng Y., vom Hagen F., Hillenbrand A., Lin J., Erber R.,
Vajkoczy P., Gourzoulidou E., Waldmann H., Giannis A., Wolburg
H., Shani M., Jaeger V., Weich H.A., Preissner K.T., Hoffmann S.,
Deutsch U. and Hammes H.P. (2005). Endothelial survival factors
and spatial completion, but not pericyte coverage of retinal
capillaries determine vessel plasticity. FASEB J. 19, 2035-2036.
Holash J., Maisonpierre P.C., Compton D., Boland P., Alexander C.R.,
Zagzag D., Yancopoulos G.D. and Wiegand S.J. (1999). Vessel
cooption, regression, and growth in tumors mediated by
angiopoietins and VEGF. Science 284, 1994-1998.
Inai T., Mancuso M., Hashizume H., Baffert F., Haskell A., Baluk P., Hu-
Lowe D.D., Shalinsky D.R., Thurston G., Yancopoulos G.D. and
McDonald D.M. (2004). Inhibition of vascular endothelial growth
factor (VEGF) signaling in cancer causes loss of endothelial
fenestrations, regression of tumor vessels, and appearance of
basement membrane ghosts. Am. J. Pathol. 165, 35-52.
Iruela-Arispe M.L. and Davis G.E. (2009). Cellular and molecular
mechanisms of vascular lumen formation. Dev. Cell 16, 222-231.
Jakobsson L., Franco C.A., Bentley K., Collins R.T., Ponsioen B.,
Aspalter I.M., Rosewell I., Busse M., Thurston G., Medvinsky A.,
Schulte-Merker S. and Gerhardt H. (2010). Endothelial cells
dynamically compete for the tip cell position during angiogenic
sprouting. Nat. Cell Biol. 12, 943-953.
Jin S.W. and Patterson C. (2009). The opening act: vasculogenesis and
the origins of circulation. Arterioscler. Thromb. Vasc. Biol. 29, 623-
629.
Jin S.W., Beis D., Mitchell T., Chen J.N. and Stainier D.Y. (2005)
Cellular and molecular analyses of vascular tube and lumen
formation in zebrafish. Development 132, 5199-5209.
Kale S., Hanai J., Chan B., Karihaloo A., Grotendorst G., Cantley L. and
Sukhatme V.P. (2005). Microarray analysis of in vitro pericyte
differentiation reveals an angiogenic program of gene expression.
FASEB J. 19, 270-271.
Kalka C., Masuda H., Takahashi T., Kalka-Moll W.M., Silver M.,
Kearney M., Li T., Isner J.M. and Asahara T. (2000). Transplantation
of ex vivo expanded endothelial progenitor cells for therapeutic
neovascularization. Proc. Natl. Acad. Sci. USA 97, 3422-3427.
Kanbe N., Kurosawa M., Nagata H., Yamashita T., Kurimoto F. and
Miyachi Y. (2000). Production of fibrogenic cytokines by cord blood-
derived cultured human mast cells. J. Allergy Clin. Immunol. 106,
S85-S90.
Kandemir N.O., Narli Z.I., Kalayci M. and Ozdamar S.O. (2014). A rare
pattern of angiogenesis in meningiomas: glomeruloid microvascular
proliferation. Turk. Neurosurg. 24, 765-769.
Kashiwagi S., Izumi Y., Gohongi T., Demou Z.N., Xu L., Huang P.L.,
Buerk D.G., Munn L.L., Jain R.K. and Fukumura D. (2005). NO
mediates mural cell recruitment and vessel morphogenesis in
murine melanomas and tissue-engineered blood vessels. J. Clin.
Invest. 115, 1816-1827.
Kilarski W.W., Samolov B., Petersson L., Kvanta A. and Gerwins P.
(2009). Biomechanical regulation of blood vessel growth during
tissue vascularization. Nat. Med. 15, 657-664.
Kim E.S., Serur A., Huang J., Manley C.A., McCrudden K.W., Frischer
J.S., Soffer S.Z., Ring L., New T., Zabski S., Rudge J.S., Holash J.,
Yancopoulos G.D., Kandel J.J. and Yamashiro D.J. (2002). Potent
VEGF blockade causes regression of coopted vessels in a model of
neuroblastoma. Proc. Natl. Acad. Sci. USA 99, 11399-11404.
Kim H.K., Song K.S., Kim H.O., Chung J.H., Lee K.R., Lee Y.J., Lee
1273
D.H., Lee E.S., Kim H.K., Ryu K.W. and Bae J.M. (2003). Circulating
numbers of endothelial progenitor cells in patients with gastric and
breast cancer. Cancer Lett. 198, 83-88.
Kim M., Lee C., Payne R., Yue B.Y., Chang J.H. and Ying H. (2015).
Angiogenesis in glaucoma filtration surgery and neovascular
glaucoma: A review. Surv. Ophthalmol. 60, 524-535.
Kim S.O., Trau H.A. and Duffy D.M. (2017). Vascular endothelial growth
factors C and D may promote angiogenesis in the primate ovulatory
follicle. Biol. Reprod. 96, 389-400.
Kochhan E., Lenard A., Ellertsdottir E., Herwig L., Affolter M., Belting
H.G. and Siekmann A.F. (2013). Blood flow changes coincide with
cellular rearrangements during blood vessel pruning in zebrafish
embryos. PLoS One 8, e75060.
Konerding M.A., Turhan A., Ravnic D.J., Lin M., Fuchs C., Secomb
T..W, Tsuda A and Mentzer SJ. (2010) Inflammation-induced
intussusceptive angiogenesis in murine colitis. Anat. Rec.
(Hoboken). 293, 849-857.
Kreuger J. and Phillipson M. (2016). Targeting vascular and leukocyte
communication in angiogenesis, inflammation and fibrosis. Nat. Rev.
Drug Discov. 15, 125-142.
Krishna Priya S., Nagare R.P., Sneha V.S., Sidhanth C., Bindhya S.,
Manasa P. and Ganesan T.S. (2016). Tumour angiogenesis-Origin
of blood vessels. Int. J. Cancer 139, 729-735.
Kugler A, Koelblinger P, Zelger B, Ahlgrimm-Siess V. and Laimer M.
(2016). Papillary intralymphatic angioendothelioma (PILA), also
referred to as Dabska tumour, in an 83-year-old woman. J. Eur.
Acad. Dermatol. Venereol. 30, e59-e61.
Kuo T., Sayers C.P. and Rosai J. (1976). Masson's "vegetant
intravascular hemangioendothelioma:" a lesion often mistaken for
angiosarcoma: study of seventeen cases located in the skin and soft
tissues. Cancer 38, 1227-1236.
Kuo C.L, Chen P.C, Li W.Y. and Chu P.Y. (2015). Retiform
hemangioendothelioma of the neck. J. Pathol. Transl. Med. 49, 171-
173.
Kurz H., Gärtner T., Eggli P.S. and Christ B. (1996). First blood vessels
in the avian neural tube are formed by a combination of dorsal
angioblast immigration and ventral sprouting of endothelial cells.
Dev. Biol. 173, 133-147.
Kurz H., Burri P.H. and Djonov V.G. (2003). Angiogenesis and vascular
remodeling by intussusception: from form to function. News Physiol.
Sci. 18, 65-70.
Lamagna C. and Bergers G. (2006). The bone marrow constitutes a
reservoir of pericyte progenitors. J. Leukoc. Biol. 80, 677-681.
Lange C., Ehlken C., Stahl A., Martin G., Hansen L. and Agostini H.T.
(2009). Kinetics of retinal vaso-obliteration and neovascularisation in
the oxygen-induced retinopathy (OIR) mouse model. Graefes Arch.
Clin. Exp. Ophthalmol. 247, 1205-1211.
Langlois S., Gingras D. and Béliveau R. (2004). Membrane type 1-
matrix metalloproteinase (MT1-MMP) cooperates with sphingosine
1-phosphate to induce endothelial cell migration and morphogenic
differentiation. Blood 103, 3020-3028.
Larrivée B., Niessen K., Pollet I., Corbel S.Y., Long M., Rossi F.M.,
Olive P.L. and Karsan A. (2005). Minimal contribution of marrow-
derived endothelial precursors to tumor vasculature. J Immunol.
175, 2890-2899.
le Noble F., Moyon D., Pardanaud L., Yuan L., Djonov V., Matthijsen R.,
Bréant C., Fleury V. and Eichmann A. (2004). Flow regulates
arterial-venous differentiation in the chick embryo yolk sac.
Development 131, 361-375.
1274
Angiogenesis and related secondary structures
le Noble F., Fleury V., Pries A., Corvol P., Eichmann A. and Reneman
R.S. (2005). Control of arterial branching morphogenesis in
embryogenesis: go with the flow. Cardiovasc. Res. 65, 619-628.
Leavesley D.I., Schwartz M.A., Rosenfeld M. and Cheresh D.A. (1993).
Integrin beta 1- and beta 3-mediated endothelial cell migration is
triggered through distinct signalling mechanisms. J. Cell Biol. 121,
163-170.
Lee J.F., Ozaki H., Zhan X., Wang E., Hla T. and Lee M.J. (2006).
Sphingosine-1-phosphate signaling regulates lamellipodia
localization of cortactin complexes in endothelial cells. Histochem.
Cell Biol. 126, 297-304.
Lee G.S., Filipovic N., Miele L.F., Lin M., Simpson D.C, Giney B.,
Konerding M.A., Tsuda A. and Mentzer S.J. (2010). Blood flow
shapes intravascular pillar geometry in the chick chorioallantoic
membrane. J. Angiogenes. Res. 2, 11.
Lee G.S., Filipovic N., Lin M., Gibney B.C., Simpson D.C., Konerding
M.A., Tsuda A. and Mentzer S.J. (2011). Intravascular pillars and
pruning in the extraembryonic vessels of chick embryos. Dev. Dyn.
240, 1335-1343.
Lenard A., Ellertsdottir E., Herwig L., Krudewig A., Sauteur L., Belting
H.G. and Affolter M. (2013). In vivo analysis reveals a highly
stereotypic morphogenetic pathway of vascular anastomosis. Dev.
Cell 25, 492-506.
Lenard A., Daetwyler S., Betz C., Ellertsdottir E., Belting H.G., Huisken
J. and Affolter M. (2015). Endothelial cell self-fusion during vascular
pruning. PLoS Biol. 13, e1002126.
Leslie J.D., Ariza-McNaughton L., Bermange A.L., McAdow R., Johnson
S.L. and Lewis J. (2007). Endothelial signalling by the Notch ligand
Delta-like 4 restricts angiogenesis. Development 134, 839-844.
Li Q., Fukuda K., Lu Y., Nakamura Y., Chikama T., Kumagai N. and
Nishida T. (2003). Enhancement by neutrophils of collagen
degradation by corneal fibroblasts. J. Leukoc. Biol. 74, 412-419.
Li B., Li Y., Tian X.Y. and Li Z. (2013). Unusual multifocal intraosseous
papillary intralymphatic angioendothelioma (Dabska tumor) of facial
bones: a case report and review of literature. Diagn. Pathol. 8, 160.
Liaw L. and Schwartz S.M. (1993). Microtubule disruption
stimulatesDNA synthesis in bovine endothelial cells and potentiates
cellular response to basic fibroblast growth factor. Am. J. Pathol.
143, 937-948.
Lin C.S. and Lue T.F. (2013). Defining vascular stem cells. Stem Cells
Dev. 22, 1018-1026.
Lin Y., Weisdorf D.J., Solovey A. and Hebbel R.P. (2000). Origins of
circulating endothelial cells and endothelial outgrowth from blood. J.
Clin. Invest. 105, 71-77.
Lin C.S., Xin Z.C., Deng C.H., Ning H., Lin G. and Lue T.F. (2010).
Defining adipose tissue-derived stem cells in tissue and in culture.
Histol. Histopathol. 25, 807-815.
Liu Q., Ouyang R., Chen P. and Zhou R. (2015). A case report of
retiform hemangioendothelioma as pleural nodules with literature
review. Diagn. Pathol. 10, 194.
Lobov I.B., Rao S., Carroll T.J., Vallance J.E., Ito M., Ondr J.K., Kurup
S., Glass D.A., Patel M.S., Shu W., Morrisey E.E., McMahon A.P.,
Karsenty G. and Lang R.A. (2005). WNT7b mediates macrophage-
induced programmed cell death in patterning of the vasculature.
Nature 437, 417-421.
Lobov I.B., Renard R.A., Papadopoulos N., Gale N.W., Thurston G.,
Yancopoulos G.D. and Wiegand S.J. (2007). Delta-like ligand 4
(Dll4) is induced by VEGF as a negative regulator of angiogenic
sprouting. Proc. Natl. Acad. Sci. USA 104, 3219-3224.
Lu X., Le Noble F., Yuan L., Jiang Q., De Lafarge B., Sugiyama D.,
Bréant C., Claes F., De Smet F., Thomas J.L., Autiero M., Carmeliet
P., Tessier-Lavigne M. and Eichmann A. (2004). The netrin receptor
UNC5B mediates guidance events controlling morphogenesis of the
vascular system. Nature 432, 179-186.
Lubarsky B. and Krasnow M.A. (2003). Tube morphogenesis: making
and shaping biological tubes. Cell 112, 19-28.
Macchiarelli G., Jiang J.Y., Nottola S.A. and Sato E. (2006).
Morphological patterns of angiogenesis in ovarian follicle capillary
networks. A scanning electron microscopy study of corrosion cast.
Microsc. Res. Tech. 69, 459-468.
Maden C.H., Fantin A. and Ruhrberg C. (2009). Neuropilin ligands in
vascular and neuronal patterning. Biochem. Soc. Trans. 37, 1228-
1232.
Madri J.A. and Stenn K.S. (1982). Aortic endothelial cell migration. l.
Matrix requirements and composition. Am. J. Pathol. 106, 180-1 86.
Madri J.A. and Pratt B.M. (1986). Endothelial cell-matrix interactions: in
vitro models of angiogenesis. J. Histochem. Cytochem. 34, 85-91.
Madrid J.F., Diaz-Flores L., Gutierrez R., Varela H., Valladares F.,
Rancel N. and Rodriguez F. (1998). Participation of angiogenesis
from rat femoral veins in the neovascularization of adjacent
occluded arteries. Histol. Histopathol 13, 1-11.
Makanya A.N., Stauffer D., Ribatti D., Burri P.H. and Djonov V. (2005).
Microvascular growth, development, and remodeling in the
embryonic avian kidney: the interplay between sprouting and
intussusceptive angiogenic mechanisms. Microsc. Res. Tech. 66,
275-288.
Makanya A.N., Hlushchuk R., Baum O., Velinov N., Ochs M. and Djonov
V. (2007). Microvascular endowment in the developing chicken
embryo lung. Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L1136-
L1146.
Makanya A.N., Hlushchuk R. and Djonov V.G. (2009). Intussusceptive
angiogenesis and its role in vascular morphogenesis, patterning,
and remodeling. Angiogenesis 12, 113-123.
Makrilia N., Lappa T., Xyla V., Nikolaidis I. and Syrigos K. (2009). The
role of angiogenesis in solid tumours: an overview. Eur. J. Intern.
Med. 20, 663-671.
Maltby S., Khazaie K. and McNagny K.M. (2009). Mast cells in tumor
growth: angiogenesis, tissue remodelling and immune-modulation.
Biochim. Biophys. Acta 1796, 19-26.
Manetti M., Guiducci S. and Matucci-Cerinic M. (2016). The crowded
crossroad to angiogenesis in systemic sclerosis: where is the key to
the problem? Arthritis. Res. Ther. 18, 36.
Maniotis A.J., Folberg R., Hess A., Seftor E.A., Gardner L.M., Pe'er J.,
Trent J.M., Meltzer P.S. and Hendrix M.J. (1999). Vascular channel
formation by human melanoma cells in vivo and in vitro:
vasculogenic mimicry. Am. J. Pathol. 155, 739-752.
Maquart F.X., Bellon G., Pasco S. and Monboisse J.C. (2004).
Matrikines in the regulation of extracellular matrix degradation.
Biochimie 87, 353-360.
Matsuki M., Kabara M., Saito Y., Shimamura K., Minoshima A.,
Nishimura M., Aonuma T., Takehara N., Hasebe N. and Kawabe J.
(2015). Ninjurin1 is a novel factor to regulate angiogenesis through
the function of pericytes. Circ. J. 79, 1363-1371.
Maumus M., Peyrafitte J.A., D'Angelo R., Fournier-Wirth C., Bouloumié
A., Casteilla L., Sengenès C. and Bourin P. (2011). Native human
adipose stromal cells: localization, morphology and phenotype. Int.
J. Obes. (Lond) 35, 1141-1153.
Mazzone M., Dettori D., Leite de Oliveira R., Loges S., Schmidt T.,

Angiogenesis and related secondary structures
Jonckx B., Tian Y.M., Lanahan A.A., Pollard P., Ruiz de Almodovar
C., De Smet F., Vinckier S., Aragonés J., Debackere K., Luttun A.,
Wyns S., Jordan B., Pisacane A., Gallez B., Lampugnani M.G.,
Dejana E., Simons M, Ratcliffe P., Maxwell P. and Carmeliet P.
(2009). Heterozygous deficiency of PHD2 restores tumor
oxygenation and inhibits metastasis via endothelial normalization.
Cell 136, 839-851.
McCracken J.S., Burger P.C. and Klintworth G.K. (1979). Morphologic
observations on experimental comeal vascularization in the rat. Lab.
Invest. 41, 519-530.
McDonald D.M. and Choyke P.L. (2003). Imaging of angiogenesis: from
microscope to clinic. Nat. Med. 9, 713-725.
Mehta D. and Malik A.B. (2006). Signaling mechanisms regulating
endothelial permeability. Physiol. Rev. 86, 279-367.
Mentzer S.J. and Konerding M.A. (2014). Intussusceptive angiogenesis:
expansion and remodeling of microvascular networks. Angiogenesis
17, 499-509.
Mihic-Probst D., Ikenberg K., Tinguely M., Schraml P., Behnke S.,
Seifert B., Civenni G., Sommer L., Moch H. and Dummer R. (2012).
Tumor cell plasticity and angiogenesis in human melanomas. PLoS
One 7, e33571.
Miranville A., Heeschen C., Sengenès C., Curat C.A., Busse R. and
Bouloumié A. (2004). Improvement of postnatal neovascularization
by human adipose tissue-derived stem cells. Circulation 110, 349-
355.
Moldovan L. and Moldovan N.I. (2005). Role of monocytes and
macrophages in angiogenesis. EXS 94, 127-146.
Moldovan N.I., Goldschmidt-Clermont P.J., Parker-Thornburg J.,
Shapiro S.D. and Kolattukudy P.E. (2000). Contribution of
monocytes/macrophages to compensatory neovascularization: the
drilling of metalloelastase-positive tunnels in ischemic myocardium.
Circ Res. 87, 378-384.
Monboisse J.C., Oudart J.B., Ramont L., Brassart-Pasco S. and
Maquart F.X. (2014). Matrikines from basement membrane
collagens: a new anti-cancer strategy. Biochim. Biophys. Acta 1840,
2589-2598.
Moore M.A., Hattori K., Heissig B., Shieh J.H., Dias S., Crystal R.G. and
Rafii S. (2001). Mobilization of endothelial and hematopoietic stem
and progenitor cells by adenovector-mediated elevation of serum
levels of SDF-1, VEGF, and angiopoietin-1. Ann. NY Acad. Sci. 938,
36-45.
Moreau V., Tatin F., Varon C., Anies G., Savona-Baron C. and Génot E.
(2006). Cdc42-driven podosome formation in endothelial cells. Eur.
J. Cell Biol. 85, 319-325.
Morikawa S., Baluk P., Kaidoh T., Haskell A., Jain R. and McDonald D.
(2002). Abnormalities in pericytes on blood vessels and endothelial
sprouts in tumors. Am. J. Pathol. 160, 985-1000.
Moscatelli D. and Rifkin D.B. (1988). Membrane and matrix localization
of proteinases: A common theme in tumor invasion and
angiogenesis. Biochim. Biophys. Acta 948, 67-85.
Moscatelli D., Gross J.L. and Rifkin D.B. (1981). Angiogenic factors
stimulate plasminogen activator and collagenase production
bycapillary endothelial cells. J. Cell Biol. 91, 201a
Mota A., Argenziano G., Zalaudek I., Piana S., Longo C., Moscarella E.
and Lallas A. (2013). Clinical, dermoscopic and histopathologic
findings of retiform hemangioendothelioma. Dermatol. Pract.
Concept. 3, 11-14.
Moya I.M., Umans L., Maas E., Pereira P.N., Beets K., Francis A., Sents
W., Robertson E.J., Mummery C.L., Huylebroeck D. and Zwijsen A.
1275
(2012). Stalk cell phenotype depends on integration of Notch and
Smad1/5 signaling cascades. Dev. Cell 22, 501-514.
Mulligan-Kehoe M.J. and Simons M. (2008). Vascular disease in
scleroderma: angiogenesis and vascular repair. Rheum. Dis. Clin.
North Am. 34, 73-79.
Myers K.A., Applegate K.T., Danuser G., Fischer R.S. and Waterman
C.M. (2011). Distinct ECM mechanosensing pathways regulate
microtubule dynamics to control endothelial cell branching
morphogenesis. J. Cell Biol. 192, 321-334.
Nagy J.A., Brown L.F., Senger D.R., Lanir N., Van de Water L., Dvorak
A.M. and Dvorak H.F. (1989). Pathogenesis of tumor stroma
generation: a critical role for leaky blood vessels and fibrin
deposition. Biochim. Biophys. Acta 948, 305-326.
Nakayasu K. (1988). Origin of pericytes in neovascularization of rat
cornea. Jpn. J. Ophthalmol. 32, 105-112.
Nehls V., Denzer K. and Drenckhahn D. (1992). Pericyte involvement in
capillary sprouting during angiogenesis in situ. Cell Tissue Res. 270,
469-474.
Neves R.I., Stevenson J., Hancey M.J., Vangelisti G., Miraliakbari R.,
Mackay D. and Clarke L. (2011). Endovascular papillary
angioendothelioma (Dabska tumor): underrecognized malignant
tumor in childhood. J. Pediatr. Surg. 46, e25-28.
Nico B., Crivellato E., Guidolin D., Annese T., Longo V., Finato N.,
Vacca A. and Ribatti D. (2010). Intussusceptive microvascular
growth in human glioma. Clin. Exp. Med. 10, 93-98.
Nicosia R.F. and Madri J.A. (1987). The microvascular extracellular
matrix. Developmental changes during angiogenesis in the aortic
ring-plasma clot model. Am. J. Pathol. 128, 78-90.
Nicosia R.F. and Villaschi S. (1995). Rat aortic smooth muscle cells
become pericytes during angiogenesis in vitro. Lab. Invest. 73, 658-
666.
Ohki Y., Heissig B., Sato Y., Akiyama H., Zhu Z., Hicklin D.J., Shimada
K., Ogawa H., Daida H., Hattori K. and Ohsaka A. (2005).
Granulocyte colony-stimulating factor promotes neovascularization
by releasing vascular endothelial growth factor from neutrophils.
FASEB J. 19, 2005-2007.
Ohtani H. (1992). Glomeruloid structures as vascular reaction in human
gastrointestinal carcinoma. Jpn. J. Cancer Res. 83, 1334-1340.
Okada H., Tsuzuki T., Shindoh H., Nishigaki A., Yasuda K. and Kanzaki
H. (2014). Regulation of decidualization and angiogenesis in the
human endometrium: mini review. J. Obstet. Gynaecol. Res. 40,
1180-1187.
Orlidge A. and D'Amore P.A. (1987). lnhibition of capillary endothelial
cell growth by pericytes and smooth muscle cells. J. Cell Biol. 105,
1455-1462.
Osiak A.E., Zenner G. and Linder S. (2005). Subconfluent endothelial
cells form podosomes downstream of cytokine and RhoGTPase
signaling. Exp. Cell Res. 307, 342-353.
Ozerdem U and Stallcup W.B. (2004). Pathological angiogenesis is
reduced by targeting pericytes via the NG2 proteoglycan.
Angiogenesis 7, 269-276.
Ozerdem U., Grako K.A., Dahlin-Huppe K., Monosov E. and Stallcup
W.B. (2001). NG2 proteoglycan is expressed exclusively by mural
cells during vascular morphogenesis. Dev. Dyn. 222, 218-227.
Paku S., Dezso K., Bugyik E., Tóvári J., Tímár J., Nagy P., Laszlo V.,
Klepetko W. and Döme B. (2011). A new mechanism for pillar
formation during tumor-induced intussusceptive angiogenesis:
inverse sprouting. Am. J. Pathol. 179, 1573-1585.
Patan S. (1998). TIE1 and TIE2 receptor tyrosine kinases inversely
1276
Angiogenesis and related secondary structures
regulate embryonic angiogenesis by the mechanism of
intussusceptive microvascular growth. Microvasc. Res. 56, 1-21.
Patan S. (2000). Vasculogenesis and angiogenesis as mechanisms of
vascular network formation, growth and remodeling. J. Neurooncol.
50, 1-15.
Patan S., Munn L.L. and Jain R.K. (1996). Intussusceptive
microvascular growth in a human colon adenocarcinoma xenograft:
a novel mechanism of tumor angiogenesis. Microvasc. Res. 51, 260-
372.
Patan S., Haenni B. and Burri P.H. (1997). Implementation of
intussusceptive microvascular growth in the chicken chorioallantoic
membrane (CAM). Microvasc. Res. 53, 33-52.
Patan S., Munn L.L., Tanda S., Roberge S., Jain R.K. and Jones R.C.
(2001a). Vascular morphogenesis and remodeling in a model of
tissue repair: blood vessel formation and growth in the ovarian
pedicle after ovariectomy. Circ. Res. 89, 723-731.
Patan S., Tanda S., Roberge S., Jones R.C., Jain R.K. and Munn L.L.
(2001b). Vascular morphogenesis and remodeling in a human tumor
xenograft: blood vessel formation and growth after ovariectomy and
tumor implantation. Circ. Res. 89, 732-739.
Paulis Y.W., Soetekouw P.M., Verheul H.M., Tjan-Heijnen V.C. and
Griffioen A.W. (2010). Signalling pathways in vasculogenic mimicry.
Biochim. Biophys. Acta 1806, 18-28.
Peichev M., Naiyer A.J., Pereira D., Zhu Z., Lane W.J., Williams M., Oz
M.C., Hicklin D.J., Witte L., Moore M.A. and Rafii S. (2000).
Expression of VEGFR-2 and AC133 by circulating human CD34(+)
cells identifies a population of functional endothelial precursors.
Blood 95, 952-958.
Pelosi E., Valtieri M., Coppola S., Botta R., Gabbianelli M., Lulli V.,
Marziali G., Masella B., Müller R., Sgadari C., Testa U., Bonanno G.
and Peschle C. (2002). Identification of the hemangioblast in
postnatal life. Blood 100, 3203-3208.
Petrovic N., Schacke W., Gahagan J.R., O'Conor C.A., Winnicka B.,
Conway R.E., Mina-Osorio P. and Shapiro L.H. (2007). CD13/APN
regulates endothelial invasion and filopodia formation. Blood 110,
142-150.
Pezzella F., Pastorino U., Tagliabue E., Andreola S., Sozzi G.,
Gasparini G., Menard S., Gatter K.C., Harris A.L., Fox S., Buyse M.,
Pilotti S., Pierotti M. and Rilke F. (1997). Non-small-cell lung
carcinoma tumor growth without morphological evidence of neo-
angiogenesis. Am. J. Pathol. 151, 1417-1423.
Phng L.K., Potente M., Leslie J.D., Babbage J., Nyqvist D., Lobov I.,
Ondr J.K., Rao S., Lang R.A., Thurston G. and Gerhardt H. (2009).
Nrarp coordinates endothelial Notch and Wnt signaling to control
vessel density in angiogenesis. Dev. Cell 16, 70-82.
Polverini P.J., Cotran R.S. and Sholley M.M. (1977). Endothelial
proliferation in the delayed hypersensitivity reaction: an
autoradiographic study. J. Immunol. 118, 529-537.
Potente M., Gerhardt H. and Carmeliet P. (2011). Basic and therapeutic
aspects of angiogenesis. Cell 146, 873-887.
Primo L., Seano G., Roca C., Maione F., Gagliardi P.A., Sessa R.,
Martinelli M., Giraudo E., di Blasio L. and Bussolino F. (2010).
Increased expression of alpha6 integrin in endothelial cells unveils a
proangiogenic role for basement membrane. Cancer Res. 70, 5759-
5769.
Rai A. and Cross J.C. (2014). Development of the hemochorial maternal
vascular spaces in the placenta through endothelial and
vasculogenic mimicry. Dev. Biol. 387, 131-141.
Rajantie I., Ilmonen M., Alminaite A., Ozerdem U., Alitalo K. and Salven
P. (2004). Adult bone marrow-derived cells recruited during
angiogenesis comprise precursors for periendothelial vascular mural
cells. Blood 104, 2084-2086.
Randles M.J., Humphries M.J. and Lennon R. (2017). Proteomic
definitions of basement membrane composition in health and
disease. Matrix Biol. 58, 12-28.
Reynolds L.P., Grazul-Bilska A.T. and Redmer D.A. (2000).
Angiogenesis in the corpus luteum. Endocrine 12, 1-9.
Rhodin J.A.G. and Fujita H. (1989). Capillary growth in the mesentery of
normal young rats. lntravital video and electron microscope
analyses. J. Submicrosc. Cytol. Pathd. 21, 1-34.
Ribatti D and Crivellato E. (2012a). Mast cells, angiogenesis, and
tumour growth. Biochim. Biophys. Acta 1822, 2-8.
Ribatti D. and Crivellato E. (2012b). "Sprouting angiogenesis", a
reappraisal. Dev. Biol. 372, 157-165.
Ribatti D. and Djonov V. (2012). Intussusceptive microvascular growth
in tumors. Cancer Lett. 316, 126-131.
Ribatti D., Nico B. and Crivellato E. (2011). The role of pericytes in
angiogenesis. Int. J. Dev. Biol. 55, 261-268.
Ricard N. and Simons M. (2015). When it is better to regress: dynamics
of vascular pruning. PLoS Biol. 13, e1002148.
Risau W. and Flamme I. (1995). Vasculogenesis. Annu. Rev. Cell Dev.
Biol. 11, 73-91.
Rizov M., Andreeva P. and Dimova I. (2017). Molecular regulation and
role of angiogénesis in reproduction. Taiwan J. Obstet. Gynecol. 56,
127-132.
Rojiani A.M. and Dorovini-Zis K. (1996). Glomeruloid vascular structures
in glioblastoma multiforme: an immunohistochemical and
ultrastructural study. J. Neurosurg. 85, 1078-1084.
Ronca R., Benkheil M., Mitola S., Struyf S. and Liekens S. (2017).
Tumor angiogenesis revisited: Regulators and clinical implications.
Med. Res. Rev. (in press).
Rong Y., Durden D.L., Van Meir E.G. and Brat D.J. (2006).
'Pseudopalisading' necrosis in glioblastoma: a familiar morphologic
feature that links vascular pathology, hypoxia, and angiogenesis. J.
Neuropathol. Exp. Neurol. 65, 529-539.
Ruhrberg C., Gerhardt H., Golding M., Watson R., Ioannidou S.,
Fujisawa H., Betsholtz C. and Shima D.T. (2002). Spatially restricted
patterning cues provided by heparin-binding VEGF-A control blood
vessel branching morphogenesis. Genes Dev. 16, 2684-2698.
Sabin F.R. (1920). Studies on the origin of the blood vessels and of
redblood corpuscles as seen in the living blastodenn of chick during
thesecond day of incubation. Contr. Embryol. 9, 215-262.
Sainson R.C., Johnston D.A., Chu H.C., Holderfield M.T., Nakatsu M.N.,
Crampton S.P., Davis J., Conn E. and Hughes C.C. (2008). TNF
primes endothelial cells for angiogenic sprouting by inducing a tip
cell phenotype. Blood 111, 4997-5007.
Sanz-Trelles A., Rodrigo-Fernandez I., Ayala-Carbonero A. and
Contreras-Rubio F. (1997). Retiform hemangioendothelioma. A new
case in a child with diffuse endovascular papillary endothelial
proliferation. J. Cutan. Pathol. 24, 440-444.
Sato Y. and Rifkin D.B. (1989). lnhibition of endothelial cell movement
by pericytes and smooth muscle cells: Activation of a latent
transforrning growth factor-bl-like molecule by plasmin during
coculture. J. Cell Biol. 109, 309-315.
Sauteur L., Krudewig A., Herwig L., Ehrenfeuchter N., Lenard A.,
Affolter M. and Belting H.G. (2014). Cdh5/VE-cadherin promotes
endothelial cell interface elongation via cortical actin polymerization
during angiogenic sprouting. Cell Rep. 9, 504-513.

Angiogenesis and related secondary structures
Sawamiphak S., Seidel S., Essmann C.L., Wilkinson G.A., Pitulescu
M.E., Acker T. and Acker-Palmer A. (2010). Ephrin-B2 regulates
VEGFR2 function in developmental and tumour angiogenesis.
Nature 465, 487-491.
Schioppa T., Uranchimeg B., Saccani A., Biswas S.K., Doni A.,
Rapisarda A., Bernasconi S., Saccani S., Nebuloni M., Vago L.,
Mantovani A., Melillo G. and Sica A. (2003). Regulation of the
chemokine receptor CXCR4 by hypoxia. J. Exp. Med. 198, 1391-
1402.
Schlingemann R.O., Rietveld F.J.R., de Waal R.M.W., Ferrone S. and
Ruiter D.J. (1990). Expression of the high molecular weight
melanoma-associated antigen by pericytes during angiogenesis in
tumors and in healing wounds. Am. J. Pathol. 136, 1393-1405.
Schlingemann R.O., Rietveld F.J.R., Kwaspen F., van de Kerkhof
P.C.M., de Waal R.M.W. and Ruiter D.J. (1991). Differential
expresion of markers for endothelial cells, pericytes, and basal
lamina in the microvasculature of tumors and granulatlon tissue.
Am.J. Pathol. 138, 1335-1347.
Schlingemann R.O., Oosterwijk E., Wesseling P., Rietveld F.J. and
Ruiter D.J. (1996). Aminopeptidase a is a constituent of activated
pericytes in angiogenesis. J. Pathol. 179, 436-442.
Schor A.M., Schor S.L. and Allen T.D. (1983). Effects of culture
conditions on the proliferation morphology and migration of bovine
aortic endothelial cells. J. Cell Sci. 62, 267-285.
Schruefer R., Sulyok S., Schymeinsky J., Peters T., Scharffetter-
Kochanek K. and Walzog B. (2006). The proangiogenic capacity of
polymorphonuclear neutrophils delineated by microarray technique
and by measurement of neovascularization in wounded skin of
CD18-deficient mice. J. Vasc. Res. 43, 1-11.
Seano G. and Primo L. (2015). Podosomes and invadopodia: tools to
breach vascular basement membrane. Cell Cycle 14, 1370-1374.
Seano G., Chiaverina G., Gagliardi P.A., di Blasio L., Puliafito A.,
Bouvard C., Sessa R., Tarone G., Sorokin L., Helley D., Jain R.K.,
Serini G., Bussolino F. and Primo L. (2014). Endothelial podosome
rosettes regulate vascular branching in tumour angiogenesis. Nat.
Cell Biol. 16, 931-941.
Seftor E.A., Meltzer P.S., Kirschmann D.A., Pe'er J., Maniotis A.J., Trent
J.M., Folberg R. and Hendrix M.J. (2002). Molecular determinants of
human uveal melanoma invasion and metastasis. Clin. Exp.
Metastasis 19, 233-246.
Seftor R.E., Hess A.R., Seftor E.A., Kirschmann D.A., Hardy K.M.,
Margaryan N.V. and Hendrix M.J. (2012). Tumor cell vasculogenic
mimicry: from controversy to therapeutic promise. Am. J. Pathol.
181, 1115-1125.
Shi Q., Rafii S., Wu M.H., Wijelath E.S., Yu C., Ishida A., Fujita Y.,
Kothari S., Mohle R., Sauvage L.R., Moore M.A., Storb R.F. and
Hammond W.P. (1998). Evidence for circulating bone marrow-
derived endothelial cells. Blood 92, 362-367.
Sholley M.M., Cavallo T. and Cotran R.S. (1977a). Endothelial cell
proliferation in inflammation. l. Autoradiographic studies following
themal injured to the skin of nomal rats. Am. J. Pathol. 89, 277-296.
Sholley M.M., Gimbrone M.A. and Cotran R.S. (1977b). Cellular
migration and replication in endothelial regeneration: a study using
irradiated endothelial cultures. Lab. Invest. 36, 18-25.
Sholley M.M., Gimbrone M.A. and Cotran R.S. (1978). The effects of
leukocyte depletion on corneal neovascularization. Lab. Invest. 38,
32-40.
Sholley M.M., Ferguson G.P., Seibel H.R., Montuor J.L. and Wilson J.D.
(1984). Mechanisms of neovascularization. Vascular sprouting can
1277
occur without proliferation of endothelial cells. Lab. Invest. 51, 624-
634.
Siekmann A.F. and Lawson N.D. (2007). Notch signalling and the
regulation of angiogenesis. Cell Adh. Migr. 1, 104-106.
Soda Y., Myskiw C., Rommel A. and Verma I.M. (2013). Mechanisms of
neovascularization and resistance to anti-angiogenic therapies in
glioblastoma multiforme. J. Mol. Med. (Berl) 91, 439-448.
Sprague A.H. and Khalil R.A. (2009). Inflammatory cytokines in vascular
dysfunction and vascular disease. Biochem. Pharmacol. 78, 539-
552.
Stratman A.N., Malotte K.M., Mahan R.D., Davis M.J. and Davis G.E.
(2009). Pericyte recruitment during vasculogenic tube assembly
stimulates endothelial basement membrane matrix formation. Blood
114, 5091-5101.
Straume O., Chappuis P.O., Salvesen H.B., Halvorsen O.J., Haukaas
S.A., Goffin J.R, Bégin L.R., Foulkes W.D. and Akslen L.A. (2002).
Prognostic importance of glomeruloid microvascular proliferation
indicates an aggressive angiogenic phenotype in human cancers.
Cancer Res. 62, 6808-6811.
Styp-Rekowska B., Hlushchuk R., Pries A.R. and Djonov V. (2011).
Intussusceptive angiogenesis: pillars against the blood flow. Acta
Physiol. (Oxf) 202, 213-223.
Suchting S., Freitas C., le Noble F., Benedito R., Bréant C., Duarte A.
and Eichmann A. (2007). The Notch ligand Delta-like 4 negatively
regulates endothelial tip cell formation and vessel branching. Proc.
Natl. Acad. Sci. USA 104, 3225-3230.
Suciu L., Popescu L.M., Gherghiceanu M., Regalia T., Nicolescu M.I.,
Hinescu M.E. and Faussone-Pellegrini M.S. (2010). Telocytes in
human term placenta: morphology and phenotype. Cells Tissues
Organs 192, 325-339.
Sundberg C., Nagy J.A., Brown L.F., Feng D., Eckelhoefer I.A.,
Manseau E.J., Dvorak A.M. and Dvorak H.F. (2001). Glomeruloid
microvascular proliferation follows adenoviral vascular permeability
factor/vascular endothelial growth factor-164 gene delivery. Am. J.
Pathol. 158, 1145-1160.
Suri C., Jones P.F., Patan S., Bartunkova S., Maisonpierre P.C., Davis
S., Sato T.N. and Yancopoulos G.D. (1996). Requisite role of
angiopoietin-1, a ligand for the TIE2 receptor, during embryonic
angiogenesis. Cell 87, 1171-1180.
Szade A., Grochot-Przeczek A., Florczyk U., Jozkowicz A. and Dulak J.
(2015). Cellular and molecular mechanisms of inflammation-induced
angiogenesis. IUBMB Life 67, 145-159.
Szekanecz Z., Besenyei T., Szentpétery A. and Koch A.E. (2010).
Angiogenesis and vasculogenesis in rheumatoid arthritis. Curr.
Opin. Rheumatol. 22, 299-306.
Takahashi T., Kalka C., Masuda H., Chen D., Silver M., Kearney M.,
Magner M., Isner J.M. and Asahara T. (1999). Ischemia- and
cytokine-induced mobilization of bone marrow-derived endothelial
progenitor cells for neovascularization. Nat. Med. 5, 434-438.
Tammela T., Zarkada G., Wallgard E., Murtomäki A., Suchting S.,
Wirzenius M., Waltari M., Hellström M., Schomber T., Peltonen R.,
Freitas C., Duarte A., Isoniemi H., Laakkonen P., Christofori G., Ylä-
Herttuala S., Shibuya M., Pytowski B., Eichmann A., Betsholtz C.
and Alitalo K. (2008). Blocking VEGFR-3 suppresses angiogenic
sprouting and vascular network formation. Nature 454, 656-660.
Taylor A.C., Seltz L.M., Yates P.A. and Peirce S.M. (2010). Chronic
whole-body hypoxia induces intussusceptive angiogenesis and
microvascular remodeling in the mouse retina. Microvasc. Res. 79,
93-101.
1278
Angiogenesis and related secondary structures
Tillet E., Vittet D., Féraud O., Moore R., Kemler R. and Huber P. (2005).
N-cadherin deficiency impairs pericyte recruitment, and not
endothelial differentiation or sprouting, in embryonic stem cell-
derived angiogenesis. Exp. Cell Res. 310, 392-400.
Tirziu D., Jaba I.M., Yu P., Larrivée B., Coon B.G., Cristofaro B.,
Zhuang Z.W., Lanahan A.A., Schwartz M.A., Eichmann A. and
Simons M. (2012). Endothelial nuclear factor-κB-dependent
regulation of arteriogenesis and branching. Circulation 126, 2589-
2600.
Todorova D., Simoncini S., Lacroix R., Sabatier F. and Dignat-George
F. (2017). Extracellular vesicles in angiogenesis. Circ. Res. 120,
1658-1673.
Tsuda A., Turhan A., Konerding M., Ravnic D., Hanidziar D., Lin M. and
Mentzer S.J. (2009). Bimodal oscillation frequencies of blood flow in
the inflammatory colon microcirculation. Anat. Rec. (Hoboken) 292,
65-72.
Tsuzuki H. and Sasa S. (1994). Ultrastructural observation of capillary
sprouts in the dental organs of rat molars. Kaibogaku Zasshi 69,
684-696.
Tzima E., Kiosses W.B., del Pozo M.A. and Schwartz M.A. (2003).
Localized cdc42 activation, detected using a novel assay, mediates
microtubule organizing center positioning in endothelial cells in
response to fluid shear stress. J. Biol. Chem. 278, 31020-
31023.
Tzima E., Irani-Tehrani M., Kiosses W.B., Dejana E., Schultz D.A.,
Engelhardt B., Cao G., DeLisser H. and Schwartz M.A. (2005). A
mechanosensory complex that mediates the endothelial cell
response to fluid shear stress. Nature 437, 426-431.
Udan R.S., Vadakkan T.J. and Dickinson M.E. (2013). Dynamic
responses of endothelial cells to changes in blood flow during
vascular remodeling of the mouse yolk sac. Development 140,
4041-4050.
Vajkoczy P., Blum S., Lamparter M., Mailhammer R., Erber R.,
Engelhardt B., Vestweber D. and Hatzopoulos A.K. (2003). Multistep
nature of microvascular recruitment of ex vivo-expanded embryonic
endothelial progenitor cells during tumor angiogenesis. J. Exp. Med.
197, 1755-1765.
van Hinsbergh V.W. and Koolwijk P. (2008). Endothelial sprouting and
angiogenesis: matrix metalloproteinases in the lead. Cardiovasc.
Res. 78, 203-212.
Van Steenkiste C., Trachet B., Casteleyn C., van Loo D., Van
Hoorebeke L., Segers P., Geerts A., Van Vlierberghe H. and Colle I.
(2010). Vascular corrosion casting: analyzing wall shear stress in the
portal vein and vascular abnormalities in portal hypertensive and
cirrhotic rodents. Lab. Invest. 90, 1558-1572.
VanKlompenberg M.K., Manjarín R., Donovan C.E., Trott J.F. and
Hovey R.C. (2016). Regulation and localization of vascular
endothelial growth factor within the mammary glands during the
transition from late gestation to lactation. Domest. Anim. Endocrinol.
54, 37-47.
Vitorino, P. and T. Meyer. (2008). Modular control of endothelial sheet
migration. Genes Dev. 22, 3268-3281.
von Tell D., Armulik A. and Betsholtz C. (2006). Pericytes and vascular
stability. Exp. Cell Res. 312, 623-629.
Wacker A. and Gerhardt H. (2011). Endothelial development taking
shape. Curr. Opin. Cell Biol. 23, 676-685.
Wakui S. (1988). Two and three dimensional ultrastructural observation
of two cell type angiogenesis in human granulation tissue. Virchows
Arch. (B) 56, 127-139.
Wall R.T., Harker L.A., Quadracci L.J. and Striker G.E. (1978). Factors
influencing endothelial cell proliferation in vitro. J. Cell Physiol. 96,
203-213.
Walsh T.G., Metharom P. and Berndt M.C. (2015). The functional role of
platelets in the regulation of angiogenesis. Platelets 26, 199-211.
Weavers H. and Skaer H. (2014). Tip cells: master regulators of
tubulogenesis? Semin. Cell Dev. Biol. 31, 91-99.
Wesseling P., van der Laak J.A., de Leeuw H., Ruiter D.J. and Burger
P.C. (1994). Quantitative immunohistological analysis of the
microvasculature in untreated human glioblastoma multiforme.
Computer-assisted image analysis of whole-tumor sections. J.
Neurosurg. 81, 902-909.
Wesseling P., Schlingemann R.O., Rietveld F.J., Link M., Burger P.C.
and Ruiter D.J. (1995). Early and extensive contribution of
pericytes/vascular smooth muscle cells to microvascular proliferation
in glioblastoma multiforme: an immuno-light and immuno-electron
microscopic study. J. Neuropathol. Exp. Neurol. 54, 304-310.
Wietecha M.S., Cerny W.L. and DiPietro L.A. (2013). Mechanisms of
vessel regression: toward an understanding of the resolution of
angiogenesis. Curr. Top. Microbiol. Immunol. 367, 3-32.
Williams C.K., Li J.L., Murga M., Harris A.L. and Tosato G. (2006a). Up-
regulation of the Notch ligand Delta-like 4 inhibits VEGF-induced
endothelial cell function. Blood 107, 931-939.
Williams J.L., Weichert A., Zakrzewicz A., Da Silva-Azevedo L., Pries
A.R., Baum O. and Egginton S. (2006b). Differential gene and
protein expression in abluminal sprouting and intraluminal splitting
forms of angiogenesis. Clin. Sci. (Lond) 110, 587-595.
Woad K.J. and Robinson R.S. (2016). Luteal angiogenesis and its
control. Theriogenology 86, 221-228.
Yana I., Sagara H., Takaki S., Takatsu K., Nakamura K., Nakao K.,
Katsuki M., Taniguchi S., Aoki T., Sato H., Weiss S.J. and Seiki M.
(2007). Crosstalk between neovessels and mural cells directs the
site-specific expression of MT1-MMP to endothelial tip cells. J. Cell
Sci. 120, 1607-1614.
Yu J.A., Castranova D., Pham V.N. and Weinstein B.M. (2015). Single-
cell analysis of endothelial morphogenesis in vivo. Development
142, 2951-2961.
Yuri T., Yamazaki F., Takasu K., Shikata N. and Tsubura A. (2008).
Glomeruloid hemangioma. Pathol. Int. 58, 390-395.
Zeng G., Taylor S.M., McColm J.R., Kappas N.C., Kearney J.B.,
Williams L.H., Hartnett M.E. and Bautch V.L. (2007). Orientation of
endothelial cell division is regulated by VEGFsignaling during blood
vessel formation. Blood 109, 1345-1352.
Zengin E., Chalajour F., Gehling U.M., Ito W.D., Treede H., Lauke H.,
Weil J., Reichenspurner H., Kilic N. and Ergün S. (2006). Vascular
wall resident progenitor cells: asource for postnatal vasculogenesis.
Development 133, 1543-1551.
Zhang L., Yang N., Park J.W., Katsaros D., Fracchioli S., Cao G.,
O'Brien-Jenkins A., Randall T.C., Rubin S.C. and Coukos G. (2003).
Tumor-derived vascular endothelial growth factor up-regulates
angiopoietin-2 in host endothelium and destabilizes host
vasculature, supporting angiogenesis in ovarian cancer. Cancer
Res. 63, 3403-3412.
Zhang S., Zhang D. and Sun B. (2007). Vasculogenic mimicry: current
status and future prospects. Cancer Lett. 254, 157-164.
Zhang Y., Lin X., Dai Y., Hu X., Zhu H., Jiang Y. and Zhang S. (2016).
Endometrial stem cells repair injured endometrium and induce
angiogenesis via AKT and ERK pathways. Reproduction 152, 389-
402.

Angiogenesis and related secondary structures
Zheng Y., Chen X., Qian M., Zhang M., Zhang D., Bai C., Wang Q. and
Wang X. (2014a). Human lung telocytes could promote the
proliferation and angiogenesis of human pulmonary microvascular
endothelial cells in vitro. Mol. Cell. Ther. 1, 2-3.
Zheng Y., Cretoiu D., Yan G., Cretoiu S.M., Popescu L.M., Fang H. and
Wang X. (2014b). Protein profiling of human lung telocytes and
microvascular endothelial cells using iTRAQ quantitative proteomics.
J. Cell. Mol. Med. 18, 1035-1059.
Zheng Y., Cretoiu D., Yan G., Cretoiu S.M., Popescu L.M. and Wang X.
(2014c). Comparative proteomic analysis of human lung telocytes
with fibroblasts. J. Cell. Mol. Med. 18, 568-589.
1279
Zhou A., Egginton S., Hudlická O. and Brown M.D. (1998). Internal
division of capillaries in rat skeletal muscle in response to chronic
vasodilator treatment with alpha1-antagonist prazosin. Cell Tissue
Res. 293, 293-303.
Zhou Z., Apte S.S., Soininen R., Cao R., Baaklini G.Y., Rauser R.W.,
Wang J., Cao Y. and Tryggvason K. (2000). Impaired endochondral
ossification and angiogenesis in mice deficient in membrane-type
matrix metalloproteinase I. Proc. Natl. Acad. Sci. USA 97, 4052-
4057.
Accepted July 19, 2017