THE ANATOMICAL RECORD 291:721–734 (2008)

Vascular Changes in Hepatocellular

Carcinoma
ZHEN FAN YANG AND RONNIE T.P. POON*
Centre for Cancer Research, Department of Surgery, Queen Mary Hospital,
The University of Hong Kong, Hong Kong, China

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most vascular solid
tumors, in which angiogenesis plays an important role. The status of
angiogenesis in HCC correlates with the disease progression and progno-
sis, and thus provides a potential therapeutic target. This review summa-
rizes the vascular changes and molecular and cellular basis of angiogene-
sis in HCC. Development of HCC is characterized by arterialization of its
blood supply and sinusoidal capillarization. Vascular endothelial growth
factor (VEGF) is a potent angiogenic factor that plays a critical role in
mediating angiogenesis in HCC. The VEGF can function on various types
of cells, such as endothelial cells, hepatic stellate cells, endothelial pro-
genitor cells and hemangiocytes, to induce vascular changes in HCC.
Therefore, blockade of VEGF-mediated pathways, either by anti-VEGF
neutralizing antibody or tyrosine kinase inhibitors that target VEGF
receptors, suppresses carcinogenesis and angiogenesis in HCC. In addi-
tion to VEGF, several other angiogenic factors in HCC have recently been
identified. These factors can also regulate angiogenic processes through
interaction with VEGF or VEGF-independent pathways. Despite the fact
that treatment of HCC remains a tough task due to lack of effective sys-
temic therapy, antiangiogenic therapy has already entered clinical trials
in HCC patients and sheds light on a promising novel treatment for this
disease. Anat Rec, 291:721–734, 2008. Ó 2008 Wiley-Liss, Inc.

Key words: neoangiogenesis; vascular endothelial growth fac-

tor; hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the five
most common malignancies worldwide, with an increas-
ing incidence due to hepatitis B and C viral infection
(Llovet et al., 2003; McMahon, 2005; El-Serag, 2004).
HCC is characterized as a highly vascularized solid tu-
mor with rapid growth rate and poor prognosis. Hepatic
resection and liver transplantation are the two main-
stays that may cure HCC, but the long-term patient sur-
vival is dependent on the stage of tumor at the time of
diagnosis (Llovet et al., 1999; Arii et al., 2000). Most
patients with HCC present with advanced stage tumors
beyond surgical treatment, and chemotherapy and radio-
therapy have limited efficacy in these cases (Groupe
d’Etude et de Traitement du Carcinome Hépatocellu-
laire, 1995; Lo et al., 2002; Raoul et al., 1997). Cur-
rently, there is no proven effective systemic chemother-
apy for HCC patients with metastasis (Bruix et al.,
2005). Therefore, understanding the molecular and cel-
lular mechanisms that modulate the development and
progression of HCC is crucial for the design of therapeu-
tic strategies.
Hepatocellular carcinoma is one of the most vascular
solid tumors, in which angiogenesis plays an important
role in its development, progression, and metastasis.
Vascular changes in HCC are characterized by arteriali-
Grant sponsor: The University of Hong Kong.
*Correspondence to: Ronnie T.P. Poon, Department of Sur-
gery, The University of Hong Kong, Queen Mary Hospital, 102
Pokfulam Road, Hong Kong, China. Fax: 852-2817-5475.
E-mail: poontp@hkucc.hku.hk
Received 29 April 2007; Accepted 19 December 2007
DOI 10.1002/ar.20668
Published online in Wiley InterScience (www.interscience.wiley.
com).

Ó 2008 WILEY-LISS, INC.

722 YANG AND POON

Fig. 1. Determination of microvessel density (MVD) in hepatocellular carcinoma (HCC) by CD34 immu-
nohistochemical staining. A significant higher level of MVD was detected in the tumor tissue than the
adjacent nontumorous tissue. T, tumor tissue; NT, nontumorous tissue. Arrows point to microvessels
formed in the tumor tissue. Copyright from Poon et al. (2002).

zation and sinusoidal capillarization. Recently, accumu-
lating data have provided insights into the molecular
mechanisms that are related to angiogenesis in HCC
(Zhao et al., 2003; Cervello and Montalto, 2006; Lee
et al., 2006). Antiangiogenic strategies that target angio-
genic molecules and inhibit activation of various types of
cells that participate in the angiogenic processes become
potential therapies for HCC. In this review, we summa-
rize the recent findings in molecular and cellular mecha-
nisms of angiogenesis in HCC, and the potential implica-
tions on the design of therapeutic strategies.
SIGNIFICANCE OF ANGIOGENESIS IN HCC
Angiogenesis is switched on when precancerous
lesions, also named dysplastic nodules, develop into
HCC, and the process of angiogenesis carries on with
the progression of the tumor (Poon et al., 2001). Due to
the rapid growth pattern of HCC, tumor cells require a
substantial amount of nutrient and oxygen from the cir-
culation. Hence, neoangiogenesis is required to provide
the nutrient and oxygen for tumor cell survival. Another
consequence of neoangiogenesis is that tumor cells may
enter into the circulation and develop distal metastasis
(Gwak et al., 2005; Tanaka et al., 2006). In fact, the
expression of markers for microvessel density (MVD)
has been reported to be associated with the development
and progression of HCC (Fig. 1). A study has demon-
strated that the expression of CD31, CD34, and BNH9
in endothelial cells was absent in cirrhotic liver and dys-
plastic nodules, but became detectable in human HCC
tumor tissues (Frachon et al., 2001). However, in an ex-
perimental cirrhotic model induced by bile duct ligation,
up-regulation of CD31 was detected, suggesting that
angiogenesis might also be present in the premalignant
lesions (Geerts et al., 2006). In established HCC, MVD
has been shown to impact on prognosis of patients. In a
previous study from our group, high tumor MVD was a
 VASCULAR CHANGES IN HCC 723

Fig. 2. Contrast computed tomography scans showing the typical enhancement pattern of HCC. A: A
hypodense lesion in the right lobe of the liver in noncontrast scan. B: The lesion is densely enhanced in
arterial phase, indicating its predominant arterial blood supply. C: The lesion becomes hypoattenuated in
the portal venous phase. An arrow points to a tumor nodule in the right lobe of the liver.

significant prognostic factor for tumor recurrence after
resection of early HCC < 5 cm in diameter (Poon et al.,
2002).
VASCULAR CHANGES ASSOCIATED WITH
DEVELOPMENT OF HCC
The liver has dual blood supply from the portal vein
and the hepatic artery. HCC usually develops blood sup-
ply predominantly from the arterial system due to its
high requirement of oxygen during progression. The
presence of new unpaired arteries not accompanied by
bile duct has been shown to be able to differentiate
neoplastic nodules from regenerative cirrhotic nodules
(Himeno et al., 1994; Ueda et al., 1992; Matsui, 2001).
The number of unpaired arteries gradually increases
from cirrhotic nodules (lowest) to low- and high-grade
dysplastic nodules and with the greatest numbers in
HCC, particularly in HCC smaller than 3 cm in diame-
ter. Using smooth muscle actin as a marker to label
arteries in the liver parenchyma, a study demonstrated
that unpaired arteries were histologically useful for dis-
tinguishing dysplastic nodules from large cirrhotic nod-
ules (Park et al., 1998). The arterialization of HCC pro-
vides a characteristic vascular enhancement pattern in
contrast computed tomography (CT) scan, which is use-
ful as a diagnostic feature of HCC to differentiate it
from other liver tumors such as secondary metastatic
tumors. Typically, HCC manifests as a hyperattenuated
lesion against a background of minimally enhanced liver
parenchyma in the arterial phase because the tumor is
supplied by the hepatic artery and is hypervascular,
whereas on delayed phase images, the HCC becomes low
attenuating compared with the surrounding liver paren-
chyma owing to early washout of contrast (Figures 2A,
B, and C). However, not all HCCs develop the typical ar-
terial blood supply. Some small HCCs can remain ‘‘hypo-
vascular’’ on CT scan. A previous study has shown that
the degree of arterialization correlated with tumor dif-
ferentiation of HCC (Yamamoto et al., 2001). The study
showed that well-differentiated HCC with a diameter
less than 3 cm often received preferential portal venous
blood, whereas moderately and poorly differentiated
ones were all supplied with arterial blood. When tumors
grow in size beyond 3 cm, most HCCs including well-dif-
ferentiated ones generally develop a predominant arte-
rial blood supply.
Sinusoidal capillarization, another neoangiogenic
process, has also been found to be common in HCC and
occurs even in early small HCC (Kin et al., 1994).
Sinusoidal capillarization involves transformation of
fenestrated hepatic sinusoids into continuous capillaries,
coupled with collagenization of the extravascular spaces
of Disse and deposition of laminin and basement mem-
branes near the endothelial cells and hepatocytes
(Schaffner and Poper, 1963; Kin et al., 1994). This vas-
cular change also accompanies the development of cir-
rhosis. The sinusoidal capillarization was characterized
by the expression of CD34, a marker which is absent in
the normal sinusoidal endothelium (Couvelard et al.,
1996; Frachon et al., 2001). While arterialization and si-
nusoidal capillarization both occur in HCC, no correla-
tion has been identified between the location of arteriali-
zation and sinusoidal capillarization (Park et al., 1998).
The process of sinusoidal capillarization also appears to
be related to tumor differentiation, with mixed sinusoi-
dal and capillary types of microvessels present in well-
differentiated HCC, whereas capillary type is the only
type observed in poorly differentiated HCC (Yamamoto
et al., 2001). A recent study evaluated the correlation of
arterialization and sinusoidal capillarization by immuno-
staining histologically with the degree of arterial con-
trast enhancement in dynamic CT scan in HCC (Kim
et al., 2005). The study identified a positive correlation
between the number of unpaired arteries in nodular
HCC and the degree of contrast enhancement in the ar-
terial phase by CT imaging. In contrast, there was no
correlation between the degree of sinusoidal capillariza-
tion and the degree of contrast enhancement in the arte-
rial phase.
724 YANG AND POON
The exact significance of arterialization and sinusoidal
capillarization in the process of angiogenesis of HCC
remains to be elucidated in further studies. Other vascu-
lar changes associated with neovascularization include
increase in MVD and development of heterogeneous
immature vessels with increased permeability, all con-
tributing to the contrast enhancement pattern of HCC
in imaging studies. It is now possible to use perfusion
magnetic resonance imaging (MRI) to study changes in
these parameters (Huwart et al., 2007). A dynamic mon-
itoring of vascular changes may play an important role
in monitoring the response of the tumor to antiangio-
genic therapy.
MAJOR MOLECULAR PATHWAYS THAT
MEDIATE ANGIOGENESIS IN HCC
Tumor cells release angiogenic factors to initiate the
process of angiogenesis (Sun and Tang, 2004). Endothe-
lial cells express a variety of receptors that bind to
angiogenic molecules secreted by the tumor cells. It has
been postulated for many years that neoangiogenesis
originates from migration of endothelial cells in the pre-
existing vessels in tumor-surrounding tissues. Accumu-
lated evidence suggests that some of the tumor neoves-
sels may be derived from circulating bone marrow-
derived endothelial progenitor cells (EPCs; Shi et al.,
1998; Asahara et al., 1999), although some other studies
propose a limited role of EPCs in tumor neovasculariza-
tion (Yung et al., 2004; Peters et al., 2005).
In addition to tumor cells, other types of cells, such as
endothelial cells and infiltrating lymphocytes, can also
secrete angiogenic factors. On the other hand, endoge-
nous presence of antiangiogenic factors has also been
identified. Therefore, tumor angiogenesis is a dynamic
process regulated by the balance between angiogenic
and antiangiogenic pathways. In our previous review,
we have listed some of the relatively well-characterized
angiogenic and antiangiogenic factors, with a focus on
their potential roles in HCC (Pang and Poon, 2006). The
roles of a few more important factors are briefly
described as follows, including updated information from
the recent literature.
Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) is the most
well-studied angiogenic factor in HCC. The angiogenic
effects of VEGF are achieved by binding to its receptors,
VEGFR1 (Flt-1) and VEGFR2 (Flk-1), expressed on en-
dothelial cells. Interaction between VEGF and VEGFR
initiate several signaling pathways, such as Akt/PI3K/
MAPK pathway, resulting in proliferation, migration,
and invasion of endothelial cells (Gerber et al., 1998;
Ahmad et al., 2006; Vogel et al., 2007). In addition to en-
dothelial cells, recent findings have demonstrated the
expression of VEGFR in tumor cells, proposing an auto-
crine loop of VEGF/VEGFR in tumor cells (Masood
et al., 2001; Weigand et al., 2005).
Mise et al. (1996) first reported the expression of
VEGF in HCC and detected significantly higher levels of
VEGF mRNA and protein in the tumor tissues than the
nontumorous counterpart. Subsequent studies reported
similar findings of high VEGF expression at both mRNA
and protein levels in HCC (Torimura et al., 2004; Yasuda
et al., 2004). The levels of VEGF expression during the
development of HCC have been shown to be associated
with the increase of unpaired arteries and sinusoidal
capillarization (Park et al., 2000). Increasing evidence of
up-regulated expression of VEGF in small HCC suggests
the importance of VEGF not only in tumor progression
but also in hepatocarcinogenesis (Iavarone et al., 2007).
This hypothesis has been proven in a diethylnitros-
amine-induced murine hepatocarcinogenesis model
(Yoshiji et al., 2004). The expression of VEGF in HCC
has been found to be associated with the clinicpathologi-
cal features of HCC patients in several studies. Tori-
mura et al. (1998) showed a significant association
between higher levels of VEGF protein by immunostain-
ing and poorly differentiated HCC. A higher level of
VEGF expression in HCC is also associated with a
higher proliferation index, poor encapsulation of tumors,
and venous tumor emboli or portal vein thrombosis
(Chow et al., 1997; Li et al., 1998). In addition, circulat-
ing VEGF levels before treatment could predict disease
outcome after a variety of treatments, including hepatic
resection, radiofrequency ablation, transcatheter arterial
chemoembolization (TACE; Poon et al., 2003a, 2004a,
2007). Parallel to VEGF, VEGFR expression is also asso-
ciated with the clinical outcome of HCC patients,
although only the level of VEGFR1 mRNA significantly
correlated with the VEGF mRNA level in tumor tissues
and intrahepatic metastasis in HCC (Ng et al., 2001).
Another study also reported that VEGFR1 expression in
tumor tissues correlated with CD31 expression, a
marker for tumor microvessels (Dhar et al., 2002). The
above findings suggest that VEGFR1 may play a more
important role in VEGF/VEGFR pathway in mediating
angiogenesis of HCC.
In addition to VEGFR1 and VEGFR2, VEGF also
interacts with the members of the neuropilin family,
neuropilin-1 and neuropilin-2, to regulate angiogenesis.
Moreover, these neuropilin members can act together
with VEGFR1 and VEGFR2 to mediate signal transduc-
tion (Staton et al., 2007). Little evidence has shown how
neuropilins participate in the angiogenic process of
HCC. However, some studies on other types of cancers
reported that neuropilins that are expressed on tumor
cells could directly modulate cell survival, proliferation,
and invasion (Hong et al., 2007; Fukasawa et al., 2007).
In endothelial cells, neuropilin-1 has been identified to
be a coreceptor of VEGFR2 and function on angiogenic
behavior by means of an enhancement of the VEGFR-2
phosphorylation threshold (Favier et al., 2006). Hence,
exploring the potential role of the neuropilin family on
HCC, either alone or combined with other VEGFRs, is
warranted, and may shed light on a novel antiangio-
genic therapy for this malignancy.
The VEGF promoter contains a hypoxia-responsive
element, which is regulated by hypoxia inducible factor-
1a (HIF-1a), a transcription factor. Activated HIF-1a
under stress, such as hypoxia, can bind to hypoxia-re-
sponsive elements expressed in several pro-survival and
pro-proliferation genes, leading to up-regulation of HIF-
1a downstream proteins (Harris, 2002; Brown and Wil-
son, 2004). HIF-1a–mediated up-regulation of VEGF is
pivotal for hypoxia-induced angiogenesis (von Marschall
et al., 2001; Yang et al., 2004), and a higher level of
HIF-1a is also associated with a poorer prognosis of
HCC patients (Huang et al., 2005). Transcriptional acti-
 VASCULAR CHANGES IN HCC
vation of HIF-1a can also be induced by hepatitis B vi-
rus X protein (Yoo et al., 2003), indicating the potential
interaction among viral infection, HIF-1a and VEGF up-
regulation, and hepatocarcinogenesis.
VEGF can directly function on HCC tumor cells, as
VEGFRs are also expressed by tumor cells. Our previous
study demonstrated that stimulation of HCC cell lines
by exogenous VEGF recombinant protein could stimu-
late tumor cell proliferation (Liu Y et al., 2005), suggest-
ing the presence of an autocrine loop in HCC tumor
cells. Moreover, VEGF may also have an angiogenesis-
independent effect on HCC cell invasion. A study by
Schmitt et al. (2004) showed that VEGF induced a
marked loss of pseudocanaliculi and disruption of occlu-
din-delineated tight junctions in tumor cells. Sections
from surgically removed tumor specimens showed VEGF
expression in the tumor and occludin disassembly in
normal liver parenchyma next to the tumor, suggesting
that VEGF may enhance invasion of HCC cells into nor-
mal liver parenchyma.
VEGF has been reported to be overexpressed in cirrho-
sis, a well-recognized precancerous lesion of HCC. There-
fore, VEGF is also postulated to play an important role
in the development of liver cirrhosis (Deli et al., 2005;
Medina et al., 2005). However, there is no solid evidence
showing the relationship among VEGF expression, cir-
rhosis and the development of HCC, although a recent
study demonstrated that a higher level of VEGF165
mRNA in noncancerous liver tissue correlated signifi-
cantly with a higher risk of HCC recurrence (Sheen
et al., 2005). The significance of angiogenesis in cirrhosis
and its relationship with hepatocarcinogenesis in cir-
rhotic liver are areas that need further investigation.
Angiopoietins
Angiopoietins constitute another group of angiogenic
molecules. Different members in the angiopoietin family
play different roles in the process of angiogenesis. For
instance, angiopoietin-1 provides survival signals to
endothelial cells and promotes recruitment of pericytes
and smooth muscle cells to form mature blood vessels,
whereas angiopoietin-2 functions as a natural antagonist
of angiopoietin-1 (Tanaka et al., 1999). The presence of
angiopoietin-2 enhances the sensitivity of endothelial
cells to other angiogenic factors, such as VEGF. Higher
levels of angiopoietin-2 expression have been detected in
the tumor tissues than in the nontumorous tissues in
HCC. The level of angiopoietin-2 was found to be associ-
ated with the clinicopathological parameters of HCC
patients, and the ratio between angiopoietin-2 and
angiopoietin-1 indicated the status of tumor angiogene-
sis (Zhang et al., 2006). A study by Torimura et al.
(2004) also showed a positive correlation between angio-
poietin-1 and angiopoietin-2 expression with tumor de-
differentiation. By using immunohistochemistry, angio-
poietin-1 and angiopoietin-2 were detected in HCC cells,
hepatic stellate cells, and smooth muscle cells, whereas
their receptor Tie-2 was detected in endothelial cells, he-
patic stellate cells and smooth muscle cells, suggesting
that multiple cell types are involved in the angiopoietin/
Tie-2 signaling pathways to mediate tumor angiogenesis.
Direct interaction between angiopoietin-2 and VEGF has
been identified using a murine HCC model (Yoshiji
et al., 2005), suggesting a synergistic effect between
725
angiopoietin-2 and VEGF. Based on the above findings,
the expression of angiopoietins might be a potent param-
eter to predict the outcome of HCC patients. Indeed,
Mitsuhashi et al. (2003) reported that HCC patients har-
boring tumors with high angiopoietin-2/1 mRNA ratio
had a shorter survival than those with tumors having
low angiopoietin-2/1 mRNA ratio. A study by Wada et al.
(2006) also revealed that a higher level of angiopoietin-2
in the tumors was associated with a shorter disease-free
survival in HCC patients with hepatic resection.
However, the mechanism responsible for the regula-
tion of angiopoietin-2 expression appears to be different
from that of VEGF in HCC. Unlike VEGF, hypoxia does
not regulate the expression of angiopoietin-2 in HCC
(Sugimachi et al., 2003). In addition, the role of angio-
poietin-1 in HCC is controversial. Sugimachi et al.
(2003) showed that angiopoietin-1 was more frequently
expressed in normal liver, whereas angiopoietin-2 was
more frequently expressed in HCC. Hence, a balance
between angiopoietin-1 and angiopoietin-2 may play a
more important role in mediating angiogenesis in HCC.
Epidermal Growth Factor
The epidermal growth factor (EGF) family contains six
ligands, that is, EGF, tumor growth factor (TGF) -a,
amphiregulin, betacellulin (BTC), heparin-binding EGF-
like growth factor and epiregulin, and four receptors,
that is, EGFR, ErbB2, ErbB3, and ErbB4 (Salomon
et al., 1995; Riese and Stern, 1998). All the EGF family
members share common features including an extracellu-
lar ligand binding domain, a transmembrane domain,
and an intracellular tyrosine kinase domain. The interac-
tion between any of the six ligands to EGFR activates
cell signaling pathways that lead to cell proliferation and
differentiation. HCC tumor cells secrete EGF, which may
act on multiple EGFRs expressed by tumor cells, and
thus achieve an autocrine cascade (Zhang et al., 2004).
Recent evidence has shown the potential role of EGF
family in angiogenesis of HCC. Moon et al. (2006) dem-
onstrated that BTC mRNA was prominently expressed
by tumor cells in human HCC specimens, whereas
EGFR was mainly expressed by sinusoidal endothelial
cells. In addition, a strong correlation between BTC
expression in tumor cells and EGFR expression in tumor
endothelial cells was identified. Furthermore, BTC was
secreted by HCC cell lines, suggesting the potential
interaction between BTC and EGFR in tumor angiogene-
sis. Targeting EGFR signaling has been recently demon-
strated to be effective in blockade of angiogenesis in ani-
mal models (Ueda et al., 2006).
Platelet-derived Endothelial Cell
Growth Factor
Platelet-derived endothelial cell growth factor (PD-
ECGF) is another angiogenic molecule that can promote
endothelial cell migration, but it has little effect on cell
proliferation. Its angiogenic effect is mediated by the
release of 2-deoxy-d-ribose as a result of breakdown of
thymidine by its thymidine phosphorylase activity (Grif-
fiths and Stratford, 1997). Up-regulation of PD-ECGF
has been identified in HCC tumor tissues compared with
the adjacent nontumorous tissues, suggesting the poten-
tial role of PD-ECGF in tumor angiogenesis. However,
726 YANG AND POON
in a study, the expression of PD-ECGF in tumor tissues
did not correlate with the outcome of HCC patients
(Morinaga et al., 2003). Interestingly, another study
revealed that a higher level of thymidine phosphorylase
activity in nontumorous tissues adjacent to tumor tis-
sues was associated with an earlier tumor recurrence
after hepatic resection, suggesting a prognostic value of
thymidine phosphorylase activity in nontumorous livers
(Ezaki et al., 2005). Another two studies have reported a
positive correlation between PD-ECGF expression in the
tumor tissue and portal vein tumor thrombosis in HCC
(Zhou et al., 2000; Guo et al., 2001).
Other Angiogenic Factors
Several other factors secreted by tumor cells have also
been reported to participate in the angiogenic process.
Fibroblast growth factors, a family of heparin-binding
growth factors, exhibit potent angiogenic properties. Ba-
sic fibroblast growth factor (bFGF) could be secreted by
both tumor cells and endothelial cells, suggesting the
presence of both autocrine and paracrine cascades in
bFGF-mediated angiogenic activities (Schweigerer et al.,
1987). El-Assal et al. (2001) demonstrated that the
expression of bFGF protein in human HCC specimens
correlated with tumor angiogenesis. HCC cells also
secrete platelet-derived growth factor (PDGF), another
angiogenic factor, to function on endothelial cells which
express PDGFR, resulting in an increased metastatic
potential (Zhang et al., 2005). A recent report demon-
strated that PDGF links TGF-beta signaling to nuclear
beta-catenin accumulation for HCC progression (Fischer
et al., 2007). Inducible nitric oxide synthase (iNOS) is
also regarded to be an angiogenic factor, supported by
the evidence that its expression was positively correlated
with MVD and tumor recurrence after hepatic resection,
and its angiogenic effects seemed to be mediated by ma-
trix metalloproteinase related pathways (Sun et al.,
2005). However, detailed mechanism studies are still
required to investigate the role of inducible nitric oxide
synthase in HCC angiogenesis.
Our previous study showed that the expression of tis-
sue factor, a hemostatic protein, correlated with tumor
angiogenesis and tumor invasiveness (Poon et al.,
2003b), suggesting that clotting-dependent induction of
tumor angiogenesis is mediated by tissue factor-induced
generation of thrombin and subsequent deposition of a
cross-linked fibrin network, which provides a proangio-
genic matrix that facilitates blood vessel infiltration.
Interleukin-8 (IL-8) is another potential angiogenic
factor that is expressed by various types of tumor cells
including HCC (Shin et al., 2002; Azenshtein et al.,
2005). However, controversial findings have been
reported about the angiogenic property of IL-8 in HCC.
One study demonstrated that increased expression of IL-
8 in human HCC specimens was associated with venous
and bile duct invasion, and IL-8 produced by HCC cell
lines stimulated proliferation of human umbilical vein
cells (Akiba et al., 2001). In contrast, a more recent
study reported that the expression of IL-8 in human
HCC was related to vascular invasion but not tumor
angiogenesis as assessed by MVD (Kubo et al., 2005).
Cyclo-oxygenase (COX-2) contributes to several physi-
ological and pathological processes. COX-2 expression
has been found to be associated with VEGF expression
and MVD in HCC tumor tissue (Cheng et al., 2004). Our
data also showed that tumor cytosolic COX-2 levels in
HCC correlated with VEGF levels and features of inva-
siveness such as venous invasion and microsatellite
lesions (Tang et al., 2005). These findings suggest an
angiogenic effect of COX-2 in HCC.
Other angiogenic factors, such as macrophage migra-
tion inhibitory factor (Ren et al., 2003; Hisai et al.,
2003) and thrombospondin-1 (Poon et al., 2004b), might
also play important roles in mediating tumor angiogene-
sis in HCC, though further studies are needed to clarify
the molecular pathways of these molecules in angiogene-
sis. With the development of gene and protein expres-
sion profiling technologies, novel angiogenic factors,
such as pituitary tumor transforming gene 1 (PTTG1),
brain-derived neurotrophic factor (BDNF) and stromal-
derived factor-1 (SDF-1), have been identified. Fujii
et al. (2006) reported that PTTG1 was remarkably over-
expressed in HCC tumor tissues, as compared with non-
tumorous tissues. In addition, PTTG1 mRNA expression
significantly correlated with MVD. Finally, PTTG1
mRNA was an independent prognostic factor for disease-
free survival of HCC patients. These findings suggest
that PTTG1 might be involved in the development of
HCC through promotion of angiogenesis.
Our previous animal study has detected an increasing
production of BDNF by tumor cells during HCC tumor
development (Yang et al., 2005). As endothelial cells and
bone marrow-derived progenitor cells express BDNF re-
ceptor, tyrosine kinase receptor B (TrkB), it is reasonable
to hypothesize that BDNF might exhibit angiogenic
effects through recruitment of endothelial cells and bone
marrow-derived progenitors. In fact, some studies have
shown the presence of autocrine and paracrine loops of
BDNF in endothelial cells (Kim et al., 2004), and BDNF
could stimulate mobilization of TrkB1 progenitor cells to
the site of hypoxia to promote revascularization (Kermani
et al., 2005). In addition, BDNF/TrkB interaction could
up-regulate HIF-1a and VEGF expression in tumor cells
(Nakamura et al., 2006), providing another clue that
BDNF might be a tumor angiogenic factor. Therefore, fur-
ther studies are warranted to explore the expression of
BDNF in relation to angiogenic parameters in HCC.
SDF-1 is a recently identified angiogenic factor that
could recruit endothelial progenitors to the site where
neovasculature is formed (Aghi et al., 2006). Because
bone marrow-derived progenitors (CXCR41Flt-11
hemagiocytes) express CXCR4, a receptor of SDF-1, the
chemotaxic role of SDF-1 seems to be more prominent in
these endothelial progenitors. However, available studies
about SDF-1 and CXCR4 in HCC mainly focused on
CXCR41 tumor cell migration and metastasis (Schi-
manski et al., 2006; Sutton et al., 2007). The role of
SDF-1/CXCR4 interaction in angiogenesis of HCC
deserves further studies.
Antiangiogenic Factors
It has been postulated that physiological angiogenesis
needs a balanced network between angiogenic and anti-
angiogenic cascades, whereas tumor angiogenesis is an
imbalance between them (Zhao et al., 2003). However,
there are few data about antiangiogenic factors in tumor
angiogenesis in HCC. A study has shown that angiogen-
esis in HCC depends on a net balance between VEGF
 VASCULAR CHANGES IN HCC
and matrix metalloelastase, a matrix metalloproteinase
responsible for the generation of the natural angiogene-
sis inhibitor angiostatin (Gorrin-Rivas et al., 2000).
Endostatin is another important endogenous angiogene-
sis inhibitor. Musso et al. (2001) demonstrated that both
long form and short form of endostatin could be detected
in HCC cells and cancerous stromal cells. Lower mRNA
level of tissue collagen XVIII (the precursor of endosta-
tin) in tumor tissues was associated with larger tumor
size and higher MVD, suggesting that low endostatin
expression in HCC may be associated with increased
angiogenic activity and tumor progression.
CELLULAR BASIS OF ANGIOGENESIS IN HCC
Tumor cells secrete pro-angiogenic factors that attract
various types of cells to the site where angiogenesis is
needed. Previous studies have suggested that migration
of endothelial cells from adjacent nontumorous tissues to
tumor tissues contribute mostly to the formation of tumor
neovasculature. However, recent studies have identified
the potential role of other cell types, such as circulating
endothelial cells, hepatic stellate cells (HSCs), bone mar-
row-derived EPCs and hemangiocytes, in neoangiogene-
sis, though molecular mechanisms that mediate the
recruitment of these types of cells remain largely unclear.
Tumor Endothelial Cells
Endothelial cells express a variety of receptors of
angiogenic factors, including VEGFRs (Ryschich et al.,
2006), Tie-2 (angiopoietin receptor; Yu, 2005), EGFR
(Amin et al., 2006), PDGFR (Rolny et al., 2006) and
CXCRs (Ryschich et al., 2006). The interaction between
ligands and their corresponding receptors initiates sev-
eral signaling pathways that regulate survival, prolifera-
tion, mobilization and invasion of endothelial cells. MVD
is a commonly used parameter to assess the number of
neovessels formed in the tumor tissues. The level of
MVD is usually quantified by immunohistochemical
staining using endothelial cell markers. The commonly
used endothelial cell markers for HCC include CD31,
CD34 and von Willebrand factor (vWF; Poon et al.,
2002; Wang et al., 2006; Lau et al., 2006). Previous stud-
ies by our group (Poon et al., 2002) and Tanigawa et al.
(1997) showed that CD34 stained mainly larger vessels
with wider lumen in the fibrous tissue within the tumor
tissues, rather than the capillary-like vessels that are
stained by CD31, suggesting that CD34 might more
accurately represent MVD in HCC. In the study by
Tanigawa et al. (1997), MVD by CD34 staining, but not
vWF staining, was an independent prognostic factor of
patient survival after resection of HCC. However,
another study of MVD by CD34 staining in 40 HCC
patients with hepatic resection did not show any signifi-
cant correlation between tumor MVD and pathological
features of the tumor, such as portal vein invasion and
tumor stage (Kimura et al., 1998). Two other studies
reported that MVD stained by vWF, but not CD34, could
predict HCC patient outcome after hepatic resection
(Yamamoto et al., 1998; Ker et al., 1999). However, our
study and others’ showed that CD34-MVD could achieve
a significant prognostic value for tumor recurrence after
resection of small HCC (
5 cm in diameter; Sun et al.,
1999; Poon et al., 2002). Therefore, it remains controver-
727
sial whether MVD evaluated by CD34 or vWF staining
provides better prognostic information in HCC. The vari-
ation in results among the studies may be partly related
to the subjective bias in counting microvessels irrespec-
tive of the endothelial markers used.
Recently, CD105 (endoglin), a new endothelial cell
marker which may represent the proliferation status of
endothelial cells, has been shown to be superior to CD34
in other cancers as an angiogenesis marker (Tanaka
et al., 2001). However, a study by our group showed that
CD105 was positively stained only in a subset of micro-
vessels in HCC compared with CD34, and MVD by
CD105 staining did not provide significant prognostic in-
formation in HCC (Ho et al., 2005a). In contrast, other
studies reported a significant correlation between CD105
staining and VEGF expression in HCC tumor tissues,
and suggested that CD105 could serve as an independ-
ent prognostic marker for patient survival (Yang et al.,
2006; Yao et al., 2007). There are several drawbacks of
using MVD in quantifying angiogenesis in tissue sec-
tions that have hindered its potential for clinical appli-
cation. They include bias in selecting the areas for
counting, subjective errors in counting, conflicting data
on the predictive value of various MVD markers, and
lack of universally accepted criteria of assessment.
Hepatic Stellate Cells (HSCs)
HSCs play an essential role in the development of
liver fibrosis. Some studies demonstrated that this type
of cells might also participate in the development of dys-
plastic nodules and hepatocarcinogenesis (Park et al.,
1997; Sano et al., 2005). In the study by Park et al.
(1997), the expression of a-smooth muscle actin (a
marker for activated HSCs) was found to be significantly
lower in dysplastic nodules than that in cirrhotic nod-
ules or in HCCs, leading to a hypothesis that dysplastic
nodules develop from clonal expansions of neoplastic he-
patocytes in advance of or parallel to the development of
cirrhosis. In a rat HCC model, blockade of HSC activa-
tion significantly prevent the development of fibrogene-
sis and HCC, suggesting a sequential change from liver
injury, fibrosis and HCC (Sano et al., 2005).
HSCs share various types of endothelial receptors,
such as VEGFRs (Ankoma-Sey et al., 2000), PDGFR
(Borkham-Kamphorst et al., 2007), and Tie-2 (Novo
et al., 2007). Therefore, the angiogenic factors secreted
by tumor cells may also function on HSCs. Olaso et al.
(2003) reported that hypoxic induction of VEGF in tu-
mor-activated HSCs may create a proangiogenic micro-
environment that favors recruitment and survival of en-
dothelial cells during hepatic metastasis transition from
an avascular to a vascular stage. An in vitro experiment
revealed that tumor cells could directly activate HSCs,
and the activation process depended on the PDGF/
PDGFR pathway (Faouzi et al., 1999).
Bone Marrow-Derived Endothelial
Progenitor Cells
Recent research interest has moved to another poten-
tial angiogenic cell type, namely EPCs. EPCs express
some cell surface markers, such as CD31 and VEGFR2,
that are similar to mature endothelial cells, but display
more abundant phenotypes of stem/progenitor cells, such
728 YANG AND POON
Fig. 3. Measurement of circulating endothelial progenitor cells
(EPCs) in hepatocellular carcinoma (HCC). A: The level of circulating
EPCs was assessed by colony formation unit (a) and (b), and the con-
firmation of endothelial cell lineage was evaluated by colony’s positive
uptake of (c) DiI-labled acetylated low-density lipoprotein and (d) fluo-
as CD133 (Bertolini et al., 2006). The first study on the
role of EPCs in tumor angiogenesis was reported by
Lyden et al. in 2001. Several other studies have subse-
quently demonstrated the contribution of EPCs to tumor
vasculature (De Palma and Naldini, 2006; Li et al.,
2006). However, these studies are mainly performed in
animal models of tumors. A recent report showed that
by using the fluorescence in situ hybridization technol-
ogy, EPCs were found in approximately 4.9% of vascula-
ture in human tumor specimens, suggesting a less prom-
inent role of EPCs in tumor angiogenesis than previ-
ously suggested (Peters et al., 2005).
Our previous clinical data showed that the level of cir-
culating EPCs was significantly higher in HCC patients
than that in patients with cirrhosis and normal subjects
(Ho et al., 2006; Fig. 3). In addition, patients with an
advance stage HCC had a higher level of circulating
EPCs. Moreover, circulating EPC level was significantly
correlated with serum AFP levels, plasma VEGF and IL-
8 levels, suggesting that VEGF and IL-8 might contrib-
ute to the mobilization of circulating EPCs to the tumor
tissues. However, direct evidence to show the localiza-
tion of EPCs in the endothelia of tumor tissues is needed
to more strongly support the role of EPCs in angiogene-
sis of HCC. In addition to natural course of tumor devel-
opment, some therapeutic approaches, such as vascula-
ture destruction agents, could also induce acute recruit-
ment of EPCs to the tumor tissues (Shaked et al., 2006),
suggesting the potential role of EPC mobilization in tu-
mor angiogenesis in response to anti-cancer treatment.
However, more clinical investigation is needed to prove
this hypothesis.
Bone Marrow-Derived Hemangiocytes
Another type of bone marrow-derived stem/progenitor
cells, which express Flt-1 and CXCR4, has recently been
rescein isothiocyanate-labeled Ultex europaeus agglutinin-1. B: Levels
of circulating EPCs were significantly higher in HCC patients, particu-
larly unresectable HCC, than that in normal controls and patients with
cirrhosis. Copyright from Ho et al. (2006).
identified in the premetastatic niche (Kaplan et al.,
2005). Jin et al. (2006) further characterized the pheno-
types of bone marrow-derived Flt-11 cells and found
that these cells also simultaneously expressed CXCR4, a
chemokine receptor of SDF-1. The mobilization of
CXCR41Flt-11 cells (hemangiocytes) to the site of is-
chemia could be inhibited by CXCR4 blockade. These
studies provide a hint that tumor cell-secreted VEGF
may recruit hemangiocytes for tumor angiogenesis.
Because HCC tumor cells secrete a high level of VEGF,
it is reasonable to postulate that VEGF-mediated pro-
genitor cell migration might contribute to tumor angio-
genesis in HCC, though further experimental and clini-
cal evidence is needed to explore this hypothesis.
Other Types of Cells
HCC tumor cells are also one of the key players in the
process of angiogenesis by secreting various types of
angiogenic factors that function on endothelial cells-
expressed corresponding receptors. Some of the major
angiogenic factors secreted by the tumor cells have been
discussed in the section Major Molecular Pathways That
Mediate Angiogenesis in HCC earlier in this review. On
the other hand, a recent work suggested that tumor cells
might be directly involved in the process of vasculogenic
mimicry (Sun et al., 2006), another type of angiogenesis,
though limited evidence demonstrated its mechanism.
Pericytes/smooth muscle cells are another type of cells
that participate in the maturation of tumor neovascula-
ture (Dickson et al., 2007). However, little evidence has
shown the presence of this type of cells in HCC and
their effects on tumor angiogenesis. The major concern
is that the marker, smooth muscle actin, which is used
to label smooth muscle cells, can also be used to recog-
nize HSCs, leading to the hypothesis that HSCs belong
to the pericyte/smooth muscle cell family.
 VASCULAR CHANGES IN HCC
ANTIANGIOGENIC THERAPY FOR HCC
Targeting endothelial cells as an anticancer strategy
may have the following advantages: (i) drugs targeted at
the endothelial cells are less likely than cytotoxic drugs
to induce drug resistance due to a relatively stable
genetic background of the microvascular endothelial
cells; (ii) low toxicity has been found when targeting tu-
mor vasculature, which is characterized by a relatively
immature structure compared with normal quiescent
vasculature; (iii) endothelial cells are a major obstacle to
conventional anticancer therapy as they may hinder
drug delivery to tumor cells. Therefore, targeting endo-
thelial cells may enhance delivery of cytotoxic drugs to
tumor cells.
Several preclinical studies have demonstrated the effi-
cacy of antiangiogenesis as a potent anticancer strategy
for HCC. Blockade of VEGF/VEGFR pathway by gene
delivery vehicle encoding antisense VEGF could inhibit
the growth of HCC xenograft in nude mice (Kang et al.,
2000). Our group also demonstrated that using a tyro-
sine kinase inhibitor, PTK 787, targeting VEGFRs could
suppress HCC tumor cell growth both in vitro and in
vivo, and the significant inhibition of tumor growth in
vivo corresponded to the decreased expression of MVD
(Liu Y. et al., 2005). Moreover, inhibition of VEGFR ac-
tivity using PTK787 was also effective in blocking revas-
cularization in tumor tissues after hepatic artery liga-
tion, suggesting that blockade VEGF/VEGFR pathway
by molecular targeted therapy may enhance the thera-
peutic efficacy of TACE for the treatment of unresectable
HCC (Yang et al., 2006).
Another approach of antiangiogenic therapy is to use
drugs that directly inhibit the proliferation of endothe-
lial cells. TNP-470, a fumagillin analogue that can in-
hibit endothelial cell proliferation, has been shown to
suppress the growth and metastasis of human HCC xen-
otransplant in an animal model (Xia et al., 1997). Inter-
feron-alpha, an immunomodulatory agent, has been
shown to directly inhibit endothelial cell proliferation
and suppress angiogenesis in vivo (Wang et al., 2000).
More recently, our group has shown that a novel immu-
nomodulatory agent FTY720 could also inhibit the
growth of HCC xenotransplant in nude mice by means
of an antiangiogenic effect (Ho et al., 2005b).
Researchers have also used agents that can prevent
the degradation of extracellular matrix and basement
membrane, a step essential for angiogenesis, for antian-
giogenic treatment of cancers (Qin et al., 1999). Gene
therapy by delivery of natural antiangiogenic factors,
such as angiostatin and endostatin, can also achieve
antiangiogenic effects in HCC xenograft in nude mice
(Ishikawa et al., 2003; Liu H. et al., 2005). Analysis of
the vascular density in the tumor tissues showed that
inhibition of tumor growth by angiostatin correlated
with suppression of tumor vascularity. Some analogues
of VEGF receptors, such as soluble Flt-1 recombinant
protein, could competitively bind to Flt-1 expressed on
endothelial cells, and thus inhibit the formation of neo-
vasculature (Graepler et al., 2005).
On-going clinical trials have demonstrated the efficacy
of VEGF/VEGFR blockade using anti-VEGF antibody,
bevacizumab, in combination with chemotherapy in
treating various types of human cancers including HCC
(Zhu et al., 2006; Fanale and Younes, 2007), leading to
729
intense interest in research on antiangiogenic therapy
for cancers. There are reports of some clinical trials
using antiangiogenic agents, such as Thalidomide, for
the treatment of HCC, but the tumor response rate is
low (Patt et al., 2005). Preliminary results from clinical
trials of single-agent antiangiogenic therapy in advanced
solid cancers showed poor efficacy (Chen et al., 2005).
Current evidence suggests that antiangiogenic therapy
works better in combination with cytotoxic chemother-
apy (Gasparini et al., 2005). In addition, antiangiogenic
therapy may also have a synergistic effect with other
molecular targeted therapies (Feng et al., 2005). Our
group is currently conducting a phase I/II trial on combi-
nation of systemic doxorubicin and PTK787 for patients
with advanced or metastatic HCC. Antiangiogenic ther-
apy may also be used as an adjunctive therapy with
established therapies for HCC. Our animal model has
shown that by using the combination of hepatic artery
ligation with PTK787, significant inhibition of tumor
growth and prolongation of animal survival could be
achieved (Yang et al., 2006), suggesting that addition of
antiangiogenic therapy may enhance the therapeutic ef-
ficacy of chemoembolization in clinical setting. Another
group also demonstrated that VEGF antisense oligodeox-
ynucleotides mixed with lipiodol more significantly
inhibited HCC growth in rats than arterial embolization
with lipiodol alone (Wu et al., 2004). These pre-clinical
data suggest that it is reasonable to conduct a clinical
trial to test whether combined anti-VEGF therapy can
enhance the efficacy of TACE for the treatment of HCC.
CONCLUSIONS
Angiogenesis plays a crucial role in carcinogenesis,
growth and progression of HCC, which is one of the
most vascularized human cancers. Morphologically,
arterialization and sinusoidal capillarization are two
important vascular changes associated with tumor
angiogenesis in HCC. VEGF appears to be the most crit-
ical angiogenic factor regulating angiogenesis in HCC.
Recently, other cell types in addition to endothelial cells
have been identified to play important roles in tumor
angiogenesis. However, the molecular and cellular mech-
anisms that regulate angiogenesis in HCC still remain
largely unclear. Further studies to elucidate the mecha-
nisms involved in HCC angiogenesis are not only crucial
to our understanding of tumor biology of this disease,
but may also provide guidance to the use of appropriate
antiangiogenic agents for HCC.
ACKNOWLEDGMENTS
The study was supported by the Seed Funding Pro-
gram for Basic Research 2007 of the University of Hong
Kong.
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