Vascular Endothelial Growth Factor Signaling

Vascular Endothelial Growth Factor Signaling

David Semela, Jean-François Dufour

8

8.1

Introduction

Vascular endothelial growth factor (VEGF) is the main driving force for angiogenesis and vasculo- genesis. Identified as a vascular endothelial cell mitogen and survival factor, it has been sequenced and cloned by Ferrara and Connolly in 1989 [74, 84].

Intense research over the past years has deciphered the gene, molecular pathways, receptors and func- tions of this angiogenic factor [35]. VEGF is thought to play an important role in liver regeneration, he- patic fibrogenesis, portal hypertension, hepatocar- cinogenesis and malignant ascites formation.

8.2 VEGF

8.2.1

Biological Functions of VEGF

Vascular endothelial growth factor is the key ang- iogenic factor of developmental, physiological and pathological angiogenesis, which is the formation of new microvessels from a pre-existing vascular bed.

VEGF is a glycoprotein that can be produced and secreted by most cells in mammals. The main tar- gets of VEGF are vascular endothelial cells. VEGF has been shown to promote proliferation [34] and survival [9, 45, 46, 175] in vascular endothelial cells in vivo and in vitro. It acts as an endothelial survival factor by inducing expression of the anti-apoptotic proteins Bcl-2 and A1 in vascular endothelial cells [45]. Furthermore, VEGF induces the expression of proteases such as collagenase [156], matrix met- alloproteinases [178], and urokinase- and tissue- type plasminogen activators [120], which enable endothelial cells to break down the surrounding basal membrane and extracellular matrix in order to migrate and form new blood vessels. Initially de-

scribed as vascular permeability factor (VPF) [139], VEGF increases the permeability of blood vessels up to 50,000 times more than histamine [28]. Trans- mission and scanning electron microscopy studies showed that VEGF regulates hepatic sinusoidal per- meability by inducing fenestration in hepatic sinu- soidal endothelial cells possibly through caveolin-1 protein [170].

During embryonic development, liver organo- genesis and vasculogenesis, which is defined as the de novo formation of blood vessels from heman- gioblasts, are regulated through the VEGF signal- ing system [15, 33, 100]. The importance of VEGF signaling during embryonic development is high- lighted by the fact that lack of a single VEGF gene allele results in abnormal blood vessel development and embryonic lethality in mice [15, 33]. Knockout of the genes for the VEGF receptors VEGFR-1 or -2 also results in embryonic lethality [38, 140]. Kidney development, skeletal growth, enchondral bone for- mation, wound healing and ovarian angiogenesis are further fundamental physiological processes regulated by VEGF [36].

Recent evidence suggests that elevated levels of systemically circulating VEGF lead to recruitment of hematopoietic stem cells and endothelial pro- genitor cells from bone marrow to sites of neovas- cularization (reviewed in [127]). These cells home to sites of neovascularization such as tumor microvas- culature and the regenerating liver, where they are incorporated and contribute to angiogenesis [23, 41, 94, 129].

The level of circulating VEGF in healthy individ-

uals is low and depends on the method and sample

used for measurement (free vs. total VEGF, plasma

vs. serum) [64]. Significant amounts of VEGF are

bound to plasma proteins such as α2-microglobulin

and are stored in platelets, which secrete VEGF from

their α granules upon activation during blood clot-

ting [162]. The complex biology of VEGF, its release

from platelets and the different isoforms (see below)

have to be considered when choosing a VEGF detec-

tion test [7, 64, 162].

8.2.2

VEGF Gene and Splice Variants

The human VEGF gene is localized on chromosome 6p21.3 [159] and contains eight exons (Fig. 8.1) [155].

VEGF is highly conserved across species with a ho-

mology of approximately 85% between human and rat VEGF [35]. Alternative exon splicing of the hu- man VEGF pre-mRNA produces six VEGF isoforms containing 121, 145, 165, 183, 189 or 206 amino acids [155]. All VEGF isoforms are secreted as covalently linked homodimers but display differences in the basic amino rich domains encoded by exons 6 and 7 [155, 132]. These domains are important sites for molecular interaction and mediate binding of VEGF to heparin, heparan sulfate proteoglycans and to el- ements of the extracellular matrix, which results in sequestration of certain VEGF isoforms in the ex- tracellular matrix or at the cell surface: VEGF

, an isoform that lacks the domains encoded by exons 6 and 7, does not bind to heparin and is therefore freely diffusible after secretion [62]. In contrast, the high- ly basic isoforms VEGF

and VEGF

containing exons 6 and 7 remain almost entirely bound to the extracellular matrix and cell surface [62]. VEGF

(exon 6 absent), the most abundant isoform (46 kDa as homodimer), displays an intermediate behav- ior with 50%–70% sequestration [62]. Sequestered VEGF can be released by heparin, heparan sulfate, and heparinase [62] and proteases such as plasmin or urokinase-type plasminogen activator [122].

8.2.3

VEGF Protein Family

Vascular endothelial growth factor is member of a gene family of growth factors consisting of VEGF it- self (also named VEGFA), VEGFB, VEGFC, VEGFD, VEGFE and placenta growth factor (PlGF, also known as PGF, consisting of three isoforms PlGF-1, -2 and -3). They share significant sequence homol- ogy and are ligands to the same receptors as VEGF (see below and Fig. 8.2) [132]. PlGF expression is not only restricted to the placenta [96] but can be up- regulated in different cell populations such as en- dothelial, smooth muscle, inflammatory and malig- nant cells [16]. VEGFB is mainly expressed in heart and skeletal muscle [115]. VEGFC, also referred to as VEGF-related protein [68, 82], and VEGFD [116]

are involved in lymphangiogenesis [73] and fetal lung development [30]. Overexpression of VEGFC induces selective proliferation of lymphatic, but not vascular, endothelial cells and lymphatic vessel en- largement [65]. However, recent evidence suggests

that VEGFC might play a role in angiogenesis by inducing fenestrations in vascular endothelial cells and by inducing blood capillaries under certain circumstances [14]. VEGFE has been found in the genome of orf virus, which affects sheep, goats and humans [95]. It is believed that the gene has been originally acquired from a mammalian host [95].

Interestingly, skin lesions induced by the orf virus show extensive dermal vascular endothelial cell proliferation and vasodilatation [95].

Fig. 8.1. The six different splice variants of the VEGF (VEGFA) gene ordered by length of the amino acid sequence (* trunca- tion in exon 6, ** additional sequence encoded by exon 6)

Fig. 8.2. VEGF receptors (VEGFR)-1, -2, -3 and the neuropilin (NRP) receptors 1 and 2 with their function in VEGF signaling

8.2.4

VEGF Gene Expression

The regulatory factors for VEGF expression can be divided into two main categories: hypoxia through hypoxia-inducible factor (HIF)-1 and a group of cy- tokines, growth factors and transcription factors other than HIF-1. Additionally, mutations in tumor suppressor genes and oncogenes such as p53, ras,

regulate VEGF expression [49, 67, 76, 91, 109].

Hypoxia

Hypoxia is a potent stimulator of VEGF expression.

HIF-1 is the key transcription factor in hypoxic tis- sues and induces the expression of several hypoxia- response genes such as VEGF and VEGFR-1 [44, 57].

The detailed molecular mechanisms of HIF-1 and the regulation of VEGF expression by HIF-1 are re- viewed in Chap. 26.

Hypoxia not only induces and upregulates the expression of VEGF but also stabilizes the intrinsi- cally labile VEGF mRNA, which contains destabiliz- ing elements in its 3’ and 5’ untranslated and coding regions [25, 85, 90, 144]. The hypoxia-induced RNA- binding protein HuR binds with high affinity and specificity to regulatory elements of VEGF mRNA and prevents its degradation [86].

Cytokines and Growth Factors

Many cytokines such as interleukin (IL)-1 α, IL-1β, IL-6, nitric oxide (NO) and growth factors such as fibroblast growth factor (FGF), transforming growth factor (TGF)- α, TGF-β, tumor necrosis fac- tor (TNF)- α, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and insulin-like growth factor-1 up- regulate VEGF, highlighting the complex regulation and redundancy of the angiogenesis network [36, 67, 132].

8.3

VEGF Receptors

The effects of VEGF are mediated mainly through two cell surface receptor tyrosine kinases, namely VEGFR-1 and VEGFR-2. Neuropilin-1 (NRP1) and neuropilin-2 (NRP2) are recently discovered VEGF receptors belonging to the semaphorin sub-family.

VEGFR-3 is a receptor for VEGFC and VEGFD but not for VEGF. VEGFR-1, -2 and -3 belong to the flt

sub-family of receptor tyrosine kinases [132] and consist of seven extracellular immunoglobulin- like domains, one transmembrane region and a conserved tyrosine kinase domain intracellularly, which is interrupted by a kinase insert domain [102, 142, 153]. The functions of the different VEGF recep- tors and their ligands are summarized in Fig. 8.2.

8.3.1 VEGFR-1

Vascular endothelial growth factor receptor-1 (also known as fms-like-tyrosine kinase [Flt]-1 or FLT-1 in humans [24]) is a 180-kDa glycoprotein bind- ing VEGF, VEGFB and PlGF [132, 137]. In contrast to VEGFR-2, VEGFR-1 is upregulated by hypoxia through HIF-1 (see Chap. 26) [44]. It is highly ex- pressed on the surface of the different endothelial cell populations (quiescent and cycling) such as en- dothelial cells of liver sinusoids and hepatic arteri- oles [81, 134, 168], but also on hepatocytes during liver regeneration [107, 134] and on a few other cells such as hepatic stellate cells [2, 99], monocyte-mac- rophages including Kupffer cells [22, 136], pericytes [114], smooth muscle cells [21], nerve cells [147] and hematopoietic cells [36]. The exact role of VEGFR-1 is still under debate and depends on the biological situation (i.e. physiological vs. pathological angio- genesis). Although a knockout of the VEGFR-1 gene results in the formation of abnormal vascular chan- nels, excessive angioblast proliferation and embry- onic lethality by day E8.5 in mice [38, 39], VEGFR-1 lacking only the tyrosine kinase domain is sufficient for normal development and angiogenesis [59]. In contrast, VEGFR-1 tyrosine kinase-deficient mice showed impaired angiogenesis during carcinoma growth [60]. No proliferative or migratory response in endothelial cells or hepatocytes has been attrib- uted to VEGFR-1 [118, 134, 160]. Signaling through VEGFR-1 was shown to contribute to the regulation of endothelial cell permeability [148]. Activation of VEGFR-1 by the VEGF homolog PlGF enhances VEGF-driven angiogenesis through VEGFR-2 [6, 16] (see below). Further, VEGFR-1 is involved in the recruitment of endothelial progenitor cells [94]

and bone marrow-derived myeloid progenitors [93]

and promotes survival of hematopoietic stem cells [47]. Matrix metalloproteinase 9 (MMP-9) has been shown to be induced specifically via VEGFR-1 in endothelial cells of the lung promoting pulmonary metastasis [61].

Recent evidence suggests an angiogenesis-in-

dependent endothelial protection of hepatocytes

through VEGFR-1 [81]. Selective activation of liver

sinusoidal endothelial cells via VEGFR-1 induces

paracrine secretion of the potent mitogens HGF and IL-6 (see Chap. 7), which promote hepatocyte prolif- eration and reduce liver damage in mice exposed to the hepatotoxin CCl

.

Besides a membrane-bound form of VEGFR-1, a soluble form of VEGFR-1 (sVEGFR-1, sFlt-1), which is produced by endothelial cells and monocytes by alternative splicing, has been identified [8, 75].

sVEGFR-1 has been detected in human serum and plasma of normal male and female donors [8]. By binding VEGF, sVEGFR-1 is a naturally occurring VEGF antagonist. The function of sVEGFR-1 in physiologic angiogenesis and malignant neovascu- larization is unclear.

8.3.2 VEGFR-2

Vascular endothelial growth factor receptor-2 (also known as mouse fetal liver kinase [Flk]-1 or kinase domain region [KDR] in humans [154]) is a 200–230- kDa VEGF receptor, which shares 85% of sequence identity with VEGFR-1 [102] but binds VEGFC, VEGFD and VEGFE instead of VEGFB or PlGF [132].

In contrast to the non-mitogenic and non-moto- genic VEGFR-1, VEGFR-2 induces endothelial cell proliferation, migration and survival [34, 46, 126].

VEGFR-2 is involved in the mechanotransduction of blood flow shear stress to the vascular endothelium by nuclear translocation of VEGFR-2 and consecu- tive binding to the cytoskeleton together with VE- cadherin and β-catenin [141]. VEGFR-2 is highly expressed on adult and embryonic endothelial cells, embryonic angioblasts and hematopoietic stem cells [36, 132]. In resting liver, VEGFR-2 expression is limited to endothelial cells of the larger hepatic vessels [134], although earlier reports described VEGFR-2 mRNA expression also in liver sinusoi- dal endothelial cells [168]. During liver regenera- tion, VEGFR-2 expression predominantly increases on endothelial cells of large vessels (portal venules, arterioles, central venules) and to a lesser extent on sinusoidal endothelial cells often in close proximity to large vessels [134]. Additionally, hepatic stellate cells have been found to express VEGFR-2 in vitro [99]. Homozygous loss of VEGFR-2 results in lack of endothelial cells, impaired liver organogenesis [100] and embryonic lethality at day E9.5 to E10.5 [140], whereas heterozygous mice are normal [140].

VEGFR-2 is phosphorylated in resting liver, but has been shown to increase in activation during liver re- generation [134].

8.3.3 VEGFR-3

Vascular endothelial growth factor receptor-3 (or fms-like-tyrosine kinase [Flt]-4) is a receptor for VEGFC and VEGFD and does not bind VEGF [37, 117]. VEGFR-3 is mainly expressed on lymphatic endothelium and is involved in lymphangiogenesis [65, 70]. VEGFR-3 could not be detected on endothe- lial cells in resting or regenerating liver [134].

8.3.4

Neuropilin-1 and -2

Neuropilin-1 (NRP1) and NRP2 are additional VEGF receptors unrelated to VEGFR-1, -2 and -3 [146]. They are isoform-specific receptors binding VEGF

but not VEGF

and are involved in neu- ronal guidance [112]. Coexpression of VEGFR-2 and NRP1 enhances binding of VEGF to VEGFR-2 [113].

Neuropilins can form complexes with VEGFR-1 and probably with VEGFR-2 [113]. No intrinsic recep- tor signaling has been shown after binding of VEGF to NRP1 or NRP2 [113]. To date, nothing is known about the presence or function of NRP1 and NRP2 in liver biology.

8.3.5

VEGF Receptor Signaling

After binding, soluble VEGF dimers induce VEGF receptor dimerization leading to homo- or het- erodimers [6]. The juxtaposed cytoplasmic tyrosine kinase domains of the VEGFR molecules transphos- phorylate several tyrosine residues in the neighbor molecule [6, 40, 101, 137]. Activated receptors in turn activate proteins of different signaling pathways by phosphorylation (reviewed in [101] and Chap. 20 in this book): phospholipase C (PLC), phosphatidyli- nositol 3’-kinase (PI-3 kinase)/Akt, Ras GTPase-ac- tivating protein (GAP), Src family kinases and Raf [1, 29, 46, 53].

A recent study showed that although VEGF and

PlGF both bind VEGFR-1, they activate this recep-

tor differently, leading to a distinct gene expression

profile, where only PlGF was capable of switch-

ing on downstream target genes [6]. Many of these

PlGF-regulated genes have a role in cell cycle (Ets2,

authors were able to show that there is an intra- and

intermolecular crosstalk between VEGFR-1 and

VEGFR-2: activation of VEGFR-1 by PlGF resulted in intermolecular transphosphorylation of VEGFR- 2, thereby amplifying VEGF-driven angiogenesis through VEGFR-2 [6]. Further, VEGF/PlGF het- erodimers activated intramolecular VEGF receptor crosstalk through formation of VEGFR-1/VEGFR-2 heterodimers [6].

8.4

VEGF Signaling in Specific Liver Conditions

8.4.1

Liver Organogenesis

During embryonic development of the liver, hepatic cells are induced within the endoderm by day E8.5 of mouse gestation [51]. Interaction between these cells and surrounding endothelial cells or angiob- lasts induces outgrowth of the liver bud into the mesenchyme [100]. This stage is then followed by the formation of a de novo local vascular network (vasculogenesis) and the recruitment of hematopoi- etic cells [100]. Mice with homozygous deficiency in

vessels show normal thickening of the hepatic endo- derm but lack liver bud emergence [100]. Vasculo- genic endothelial cells with intact signaling through VEGFR-2 are therefore critical already in the earli- est stages of liver organogenesis, even prior to blood vessel function [100]. Similar interactions between endothelial cells and hepatocytes are at play in adult liver tissue, i.e. during liver regeneration.

8.4.2

Liver Regeneration

Liver regeneration is angiogenesis-dependent [27];

inhibition of angiogenesis by anti-angiogenic sub- stances such as angiostatin or TNP-470 impairs liver regeneration [27, 48]. In the course of liver regeneration after hepatectomy, there is an initial wave of hepatocyte proliferation with formation of avascular hepatocellular islands [134]. This stage is followed by endothelial cell proliferation [135] and consecutive penetration of these avascular islands by endothelial cells with the formation of new sinu- soids [97]. Recent evidence suggests that endothelial progenitor cells are mobilized from bone marrow and participate in this neovascularization by com- mitting to sinusoidal endothelial cells [41]. HIF-1 α and VEGF as key regulators have been shown to be upregulated during this process [97, 128]: hepatocel-

lular production of VEGF peaks 48–72 hours after hepatectomy and is detected mainly in periportal hepatocytes [97, 151]. VEGF production is accompa- nied by an increase in the expression of VEGFR-1 on hepatocytes and of VEGFR-1 and VEGFR-2 on sinusoidal endothelial cells [107, 128, 134, 135]. Ad- ministration of VEGF in hepatectomized rodents increases hepatocyte and sinusoidal endothelial cell proliferation [5, 151], accelerates gain in liver mass [81] and improves functional hepatic recovery [128]. Neutralizing antibodies against VEGF inhibit hepatocyte and endothelial cell proliferation after partial hepatectomy [151]. LeCouter and coworkers showed that this effect is likely due to a VEGF-in- duced release of HGF by sinusoidal endothelial cells [81, 168]. Additional effects of VEGF on functional hepatic recovery could be a stimulatory effect on the formation of new blood vessels and/or a direct ef- fect on the hepatocytes, which express VEGF recep- tors after partial hepatectomy [128, 134]. Transduc- tion of VEGF before hepatic resection also hastens functional hepatic recovery in mice with fatty liver, which is known for its impaired regenerative capac- ity [128].

8.4.3

Liver Fibrosis and Cirrhosis

Accumulating evidence suggests that VEGF and its receptors are also involved in fibrogenesis and cirrhotic remodeling. The concept of an interplay between fibrosis and angiogenesis probably medi- ated through the stimulus of hypoxia is supported by two facts. First, the deposited matrix proteins – a hallmark of fibrosis – contain and sequester differ- ent angiogenic factors such as VEGF, which are lib- erated during remodeling of the connective tissue framework by proteolytic enzymes such as matrix metalloproteinases [71, 72]. Second, an abundant vascular network develops in the fibrotic tissue around regenerative nodules [56, 167], leading to a remodeled and abnormal hepatic microcirculation in cirrhotic liver [63, 158].

Experimental liver fibrogenesis after common

bile duct ligation in rats induces VEGF expression

in hepatocytes and angiogenesis [133]. The authors

of this study showed that this process is hypoxia-

driven and that the percentage of VEGF-expressing

hepatocytes increases from 3% at the time of liga-

tion to >95% 2 weeks after ligation. The upregula-

tion of VEGF is followed by vascular endothelial cell

proliferation and angiogenesis in fibrotic areas by

week 3 [133]. A similar animal study with diethyl-

nitrosamine-induced cirrhosis confirmed the con-

cept that hepatocellular hypoxia and angiogenesis

progress together with fibrogenesis after liver inju- ry [20]. Using neutralizing monoclonal antibodies of VEGFR-1 and VEGFR-2 in murine CCl

-induced liver fibrosis, both significantly attenuated the de- velopment of fibrosis and suppressed neovasculari- zation in the liver [174]. Fibrosis markers (hepatic hydroxyproline, serum hyaluronic acid and procol- lagen III-N-peptide), the number of smooth muscle actin positive cells and procollagen mRNA expres- sion were also suppressed by this treatment [174].

The inhibitory effect of the anti-VEGFR-2 antibody was more potent than that of anti-VEGFR-1 and combination treatment with both almost completely attenuated fibrosis development [174].

Hypoxia directly contributes to the progression of liver fibrosis by inducing the expression of VEGF, VEGFR-1 and type I collagen in activated hepatic stellate cells, the key player cell in the pathogenesis of hepatic fibrosis [20, 161]. VEGF on the other hand increases α1(I)-procollagen mRNA expression and stimulates proliferation of activated hepatic stellate cells [174]. VEGF expression in hypoxic hepatic stel- late cells has been shown to be mediated by cyclo- oxygenase-2 (COX-2) protein and COX-2 inhibitors significantly blocked VEGF production via the HIF- 1 α pathway [161]. VEGF signaling through VEGFR- 1 was shown to inhibit hepatic stellate cell contrac- tion, probably through attenuation of smooth mus- cle α-actin expression [99].

8.4.4

Portal Hypertension

A major complication of chronic liver diseases is portal hypertension. The development of portal- systemic collateral vessels in portal hypertension is classically explained as a mechanical consequence of increased portal pressure with subsequent open- ing of collateral vessels [11]. Recent evidence sug- gests that active, VEGF-dependent angiogenesis is also involved in this process: VEGF, VEGFR-2 and CD31 (as specific endothelial cell marker) protein levels in splanchnic organs increased after partial portal vein ligation in mice in a time-dependent fashion during the evolution of portal hyperten- sion [32]. A monoclonal antibody against VEGFR-2 given to these animals after ligation decreased the expression of VEGFR-2 and CD31 significantly and inhibited the formation of portal-systemic collateral vessels measured with labeled microspheres [32].

Experiments using partial portal vein-ligated rats and a VEGFR-2-specific tyrosine kinase inhibitor (SU5416) confirmed that the formation of portal- systemic collateral vessels is an angiogenesis-de- pendent process, which can be inhibited by antago-

nization of the VEGF/VEGFR-2 signaling pathway [32].

8.4.5

Viral Hepatitis

Hepatitis B virus X protein (HBx) is a hepatitis B virus-encoded transcriptional activator, which is involved in hepatocarcinogenesis and hypoxia- induced angiogenesis [83]. Recent studies have shown that HBx protein stabilizes HIF-1 α and en- hances transcriptional activity of HIF-1 α through activation of the mitogen-activated protein kinase (MAPK) pathway under normoxic and hypoxic conditions [108, 171]. The expression of HIF-1 α and VEGF was increased in the liver of HBx-transgenic mice [171] and in HBx-transfected HCC cell lines [83, 152]. Immunohistochemical staining for VEGF in chronic hepatitis B correlated with the degree of injury (grade) and amount of fibrosis (stage) [169].

In chronic hepatitis C liver samples, an increase in VEGF expression and angiogenesis in portal tracts has been described [103]. Another study found no difference in VEGF mRNA expression ratios among steatohepatitis, chronic hepatitis C and hepatocel- lular carcinoma [145]. Hepatitis C virus (HCV) core protein has been found to activate expression of VEGF in HepG(2) cells [89].

8.4.6

Hepatocellular Carcinoma

Tumor growth beyond the size of 1–2 mm

requires

the formation of new blood vessels in order to supply

the malignant tissue with nutrients and oxygen [55,

138]. Central hypoxia is the main driving force of

tumor angiogenesis and upregulates proangiogenic

growth factors like VEGF (Fig. 8.3) [57]. Therefore it

is not surprising that VEGF is upregulated in most

human tumors and that direct correlation with in-

tratumoral microvessel density exists [34]. The

angiogenic switch, which describes the acquisition

of the capacity to stimulate angiogenesis by shift-

ing the balance between stimulatory and inhibi-

tory factors of angiogenesis towards proangiogenic

factors, is a rate-limiting step in tumoral develop-

ment [54]. VEGF and other growth factors promote

survival, proliferation and migration of endothelial

cells, which will finally result in the formation of

new tumoral blood vessels enhancing further tu-

mor growth. These growth factors are secreted by

neoplastic cells, adjacent stroma, hepatocytes, stel-

late cells and tumor-infiltrating inflammatory cells

[42, 54, 69, 119]. Besides hypoxia, mutations in tu-

mor suppressor genes and oncogenes and certain viral proteins are also involved in the upregulation of VEGF during hepatocarcinogenesis (see above).

In vitro studies have shown that different inflam- matory cytokines (IL-1 β, interferon-α, interferon-γ, TNF- α) and growth factors (EGF, PDGF, basic fi- broblast growth factor, TGF- α) increase the secre- tion of VEGF in HCC cell lines [165].

Hepatocellular carcinoma (HCC) is a hypervas- cular tumor [4, 78, 165] and arterial hypervascu- larization is included in the non-invasive criteria to establish HCC diagnosis [12]. Several studies report on the overexpression of VEGF in HCC [17, 19, 105, 106, 108, 110, 165, 166, 173, 177]. Grafting HCC tis- sue onto chick embryo chorioallantoic membrane, which is a classical angiogenesis assay, stimulates neovascularization [98]. It has been shown that dur- ing hepatocarcinogenesis, expression of VEGF in- creases gradually from low-grade dysplastic nodules to high-grade dysplastic nodules to early HCC [119].

The degree of VEGF expression during development of HCC correlates with the density of vessels, un- paired arteries (i.e. arteries not accompanied by bile ducts, indicative of angiogenesis), CD34 staining (as a marker of sinusoidal capillarization) and the pro- liferation of hepatocytes assessed by staining with PCNA [119]. Moreover, tumor expression of VEGF (mRNA and protein expression) significantly corre- lated with serum VEGF level per platelet in patients with HCC, providing the basis for using circulating VEGF as a prognostic marker [125]. Furthermore, hepatocytes and HCC cells adjacent to peliosis and fibrous septa showed stronger VEGF expression [119]. Small HCCs showed a higher status of neoan- giogenesis and cell proliferation activity than ad- vanced HCCs [119].

The circulating concentration of VEGF increases with the stage of HCC, the highest levels being in patients with metastasis [66]. A prospective study of 100 patients suffering from HCC found that high serum levels of VEGF significantly correlated with absence of tumor capsule, presence of intrahepatic metastasis, presence of microscopic venous inva- sion, advanced stage and postoperative recurrence [124]. Similar results have been found using serum VEGF per platelet count in 52 HCC patients [77].

In a recent study, preoperative serum VEGF in 98 patients with resectable HCC was a significant and independent predictor of tumor recurrence, dis- ease-free survival and overall survival [17]. In 80 patients with inoperable HCC undergoing transar- terial chemoembolization (TACE), Poon and cow- orkers evaluated the prognostic significance of pre- treatment serum VEGF levels prospectively [123]:

pretreatment serum VEGF levels were significantly higher in patients with progressive disease than in

those with stable or responsive disease. Patients with serum VEGF >240 pg/ml had significantly worse survival than those with serum VEGF <240 pg/ml (median survival 6.8 vs. 19.2 months, p=0.007). In a Cox multivariate analysis, serum VEGF >240 pg/

ml was an independent prognostic factor of survival [123]. Expression of VEGF in patients and animals with HCC increased significantly after TACE [87, 88, 150]. VEGF antisense oligodeoxynucleotides mixed with lipiodol inhibited HCC growth in rats signifi- cantly more than arterial embolization with lipiodol alone [163]. These studies suggest that the VEGF sig- naling pathway plays an important role in tumor re- sponse to hypoxia after arterial embolization treat- ment and that anti-VEGF strategies might enhance the efficacy of arterial embolization in HCC.

8.4.7

Malignant Ascites Formation

In 1983, Senger and coworkers reported that hepa- tocarcinoma cells secrete a vascular permeability factor (VPF, later named VEGF), which promotes accumulation of ascites fluid [139]. Ascites, which is defined as accumulation of excess fluid within the peritoneal cavity, is encountered in many patients

Fig. 8.3. VEGF signaling and its effect on tumoral endothelial cells and HCC growth

with cirrhosis, other forms of liver disease and ma- lignancies. The pathogenesis of ascites depends on the underlying disease. An important component in the formation of ascites in patients with malignan- cies of the liver and abdominal cavity is microvas- cular hyperpermeability of tumor vessels due to tu- mor-secreted VEGF with consecutive extravasation of plasma and plasma proteins. As a matter of fact, levels of biologically active VEGF in patients with malignant ascites are higher in comparison to pa- tients with ascites due to non-malignant or cirrhotic causes [26, 80, 157, 176].

Four pathways for macromolecular extravasation have been described: endothelial fenestrae [130, 131], interendothelial cell gaps [58], transendothelial cell pores [111] and vesiculo-vacuolar organelles [31, 79].

VEGF has been shown to induce fenestration and in- crease permeability in normal and tumoral microv- ascular endothelium [14, 50, 130, 131, 170]: within 10 minutes of VEGF application fenestrations appear even in vascular beds that do not have fenestrated endothelium under physiological circumstances [130]. Liver sinusoidal endothelial cells incubated with 100 ng VEGF/ml increased the number of fen- estrations and cell pores in vitro [43, 170]. Neutral- izing antibodies against VEGF or against VEGFR-2 significantly suppressed the volume of ascites, the number of tumor cells in ascites and the peritoneal capillary permeability and prolonged the survival of ascites-bearing mice suffering from HCC [172].

Soluble VEGFR-1 and VEGF-trap (both soluble de- coy receptors for endogenous VEGF), monoclonal antibody against VEGF or VEGFR-2 and the VEGF receptor tyrosine kinase inhibitor PTK 787 all pre- vented the formation of malignant ascites in animal tumor models [13, 104, 143, 149, 164]. These results suggest that the VEGF/VEGFR-2 interaction is a ma- jor regulator of malignant ascites formation.

Interestingly, ascites VEGF levels are higher in cirrhotic patients with spontaneous bacterial peri- tonitis, which is a frequent complication of cirrhotic patients with ascites, than in non-infected cirrhotic patients [121]. VEGF is thereby produced in perito- neal macrophages of cirrhotic patients and is mark- edly upregulated by bacterial lipopolysaccharide and cytokines such as IL-1 [121].

8.4.8

Liver Transplantation

Hepatocyte and endothelial cell damage due to ischemia/reperfusion injury in liver transplantation after cold preservation is an important determinant of graft function. Activation of sinusoidal endothe- lial cells by cold ischemia alters expression of differ-

ent adhesion molecules and sequesters leukocytes and platelets during reperfusion, leading to micro- circulatory disturbance and liver injury. VEGF ex- pression is upregulated in hepatocytes of rat livers preserved in University of Wisconsin (UW) solution for orthotopic liver transplantation probably due to hypoxic stress [3]. Another study showed that VEGF is expressed and released in a biphasic pattern by Kupffer cells and hepatocytes during the early post- operative period after transplantation in a syn- geneic rat orthotopic liver transplantation model [10]. Anti-VEGF antibody treatment, administered during reperfusion, decreased the degree of dam- age (measured as liver function tests, lipid peroxi- dation, and metalloproteinase activity), suggesting that VEGF may have a role in ischemia/reperfusion injury to liver grafts [10].

Cyclosporin A and sirolimus (also known as rapamycin) are potent immunosuppressive drugs used after liver transplantation. Both drugs have been shown to downregulate VEGF expression un- der certain circumstances [18, 52, 92].

8.5 Outlook

Years of intense basic and preclinical research and the recent FDA approval of the first angiogenesis in- hibitor drug bevacizumab have now opened a wide range of clinical applications for VEGF-based strat- egies in liver diseases. Anti-VEGF strategies might have a potential in the treatment of HCC, liver fibro- sis, portal hypertension and in the inhibition of ma- lignant ascites, whereas pro-VEGF strategies might improve outcome after liver resection and stimulate liver regeneration. VEGF expression levels may be useful as prognostic markers in HCC patients and as predictors of tumor response to treatment.

Selected Reading

Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its re- ceptors. Nat Med 2003;9:669–676. (This review [36] provides a detailed and comprehensive description of the different VEGF isoforms and receptors and discusses their role under physiological and pathological conditions.)

Matsumoto T, Claesson-Welsh L. VEGF receptor signal transduc- tion. Sci STKE 2001;2001:RE21. (This review [101] outlines the current knowledge about the signal transduction proper- ties of VEGF receptors, with focus on VEGF receptor-2.) Autiero M, Waltenberger J, Communi D et al. Role of PlGF in the

intra- and intermolecular cross talk between the VEGF re-

ceptors Flt1 and Flk1. Nat Med 2003;9:936–943. (This study [6] provides new insight into the poorly understood role of VEGFR-1 signaling and the interaction between VEGFR-1 and VEGFR-2.)

LeCouter J, Moritz DR, Li B et al. Angiogenesis-independent endothelial protection of liver: role of VEGFR-1. Science 2003;299:890–893. (This study [81] describes the paracrine crosstalk between hepatocytes and liver sinusoidal en- dothelial cells during hepatocyte growth.)

http://www.nature.com/focus/angiogenesis/ (This web focus on angiogenesis with a special section on VEGF signaling is a joint project of the journals Nature Medicine and Nature Reviews Cancer. The web site provides review articles and a selection of “classic” papers nominated by experts in the field of angiogenesis.)

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