I. Introduction and literature review
1.4 PlGF and cancers
1.4.1. Placenta growth factor, PlGF
PlGF, shares 53% identity with the platelet-derived growth factor (PDGF)-like region of
VEGF (Nissen et al., 1998). The human PlGF gene has been mapped to chromosome 14q24 (Mattei
et al., 1996). PlGF is anticipated to have 149 amino acids and is encoded by 7 exons that span 800
kb (Roy et al., 2006). By alternative splicing, 4 forms of PlGF protein are generated: PlGF-1,
PlGF-2, PlGF-3, and PlGF-4 (Maglione et al., 2000). Only PlGF-2 is capable of binding heparin
(Hauser and Weich, 1993). Unlike VEGFA, which binds to both FLT1 and FLK1, PlGF binds to
FLT1 but not FLK1, and it also binds to neuropilin 1 and 2 (Persico et al., 1999; Migdal et al.,
1998)
1.4.2. Distribution of PlGF in tissues
PlGF is abundant in the trophoblasts of the placenta at the middle to late stages of pregnancy.
The amount of PlGF persistently increases toward the terminal development of the placenta, and its
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biosynthesis seems to be limited to trophoblasts and stromal cells (Maglione et al., 1993; Hauser et
al., 1993). This temporal pattern of PlGF production may reflect a role in preventing excessive
neovascularization and overgrowth of the placenta tissue by downregulating angiogenesis. In
addition to the placenta, the gene is abundantly expressed in the thyroid under normal physiological
conditions (Viglietto et al., 1995).
1.4.3. Impact of PlGF gene knockout mice
Knockout of only one allele of the Vegfa gene, encoding VEGF-A, is lethal in the mouse
embryo (heterozygous lethality) with impaired angiogenesis and blood island formation leading to
developmental abnormalities (Ferrara et al., 1996; Carmeliet et al., 1996). In contrast, several
studies suggest that genetic deletion of either Vegfb or PlGF genes does not result in obvious
impairment of the vascular system (Luttun et al., 2002; Aase et al., 2001; Bellomo et al., 2001).
Although PlGF knockout mice under normal physiological conditions do not exhibit obvious
phenotypic changes, these animals show vascular defects under several pathological settings
(Luttun et al., 2002; Fischer et al., 2007; Carmeliet et al., 2001; Luttun et al., 2002). The response to
VEGF-A is also impaired, including reduced angiogenesis and vascular permeability under
ischemic insult in a hind limb model and recruitment of monocytes and macrophages in a skin
wound assay. Transplantation of wild-type bone marrow restores angiogenesis and collateral growth
in this knockout model under ischemic conditions, which suggests that PlGF may contribute to
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vessel growth in adult animals through the mobilization of bone marrow–derived cells (Carmeliet et
al., 2001).
1.4.4 Mechanisms of PlGF
Several studies have analysed the molecular mechanisms of PlGF. This growth factor
stimulates angiogenesis, in part by enhancing VEGF signalling. It displaces VEGFA from FLT1,
which liberates VEGFA and allows it to activate FLK1 and enhance VEGF-driven angiogenesis
(Park et al., 1994). PlGF also upregulates the expression of VEGFA, fibroblast growth factor 2
(FGF2), platelet-derived growth factor-β (PDGFB), matrix metalloproteinases (MMPs), and other
angiogenic factors (Marcellini et al., 2006; Roy et al., 2005). Moreover, the binding of PlGF to
FLT1 leads to intermolecular crosstalk between FLT1 and FLK1, which amplifies FLK1 signalling
and consequently enhances VEGF-driven responses (Autiero et al., 2003). These effects of PlGF
suggest that endothelial cells enhance their own responsiveness to VEGFA by producing PlGF
(Autiero et al., 2003). However, PlGF is also capable of inducing its own signals through FLT1
independently of VEGFA. Indeed, gene expression profiling of endothelial cells has revealed that
PlGF signals directly through FLT1 and switches on a number of pro-angiogenic genes (Autiero et
al., 2003; Schoenfeld et al., 2004).
1.4.5. PlGF in primary human tumors
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The first investigation conducted on PlGF expression in tumors was undertaken by Takahashi
et al. (1994), who demonstrated that PlGF is expressed in hypervascular renal cell carcinoma. PlGF
levels correlate with serosal invasion, lymph-node metastasis, tumor stage, and survival in gastric
cancer (Chen et al., 2004); with disease progression and survival in colorectal cancer (Wei et al.,
2005 ); with tumor stage in non-small cell lung carcinoma (Zhang et al., 2005 ); with recurrence,
metastasis and mortality in breast cancer (Parr et al., 2005); and with post-surgical early recurrence
of hepatocellular carcinoma (Ho et al., 2007). Plasma PlGF protein levels are also upregulated and
correlate with tumor grade and survival in patients with renal cell carcinoma (Matsumoto et al.,
2003). PlGF is not only produced by malignant cells, but also by endothelial cells, smooth muscle
cells, pericytes, cancer-associated fibroblasts, tumour-associated macrophages and various other
inflammatory cells in the tumor stroma (Fischer et al., 2007; Luttun et al., 2002; Carmeliet et al.,
2001; Yonekura et al., 1999). Tumor cells can also induce PlGF expression by fibroblasts via
crosstalk between tumor cells and the stroma. However, not all studies have found increased PlGF
levels in tumors. PlGF mRNA is downregulated in thyroid tumors (Viglietto et al., 1995), and is
undetectable in ovarian tumors (Sowter et al., 1997). PlGF expression is apparently lower in
prostatic tumors than in normal prostatic tissue (Matsumoto et al., 2003). A similar situation occurs
in the thyroid, where strong PlGF expression has been noted in normal tissue and its production is
decreased in thyroid tumors (Viglietto et al., 1995).
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1.4.6 PlGF as a positive regulator of tumorigenesis and metastasis
In the transgenic PLGF-expressing mice, overexpression of PlGF in keratinocytes is also
linked to an increase in tumor growth, invasiveness, and the numbers and sizes of metastases in
animals inoculated intradermally with B16-BL6 melanoma cells (Marcellini et al., 2006).
Experiments with cultured cancer cell lines suggest that PlGF has a role in controlling cell motility
and invasiveness. In breast cancer cell lines, exogenous addition of PlGF-2, but not VEGF-A, to
culture medium stimulates two key characteristics associated with metastatic potential: motility
(detected by cell migration assay) and invasiveness (detected by Matrigel spheroid assay) (Taylor et
al., 2007). The PlGF-2–associated stimulation of motility and invasion is suppressed in an in vitro
system by the addition of a peptide that blocks the heparin-binding site of VEGFR1 or by an
antibody to PlGF (Taylor et al., 2007).
1.4.7 PlGF as a negative regulator of tumor growth angiogenesis.
Xu and colleagues (2006) reported that overexpression of PlGF impairs tumor growth in
xenograft models. In above study, a full-length PlGF-2 plasmid expression construct was stably
transfected into three human cell lines (lung carcinoma, colon carcinoma, and glioblastoma) that
produced high amounts of VEGF-A, and clones with the highest amounts of PlGF-2 were selected
for implantation (Xu et al., 2006). Although there was no effect on the growth rate of the cell lines
in culture, subcutaneous or orthotopic implantation of the PlGF-overexpressing cell lines into mice
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revealed an inhibition of tumor growth and a reduction in tumor-associated angiogenesis. The
antagonism of VEGF-induced angiogenesis by production of PlGF within the same population of
cells has also been demonstrated in a murine model of fibrosarcoma (Eriksson et al., 2002).
VEGF-A–PlGF-1 heterodimers fails to activate VEGFR2-mediated signaling and fails to induce
angiogenesis in vitro and in vivo. Although overexpression of human PlGF-1 in murine
fibrosarcoma cells does not alter the growth rate of the cells in culture, forced production of PlGF-1
markedly reduces the rate of growth of tumors arising from subcutaneously implanted inoculates.
1.4.8 Epression of PlGF in non-cancerous disease
Pre-eclampsia is characterized by high blood pressure and elevated protein levels in urine
during pregnancy. In a normal pregnancy, angiogenesis, and vascular transformation lead to normal
placental development, whereas pre-eclamptic pregnancies are subject to abnormal angiogenesis
and vascular transformation. Since PlGF is a factor in angiogenesis, testing of the PlGF levels
between 15 and 18 weeks allows a doctor to determine whether a patient is at high risk, since the
PlGF level in a patient with pre-eclampsia will be lower than average PlGF levels during a normal
pregnancy (Schmidt et al., 2009). The new tests identify PlGF in blood earlier and more effectively
than do traditional testing measures.
The pathogenesis of chronic obstructive pulmonary disease (COPD) is hypothesized to result
from an imbalance of proteases and antiproteases in the lung. The expression of PlGF increases as a
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response of airway epithelial cells to proinflammatory cytokines. The sustained stimuli of cytokines
and PlGF subsequently reduce VEGF expression and promote the apoptosis of airway epithelial
cells through VEGFR. The apoptosis of epithelial cells is considered essential for the pathogenesis
of pulmonary emphysema (Cheng et al., 2008).
As a risk-predicting biomarker in patients with acute coronary syndrome (ACS), placental
growth factor (PlGF) (Lenderink et al., 2006) was recently shown to be important in early and
advanced atherosclerotic lesions and coronary plaque rupture. This biomarker could be up-regulated
in the crucial mechanism of atherosclerosis in ACS patients (Heeschen et al., 2004) based on
subsequent platelet aggregation and systematic thrombosis, which may lead to the acute myocardial
infarction (AMI) or sudden cardiac death. Hence, as a member of the vascular endothelial growth
factor family (VEGF) (Carmeliet et al., 2001) PlGF may be a primary inflammatory instigator of
atherosclerotic plaque instability during the acute phase of ACS.
In our previous study, we had used an immunohistochemical technique to examine the
expression of PlGF in 100 specimens of oral squamous cell carcinoma (OSCC) (Cheng et al., 2010).
We found that the higher mean PLGF labeling index was significantly associated with OSCCs with
positive lymph node metastasis or with more advanced clinical stages. Positive lymph node
metastasis and PlGF labeling index > 40% are identified as independent unfavorable prognosis
factors. Several previous studies have reported an increased PlGF mRNA expression in tumor
tissues and increased serum PlGF protein levels in patients with several human cancers.Thus, in
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this study we assessed whether the PlGF mRNA expression in OSCC tissues and serum PlGF
protein level in OSCC patients could be valuable biomarkers to predict the therapeutic effect,
progression, recurrence and prognosis of OSCC patients.
Specific goals:
1. To assess whether the expression of PlGF mRNA in OSCC tissues could be used to predict the
progression, recurrence and prognosis of OSCCs in Taiwan.
2. To evaluate whether the serum PlGF protein level in OSCC patients could be used to predict the
progression, recurrence and prognosis of OSCCs in Taiwan.This work was published by Oral
Oncology and the article is included as appendix B1.
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