Activated hepatic stellate cells promote angiogenesis via interleukin-8 in hepatocellular carcinoma

A-HSCs secreted high levels of the inflammatory chemokine IL-8

To confirm that a-HSCs facilitate tumour angiogenesis, we first isolated a-HSCs from
HCC tissues. A-HSCs were identified based on the high expression of the fibroblast-specific
markers ?-SMA and vimentin using fluorescence microscopy (Fig. 1A). Using the Multiplex bead-based Enzyme–Linked Immunosorbent Assay system, the levels
of various inflammatory chemokines that are closely associated with angiogenesis,
including GRO (?, ?, and ?), CXCL-5, CXCL-6, CXCL-7 and IL-8, were detected in the
50 % a-HSC conditioned medium (HSC-CM) (CM:HSC-CM = 1:1) 23]. A-HSCs secreted significantly higher levels of IL-8 than of any other inflammatory
chemokines (Fig. 1B).

Fig. 1. A-HSCs expressed high levels of IL-8. A Immunofluorescent staining of primary human a-HSCs isolated from a representative
sample of HCC with an anti-?-SMA antibody,anti-vimentin antibody, and IgG. Scale bar,
50 ?M. B The levels of various angiogenic chemokines in the cell-free culture supernatants
of the a-HSCs were measured by Multiplex bead–based enzyme–linked immunosorbent assay
at day 2. The data are expressed as mean ± SEM of triplicates. (C) The concentrations of IL-8 (pg/mL) in the supernatants of a-HSCs and hepatoma cells
were determined by ELISA. The concentration of IL-8 in the a-HSCs culture medium was
markedly higher compared with hepatoma cells. (D) The expression of IL-8 in the abovementioned cells was assessed by Western blotting.
A-HSCs spontaneously released large amounts of IL-8, whereas hepatoma cells produced
low levels of IL-8. (E) Hepatocarcinoma samples were stained with various antibodies, and different levels
of antibodies expression can be seen on the same section. E (a) Immunohistochemical staining for ?-SMA (1:1000) to identify a-HSCs in the tumour
stromal region. E (b) The IL-8 distribution in hepatocarcinoma samples was visualized by immunohistochemical
staining using the IL-8 neutralizing antibody (1:400). E (c) Neovascularization in hepatocarcinoma samples was visualized by immunohistochemical
staining for CD31 (1:400). Scale bar 200 ?M. One of the 22 representative micrographs is shown

It was reported that tumour cells also secret the angiogenic factor IL-8 12]. Therefore, we compared the levels of IL-8 secreted by a-HSCs with those secreted
by hepatoma cells. The ELISA assay revealed that the concentration of IL-8 in the
a-HSC culture medium was markedly higher than that in the culture medium of hepatoma
cells (Fig. 1C). Consistently, IL-8 production by a-HSCs and hepatoma cells was further confirmed
by Western blotting (Fig. 1D).

IL-8 was mainly enriched in the HCC stroma in vivo

To further study the role of IL-8 in tumour angiogenesis, we detected the distribution
of IL-8 in tumour tissues from patients with HCC by immunohistochemistry. As shown
in Fig. 1E (a and b), IL-8 was mainly enriched in the stroma surrounding the tumour, where
numerous a-HSCs, as detected based on the fibroblast-specific marker ?-SMA, were also
present. This finding further confirmed that a-HSCs were the main source of IL-8 in
HCC tissues. Furthermore, immunohistochemical staining for CD31 (Abcam, Cambridge,
MA, USA), a microvessel marker, revealed that neovascularization occurred largely at
the invading tumour edge, and close to the tumour stroma (Fig. 1E (c)).

IL-8 neutralizing antibody suppresses tumour angiogenesis in vitro and in vivo

To study the effect of IL-8 secreted by a-HSCs on angiogenesis, we collected the supernatants
of a-HSCs and hepatoma cells cultured in the 50 % serum-free medium. Supernatants
from untreated hepatoma cells afterculture for 24 h had only a slight effect on HUVEC
tube formation. Supernatants from hepatoma cells that had been exposed to HSC-CM for
24 h significantly promoted angiogenic tube formation (Fig. 2a, b). Furthermore, the number of branch points within the capillary-like structures
was dramatically reduced by the IL-8 neutralizing antibody (Fig. 2d, e).

Fig. 2. IL-8 neutralizing antibody repressed tumour angiogenesis in vitro and in vivo. a, b Soluble factors derived from HSC-CM-treated hepatoma cells induced angiogenic tube
formation in vitro. The tube formation assay was done using HUVECs in the presence
of serum-free conditioned medium from hepatoma cells, HSC-CM-treated hepatoma cells
alone or supplemented with an IL-8 neutralizing antibody, and the IL-8 neutralizing
antibody dramatically inhibited HUVEC tube formation. The illustrated results represent
six separate experiments. c In the CAM assay, more capillary-like structures developed in the presence of serum-free
supernatants from Hep3B cells exposed to the 50 % serum-free HSC-CM compared with
supernatants from untreated Hep3B cells. The number of these capillary-like structures
was significantly reduced by the IL-8 neutralizing antibody. d, e Representative images of the number of branch points generated by HUVECs in vitro.
The data are presented as mean ± SEM of four independent experiments. *p  0.05; **p  0.01

A similar effect of a-HSCs and IL-8 on tumour angiogenesis was also observed in the
CAM animal model (Fig. 2c). The group of eggs that was treated with HSC-CM showed more marked signs of angiogenesis
compared to the other two groups in the CAM assay. Consistently, treatment with the
IL-8 neutralizing antibody markedly inhibited angiogenesis.

IL-8 neutralizing antibody down-regulated the expression of angiogenic factors in
HSC-CM-treated hepatoma cells

We next investigated the secretion of angiogenic factors in hepatoma cells under various
conditions. VEGF-A levels were higher in the medium from HSC-CM-treated hepatoma cells
(Hep3B and Huh-7) than from normal medium-treated hepatoma cells (Hep3B: p = 0.0193; Huh-7: p = 0.0155). Notably, the concentration of VEGF-A was dramatically decreased when the
HSC-CM-treated hepatoma cells were treated with the IL-8 neutralizing antibody (Hep3B:
p = 0.0152; Huh-7: p = 0.0065) (Fig. 3). Consistent with the above findings, HSC-CM up-regulated the levels of VEGF-A mRNA
(p = 0.010) and VEGF-B mRNA (p = 0.034) in Hep3B cells compared to the normal medium (Fig. 4). Additionally, the IL-8 neutralizing antibody effectively down-regulated VEGF-A
mRNA (p = 0.034) and VEGF-B mRNA (p = 0.048) levels. However, the mRNA levels of other angiogenic factors (PDGF-A, PDGF-B,
PDGF-C and Angiopoietin-1 (Ang-1)) did not change significantly. In Huh-7 cells, the
mRNA expression of angiogenic factors (VEGF-A (p = 0.008), VEGF-B (p = 0.031), PDGF-B (p = 0.007), and PDGF-C (p = 0.047)) was up-regulated in response to stimulation with HSC-CM. The mRNA levels
of these angiogenic factors (VEGF-A (p = 0.005), VEGF-B (p = 0.0244), PDGF-B (p = 0.001), and PDGF-C (p = 0.022)) were significantly reduced by the IL-8 neutralizing antibody. Nevertheless,
PDGF-A mRNA and Ang-1 mRNA levels remained unchanged in response to various conditioned
media.

Fig. 3. VEGF-A levels in the supernatant of hepatoma cells subjected to various treatments.
The concentrations of VEGF-A (pg/mL) were assessed in the presence of serum-free conditioned
medium from hepatoma cells, HSC-CM-treated hepatoma cells alone or supplemented with
an IL-8 neutralizing antibody by ELISA. The IL-8 neutralizing antibody significantly
down-regulated VEGF-A levels in the supernatant of hepatoma cells that were treated
with 50 % HSC-CM for 24 h. The data are expressed as mean ± SEM of triplicates. *p  0.05; **p  0.01

Fig. 4. IL-8 neutralizing antibody down-regulated the mRNA expression of angiogenic factors
in HSC-CM-treated hepatoma cells. a–f mRNA expression of angiogenic factors (VEGF-A, VEGF-B, PDGF-A, PDGF-B, PDGF-C, and
Ang-1) in hepatoma cells after various treatments. In Hep3B cells, the mRNA levels
of VEGF-A and VEGF-B were up-regulated in response to stimulation with HSC-CM and
were down-regulated by IL-8 neutralizing antibody. The mRNA levels of PDGF-A, PDGF-B,
PDGF-C, and Ang-1 remained unchanged in response to various conditioned media. In
Huh-7 cells, the mRNA levels of VEGF-A, VEGF-B, PDGF-B, and PDGF-C were up-regulated
in response to stimulation with HSC-CM and were down-regulated by IL-8 neutralizing
antibody. The mRNA levels of PDGF-A and Ang-1 remained unchanged in response to various
conditioned media. The data are presented as mean ± SEM of three independent experiments.
*p  0.05; **p  0.01; ***p  0.001

IL-8 neutralizing antibody down-regulated Ser727-phosphorylated STAT3 levels in HSC-CM-treated
hepatoma cells

Evidence suggests that IL-8 modulates tumour angiogenesis by up-regulating the expression
of the HIF-1, NF-?B, and STAT3 transcription factors 12]. We used western blotting to analyse these signalling pathways in hepatoma cells
exposed to HSC-CM (Fig. 5a). Amongst these pathways, only Ser727-phosphorylated STAT3 was activated in HSC-CM-treated
hepatoma cells (Fig. 5b, c). Moreover, Ser727-phosphorylated STAT3 was effectively inhibited in HSC-CM-treated
hepatoma cells by the IL-8 neutralizing antibody, whereas HIF-1? and NF-?B p65 activation
was not affected (Fig. 5d–f). Taken together, these results demonstrated that a-HSCs partially exerted their
angiogenic function via IL-8, which up-regulated Ser727-phosphorylated STAT3 levels
in hepatoma cells.

Fig. 5. IL-8 neutralizing antibody exerted its function by suppressing Ser727-phosphorylated
STAT3 signalling. a Hepatoma cells were cultured in CM alone (lanes 1 and 4), 50 % HSC-CM (lanes 2 and 5), or 50 % HSC-CM and the IL-8 neutralizing antibody (lanes 3 and 6). Twenty-four hours after treatment, HIF-1?, Ser727-phosphorylated STAT3, and Ser536-phosphorylated
NF-?B p65 levels were determined by Western blotting. b–f The results of Western blot analyses were quantified by densitometry (relative ratio
to GAPDH). Western blotting showed differences in Ser727-phosphorylated STAT3 expression
among the hepatoma cells after various treatments. Data are presented as mean ± SEM
of three independent experiments. *p  0.05; **p  0.01. GAPDH was used as the internal control