Visfatin induces human endothelial VEGF and MMP-2/9
production via MAPK and PI3K/Akt signalling pathways:
novel insights into visfatin-induced angiogenesis
Raghu Adya, Bee K. Tan, Anu Punn, Jing Chen, and Harpal S. Randeva*
Endocrinology and Metabolism Group, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick,
Coventry CV4 7AL, UK
Received 17 July 2007; revised 11 December 2007; accepted 17 December 2007; online publish-ahead-of-print 18 December 2007
Time for primary review: 25 days
Aims Visfatin is a novel adipokine whose plasma concentrations are altered in obesity and obesity-
related disorders; these states are associated with an increased incidence of cardiovascular disease.
We therefore investigated the effect of visfatin on vascular endothelial growth factor (VEGF) and
matrix metalloproteinases (MMP-2, MMP-9) production and the potential signalling cascades.
Methods and results In human umbilical vein endothelial cells (HUVECs), visfatin significantly and dose-
dependently up-regulated gene expression and protein production of VEGF and MMPs and down-
regulated expression of tissue inhibitors of MMPs (TIMP-1 and TIMP-2). The gelatinolytic activity of
MMPs (analysed by zymography) correlated with mRNA and western blot findings. Interestingly, visfatin
significantly up-regulated VEGF receptor 2 expression. Inhibition of VEGFR2 and VEGF [by soluble FMS-
like tyrosine kinase-1 (sFlt1)] down-regulated visfatin-induced MMP induction. Visfatin induced dose-
and time-dependent proliferation and capillary-like tube formation. Importantly, visfatin was noted
to have anti-apoptotic effects. In HUVECs, visfatin dose-dependently activated PI3K/Akt (phosphatidyl-
inositol 3-kinase/Akt) and ERK1/2 (extracellular signal-regulated kinase) pathways. The functional
effects and MMP/VEGF induction were shown to be dependent on the MAPK/PI3K-Akt/VEGF signalling
pathways. Inhibition of PI3K/Akt and ERK1/2pathways led to significant decrease of visfatin-induced
MMP and VEGF production and activation, along with significant reduction in endothelial proliferation
and capillary tube formation.
Conclusion Our data provide the first evidence of visfatin-induced endothelial VEGF and MMP pro-
duction and activity. Further, we show for the first time the involvement of the MAPK and PI3K/Akt sig-
nalling pathways in mediating these actions, as well as endothelial cell proliferation. Collectively, our
findings provide novel insights into visfatin-induced endothelial angiogenesis.
The increasing incidence of atherosclerotic cardiovascular
disease (CVD) associated with obesity and the metabolic
syndrome is one of the leading causes of mortality and mor-
bidity.1Recently, there has been significant interest in bio-
active molecules secreted from adipose tissue, termed
A recently identified adipocytokine, visfatin (initially
described as pre-B-cell colony-enhancing factor, PBEF), has
been shown to be elevated in obesity, insulin resistance,
type II diabetes mellitus, and pro-inflammatory states4;
however, others have reported the contrary.5,6We have pre-
viously shown elevated circulating visfatin levels in women
with polycystic ovary syndrome, a pro-inflammatory con-
dition known to predispose to cardiovascular risk and prema-
ture atherosclerosis.7This is of interest, as it is increasingly
evident from the literature that adipocytokines play a sig-
nificant role in the induction of atherogenesis and dysregu-
Matrix metalloproteinases (MMPs) are proteolytic enzymes
that remodel the extra-cellular matrix as part of an inflam-
matory response. Increased activity of MMPs have been
implicated in atherosclerosis and CVD.10–13One of the
major MMP species in the vasculature are the gelatinases
(MMP-2 and -9), regulating vascular matrix remodelling,14
with raised peripheral concentration of these MMPs reported
in patients with acute coronary syndromes,15,16and in cer-
ebral ischaemia.12MMPs are up-regulated by a variety of
hormones, cytokines, and growth factors, including vascular
endothelial growth factor (VEGF).17VEGF, a homodimeric
glycoprotein, plays an important role in vasculogenesis,
*Corresponding author. Tel: þ44 2476 572552; fax: þ44 2476 523701.
E-mail address: email@example.com
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2007.
For permissions please email: firstname.lastname@example.org.
Cardiovascular Research (2008) 78, 356–365
by guest on June 12, 2013
atherogenesis, and vascular remodelling in response to
With the aforementioned, we sought to study the possible
interplay between visfatin and the pro-angiogenic mol-
ecules, VEGF and MMPs. In the present study, we found
and report for the first time that visfatin dose-dependently
increased MMP-2 and -9 and VEGF production. Visfatin’s pro-
up-regulation of VEGF and MMPs, were dependent on the
MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt and
VEGF/VEGF type-II receptor (VEGFR2) signalling cascades.
2. Materials and methods
2.1 Endothelial cell culture
Ethical approval for the procurement of human umbilical veins from
healthy-term pregnancies during elective caesarean sections was
obtained from the Local Research Ethics Committee, and all
patients involved gave their informed consent, in accordance with
the guidelines in The Declaration of Helsinki 2000. Human umbilical
vein endothelial cells (HUVECs) were isolated and cultured as
described previously18(for details, see Supplementary material
online). Similar experiments were performed in human micro-
vascular endothelial cells (HMECs, Supplementary material online).
2.2 RNA isolation and real-time quantitative
reverse transcription PCR
Quantitative PCR was performed on a Roche Light Cycler system
(Roche Molecular Biochemicals, Manheim, Germany). For analysis,
quantitative amounts of MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF, and
VEGFR2 were standardized against the housekeeping gene GAPDH
(see Table 1 for primers for genes of interest). The mRNA levels
were expressed as a ratio, using delta–delta method for comparing
relative expression results between treatments in real-time PCR19
(for protocol conditions, see Supplementary material online).
2.3 Cell apoptosis
2.3.1 Annexin V staining: flow cytometry protocol
Annexin V-fluorescein isothiocyanate (FITC) staining was performed
using annexin V-FITC apoptosis detection kit (Immunotech, Beckman
Coulter, Marseille, France) according to the manufacturer’s instruc-
tions (for details of protocol conditions, see Supplementary material
2.4 Cell proliferation
2.4.1 Alamar blue cytotoxicity/proliferation assay
To determine the effect of visfatin on cell proliferation, the vital
dye alamar blue was used. HUVECs were incubated at various time
points (4–72 h) with varying concentrations of visfatin (0–100 nM)
with or without VEGF at the optimized dose of 10 ng/mL (for
details, see Supplementary material online).
2.5 MTS proliferation assay
Cell proliferation was also determined with CellTiter 96 AQueous
One Solution Cell Proliferation Assay (MTS) kit (Promega, UK). Fol-
lowing visfatin treatment and addition of MTS reagent, absorbance
was recorded. The percentage of the absorbance was calculated
against untreated cells according to the manufacturer’s instructions
(for details, see Supplementary material online).
2.6 In vitro angiogenesis assay
Angiogenesis was assessed by studying the formation of capillary-
like structures by HUVEC on a Matrigel (BD Biosciences, San Jose,
CA, USA) according to the manufacturer’s protocol. Following visfa-
tin treatment for 24 h, capillary tube formation assay was per-
formed (for details, see Supplementary material online).
2.7 Migration assay
Endothelial cell migration was performed using a protocol obtained
from BD BioCoat Angiogenesis System and using a modified Boyden
chamber. Briefly serum starved, Calcein-AM-labelled HUVECs were
treated with or without visfatin. The migration induced was quanti-
fied by a fluorescence plate reader (for details, see Supplementary
2.8 Gelatin zymography
Following optimization experiments (data not shown), the gelatino-
lytic activity of secreted MMP-2 and MMP-9 in the culture super-
natants was measured by gelatin zymography, following 24 h
visfatin treatment (for details, see Supplementary material online).
2.9 Western blot analysis
For MMPs, tissue inhibitors of MMP (TIMPs), and VEGF protein ana-
lyses, and their regulation by visfatin, HUVECs were serum starved
overnight and then pre-treated with or without an MEK inhibitor,
U0126 (Calbiochem), a PI3K inhibitor, LY 294002 (Calbiochem), a
VEGF antagonist, soluble FMS-like tyrosine kinase-1 (sFlt1) (Calbio-
chem), and a VEGF receptor blocker, SU1498 (Calbiochem), followed
by treatment with human recombinant visfatin [(0–100 nM); Axxora
Ltd, Nottingham, UK (ALX-201-336)] (for detailed protocol and anti-
body concentrations, see Supplementary material online).
2.10 Immunoprecipitation and PI3K activity assay
Serum-starved HUVECs were treated with or without visfatin (0–
100 nM) or insulin (1 mM) (positive control) for various time points
(0, 2, 5, 15, 20, 30 min). Cells were washed twice with ice-cold Tris-
buffered saline, followed by Buffer A (for details, see Supplemen-
tary material online) and lysis buffer (Buffer A plus 1% NP-40 and
1 mM PMSF). The cells were scrapped and centrifuged for 10 min.
Primers used for RT-PCR analysis
Gene/product size (bp)Sense primerAntisense primer
Visfatin induces VEGF and MMP in human endothelial cells357
by guest on June 12, 2013
The supernatants were incubated with anti-PI 3 kinase antibody
(Upstate Biotechnology, catalogue 06–195). Sixty millilitres of 50%
slurry of Protein A-agarose beads in PBS were added to each of
the tubes and incubated by constant mixing. Immunoprecipitated
enzyme was collected by centrifugation and washed three times
by each these buffers (A, B, and C) in sequence.
PI3K activity in these prepared cell lysates was evaluated using
PI3 kinase ELISA kit (K1000, Echelon Biosciences, UT, USA) according
to the manufacturer’s protocol (for details, refer to Supplementary
2.11 Statistical analysis
All of the data in the present study are expressed as mean+SEM.
Differences between two groups were assessed using the Mann–
Whitney U test. Comparisons among groups were made by ANOVA
(non-parametric). When significance (P , 0.05) was detected, a
post hoc Dunns multiple-comparison test was performed. All stat-
3.1 Visfatin enhances MMP-2 and MMP-9 and
decreases TIMP-1 and TIMP-2 mRNA levels in
The angiogenic potential of HUVECs is greatly enhanced by
the degradation of the extra-cellular matrix, where gelati-
nases MMP-2 and -9, and VEGF play a vital role. Enhanced
MMP-2 and MMP-9 and a decrease in TIMP-2 and TIMP-1
mRNA were observed as early as 1 h but peaked at 4 h and
then declined thereafter (data not shown). At 4 h, visfatin
induced a dose-dependent increase in mRNA expressions of
MMP-2 and MMP-9, which was significant at the maximum
dose (Figure 1A and B: MMP-2 and -9, P , 0.01).
At 4 h, visfatin induced a significant dose-dependent
increase, maximal response at 100 nM, in mRNA expressions
of MMP-2 and MMP-9 (Figure 1A and B: MMP-2 and 9, P ,
0.01). However, mRNA levels of TIMPs, TIMP-1 and TIMP-2,
regulators of MMP-9 and MMP-2 activity, respectively, con-
currently decreased dose-dependently following visfatin
treatment, with maximal effect at 100 nM (Figure 1C:
TIMP-2, P , 0.05; Figure 1D: TIMP-1, P , 0.001). VEGF,
used as a positive control, revealed significant up-regulation
of MMPs and down-regulation of TIMPs (Figure 1A–D: P ,
3.2 Effect of visfatin on MMP-2 and MMP-9 protein
3.2.1 Western blotting
Further, we measured protein expression of MMP-2 and
MMP-9 following 24 h visfatin treatments (0–100 nM), by
western blot analyses, and observed similar results as our
mRNA findings (Figure 1E: MMP-2, six-fold increase, P ,
0.001; Figure 1F: MMP-9, 5.2-fold increase, P , 0.001).
treated with or without visfatin (0–100 nM) or VEGF (10 ng/mL) for 4 h. mRNA levels of (A) MMP-2, (B) MMP-9, (C) TIMP2, and (D) TIMP1 were analysed by real-
time PCR and normalized with the housekeeping gene GAPDH. HUVECs were treated with or without visfatin (0–100 nM) or VEGF (10 ng/mL) for 24 h. (E) and (F)
Representative western blot and densitometric analyses of MMP-2, MMP-9, and their respective b-actin protein. Increasing MMP-2 and MMP-9 protein levels in the
presence of visfatin (0–100 nM) are normalized to b-actin and expressed as a fold increase over basal. (G) and (H) Representative zymograms and densitometric
analysis of MMP-2 and MMP-9 protein activities in conditioned media for 24 h, respectively. Data shown are means+SEM of triplicates. The values represented
are relative to basal. ***P , 0.001, **P , 0.01, *P , 0.05 vs. control, n ¼ 6 per group.
Visfatin enhances MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA, protein levels, and gelatinolytic activity in endothelial cells. Serum-starved HUVECs were
R. Adya et al. 358
by guest on June 12, 2013
Likewise, there was a significant decrease in TIMP-1 and
TIMP-2 protein expressions (TIMP-1: P , 0.001; TIMP-2:
P , 0.01; data not shown).
Zymographic assessment of gelatinolytic activity of MMPs
treatment, dose and time dependent; MMP-2 and MMP-9
activities in the conditioned
up-regulated, with maximal activity at 24 h and at 100 nM
of visfatin (Figure 1G: MMP-2, 4.5-fold, P , 0.001 and
Figure 1H: MMP-9, seven-fold, P , 0.001, when compared
media was significantly
3.3 Visfatin induces VEGF and VEGFR2 levels in
HUVECs: role of VEGF/VEGFR2 in visfatin-induced
MMP-2 and -9 production
Serum-starved HUVECs were treated with visfatin (0–
100 nM) for 4 and 24 h. Visfatin induced a dose-dependent
increase in VEGF mRNA (Figure 2A: P , 0.001). In a similar
set of experiments, visfatin induced a dose-dependent (0–
100 nM) increase of VEGF protein levels in cell lysates
P , 0.001)and VEGFprotein levels in
conditioned media (Figure 2C: P , 0.001). Given that
VEGF, acting through its receptor VEGFR2, is a known reg-
ulatorof MMPs, wesought
visfatin-induced MMP production was VEGF dependent.
VEGF significantly up-regulated VEGFR2 mRNA expression
in HUVECs at 4 h (Figure 2D: P , 0.001), and interestingly
the same was true for visfatin (Figure 2D: P , 0.001). In
the light of visfatin’s stimulatory effects on VEGF, we
employed sFlt1 and found a significant reduction in
visfatin-induced VEGFR2 mRNA levels by 50% (Figure 2D:
P , 0.01),suggestingboth
VEGF-independent effects of visfatin on VEGFR2. Interest-
ingly, prior to visfatin treatment of HUVECs, pre-incubation
with sFlt1 resulted in significant reduction in both MMP-2
and -9 mRNA levels (Figure 2E and F: P , 0.05) and,
more importantly, the gelatinolytic activity (Figure 2G
and H: P , 0.01). Furthermore, HUVECs pre-incubated
with VEGFR2 blocker, SU1498, for 1 h, followed by visfatin
treatment, resulting in a significant down-regulation of
mRNA levels (Figure 2E and F: MMP2, P , 0.01; MMP9,
P , 0.05), and gelatinolytic activity (Figure 2G and H:
P , 0.001). SU1498 (10 mM) was more efficacious at lower-
ing MMP-9 than sFlt1 (P , 0.05), whereas in relation to
MMP-2, this just failed to reach significance (P ¼ 0.07).
starved HUVECs were treated with visfatin (0–100 nM) for 4 h. (A) VEGF mRNA levels were measured. Following 24 h treatments with visfatin (0–100 nM), (B)
VEGF protein levels in cell lysates and (C) VEGF protein levels in conditioned media were measured. Serum-starved HUVECs were pre-incubated with or
without sFlt1 and SU1498 (10 mM each). On treatment with visfatin (100 nM) or VEGF (10 ng/mL) for 4 h, (D) VEGFR2 (E) MMP-2, and (F) MMP-9 mRNA levels
were measured. Similarly, HUVECs were pre-incubated with or without the aforementioned inhibitors, followed by visfatin (100 nM) treatment for 24 h, and
(G) MMP-2 and (H) MMP-9 gelatinolytic activities were measured in the conditioned media. Results are means+SEM of six independent experiments. *P ,
0.05, **P , 0.001, ***P , 0.001 vs. basal; #P , 0.05, ##P , 0.01, and ###P , 0.001 vs. visfatin treatment; þP , 0.05 vs. sFlt1 treatment, n ¼ 6 experiments
Effect of sFlt1 and SU1498 on visfatin-induced VEGFR2, MMP-2, MMP-9, VEGF mRNA expressions, and MMP-2 and MMP-9 gelatinolytic activities. Serum-
Visfatin induces VEGF and MMP in human endothelial cells 359
by guest on June 12, 2013
These findings highlight the involvement of VEGF and its
receptor in visfatin-induced MMP up-regulation.
3.4 Visfatin-induced endothelial cell proliferation
HUVEC proliferation was studied in a time-dependent
manner (4–48 h), maximal response being noted at 24 h
(data not shown). Treatment with visfatin (0–100 nM) or
10 ng/mL VEGF (positive control), at 24 h, led to a dose-
dependent proliferative effect in visfatin/VEGF-treated
samples [Figure 3A: 1.85-fold for VEGF (P , 0.001), and
1.65-fold at the maximal dose of visfatin (100 nM) compared
with control; P , 0.01; n ¼ 6 experiments]. These prolifera-
tive effects were confirmed by colorimetric Alamar blue
cytotoxicity/proliferation assay (Supplementary material
online, Figure S1C; 1.75-fold for VEGF (P , 0.001) and
1.55-fold at the maximal dose of visfatin, 100 nM, compared
with control; P , 0.01; n ¼ 6 experiments). Given that
endothelial migration, similar to proliferation, is a critical
step in angiogenesis, we assessed the migratory potential
3.5 Visfatin-induced endothelial cell migration
Serum-starved, Calcein-labelled HUVECs were subjected to
migration assay, which were treated with dose-dependent
visfatin (0–100 nM) and VEGF (10 ng/mL). The time points
were 4, 8 12, and 24 h. Visfatin increased migration in a dose-
dependent and time-dependent manner, with a maximal
effect at 100 nM and 24 h (Figure 3B: **P , 0.01, ***P ,
0.001; visfatin treated vs. basal); VEGF, used as a positive
control, also significantly increased HUVEC migration. Our
data confirm the effect of visfatin on migration in HUVECs.
3.6 Anti-apoptotic effect of visfatin on endothelial
To evaluate whether the visfatin-induced proliferative effect
was due to its anti-apoptotic activity, early- and late-stage
apoptotic cells were analysed by Annexin V staining. Using
the maximal proliferative dose of visfatin (100 nM), endo-
thelial cells treated with both visfatin and H2O2were sub-
jected to flow cytometric analysis. Both, early- and
late-stage apoptotic cells were sparse after combined treat-
ment of visfatin and H2O2(4%), compared with H2O2-treated
(0–100 nM)/VEGF (10 ng/mL) (positive control) for 24 h, and cell growth was assessed by MTS assay. Results were expressed as percentage of cells in relation to
basal (untreated) and represent the mean of triplicates. ***P , 0.001, **P , 0.01, *P , 0.05 vs. control, n ¼ 6. (B) Endothelial cell migration assay. Serum-starved
Calcein-AM-labelled HUVECs were treated with visfatin (0–100 nM) for 4, 8, 12, and 24 h. VEGF (10 ng/mL) was used as a positive control. Migrated cells were
quantified using a fluorescence plate reader. The migrated cells were expressed as the ratio of the fluorescence compared with the control. Results are means+
SE of six independent experiments. **P , 0.01, ***P , 0.001, vs. control. (C) Apoptosis assay. Annexin V staining for the assessment of apoptosis. (C1) and (C2)
Flow cytometric analysis of HUVECs treated with H2O2(200 mM) and visfatin (100 nM) þ H2O2(200 mM) for 8 h, respectively, and analysed by a four-colour flow
cytometer. (C3) Percentage of treated cells that are live, early, and late apoptotic. (D)–(E) Visfatin-induced activation of ERK1/2. Serum-starved HUVECs were
treated with (D) time (0–30 min) and (E) dose (0–100 nM) dependent visfatin, and phosphorylation of ERK1/2was measured by western blot analysis. The results
are represented as a ratio of phosphorylated to total protein and expressed as fold changes over basal. Results are means+SEM of six independent experiments.
***P , 0.001, **P , 0.01, *P , 0.05 vs. basal, n ¼ 6 experiments per group.
Proliferative, migratory, and anti-apoptotic actions of visfatin. (A) Proliferation assay—MTS assay. Serum-starved HUVECs were treated with visfatin
R. Adya et al. 360
by guest on June 12, 2013
cells (65%; Figure 3C). These novel anti-apoptotic effects
were further confirmed using DNA fragmentation assay
(data not shown).
3.6 Visfatin-induced activation of ERK1/2
MAPK signalling pathways are involved in HUVEC prolifer-
ation. Interestingly, visfatin significantly phosphorylated
both p42 and p44 (ERK1/2—extracellular signal-regulated
kinase), maximally at 20 min, decreasing thereafter (Figure
3D: P , 0.001). More importantly, visfatin dose-dependently
(0–100 nM) phosphorylated ERK1/2(Figure 3E: 23-fold, com-
pared with controls; P , 0.001). Visfatin-activated ERK1/2
was completely inhibited by U0126 (Supplementary material
online, Figure S1A). Interestingly, visfatin had no effect on
JNK activity (data not shown).
3.8 Visfatin-induced activation of PI3K/Akt
The PI3K/Akt pathway is known to regulate MMPs and VEGF,
and play a role in angiogenesis. In order to address whether
visfatin signals via this pathway in HUVECs, we treated
HUVECs with visfatin and insulin (positive control for
PI3K induction) compared with basal (untreated). Visfatin
(0–100 nM) treated cell lysates showed a dose-dependent
significant up-regulation in PI3K activity following 5 min of
incubation [Figure 4A: visfatin (100 nM) showed a 2.1-fold
increase compared with untreated cell lysates; P , 0.01].
Treatment with insulin 1 mM (positive control) also showed
a significant rise in PI3K activity (Figure 4A: 2.8-fold
increase compared with untreated cell lysates; P , 0.001).
Moreover, to investigate the downstream PI3K signalling
pathway, we probed visfatin-treated cell lysates for Akt
phosphorylation. On western blot analysis, we observed
that visfatin time- and dose-dependently increased phos-
phorylation of Akt, with maximal response at 15 min
(Figure 4B: P , 0.001) of incubation with 100 nM visfatin
(Figure 4C: 11.1-fold increase compared with basal; P ,
0.001). Both ERK1/2phosphorylation and visfatin-activated
Akt were significantly inhibited by the MEK inhibitor
(U0126) and by the PI3K inhibitor, LY294002 (Supplementary
material online, Figure S1A and B).
3.9 Involvement of ERK1/2and PI3K/Akt signalling
pathways in visfatin-induced MMP up-regulation and
Given the involvement of MAPK and PI3K signalling in angio-
genesis and visfatin’s pro-inflammatory effects, we sought
Cells were lysed, and PI3K activity was measured according to the manufacturer’s protocol (Echelon Inc.) (B) Time (0–30 min) and (C) dose (0–100 nM) dependent
visfatin treatment with changes in Akt phosphorylation. This is represented as a ratio of phosphorylated to total protein in relation to basal (untreated) and
expressed as fold increase over basal. Results are means+SEM of six independent experiments. ***P , 0.001, **P , 0.01, *P , 0.05 vs. control, n ¼ 6 per group.
Visfatin-induced activation of PI3K/Akt signalling pathways. Serum-starved HUVECs were treated with (A) visfatin (0–100 nM)/insulin (1 mM) for 5 min.
Visfatin induces VEGF and MMP in human endothelial cells361
by guest on June 12, 2013
to elucidate the involvement of MAPK and PI3K signalling in
visfatin-induced MMP up-regulation and VEGF production.
HUVECs pre-incubated with an MEK inhibitor (U0126) and
a PI3K inhibitor (LY294002), prior to visfatin treatment
(100 nM),showed significant
MMP-2 and MMP-9 mRNA (Figure 5A and B: P , 0.001 vs.
visfatin-only treated). Moreover, the MMP-2 and MMP-9 gela-
tinolytic activities were significantly attenuated (Figure 5C
and D: P , 0.001 vs. visfatin-only treated). Similar findings
were noted for VEGF mRNA expression, where both MEK
and PI3K inhibitors significantly attenuated visfatin-induced
VEGF induction (Figure 5E: P , 0.001). VEGF protein pro-
duction in cell lysates and conditioned media was signifi-
cantly reduced by both MEK and PI3K inhibitors (Figure 5F
and G: P , 0.001). Similar down-regulatory effect was
observed with VEGFR2 mRNA expression (Figure 5H: P ,
3.10 Involvement of VEGF, PI3K, and MAPK
signalling pathways in visfatin-induced gelatinolytic
activity, endothelial cell proliferation, and capillary
Further experiments were conducted to elucidate the invol-
vement of VEGF, MAPK, and PI3K signalling pathways on
visfatin-induced gelatinolyticactivity, endothelial
proliferation, and capillary tube formation by using specific
inhibitors. Visfatin-induced endothelial cell proliferation
was significantly inhibited when HUVECs were pre-incubated
with inhibitors: 10 mM of MAPK, PI3K, VEGF, and a global
MMPinhibitor-GM6001 (Figure 6A: P , 0.001 vs. visfatin-only
visfatin-induced capillary tube formation was significantly
attenuated (Figure 6B1; P , 0.001 vs. visfatin treated).
When the condition media of the aforementioned prolifer-
ation assay was subjected to gelatin zymography, similar
observations were noted (Figure 6C and D: P , 0.001 vs.
visfatin-only treated), implicating MMPs and VEGF in
visfatin-induced HUVEC proliferation.
Dysregulated angiogenesis is involved in conditions such as
ischaemic heart disease, diabetes, or chronic inflammation,
including atherosclerosis, and involves the VEGF–MMP
system.20Despite significant advances in therapeutic angio-
genesis, the treatment of both macro- and micro-vascular
ischaemic diseases remains a major concern. Extending dia-
metrically opposed therapeutic advancements is necessary
to deal with complex diseases such as diabetes and athero-
Although inhibition of endogenous growth
without U0126, LY294002, and sFLT1 (10 mM each). On treatment with visfatin (100 nM) for 4 h, (A) MMP-2, (B) MMP-9, (E) VEGF, and (H) VEGFR2 mRNA levels
were measured respectively. In the similar set of experiments on treatment with visfatin (100 nM) for 24 h, (C) MMP-2 and (D) MMP-9 gelatinolytic activities were
measured respectively. (F) and (G) Representative western blot analysis of VEGF in protein lysates and conditioned media, respectively. Results are means+SEM
of six independent experiments. ***P , 0.001, **P , 0.01, *P , 0.05 vs. basal, #P , 0.05 and ##P , 0.001 vs. visfatin treatment, n ¼ 6 per group.
Effects of MEK (U0126) and PI3K (LY294002) inhibition on visfatin-induced MMP and VEGF induction. Serum-starved HUVECs were pre-incubated with or
R. Adya et al. 362
by guest on June 12, 2013
factors seems beneficial in certain ophthalmic conditions,
atherosclerotic plaques, and so on, the reverse is required
in inducing angiogenesis for improving macro-vascular insuf-
ficiency, for example in peripheral limb ischaemia.22There-
fore, the study of VEGF–MMP system assumes crucial
In the present study, we describe visfatin-induced pro-
duction of VEGF and MMP-2 and -9 in both human micro-
and macro-vascular endothelial cells. More importantly, we
report that visfatin, whose plasma concentrations are
alteredin obesityand obesity-related
induces migration, tube formation, and angiogenesis poss-
ibly through activation of VEGF–MMP pathways. In addition,
we demonstrate for the first time the role of VEGF/VEGFR2
signalling, transient phosphorylation of ERK1/2, and PI3K/Akt
pathways, in visfatin-induced VEGF/MMP up-regulation and
endothelial angiogenesis. During the preparation of our
manuscript, a terse report by Kim et al.25has demonstrated
visfatin’s in vivo angiogenic potential in HUVECs via the
ERK1/2pathway. However, maintenance of capillary tube-
like structures, a prerequisite for angiogenesis, requires
In our study, we extensively elucidate that ERK1/2plays a
role in visfatin-induced angiogenesis. In relation to its
pro-angiogenic actions where visfatin activates PI3K/Akt sig-
nalling pathways, known to play a crucial role in angiogen-
esis, we also illustrate this function in tandem with
MMP-2/9 and VEGF production. When HUVECs were pre-
incubated with inhibitors of MEK and PI3K, visfatin-induced
MMP expression and gelatinolytic activity were significantly
reduced, supporting the involvement of these pathways in
MMP induction. Likewise, these inhibitors decreased VEGF
production and secretion. In addition, these inhibitors com-
pletely blocked visfatin-induced HUVEC proliferation and
capillary-like tube formation. However, it is important to
note that the observed reduction of visfatin-induced MMPs
and VEGF by some of the kinase inhibitors resulted in
below basal levels, possibly suggesting that the observed
effects are not specific for visfatin. In relation to this, it is
interesting to note that activation of Akt alone is sufficient
for angiogenesis.27Collectively, our observations highlight
the importance of MAPK, and PI3K/Akt pathways in
visfatin-induced VEGF production, MMP up-regulation, and
formation. (A) Visfatin-induced endothelial cell proliferation was significantly inhibited when HUVECs were pre-incubated with inhibitors: 10 mM of MAPK, PI3K,
VEGF, and a global MMP inhibitor, GM6001. (B1 and B2) Again, using the same inhibitors, capillary tube formation induced by visfatin was significantly attenuated
(#P , 0.001 vs. visfatin treated). (C) and (D) When the condition media of the aforementioned proliferation assay was subjected to gelatine zymography, similar
observations were noted for visfatin-induced gelatinolytic activity of both MMP-2 and MMP-9 (#P , 0.001 vs. visfatin-only treated). It is clear that VEGF directly
influences visfatin-induced MMP effects. Results are means+SEM of six independent experiments. ***P , 0.001, **P , 0.01, *P , 0.05 vs. basal, #P , 0.001 vs.
visfatin-only treated, n ¼ 6 per group.
Involvement of VEGF, PI3K, and MAPK signalling pathways in visfatin-induced gelatinolytic activity, endothelial cell proliferation, and capillary tube
Visfatin induces VEGF and MMP in human endothelial cells 363
by guest on June 12, 2013
Bearing our observations in mind, it is likely that the
angiogenic property of visfatin may play a pathological
role in the recruitment of blood inflammatory cells and
the infiltration of macrophages and T-lymphocytes. Interest-
ingly, Dahl et al.28have recently suggested that visfatin may
play a role in plaque destabilization, given that macro-
phages are laden with visfatin. Moreover, they have shown
that visfatin induced an increase in MMP-9 activity in
THP-1 monocytes. Our findings are therefore very timely
and have revealed for the first time that visfatin directly
induces VEGF production and secretion, as well as MMP-2
and MMP-9 production and gelatinolytic activity in HUVECs
and HMECs. Both VEGF and MMP-2/-9 play critical roles in
the initiation and progression of vascular pathology.29,30
A feature of early vascular remodelling is enhanced pro-
duction of MMPs, in particular, MMP-2 and MMP-9, which
are regulated by TIMP-2 and TIMP-1, respectively.31–34Fur-
thermore, activated MMPs contribute to a decrease in endo-
thelial barrier function.35Therefore, our observations of
visfatin up-regulation of MMP-2/-9, with concurrent and
dose-dependent decrease in TIMP-2 and TIMP-1, is of import-
ance, as this would tip the MMP/TIMP balance in favour of
matrix degradation. Furthermore, when the MMP inhibitor,
GM6001, was pre-incubated with visfatin, there was a sig-
nificant decrease in HUVEC proliferation and capillary tube
formation, suggestinga potential
between visfatin-induced MMP activity and angiogenesis. In
addition to MMPs, we have shown for the first time the
effect of visfatin on the VEGF/VEGFR2 system. Collectively,
in tandem with Kim et al.25and Dahl et al.,28our findings
would tentatively support a role of visfatin in vascular
MMPs are regulated by a number of cytokines and growth
factors.36VEGF is a specific endothelial mitogen,37,38known
to initiate and accelerate atherosclerosis,39that acts via the
VEGF type-II receptor (VEGFR2).40We provide novel evi-
dence that visfatin not only increases VEGF production,
but significantly up-regulates VEGFR2. It is known that
VEGF up-regulates VEGFR2 by a positive feedback mechan-
ism28; however, when pre-incubated with sFlt1, visfatin
induction of VEGFR2 was attenuated by 50%, suggesting
both VEGF-dependent and -independent mechanisms. In
addition, visfatin-induced MMP expression and gelatinolytic
activity were significantly reduced when pre-incubated
with sFlt1 and SU1498; however, the VEGFR2 blocker com-
pared with sFlt1 was significantly more potent at decreasing
MMP-9 gelatinolytic activity. These observations were
further extended to visfatin-induced endothelial prolifer-
ation and capillary tube formation. Our findings illustrate
visfatin-induced MMP up-regulation and angiogenesis. It
should be emphasized that the inhibition of gelatinase
activity by either the SU compound or sFlt1 is not selective,
as sFlt1 scavenges VEGF and thereby affects activation of
both R1 and R2; the SU compound is known to have
limited selectivity for growth factor receptor tyrosine
kinases and also blocks MAPK in a manner that is dependent
on Raf-B or MEK activation, but is not necessarily growth
At least two functionally distinct endothelial cell types,
macro-vascular and micro-vascular, exist within most vascu-
larized tissues. However, there are distinct morphogenetic,
antigenic, and functional characteristics between micro-
vascular and macro-vascular (HUVEC) endothelial cells,42,43
with differences in the secretion of vasoactive substances
and the functional responses to external stimuli between
these different cell types,44which are likely to reflect
differences in the activation of transcription factors that
mediate signal transduction mechanisms in the two cell
types. These differences notwithstanding, our functional
effects of visfatin in both endothelial cells were similar.
In conclusion, we demonstrate for the first time the
effects of visfatin on MMP up-regulation and VEGF pro-
duction in human micro- and macro-vascular endothelial
cells. Our findings suggest a functional interplay between
visfatin and these pro-angiogenic molecules, via multiple
VEGFR2. Finally, our data add to the diverse effects of visfa-
tin, but more importantly reveal novel insights into the
potential role(s) of visfatin in human CVD.
Supplementary material is available at Cardiovascular
H.S.R. would like to acknowledge the continual support of
S. Waheguru, University of Warwick.
Conflict of interest: none declared.
The General Charities of the City of Coventry funded this
1. Braunwald E. Shattuck lecture. Cardiovascular medicine at the turn of
the millennium: triumphs, concerns, and opportunities. N Engl J Med
2. Mather K, Anderson TJ, Verma S. Insulin action in the vasculature: physi-
ology and pathophysiology. J Vasc Res 2001;38:415–422.
3. Shuldiner AR, Yang R, Gong DW. Resistin, obesity and insulin resistance:
the emerging role of the adipocyte as an endocrine organ. N Engl J
4. Chen MP, Chung FM, Chang DM, Tsai JC, Huang HF, Shin SJ et al. Elevated
plasma level of visfatin/pre-B cell colony-enhancing factor in patients
with type 2 diabetes mellitus. J Clin Endocrinol Metab 2006;91:295–299.
5. Haider DG, Schindler K, Schaller G, Prager G, Wolzt M, Ludvik B.
Increased plasma visfatin concentrations in morbidly obese subjects are
reduced after gastric banding. J Clin Endocrinol Metab 2006;91:
6. Chan TF, Chen YL, Lee CH, Chou FH, Wu LC, Jong SB et al. Decreased
plasma visfatin concentrations in women with gestational diabetes melli-
tus. J Soc Gynecol Investig 2006;13:364–367.
7. Tan BK, Chen J, Digby JE, Keay SD, Kennedy CR, Randeva HS. Increased
visfatin messenger ribonucleic acid and protein levels in adipose tissue
and adipocytes in women with polycystic ovary syndrome: parallel
increase in plasma visfatin. J Clin Endocrinol Metab 2006;91:5022–5028.
8. Lau DC, Schillabeer G, Li ZH, Wong KL, Varzaneh FE, Tough SC. Paracrine
interactions in adipose tissue development and growth. Int J Obes Relat
Metab Disord 1996;20:S16–S25.
9. Mohamed-Ali V, Pinkney JH, Coppack SW. Adipose tissue as an endocrine
and paracrine organ. Int J Obes Relat Metab Disord 1998;22:1145–1158.
10. Komorowski J, Pasieka Z, Jankiewicz-Wika J, Stepien H. Matrix metallo-
proteinases, tissue inhibitors of matrix metalloproteinases and angio-
genic cytokines in peripheral blood of patients with thyroid cancer.
R. Adya et al. 364
by guest on June 12, 2013
11. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix Download full-text
metalloproteinases and matrix degrading activity in vulnerable regions
of human atherosclerotic plaques. J Clin Invest 1994;94:2493–2503.
12. Mun-Bryce S, Rosenberg GA. Matrix metalloproteinases in cerebrovascu-
lar disease. J Cerebral Blood Flow Metab 1998;18:1163–1172.
13. Wang W, Schulze CJ, Suarez-Pinzon WL, Dyck JR, Sawicki G, Schulz R.
Intracellular action of matrix metalloproteinase-2 accounts for acute
myocardial ischemia and reperfusion injury. Circulation 2002;106:
14. Creemers EE, Cleutjens JP, Smits JF, Daemen MJ. Matrix metalloprotei-
nase inhibition after myocardial infarction: a new approach to prevent
heart failure? Circ Res 2001;89:201–210.
15. Shah PK. Plaque disruption and thrombosis: potential role of inflam-
mation and infection. Cardiol Rev 2000;8:31–39.
16. Kai H, Seki Y, Kuwahara F, Ueno T, Sugi K, Imaizumi T. Peripheral blood
levels of matrix metalloproteases-2 and -9 are elevated in patients
with acute coronary syndromes. J Am Coll Cardiol 1998;32:368–372.
17. Wang H, Keiser JA. Vascular endothelial growth factor upregulates the
expression of matrix metalloproteinases in vascular smooth muscle
cells: role of flt-1. Circ Res 1998;83:832–840.
18. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endo-
thelial cells derived from umbilical veins. Identification by morphologic
and immunologic criteria. J Clin Invest 1973;52:2745–2756.
19. Pfaffl MW. A new mathematical model for relative quantification in real-
time RT-PCR. Nucleic Acids Res 2001;29:e45.
20. Samal B, Sun Y, Stearns G, Xie C, Suggs S, McNiece I. Cloning and charac-
colony-enhancing factor. Mol Cell Biol 1994;14:1431–1437.
21. Ye SQ, Simon BA, Maloney JP, Zambelli-Weiner A, Gao L, Grant A et al.
Pre-B-cell colony-enhancing factor as a potential novel biomarker in
acute lung injury. Am J Respir Crit Care Med 2005;171:361–370.
22. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature
23. Duh E, Aiello LP. Vascular endothelial growth factor and diabetes: the
agonist versus antagonist paradox. Diabetes 1999;48:1899–1906.
24. Aiello LP. Keeping in touch with angiogenesis. Nat Med 2000;6:379–381.
25. Kim SR, Bae SK, Choi KS, Park SY, Jun HO, Lee JYet al. Visfatin promotes
angiogenesis by activation of extracellular signal-regulated kinase 1/2.
Biochem Biophys Res Commun 2007;357:150–156.
26. Pollman MJ, Naumovski L, Gibbons GH. Endothelial cell apoptosis in capil-
lary network remodeling. J Cell Physiol 1999;178:359–370.
27. Ackah E, Yu J, Zoellner S, Iwakiri Y, Skurk C, Shibata R et al. Akt1/protein
kinase Balpha is critical for ischemic and VEGF-mediated angiogenesis.
J Clin Invest 2005;115:2059–2064.
28. Dahl TB, Yndestad A, Skjelland M, Oie E, Dahl A, Michelsen A et al.
Increased expression of visfatin in macrophages of human unstable
carotid and coronary atherosclerosis: possible role in inflammation and
plaque destabilization. Circulation 2007;115:972–980.
29. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth
factor. Endocr Rev 1997;18:4–25.
a novel humanpre-B-cell
30. Moses MA. The regulation of neovascularization of matrix metalloprotei-
nases and their inhibitors. Stem Cells 1997;15:180–189.
31. Olson MW, Gervasi DC, Mobashery S, Fridman R. Kinetic analysis of the
binding of human matrix metalloproteinase-2 and -9 to tissue inhibitor
of metalloproteinase TIMP-1 and TIMP-2. J Biol Chem 1997;272:
32. Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N
Engl J Med 1994;330:1431–1438.
33. Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle
cell migration and matrix metalloproteinase expression after arterial
injury in the rat. Circ Res 1994;75:539–545.
34. Godin D, Ivan E, Johnson C, Magid R, Galis ZS. Remodeling of carotid
artery is associated with increased expression of matrix metalloprotei-
nases in mouse blood flow cessation model. Circulation 2000;102:
35. Rosenberg GA, Estrada EY, Dencoff JE. Matrix metalloproteinases and
TIMPs are associated with blood-brain barrier opening after reperfusion
in rat brain. Stroke 1998;29:2189–2195.
36. Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Unemori EN,
Lark MW et al. Cytokine-stimulated human vascular smooth muscle
cells synthesize a complement of enzymes required for extracellular
matrix digestion. Circ Res 1994;75:181–189.
37. Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization
of a vascular endothelial cell mitogen produced by pituitary-derived fol-
liculo stellate cells. Proc Natl Acad Sci USA 1989;86:7311–7315.
38. Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-
binding growth factor specific for vascular endothelial cells. Biochem
Biophys Res Commun 1989;161:851–858.
39. Celletti FL, Waugh JM, Amabile PG, Brendolan A, Hilfiker PR, Dake MD.
Vascular endothelial growth factor enhances atherosclerotic plaque pro-
gression. Nat Med 2001;7:425–429.
40. Terman BI, Dougher-Vermazen M, Carrion ME, Dimitrov D, Armellino DC,
Gospodarowicz D et al. Identification of the KDR tyrosine kinase as a
receptor for vascular endothelial cell growth factor. Biochem Biophys
Res Commun 1992;187:1579–1586.
41. Boguslawski G, McGlynn PW, Harvey KA, Kovala AT. SU1498, an inhibitor
of vascular endothelial growth factor receptor 2, causes accumulation
of phosphorylated ERK kinases and inhibits their activity in vivo and in
vitro. J Biol Chem 2004;279:5716–5724.
42. Dye J, Lawrence L, Linge C, Leach L, Firth J, Clark P. Distinct patterns of
microvascular endothelial cell morphology are determined by extracellu-
lar matrix composition. Endothelium 2004;1:151–167.
43. Thorin E, Sheeve SM. Heterogeneity of vascular endothelial cells in
normal and disease states. Pharmacol Ther 1998;78:155–166.
44. Lang I, Pabst MA, Hiden U, Blaschitz A, Dohr G, Hahn Tet al. Heterogen-
eity of microvascular endothelial cells isolated from human term pla-
centa and macrovascular umbilical vein endothelial cells. Eur J Cell
Visfatin induces VEGF and MMP in human endothelial cells365
by guest on June 12, 2013