Cell culture. HUVECs were purchased from PromoCell (Heidelberg, Germany) and grown according to the manufacturer’s instructions.
RNA Isolation and Real-Time Quantitative Reverse Transcription Polymerase Chain
Reaction. Total cellular RNA was extracted using the RNeasy Mini Kit (Qiagen Ltd, UK) according to the manufacturer’s protocol. The purity of the extracted RNA was measured by a
NanoDrop spectrophotometer. A set concentration of RNA was reverse transcribed into cDNA, by using 5IU/ RevertAid H Minus M-MuLV Reverse Transcriptase (Fermentas, York,
UK).
The concentrations of target mRNAs were measured by real-time PCR performed on a
Roche Light Cycler system (Roche Molecular Biochemicals, Mannheim, Germany). RTQ-
PCRs were carried out using 2.5 μl cDNA in 5.5 μl PCR SYBR Green-1 Light Cycler
‘Master Mix’ (Biogene, Kimbolton, Cambridgeshire, UK) and 1 μl each of sense and antisense primers.
Protocol conditions consisted of denaturation at 95ºC for 15 sec, followed by 40 cycles of
94ºC for 1 sec, 60ºC for 5 sec, and 72ºC for 12 sec, followed by melting curve analysis. For analysis, quantitative amounts of MMP-2 and MMP-9 were standardized against the housekeeping gene GAPDH. (Primers for genes of interest; Table-1) As a negative control for all reactions, preparations lacking RNA or reverse transcriptase were used in place of sample cDNA.
RNAs were assayed from three independent replicates.
The PCR products from all samples were purified from the 1% agarose gel using the
QIAquick Gel Extraction Kit (QIAGEN).
PCR products were then sequenced in an automated
DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database
Searches from the National Centre for Biotechnology Information, confirming the identity of our products.
Western Blot Analysis. For MMP-2 and MMP-9 protein analyses, and their regulation by visfatin, HUVECs (and EaHY926 cells) were serum starved overnight, and then pre-treated with or without the NF-kB inhibitor (BAY 11-7085) and followed by treatment with human recombinant visfatin [(0 - 100nM); Axxora Ltd, Nottingham, UK (ALX-201-336)]. Cells were then lysed with Laemmli buffer [5M urea, 0.17M SDS, 0.4M dithiothreitol, and 50mM
Tris-HCl (pH 8.0)], mixed, sonicated, boiled, centrifuged (5,000 rpm for 2 minutes), and stored at -80º C until use.
Eighty micrograms of each sample were separated on a 10% Sodium Dodecyl Sulfate
(SDS)-polyacrylamide gel, and electro-blotted onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The PVDF membrane was then incubated with
5% Bovine Serum Albumin (Sigma) [BSA] in 1M Trizma/base, 1.54M NaCl, 0.05% Tween
20 (Tris buffered solution plus Tween 20, TBST, pH 7.4) for one hour at room temperature, and then exposed overnight at 4 °C to TBST containing primary antibodies [MMP-2 (mouse monoclonal IgG, AB3158, dilution 1:200) and MMP-9 (mouse monoclonal AB2463, dilution
1:200). The membranes were then washed thoroughly for 60 min with TBS/0.1% Tween before incubation with antimouse secondary antibody, horseradish-peroxidase-conjugated Ig
(1:2000) (Dako Ltd, Cambridge, UK) for 1 hour at room temperature. Antibody complexes were visualized using chemiluminescence (ECL; GE Healthcare, Little Chalfont, UK). Band densities were measured using a scanning densitometer coupled to scanning software Scion
Image™ (Scion Corporation, Maryland, U.S.A). In addition to quantitative loading of gels, membranes were also re-probed with the β-actin antibody (Cell Signalling Technology Inc.,
Beverly, MA, USA; 1 in 10,000 dilution) to determine equal protein loading
Gelatin Zymography.
The gelatinolytic activity of secreted MMP-2 and MMP-9 in the culture supernatants was measured by gelatin zymography, following 24-hour visfatin

treatment. We performed BCA protein quantification assay as per manufacturer protocol
(Pierce Biotechnology, USA) in the cultured supernatants to standardize protein concentrations loaded on the gel for zymography. Ten microlitres of culture supernatant was mixed with 10µl zymography sample buffer and resolved in 10% SDS-PAGE containing 1 mg/ml of gelatin (Sigma, St. Louis, USA) under non-reducing conditions. Following electrophoresis at 4º C, gels were washed twice for 30 minutes with renaturation buffer (2.5%
Triton X-100) at room temperature, and then incubated overnight in incubation buffer (50mM
Tris-HCl pH 7.5, 200mM NaCl, 10mM CaCl
2
, 1µM ZnCl
2
) at 37º C. Characterization of
MMP activity was determined by inhibition with EDTA (10mM). Following incubation, gels were stained for 1 hour (0.25% Coomassie brilliant blue R-250 in 45% methanol, 10% acetic acid), and destained in the same buffer without Coomassie. White bands against a blue background was observed following de-staining which indicated gelatinolytic activity of the expressed MMPs, which was quantified by measuring the band intensity [Gel Pro image analysis (Gel Pro 4.5, Media Cybernetics, USA) ]
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 statistical analysis was performed using Graph Pad software (version 4.0).

TABLE 1
Gene/product size Sense primer Antisense primer
MMP2 (179 bp) 5′-tggcaagtacggcttctgtc -3′ 5′-ttcttgtcgcggtcgtagtc-3′
MMP9 (200 bp)
5′-tgcgctaccacctcgaactt-3′ 5′-gatgccattgacgtcgtcct-3′

Figure A . Serum-starved HUVECs transiently transfected with pNF B-Luciferase were treated with or without visfatin (0-1600 ng/ml) for 2 hours. Cells were lysed and luciferase activities were measured. Visfatin induced a dose dependent increase in luciferase activity at
2 hours. * P < 0.05, ** P < 0.01, *** P <0.001 vs.
basal, respectively. Data are means

SEM of three experiments. Each experiment was carried out in triplicates
Figure B1 & B2.
Serum starved ECs treated with visfatin (0-1600 ng/ml) or TNFα (10ng/ml) for 24 hours showed a significant dose-dependent increase in MMP-2/9 protein levels
(* P < 0.05 ** P < 0.01, *** P <0.001 vs.
basal) respectively. Furthermore, serum starved ECs treated with visfatin (1600 ng/ml) or TNFα (10ng/ml) [24 hours] pre-incubated with BAY 11-
7085 (10µM) for 1 hour significantly decreased MMP-2/9 protein levels. ###
P <0.001 vs . visfatin treated. Data are means

SEM of three experiments. Each experiment was carried out in triplicates.

FIGURE 1
A.
B-1
72kD
10.0
7.5
**
***
5.0
* ##
#
2.5
##
#
0.0
Basal 400 800 1600 BAY+ BAY+
Visfatin (ng/ml)
Vis TN
F
B-2
92kD
10.0
***
7.5
**
5.0
2.5
*
### ###
0.0
Basal 400 800 1600 BAY+ BAY+
Vis TNF
Visfatin (ng/ml)