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SUPPLEMENTARY METHODS
Isolation and culture of vascular smooth muscle cells (VSMC) derived from human temporal arteries (TA)
Cell culture
Serial, 1 mm thick sections of fresh TA fragments, obtained for diagnostic purposes, were placed onto ice-cold reconstituted basement membrane Matrigel TM (BD
Biosciences, San Jose, California, USA) and allowed to gelify at 37 oC for 30 minutes.
Explants were cultured in Dulbecco modified Eagle medium (DMEM) (Lonza,
Hopkinton, MA USA) supplemented with 10% fetal bovine serum (FBS) at 37ºC in 5%
CO 2. VSMC emerged from the explanted sections after 1 week and reached confluence in 3 weeks. Cells were then released with trypsin/EDTA (Invitrogen, Waltham,
Massachusetts, USA), transferred to uncoated flasks, and split 1:2 upon confluence.
Cells were used up to passage 7. Cells were characterized according to αSMA expression and homogeneity was confirmed by flow citometry.[1]
Chemical reagents
Human ET-1 was purchased from MP Biomedical (Aurora, Ohio, USA). Fibronectin
(FN) was obtained by Calbiochem (EMD Chemicals Inc., Gibbstown, NJ, USA) and used at 5µg/cm 2. Polylysine was acquired by Sigma-Aldrich (St. Louis, MO, USA) and used at 10µg/mL.
ET AR antagonist BQ123 was chemically synthesized through solid phase peptide synthesis and confirmed to be chromatographically identical to a commercial sample obtained from Sigma-Aldrich. ET BR antagonist BQ788 and PF-573228 (FAK inhibitor) were purchased from Sigma-Adrich. LY294002 (PI3K inhibitor), PP2 (Src inhibitor) 2 and PD98059 (ERK inhibitor) were acquired from Calbiochem. Pertussis toxin (PT) from Bordetella pertussis was acquired from Sigma-Aldrich.
Cell viability upon exposure to chemicals.
5000 cells per well were seeded into 96 well plates. After 12 hours, fresh DMEM medium supplemented with or without Src inhibitor PP2 (10µmol/L), ERK inhibitor
PD98059 (20µmol/L), PI3Kinase inhibitor LY294002 (20µmol/L) or FAK inhibitor
PF573228 (20µmol/L) was added to quadruplicates. After 3 or 6 hours, cells were removed from the plate, stained with Trypan Blue and counted using a Neubauer
Chamber.
Immunofluorescence staining
Frozen temporal artery biopsies
Temporal artery biopsy fragments were fixed with 4% PFA and incubated with increasing concentrations of saccharose (15 and 30%) before being frozen in optimal cutting temperature (OCT, Sakura, Flemingweg, The Netherlands) and stored at -80ºC.
10µm cryostat sections were obtained, refixed with 4% PFA, permeabilized with 0.1% triton X, and blocked with 1%BSA and 5% Donkey Serum (Sigma-Aldrich) in PBS.
Immunofluorescence staining was performed as detailed above for VSMC. Primary and secondary antibodies used and the corresponding sources and dilutions are detailed in the supplementary table S3. Hoechst (Molecular Probes, Life Technologies) (1:1000) was used for staining nuclei. Slides were mounted with Prolong Gold Antifade Reagent
(Molecular Probes, Life Technologies). Control for background non-specific staining was performed by omitting the primary antibody from the procedure.
Immunofluorescence stained VSMC or temporal artery sections were examined using a 3 laser scanning confocal Leica TCS SP5 microscope (Leica Microsystems, Heildelberg,
Germany). Images were processed with Leica Confocal software (LAS-AF Lite) and
Image J software (Wayne Rasband, Bethesda, Maryland, USA).
VSMC
For immunofluorescence staining VSMC were cultured on 16 well chamber slides
(Nunc). Cells were fixed with 4% PFA (10 minutes at room temperature), permeabilized with 0.1% triton X, and blocked with 1%BSA and 5% Donkey Serum
(Sigma-Aldrich) in PBS.
Dilutions and sources of the primary and secondary antibodies used are detailed in the supplementary table S3. Primary antibody incubation was performed at 4ºC overnight.
After 3 washes with PBS, secondary antibodies were incubated at room temperature for
2 hours. F-actin was stained with rhodamine-phalloidin (Molecular Probes, Life
Technologies) at 1:3000 dilution. Hoechst (Molecular Probes, Life Technologies)
(1:1000) was used for nuclear staining. Slides were mounted with Prolong Gold
Antifade Reagent (Molecular Probes, LifeTechnologies).
PBMC, CD4+T cells, monocytes (CD14+) and VSMC co-culture
VSMC were seeded at 90% confluence onto 6-well plates and cultured with DMEM
(10% FBS) overnight. Peripheral Blood Mononuclear Cells (PBMCs) were counted using a Neubauer Chamber and Untouched human CD4+T or monocyte Dynabead Kits
(Invitrogen) were used to obtain purified cells. Efficiency of purification was tested using flow cytometry. Media from the VSMC was replaced by 1 million isolated
CD4+TCells or CD14+ in RPMI (10% FBS) (Lonza). After 24h supernatants were collected and RNA and protein were extracted. The co-cultured cell populations were processed separately. Supernatants were centrifuged to obtain lymphocytes and 4 monocytes and VSMC were treated with cold Versene (Invitrogen) under microscope monitoring to remove attached white blood cells. VSMC were released using Trypsin-
EDTA (Invitrogen).
Quantitative real-time reverse transcription q(RT)-PCR
Total RNA (1 µg) was reverse transcribed using High Capacity cDNA Reverse
Transcription kit (Applied Biosystems, Foster City, California, USA). Pre-developed
TaqMan gene expression assays were used according to the manufacturer instructions and fluorescence was detected with an ABI PRISM 7900 Sequence Detection system and results were analyzed with the Sequence Detection Software V 2.3 (Applied
Biosystems). All samples were normalized to the expression of the endogenous control
GUSb. Comparative CT method was used to assess relative gene expression.
Assessment of protein secretion by immunoassay
Cell supernatants were centrifuged in order to remove cell debris. R&D Quantikine
ELISA Kit (Minneapolis, Minnesota, USA) was used to measure mature ET-1 following the manufacturer instructions.
Migration Assay
VSMC migration was assessed using Boyden chambers with 10 µm-pore polyester filters (Poretics Osmonics Inc., Minnesota, MN, USA). VSMC in DMEM (1% FBS) were placed in the upper chambers (5000 cells/well). Cells were pre-incubated with medium alone or supplemented with BQ123, BQ788, or combination of both (20
µmol/L) for 1 hour and then ET-1 was added at various concentrations either in the bottom or the upper chambers in quadruplicate wells. VSMC were allowed to migrate 5
for 6 hours (5% CO 2 at 37ºC). Filters were removed and after eliminating cells from the upper surface with cotton swap, were fixed with 20% methanol and stained with haematoxylin. Four randomly selected fields/ filter were counted under a microscope at
4X o 10X magnification.
Scratch-wound healing Assay
VSMC were seeded at 80% confluence onto 0.1% gelatin pre-coated 12-well plates and cultured overnight. One scratch per well was done using a sterile 200 µL pipette tip.
Cells were rinsed with PBS to release possible debris and cultured in DMEM (10%FBS) supplemented with 50mM Hepes (Sigma-Aldrich). After 12-hour culture, medium was removed and replaced by fresh medium with the corresponding additives and molecules. Three pictures per well were automatically taken using phase contrast microscope (Leica, Microsystems, Heildelberg, Germany) every 15 minutes during 24 hours Image J was used to measure the width of each wound at 10 different levels/ picture. The composed video is displayed as supplementary movie.
Proliferation Assay
VSMC were plated in 7 flat-bottom 96-well plates at 4000 cells/well in quadruplicate wells, serum starved for 24 hours and incubated at 37° C in 5% CO 2 for 1 to 7 days with fresh DMEM (1% FBS) with or without ET-1 (10 -9mol/L). Cells were fixed and stained with 0.2% crystal violet (Sigma-Aldrich) in 20% methanol for 20 min. Wells were washed, air-dried, and solubilized in 1% sodium dodecyl sulfate (SDS). Optical density was measured with a plate reader (Multiskan Ascent, Thermo Scientific) at 620 nm wavelength.
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Western blot
Cell lysates were obtained using modified radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Complete, Boehringer Mannheim,
Mannheim, Germany), NaF (50mM), PMSF (1mM), protinine, leupeptine, pepstatine
(1ng/ml) and orthovanadate (2mM) (Sigma-Aldrich).
The following primary antibodies were used for immunodetection: rabbit polyclonal anti-human phospo-ERK, total ERK, phospo Y416-Src, total Src, phospho-Y397FAK and Y925 FAK (purchased from Cell Signaling (Boston, MA, USA) and used at 1:1000 dilution), goat polyclonal anti-ET-1, rabbit polyclonal anti-human FAK obtained from
Santa Cruz Biotechnology (Dallas, Texas) and used at 1:500 dilution, mouse monoclonal anti-αSMA (1:1000) and rabbit monoclonal anti-human β-actin (1:500) were purchased from Abcam (Cambridge, UK). Goat anti-rabbit IgG HRP-linked (Cell
Signaling), goat anti-mouse IgG HRP-conjugated (Bio-Rad) and donkey anti-goat IgG
HRP conjugated (Santa Cruz Biotechnologies) were used as secondary antibodies at
1:2000 dilution.
Transient transfection
FAK wild-type cDNA and FAK point mutants Y397F and Y925F, were expressed using the pcDNA3 vector containing the hemagglutinin (HA) epitope generated as previously described.[1] Lipofectamin 2000 Reagent (Invitrogen) was used for transient transfection of VSMC following the protocol advised by the manufacturer and using 2
µg of cDNA/well in 12-well plates. Transfected cells were used for experimental procedures 3 days post-transfection, and in all experimental conditions were compared with VSMC transfected with the empty vector pcDNA3 (MOCK). Efficiency of transfection was verified both by detecting total FAK by Western Blot and by 7 immunofluorescence of simultaneous trasfected cells with the plasmid pEGFP-C3 encoding green fluorescent protein (Supplementary Figure S2).
Gelatin zymography
6 hours after exposure of VSMC to the inducers or inhibitors of interest, conditioned media were collected, centrifuged, and stored at −80°C. 100 µL of each supernatant was subjected to SDS-PAGE through 10% polyacrylamide gels copolymerized with 0.2 g/mL gelatin (Bio-Rad Laboratories, Hercules, CA). Gels were washed with 2.5%
Triton-X-100, rinsed with 10 mmol/L Tris and incubated overnight at 37°C in 50 mmol/L Tris, 5 mmol/L CaCl 2, and 1 µmol/L ZnCl 2. Gels were fixed and stained with
0.2% Coomassie blue R250. In inhibitory experiments, zymogram gels were developed for 24hours.
VSMC outgrowth from ex vivo cultured TA sections from patients with GCA
To analyze VSMC outgrowth from cultured TA in Matrigel TM daily photographs were taken from day 3 to day 12. Outgrowth of VSMC was scored by two investigators blinded to the conditions tested, as follows: 1=visible outgrowth of scattered cells;
2=cells growing from the entire ring; 3=cells expanding at least two times the length of
2; and 4=cells covering the entire surface of the well. Agreement was achieved in 96% of measurements. [2]
REFERENCES
1.- Segarra M, Vilardell C, Matsumoto K, Esparza J, Lozano E, Serra-Pages C, Urbano-
Márquez A, Yamada KM, Cid MC. Dual function of focal adhesion kinase in regulating 8 integrin-induced MMP-2 and MMP-9 release by human T lymphoid cells. FASEB J
2005;19:1875-1877.
2.- Lozano E, Segarra M, Garcia-Martinez A, Hernandez-Rodriguez J, Cid MC.
Imatinib mesylate inhibits in vitro and ex vivo biological responses related to vascular occlusion in giant cell arteritis. Ann Rheum Dis 2008;67:1581-1588.
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Supplementary table S1 : Clinical findings and blood tests at diagnosis in the 5 patients with GCA included
Age, median (±SD) years 73,07 ± 4,76 Gender (male/female) ratio 1/4 Clinical data at diagnosis Cranial Symptoms Headache 3 Scalp tenderness 4 Jaw claudication 3 Diplopia 1 Visual loss 1 Otalgia 2 Systemic Symptoms Fever 3Weight loss Polymyalgia2 rheumatica 1 Laboratory findings at diagnosis median ± SD ESR, mm/h 104,73 ± CRP,13,31 mg/dl 16,99 ± 4,79Haemoglobin, mg/dl 100,95 ± 14,95 ESR= erythrocyte sedimentation rate; CRP= C-reactive protein
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Supplementary table S2: Demographic data and final diagnosis in the 5 control patients included.
Demography
Age, median (±SD) years 81,68 ± 4,81 Gender (male/female) ratio 0/5 Diagnosis
Non-arteritic anterior ischemic neuritis 1 Chronic multifactorial anemia 1 Isolated polymyalgia rheumatica* 2 Rheumatoid arthritis 1
The 2 patients diagnosed with polymyalgia rheumatica were treated with low-dose prednisone and did not develop GCA symptoms or complications during 2 and 4 years of follow-up, respectively.
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Supplementary Table S3. Detailed information for primary and secondary antibodies used for immunofluorescence staining of cells or tissues.
Primary antibodies used for immunofluorescence
Target molecule Antibody type Company Working
dilution
Pi3Kinase (p85) Rabbit polyclonal Cell Signaling 1/100 p-FAK (Tyr397) Rabbit polyclonal Cell Signaling 1/100
FAK Mouse monoclonal Santa Cruz Biotechnology 1/50
FAK Rabbit polyclonal Santa Cruz Biotechnology 1/50
ET-1 Goat polyclonal Santa Cruz Biotechnology 1/50
ET AR Mouse monoclonal BD Transduction 1/100
Laboratories
ET BR Rabbit polyclonal Abcam 1/100
CD45 Mouse monoclonal Dako 1/100 (ready
to use)
αSMA Mouse monoclonal Abcam 1/100
Secondary antibodies used for immunofluorescence
Fluorochrome Antibody type Company Working dilution
Alexa Fluor 488 Donkey polyclonal Molecular Probes 1/300
Alexa Fluor 555 Donkey polyclonal Molecular Probes 1/300
Alexa fluor 647 Donkey polyclonal Molecular Probes 1/300 9
SUPPLEMENTARY VIDEO LEGEND
Time-lapse video microscopy applied to record cell movement comparing VSMC cultured with ET-1 (10 -9 mol/L) or fresh medium. Cells were seeded at 80% confluence onto 0.1% gelatin pre-coated 12-well plates and cultured overnight. One scratch per well was performed.
0,45 Untreated 0,4 ET-1 0,35
) 0,3 m n
0 0,25 2 6 (
0,2 s
b 0,15 A 0,1 0,05 0 0 1 2 3 5 7 (days)
Supplementary Figure S1. Effect of endothelin-1 (ET-1) on human temporal artery (TA)- derived vascular smooth muscle cell (VSMC) proliferation. VSMC were cultured at 4000 c/well in quadruplicate wells and in seven 96-well plates. Cells were serum starved for 24 hours and medium was replaced with fresh DMEM (1% FBS) with or without ET-1 10-9 mol/L. From day 1 to 7, a plate was daily fixed and stained with 0.2% in crystal violet in 20% methanol and absorbance was measured at 620nm wavelength. One out of 3 independent experiments is displayed. A 30% Transfection
Mock-Transfected VSMC 50µm pEGFP-C3 transfected VSMC
B 2 FAK/ β-Actin Untreated MOCK FAK Y397F FAK Y925F FAK y t
i 1,5 s n
FAK e t 1 n i d
n 0,5 a
βActin B 0 Untreated MOCK FAK Y397FAK Y925FAK
Supplementary Figure S2. Transient transfection of Focal adhesion kinase (FAK) and FAK mutated at tyrosine phosphorylation sites. (A) Green fluorecence protein detection (GFP) (green) of non-transfected vascular smooth muscle cells (VSMC) vs. GFP pcDNA3 transfected cells. 30% transfection efficiency was obtained after 48hours. (B) Immunoblot of total FAK in untreated VSMC, mock-transfected VSMC or VSMC transfected with vectors containing wild type FAK, FAK mutated at Y397F or FAK mutated at Y925F. 7
A ET-1 (10-9mol/L) - - - - + + + +
PF-573228 (µmol/L) 0 1 10 20 0 1 10 20
Y397FAK
FAK
Y925FAK
FAK
B 1,6 Y397/ FAK 1,2 Y925/ FAK
1 y y t t i
i 1,2 s s
n 0,8 n e e t t n n i i 0,8 0,6 d d n n a a 0,4 B B 0,4 0,2
0 0 0 1 10 20 0 1 10 20 0 1 10 20 0 1 10 20 PF-573228 (µmol/L) PF-573228 (µmol/L)
Untreated ET-1 (10-9 mol/L)
Supplementary Figure S3. Effect of FAK inhibitor PF-573228 on FAK phosphorylation at residues Y397 or Y925. Human TA-derived VSMC were exposed to various concentrations of PF- 573228 for 90 minutes and cell lysates were subjected to immunoblot.(A) Immunoblot of phosphorylated forms of FAK (Y397 and Y925) vs. total FAK. (B) Quantification of pY397/total FAK and pY925/ total FAK band intensity ratios Untreated PP2(Src inhibitor) 120 PD98059(ERK inhibitor) 0
0 ** PF-573228(FAK inhibitor) 1 100 x LY294002(PI3K inhibitor) s l l
e 80 c
l a t o
t 60
/ s l l
e 40 c
e l b
a 20 i V
0
Supplementary Figure S4. Cell viability assay. After 3 or 6 hours of culture with the corresponding inhibitors as labeled, cells were collected, stained with Tryplan blue and counted using a Neubauer Chamber. Bars represent mean and SEM of quadruplicates corresponding to the percentage of the alive cells counted vs total number of cells. (**) p<0.005 of untreated cells vs. PD98059 treated cells