Online Supplement
Online Supplement contains supplementary figures important to the manuscript and detailed description of materials and methods.
Supplementary Figures
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Figure 1. Validation of co-culture model of ECs and SMCs and removal of endothelium from isolated rat aorta.
A, Representative blots with specific markers of ECs (PECAM) and
SMCs (Desmin). The markers in cell lysates were detected using Western blot. In ECs
PECAM, but not Desmin was detected both in co-culture and pure culture, whereas SMCs showed Desmin, but not PECAM. B, Representative fluorescent images of immunocytochemical staining with specific mar kers of EC (Factor VIII) and SMC (α-SMCactin). ECs stained positively for Factor VIII, but negatively for α-SMC-actin, whereas SMCs stained positively for α-SMC-actin, but negatively for Factor VIII. C, Histological images of the cell extensions from ECs and SMCs, forming cell-cell contacts through the pores of the insert
(upper, with cells). Image of another insert without cultured cells, where extensions are missing (lower, without cells). The inserts were prepared and imaged after 72 hours of culture. D, Representative images of immunocytochemical staining after biocytin assay.
Byocytin was transferred from ECs to SMCs. This transfer was abolished with gap-junction inhibitors 18 alpha GA (100 µmol/Ll) and FFA (150 µmol/L). E, Histological images of HE staining comparing denuded aorta (without endothelium, left) and intact aorta (with endothelium, right). F, Functional assessment of efficient endothelial removal from isolated rat aorta. As assessed with a myograph, endothelium-dependent relaxation with ACH
(acetylcholine , 10 µmol/L) was abolished in aortas without endothelium. SMCs-dependent relaxation with SNP (sodium nitroprusside, 3.5 µmol/L) was not different between denuded and intact aortas. G, In addition, there was no difference in development of tension in response to K + (123 mmol/L) and NE (norepinephrine, 3 µmol/L) between denuded and intact aortas. Scale bar - 10 µm (B, C, D), 50 µm (E). ## P< 0.01; n=4 independent experiments on the co-culture; n=5 independent experiments on aortic rings isolated from 5 rats. Two rings
(one with- and another without endothelium) from each animal were used per experiment.
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92 kDa
ECs
92 kDa
SMCs
Crtl ANGII
Pro-MMP9
Pro-MMP9
Figure 2. ANGII (1 µmol/L) did not increase pro-MMP9 neither in ECs nor SMCs. Active
MMP9 (82 kDa) was not detected.
72 kDa
62 kDa
Band 1 Band 2
Band 1 – Untreated control
Band 3
Pro-MMP2
MMP2
Band 2 – ANGII applied on both, EC and SMC, compartments
Band 3 – ANGII applied only to EC compartment
Figure 3. Selective treatment of ECs and SMCs with ANGII in co-culture. The experiment was performed to rule out a transfer of substance between compartments of ECs and SMCs in co-culture. For this, EC compartment of the coculture was treated with 1 µmol/L ANGII.
Pro-MMP2 and active MMP2 was assessed in supernatants of SMC compartment using gelatin zymography. Compared to the untreated control (band 1), treatment of both EC and
SMC compartments with ANGII resulted in increase of both latent pro-MMP2 and active
MMP2 (band 2) in SMC supernatant. Selective treatment of only the EC compartment, however, did increase neither latent pro-MMP2 nor active MMP2 in SMC supernatant.
3

Figure 4. ET
A
inhibitor dose-dependently inhibited ANGII-stimulated activation of latent pro-
MT1MMP (A) and pro-MMP2 (B) in SMCs.
# P< 0.05 vs. without inhibitor set to 100 %; n=4.
Figure 5. Co-stimulation with ANGII and Endothelin-1 dose-dependently increased activation of latent pro-MT1MMP and pro-MMP2 in pure culture of SMC (A), as well as denuded rat aorta (B). Abbreviations: ET-1 – Endothelin 1, ANGII – angiotensin II. n=4.
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72 kDa
62 kDa
Pro-MMP2
MMP2
Band 1 Band 2 Band 3
Band 1 – Untreated control – ECs and SMCs were cultured on upper and lower surfaces of the transwell membrane, respectively.
Band 2
– ANGII treated, both ECs and SMCs – ECs and SMCs were cultured on upper and lower surfaces of the same transwell membrane, respectively.
Band 3
– ANGII treated, both ECs and SMCs – ECs were cultured on the surface of the transwell membrane, while SMCs were cultured on the bottom of the culture well. Distance between transwell membrane and culture well surface was 4 mm according to the manufacturer.
Figure 6. In this experiments importance of cell-cell contacts and/or cell distance in activation of pro-MMP2 in SMCs was tested. For this, SMCs were cultured on the surface of the culture well plate instead of the lower surface of the transwell membrane. This most likely prohibited development of cell-cell contacts between ECs and SMCs. Band 3 shows that this condition did not allow activation of latent pro-MMP2. This is demonstrated by absence of the active MMP2, compared to band 2 which is from conditions where ECs and SMCs were cultured on both sides of the transwell membrane (thickness 10 µm) and connect with cell extensions through membrane pores (see Figure 1C).
72 kDa
62 kDa
Ctrl ANGII
Pro-MMP2
MMP2
ANGII
+Oligo
(scrambled)
Figure 7. Scrambled furin oligo did not inhibit ANGII stimulated gelatinolytic activity 62 kDa
MMP2.
5

Figure 8.
Localization of total (pro- and active) MT1MMP and MMP2 (brown colored signal) in medial layer of aorta after immunostaining. Arrows indicate endothelium.
6

Figure 9.
Ineffectiveness of gap-junction inhibitors in rat aorta on ANGII-mediated increase in activation of latent pro-MMP-2 (A) and pro-MT1MMP (B). 1µmol/L – ANGII, 100 µmol/L –
18α GA, 150 µmol/L – FFA. Ctrl=untreated control; * P< 0.01 vs. Control; # P< 0.05 vs. ANGII stimulation without inhibitor; n=3.
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Materials and methods
Antibodies and chemicals
Anti Desmin (D-1033 Sigma), anti-PECAM (SC-1506 Santa Cruz) and anti-GAPDH (A5316) antibodies, as well as gap junction inhibitors - 18α-glycyrrhetinic acid (18-α-GA, Sigma) and flufenamic acid (FFA, Sigma) were kindly donated by the research group of Prof. Thomas
Noll from Department of Physiology, TU Dresden. Anti-factor VIII (sc-14014) was purchased from Santa Cruz, while antiαSMC actin (ab7817), anti-MT1MMP (ab51074), anti-endothelin-
1 (ab88093), anti-integrin beta 3 (ab119992) and anti-albumin (ab137885) antibodies from
Abcam. Anti-actin (MAB1501) antibody was from Millipore. Furin inhibitor (Decanoyl-Arginin-
Valin-Lysin-Arginin-Chloromethylketon) was purchased from Calbiochem. Losartan and endothelin was obtained from Sigma. Materials for biocytin assay (see below) – biocytin (B-
1592), pinocytic cell-loading Reagent (I14402), Alexa Fluor 488 Streptavidin (S-11223) was purchased from Molecular Probes.
Animals
Aorta was isolated from 10-weeks old male Wistar rats (Charles River Laboratories
®
,
Germany, total n=18). Animals were handled with approvals of institutional ethics committee and local authorities (permission: 24-9168.24-1/2012-16). Animals were anesthetized with intraperitoneal injection of ethyl carbamate (1.3 g/kg body weight). Anaesthesia was monitored by assessment of pain and corneal reflexes. Isolation of aorta was started after both reflexes completely disappeared.
Cells
Cryopreserved rat aortic endothelial (RAOEC) and smooth muscle cells (RASMC) were purchased from Cell Applications Inc. (California, USA) in two separate complex kits (R304K-
05, R354K-05), each containing >500,000 3rd passage cryopreserved RAOEC or >500,000
2nd passage cryopreserved RASMC, 500 mL of rat endothelial cell growth medium
(RECGM), 500 mL of rat smooth muscle cell growth medium (RSMCGM), 100 mL of attachment factor sol ution, and a subculture reagent kit containing Hank’s buffered salt solution (HBSS), Trypsin/EDTA solution and trypsin neutralizing solution. The cells were stored in a liquid nitrogen storage tank immediately upon arrival and then cultured according to th e company’s instructions (see below).
Co-culture
The model was established based on a previous report.
1 Briefly (details follow in the subsequent paragraphs), the RASMC were cultured on the lower surface of the 10 µm thik polyester membrane of the transwell insert (Corning) by inverting the insert. The cells were let to attach to the membrane for 2 hours in the incubator. After this, the insert was inverted
8

again and placed in a 12 well culture plate. 2 ml of supplemented DMEM was added to the cells. The next day, RAOEC were cultured on the upper surface of the insert membrane and the same medium. As a control to the co-culture model, pure cultures of SMCs were used by culturing only SMCs on the lower side of the inserts. The cells were grown for at least 72 hours to ensure the formation of cell-cell contacts. Detailed procedure is given in the following paragraphs.
Culturing of RAOEC and RASMC was performed under strict sterile conditions. After removing the cryopreserved cells from the liquid nitrogen tank, cells were thawed quickly by placing the lower half of the vial containing the cells in a 37° C water bath for ~ 1 min. Then the vial exterior was decontaminated with 70 % ethanol. The cells were re-suspended in the vial by gently pipetting the cells 3-times with a 2 mL pipette avoiding foaming. The cells were then re-suspended in 15 mL of RECGM and RSMCGM growth medium, respectively, using
T-75 culture flasks coated with attachment factor solution. The cel ls were grown at 37° C in a humidified incubator with 5 % CO
2 atmosphere. After 24 hours the cells were washed twice with PBS and the medium was renewed. Consecutively, the medium was changed every other day. When the cells reached 60 % confluence the volume of the medium was doubled.
After RAOEC and RASMC cells reached 80 % of confluence, they were co-cultured on transwell-inserts (3460 Corning).
The cells were co-cultured using the sub-culture reagent kit. After the medium was removed from T-75 culture flasks by aspiration the cell monolayer was washed twice with 10 mL of
HBSS. After removing the HBSS, 6 mL of 0.05 % trypsin/EDTA solution was added into a T-
75 culture flask. The flask was gently shaken to ensure the solution covered the entire surface of the flask. After removal of 5 mL of trypsin/EDTA solution the effect of trypsinisation was observed during ~ 1 to 2 min at room temperature under an inverted microscope.
Complete detachment of cells was achieved by gently hitting the flask once or twice against the palm. To inhibit further trypsin activity 5 mL of trypsin neutralizing solution was added into the flask. Cell suspension was transferred to a 50 mL sterile Falcon tube. The flask was rinsed again with 5 mL of trypsin neutralizing solution and the solution with remaining cells was transferred to the same tube. The tube was centrifuged at 220 x g for 5 min to pellet the cells. The supernatant was aspirated without disturbing the cell pellet. Then, the cells were re-suspended in respective media and seeded on inserts for co-culture.
Co-culture validation
Before performing experiments on co-culture, the model was validated in three major directions. First, purity of ECs and SMCs was confirmed with assessment of specific markers
– PECAM (ECs) and Desmin (SMCs) by western blot and Factor VIII (ECs) and α-SMC-actin
(SMCs) by immunocytochemistry, respectively. Second, polyester membranes of the
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transwell inserts were evaluated to detect EC and SMC elongations – cell-cell contacts. For this, paraffin sections of the membranes were examined with phase-contrast microscopy.
Third, functional validation of the model was performed with dye transfer assay. Removal of endothelium from aorta was validated with HE staining as well as functional assessment of aortic segments with a Mulvany myograph. Detailed description of the methods used in the validation is described below.
Immunocyto- and histochemistry
Cells were fixed with 4 % paraformaldehyde directly on the membrane of the transwell insert.
Then the part of the membrane was gently cut out and moved to the culture slide (Falcon) where stainings with primary and secondary antibodies were performed. In case of aorta, immunohistochemistry was performed as previously described.
2 4% formalin fixed aorta was embedded in paraffin. Briefly, slides with 5 µm thick paraffin sections were microwave irradiated twice for 5 min each. After cooling the slides down, they were rinsed twice with distilled water and treated with 0.3 % H
2
O
2
for 30 min. Then the slides were washed twice 5 min each with PBS and blocked in 1.5 % goat serum for 20 min.
Histology
Preparation of aorta for hematoxylin-eosin staining (HE) was performed as previously described.
3 Briefly, isolated aorta was fixed with 4 % formalin. After series of dehydration
(with ethanol, once 20 min in 40%, overnight in 70%, twice 20 min each in 96%, three times
20 min in 100% and twice 20 min in xylene), aorta was embedded in paraffin. 5 µm thick paraffin sections were dried overnight at
37°C. After dewaxing with xylene followed by series of hydration (with ethanol, three times 2 min each in 100%, twice 2 min each in 96%, once 2 min in 70%, once 2 min in 40% and once 2 min in distilled water) HE staining was performed by incubating for 10 min in haematoxylin followed by rinsing in water for 10 min and 2 min incubation in eosin solution and finally rinsing three times with distilled water.
Experiments on co-culture
Experiments on confluent co-culture of RAOEC and RASMC was performed in nonsupplemented DMEM. Stimulation with 1 µmol/L of ANGII or 0.01, 0.1 and 1 µmol/L
Endothelin-1 was performed for 8 hours. Pre-treatments with various inhibitors were done 30 min prior to ANGII or Endothelin-1 stimulations. In case of furin antisense oligo ( 40 µmol/L,
5’-AGC TCC ATG GGG GGG A-3’), pre-treatment for 24 hours was performed. In the complementary experiments on isolated rat aorta a similar procedure was applied.
Cell lysis
Cell lysis was performed with RIPA buffer (Sigma) according to user instructions. Briefly, after removal of the medium cells were incubated in 200 μL of lysis buffer containing
10

phosphatase and protease (Roche) inhibitors for 5 min at 4° C. At the end of this incubation period still attached cells were scraped off to remove cell material completely. The samples were divided and used in part for specific experimental measurements and in part for the protein assay.
Cytotoxicity assay
To make sure that the compounds used throughout the work did not affect cell survival and caused cell death, besides microscopic evaluation, lactate dehydrogenase (LDH) release from cells into the culture supernatant as an indicator of cell death was measured. For this purpose, cytotoxicity (LDH) detection kit (Roche) was used and the measurements were performed according to the instructions of the manufacturer. Briefly, 50 µl of reaction mix solution was added to each well of a 96well plate. Wells contained either 50 μL of cell culture supernatant or cell lysate as a positive control or fresh cell medium as a negative control. The plate was incubated for 30 min at room temperature in a dark place. After that
50 μL of stopping solution was added avoiding formation of air bubbles. Before measurements the plate was shaken for about 10 s and the measurement was performed in an Elisa reader at 490 nm and 660 nm wavelengths. The data was processed according to the instructions of the manufacturer.
Biocytin assay
Functional validation of the co-culture model was performed with dye transfer assay based on previous report.
1 Briefly, biocytin (5 mg/mL, Life Technologies, B1592) was loaded on
ECs and the uptake by ECs was ensured with a pinocytotic uptake method (Life
Technologies, I14402). Biocytin on SMCs, as well as ECs was detected with Streptavidin
Alexa Flour 488 (Life Technologies, S32354).
Experiments on isolated rat aorta
Removal of endothelium was performed as previously described.
4 Briefly, after preparation
(removal of surrounding fat) each isolated aorta was divided in two (4 mm) pieces. Prior to incubation, endothelium was removed from one piece of aorta with the following procedure: a wire (almost with a diameter of the lumen of the aorta) was introduced into the aorta, and gently moved back and forth (5-7 times). Aortic vessel segments were incubated in nonsupplemented DMEM. Two millilitres of respective medium were used. Aortas were stimulated with ANGII (1 μmol/L) for 8 hours.
Validation of endothelial removal with myograph
Validation of endothelial removal was performed as previously described. 4 Four millimetres wide intact and endothelium-denuded aortic rings were suspended in 5 mL oxygenated phosphate-buffered saline solution (119 mmol/L NaCl, 4.7 mmol/L KCl, 1.17 mmol/L MgSO
4
,
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1.18 mmol/L KH
2
PO
4
, 25 mmol/L NaHCO
3
, 5.5 mmol/L Glucose, 2,5 mmol/L CaCl
2
and 0.027 mmol/L EDTA) for assessment of endothelium-dependent and –independent relaxations.
Maximum precontraction was achieved with potassium enriched (123 mmol/L) phosphate saline solution and maintained with 10 μmol/L noradrenaline. After maximum precontraction was reached, 10 μmol/L acetylcholine and 3.5 μmol/L sodium (Na) nitroprusside was added to assess endothelium-dependent and -independent relaxations, respectively.
Protein extraction
Active and latent forms of MMP-2 and MT1-MMP were extracted from aorta according to a previous report.
4 One millilitre of freshly prepared, ice cold extraction buffer (10 mmol/L cacodylic acid pH 5.0, 150 mmol/L NaCl, 20 mmol/L CaCl
2
, 10 µmol/L ZnCl) was added to each piece of aorta. The tissues were homogenized and incubated overnight at 4° C on an agitator. After incubation, the extract was centrifuged at 1200 x g for 15 min at 4° C.
Supernatant was collected and pH was raised to 7.0 by 0.1 M Tris HCl pH 8.8. The extracts were concentrated using Amicon
©
filters (UFC803096) and finally the samples were no rmalized to ~650 μg/mL of total protein concentration.
Protein assay
Protein concentration in the samples was determined using Pierce BCA Protein Assay Kit
(Thermo Scientific) as previously described 4 and following the user manual. Briefly, bovine serum albumin standards were prepared in cell lysis buffer (RIPA) with concentrations of 0,
25, 125, 250, 500, 750, 1000, 1500 and 2000 μg/mL, respectively. Samples were incubated in a working reagent (mixture of protein assay reagents A and B) for 30 min at 37° C.
Reaction between protein and the working reagent solution was detected by an UV recording spectrophotometer (Schimadzu 2100) at an absorbance wavelength of 562 nm. Protein concentration of the samples was calculated using regression functions.
Western blot
Western blot was performed based on previous reports.
4 Briefly, the samples were loaded on
10 % SDS polyacrylamide gel. After loading the appropriate concentration of protein (10 to
40 μg) electrophoresis was started with 80 V and after the sample left the stacking gel proceeded with 125 V. Electrophoresis was stopped after bromophenol blue left the gel (90 min). Electrophoretically separated proteins in the gel were transferred to nitrocellulose membrane using a semi-dry blotting system at 20 V for 60 min. Then the membranes were blocked in appropriate blocking solutions for non-phosphorylated (5 % non-fat milk) or phosphorylated (5 % BSA) proteins. After blocking, the membranes were incubated with primary antibodies. MT1MMP, αvβ3, Desmin, PECAM, GAPDH and actin was detected using western blot. A Kodak LAS-3000
©
imager was used to visualize the protein band on the membrane with appropriate exposure time. The blot images were quantified densitometrically
12

using ImageJ software. The amounts of the proteins of interest were normalized to the
GAPDH or actin, which served as a loading control.
Gelatin zymography
Latent (72 kDa) and active (62kDa) forms of MMP-2 in cell supernatants and aortic tissue extracts were detected using gelatin zymography according to a previous report.
4 10 % SDS polyacrylamide gels were co-polymerized with 0.1 % gelatin. After loading the samples with an equal concentration of total protein (10 μg) electrophoresis was started with 80 V. After the samples had left the stacking gel, the voltage was increased to 125 V. The duration of electrophoresis was 90 min. To maintain enzymatic activity of MMP-2, samples were separated electrophoretically under non-reducing conditions. After electrophoresis, gels were removed and incubated in washing buffer (2.5 % Triton X-100 to wash out SDS) for 1 hour at room temperature on a shaker. Then the gels were gently rinsed 3-times in ddH
2
O (two times distilled water to wash out Triton X-100) and incubated in ddH2O (final wash out of Triton X-
100) for 30 min at room temperature on a shaker. After completing the washing step, the gels were incubated in developing buffer (50 mmol/L Tris pH 7.5, 10 mmol/L CaCl
2
, 200 mmol/L
NaCl, 5 μmol/L ZnCl
2
, 0.1 % Brij-35, 1.5 mmol/L NaN
3
) for 20 hours at 37° C on a shaker.
The gels were stained with a staining buffer (0.1 % Coomassie brilliant blue R-250) for 1 hour and then distained (10 % methanol, 5% acetic acid) for another hour. Images were captured using Kodak imager model LAS-3000. Unstained band intensity was quantified densitometrically against the homogenously stained background using ImageJ software.
Furin activity assay
The assay was performed based on previous report with modifications.
5 Briefly, 30 µg of total protein was inoculated with 2 µmol/L of furin specific fluorogenic substrate Pyr-Arg-Thr-Lys-
ArgAMC (Calbiochem) at 37° C for 8 hours. After terminating the reaction with 2 mmol/L
ZnCl
2 fluorescence was measured using a microplate reader (BMG Labtech
©
) at 365 nm excitation and 450 nm emission. A standard curve (0.05 to 2 µmol/L) was used to calculate furin activity in the sample. Furin antisense oligo treated samples were used to assess specificity of the furin substrate.
RNA isolation and Reverse Transcription qPCR (RT-qPCR)
RT-qPCR following total RNA isolation from cells and aorta RT-qPCR was performed as previously described.
6 Briefly, total mRNA from SMCs and aortic rings for RT-qPCR was isolated using TRIzol Reagent (Life Technologies) according to the user manual. In case of aortic rings, total RNA was isolated by homogenization using a power homogenizer
(Polytron). 1 µg of total RNA was reverse transcribed to cDNA using reverse transcription kit
(Invitrogen). The amplification of cDNA templates was performed in presence of SensiMix
SYBR (Invitrogen) and furin primers – 5’-CAG ATC TTCGGGGAC TAT TAC CAC-3’ (sense)
13

and 5’-CCT GTT GTC ATT CAT CTG TGT GTA CC-3’ (antisense). All kits were used as instructed by the manufacturer.
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