In vivo experimentsMice

10–12 week-old male C57/BL6-J (wildtype) mice were purchased from Janvier Labs, France. TLR3−/−mice on a C57/BL6-J background were obtained through our own breeding program. Knockouts were confirmed via PCR analyses of ear punches. All animals were maintained in a room at 22 °C, with a 12 h light/dark cycle, and received chow and drinking water ad libitum. Mice were held in cages with 4–5 animals. Randomization was ensured by arbitrarily assigning an animal to a cage. All animals in one cage were either assigned to the control or the treatment group, the ratio of control to treatment animals was 1:1. Only male mice were used for the experiments because female animals have significantly lower trans-aortic valve peak blood flow velocity at baseline and display different hemodynamic changes following wire injury.

All animal experiments were performed according to institutional guidelines, the German animal protection law and were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen (81-02.04.2018.A250, 84-2.04.2013.A103 and 84-02-04-2013.A197.

Echocardiography

For all functional analyses, a Fujifilm Visualsonics Vevo 2100 or 3100 Ultra High Frequency Imaging Platform was used. Echocardiography was performed as previously described [20]. Briefly, the mice were anesthetized and their chest hair was removed. The aortic valve peak velocity was measured in the suprasternal view with a pulse-wave Doppler. Left ventricular (LV) function, wall thickness, and LV volume were each measured in parasternal long-axis and short-axis views in B- and M-mode images. The sonographer was blinded to the specifics of the experiments including the genotype and/or the medical treatment of the mice. All analyses were performed with Fujifilm Visualsonics VevoLab software.

Aortic valve injury

For the induction of a severe aortic valve stenosis in mice, standard protocols have been established in our lab: a conventional guide wire with a 15° angled tip (ASAHI INTECC MIRACLEbros 6) was inserted into the right carotid artery and then advanced into the left ventricle under echocardiographic guidance. The wire was pushed into the apex and pulled back just below the aortic valve level ten times, followed by 200 rotations of the wire at level of the aortic valve [20, 25]. The operating team alternated between mice from the intervention control group. The surgeon was unaware of the genotype or the medical treatment of the mice. Animals that did not survive the procedure were excluded from the analyses. No other exclusion or inclusion criteria were determined.

Medical treatment

For in vivo stimulation of TLR3, 100 µg polyIC (Sigma-Aldrich) was dissolved in 0.9% sodium chloride solution (NaCl) (Fresenius Kabi, Germany) and injected subcutaneously every day for 6 weeks (initial n = 20, 5 animals died during surgery). TLR3 was inhibited using the TLR3/dsRNA-complex inhibitor Compound 4a (C4a, Millipore Sigma). 27.5 µg C4a were dissolved in 200 µl phosphate buffered saline (PBS) and was injected subcutaneously. Control animals received similar amounts of NaCl or PBS, respectively (initial n = 40, 7 mice died during the procedure).

Histological analysis

The mice were euthanized via cervical dislocation six weeks after the aortic-valve injury. Hearts were isolated and blood was flushed by injecting 0.9% sodium chloride solution into the LV. Afterwards, the collected hearts were embedded in a tissue-freezing medium and sectioned (8 μm thickness). The sections were stained with hematoxylin and eosin according to standard protocols. Tissue calcification was measured using von-Kossa staining (Abcam Staining Kit). Collagen was visualized using Pico-Sirius-Red staining (Sigma Aldrich). Images were obtained using light/polarization (Sirius-Red) microscopy at 10x (Axio Observer, Zeiss, Germany). Aortic valve area, collagen, and calcium deposits were measured with Zeiss ZEN Imaging Software.

Immunofluorescence

CD68 staining was used to visualize macrophage infiltration into the stenotic aortic tissue. Aortic sections were fixed in acetone for 20 min, washed with PBS, and blocked with 1% bovine serum albumin (BSA) for 30 min. The primary antibody was diluted 1:100 (anti-CD68 rat IgG2a, Acris Antibodies, Germany) and incubated at 4 °C overnight. After washing with PBS, the sections were incubated with the secondary antibody, which was diluted 1:50 (Cy3 AffiniPure Donkey anti Rat IgG, Jackson ImmunoResearch Laboratories Inc), for 1 h at 4 °C. Vectashield mounting medium containing 4’,6’-diamidino-2-phenylindole (DAPI) (Vector Laboratories) was used for counterstaining of the nuclei.

Images were taken with an Axio Observer (Zeiss, Germany). The CD68 positive areas were automatically quantified with Zeiss ZEN Imaging Software.

Cytokine-analyses

After cervical dislocation of the mice, whole blood was drawn from the retroorbital plexus with heparin-coated glass pipettes. After centrifugation at 7500 g for 20 min at 4 °C, plasma was collected. Plasma-levels of IL-6, IL-10 and TNFα were measured using a Mesoscale V-plex “mouse-proinflammatory panel 1” Assay-Kit (MSD). Samples were measured in duplicates Assays were analyzed using a MESO Sector S 600 Plate reader (MSD).

Flow cytometry

For quantification of peripheral blood cells, 50 µl of whole blood was collected. Red blood cells were lysated and Fc- blockage was performed (Fc-Block, Pharmingen). After washing cells were stained with anti-B220 (BD Pharmingen), anti-CD8a (BioLegend), anti-CD11b (BD-Pharmingen), anti-CD4, anti-CD115, anti-Nk1.1, and anti-Ly6c (all ThermoFisher Scientific). To collect absolute numbers, CountBright Absolute Counting Beads (ThermoFisher Scientific) were added. Samples were measured on a FACSCanto II (BD Bioscience, Franklin Lakes, USA) and analyzed with FlowJo (Tree Star, Ashland, USA).

In vitro experimentsValvular interstitial cell culture and calcification

Valvular interstitial cells (VICs) were purchased from Lonza Bioscience. Cells were cultured in culturing medium (Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher) containing 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin (P/S)) until passage six. Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere and fed every second day. When the cells reached 80–90% confluence, they were treated with polyIC (10 µg/ml) (Sigma Aldrich) as well as 10 µg/ml compound C4a (Millipore Sigma) or both for 72 h. Calcification was induced in conditioning medium (DMEM containing 5% FBS, 1% P/S, 2 mmol/l disodium hydrogen phosphate, 50 µg/ml ascorbic acid).

TLR3 knockdown

VICs in passage 6 were seeded on 12-well plates for gene expression and 48-well plates for alizarin red staining. When cells reached 80–90% confluence, TLR3 siRNA (siRNA TLR3 107055: sense: GGUACCUGAAUUUGAAACGtt antisense: CGUUUCAAAUUCAGGUACCtc; siRNA TLR3 107056: sense: GGUACAUCAUGCAGUUCAAtt antisense: UUGAACUGCAUGAUGUACCtt) or scrambled siRNA (Thermo Fisher) as negative control were transfected into cells using Lipofectamine 2000 (4 µl/ml) (Thermo Fisher). The transfection concentration of siRNA against TLR3 was 10 nM. Cells were incubated at 37 °C in a humidified 5% CO2 atmosphere for 48 h after transfection. Subsequently, calcification was induced by changing to conditioning media.

qPCR was performed to analyze calcification using the osteogenic markers BMP2 (Hs00154192_m1) and Runx2 (Hs01047973_m1). Further, the pro-inflammatory cytokine IL6 (Hs00174131_m1) was measured. Activation and inhibition of TLR3 was measured using TaqMan primers for the respective gene (TLR3 Hs01551079_g1). Gapdh (Hs02758991_g1) was used as a housekeeping gene.

Valvular endothelial cell (VEC) culture

Human valvular endothelial cells were obtained from the Lonza Bioscience company.

VECs were cultured in culturing medium containing EBM-2 basal media supplemented with 5% FBS, 0.04% hydrocortisone, 0.4% human basic fibroblast growth factor (hFGF-β), 0.1% vascular endothelial growth factor (VEGF), 0.1% insulin like growth factor (R3-IGF-1), 0.1% ascorbic acid, 0.5 ml antibiotics (GA-1000), and 0.1% human epidermal growth factor (hEGF) (Lonza) until passage six. Cells were incubated at 37 °C in a humidified 5% CO2 air atmosphere and fed every second day. VECs in passage 6 were seeded on 12-well plates for gene expression. When the cells reached 80–90% confluence, VECs were treated with polyIC (10 µg/ml) (Sigma Aldrich) as well as 10 µg/ml compound C4a (Millipore Sigma) or both in hunger medium (without any supplements) followed by incubation of 24 h at 37 °C in a humidified 5% CO2 air atmosphere. Afterwards, cells were incubated in medium containing EBM-2- supplements: Basal media was supplemented with 5% FBS, 0.4% hFGF-B, 0.1% R3-IGF-1, 0.1% ascorbic acid, and 0.1% hEGF. After 24, 36, and 72 h, cells were harvested and RNA was isolated as described in the following.

qPCR was used to analyze endothelial as well as mesenchymal markers in VECs. Therefore, CD31 (Hs01065279_m) and von Willebrand factor (VWF) (Hs01109446_m1) were used as endothelial markers, whereas actin alpha2 (ACTA2) (Hs00426835_g1), Vimentin (Hs00418522_m), and Vascular cell adhesion protein 1 (VCAM1) (Hs00365486_m1) were used as mesenchymal markers. Activation and inhibition of TLR3 was measured using TaqMan primers for the respective gene (TLR3 Hs01551079_g1). Gapdh (Hs02758991_g1) was used as a housekeeping gene.

Gene expression

Cells were isolated using Trizol (Thermo Fisher) according to the manufacturer’s instructions. 200 ng of total RNA was reverse transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Quiagen), according to the manufacturer’s protocol.

For amplification of specific genes, the TaqMan Gene Expression Assay (FAM) was used containing a combination of forward and reverse primers that match the cDNA sequences of the gene of interest. For data analysis, the relative levels of target gene expression were calculated by the comparative CT method (ΔΔCT Method).

Alizarin red S staining

Cells were fixed for 15 min in 4% paraformaldehyde (Millipore) followed by a washing step with ultrapure distilled water. Cells were then incubated with 1% alizarin red S solution (Sigma Aldrich) (pH 4.2) for 15–30 min. The dye was removed by washing twice with ultrapure distilled water. Alizarin red S staining was photographed with a Zeiss Axio Observer microscope.

Alamar blue assay

Alamar blue assay indicates metabolic active cells and therefore, it was used as a viability assay. The pre-mixed alamarBlue reagent (TermoFisher) was added to the cells and incubated for four hours at 37 °C. Afterwards, fluorescence (using an excitation of 560 nm and an emission at 590 nm) was measured using the plate reader Tecan Infinite M Plex.

Cell counts

To count the cell number and cell budding, microscopic images were divided in 4 quadrants. Cells and cell budding were counted in each section. The number of cells or cell buds are shown per field of view.

Human aortic valve samples

Aortic valve specimens were collected from patients undergoing surgical aortic valve replacement for either severe aortic stenosis or aortic regurgitation. All patients provided written informed consent. The study protocols were approved by the local ethics committee (Lfd. Nr. 078/17).

After fixation in formaldehyde for 24 h, the valves were decalcified using Titriplex III- buffer (Merck) for 72 h, embedded in paraffin, and sectioned (4 μm thickness). The sections were stained with hematoxylin and eosin according to standard protocols.

Immunofluorescence

Samples were deparaffinized with xylene and slowly rehydrated by putting them into decreasing concentrations of ethanol, followed by washing steps. For antigen retrieval, the slides were boiled in 10 nM citrate buffer and then washed again with distilled water. Sections were incubated for 10 min at room temperature with PBS containing 0.25% Triton X-100 (PBS-T) to permeabilize the cells, followed by three washing steps with PBS. For blocking, slides were incubated 30 min at room temperature in 5% BSA in PBS-T and 0.3 M glycine. Afterwards, the primary antibody (20 µg/ml; anti-TLR3 antibody (Abcam)) diluted in 1% BSA PBS-T) was added and incubated overnight at 4 °C. The next day, slides were washed twice with PBS-T, followed by a one-hour incubation with the secondary antibody (Goat anti-rabbit Cy3 (Dianova)) diluted 1:100 in 1% BSA PBS-T) at room temperature. Finally, slides were mounted with DAPI and analyzed with a confocal microscope using 20x and 40x objectives.

Statistical analyses

All data are presented as the mean ± SEM. Statistical significance was calculated by two-way ANOVA followed by a Tukey Test or a mixed-model followed by a Bonferroni Test for multiple comparisons. Two groups were compared using the Student t test. P values of 0.05 or less were considered to be statistically significant.