We examined 50 aortic valves that were explanted from patients at the time of aortic valve replacement for CAVD. Control non-calcified aortic valves (n
28) with normal echocardiographic analyses were obtained during heart transplant procedures. Patients with a history of rheumatic disease, endocarditis, and inflammatory diseases were excluded. Valves with an aortic valve regurgitation grade >2+ were excluded. Patients with reduced left ventricular ejection fraction (LVEF) (<40%) were excluded. All patients underwent a comprehensive Doppler echocardiographic examination preoperatively. Doppler echocardiographic measurements included the left ventricular stroke volume and transvalvular gradients using the modified Bernoulli equation. The protocol was approved by the local ethical committee and informed consent was obtained from the subjects.
Immunohistologic analysis for SLC20A1 was performed using the rabbit antibody anti-SLC20A1 (Novus Biologicals, Oakville, ON, Canada). Slides were then incubated with HRP-conjugated and AEC substrate (Dako, Mississauga, ON, Canada). Tissue sections were counterstained with hematoxylin. Rabbit serum was used as a negative control in immunohistologic experiments.
Valve Interstitial Cells Isolation and in vitro Analyses of Calcification
Human VICs were isolated by collagenase digestion. To promote calcification, cells were incubated for 7 days with a pro-calcifying medium containing: DMEM +5% FBS, 10−7 M insulin, 50 µg/ml ascorbic acid and NaH2PO4 at 2 mM. In some experiments, phosphonoformic acid (PFA) (1 mM) or LY294002 (50 µM) (Sigma, Oakville, ON, Canada) was added as specified. The calcium content was determined by the Arsenazo III method (Synermed, Monterey Park, CA, USA), which relies on the specific reaction of Arsenazo III with calcium to produce a blue complex. The results were measured at 650 nm on the Roche Diagnostics Modular P800 Elecsys (Roche Diagnostics, Laval, QC, Canada). This reaction is specific for calcium. Magnesium is prevented from forming a complex with the reactive. The results were normalized for protein contents and reported as percent changes.
RNA was extracted from valves explanted from 78 patients (50 CAVD and 28 controls). RNA was also extracted from cells during in vitro experiments. Total RNA was isolated with RNeasy micro kit from Qiagen (Qiagen, Mississauga, ON, Canada). The RNA extraction protocol was performed according to the manufacturer’s instructions using 100 mg of tissue. The quality of total RNA was monitored by capillary electrophoresis (Experion, Biorad, Mississauga, ON, Canada). One µg of RNA was reverse transcribed using the Quantitec Reverse Transcription Kit from Qiagen. Quantitative real-time PCR (q-PCR) was performed with Quantitec SYBR Green PCR kit from Qiagen on the Rotor-Gene 6000 system (Corbett Robotics Inc, San Francisco, CA, USA). Primers for the following transcripts were obtained from Qiagen (Mississauga, ON, Canada): SLC20A1, SLC20A2, SLC17A1, SLC34A1, SLC34A2, Akt-1, Akt-2, and Akt-3. The expression of hypoxanthine guanine phosphoribosyl transferase (HPRT) was used as a reference gene to normalize the results.
Transfection of Valve Interstitial Cells with pCMVAkt-1
VICs were seeded in 6-well plates (1×10 5 cells/well) for RNA extraction and in 12-well plates (5×104 cells/well) for analysis of calcification. After 24 hours, the cells were transfected with 1 µg of Akt-1 human cDNA ORF clone incorporated into the vector pCMV6-AC from Origene (Rockville, MD, USA). The transfection was done using the Turbofectin 8 system from Origene. After 48 hours, cells were harvested for RNA extraction or exposed to the mineralizing medium.
Transfection of Valve Interstitial Cells with siRNAs
VICs were cultured into 12-well plates, at a density of 6×104 cells per well, for determination of calcification, and into 6-well plates, at a density of 1×105 cells per well, for real-time PCR analysis performed in the transfection experiment. VICs were grown in a volume of 1 ml and allowed to adhere overnight in serum-containing antimicrobial-DMEM (5% CO2 and 37°C). The next day, VICs were transfected by incubation in a HiPerfect reagent containing 2700 ng siRNA (either negative control or SLC20A1, SLC20A2 and Akt-1 sequences) (Qiagen, Mississauga, ON, Canada). After two days, the medium was changed and a second transfection was done with either negative control or SLC20A1, SLC20A2 and Akt-1 siRNAs. After 24 hours, the transfection medium was replaced by a serum-containing antimicrobial-DMEM calcification medium supplemented with 2 mM NaH2PO4 (Sigma, Oakville, ON, Canada). VIC cultures were maintained for seven days in a calcification medium that was changed every two days. A third transfection was done three days after the second transfection, and cells were collected on the seventh day of the mineralization process. The transfection efficiency was verified by reduction of the target gene measured by real-time PCR, using SLC20A1, SLC20A2 and Akt-1 primers (Qiagen, Mississauga, ON, Canada).
Detection of Apoptosis
Apoptosis was documented in human VIC culture by TUNEL assay using an Apoptag Plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA). For the quantitative analysis, we counted 300 cells of each well and each condition. Then the percentage of apoptotic cells over the total counted cells was calculated. In some of the experiments with VICs, apoptosis was also confirmed using the Apopercentage apoptosis assay (Biocolor, Carrickfergus, UK), and the apoptosis levels were analysed using Image Pro Plus Version 6.1 image analysis software and expressed as pixel units. In some of the experiments, cyclosporine A (0.05 µM) or PFA (1 mM) were added to the growth medium to inhibit the mitochondrial permeability transition pore (MTP) or Pi transporters respectively.
Release of Cytochrome c in Valve Interstitial Cells
The cells were fixed with paraformaldehyde +0.1% Triton and washed in phosphate-buffered saline (PBS), before being incubated with rabbit antibody against cytochrome c (Cell Signaling Technology, Danvers, MA, USA) diluted in 5% BSA/PBS. After being washed, the cells were treated with an autofluorescence eliminator reagent and incubated with a secondary anti-rabbit antibody (Abcam, Cambridge, MA, USA).They were also treated with 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/ml). Images were acquired with an Olympus 1X81-UCB microscope using Image Pro Plus Version 6.1 and processed using ImageJ 1.44p.
Measurement of the Mitochondrial Membrane Potential (ΔΨm) in Valve Interstitial Cells
Valve interstitial cells were grown in a normal or mineralizing medium for four days, in the presence of 1 mM PFA where indicated. On the fourth day, cells were incubated for 20 minutes with 200 nM of MitoPT TRME (ImmunoChemistry technologies, Bloomington, MN, USA). The medium was replaced with a complete medium without phenol red, to remove excess dye. The cells with intact mitochondrial membrane potential were counted in comparison to the total population. Epifluorescence microscopy was performed with an Olympus IX81 inverted microscope (40X) and an Olympus IEQ camera. Images were corrected for background, and subjected to fast iteration and a fine noise filter using Volocity 6.0.1 (Perkin Elmer).
Quantification of Akt and pAkt by ELISA
VICs were collected (1 ml from each well, 1.106 per condition) and centrifuged at 12,000 rpm for 10 minutes to pellet off dead cells and debris. The quantification of Akt or pAkt was determined in accordance with the manufacturer’s instructions (EMD Millipore, Billerica MA, USA) and normalized with protein contents.
Results were expressed as means ±SEM. For continuous data, values were compared between groups with Student t-test or a one-way ANOVA to test the group effect (when more than two groups were compared). Post hoc Tukey analyses were done when the p value of the ANOVA was <0.05. Categorical data were expressed as a percentage and compared with the chi-square test. A p value <0.05 was considered as statistically significant. Statistical analysis was performed with a commercially available software package (JMP IN 8.1).