Gene silencing of endothelial von Willebrand Factor attenuates angiotensin II-induced endothelin-1 expression in porcine aortic endothelial cells

Expression of endothelin (ET)-1 is increased in endothelial cells exposed to angiotensin II (Ang II), leading to endothelial dysfunction and cardiovascular disorders. Since von Willebrand Factor (vWF) blockade improves endothelial function in coronary patients, we hypothesized that targeting endothelial vWF with short interference RNA (siRNA) prevents Ang II-induced ET-1 upregulation. Nearly 65 ± 2% silencing of vWF in porcine aortic endothelial cells (PAOECs) was achieved with vWF-specific siRNA without affecting cell viability and growth. While showing ET-1 similar to wild type cells at rest, vWF-silenced cells did not present ET-1 upregulation during exposure to Ang II (100 nM/24 h), preserving levels of endothelial nitric oxide synthase activity similar to wild type. vWF silencing prevented AngII-induced increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity and superoxide anion (O2−) levels, known triggers of ET-1 expression. Moreover, no increase in O2− or ET-1 levels was found in silenced cells treated with AngII or NOX-agonist phorbol ester (PMA 5 nM/48 h). Finally, vWF was required for overexpression of NOX4 and NOX2 in response to AngII and PMA. In conclusion, endothelial vWF knockdown prevented Ang II-induced ET-1 upregulation through attenuation of NOX-mediated O2− production. Our findings reveal a new role of vWF in preventing of Ang II-induced endothelial dysfunction.

The analysis was performed in three independent experiments performed in triplicate.

Real-Time PCR assay
Total RNA was extracted using an RNAesy kit (Qiagen, Hilden, Germany) and retrotranscribed with Reverse iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer's instructions. The trascriptomic profile of endothelial vWF and angiotensin II receptor type 1 (ATR1) was assessed by Real-Time PCR assay. The reactions were performed in duplicate in the Bio-Rad C1000™ thermal cycler (CFX-96 Real-Time PCR detection systems, Bio-Rad Laboratories Inc., Hercules, CA, USA) as previously described 3,4 . EvaGreen, a third-generation fluorophore, was used to monitor cDNA amplification (SsoFAST EvaGreen Supermix, Bio-Rad Laboratories Inc., Hercules, CA, USA). PCR was performed in a volume of 20µl per reaction; to minimize the influence of PCR inhibitors in Real-Time applications, all cDNA samples were diluted 1:5 and 0.2µM of each primer (Eurofins Genomics, Germany), 1X SsoFAST EvaGreen SuperMix (Bio-Rad Laboratories S.r.l., Milan, Italy) and sterile water were added.
Amplification protocol started with 98°C for 30s followed by 40 cycles at 95°C for 5s and 60°C for 30s. To assess the product specificity, melting curve analysis systematically checked amplicons. Melting curves were generated from 65°C to 95°C with increments of 0.5°C/cycle. The primer pairs specific for both target and reference genes (Suppl. Data: Table 2) were designed with Primer Express Version 2.0 (Applied Biosystems); whenever possible, intron-spanning primers were selected to avoid amplification of genomic DNA.
A standard curve obtained by scalar dilution of a cDNA pool (1:5, 1:25, 1:125, 1:625) was generated to verify PCR efficiency and a linear standard curve, R 2 , greater than │0.990│was generated, as reported in Supplemental Figure 1.
In an effort to provide greater transparency of our results between research laboratories, this study was carried out to conform to the Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE) 5

. A MIQE checklist was listed in
Supplemental Table 2.
To verify vWF presence in samples, the amplified products of both vWF and one of the reference genes analyzed (TOP2B, Topoisomerase II beta) were then electrophoresed on 1.5% (w/v) agarose gel, making parallel with a DNA Ladder both 50 than 100 bp (Promega Corporation, USA), stained with Gel Star Stain (Lonza, Switzerland, CH) and displayed on an ultraviolet transilluminator at 260nm (Benchtop UV transilluminator, UVP, Euroclone).
GeNorm software was used to define the most stably expressed gene set, as previously described 6 . Two stably expressed reference genes were selected among ten of the most studied in literature and the geometric mean of these was used for normalization of mRNA expression (Top2B and HPRT-1, M value< 1) ( Table 2). The relative quantification was performed by ∆∆Ct method using BioRad's CFX96 manager software.

Immunofluorescent detection of NOX4
PAOECs were grown on chamber slides, fixed, and incubated twice for 10 min with PBS 0.1% Triton X-100 to permeabilize plasma membranes, then extensively washed in PBS; after 1h incubation with blocking buffer (PBS 2% BSA), cells were incubated with primary antibody (anti-NOX4 antibody, 1:100) for 2h at 37°C. Cells were then washed in PBS 0.5% BSA and incubated with secondary antibody (goat anti-rabbit, Alexa-488 conjugated, 1:500, Santa Cruz Biotechnology, Inc, USA) for 1h at 37°C. Slides were mounted with Vectashield antifade mounting medium containing DAPI (Vectorlabs, CA, US) and analyzed. All antibodies were diluted in PBS 1% BSA. In general, 5 images were acquired for each condition with a Leica DM2500 fluorescence microscope with a setting of 40X magnification. Quantification of relative fluorescence intensity per cell was performed with ImageJ software and obtained data were analyzed for statistical significance with graphPad PRISM. The experiments were performed in triplicate.