Histological, protein and RNA studies in aorta.
Aortic Histochemistry Staining: Paraffin-embedded aortic sections (4 µm thick) were placed in coated slides, deparaffinized, rehydrated through alcohol gradients (100-95-70% alcohol) and washed in distilled water. To visualize the aortic structure, Hematoxylin-Eosin and Van Gieson staining were performed as previous described (Esteban et al., 2011). Images were obtained using a Leica DMD108 microscope (Leica Microsystems).
Transmission electronic microscopy (TEM). Samples were fixed with a 1% glutaraldehyde-4% formaldehyde solution in 0.1M cacodylate buffer. Post-fixation process consisted on one hour and forty minutes of 1% osmium tetraoxide solution incubation at room temperature. Afterwards, samples were washed in distilled water, stained with 0.5% uranyl acetate solution along ten minutes, dehydrated in increasing graded alcohol solutions (30%, 50%, 70%, 95% and 100%) and incubated in acetone. Samples were included into epoxy Durcupan ACM resin (Sigma-Aldrich, Darmstadt, Germany) with increasing proportions of Resin/acetone (1:3, 1:1, 3:1 and pure resin). Samples were polymerized in 60ºC oven during 48 hours. Ultrathin slides were obtained by using Leica Ultracut S (60nm), which were included into 200 mesh cupper grids and counterstained with uranyl acetate and plump citrate. Slides were visualized by transmission electron microscopy JEOL JEM1010 (100 kV).
Multiple reaction monitoring (MRM) mass spectrometry (MS) analysis. Whole mouse aortas were homogenized, denatured, resolubilized and trypsinized in 50 mmol/L ammonium bicarbonate at 37°C overnight. Tryptic peptides were purified on custom made Poros R2/R3 (Thermo Scientific) micro columns, dried, reconstituted and finally resuspended in 2% acetonitrile, 0.1% formic acid. Tryptic peptide concentration was determined and normalized across samples. Heavy isotope-labelled standard peptides (JPT) were then added to each sample in a 1:1 volume ratio. 2.5 ug endogenous peptide was run for each sample on an Easy-nLC II nano liquid chromatography (LC) system using a C18 trapping column for desalting and a C18 analytical column for peptide separation (Thermo Scientific). Peptides were eluted, ionised and analyzed on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific) in a selected reaction monitoring mode. MRM raw files were processed using Pinpoint 1.3 (Thermo Scientific). The peak area ratio between endogenous and heavy isotope labelled spiked peptide (1-3 peptides per protein) was used for data analysis. Peak area ratios for individual proteins were normalized to GAPDH and ACTB.
Western blot studies were done in whole aorta samples as described (Rodriguez-Vita et al., 2005). Protein content was determined by the bicinchoninic acid method (BCA; ThermoFisher), and protein were loaded and separated on 10-12% polyacrylamide-SDS gels under reducing conditions. Afterwards, proteins were blotted onto nitrocellulose membranes (BioRad), incubated with the corresponding primary and secondary antibodies and developed using the ECL substrate (Millipore). Results were analyzed by LAS 4000 (GE Healthcare) and obtained bands quantified by using the QuantityOne software (BioRad). The primary antibodies employed were MMP-8 (1/500; sc-514803; Santa Cruz Biotechnologies) and GAPDH (1/5000; CB1001; Millipore,).
Zymography. Protein samples were loaded and separated onto 10% polyacrylamide-SDS gels without reducing conditions with 1% of gelatin (Sigma Aldrich). Gels were washed with 2,5% Triton-X 100 three times for 30 minutes, followed by a final distilled water washing for 30 minutes. Gels were incubated in a reaction buffer (50 mmol/L Tris-HCl pH 7.5, 200 mmol/L NaCl, 10 mmol/L CaCl2) overnight at 37oC with agitation, stained with Coomassie Brilliant-Blue (Sigma Aldrich) during 30 minutes and incubated in a bleaching buffer to release the leftover Coomassie. Results were analyzed by BioRad Gel DocTM EZ Imager in fresh and band densitometry was quantified by using the QuantityOne software (BioRad).
In situ Zymography. For localization of gelatinolytic activity, in situ zymography was performed as previously described (Lindblad, 2001). Briefly, 8 µm thick aortic sections were deparaffinized and rehydrated through graded alcohols. Each sample was incubated with substrate (DQgelatin, Invitrogen) overnight in a dark humidity chamber at 37oC. Nuclei were stained with DAPI (1/10000, Sigma-Aldrich) and samples mounted by using ProlongTM Gold antifade reagent (Invitrogen). Fluorescence (Ex/Em 495/515 nm) was visualized in a Leica DM-IRB confocal microscope. In order to verify the contribution of MMPs, control slides were preincubated with 20mM EDTA for 1 hour. 20 mmol/L EDTA was added to substrate solution to be used also as a technique control.
RNA extraction and RNA-sequencing studies. Aortic total mRNA as obtained by the TRIzol method (Invitrogen, Life Technologies, Philadelphia, PA) as previously described (Lavoz et al., 2020). Libraries were prepared according to the instructions of the Kit “NEBNext Ultra Directional RNA Library Prep kit for Illumina” (New England Biolabs), following the protocol “Poly(A) mRNA Magnetic Isolation Module”. RNA quality was evaluated according to RIN numbers, quantified using a RNA 6000 nanoLabChipin an Agilent 2100 Bioanalyzer (ranges 6-7). The input yield of total RNA to start the protocol was 900 ng. The fragmentation time used was 8-15 minutes according to RIN values. The rest of the protocol was performed according to manufacturer instructions. Obtained libraries were validated and quantified by DNA7500 LabChip kit (Agilent 2100 Bioanalyzer). An equimolecular pool (8-10 aortas per group) of libraries were titrated by quantitative PCR using the “Kapa-SYBR FAST qPCR kit forLightCycler480” (KapaBioSystems) and a reference standard for quantification. The pool of libraries was denatured prior to be seeded on a flowcell at a density of 2,2 pmol/L, where clusters were formed and sequenced using a “NextSeq™ 500 High Output Kit”, in a 1x75 single read sequencing run on a NextSeq500 sequencer. Approximately 50 million pass-filter reads (range 42-54) were produced for each pool of samples and used for further bioinformatics analysis. FASTq files were analyzed using the XploreRNA service from Exiqon-Quiagen. The obtained results were studied using the Functional Annotation Tool of David database (49,50). *RNA-seq data are available at the NCBI SRA archive with BioProject record PRJNA669604, and BioSample records SAMN16442923; SAMN16442924; SAMN16442925 and SAMN16442926.