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.