Methods
Animal models
Experiments were performed on mesenteric arteries from Male Wistar rats
(Charles River, Margate, UK) ages 11-14 weeks (200-350 g) from the
Biological Research Facility, St George’s, London, UK; and from the
Animal Resources Center, Perth, Australia. Animals were housed in cages
with free access to water and food (RM1; Dietex Inter-national, UK)ad libitum , with a 12-hour light/dark cycle and constant
temperature and humidity (21 ± 1°C; 50% ± 10% humidity) in accordance
with the Animal (Scientific Procedures) Act 1986, the guidelines of the
National Health and Medical Research Council of Australia and the UNSW
Animal Ethics and Experimentation Committee (AEEC #18/86B). Animals
were kept in a bedding of LSB Aspen woodchip. Animals were culled by
either cervical dislocation with secondary confirmation via cessation of
the circulation by femoral artery severance or were anaesthetized with
sodium pentathol (intraperitoneal, 100 mg/kg) in accordance with
Schedule 1 of the ASPA 1986.
Either whole mesenteric plexus or
2nd/3rd/4th order
MA were used with vessel order identified from the second bifurcation of
the superior mesenteric artery. Arteries were dissected, cleaned of fat
and adherent tissue and stored on ice within physiological salt solution
(PSS) of the following composition (mmol-L-1); 119
NaCl, 4.5 KCl, 1.17 MgSO4.7H20, 1.18
NaH2PO4, 25 NaHCO3, 5
glucose, 1.25 CaCl2.
Reverse transcription quantitative polymerase chain
reaction
To investigate gene expression in fresh ECs we used a subtractive
approach rather than generating pure native EC samples. Relative
fold-changes in expression levels of VSMC/EC markers and Kcnqtranscript was determined in denuded MA samples (an EC(-) population)
compared to whole MA samples (an EC(+) population) via Reverse
transcription quantitative polymerase chain reaction (RT-qPCR).
EC(-) MA samples were prepared as described previously (Askew Page et
al., 2019); Briefly, vessels were cut open longitudinally and pinned on
a Sylgard dish, the lumen of the vessel was then rubbed with human hair,
and vessels washed in 0.1% (phosphate buffered saline) PBS-Triton X for
1 x 1 min, then 3 times in PBS (1 minute each) on ice. EC(+) MA samples
were whole MA plexus that had not undergone EC removal as above.
mRNA from both whole MA EC(+) and MA EC(-) was extracted using Monarch
Total RNA Miniprep Kit (New England BioLabs, Ipswich, Massachusetts,
USA) with a LunaScript RT SuperMix Kit (New England BioLabs, Ipswich,
Massachusetts, USA). Quantitative analysis of relative gene expression
was assessed via CFX-96 Real-Time PCR Detection System (BioRad,
Hertfordshire, UK). Samples were run in duplicate to account for
variation. Samples were run in BrightWhite qPCR plate (Primer Design,
Camberley, UK), with each well containing 20 µL of reaction solution
containing: 10 µL of PrecisionPLUS qPCR Master Mix (Primer Design,
Camberley, UK), 300 nmol-L-1 of gene specific target
primer (Thermofisher scientific, Waltham, Massachusetts, USA) and 10 ng
of cDNA sample made up to 20 µL total volume with nuclease free water.
Run protocol: 1. activation step (15 min:95°C), 2. denaturation step (15
sec: 94°C), 3. annealing step (30 sec: 55°C) and 4. extension step (30
sec: 70°C). Steps 2- 4 were repeated x 40. Quantification cycle (Cq) was
determined via Bio-Rad CFX96 Manager 3.0. Values are Cq values
normalised to housekeeper genes expressed as a 2-ΔΔCqof MA EC(-) compared to MA EC(+) samples. Appropriate reference genes
including ubiquitin C (UBC), cytochrome C1 (CYC1) and
Beta-2-microglobulin (B2M; Primer design, UK) were determined using
qbase+ PCR analysis system (Biogazelle, Ghent, Belgium) geNorm
programme. See Table 1.1 for a list of the primers used in the following
(Askew Page et al., 2019; Thomas A. Jepps et al., 2011); ThermoFisher
Scientific).
Immunocytochemistry
2nd and 3rd order MA segments were
enzymatically digested to obtain freshly isolated ECs as previous
(Greenberg et al., 2016). Briefly, vessels were washed in Hanks’
Balanced Salt Solution (HBSS; ThermoFisher Scientific, GIBCO, 14170-088)
containing 50 μmol-L-1 CaCl2 for 5 min
at 37 °C and then placed in 1mg/mL collagenase IA (Sigma Aldrich, C9891,
UK) in the same solution for 15 min at 37°C. Vessel were washed in HBSS
containing 50 μmol-L-1 CaCl2 for 10
min at 37°C. The supernatant was removed and the vessels cells suspended
in fresh HBSS containing 0.75 mmol-L-1CaCl2. ECs were dissociated using a wide-bore
smooth-tipped pipette. The cell-containing solution was plated onto
coverslips and left at RT for 1 h before use.
Freshly dispersed ECs, together with residual VSMC, were fixed in 4%
paraformaldehyde (Sigma-Aldrich, UK ) in PBS for 20 min at RT as
previously described (Barrese, Stott, Figueiredo, et al., 2018). Cells
were treated with 0.1 mol-L-1 glycine for 5 min and
incubated for 1 h with blocking solution (PBS-0.1% Triton X-100-10%
bovine serum albumin) at RT. Following the incubation overnight at 4°C
with primary antibodies (Table 1.2) diluted in blocking solution
(anti-PECAM-1 for ECs, anti-α-actin for VSMCs and
anti-KV7.4 or KV7.5 channel for
ECs/VSMCs), cells were then washed for 20 min with PBS, incubated for 1
hr at RT with the secondary conjugated antibodies diluted in blocking
solution. Excess secondary antibody was removed by washing with PBS and
mounted using media containing DAPI for nuclei counterstaining. Using
triple staining, ECs and VSMC were differentiated via the following: ECs
were positive for anti-CD31 (endothelial cell-specific marker) and
negative for anti-α-actin (data not showed); while VSMC was positive for
anti-α-actin and negative for anti-PECAM-1 (data not showed). Cells were
analysed using a Zeiss LSM 510 Meta argon/krypton laser scanning
confocal microscope (Image Resource Unit St George’s University of
London).
Immunohistochemistry
Animals were anaesthetized with sodium pentathol (intraperitoneal, 100
mg/kg) and perfusion fixed (Sandow, Goto, Rummery, & Hill, 2004) in 2%
paraformaldehyde in 0.1 mol-L-1 PBS. Third to
4th order mesenteric artery segments were dissected,
opened laterally and pinned as a sheet to a Sylgard dish. Segments were
washed in PBS (3 x 5 min), incubated in blocking buffer (PBS with 1%
BSA and 0.2% Triton) at room temperature (RT) for 2 h and then
overnight with primary antibody (Table 1.2) in blocking buffer at
4oC, washed again (3 x 5 min with gentle agitation),
and incubated in secondary antibody (Table 1.2; matched to the
respective primary) in PBS with 0.1% Triton in PBS for 2 h at RT.
Tissue was mounted on slides in anti-fade media containing propidium
iodide (PI) or 4′,6-diamidino-2-phenylindole (DAPI; Table 1.2) and
imaged with uniform confocal settings. Incubation of tissue with
secondary only was used as a ‘zero’ setting for confocal imaging.
Controls involved substitution of primary with isotype control, with
concentration (where provided by manufacturer) matched, or 10-fold
higher than the respective antibody of interest (Table 1.2). Working Ab
dilutions were prepared in accordance with previous work (Chadha et al.,
2012; Thomas A. Jepps, Greenwood, Moffatt, Sanders, & Ohya, 2009).
Confocal image stacks were collected at 0.2 µm intervals. The optimal
rinsing protocol was determined by incubating in secondary only; and
rinsing after successive 5 min incubations until fluorescence was
reduced to background. Note that if this was not done secondary alone
was specifically highly localized to IEL hole sites; as potential false
positives at such sites; suggesting that such sites have an affinity for
IgG-secondary label alone.
Immunoelectron microscopy
Animals were anaesthetized as above and perfusion fixed in 0.2%
glutaraldehyde and 2% paraformaldehyde in 0.1 mol-L-1PBS (pH 7.4). Mesenteric artery segments (~2 mm in
length) were washed (3 x 5 min) and processed in a Leica EMPACT 2
high-pressure freezer using 0.7% low melting agarose as a
cryoprotectant. Samples were then freeze-substituted in a Leica AFS2
into 0.2% uranyl acetate in 95% acetone (from -85 to -50oC) and infiltrated with Lowicryl (at -50oC), before UV polymerization (2 d each at -50 and 20oC; (Zechariah et al., 2020).
Individual serial transverse sections (~100 nm) were
mounted on Formvar-coated slot grids and processed for antigen
localization as for confocal immunohistochemistry (per above and Table
1.2). The secondary used was 5 or 10 nmol-L-1colloidal gold-conjugated antibody (1:40; 2 h) in 0.01% Tween-20.
Sections were imaged at x10-40,000 on a JEOL transmission electron
microscope at 16 MP (Emsis, Morada G3). Background gold label density
was determined from randomly selected (4 x) 1 x 1 µm regions per sample
of lumen and IEL, compared to the same sized regions of interest in the
endothelium.
Wire Myography
2nd order MA segments (~2 mm in
length) were mounted on 40 µm tungsten wire in a tension myograph
chamber (Danish Myo Technology, Arhus, Denmark) containing 5 mL of PSS
(composition, as above) oxygenated with 95% O2 and 5%
CO2 at 37°C. Vessels then underwent a passive force
normalization process to achieve an internal luminal circumference at a
transmural pressure of 100 mmHg (13.3 kPa) to standardize
pre-experimental conditions (Mulvany, 1977). Force generated was first
amplified by a PowerLab (ADInstruments, Oxford, UK), and recorded by
LabChart software (ADInstruments, Oxford, UK). Vessels were then
challenged with 60 mM [K+] to determine viability.
Vessels were then constricted with 10 µmol-L-1methoxamine, an α-1 adrenoreceptor agonist, and EC integrity determined
via addition of 10 µmol-L-1 CCh. Vessels displaying
≥90% vasorelaxation in response to CCh (10 µmol-L-1)
were considered EC positive (EC+). Vessels were denuded of ECs by gently
passing a human hair through the lumen. Vessels expressing ≤10%
vasorelaxation in response to CCh (10 µmol-L-1) were
considered EC negative (EC-). During functional investigations, all
vessels were pre-constricted with the thromboxane A2 receptor agonist
U46619 (300 nmol-L-1) to elicit an
EC80 contraction. Concentration-dependent relaxant
responses to S-1 (0.1-10 µmol-L-1), ML213 (0.1-10
µmol-L-1), ML277 (0.03-1 µmol-L-1),
CCh (0.3-10 µmol-L-1) and S-nitroprusside (SNP; 0.01-3
µmol-L-1) were determined in the presence and absence
of ECs, linopirdine (10 µmol-L-1), HMR-1556 (10
µmol-L-1), ML133 (20 µmol-L-1),
barium chloride (BaCl; 100 µmol-L-1), L-nitroarginine
methyl ester (L-NAME; 100 µmol-L-1), TRAM34 (1
µmol-L-1), Apamin (10 nmol-L-1),
4-aminopyridine (4-AP; 1 mmol-L-1) and
tetraethylamonium (TEA; 1 mmol-L-1).
Dara and statistical
analysis
All functional figures express mean data from at least 5 animals
±standard error of the mean (SEM). For functional experiments involving
cumulative concentrations, a transformed data set was generated using;
X=Log(X), to reduce representative skew. A four parametric linear
regression analysis was then performed using the following equation;
(Log(Agonist) vs. response – variable slope (four parameters
bottom/hillslope/top/EC50)) using GraphPad Prism
(Version 8.2.0) to fit a CEC to the figure. For data comparing multiple
groups, a two way-ANOVA followed by a post hoc Bonferonni test,
to account for type 1 errors in multiple comparisons was performed for
comparison of mean values. Significance values are represented as
follows; P <0.05 (*). The data and statistical analysis
comply with the recommendations on experimental design and analysis in
pharmacology (Curtis et al., 2018).