healthmedicinet

Mice

Female low-density lipoprotein-receptor-deficient (Ldlr ^?/?
) mice on a C57BL/6 background (13 times backcrossed, stock #002207, Jackson Laboratory
mice; 2–3 months of age; total of 57 females) were bred in-house. To promote adult
AAA susceptibility, female mice were administered (subcutaneously) 1 dose of testosterone
(400 ?g per mouse) at 1 day of age. At 2 months of age, females were fed a Western
diet (Teklad, TD88137, 42 % kcal from fat, 0.2 % cholesterol) and were infused with
AngII (1000 ng/kg/min; Bachem) by micro-osmotic pump (Model 1004, Alzet Inc.) for
28 days. The Western diet was continued through study duration. Ultrasound was used
as described below to quantify suprarenal aortic lumen diameters in anesthetized mice
(isoflurane, 2–3 %). Mice (total of 44) exhibiting an increase (33 %) in aortic lumen
diameter compared to baseline (day 0) were classified as having an AAA. Mice were
stratified to three groups: sham surgery (?=?14), ovariectomized (Ovx) plus vehicle (corn oil; ?=?14), or Ovx plus E2 (36 ?g/mL, equivalent to a dose of 1.2 ?g/kg/day via silastic
tubing; ?=?16; Sigma) 14]. We stratified AAAs by aortic lumen diameter to each group such that mean diameters
of the three groups (at day 28 of AngII infusions) were similar. After surgery (sham
or Ovx), mice were implanted with minipumps containing AngII or silastic implants
containing vehicle or E2, with replacement of minipumps and silastic implants every
28 days for two more months. During the final 2 months of AngII infusions, aortic
ruptures occurred in each group (sham, ?=?2; Ovx?+?vehicle, ?=?3; Ovx?+?E2, ?=?4). Therefore, the numbers completing the study were sham-operated (?=?12), Ovx, vehicle (?=?11), and Ovx, E2 (?=?12). All procedures follow the National Institutes of Health Guide for the Care
and Use of Laboratory Animals and were approved by the University of Kentucky Institutional
Animal Care and Use Committee.

Blood cell content

Whole blood (20 ?L) was placed in EDTA-coated tubes (cat#20.1278.100, Sarstedt) and
placed on a rotator until all samples were collected. Samples were analyzed using
a Hemavet 950FS (ERBA Diagnostics Inc.), and results were averaged (K or 1000 cells
per microliter)(K/?L).

Ovariectomy

Surgeries were performed on anesthetized female mice (2–3 % isoflurane) administered
pre- (30 min prior) and post-analgesic (48 h after surgery; flunixin; 2.5 mg/kg).
Mice were shaved on each flank and a depilatory cream was used to remove hair, followed
by sterilizing with povidone-iodine/ethanol (three times). An incision was made through
the skin to visualize the abdominal wall, where an additional incision was made (1–3 mm)
to locate the ovaries. Fallopian tubes were collapsed using a hemostat and the ovaries
were removed. The wound site was then cauterized using a high-temperature fine-tip
loop and the hemostat was released. The wound site was monitored for bleeding, and
the abdominal wall was sutured (absorbable, Vicryl 5.0) and skin was stapled (Autoclip
stapler). The site was then treated with povidone-iodine, and mice were allowed to
recover in a clean cage on a heating pad.

Quantification of AAAs by ultrasound and ex vivo measurements

Ultrasound was performed using a 55-MHz probe with a Vevo 2100 high-resolution imaging
system (VisualSonics, Inc.) to quantify suprarenal aortic lumen diameters 15]. Mice were anesthetized (2–3 % isoflurane), and abdominal hair was removed by shaving
and applying a hair depilatory cream (Nair, Inc.). Ultrasound was used to quantify
aortic lumen diameters once/week during months 2 and 3 of AngII infusions by two independent
observers blinded to the experimental design. To quantify AAA maximal external diameters
at study endpoint, aortas were removed, placed in fixative (10 % formalin), cleaned
of extraneous tissue, and mounted on a black wax background. Images were taken with
a Nikon SMZ800 dissecting microscope with camera, and a ruler was included in the
frame. Image analysis was performed using Nikon Elements Version 3.2.

Characterization of AAA tissue sections

Immersion-fixed AAAs were incubated in 30 % sucrose, placed on wax, and pinned. Serial
sections of AAAs (at 10 ?m intervals) covering a distance of 4–8 mm were placed on
each slide, which was stored at ?20 °C. For immunostaining, tissue sections were incubated
at 60 °C for 1–2 h to remove water, then cleared with 100 % xylene for 5 min, followed
by alcohol dehydration at 100, 95, and 75 %, and then distilled water (2 min each).
Redusol (0.05 % chromic acid) was incubated with AAA sections for 2 min at 40 °C.
Sections were washed with automation buffer (GeneTex, Inc.) and incubated in hydrogen
peroxide (1 % in methanol) to extinguish endogenous peroxidase activity, and then
a blocking agent (1.5 % goat or rabbit serum in PBS) was included for 5 min at 40 °C.
AAA tissue sections were incubated (30 min at 40 °C) with the following antibodies:
alpha(?)-actin (Abcam cat#5694 rabbit, 1.4 ?g/mL), Ly-6G/-6C (NIMP-R14 rat, neutrophil
marker) (Abcam, cat#2557 rat, 5 ?g/mL), striatin (index of E2 receptor signaling;
EMD Millipore, cat#AB5779 rabbit, 5 ?g/mL), or ER-TR7 (fibroblast marker; Abcam cat#51824
rat, 2 ?g/mL). Sections were washed and then a secondary antibody (biotinylated anti-rabbit;
BA-1000, Vector Labs, 7.5 ?g/mL), biotinylated anti-rat (BA-4001, Vector Labs, 2.5 ?g/mL),
was incubated for 30 min at 40 °C. Tissue sections were washed, incubated with Vectastain
and rinsed to detect peroxidase activity using an AEC chromagen substrate. Sections
were counterstained with hematoxylin unless utilized for image quantification. Image
quantification of ?-actin and striatin were done using the hue, saturation, and intensity
(HSI) method with ImagePro Plus v.7 13]. Briefly, sham sections (200× magnification) were utilized to set up the baseline
measurements for HSI. Next, a mask was created for each image and an area was boxed
for each section. For ?-actin, the region of interest was the thrombus region within
the medial break of the AAA. For striatin, the region of interest was the medial smooth
muscle layer of the abdominal aorta. Neutrophil counts were performed in areas of
aneurysm medial break in a blinded fashion. At least 5–6 sections were quantified
per mouse with N?=?3 mice per group.

Quantification of atherosclerosis

After quantification of external diameters of AAAs, aortas were cut open and pinned
to quantify atherosclerosis in the aortic arch. Arch areas were defined by drawing
a 3-mm line from the left subclavian artery and every lesion within this area was
summed and divided by the total arch area to calculate the percent lesion area 16].

Primary abdominal aorta-derived smooth muscle cell (SMC) isolation and culture

Primary abdominal aorta-derived SMC were isolated from C57BL/6 female mice (6–8 weeks
of age) using collagenase/elastase with soybean trypsin inhibitor digestion (1 mg/mL
collagenase, cat#LS004174, 0.744 units/mL elastase, cat#LS002279, 1 mg/mL soybean
trypsin inhibitor, cat#LS003570, Worthington) of the suprarenal and infrarenal aorta
5]. Abdominal aortic SMC were grown in DMEM/F12 media without phenol red with 2 % penicillin/streptomycin,
1 % fungizone (Invitrogen), and 20 % female bovine serum (Tissue Biologics) until
cells were confluent, and then trypsinized (0.25 %). All studies in SMCs were performed
on cells from passage 3–10. Cells past passage 3 were grown in 10 % female bovine
serum in culture media.

Real-time PCR (RT-PCR)

Cells were grown in six-well plates at a density of 5?×?10
⁵
 cells/mL in 1 % charcoal-stripped FBS media (Invitrogen) for 24 h prior to the start
of each experiment. Cells were incubated with vehicle (dimethyl sulfoxide, DMSO, final
0.1 %) or E2 (0, 1, 10, 50, or 100 nM) for 48 h. RNA was extracted from cells using
the Promega SV Total RNA Isolation System for Tissues and reverse transcribed (0.2 ?g)
using the Quanta qScript cDNA supermix (Quanta Biosciences). cDNA was diluted (1:10)
and amplified using the Perfecta SYBR Green FastMix (Quanta Biosciences). Groups were
normalized to vehicle using the delta delta Ct method (ddCt). Glyceraldehyde phosphate
dehydrogenase (GAPDH) mRNA abundance was used to control for DNA template concentrations.
Primers for proliferating cell nuclear antigen (PCNA) 17], forward 5?-CTAGCCATGGGCGTGAAC-3? reverse 5?-GAATACTAGTGCTAAGGTGTCTGCAT-3? and GAPDH,
forward 5?-GCCAAAAGGGTCATCATCTC-3? reverse 5?-GGCCATCCACAGTCTTCT-3? were utilized
for this study.

SMC proliferation assay

Abdominal aortic SMCs were cultured in 12-well plates and seeded at a density of 1?×?10
⁴
 cells/mL for 48 h in 1 % charcoal-stripped FBS plus complete media. After washing,
cells were incubated with vehicle (DMSO, final 0.1 %) or E2 (1–100 nM) for 48 h. Cells
were harvested and a fluorescent-based assay (CyQUANT, Invitrogen) was used to quantify
nucleic acid concentrations. A standard curve was generated using thoracic or abdominal
cells at a starting concentration of 1?×?10
⁶
 cells/mL. Detection was based on an excitation of 480 nm and an emission of 520 nm.

Western blot analyses

Abdominal aortic SMCs were incubated in six-well plates with vehicle (DMSO, final
0.1 %) or E2 (1–100 nM) for 48 h prior to protein extraction. Extraction was performed
with M-PER with protease (Roche-Mini tablets) and phosphatase inhibitors (Thermo Fisher
phosphatase inhibitor tablets; 200 ?L per well) and samples were sonicated for 10 s
on a setting of 10 (Misonix, XL-2000) and then incubated on ice for 30 min. Cell extracts
were centrifuged (10,000g?s) for 10 min to pellet cellular debris, and protein concentration was quantified
in supernatants (BCA assay; ThermoFischer). For Western analyses, protein (12 ?g)
was electrophoresed on a 12 % reducing SDS-PAGE gel. Antibodies utilized for Western
analyses were ?-actin (Abcam, cat#5694), ?-actin (Sigma mouse, cat#A5441), SM22?/transgelin
(Abcam rabbit, cat#14106), and TGF-? (Abcam rabbit, cat which recognizes all
three isoforms of TGF-?). Protein content was quantified using background subtraction
(50 pixels per blot subtracted) followed by the gel plug-in for ImageJ 1.48v and was
normalized to GAPDH (Sigma mouse, cat#G9295).

Wound healing assay

Abdominal aortic SMCs were grown in ibidi ?-Dishes (ibidi, Inc.) at a cell density
of 5?×?10
⁵
 cells/mL. After 21–24 h, inserts were removed with sterile forceps and incubated
with 3 % charcoal-stripped serum media containing either vehicle (DMSO, final 0.1 %)
or E2 (100 nM). Phase-contrast images were taken at time 0 and 21–24 h under 40× magnification.
Image analyses on areas that were not occupied by cells were summed and data were
averaged (Nikon Elements v3.2).

Quantification of plasma and serum components

Serum E2 concentrations were quantified with a rat/mouse serum estrogen ELISA (cat#ES180S-100,
Calbiotech, sensitivity of the assay = 3 pg/ml). Cholesterol was quantified in sera
using an enzymatic, colorimetric kit for esterified serum cholesterol (WAKO, cat#439-17501).
Fast performance liquid chromatography (FPLC, BioRad) was utilized to separate lipoprotein
particles using 50 ?Ls of serum from each mouse (N?=?3–4 mice per group). In order to analyze areas under the curve for each FPLC, software
was utilized (PeakFit v4.12) to determine chylomicron (CM) CM/very-low-density lipoprotein
(VLDL), intermediate and low-density lipoprotein (LDL), and high-density lipoprotein
(HDL) areas, and cholesterol concentrations (mg/dl) were determined from each area
18]. Plasma renin concentrations (PRC) were quantified by measuring angiotensin I generation
in the presence of an excess of exogenous angiotensinogen. Plasma was harvested from
mice in ice-cold EDTA (0.2 M). Mouse plasma (8 ?l) was incubated in buffer (Na
₂
HPO
₄
, 0.1 M; EDTA, 0.02 M; maleate buffer, pH 6.5; total volume of 250 ?l) containing
phenylmethylsulfonyl fluoride (2?/250 ?l reaction volume) for 30 min at 37 °C in a
shaking water bath. Plasma samples were incubated with an excess of exogenous rat
angiotensinogen (partially purified from nephrectomized rat plasma). The reaction
was terminated by placing samples at 100 °C for 5 min. Angiotensin I was quantified
by radioimmunoassay using a commercial kit (DiaSorin, cat# CA-1553).

Statistics

All in vitro studies were performed in duplicates or triplicates with at least three
or more replicates per assay. Aortic lumen diameters were analyzed using a linear
mixed model to compare time as a within group factor and E2 and sham/Ovx as between
group factors. Maximal external aortic AAA diameters were analyzed using a one-way
ANOVA. Quantification of immunostaining for ?-actin in AAA tissue sections was analyzed
by one-way ANOVA with Holms-Sidak post hoc analysis. Measurements in cultured abdominal
aortic VSMC incubated with E2 were analyzed by one-way ANOVA with Holms-Sidak post
hoc analysis. A t test was used for statistical analysis of wound healing results. All data were plotted
and analyzed by SigmaPlot v.12.3. Statistical significance was defined as P??0.05. Data are represented as mean?±?SEM.