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 μg 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 (n = 14), ovariectomized (Ovx) plus vehicle (corn oil; n = 14), or Ovx plus E2 (36 μg/mL, equivalent to a dose of 1.2 μg/kg/day via silastic tubing; n = 16; Sigma) . 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, n = 2; Ovx + vehicle, n = 3; Ovx + E2, n = 4). Therefore, the numbers completing the study were sham-operated (n = 12), Ovx, vehicle (n = 11), and Ovx, E2 (n = 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 μL) 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/μL).
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 . 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 μm 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(α)-actin (Abcam cat#5694 rabbit, 1.4 μg/mL), Ly-6G/-6C (NIMP-R14 rat, neutrophil marker) (Abcam, cat#2557 rat, 5 μg/mL), striatin (index of E2 receptor signaling; EMD Millipore, cat#AB5779 rabbit, 5 μg/mL), or ER-TR7 (fibroblast marker; Abcam cat#51824 rat, 2 μg/mL). Sections were washed and then a secondary antibody (biotinylated anti-rabbit; BA-1000, Vector Labs, 7.5 μg/mL), biotinylated anti-rat (BA-4001, Vector Labs, 2.5 μg/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 α-actin and striatin were done using the hue, saturation, and intensity (HSI) method with ImagePro Plus v.7 . 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 α-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 .
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 . 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 × 105 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 μg) 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) , 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 × 104 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 × 106 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 μL 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 μg) was electrophoresed on a 12 % reducing SDS-PAGE gel. Antibodies utilized for Western analyses were α-actin (Abcam, cat#5694), β-actin (Sigma mouse, cat#A5441), SM22α/transgelin (Abcam rabbit, cat#14106), and TGF-β (Abcam rabbit, cat#66043; which recognizes all three isoforms of TGF-β). 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 μ-Dishes (ibidi, Inc.) at a cell density of 5 × 105 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 μLs 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 . 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 μl) was incubated in buffer (Na2HPO4, 0.1 M; EDTA, 0.02 M; maleate buffer, pH 6.5; total volume of 250 μl) containing phenylmethylsulfonyl fluoride (2 /250 μl 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).
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 α-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.