2.1

In vitro cell culture

The human monocyte cell line THP-1 (ATCC, Manassas, VA) was cultured in Opti-MEM® I reduced serum medium (Invitrogen, San Diego, CA) supplemented with 20 U/mL penicillin (Invitrogen), 20 μg/mL streptomycin (Invitrogen), 0.05 μg/mL Fungizone® (amphotericin B) (Invitrogen), 10 μg/mL gentamycin (Sigma-Aldrich, St. Louis, MO), and 2% fetal bovine serum (FBS) (Lonza, Walkersville, MD). Primary smooth muscle cells, isolated from human coronary arteries (HCASMC), and endothelial cells, isolated from human umbilical veins (HUVEC), were each obtained from a commercial supplier (Lonza) at passage 3, and cultured according to supplier specifications. HUVEC were cultured in EGM-2 growth media supplemented with 2% FBS and a specific cocktail of hydrocortisone, hFGF-β, VEGF, R3-IGF-1, ascorbic acid, hEGF, heparin, gentamycin, and amphotericin B (Lonza) supplied by the manufacturer. HCASMC were cultured in SmGM-2 growth media supplemented with 5% FBS and a specific cocktail of insulin, hFGF-β, hEGF, gentamycin, and amphotericin B (Lonza) supplied by the manufacturer. All cells were cultured in a 37 °C and 5% CO ₂ environment, and re-fed every 2–3 days and passed upon reaching 80% confluence. All experiments were performed using cells at passage 8 or lower.

2.2

THP-1 foam cell differentiation and characterization

To induce FC differentiation, THP-1 cells were incubated with 10 ^-7 M phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 72 hours, followed by incubation with 100 μg/mL human acetylated low density lipoprotein (acLDL) (Intracel Resources, Frederick, MD) for 72 hours. To demonstrate FC formation, acLDL derived THP-1 FC as well as PMA derived THP-1 macrophages were stained with lipophilic Nile Red (1 μg/mL) (Sigma-Aldrich) for 10 minutes at room temperature. Nile Red staining allowed for visualization and imaging of FC containing intracellular lipid droplets using a Nikon Eclipse E800 microscope (Nikon Inc., Melville, NY) and photographed with a Coolsnap ES (Photometrics, Tucson, AZ) camera, using Simple PCI (Hamamatsu Corporation, Sewickley, PA) image capture software at an objective magnification of 20×. In addition, an Amplex® Red cholesterol assay (Invitrogen) was used according to manufacturer’s instructions to quantify total cholesterol levels in PMA derived THP-1 macrophages and acLDL derived THP-1 FC using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA). Total cholesterol levels were normalized to respective total protein content of cell lysates determined using a DC protein assay (Bio-Rad, Hercules, CA), with an endpoint of absorbance [A _750nm ] measured using a SpectraMax M2 microplate reader (n = 3). Lastly, a Quantigene® Plex assay (Affymetrix, Santa Clara, CA) was used according to manufacturer’s instructions to evaluate transcriptional changes in the mRNA levels of a panel of gene markers related to cholesterol efflux/lipid metabolism (FABP4, CD36, ABCA1, adipophilin, CD68) in PMA derived THP-1 macrophage and acLDL derived THP-1 FC lysates. This assay combines branched DNA (bDNA) signal amplification and multianalyte profiling bead technologies to enable detection and simultaneous quantification of multiple mRNA targets bound to fluorescent microspheres directly from cell lysates using a Bio-plex™ 100 suspension system (Bio-Rad) . The Quantigene® assay platform was selected based on a comprehensive FDA-led study showing that data generated using the Quantigene® assay platform are comparable to QPCR based methods, with both methodologies generating functionally equivalent results to a large extent when used to validate microarray platform results from the same set of samples . Background fluorescence (measured from microspheres not hybridized with mRNA) was subtracted from RNA-containing samples. Each gene marker was then normalized to a housekeeping gene, cyclophilin B (PPIB), a protein folding enzyme that is constitutively and ubiquitously expressed at high levels in cells. Data were then expressed as percentage fold change of marker levels in acLDL derived THP-1 FC normalized to PMA derived THP-1 macrophages (n = 3).

Two-step Taqman QPCR was performed to verify changes in mRNA levels obtained with the Quantigene® Plex assay. RNA from cell samples were isolated and purified using the RNeasy minikit (Qiagen, Valencia, CA). The isolated RNA was treated with RNase-free DNase I to remove genomic DNA according to the Qiagen protocol. The total RNA sample concentrations were quantified using a Nanovue spectrophotometer (GE Healthcare, Pittsburgh, PA), and the quality of each RNA sample was validated by measuring the A260/A280 ratio. First-strand complementary DNA (cDNA) was synthesized from 1 μg RNA using the high-capacity cDNA reverse transcription kit with random primers and RNase inhibitor in a final volume of 20 μL according to manufacturer’s instructions (Life Technologies, Carlsbad, CA). Reverse transcription reactions were carried out in a GeneAmp 9700 System (Life Technologies) at 25 °C for 10 minutes, 37 °C for 2 hours, and 85 °C for 5 minutes. cDNA was then stored at −20 °C. Taqman Gene Expression Assays for human FABP4 (assay ID#Hs01086177_m1), CD36 (assay ID#Hs01567185_m1), ABCA1 (assay ID#Hs01059118_m1), adipophilin (assay ID#Hs00605340_m1), CD68 (assay ID#Hs02836816_g1), and PPIB (assay ID#Hs00168719_m1) primers/probes were purchased from Life Technologies, and PCR amplification was performed by using the ABI 7500 real time PCR system (Life Technologies) in 96-well plates using the Taqman Universal PCR Master Mix (Life Technologies). All samples were performed in quadruplicates with the thermal cycling profile consisting of four stages: 50 °C for 2 minutes, 95 °C for 10 minutes, and 40 cycles of 15 seconds at 95 °C and 60 °C for 1 minute. The threshold cycle (C _t ) value of each gene were determined by the instrument’s software. Relative levels of mRNA were calculated using the delta–delta C _t method, which involves comparing C _t values between samples. PPIB, as in the Quantigene® Plex assay, was used as the housekeeping gene for normalization. The relative level of target genes was then expressed as percentage fold change in acLDL derived THP-1 FC normalized to PMA derived THP-1 macrophages (n = 3).

2.3

Effects of everolimus on THP-1 derived foam cell viability – Calcein AM/DAPI staining

THP-1 derived FC were seeded in 4-chamber tissue culture treated glass slides (BD Biosciences, Bedford, MA) at a density of 2.5×10 ⁴ cells/cm ² in a 37 °C and 5% CO ₂ incubator. Cells were then treated with or without everolimus (10 ^-5 M). After 24 hours, slides were washed with 1× PBS, pH 7.4 (Invitrogen), followed by staining with calcein AM (2 μM) (Invitrogen) for 45 minutes at room temperature. Slides were then washed with 1× PBS, counterstained with Vectashield DAPI mounting media (Vector Laboratories, Burlingame, CA), and sealed with coverslip. Fluorescent images from ten randomly selected viewing fields were captured with the imaging system described above. The total number of cells judged by DAPI fluorescence as well as number of calcein AM positive live cells were counted in each image and used to calculate the average percentage of viable cells in everolimus treated and control cell populations.

2.4

Effects of everolimus on HCASMC, HUVEC, and THP-1 derived foam cell apoptosis/necrosis

Total cell necrosis and induction of apoptosis by everolimus in HCASMC, HUVEC, and THP-1 derived FC were evaluated using a Cell Death Detection ^PLUS ELISA kit (Roche Applied Science, Mannheim, Germany), with an endpoint of absorbance, [A _405nm – A _490nm ], read on a SpectraMax M2 microplate reader. In separate experiments, HCASMC, HUVEC, or THP-1 derived FC were seeded in 24-well tissue culture plates at a density of 2.5×10 ⁴ cells/cm ² in a 37 °C and 5% CO ₂ incubator. After reaching ~80% confluency, cells were then treated with various doses of everolimus (10 ^-5 M, 10 ^-7 M, 10 ^-9 M, 10 ^-11 M) for 24 hours. Cells incubated with appropriate growth media only served as controls. Following everolimus incubation, culture supernatants were immediately removed and saved for analysis. Cells were then treated with lysis buffer supplied in ELISA kit, and lysates were saved for analysis. ELISA detection of histone-associated nucleosome concentration in supernatants was used to determine necrosis, while concentration in lysates was used to determine relative induction of apoptosis, all according to manufacturer’s instruction. Total protein content of cell lysates was determined using DC Protein Assay (Bio-Rad). Apoptosis and necrosis data for each sample were normalized to respective total protein to determine relative apoptotic and necrotic indices. Data were then expressed as percentage fold change of cells treated with everolimus normalized to cell controls (n = 4).

2.5

Effects of everolimus on markers related to apoptosis, autophagy, matrix remodeling, and inflammatory cytokine secretion from acLDL-Derived THP-1 foam cells

THP-1 derived FC were seeded in 6-well tissue culture plates at a density of 2.5×10 ⁴ cells/cm ² in a 37 °C and 5% CO ₂ incubator. After reaching ~80% confluency, cells were then treated with or without everolimus (10 ^-5 M) (n = 3). After 24 hours, culture media supernatants were collected and analyzed for a panel of secreted inflammatory cytokines (MCP-1, IL-1β, IL-8, TNF-α) using a microsphere-based Procarta® immunoassay kit (Bio-Rad) according to manufacturer’s instructions. Quantigene® Plex assay (Affymetrix), as described above, was used according to manufacturer’s instructions to evaluate the mRNA levels of several gene markers related to cell viability (survivin (anti-apoptotic), clusterin, MAP1LC3 (autophagic)) and matrix remodeling (matrix metalloproteinase 1, 9 (MMP1, MMP9)) in cell lysates. PPIB was used as an internal housekeeping gene for normalization of gene markers. Data were then expressed as percentage fold change in cells treated with everolimus normalized to cell controls. QPCR, as described above, was used to verify changes in human survivin (assay ID#Hs04194392_s1), clusterin (assay ID#Hs00156548_m1), MAP1LC3 (assay ID#Hs01076567_g1), MMP1 (assay ID#Hs00899658_m1), and MMP9 (assay ID#Hs00234579_m1) mRNA levels (n = 3). QPCR was also used to assess mRNA levels of inflammatory cytokines MCP-1 (assay ID#Hs00234140_m1), IL-1β (assay ID#Hs01555410_m1), IL-8 (assay ID#Hs00174103_m1), TNF-α (assay ID#Hs01113624_g1) (n = 3). The relative level of target genes was then expressed as percentage fold change in cells treated with everolimus normalized to cell controls. Additionally, ELISA assays for human MMP1, MMP9, and survivin (all from Abcam, Cambridge, MA) were purchased and used according to manufacturer’s instructions to quantitatively assess MMP1 and MMP9 protein expression in culture media supernatants and survivin protein expression in cell lysates (n = 6). Data were normalized to respective total protein content determined using a DC protein assay (Bio-Rad) and reported as percentage fold change in cells treated with everolimus normalized to cell controls.

2.6

In vivo hypercholesterolemic rabbit model

The experimental atherosclerotic rabbit model has been previously reported . Briefly, New Zealand White rabbits (3 to 4 kg) were fed an atherogenic diet (1% cholesterol and 6% peanut oil, F4366-CHL, Bio-Serv, Inc., Frenchtown, New Jersey) for 5 weeks. Iliac artery injury was induced 1 week following induction of a high-cholesterol diet using a Fogarty catheter (3-F). Under fluoroscopic guidance, premounted XIENCE PRIME (EES, 3×12 mm, Abbott Vascular, Santa Clara, CA) were implanted over lesions in alternating iliac arteries at nominal pressures to achieve a visual target stent-to-artery ratio of approximately 1.3:1. After stent implantation, post-procedural angiography was performed to document vessel patency, and the animals were allowed to recover. The diet was then switched to a low-cholesterol diet (containing 0.025% cholesterol) for the remainder of the study. Anti-platelet therapy consisted of aspirin (40 mg/day) given orally 24 hours before catheterization with continued dosing throughout the in-life-phase of the study, while single dose intra-arterial heparin (150 IU/kg) was administered at the time of catheterization. At the designated time points, animals were anesthetized followed by euthanasia and perfusion-fixation of stented arteries.

2.7

Histology and immunohistochemistry

Samples from the atherosclerotic rabbit ilio-femoral arteries were processed and sectioned as previously described . Histologic sections proximal to the stent and from the mid-portion of the stented segments were stained for immunohistochemical identification of macrophages using RAM11, a mouse monoclonal IgG1 anti-rabbit macrophage antibody. Percent RAM11 positive area was measured using computer-assist software (IPLab; Scanalytics, BD Biosciences; San Jose, CA).

2.8

Statistical analysis

All data were expressed as mean ± standard deviation. Unless otherwise described, for each assay, each sample was assayed in triplicate. Statistical significance was determined using one-way ANOVA with multiple comparison Holm’s t-test. A p value of ≤0.05 was considered statistically significant.

2.1

In vitro cell culture

The human monocyte cell line THP-1 (ATCC, Manassas, VA) was cultured in Opti-MEM® I reduced serum medium (Invitrogen, San Diego, CA) supplemented with 20 U/mL penicillin (Invitrogen), 20 μg/mL streptomycin (Invitrogen), 0.05 μg/mL Fungizone® (amphotericin B) (Invitrogen), 10 μg/mL gentamycin (Sigma-Aldrich, St. Louis, MO), and 2% fetal bovine serum (FBS) (Lonza, Walkersville, MD). Primary smooth muscle cells, isolated from human coronary arteries (HCASMC), and endothelial cells, isolated from human umbilical veins (HUVEC), were each obtained from a commercial supplier (Lonza) at passage 3, and cultured according to supplier specifications. HUVEC were cultured in EGM-2 growth media supplemented with 2% FBS and a specific cocktail of hydrocortisone, hFGF-β, VEGF, R3-IGF-1, ascorbic acid, hEGF, heparin, gentamycin, and amphotericin B (Lonza) supplied by the manufacturer. HCASMC were cultured in SmGM-2 growth media supplemented with 5% FBS and a specific cocktail of insulin, hFGF-β, hEGF, gentamycin, and amphotericin B (Lonza) supplied by the manufacturer. All cells were cultured in a 37 °C and 5% CO ₂ environment, and re-fed every 2–3 days and passed upon reaching 80% confluence. All experiments were performed using cells at passage 8 or lower.

2.2

THP-1 foam cell differentiation and characterization

To induce FC differentiation, THP-1 cells were incubated with 10 ^-7 M phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 72 hours, followed by incubation with 100 μg/mL human acetylated low density lipoprotein (acLDL) (Intracel Resources, Frederick, MD) for 72 hours. To demonstrate FC formation, acLDL derived THP-1 FC as well as PMA derived THP-1 macrophages were stained with lipophilic Nile Red (1 μg/mL) (Sigma-Aldrich) for 10 minutes at room temperature. Nile Red staining allowed for visualization and imaging of FC containing intracellular lipid droplets using a Nikon Eclipse E800 microscope (Nikon Inc., Melville, NY) and photographed with a Coolsnap ES (Photometrics, Tucson, AZ) camera, using Simple PCI (Hamamatsu Corporation, Sewickley, PA) image capture software at an objective magnification of 20×. In addition, an Amplex® Red cholesterol assay (Invitrogen) was used according to manufacturer’s instructions to quantify total cholesterol levels in PMA derived THP-1 macrophages and acLDL derived THP-1 FC using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA). Total cholesterol levels were normalized to respective total protein content of cell lysates determined using a DC protein assay (Bio-Rad, Hercules, CA), with an endpoint of absorbance [A _750nm ] measured using a SpectraMax M2 microplate reader (n = 3). Lastly, a Quantigene® Plex assay (Affymetrix, Santa Clara, CA) was used according to manufacturer’s instructions to evaluate transcriptional changes in the mRNA levels of a panel of gene markers related to cholesterol efflux/lipid metabolism (FABP4, CD36, ABCA1, adipophilin, CD68) in PMA derived THP-1 macrophage and acLDL derived THP-1 FC lysates. This assay combines branched DNA (bDNA) signal amplification and multianalyte profiling bead technologies to enable detection and simultaneous quantification of multiple mRNA targets bound to fluorescent microspheres directly from cell lysates using a Bio-plex™ 100 suspension system (Bio-Rad) . The Quantigene® assay platform was selected based on a comprehensive FDA-led study showing that data generated using the Quantigene® assay platform are comparable to QPCR based methods, with both methodologies generating functionally equivalent results to a large extent when used to validate microarray platform results from the same set of samples . Background fluorescence (measured from microspheres not hybridized with mRNA) was subtracted from RNA-containing samples. Each gene marker was then normalized to a housekeeping gene, cyclophilin B (PPIB), a protein folding enzyme that is constitutively and ubiquitously expressed at high levels in cells. Data were then expressed as percentage fold change of marker levels in acLDL derived THP-1 FC normalized to PMA derived THP-1 macrophages (n = 3).

Two-step Taqman QPCR was performed to verify changes in mRNA levels obtained with the Quantigene® Plex assay. RNA from cell samples were isolated and purified using the RNeasy minikit (Qiagen, Valencia, CA). The isolated RNA was treated with RNase-free DNase I to remove genomic DNA according to the Qiagen protocol. The total RNA sample concentrations were quantified using a Nanovue spectrophotometer (GE Healthcare, Pittsburgh, PA), and the quality of each RNA sample was validated by measuring the A260/A280 ratio. First-strand complementary DNA (cDNA) was synthesized from 1 μg RNA using the high-capacity cDNA reverse transcription kit with random primers and RNase inhibitor in a final volume of 20 μL according to manufacturer’s instructions (Life Technologies, Carlsbad, CA). Reverse transcription reactions were carried out in a GeneAmp 9700 System (Life Technologies) at 25 °C for 10 minutes, 37 °C for 2 hours, and 85 °C for 5 minutes. cDNA was then stored at −20 °C. Taqman Gene Expression Assays for human FABP4 (assay ID#Hs01086177_m1), CD36 (assay ID#Hs01567185_m1), ABCA1 (assay ID#Hs01059118_m1), adipophilin (assay ID#Hs00605340_m1), CD68 (assay ID#Hs02836816_g1), and PPIB (assay ID#Hs00168719_m1) primers/probes were purchased from Life Technologies, and PCR amplification was performed by using the ABI 7500 real time PCR system (Life Technologies) in 96-well plates using the Taqman Universal PCR Master Mix (Life Technologies). All samples were performed in quadruplicates with the thermal cycling profile consisting of four stages: 50 °C for 2 minutes, 95 °C for 10 minutes, and 40 cycles of 15 seconds at 95 °C and 60 °C for 1 minute. The threshold cycle (C _t ) value of each gene were determined by the instrument’s software. Relative levels of mRNA were calculated using the delta–delta C _t method, which involves comparing C _t values between samples. PPIB, as in the Quantigene® Plex assay, was used as the housekeeping gene for normalization. The relative level of target genes was then expressed as percentage fold change in acLDL derived THP-1 FC normalized to PMA derived THP-1 macrophages (n = 3).

2.3

Effects of everolimus on THP-1 derived foam cell viability – Calcein AM/DAPI staining

THP-1 derived FC were seeded in 4-chamber tissue culture treated glass slides (BD Biosciences, Bedford, MA) at a density of 2.5×10 ⁴ cells/cm ² in a 37 °C and 5% CO ₂ incubator. Cells were then treated with or without everolimus (10 ^-5 M). After 24 hours, slides were washed with 1× PBS, pH 7.4 (Invitrogen), followed by staining with calcein AM (2 μM) (Invitrogen) for 45 minutes at room temperature. Slides were then washed with 1× PBS, counterstained with Vectashield DAPI mounting media (Vector Laboratories, Burlingame, CA), and sealed with coverslip. Fluorescent images from ten randomly selected viewing fields were captured with the imaging system described above. The total number of cells judged by DAPI fluorescence as well as number of calcein AM positive live cells were counted in each image and used to calculate the average percentage of viable cells in everolimus treated and control cell populations.

2.4

Effects of everolimus on HCASMC, HUVEC, and THP-1 derived foam cell apoptosis/necrosis

Total cell necrosis and induction of apoptosis by everolimus in HCASMC, HUVEC, and THP-1 derived FC were evaluated using a Cell Death Detection ^PLUS ELISA kit (Roche Applied Science, Mannheim, Germany), with an endpoint of absorbance, [A _405nm – A _490nm ], read on a SpectraMax M2 microplate reader. In separate experiments, HCASMC, HUVEC, or THP-1 derived FC were seeded in 24-well tissue culture plates at a density of 2.5×10 ⁴ cells/cm ² in a 37 °C and 5% CO ₂ incubator. After reaching ~80% confluency, cells were then treated with various doses of everolimus (10 ^-5 M, 10 ^-7 M, 10 ^-9 M, 10 ^-11 M) for 24 hours. Cells incubated with appropriate growth media only served as controls. Following everolimus incubation, culture supernatants were immediately removed and saved for analysis. Cells were then treated with lysis buffer supplied in ELISA kit, and lysates were saved for analysis. ELISA detection of histone-associated nucleosome concentration in supernatants was used to determine necrosis, while concentration in lysates was used to determine relative induction of apoptosis, all according to manufacturer’s instruction. Total protein content of cell lysates was determined using DC Protein Assay (Bio-Rad). Apoptosis and necrosis data for each sample were normalized to respective total protein to determine relative apoptotic and necrotic indices. Data were then expressed as percentage fold change of cells treated with everolimus normalized to cell controls (n = 4).

2.5

Effects of everolimus on markers related to apoptosis, autophagy, matrix remodeling, and inflammatory cytokine secretion from acLDL-Derived THP-1 foam cells

THP-1 derived FC were seeded in 6-well tissue culture plates at a density of 2.5×10 ⁴ cells/cm ² in a 37 °C and 5% CO ₂ incubator. After reaching ~80% confluency, cells were then treated with or without everolimus (10 ^-5 M) (n = 3). After 24 hours, culture media supernatants were collected and analyzed for a panel of secreted inflammatory cytokines (MCP-1, IL-1β, IL-8, TNF-α) using a microsphere-based Procarta® immunoassay kit (Bio-Rad) according to manufacturer’s instructions. Quantigene® Plex assay (Affymetrix), as described above, was used according to manufacturer’s instructions to evaluate the mRNA levels of several gene markers related to cell viability (survivin (anti-apoptotic), clusterin, MAP1LC3 (autophagic)) and matrix remodeling (matrix metalloproteinase 1, 9 (MMP1, MMP9)) in cell lysates. PPIB was used as an internal housekeeping gene for normalization of gene markers. Data were then expressed as percentage fold change in cells treated with everolimus normalized to cell controls. QPCR, as described above, was used to verify changes in human survivin (assay ID#Hs04194392_s1), clusterin (assay ID#Hs00156548_m1), MAP1LC3 (assay ID#Hs01076567_g1), MMP1 (assay ID#Hs00899658_m1), and MMP9 (assay ID#Hs00234579_m1) mRNA levels (n = 3). QPCR was also used to assess mRNA levels of inflammatory cytokines MCP-1 (assay ID#Hs00234140_m1), IL-1β (assay ID#Hs01555410_m1), IL-8 (assay ID#Hs00174103_m1), TNF-α (assay ID#Hs01113624_g1) (n = 3). The relative level of target genes was then expressed as percentage fold change in cells treated with everolimus normalized to cell controls. Additionally, ELISA assays for human MMP1, MMP9, and survivin (all from Abcam, Cambridge, MA) were purchased and used according to manufacturer’s instructions to quantitatively assess MMP1 and MMP9 protein expression in culture media supernatants and survivin protein expression in cell lysates (n = 6). Data were normalized to respective total protein content determined using a DC protein assay (Bio-Rad) and reported as percentage fold change in cells treated with everolimus normalized to cell controls.

2.6

In vivo hypercholesterolemic rabbit model

The experimental atherosclerotic rabbit model has been previously reported . Briefly, New Zealand White rabbits (3 to 4 kg) were fed an atherogenic diet (1% cholesterol and 6% peanut oil, F4366-CHL, Bio-Serv, Inc., Frenchtown, New Jersey) for 5 weeks. Iliac artery injury was induced 1 week following induction of a high-cholesterol diet using a Fogarty catheter (3-F). Under fluoroscopic guidance, premounted XIENCE PRIME (EES, 3×12 mm, Abbott Vascular, Santa Clara, CA) were implanted over lesions in alternating iliac arteries at nominal pressures to achieve a visual target stent-to-artery ratio of approximately 1.3:1. After stent implantation, post-procedural angiography was performed to document vessel patency, and the animals were allowed to recover. The diet was then switched to a low-cholesterol diet (containing 0.025% cholesterol) for the remainder of the study. Anti-platelet therapy consisted of aspirin (40 mg/day) given orally 24 hours before catheterization with continued dosing throughout the in-life-phase of the study, while single dose intra-arterial heparin (150 IU/kg) was administered at the time of catheterization. At the designated time points, animals were anesthetized followed by euthanasia and perfusion-fixation of stented arteries.

2.7

Histology and immunohistochemistry

Samples from the atherosclerotic rabbit ilio-femoral arteries were processed and sectioned as previously described . Histologic sections proximal to the stent and from the mid-portion of the stented segments were stained for immunohistochemical identification of macrophages using RAM11, a mouse monoclonal IgG1 anti-rabbit macrophage antibody. Percent RAM11 positive area was measured using computer-assist software (IPLab; Scanalytics, BD Biosciences; San Jose, CA).

2.8

Statistical analysis

All data were expressed as mean ± standard deviation. Unless otherwise described, for each assay, each sample was assayed in triplicate. Statistical significance was determined using one-way ANOVA with multiple comparison Holm’s t-test. A p value of ≤0.05 was considered statistically significant.