2.1

Experimental design

The study was approved by the Institutional Animal Care and Use Committee. All animals received standard care according to the study protocol and following the act of animal welfare and the “Principles of Care of Laboratory Animals” formulated by the Institute of Laboratory Animal Resources (National Research Council, National Institutes of Health Publication No. 85-23, revised 1996). A total of five female or castrated Domestic Yorkshire swine (mean body weight, 30 kg) were included in this study. Animals were pretreated with aspirin (650 mg) and clopidogrel (300 mg) 1 day prior to the procedure and aspirin (325 mg) and clopidogrel (150 mg) on the day of the procedure. Baseline quantitative coronary angiography (QCA) and intravascular ultrasound (IVUS) were performed. Coronary segments from a total of 15 coronaries were selected according to accessibility and appropriate lumen caliber. A dilatation balloon catheter was used to achieve ~30% overstretch of baseline reference arterial diameter, according to IVUS, to produce vascular injury. The inflation was repeated twice. Two weeks after the initial balloon injury, coronary segments were randomized to two treatment groups: intramural delivery of a liposome-based formulation of complex lipids or control saline injection group. Each coronary segment received four microinjections of 250 μl for a total of 1 ml. Each coronary received only one type of solution (lipid solution injection or control saline injection) to avoid mixture between therapies. All animals were maintained under a high-cholesterol diet (2% cholesterol, 20% lard and 1.5% sodium cholate) until the termination of the study.

2.2

IVUS imaging

IVUS pullback images were obtained and analyzed using a coronary ultrasound catheter (Atlantis SR Pro 40 MHz Coronary Imaging Catheter; Boston Scientific, Natick, MA, USA) and a commercially available measurement analytic system (iLab Boston Scientific). Using fluoroscopy, the IVUS catheter was placed distal to the vascular segment selected for intervention by angiography and an automated pullback performed at a speed of 0.5 mm/s covering at least 10 mm proximal and distal to selected vascular segment. The starting position of the IVUS catheter was determined by fluoroscopy and situated by anatomical landmarks in a live image during the pullback. The morphometric analysis in each vessel was performed using standard definitions .

2.3

Lipidic solution injectate

One part cholesteryl linoleate powder (Sigma Aldrich, St. Louis, MO, USA) was added to two parts olive oil and vortex mixed. In a modification of the previously described method, human oxidized low-density lipoprotein (LDL) isoform-5 was added to the cholesterol ester injectate. The resulting turbid and viscous solution was loaded into an injection catheter and immersed in sterile hot saline, thereby increasing the temperature and decreasing the viscosity of the solution immediately prior to injection. LDL used for the injectate was isolated from homozygotic familial hypercholesterolemic (human) and separated according to charge using a LCC-500 programmer controlling two P-500 pumps on an UnoQ12 column, an anion exchange column (BioRad, Hercules, CA, USA) preequilibrated with buffer A (0.02 M Tris-HCl, pH 8.0, 0.5 mM EDTA) at 4°C. LDL protein in buffer A was loaded onto the UnoQ12 column and eluted with a multistep gradient of buffer B (1 M NaCl in buffer A): 0%, 10 min; 0–15%, 10 min; 15%–20%, 30 min; isocratic 20%, 10 min; 20%–40%, 25 min; 40%–100%, 10 min; 100%, 15 min; 100%–0%, 5 min and 0%, 25 min. LDL fractions were pooled according to NaCl concentration into five subfractions, L1 through L5 (0.08–0.17, 0.17–0.18, 0.18–0.20, 0.20, and 0.20–0.38 M, respectively). The isolates were concentrated, sterilized and stored at 4 °C.

2.4

Intramural vascular injection

Two weeks following balloon injury, endovascular intramural injection was performed using a needle injection catheter (Mercator MedSystems, San Leandro, CA, USA) customized for local drug delivery. The system contains a needle in its distal end designed to penetrate the arterial wall and allow solution infusion during balloon deployment in a symmetric fashion using a handheld control mechanism. The needle injection catheter was advanced and located within the previously balloon injured segment. Assessment by fluoroscopy was performed to have the needle expanded to a diameter assuring solution delivery. Following coronary treatment randomization, the infusion catheter system delivered approximately 250 μl of saline control or lipid infusion in each segment per injection. The procedure was repeated circumferentially for a total of four times within the target coronary segment to achieve a total of 1 cc of solution injected. Each animal receive one treatment in at least two of three coronaries. Each coronary received only one treatment. At 12 weeks following injection, terminal angiography and IVUS were obtained, and animals were euthanized while under general anesthesia by intravenous injection of a commercially available euthanasia solution. Following euthanasia, the heart was removed, and coronaries were flushed with 1 L of saline and excised from the myocardium. The coronary arteries were further harvested in 2- to 3-mm vascular rings. The vascular segments were selected in a sequential fashion to be prepared for either histology or quantitative messenger RNA (mRNA) gene expression analysis. Thus, for each vascular segment assigned to gene expression analysis, there was an adjacent vascular segment assigned to histological analysis.

2.5

Histology protocol

Arterial segments of the injected site destined for histology were retrieved from the freezer, rinse off the Tissue-Tek® (Sakura Finetek, Torrance, CA) embedding medium for frozen tissue in saline and immersed in 10% normal buffered formalin for 12 h for complete fixation. Arterial segments were processed under standard histology protocols. Arterial segments were embedded in paraffin and sliced to produce 5-μm-thick sections. Slides were stained with hematoxylin and eosin, elastin trichrome and Movat’s pentachrome. In addition, endothelial cell detection was performed in each arterial sample using Biotinylated Griffonia simplicifolia Lectin I (Vector Laboratories, Burlingame, CA, USA) under general immunohistochemistry protocols.

Digital images of the vessels were captured using Spot Advanced v 4.1.1 Software and histomorphometry was performed using BIOQUANT NOVA PRIME v6.70.10 software. Lumen (L) area (mm ² ), internal elastic lamina (IEL) and external elastic lamina (EEL) area (mm ² ) were measured. Neointimal area was calculated as IEL − L. Percentage area of stenosis was calculated as [(IEL−L)/IEL]⁎100. Following slide quadrants, a semiqualitative score was used to determine vasa vasorum (VV) quantification. The VV was quantified in the neointima, media and adventitial vascular area. The VV quantification was performed in each single histological sample. VV density was assessed by calculating VV quantification divided by the neointimal area (EEL area−L area) with the purpose of determining VV density according to the neointima formation in each arterial segment.

2.6

RNA isolation, reverse transcription polymerase chain reaction

Arterial segments of the injected site destined for gene expression were immersed in Tissue-Tek® embedding medium for frozen tissue specimens and snap frozen in liquid nitrogen. Then, a two-step reverse transcription polymerase chain reaction (RT-PCR) methodology was used to determine relative quantities of various RNA transcripts in the arterial segments destined for gene expression. Total cellular RNA was isolated from the vascular samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A polytron homogenizer was used to homogenize the tissue samples in TRIzol prior to RNA isolation. Following isolation, the RNA samples were treated with DNase to remove residual genomic DNA. First-strand complement DNA (cDNA) was synthesized from 5.0 μg RNA in 100 μl RT reactions using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The RT method used random hexamers to prime cDNA synthesis. TaqMan real-time QPCR was used to determine the relative cDNA quantities of several target genes. In addition, the relative cDNA quantity of 18S (ribosomal) was determined for use as a normalizer. All QPCR assays for target genes were designed in-house. The 18S QPCR assay was ordered predesigned (Applied Biosystems). QPCR was conducted using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) in 50-μl singleplex reactions according to the manufacturer’s default assay conditions. Serial dilutions of a representative cDNA sample were used as a standard curve for relative cDNA quantification. This methodology was used to quantify the mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) in the intercalated arterial samples destined for gene expression.

2.7

Statistical analysis

The results of each analytic method were presented as mean±S.D. The statistical analysis was performed using SigmaStat 3.11 software (2004; Sustat Inc., San Jose, California, USA). Unpaired Student’s t tests were used to test for differences between groups. A P value<.05 was considered statistically significant.

2.1

Experimental design

The study was approved by the Institutional Animal Care and Use Committee. All animals received standard care according to the study protocol and following the act of animal welfare and the “Principles of Care of Laboratory Animals” formulated by the Institute of Laboratory Animal Resources (National Research Council, National Institutes of Health Publication No. 85-23, revised 1996). A total of five female or castrated Domestic Yorkshire swine (mean body weight, 30 kg) were included in this study. Animals were pretreated with aspirin (650 mg) and clopidogrel (300 mg) 1 day prior to the procedure and aspirin (325 mg) and clopidogrel (150 mg) on the day of the procedure. Baseline quantitative coronary angiography (QCA) and intravascular ultrasound (IVUS) were performed. Coronary segments from a total of 15 coronaries were selected according to accessibility and appropriate lumen caliber. A dilatation balloon catheter was used to achieve ~30% overstretch of baseline reference arterial diameter, according to IVUS, to produce vascular injury. The inflation was repeated twice. Two weeks after the initial balloon injury, coronary segments were randomized to two treatment groups: intramural delivery of a liposome-based formulation of complex lipids or control saline injection group. Each coronary segment received four microinjections of 250 μl for a total of 1 ml. Each coronary received only one type of solution (lipid solution injection or control saline injection) to avoid mixture between therapies. All animals were maintained under a high-cholesterol diet (2% cholesterol, 20% lard and 1.5% sodium cholate) until the termination of the study.

2.2

IVUS imaging

IVUS pullback images were obtained and analyzed using a coronary ultrasound catheter (Atlantis SR Pro 40 MHz Coronary Imaging Catheter; Boston Scientific, Natick, MA, USA) and a commercially available measurement analytic system (iLab Boston Scientific). Using fluoroscopy, the IVUS catheter was placed distal to the vascular segment selected for intervention by angiography and an automated pullback performed at a speed of 0.5 mm/s covering at least 10 mm proximal and distal to selected vascular segment. The starting position of the IVUS catheter was determined by fluoroscopy and situated by anatomical landmarks in a live image during the pullback. The morphometric analysis in each vessel was performed using standard definitions .

2.3

Lipidic solution injectate

One part cholesteryl linoleate powder (Sigma Aldrich, St. Louis, MO, USA) was added to two parts olive oil and vortex mixed. In a modification of the previously described method, human oxidized low-density lipoprotein (LDL) isoform-5 was added to the cholesterol ester injectate. The resulting turbid and viscous solution was loaded into an injection catheter and immersed in sterile hot saline, thereby increasing the temperature and decreasing the viscosity of the solution immediately prior to injection. LDL used for the injectate was isolated from homozygotic familial hypercholesterolemic (human) and separated according to charge using a LCC-500 programmer controlling two P-500 pumps on an UnoQ12 column, an anion exchange column (BioRad, Hercules, CA, USA) preequilibrated with buffer A (0.02 M Tris-HCl, pH 8.0, 0.5 mM EDTA) at 4°C. LDL protein in buffer A was loaded onto the UnoQ12 column and eluted with a multistep gradient of buffer B (1 M NaCl in buffer A): 0%, 10 min; 0–15%, 10 min; 15%–20%, 30 min; isocratic 20%, 10 min; 20%–40%, 25 min; 40%–100%, 10 min; 100%, 15 min; 100%–0%, 5 min and 0%, 25 min. LDL fractions were pooled according to NaCl concentration into five subfractions, L1 through L5 (0.08–0.17, 0.17–0.18, 0.18–0.20, 0.20, and 0.20–0.38 M, respectively). The isolates were concentrated, sterilized and stored at 4 °C.

2.4

Intramural vascular injection

Two weeks following balloon injury, endovascular intramural injection was performed using a needle injection catheter (Mercator MedSystems, San Leandro, CA, USA) customized for local drug delivery. The system contains a needle in its distal end designed to penetrate the arterial wall and allow solution infusion during balloon deployment in a symmetric fashion using a handheld control mechanism. The needle injection catheter was advanced and located within the previously balloon injured segment. Assessment by fluoroscopy was performed to have the needle expanded to a diameter assuring solution delivery. Following coronary treatment randomization, the infusion catheter system delivered approximately 250 μl of saline control or lipid infusion in each segment per injection. The procedure was repeated circumferentially for a total of four times within the target coronary segment to achieve a total of 1 cc of solution injected. Each animal receive one treatment in at least two of three coronaries. Each coronary received only one treatment. At 12 weeks following injection, terminal angiography and IVUS were obtained, and animals were euthanized while under general anesthesia by intravenous injection of a commercially available euthanasia solution. Following euthanasia, the heart was removed, and coronaries were flushed with 1 L of saline and excised from the myocardium. The coronary arteries were further harvested in 2- to 3-mm vascular rings. The vascular segments were selected in a sequential fashion to be prepared for either histology or quantitative messenger RNA (mRNA) gene expression analysis. Thus, for each vascular segment assigned to gene expression analysis, there was an adjacent vascular segment assigned to histological analysis.

2.5

Histology protocol

Arterial segments of the injected site destined for histology were retrieved from the freezer, rinse off the Tissue-Tek® (Sakura Finetek, Torrance, CA) embedding medium for frozen tissue in saline and immersed in 10% normal buffered formalin for 12 h for complete fixation. Arterial segments were processed under standard histology protocols. Arterial segments were embedded in paraffin and sliced to produce 5-μm-thick sections. Slides were stained with hematoxylin and eosin, elastin trichrome and Movat’s pentachrome. In addition, endothelial cell detection was performed in each arterial sample using Biotinylated Griffonia simplicifolia Lectin I (Vector Laboratories, Burlingame, CA, USA) under general immunohistochemistry protocols.

Digital images of the vessels were captured using Spot Advanced v 4.1.1 Software and histomorphometry was performed using BIOQUANT NOVA PRIME v6.70.10 software. Lumen (L) area (mm ² ), internal elastic lamina (IEL) and external elastic lamina (EEL) area (mm ² ) were measured. Neointimal area was calculated as IEL − L. Percentage area of stenosis was calculated as [(IEL−L)/IEL]⁎100. Following slide quadrants, a semiqualitative score was used to determine vasa vasorum (VV) quantification. The VV was quantified in the neointima, media and adventitial vascular area. The VV quantification was performed in each single histological sample. VV density was assessed by calculating VV quantification divided by the neointimal area (EEL area−L area) with the purpose of determining VV density according to the neointima formation in each arterial segment.

2.6

RNA isolation, reverse transcription polymerase chain reaction

Arterial segments of the injected site destined for gene expression were immersed in Tissue-Tek® embedding medium for frozen tissue specimens and snap frozen in liquid nitrogen. Then, a two-step reverse transcription polymerase chain reaction (RT-PCR) methodology was used to determine relative quantities of various RNA transcripts in the arterial segments destined for gene expression. Total cellular RNA was isolated from the vascular samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. A polytron homogenizer was used to homogenize the tissue samples in TRIzol prior to RNA isolation. Following isolation, the RNA samples were treated with DNase to remove residual genomic DNA. First-strand complement DNA (cDNA) was synthesized from 5.0 μg RNA in 100 μl RT reactions using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The RT method used random hexamers to prime cDNA synthesis. TaqMan real-time QPCR was used to determine the relative cDNA quantities of several target genes. In addition, the relative cDNA quantity of 18S (ribosomal) was determined for use as a normalizer. All QPCR assays for target genes were designed in-house. The 18S QPCR assay was ordered predesigned (Applied Biosystems). QPCR was conducted using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) in 50-μl singleplex reactions according to the manufacturer’s default assay conditions. Serial dilutions of a representative cDNA sample were used as a standard curve for relative cDNA quantification. This methodology was used to quantify the mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and vascular endothelial growth factor (VEGF) in the intercalated arterial samples destined for gene expression.

2.7

Statistical analysis

The results of each analytic method were presented as mean±S.D. The statistical analysis was performed using SigmaStat 3.11 software (2004; Sustat Inc., San Jose, California, USA). Unpaired Student’s t tests were used to test for differences between groups. A P value<.05 was considered statistically significant.