Abstract
Purpose
The objective of this study was to investigate potential differences in vascular response to stenting of coronary arteries with bare metal (BMS) and drug-eluting (DES) stents in juvenile vs. mature swine.
Methods and materials
Twenty-one mature (>3 years) and 22 juvenile (6-9 months) Yucatan swine were implanted with 3×12-mm XIENCE V DES and ML VISION BMS in coronary arteries. After 7 and 28 days, vessels were analyzed using light microscopy ( n =5–7) and confocal and scanning electron microscopy ( n =5–10). Messenger RNA expression levels of inflammatory and endothelial gene markers were tested from stented tissue at 7 and 28 days ( n =3). A 2 × 2 analysis of variance followed by t tests compared treatment and/or age effects.
Results
No age differences in neointimal area and percentage stenosis were measured. Juvenile swine exhibited increased fibrin scores compared to mature swine (2.6±0.5 vs. 2.2±0.5, P <.05) at 7 days, with no age-related difference at 28 days. At 7 days, significant increases in para-strut inflammation ( P <.01) and in VCAM-1, ICAM-1, CD40 and MCP-1 gene expression ( P <.05) were observed in mature swine, but differences were largely resolved by 28 days. DES exhibited less endothelial coverage than BMS at 7 days, but this difference was abrogated by 28 days, with no difference between age groups.
Conclusions
Our results indicate that mature swine exhibited an increased foreign body response compared to mature swine at 7 and 28 days following stenting that may indicate marginal delays in resolution of foreign body response in aged populations. These differences are unlikely to affect methodologies for preclinical stent safety evaluations.
1
Background
Many similarities exist in the cardiovascular system between humans and swine including size, muscularity and epicardial distribution of coronary arteries . Coronary arteries of juvenile swine continue to be the most commonly used model for preclinical evaluation of coronary stents and to be recommended by the Food and Drug Administration (FDA) . Though differences in vascular response related to animal maturity are routinely cited as being a limitation of this model , to date, no studies have been conducted to investigate whether or not this limitation actually exists.
There is considerable interest in understanding differences in vascular remodeling in response to angioplasty and stenting in vessels with advanced aging. Drug-eluting stents (DES) dramatically reduce neointimal proliferation in comparison to bare metal stents (BMS) in the general percutaneous coronary intervention population . Understanding the biological differences in arterial response to injury and foreign body placement may aid device design or clinical investigations. Age-associated arterial wall remodeling and endothelial dysfunction have been demonstrated in rodents , but differences in age-related effects on vascular smooth muscle cell proliferative capacity in response to injury have been conflicting . Additionally, the vascular biology of rodents and cell culture may differ dramatically in response to age in comparison to human patients. While newer-generation devices have demonstrated significant clinical improvement in both efficacy and safety , there remains uncertainty about the ideal animal model for evaluating DES safety , particularly in the context of investigating age-related effects on vascular wall healing and neointimal composition.
In this study, we implanted the coronary arteries of juvenile and mature swine with BMS and DES. Histopathology and gene expression of the stented arteries were evaluated to observe whether age-related differences in arterial response to stenting exist in a healthy porcine model.
2
Methods and materials
A total of 43 Yucatan swine (Valleybrook Farms, Madison, GA, USA) were used, sourced from a total of three vendors randomized throughout both juvenile and mature groups (Sinclair, Columbia, MO, USA; Lonestar Laboratories, Sequin, TX. USA; and S&S Farms, Southern Michigan, USA). The study protocol was approved by the institutional animal care and use committee of the Association for Assessment and Accreditation of Laboratory Animal Care-accredited laboratory (Synecor LLC, Durham, NC, USA). All animals received care in compliance with the Animal Welfare Act and “Principles of Laboratory Animal Care” (National Research Council, NIH Publication No. 85-23, revised 1996). Prior to arrival at the testing facility, all animals received standard vaccinations and parasite control. Animals were quarantined at least 1 week prior to procedure. All animals received a complete veterinary exam during quarantine and after procedures. Twenty-two animals were juvenile (6–9 months of age), and 21 were mature (2.5–3.5 years old). A total of 123 vessels were implanted with BMS (3.0×12 mm ML VISION) or DES (3.0×12 mm everolimus-eluting XIENCE V, Abbott Vascular, Santa Clara, CA, USA) at a 1.1:1 stent:artery ratio into the left anterior descending artery (LAD), left circumflex artery (LCX) or right coronary artery (RCA) of each animal. Arteries measuring <2.6 mm in diameter were excluded due to previously mentioned stent:artery constraints. The different stent types were rotated among the RCA, LAD and LCX and were equally distributed between the arteries and groups of 7- and 28-day follow-up (7 days: n =65 total, juvenile BMS n =14, juvenile DES n =13, juvenile DES n =20, mature DES n =18; 28 days: n =58 total, juvenile BMS n =13, mature BMS n =14, juvenile DES n =16, mature DES n =15). At designated 7- and 28-day time points, vessels were analyzed using light microscopy ( n =5–7) and confocal and scanning electron microscopy ( n =5–10). Additionally, messenger RNA (mRNA) expression levels of inflammatory and endothelial gene markers were tested from stented tissue at 7 and 28 days ( n =3).
2.1
Procedure
One day prior to the procedure, animals were administered loading dose of aspirin (325 mg) and clopidogrel (150 mg). Animals were given intramuscular analgesics/anesthetics of Telazol (5–7 mg/kg). When an adequate plane of anesthesia was reached, the animals were intubated and inhaled isoflurane (2%). Electrocardiogram and blood pressure were monitored throughout the procedure. A vascular access sheath was placed in the femoral artery by surgical cut-down using sterile technique. Before catheterization, heparin was administered to maintain an activated clotting time above 250 s (5000–10,000 U). Fluoroscopy and quantitative coronary angiography (QCA) performed with digital calipers (OEC 9800, GE Healthcare, Piscataway, NJ, USA) were used to select an arterial segment that avoided “jailing” large side branches and resulted in a stent-to-artery ratio of 1.1:1. Vessel was allocated to an experimental group, and the appropriate stent was delivered to the intended site over a guide wire under fluoroscopic guidance. Quantitative coronary angiography was performed to evaluate the following: minimal lumen diameter (MLD) pre- and postdeployment and at follow-up, balloon to artery ratio (B:A), stent to artery ratio (S:A, post-MLD/pre-MLD) and percentage stenosis [(post MLD−follow-up MLD)/post-MLD].
Following stent implantation, the artery was ligated and the incision site was closed. All animals were given aspirin (81 mg) and clopidogrel (75 mg) daily and fed a regular diet throughout the remainder of the study.
2.2
Radiographs of stented arteries
Before histologic processing, intact hearts with stented vessels were imaged (Faxitron X-ray Corp, Model 43855A, Lincolnshire, IL, USA) using high-contrast X-ray film to locate and assess device placement. The stented arterial segments were then carefully dissected free from the heart, and another X-ray was repeated. The radiographs were examined under an Olympus BX41 microscope (Olympus America Inc., Center Valley, PA, USA) at 20× magnification to assess device expansion and detect strut fractures.
2.3
Histopathology
For light microscopy, the implanted vessel segments were fixed in 10% formalin, dehydrated in a graded series of ethanol and embedded in methylmethacrylate resin. After polymerization, 2–3-mm sections were sawed from the proximal, mid and distal portions of each single stent (for a total of three sections). Sections from the stents were cut on a rotary microtome at 4 to 6 µm, mounted and stained with hematoxylin and eosin and elastic Van Gieson’s stains. Segments of the native coronary arteries proximal and distal to the stents were processed for paraffin embedding. Sections of the vessels were cut on a rotary microtome at 4 to 5 µm, mounted and stained with hematoxylin and eosin and Movat Pentachrome stains. All sections were examined and scored by light microscopy for the presence of inflammation, fibrin and neointimal formation. To compare neointimal organization and healing, ordinal data were collected on each stent section and included fibrin deposition and inflammation. An overall neointimal inflammation score (0–4) and fibrin score (0–3) were scored for each section as previously described .
2.4
Morphometry
Histology slides were analyzed by a trained computer operator using digital planimetry with a National Institute of Standards and Technology-calibrated microscope system (IP Lab software, Rockville, MD, USA). The cross-sectional areas [external elastic lamina (EEL), internal elastic lamina (IEL) and lumen] of each stented section were measured. Neointimal thickness was measured as the distance from the inner surface of each stent strut to the luminal border. Area measurements were used to calculate vessel layer areas with the following formulas:
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