Abstract
Background and purpose
NOBORI biolimus A9-eluting stent (BES) is the third generation drug eluting stent (DES) with only abluminal biodegradable polymer. Recent clinical trials have indicated that the BES is non-inferior to the XIENCE V everolimus-eluting stent (EES). Meanwhile, potential superiority of biodegradable polymer BES over current generation DES has not been addressed. The aim of this preclinical study was to assess and compare the biocompatibility of both BES and EES in porcine coronary arteries.
Methods and materials
BES with length of 24-mm (n = 9) and EES with length of 23-mm (n = 9) were both implanted in porcine coronary arteries. At 28 days endothelium-dependent vasomotion was assessed by acetylcholine (Ach) and subsequently measurements of endothelial superoxide production, histological evaluations and microarray gene analyses were performed.
Results
Angiographic and histological in-stent stenoses were significantly suppressed in BES compared with EES. Histopathological assessment showed lower inflammatory score as well as fibrin and injury scores in BES as compared with EES. On the contrary, paradoxical vasoconstriction to Ach was frequently observed in EES-treated vessels compared with BES-treated vessels. Additionally, gene expressions of inflammatory cytokines and chemokines were upregulated in vessels treated with EES compared with BES in microarray pathway specific analyses.
Conclusions
Implantation of BES revealed less inflammation and foreign-body immunoreaction than EES, suggesting more enhanced biocompatibility of BES compared with EES at 28 days in porcine coronary arteries.
1
Introduction
Although drug-eluting stents (DES) have dramatically decreased the rate of target legion revascularization and in-stent restenosis after stent implantation, DES implantation is associated with vascular inflammation following delayed arterial healing . This phenomenon induces endothelial dysfunction which increases the rate of late stent thrombosis . The third generation NOBORI biolimus A9-eluting stent (BES, Terumo, Tokyo, Japan) is a stainless steel platform loaded with poly-lactic acid as a polymer eluting biolimus A9 . BES has a bioabsorbable polymer only on the abluminal side . The second generation XIENCE V everolimus-eluting stent (EES, Abbott Vascular, Santa Clara, CA) is a cobalt–chromium VISION platform loaded with durable fluorocopolymers eluting everolimus . Stent strut and polymer thickness of EES are thinner than those of BES . Superiority of biodegradable stent polymers remains still controversial, however, recent observations from a long term follow-up clinical trial showed that biodegradable polymer BES is safer than a durable polymer first generation DES . In recent prospective, randomized and multicenter trials, the BES showed non-inferiority in terms of safety and efficacy to EES . Nevertheless, mechanism of vasculitis induced by newer generation DES has not been fully addressed. Here we assessed and compared BES and EES in healthy porcine coronary arteries by angiograms, acetylcholine challenge endothelial function test, histological analysis, ex-vivo endothelial superoxide measurement, and microarray analysis.
2
Materials and methods
2.1
Animal model
Seven Yorkshire farm pigs (46.6 ± 2.6 kg) were enrolled in this study. Animal handling and care followed recommendations of the National Institute of Health guide for care and use of laboratory animal and were consistent with guidelines of the American Heart Association. All protocols were approved by our Internal Animal Care and Use Committee and were consistent with Association for Assessment and Accreditation of Laboratory Animal Care guidelines.
2.2
Coronary angiography
Cardiac catheterization and stent implantation were performed under standard operational procedure as previously described . Full heparinization (200 U/kg) was induced and quantitative coronary angiography (QCA) guidance was used to obtain a stent-to-artery diameter ratio of 1.1:1. Seven pigs underwent BES (length: 24 mm, size: 2.75 mm, 3 mm, 3.5 mm, n = 9) or EES (length: 23 mm, size: 2.75 mm, 3 mm, 3.5 mm, n = 9) implantation as well as follow-up angiographic assessment at 28 days. The stent size and the inflation time were within the manufacturer’s suggested duration range. To obtain valid information, animals were randomized to the stent type, and one pig was treated with both BES and EES in the different vessels to exclude inter animal variation. Angiographic measurements for minimal lumen diameter and mean vessel diameter were carried out immediately after implantation and at 28 days follow-up by using a computer-based quantitative coronary measurement system (Xper, Philips, Amsterdam, The Netherlands). An independent unbiased observer blinded to study assignment performed the QCA measurement. The reference target-vessel diameter was obtained by an interpolated method. Late lumen loss was calculated as the difference between post-stented and follow-up minimal lumen diameter. The percentage of area stenosis (%Stenosis) was calculated by the following formula: 100 × [1 − (follow-up mean vessel diameter/post-stented mean diameter)].
2.3
Endothelium-dependent vasomotor function test
In vivo endothelial vasomotor function test by using acetylcholine (Ach) was performed at 28 days after DES implantation as previously described . After the angiography for baseline, Ach (10 − 6 mol/l) was infused intracoronarily at 1 ml/minute for 2 minutes. Coronary angiography was performed at 30 seconds after Ach infusion by a single angiographer with consistent hand-contrast injection via a 10-ml syringe. Intracoronary nitroglycerine (NTG, 200 μg) was injected and the measurements were performed to finalize the test. The mean vessel diameters of 5-mm segment lengths at 5-mm distal to the stents were measured and the percentage of diameter change was calculated . 48 hours after Ach challenge test, all animals were terminated for the following experiments ( Fig. 1 ).
2.4
Endothelial oxidative stress test
The measurement of superoxide production was performed as previously described . A total of 11 vessels (BES: n = 6, EES: n = 5) were randomly selected and examined. Coronary arteries were equally divided into stented and the distal segments. Segments 5-mm distal to stented regions were cut into 5-mm long rings and the samples were assayed. Data were expressed in relative light units (RLU) per second for each sample.
2.5
Histology
Randomly selected nine stented vessels (BES: n = 5, EES: n = 4) were employed in the histological assessment as previously described . The stented vessels were processed and embedded in methyl methacrylate. The sections were cut from the proximal, mid, and distal stent regions using a heavy-duty microtome and collected on glass slides. Adjacent or near-adjacent sections were stained with hematoxylin–eosin and Verheoff–Masson (VM) elastin trichrome. Histologic sections were evaluated at various magnifications using brightfield microscopy by a single observer with a blind fashion. For histomorphometric analyses, low magnification digital images of VM-stained section were evaluated. The lumen, internal elastic lamina (IEL), external elastic lamina (EEL) areas were traced and quantitatively evaluated. The neointimal area was obtained by subtraction of the lumen area from the IEL area. In addition, the neointimal thickness at each stent strut site was also measured by the same subtraction of the lengths. The histologic %stenosis was calculated by 100 × [1 − (luminal area/IEL area)] formula and evaluated. For histopathological analyses, prevalent pathological criteria, such as inflammation, fibrin and injury scores were evaluated for each section using a semi-quantitative rating scale as previously described ( Online Supplemental Data ). An independent unbiased observer blinded to study assignment performed all histopathological quantification.
2.6
Microarray gene analysis
For microarray gene expression analysis, proximal and distal unstented- (n = 2), and stented-coronary artery segments (n = 4/each group) were randomly selected and dissected. The stented- and unstented-coronary artery tissue samples were lysed and total RNA was isolated followed by purification over spin columns. Agilent Feature Extraction software was used to process scanned images from arrays and the data generated for each probe on the array were analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA). To compare individual expression values across arrays, raw intensity data from each gene were normalized to the 75th percentile intensity of each array. A cut-off value was set as > 2-fold change in this analysis, therefore differentially expressed genes were identified by > 2-fold and Welch t-test p-values < 0.05 between each treatment group comparison. Pathway specific gene expression analyses were performed using GeneSpring GX v7.3.1 and DAVID Bioinformatics software packages (Laboratory of Immunopathogenesis and Bioinformatics, Frederick, MD).
2.7
Statistical analysis
All results are presented as mean ± standard deviation unless otherwise indicated. Statistical analyses were performed by SPSS (version 16.0, SPSS Inc., Chicago, IL). Comparisons between two DES groups or the stents and naïve group measurements were performed by Student unpaired 2-tailed t-test unless otherwise indicated. A p value < 0.05 was considered significant.
2
Materials and methods
2.1
Animal model
Seven Yorkshire farm pigs (46.6 ± 2.6 kg) were enrolled in this study. Animal handling and care followed recommendations of the National Institute of Health guide for care and use of laboratory animal and were consistent with guidelines of the American Heart Association. All protocols were approved by our Internal Animal Care and Use Committee and were consistent with Association for Assessment and Accreditation of Laboratory Animal Care guidelines.
2.2
Coronary angiography
Cardiac catheterization and stent implantation were performed under standard operational procedure as previously described . Full heparinization (200 U/kg) was induced and quantitative coronary angiography (QCA) guidance was used to obtain a stent-to-artery diameter ratio of 1.1:1. Seven pigs underwent BES (length: 24 mm, size: 2.75 mm, 3 mm, 3.5 mm, n = 9) or EES (length: 23 mm, size: 2.75 mm, 3 mm, 3.5 mm, n = 9) implantation as well as follow-up angiographic assessment at 28 days. The stent size and the inflation time were within the manufacturer’s suggested duration range. To obtain valid information, animals were randomized to the stent type, and one pig was treated with both BES and EES in the different vessels to exclude inter animal variation. Angiographic measurements for minimal lumen diameter and mean vessel diameter were carried out immediately after implantation and at 28 days follow-up by using a computer-based quantitative coronary measurement system (Xper, Philips, Amsterdam, The Netherlands). An independent unbiased observer blinded to study assignment performed the QCA measurement. The reference target-vessel diameter was obtained by an interpolated method. Late lumen loss was calculated as the difference between post-stented and follow-up minimal lumen diameter. The percentage of area stenosis (%Stenosis) was calculated by the following formula: 100 × [1 − (follow-up mean vessel diameter/post-stented mean diameter)].
2.3
Endothelium-dependent vasomotor function test
In vivo endothelial vasomotor function test by using acetylcholine (Ach) was performed at 28 days after DES implantation as previously described . After the angiography for baseline, Ach (10 − 6 mol/l) was infused intracoronarily at 1 ml/minute for 2 minutes. Coronary angiography was performed at 30 seconds after Ach infusion by a single angiographer with consistent hand-contrast injection via a 10-ml syringe. Intracoronary nitroglycerine (NTG, 200 μg) was injected and the measurements were performed to finalize the test. The mean vessel diameters of 5-mm segment lengths at 5-mm distal to the stents were measured and the percentage of diameter change was calculated . 48 hours after Ach challenge test, all animals were terminated for the following experiments ( Fig. 1 ).
2.4
Endothelial oxidative stress test
The measurement of superoxide production was performed as previously described . A total of 11 vessels (BES: n = 6, EES: n = 5) were randomly selected and examined. Coronary arteries were equally divided into stented and the distal segments. Segments 5-mm distal to stented regions were cut into 5-mm long rings and the samples were assayed. Data were expressed in relative light units (RLU) per second for each sample.
2.5
Histology
Randomly selected nine stented vessels (BES: n = 5, EES: n = 4) were employed in the histological assessment as previously described . The stented vessels were processed and embedded in methyl methacrylate. The sections were cut from the proximal, mid, and distal stent regions using a heavy-duty microtome and collected on glass slides. Adjacent or near-adjacent sections were stained with hematoxylin–eosin and Verheoff–Masson (VM) elastin trichrome. Histologic sections were evaluated at various magnifications using brightfield microscopy by a single observer with a blind fashion. For histomorphometric analyses, low magnification digital images of VM-stained section were evaluated. The lumen, internal elastic lamina (IEL), external elastic lamina (EEL) areas were traced and quantitatively evaluated. The neointimal area was obtained by subtraction of the lumen area from the IEL area. In addition, the neointimal thickness at each stent strut site was also measured by the same subtraction of the lengths. The histologic %stenosis was calculated by 100 × [1 − (luminal area/IEL area)] formula and evaluated. For histopathological analyses, prevalent pathological criteria, such as inflammation, fibrin and injury scores were evaluated for each section using a semi-quantitative rating scale as previously described ( Online Supplemental Data ). An independent unbiased observer blinded to study assignment performed all histopathological quantification.
2.6
Microarray gene analysis
For microarray gene expression analysis, proximal and distal unstented- (n = 2), and stented-coronary artery segments (n = 4/each group) were randomly selected and dissected. The stented- and unstented-coronary artery tissue samples were lysed and total RNA was isolated followed by purification over spin columns. Agilent Feature Extraction software was used to process scanned images from arrays and the data generated for each probe on the array were analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA). To compare individual expression values across arrays, raw intensity data from each gene were normalized to the 75th percentile intensity of each array. A cut-off value was set as > 2-fold change in this analysis, therefore differentially expressed genes were identified by > 2-fold and Welch t-test p-values < 0.05 between each treatment group comparison. Pathway specific gene expression analyses were performed using GeneSpring GX v7.3.1 and DAVID Bioinformatics software packages (Laboratory of Immunopathogenesis and Bioinformatics, Frederick, MD).
2.7
Statistical analysis
All results are presented as mean ± standard deviation unless otherwise indicated. Statistical analyses were performed by SPSS (version 16.0, SPSS Inc., Chicago, IL). Comparisons between two DES groups or the stents and naïve group measurements were performed by Student unpaired 2-tailed t-test unless otherwise indicated. A p value < 0.05 was considered significant.
3
Results
All animals tolerated interventional stent implantation procedures without any events and no premature mortalities and complications were noted until euthanasia of the animals. All vessels were angiographically patent and no thrombi were observed at 28 days follow-up as well as immediate post-implantation.
3.1
Angiographic analysis
There were no differences between BES and EES in baseline target-vessel diameter, post-stented mean vessel diameter, stent/artery ratio and post-stented minimal lumen diameter (BES: 3.04 ± 0.23 mm, EES: 2.73 ± 0.44 mm, p = NS). The representative angiographic images for BES ( Fig. 2 a–c ) and EES ( Fig. 2 d–f) at 28 days follow-up were demonstrated in Fig. 2 . BES revealed larger follow-up in-stent mean vessel diameter and smaller late lumen loss with significance as compared with EES ( Table 1 ). BES also had significantly smaller %stenosis as compared with EES.
