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Aortocoronary Conduits May Show a Different Inflammatory Response - Comparative Study at Transcript Level

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Aortocoronary Conduits May Show a Different Inflammatory Response - Comparative Study at Transcript Level Nawrocki et al. Medical Journal of Cell Biology 2020 DOI: 10.2478/acb-2020-0003 Received: 21.01.2020 Accepted: 12.03.2020 Aortocoronary conduits may show a different inflammatory response - comparative study at transcript level Mariusz J. Nawrocki1 2, Karol Jopek2, Greg Hutchings1,3 4, Marek Jemielity4 2,7, Bartosz Kempisty1,2,5,6, Paul Mozdziak8 2 , Sandra Kałużna , Bartłomiej Perek , Agnieszka Malińska , Michał Nowicki Abstract Coronary artery bypass grafting (CABG), together with percutaneous coronary intervention (PCI), are both still the most efficient procedures for myocardial revascularization to treat advanced coronary ar- tery disease (CAD). Donor blood vessels used in CABG are usually the internal thoracic artery (ITA) and saphenous vein (SV). The importance of inflammation and inflammatory pathways in graft patency is well established. Nevertheless, not all molecular mechanisms underlying the inflammatory process appear to be clear. Employing the expressive microarray approach to analyze the transcriptome of both venous and arterial grafts, five GO BP terms has been selected: “cellular response to interferon-gamma”, “inflammatory response”, “interferon-gamma-mediated signaling pathway”, “response to interferon-gamma” and “positi- ve regulation of inflammatory response”. This study aimed to evaluate potential molecular factors that could be characteristic markers for both SV and ITA conduits. Running title: Aortocoronary conduits may show a different inflammatory response Keywords: coronary artery bypass graftin, internal thoracic artery, saphenous vein, inflammation 1 2 3Department of Anatomy, Poznań University of Medical Sciences, Poznań, Poland 4Department of Histology and Embryology, Poznań University of Medical Sciences, Poznań, Poland 5The School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Aberdeen, UK 6Department of Cardiac Surgery and Transplantology, Poznań University of Medical Sciences, Poznań, Poland 7Department of Obstetrics and Gynecology, University Hospital and Masaryk University, Brno, Czech Republic 8Department of Veterinary Surgery, Institute of Veterinary Medicine, Nicolaus Copernicus University in Toruń, Toruń, Poland *Division Correspondence: of Anatomy [email protected] and Histology, University of Zielona Góra, Zielona Góra, Poland FullPhysiology list of author Graduate information Program, is North available Carolina at the State end ofUniversity, article Raleigh, North Carolina 25 Nawrocki et al. Medical Journal of Cell Biology (2020) Introduction The other target coronary arteries were usually re- Coronary artery bypass grafting (CABG) is still vascularized with SV grafts. one of the most common surgical procedures aimed All surgeries were performed through medi- at improving blood circulation in atherosclerotic cor- an sternotomy. SV grafts were obtained through a onary arteries [1]. The predominant reason for early full-length thigh incision over its course [4]. Pivotal acute coronary syndromes is graft failure. Thus, bet- points of the procedure included minimal manipula- ter understanding of the activated molecular mech- tion of the graft (“no-touch” technique), avoiding ex- anisms occurring soon after surgery may further tensive dilation of the conduits, using low-intensity allow a reduction of the rate of these adverse cardio- electrocautery and the control of the branches with vascular events. There are a number of blood vessels stainless-steel vascular clips. In all cases, the distal which can be used as grafts in this revascularization part of the obtained SV segment (at least 15–20 mm procedure. Nevertheless, the internal thoracic artery in length) was saved for further laboratory studies. (ITA) and the saphenous vein (SV) are the most com- ITA conduits were harvested as pedicled, togeth- monly applied as aortocoronary conduits. er with satellite veins and endothoracic fascia from The early occlusion of ITA grafts is observed in ap- the 2nd to 6th intercostal space. The distal end of proximately 5% of patients, whereas SV transplants the ITA segment was divided at the level of its bi- occlude within a one year period after surgery in 10– furcation. After heparinization, ITA conduits were 15% of CABG patients [2]. Thus, venous conduits are commonly used in older patients. Despite advances in solution (1 mg/mL), and allowed to pharmacologi- perioperative management and better understanding callyclipped dilate. distally, Immediately injected with before 10 anastomosismL of a papaverine of the of the vessel wall histological architecture, current distal end of ITA to the recipient coronary artery, a knowledge regarding molecular pathways’ activation 10-mm segment of the conduit was harvested for and their possible mechanisms in the perioperative further molecular and histological tests. period of CABG procedure is highly limited. There The sets of the vessel samples, both SV and ITA, are several molecular pathways indicating increased were immediately snap-frozen in liquid nitrogen inflammatory status, haemostasis activation, as well as increased oxidative stress, which seem to play a set of samples was directed for histochemical exam- pivotal role in graft patency. Surgical stress accounts ination.and stored Transcriptome at −80 °C until screening RNA isolation. analysis wasAnother per- for more protracted and marked molecular pathway formed on 18 SV and 20 ITA samples. perturbations overall. Our previous molecular stud- ies on ITA and SV grafts have shown that there are Microarray expression study and data analysis significant discrepancies between these vessels in Our experiment employed 38 GeneChip® HG- the level of expression of genes involved in osteogen- - ic changes in these vessels. According to our findings, rays to simultaneously examine thousands of tran- we have suggested that venous grafts may be more scriptsU219 (Affymetrix, for each of the Santa analyzed Clara, samples.CA, USA) In microar the first exposed to atherosclerotic changes [3]. step, the total RNA (500 ng) from each pooled sam- Bettering understanding of the activated molec- - ular mechanisms occurring in vessels soon after plification (Ambion® WT Expression Kit, provided surgery may allow a further reduction of the rate of ple was subjected to two rounds of sense cDNA am adverse cardiovascular events. Therefore, our study was performed by in vitro transcription (16 h, 40 aimed to investigate the transcriptomic profile of by Ambion, Austin, TX, USA). The synthesis of cRNA genes characterizing both ITA and SV. Employing into cDNA. Subsequently, cDNA samples were used the microarray approach, we analyzed differences for°C). biotinThen, cRNAlabeling was and purified fragmentation and re-transcribed using an at the molecular level between both blood vessels Affymetrix GeneChip® WT Terminal Labeling and that often serve as aortocoronary grafts. We de- Hybridization kit (Affymetrix). Next, the biotin-la- cided to additionally describe “cellular response beled samples were loaded onto and hybridized to to interferon-gamma”, “inflammatory response”, “interferon-gamma-mediated signaling pathway”, - “response to interferon-gamma” and “positive regu- ployingthe Affymetrix® an AccuBlock™ Human GenomeDigital Dry U219 Bath Array (Labnet Strip. lation of inflammatory response” gene ontology bi- Hybridization was conducted at 48 °C for 20 h, em ological process (GO BP) terms, because inflamma- oven. Then, microarrays were washed and stained, tory effects can affect the usefulness of all the blood accordingInternational, to technical Inc., Edison, protocol, NJ, USA) using hybridization an Affymet- vessels used as conduits in the CABG procedure. rix GeneAtlas™ Fluidics Station (Affymetrix, Santa Materials and methods Affymetrix GeneAtlas™ Imaging Station (Affymetrix, Operation procedure and sample collection Clara, CA, USA). The strips were scanned using an In most patients, the left ITA was used to bypass were saved on hard drives as *.CEL files for down- the left anterior descending coronary artery (LAD). streamSanta Clara, data analysis.CA, USA). The scans of the microarrays 26 Nawrocki et al. Medical Journal of Cell Biology (2020) Quality control (QC) studies were performed us- their symbols, gene names, and Entrez IDs, allow- ing the Affymetrix GeneAtlas™ Instrument Control ing generation of a complex gene data table. To de- Software 2.0.0.460 (Affymetrix, Santa Clara, CA, termine the statistical significance of the analyzed genes, moderated t-statistics from the empirical - Bayes method were performed. The obtained p-val- therUSA), analysis according performed to the manufacturer’susing the R statistical standards. lan- ues were corrected for multiple comparisons using guageThe generated and Bioconductor *.CEL files package were subjectedwith the relevant to fur Bioconductor libraries. To correct the background, - normalize, and summarize the results, we used the nificantlyBenjamini altered and Hochberg’s genes was false based discovery on a p-value rate andbe- robust multiarray averaging (RMA) algorithm. As- neathdescribed 0.05 as and adjusted an expression p-values. higher The selection than two-fold. of sig signed biological annotations were obtained from The differentially expressed gene list (separated the “pd.ragene.2.1.st” library and employed for the for upregulated and downregulated genes) was up- mapping of normalized gene expression values with loaded to the DAVID Bioinformatics Resources 6.8 FIGURE 1 Heatmaps presenting differentially expressed genes involved in “cellular
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