Aortocoronary conduits may show a different inflammatory response - comparative study at transcript level

Coronary artery bypass grafting (CABG) is still one of the most common surgical procedures aimed at improving blood circulation in atherosclerotic coronary arteries [1]. The predominant reason for early acute coronary syndromes is graft failure. Thus, better understanding of the activated molecular mechanisms occurring soon after surgery may further allow a reduction of the rate of these adverse cardiovascular events. There are a number of blood vessels which can be used as grafts in this revascularization procedure. Nevertheless, the internal thoracic artery (ITA) and the saphenous vein (SV) are the most commonly applied as aortocoronary conduits.

The early occlusion of ITA grafts is observed in approximately 5% of patients, whereas SV transplants occlude within a one year period after surgery in 10–15% of CABG patients [2]. Thus, venous conduits are commonly used in older patients. Despite advances in perioperative management and better understanding of the vessel wall histological architecture, current knowledge regarding molecular pathways’ activation and their possible mechanisms in the perioperative period of CABG procedure is highly limited. There are several molecular pathways indicating increased inflammatory status, haemostasis activation, as well as increased oxidative stress, which seem to play a pivotal role in graft patency. Surgical stress accounts for more protracted and marked molecular pathway perturbations overall. Our previous molecular studies on ITA and SV grafts have shown that there are significant discrepancies between these vessels in the level of expression of genes involved in osteogenic changes in these vessels. According to our findings, we have suggested that venous grafts may be more exposed to atherosclerotic changes [3].

Bettering understanding of the activated molecular mechanisms occurring in vessels soon after surgery may allow a further reduction of the rate of adverse cardiovascular events. Therefore, our study aimed to investigate the transcriptomic profile of genes characterizing both ITA and SV. Employing the microarray approach, we analyzed differences at the molecular level between both blood vessels that often serve as aortocoronary grafts. We decided to additionally describe “cellular response to interferon-gamma”, “inflammatory response”, “interferon-gamma-mediated signaling pathway”, “response to interferon-gamma” and “positive regulation of inflammatory response” gene ontology biological process (GO BP) terms, because inflammatory effects can affect the usefulness of all the blood vessels used as conduits in the CABG procedure.

We used Human Genome U219 Array Strip for the microarray gene expression analysis of internal thoracic artery (ITA) and the saphenous vein (SV). This method allowed us to study the gene expression of 49,308 transcripts. We selected genes with more than 2- fold changes and corrected p-values less than 0.05 for downstream analysis. A total of 1170 differentially expressed genes (DEGs) were identified according to the above criteria. We started the microarray gene expression analysis with subjecting the list of DEGs to DAVID software, which showed that the genes can be assigned to many gene ontology groups (GO BP terms). This paper focused on the genes involved in inflammatory response. The DAVID software indicated the following GO BP terms, which cover the above processes: “cellular response to interferon-gamma”, “inflammatory response”, “interferon-gamma-mediated signaling pathway”, “response to interferon-gamma” and “positive regulation of inflammatory response”. The 44 genes involved in those processes were clustered using hierarchical clustering and presented as heatmaps (Fig. 1).

Figure 1

It is worth mentioning that 29 genes were downregulated while 15 genes were upregulated. The 10 most significantly upregulated and downregulated genes, their symbols, fold changes and corrected p-values are shown in table 1.

In the next part of analysis, we focused on the z-scores, which tell us whether the biological process is more likely to be decreased (negative value) or increased (positive value). The z-scores were presented as bar plot (Fig. 2 ) and as segments of inner circles in the figure 3. As can be seen from the figures, only “positive regulation of inflammatory response” GO BP term is upregulated while the other biological processes are downregulated.

Figure 2

Figure 3

In the next section, we checked the interaction between selected ontological groups. One of the most visually appealing way of presenting such interaction is dendrogram (Fig. 4 ). Clusters contain functionally related genes based on their expression pattern. The middle circle represents a logarithm of fold change (logFC) of differentially expressed genes assigned to the studied GO terms. The GO terms are shown as the outer ring. The genes whose expression is downregulated form clusters marked by blue part of the middle circle and analogously, red indicates upregulated genes. Clusters of the same color over the entire width of the outer circle represent genes that are unique for a specific GO term. Clusters of different colors on the cross section of outer circle show sets of genes which are likely to be functionally related. The dendrogram showed that many genes belong simultaneously to “cellular response to interferon-gamma”, “interferon-gamma-mediated signaling pathway” and “response to interferon-gamma”. The genes that are unique for a specific GO term belong mainly to “inflammatory response”.

Figure 4

In the gene ontology database, single genes may belong to many ontological terms. For this reason, we used plots with visualization of logFC values and relationship between genes and selected GO BP terms (Fig. 5 ). The relationship was also presented as a heatmap (Fig. 6 ). The strongest upregulated genes from examined GO BP terms included, among others: CCL4L1- C-C Motif Chemokine Ligand 4 Like 1, CCL8-C-C Motif Chemokine Ligand 8 and JAK2- Janus kinase 2. The most downregulated gene is P2RX1- purinergic receptor P2X, ligand-gated ion channel, 1.

Figure 5

Figure 6

In the next part of analysis, we focused on the interaction between proteins encoded by DEGs belonging to studied GO BP terms. Firstly, we used STRING software for the interaction prediction (Fig. 7).

Figure 7

Finally, we used ReactomeFIViz app for investigation of functional interactions between proteins encoded by DEGs belonging to selected GO BP terms. Among the most significantly enriched functional interaction networks were FI networks for “Immune response” and “Interferon-gamma-mediated signaling pathway” (Fig. 8 and 9).

Figure 8

Figure 9

While CABG has been performed for decades, questions remain regarding the detection and characterization of potential markers that will be valuable to evaluate the conduits patency used in this procedure. The predominant cause of early acute coronary syndromes (ACS) is graft failure. Thus, better understanding of the activated molecular mechanisms occurring soon after surgery may allow a further reduction of the rate of these adverse cardiovascular events. The importance of inflammation and inflammatory pathways in atherosclerotic disease and ACS is well established. Nevertheless, not all molecular mechanisms underlying the inflammatory process appear to be clear. Thus, the present study aimed to compare the level of expression of genes involved in inflammatory processes in both ITA and SV conduits and describe potential molecular factors for the evaluation of ITA and SV segment quality.

Employing the expressive microarray approach to analyze the transcriptome of both venous and arterial grafts, five GO BP terms have been selected: “cellular response to interferon-gamma”, “inflammatory response”, “interferon-gamma-mediated signaling pathway”, “response to interferon-gamma” and “positive regulation of inflammatory response”. Among all selected ontological groups genes involved in the formation and maintenance of inflammatory processes can be distinguished. Overall, the genes presented on heatmaps showed differential expression patterns in both analyzed vessels.

Our results indicate the highest fold change of TNFRSF11B transcript levels from all differentially expressed genes analyzed in this study. Our data indicates higher TNFRSF11B transcript expression levels in ITA. Tumor necrosis factor receptor superfamily member 11b (TNFRSF11B, other name: osteoprotegerin (OPG)) is a glycoprotein that acts as a cytokine of the tumor necrosis factor (TNF) [7]. In mice model without expression of OPG authors have shown increased calcification of the aortic media, particularly when given a high dose of phosphate or vitamin D3. These arteries are also the sites of endogenous OPG expression in normal arteries, raising the possibility of a protective role of OPG. This protective role of OPG is particularly important for preventing vascular calcification occurring secondary to administration of warfarin and vitamin D3 [8]. A potential protective effect of OPG against dangerous rupture in advanced abdominal aortic calcification development has been described [9]. Interestingly, in human studies, a significant association between levels of circulating OPG and vascular calcification has been shown. The presence of abdominal aortic calcification, a known risk factor in the development of abdominal aortic aneurysms, was weakly associated with the progression of abdominal aortic aneurysms [10]. Other studies suggested that a pathological increase of serum TNFRSF11B levels might play an important role in promoting leukocyte/endothelial cell adhesion and endothelial cells dysfunction [11]. In the latest scientific reports authors demonstrated that plasma TNFRSF11B levels were significantly associated with the presence of obstructive sleep apnea (OSA) and its severity. Plasma TNFRSF11B levels provided higher discriminatory accuracy levels for patients with OSA. TNFRSF11B could be a biomarker with a positive diagnostic value for premature vascular endothelial dysfunction in patients with OSA [12].

Transcript levels of C-C chemokine ligand 4-like 1 (CCL4L1) also showed one of the highest fold change value among the genes analyzes, involved in the inflammatory process. Similar to TNFRSF11B transcripts, we observed higher expression levels of CCL4L1 mRNA in arterial conduits. This gene is one of several cytokine genes (together with CCL3-CCL3L1 and CCL4) that are clustered on the q-arm of chromosome 17 [13]. During emergency repair of ruptured abdominal aortic aneurysms, researchers collected aortic wall samples and investigated gene expression using microarrays [14]. The obtained results pointed to overexpression of a set of genes, including CCL4L1. High expression of CCL4L1 in end-stage of AAA disease may identify CCL4L1 as candidate gene related to processes involved in fibrosis. Studies analyzed 127 kidney biopsies obtained from HLA-sensitized (HS), non-HS patients and control individuals, and have shown CCL4L1 expression activated by antibody-dependent cellular cytotoxicity (ADCC) [15].

It is also worth noting that our results indicate a very significant participation of the human leukocyte antigen (HLA) gene complex encoding the major histocompatibility complex (MHC) proteins. We found 4 of the HLA Class I members (HLA-A, HLA-B, HLA-C and HLA-F) and 4 belonging to HLA class II transcripts (HLA-DRB1, HLA-DPB1, HLA-DPA1 and HLA-DQA1). These, highly polymorphic, cell-surface proteins are responsible for the regulation of the immune system in humans. Class I molecules play a central role in the immune system by presenting peptides derived from the endoplasmic reticulum lumen, while class II molecules are recognized by receptors on CD4-positive helper T cells when attacking and eliminating foreign substances [16]. Studies examining the pathogenesis of abdominal aortic aneurysm (AAA) have shown that both HLA-A and HLA-B may be important genetic risk factors for the development of this abdominal aorta pathology [17]. Other studies indicated that bacterial lipopolysaccharide (LPS) enhanced expression of HLA-A, HLA-B and HLA-C in cultured human endothelial cells, whereas it inhibits HLA-DR molecule expression [18]. The new approach to avoid the risk of severe adverse reactions of small diameter vascular grafts may employ HLA-matched vascular grafts. Mallis et al. proposed a solution of the limited availability of the vessels by the use of HLA-matched vascular grafts utilizing the decellularized human umbilical arteries [19]. Our results did not show one expression pattern for HLA gene complex. Most members of the HLA family showed higher transcript levels in arterial conduits, while HLA-A and HLA-DQA1 were upregulated in SV samples.

The most significantly downregulated genes was purinergic receptor P2X 1 (P2RX1). The protein encoded by this gene belongs to the P2X family of G-protein-coupled receptors [20]. Next Generation Sequencing studies analyzing epifascial lymphatic collectors of the thigh, which were isolated for autologous transplantations, found therapeutic targets, suitable for acceleration of lymphatic contractility. Among the most promising results, the authors mention P2RX1 as potential target for diagnosis and therapy [21]. Hennigs et al. [22] showed strong pulmonary endothelial staining of P2XR1, which can contribute to an unfavorable pulmonary disease phenotype found in pulmonary arterial hypertension. We found higher transcript levels of this gene in saphenous vein.

Our transcriptomic analysis of two conduits most commonly applied in coronary artery bypass grafting procedure, the internal thoracic artery (ITA) and the saphenous vein (SV), showed potential molecular markers of formation and maintenance of inflammatory changes. We hope that an analysis of the expression of genes involved in inflammatory response may help to identify patients at high risk of grafts occlusion.