In search of markers useful for evaluation of graft patency - molecular analysis of ‘muscle system process’ for internal thoracic artery and saphenous vein conduits

Graft patency is an important aspect to consider when using coronary artery bypass graft (CABG), the surgical method most commonly used to treat coronary artery disease (CAD). The aim of the procedure is to improve blood circulation in atherosclerotic coronary arteries [1,2]. CABG gives better clinical results compared to other methods, e.g. percutaneous coronary intervention (PCI) [3]. This is reflected in the reduction in mortality in some subgroups of patients, and improvement is also noticeable in patients with complex coronary lesions, e.g. in patients with recurrent myocardial infarction or angina pectoris [2]. However, the use of the CABG method is also associated with numerous perioperative and postoperative complications that may prevent the patient from fully recovering [3].

The vessels used in CABG are usually the internal thoracic (mammary) artery (ITA/IMA) and saphenous vein (SV), [1,2,4,5], which usually replace the left anterior descending (LAD) [6]. The choice of vessel may depend on the patient’s age, as SV is most often chosen in the elderly while young people undergo ITA transplantation [7]. The saphenous vein is a large, subcutaneous and surface vein of the leg which is transplanted in the reverse position so that its venous valves don’t obstruct the flow [5]. For arterial transplantation doctors use left internal mammary artery (LIMA), right internal mammary artery (RIMA) or perform bilateral transplant (BIMA) [8].

Choosing the right vessel for transplantation can be difficult, but the choice is based on the surgeon’s skills, the unit in which the surgery will take place, and the patient’s individual approach. [9] It is well known that SV is a vessel that is easier to wash and its endoscopic uptake does not affect its patency [10]. However, an advantage for ITA may be the fact that they have their own blood source from a blood vessel, they have a well-built internal elastic plaque, less myocytes in the adventitia [11], which makes them less susceptible to atherosclerosis.

Complications that are observed in patients who received SV (acute thrombosis, intimal hypertrophy, atherosclerosis) may be caused by risk factors, including smoking, increased blood cholesterol, diameter of the vessel and the surgical technique used [3]. In contrast, ITA transplants may be subject to narrowing, especially in response to vasoactive drugs [11].

Scientists and doctors are constantly trying to learn about the molecular, physiological, pathophysiological mechanisms affecting the patency of the transplant [12, 13, 14], and to develop innovative surgical methods that can extend the functionality of the transplant [15,16]. Sabik et al. using the method longitudinal analysis showed a patency index 1, 5 and 10 years after surgery. They compared the use of ITA and SV in a population of 50 278 patients. The results for ITA were 93%, 88% and 90% respectively, while for SV 78%, 65% and 57% [17]. The comparison indicates that a better patency index is obtained by using ITA during CABG, however, if ITA arteries are taken from both sides of the body it may result in mediastinitis, which is a life-threatening complication [18]. The pathophysiology of the saphenous vein is becoming better understood over the years, however, the failure of the SV transplant still achieves high results: 35-50%. SV is exposed to arterial circulation, structural changes within the vessel that include an inflammatory response with intimal hyperplasia. To prevent graft remodeling stenosis is successfully used, which reduces hypertrophy and the development of vascular lumen irregularities [15].

The success of the transplant is influenced by many factors, however it is the patency of the transplant that is still a major impediment to the full success of the surgery. This is why it is an important topic of our research, especially in the field of molecular research. Therefore, in our work we tried to determine a marker useful for assessing the patency of the graft based on the muscular system of the blood vessels in question. We focused on 5 ontological groups: “muscle cell proliferation”, “muscle contraction”, “muscle system process”, “regulation of smooth muscle cell proliferation” and “smooth muscle cell proliferation” comparing the internal thoracic artery and saphenous vein conduits.

We used Human Genome U219 Array Strip for the microarray gene expression analysis of internal thoracic artery (ITA) and the saphenous vein (SV) conduits. 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 muscle system process. The DAVID software indicated the following GO BP terms, which cover the above processes: “muscle cell proliferation”, “muscle contraction”, “muscle system process”, “regulation of smooth muscle cell proliferation” and “smooth muscle cell proliferation”. The 39 genes involved in those processes were clustered using hierarchical clustering and presented as heatmaps (Fig. 1).

Figure 1

It is worth mentioning that 12 genes were downregulated while 27 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 the 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 segments of inner circles in the figure 2. The z-scores of above-mentioned GO BP terms had positive values, so the processes described by these GO BP terms were upregulated.

Figure 2

In the next section, we checked the interaction between selected ontological groups. One of the most visually appealing ways of presenting such interaction is dendrogram (Fig. 3). 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 “muscle contraction” and “muscle system process”. The same applies to “muscle cell proliferation”, “regulation of smooth muscle cell proliferation” and “smooth muscle cell proliferation”.

Figure 3

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. 4 ). The relationship was also presented as a heatmap (Fig. 5 ). The strongest upregulated genes from examined GO BP terms included, among others: ACTN2- actinin, alpha 2; RBPMS2- RNA binding protein with multiple splicing 2 and NR4A3- nuclear receptor subfamily 4, group A, member 3. The most downregulated genes are: P2RX1- purinergic receptor P2X, ligand-gated ion channel, 1; KCNH2- potassium voltage-gated channel, subfamily H (eag-related), member 2 and DES- desmin.

Figure 4

Figure 5

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. 6).

Figure 6

In the next part of analysis, we used ReactomeFIViz app for investigation of functional interactions between proteins encoded by DEGs belonging to selected GO BP terms (Fig. 7).

Figure 7

Finally, we mapped gene expression data (ratio more than 1.5) on “calcium signaling pathway” onto pathway graph based on KEGG (Kyoto Encyclopedia of Genes and Genomes) database (Fig. 8).

Figure 8

Despite many studies, there is still little understanding of the molecular causes that affect the patency of the blood vessel used during CABG. The arterial and venous vessels differ primarily in the thickness of the middle muscle membrane, which is much more developed in the case of arteries. A comparison of the ITA and SV muscle profiles used during CABG may indicate potential markers of both blood vessels that can have a significant impact on the quality of the ducts and their usefulness in surgical intervention. By analyzing high-throughput molecular arrays, we have obtained 39 genes that have been interpreted based on ontological groups associated with the muscular system: “muscle cell proliferation”, “muscle contraction”, “muscle system process”, “regulation of smooth muscle cell proliferation” and “Smooth muscle cell proliferation”. We have obtained 27 genes with increased expression and 10 genes with reduced expression.

The highest rate of increase in gene expression was recorded for the alpha actinin 2 (ACTN2) gene, which was identified in two ontological groups: muscle concentration and muscle system process. ACTN2 together with 3 other alpha actinin genes (ACTN1, ACTN3 and ACTN4) is a member of the spectrin gene superfamily. ACTN2 is a sarcometric isoform, its expression occurs in the myocardium and in skeletal muscle is co-expressed in ACTN3 [21]. The ACTN2 protein is responsible for the anchoring and crosslinking of actin fibers in the heart band at the sarcomer boundaries, and also interacts with the ion channels of the heart [22]. In addition, ACTN2, by interacting with other proteins plays an important role in the organization of thin fibers and interactions between the sarcomere and the muscle membrane [23]. By sequencing the entire genome of patients in one family with familial hypertrophic cardiomyopathy, mutations Ala119Thr in this gene were detected in patients with clinical symptoms [22]. Chiu et al. on the other hand, reported 3 more mutations: Thr495Met, Glu583Ala and Glu628Gly in patients from other 4 families [23]. Mutations in this gene may also lead to DCM, idiopathic ventricular fibrillation, juvenile atrial arrhythmias, restrictive cardiomyopathy, and left ventricular failure and sudden unexplained death [24]. Currently, there are numerous new reports on the discovery of mutations in the ACTN2 gene associated with cardiac diseases [24], as well as their impact on the phenotype of a given disease [25]. The formation of atherosclerotic plaques in blood vessels is one of the factors affecting the patency of the vessel. Liu et al. based on cluster analysis, Gene Ontology (GO) and pathway enrichment analysis suggest that deregulation of the immune system and cytoskeleton of smooth muscle cells accelerates the progression of carotid atherosclerotic plaques [26]. This is an interesting reference to our research and may be another alternative to conducting experiments.

Another gene with a high expression increase is RNA binding protein with multiple splicing 2 (RBPSM2), which unlike ACTN2, was found in 3 other ontological groups: muscle cell proliferation, regulation of smooth muscle cell proliferation and smooth muscle cell proliferation. RBPSM2 belongs to the RRM family and is expressed in the vertebrate heart and gastrointestinal tract [27]. As a class of RBP (RNA-binding proteins), they can affect all stages of the RNA life cycle, including synthesis, translocation, translation, processing, and degradation. In contrast, the mutations present in their sequence may be the cause of cardiovascular disorders. Akerberg et al. state that in zebrafish, encoded proteins may be responsible for regulating aspects related to the differentiation, survival proliferation and contractility of cardiomyocytes [28]. Despite the fact that scientists are trying to explain the role of DNA binding proteins in transcriptome regulation during phenotype change in health and cardiovascular disease [29], there are still few sources explaining the role of RBPSM2 in connection with CABG and especially in relation to the patency of the blood vessels used.

Another gene worthy of attention is NR4A3 belonging to the nuclear receptor superfamily. NR4A includes three members: Nur77 (also called NR4A1, TR3, NGFI-B, NAK-1), Nurr1 (NR4A2, NOT) and NOR-1 (NR4A3, MINOR) and are composed of a central DNA binding domain, a C-terminal domain ligand binding and a N-terminal transactivation domain [30]. Nur77, Nurr1 and NOR-1 are induced in human smooth muscle cells (SMCs) after activation by inflammatory cytokines and growth factors, and NR4A nuclear receptors are expressed in human vascular and atherosclerotic lesions [30]. Martinez-Gonzalez et al. demonstrated that NOR-1 is transiently expressed in coronary SMCs in response to balloon dilatation [31], which may be of great importance in CABG. NR4A1 and NR4A3 have been described as receptors regulating cell differentiation, proliferation, inflammation and apoptosis, as well as being particularly important for their impact on the development of cardiovascular diseases [32]. It is known that Neuron-derived orphan receptor 1 (NOR1) regulates the proliferation of blood vessel smooth muscle cells (VSMC). One of the latest studies was based on the protective effect of exendin-4 (Ex-4) on mouse vessels, and by inhibiting NOR1 expression in VSMC. The conclusion drawn from the study suggests that Ex-4 suppression of neointima formation following vascular damage and VSMC proliferation is likely due to inhibition of NOR1 expression [33]. In turn, another research team showed that VTN, a gene coding for the well-known multifunctional glycoprotein (vimentin) of key importance for cell adhesion, migration and proliferation, but also involved in thrombosis and fibrinolysis in humans can participate in NOR-1 mediated vascular responses [34]. Studies of this syndrome have previously also shown the role of NOR-1 as a negative modulator of acute response induced by pro-inflammatory stimuli in the vascular system [35]. In our study NR4A3 identified in 4 of 5 ontological groups (muscle cell proliferation, muscle contraction, regulation of smooth muscle cell proliferation and smooth muscle cell proliferation) which indicates a broad spectrum of the gene’s impact on the muscular system of blood vessels.

We also noticed the KCNA5 and KCNH2 genes belonging to the same ontological groups: muscle contraction and muscle system process. The first observed the fourth largest increase in expression of this gene, while in the case of KCNH2 the second largest decrease in gene expression was noted. These genes encode voltage gated potassium channels (VGPC) Kv1.5 for KCNA5 and Kv11.1 for KCNH2, whose mutations may affect the occurrence of severe arrhythmias [36]. Voltage gated channels are a frequent aspect of research in relation to vascular diseases, e.g. as DHEA affects the expression of Kv1.5 channels and how it relates to remodeling of blood vessels, especially SV and cervical vessels [37]. Further studies also based on the effect on Kv1.5 led to the conclusion that said channel is sensitive to DPO-1, which plays an important role in determining the microvascular tension and response to vasoconstrictive and vasodilating agents [38]. In turn, studies on porcine coronary vessels suggest the dominant role of KV1 channels in modulating coronary output voltage and possibly vasodilatation in response to increased metabolism and transient ischemia [39]. Unlike KCNA5, the KCNH2 encoded channels (ERG channels) were examined to determine expression and their action in mouse arteries. In this case, it was concluded that ERG channels may be non-functional in contractile smooth muscles, however, they give a proliferative story [40].

Genes with the largest decrease in gene expression belong to two ontological groups: muscle contraction and muscle system process, and the largest decrease was recorded for purinergic P2X receptor (P2RX1). P2RX1 belongs to the family of P2X receptors ligated through the P2X channel (7 subtypes) that mediate the action of the purine nucleotide (ATP) as well as its metabolites. It is known that smooth muscle cells in various rat blood vessels express P2RX, including P2RX1 [41] P2X receptors (P2RX) are ion channels that increase intracellular Ca2+ concentrations by direct gating to extracellular Ca2+ [42]. The P2X receptor is of interest to researchers in relation to blood vessel smooth muscle cells (VSMC) e.g. synthetic ATP analogues and P2X receptor agonists (Bz-ATP, α, β-meATP and β, γ-meATP) have been shown to inhibit VSMC calcification in vitro [43]. However, there is little information regarding the effect of P2RX1 on the patency of the blood vessel, so this requires further study.

Also noteworthy is the DES gene with a third decrease in gene expression and MYOT with a fourth decrease in gene expression. The DES gene codes for desmine, which belongs to the family of intermediate fibers, found in smooth, skeletal and cardiac muscle tissue. Mutations occurring in DES in humans lead to autosomal cardiomyopathy, which results in heart failure, arrhythmia and aortic valve blockage. The MYOT gene coding for motilin protein, whose mutation is one of the causes of myofibrillary myopathy (MFM) [44].

In the study presented in this article, we performed a transcriptomic analysis of two major blood vessels: the internal thoracic artery and the saphenous vein used for transplantation in CABG, looking for potential molecular factors that may be useful in assessing the patency of the blood vessel. We focused on the molecular analysis of the muscular system process based on the ontological groups associated with this process: muscle cell proliferation, muscle contraction, muscle system process, regulation of smooth muscle cell proliferation and smooth muscle cell proliferation. We distinguished several genes with different expression in both vessels, described above, of which the ACTN2 and NR4A3 gene deserve special attention. ACTN2 is a gene that can affect the formation of atherosclerotic plaques, while NR4A3 occurs in 4 of the 5 ontological groups discussed and can affect the inflammatory process in the blood vessel. Both genes can have a significant impact on vascular patency but require further confirmatory testing.