Stroke is the fourth leading cause of mortality and disability in the United States (1), and 10–20% of strokes are caused by carotid artery disease (2,3). According to epidemiological data, ~7 million adults in the United States have suffered a stroke (4), of which ischemic strokes account for ~90% (5). In addition, ~10% of ischemic strokes are caused by carotid artery stenosis (CAS) (6). CAS refers to a narrowing or constriction of the lumen of the carotid artery, usually attributed to atherosclerosis (7). The dynamic and complex process of atherosclerosis remains to be fully understood; however, it is well known that atherosclerosis is characterized by the accumulation of lipid particles and fibrous elements, associated with migration and proliferation of smooth muscle cells, in the large arteries (8–10).

Over the past decade, inflammation and the immune response in atherosclerosis have garnered attention. Previous studies have indicated that low-density lipoprotein particles and their content inside the vessel wall are susceptible to oxidation by free radicals, which may initiate the accumulation and invasion of macrophages, eventually lead to a narrowing of the major arteries (9,11).

During the progression of atherosclerosis, an imbalance between anti-inflammatory and proinflammatory cytokines serves an important role. Previous studies have attempted to identify the immune-associated genes that are involved in atherosclerosis, and achievements have been made (12,13). Superoxide dismutase, which is expressed at higher levels in regions of laminar flow, may combat oxidative stress, and hence limit vascular cell adhesion molecule-1 (VCAM-1) expression and the expression of other inflammatory pathways (12). Nitric oxide arises from endothelial nitric oxide synthase, which is known to be a shear stress-regulated gene, and can inhibit VCAM gene expression through a novel pathway involving inhibition of the activation of nuclear factor-κB, the central transcription factor in vascular inflammation (13). In addition, previous studies have reported that interleukin (IL)-35 may upregulate the expression of anti-inflammatory cytokines (14–16). Huang et al (17) investigated the effects of IL-35 on atherosclerosis and hypothesized that IL-35 could be considered a novel target for the treatment of atherosclerosis. However, the majority of genes relevant to atherosclerosis remain unknown.

Mycophenolate mofetil (MMF) is an inhibitor of the enzyme inosine monophosphate dehydroxygenase (IMPDH), and exerts a powerful cytostatic effect on activated T cells by interfering with their DNA synthesis (18). In the present study, gene expression data were obtained from a Gene Expression Omnibus (GEO) dataset uploaded by van Leuven et al (19), which included 20 carotid endarterectomy samples from patients with CAS (>70% diameter stenosis on angiography) that were randomly assigned to the following treatment groups: Treatment with 1,000 mg MMF (n=9) or placebo (n=11). Patients were treated with MMF or placebo for ≥2 weeks prior to undergoing carotid endarterectomy (CEA). van Leuven et al (19) reported that the inflammatory process in human atherosclerotic plaques could be modified by short-term treatment with MMF, as determined using mRNA expression profiling. However, this previous study did not analyze the expression data in detail, nor did it determine how MMF functioned in the treatment of symptomatic CAS (SCAS) or the molecular mechanisms of SCAS.

In the present study, the gene expression data were used to identify differentially-expressed genes (DEGs) between MMF-treated and placebo-treated groups, with the aim of identifying potential genes associated with atherosclerosis, which may be considered targets for novel gene therapy. A total of 210 DEGs between the MMF and placebo groups were identified with a threshold of P<0.05. After analyzing the regulatory effects, a regulatory network was constructed based on the DEGs. Subsequently, the data were processed by bioinformatic analyses, including hierarchical clustering, Gene Ontology (GO) terms (molecular function, biological processes and cellular components) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Finally, the 19 most significant DEGs were screened; the results of these analyses indicated that apelin (APLN) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) may be valuable for characterizing the mechanism underlying immunomodulatory therapy in atherosclerosis.

The treatment of CAS includes three modalities: Medical management, carotid artery angioplasty and stenting, and CEA (26). However, there are doubts regarding the efficacy of carotid artery angioplasty and stenting, and CEA, in specific patients (27–29). Previous studies have aimed to identify novel treatments and medications for atherosclerosis. Following construction of the hypercholesterolaemic apolipoprotein E-deficient murine model, Chen et al (30) demonstrated that preimplantation factor could prevent atherosclerosis via its immunomodulatory effects without affecting serum lipids. In addition, Sun et al (31) developed trifunctional Simian virus 40 (SV40)-based nanoparticles for in vivo targeting and imaging of atherosclerotic plaques, and targeted SV40 virus-like nanoparticles were revealed to deliver a greater concentration of the anticoagulant drug Hirulog to atherosclerotic plaques.

It is well known that MMF is a T cell suppressor, which is able to reduce synthesis of guanine via IMPDH inhibition, resulting in the suppression of T cell proliferation (32). van Leuven et al (19) demonstrated that treatment with MMF was able to reduce inflammatory cell infiltration, with a concomitant decrease in proinflammatory gene expression in patients with SCAS. The present study downloaded and analyzed a GEO mRNA expression profile uploaded by van Leuven et al (19). A total of 210 DEGs were identified between MMF-treated and placebo-treated groups. Subsequently, the 19 most significant DEGs were selected to undergo GO functional analysis and KEGG pathway enrichment analysis. The results revealed that the most enriched KEGG pathway was purine metabolism, indicating that suppression of inflammatory activity served an important role in the MMF treatment of patients with SCAS. Furthermore, with a threshold of degree ≥3 and |logFC|≥0.5, three genes, APLN, creatine kinase B (CKB) and KIT, were selected as the key genes.

APLN is a peptide, which was initially identified by Tatemoto et al in 1998 (33), that functions as an endogenous ligand for the orphaned G-protein-coupled receptor (APJ) (34). Previous studies have revealed that APJ deficiency can prevent oxidative stress-associated atherosclerosis and that the APLN-APJ system is a mediator of oxidative stress in vascular tissue (33,35–37). In the present study, expression of APLN was significantly different between MMF-treated and placebo-treated groups, and APLN was a key gene in the PPI network, indicating that APLN may be a target of MMF for the treatment of SCAS. According to the results of the KEGG analysis, purine metabolism was the most enriched signaling pathway, indicating that MMF modified the atherosclerotic plaque by suppressing activated T cells. Recent studies have also reported that APLN is involved in the immune response (38,39). By analyzing the expression and function of the APLN-APJ system in tumor vasculature, Kidoya et al (38) indicated that the APLN-APJ system could induce maturation of tumor vasculature and improve the efficiency of immune therapy. The results of the GO analysis suggested that terms associated with Stat5 were the most enriched, including tyrosine phosphorylation of Stat5 protein, regulation of tyrosine phosphorylation of Stat5 protein and positive regulation of tyrosine phosphorylation of Stat5 protein (Table III). Previous studies have indicated that Stat5 has a strong association with T cells. Lindahl et al (40) provided evidence to suggest that microRNA-21 is expressed in situ in cutaneous T cell lymphomas skin lesions, as induced by IL-2 and IL-15 cytokines, and is regulated by Stat5 in malignant T cells. The APLN-APJ system is also involved in the immune response and Stat3. Han et al (41) indicated that binding of phosphorylated-Stat3 to the APLN promoter is the final step underlying proinflammatory cytokine-induced enteric APLN expression during intestinal inflammation. However, whether APLN is involved in the Stat5 signaling remains to be elucidated. It may be hypothesized that APLN affects T cells through the Stat5 signaling pathway; however, further studies are required.

Mast/stem cell growth factor receptor, also known as proto-oncogene c-Kit, tyrosine-protein kinase Kit, or cluster of differentiation 117 is a receptor tyrosine kinase protein that in humans is encoded by the KIT gene (42). The GO analysis results revealed that KIT was closely associated with tyrosine phosphorylation of Stat5 protein, thus indicating that dysregulation of Stat5 may be important in the process of SCAS.

Brain-type creatine kinase is a creatine kinase encoded by the CKB gene in humans, which is associated with creatine kinase activity and cellular monovalent inorganic anion homeostasis (43). However, to the best of our knowledge, the involvement of CKB in the process of SCAS has not yet been reported.

In conclusion, APLN and KIT may serve important roles in the MMF treatment of SCAS. The results of the present study suggested that MMF may upregulate APLN to inhibit the proliferation of T cells through the Stat5 signaling pathway. Further investigation of the function of APLN and KIT in atherosclerosis is urgently required.