Introduction

Cluster of differentiation 146 (CD146)/melanoma cell-associated molecule is an essential immunoglobulin-like protein initially discovered in metastatic melanoma [1]. It locates at endothelial tight junctions across all vessel beds, mediating physiological and pathological events under vascular dyshomeostasis [2, 3]. Pioneering researchers regard CD146 as a historical marker for isolating circulating endothelial cells that slough off inflamed vasculature [4]. Over several decades, CD146 has also been discovered in other cell types including mesenchymal stem cells [5], endothelial progenitor cells [6], macrophages [7], T helper 17 cells [8], B lymphocytes [9], T lymphocytes [9, 10], and natural killer cells [9]. The CD146+ circulating cells occupy about 2% of peripheral mononuclear cells in healthy individuals [9] and most notably, this percentage increases in certain conditions associated with vascular dysfunction like myocardial infarction, connective tissue diseases, and cancers [6, 11,12,13]. Moreover, CD146-activated T cells have shown an enhanced ability to interact with endothelium in adhesion, rolling, and transmigration, evidenced by human and murine studies [14, 15]. Given its multiple functions in vessel structure, angiogenesis, and lymphocyte activation and its enabled detection in the bloodstream, CD146 appears to be a potential target for vascular disorders [16,17,18].

Complex networks are of great interest to researchers in the fields of computational biology and bioinformatics [19,20,21]. It has been gradually extended from initial gene comparison to protein–protein network modeling, to protein–genetic investigation, and up to the disease–disease association exploration [22]. Most of the successful bioinformatics approaches that identify the initial key genes, however, have been based on only gene expression comparison and restrict the following analysis to the top differential expression genes (DEGs) without paying attention to the gene interaction rearrangement [23, 24]. Instead, the hub-structured network is an important motif that is, to our best knowledge, leading the genome-wide association characterization in complex networks [1: Figure S1B). The clustering coefficient fold-changes of NR4A3 and CCL20 were 15.7 and 10.2, respectively; and the gene expression fold-changes were 0.379 and 0.422, respectively. Gene connections are partly verified by the STRING datasets (Additional file 1: Figure S2).

Fig. 7
figure 7

CCL20 cluster and NR4A3 cluster formation in early-stage AMI. CCL20 and NR4A3 stay in the peripheral position of DCGs’ network under normal state (A). However, they shift to the primary position of DCGs’ network dominating two functional clusters under AMI stimulation (B). DCGs differential connectivity genes

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Gene enrichment

Biological process analysis showed that DCGs were involved in response to organic substrates, positive regulation of leukocyte activation, immune response, immune system process, response to cytokine, and regulation of cytokine production. The CCL20 cluster was essential to the immune response, immune system process, and regulation of localization while the NR4A3 cluster was essential to cellular response to corticotropin-releasing hormone stimulus, positive regulation of leukocyte activation, and regulation of apoptotic process (Table 2).

Table 2 Top six gene enrichment outputs of DCGs and hub gene clusters
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Reactome pathway analysis revealed that DCGs were related to the immune system with regard to tasks such as signaling by interleukins, namely interleukin-1, interleukin-4 and interleukin-13, the innate immune system, and the dectin-2 family. The CCL20 cluster was essential to immune system, innate immune system, the dectin-2 family, and neutrophil degranulation while the NR4A3 cluster was essential to RNA Polymerase II Transcription, Generic Transcription Pathway, and MyD88 cascade initiated on plasma membrane (Table 2).

Discussion

CD146 is a junction-associated adhesion molecule that participates in immune and inflammatory pathological processes in the initiation and development of vascular diseases [2]. CD146-activated leukocytes are recruited to the inflamed endothelium to induce the expression of chemokines and cytokines and, in doing so, progressively destroy the blood vessel barrier. Our study finds that following AMI stimulation, in CD146+ human blood cells, 126 out of 23,520 genes show significant differential expression (P  <  0.0001) and, among those, 27 genes show consistent connectivity changes and serve as DCGs. Unlike DEGs, DCGs are able to not only aggregate gene expression, but also encompass gene connectivity properties, internally coupling into functional gene clusters—NR4A3 cluster and CCL20 cluster—orchestrating the gene networks’ entire dynamics in CD146-associated AMI pathophysiology development. Meanwhile, NR4A3 and CCL20 are revealed as hub genes since they experienced both significant connectivity and expression changes after AMI stimuli. Furthermore, gene enrichment analysis shows that the DCGs are involved in inflammation–immune response, with CCL20 being principal to the immune response and regulation of localization; while, the NR4A3 cluster is principal to leukocyte activation, apoptotic process, and cellular response to corticotropin-releasing hormone stimulus. Such findings align with the well-known hypothesis that CD146-mediated inflammation plays an important role in the pathogenesis of AMI.

DCGs revealed by gene connectivity network

The network structural parameter analysis method is applied to weave the gene–gene correlation network. We identify DCGs which present steadily elevated connectivity under AMI conditions in both the discovery and validation cohorts, and further confirm the upregulation seen in the combination cohort. As expected, the gene expressions of DCGs are increased after AMI, but are not distinguishable from DEGs solely by expression signatures (data not shown). NR4A3 and CCL20 as Queryhighlight hub genes are also defined as AMI biomarkers after pre-filtering the comorbidity-relevant genes by the original Topol group [12]. SOCS3 tends to be the only “shared” AMI biomarker candidate revealed by other groups in which the same GSE66360 dataset is included as one of their study objects [27, 28]. Recognizing the CCL20, NR4A3, and SOCS3 as top DCGs substantiate previous outputs and, in turn, the validity of our method is enhanced. Therefore, we recommend the gene connectivity analysis, along with gene expression signature, to be used as a powerful and unbiased way for researchers to rank the importance of candidate DEGs.

The NR4A3 hub gene

NR4A3 belongs to the NR4A orphan nuclear receptor family (with NR4A2 and NR4A1), which plays an important role in AMI development. The JM Penninger group reports that NR4A3 is the highest-ranking gene in circulating human endothelial cells under atherosclerosis [37]. Transcription analysis of human left ventricular myocardium shows that NR4A3 upregulates during ischemia and reperfusion in normal and chronic ischemic myocardium [38]. Similarly, NR4A3 is found to be elevated 10 days post-left anterior descending artery ligation ischemia surgery in mice [39]. Overexpression of NR4A3 significantly reduces infarct size, preventing deterioration of left ventricular function and repression of neutrophil infiltration in the heart of mice after coronary artery ligation and relates to the activation of JAK2/STAT3 and the inhibition of STAT3-dependent NF-κB signaling pathways [40]. Additionally, it has to be pointed out that the NR4A subgroup, including NR4A3, is an immediate early response gene induced by diverse physiological stimuli, i.e., mechanical agitation, calcium, and inflammation cytokines [41]. This reinforces our data that, in very early-stage AMI, NR4A3 has a significant 16-fold clustering coefficient climb and 42% gene expression increase. Yet, despite these characteristics, implications of the nuclear factor NR4A3 in CD146+-related myocardial disorders remain a mystery.

The CCL20 hub gene

CCL20, a C–C motif chemokine, is a chemoattractant for recruiting leukocytes to sites of injury and inflammation. CCL20 secretion is induced by pro-inflammatory chemokines and cytokines, such as CXCL12, IL17, IL1β, IL6, and is in part related to JAK/STAT pathway signaling in multiple cell types [42,43,44]. IL6 and soluble IL6 receptor stimulate STAT3 binding to the CCL20 promotor, and IL17 stimulates the phosphorylated NF-κB binding to the CCL20 promoter in murine astrocytes, facilitate neuroinflammation within the central nervous system [42]. In addition, the co-expression of CCL20 receptor CCR6 and CD146 is a marker of effector memory Th17 cells which mediate migration and is thought to be essential for inflammation in human psoriasis [8]. Moreover, it is reported that CCL20 levels are elevated in the serum of clinical patients with ischemic myocardial infarction [45, 46]. In vitro study shows that CCL20 expression increases in CD146+ human mesenchymal stromal cells at the early pro-inflammatory phase in fracture healing [47]. Thus, we hypothesize that CCL20 binding its receptor CCR6 is likely what drives the CD146-mediated vessel inflammation progress in early AMI phase.

Functional gene clusters

In terms of functional models, DCGs are self-organized into two clusters, the NR4A3 and CCL20 clusters, with 18 genes and 12 genes in each cluster, respectively. All genes are directly linked to its hub gene and partly linked to adjacent genes. Protein–protein connection analyzed by STRING database produces a structure that is greatly similar to our network pattern in which CCL20 connects with CXCL16, IL1R2, MMP9, NLRP3, BCL6 LILRB2, PELI1, CLEC4E, FCER1G, and NR4A3 connects with NR4A2, FOSL2, RNF144B, CDKN1A, SOCS3. A few of the gene–gene correlations within clusters are stated in inflammatory diseases. MMP9 activation correlates with CCL20 expression in astrocytes via Notch-1/Akt/NF-κB pathway promoting leukocyte migration across the blood–brain barrier [48]. NR4A2 and NR4A3 as orphan nuclear receptors mediate neutrophil number and survival in chronic inflammatory signals in multiple hematologic disorders [49,50,51]. FOSL2 acts as an activating protein-1 transcription factor promoting hematopoietic progenitor cells transition to macrophages and neutrophils in an SOCS3-dependent manner is reported [52]. Nevertheless, most of the cluster functions are rarely reported in AMI pathogenesis.

Taken together, NR4A3 and CCL20 clusters are novel functional modules in CD146+ cell-mediated immuno­inflammatory balance, triggering increased susceptibility to vascular deterioration and accelerating myocardial injury. Meanwhile, we propose that NR4A3 and CCL20 are promising biomarkers for clinical diagnosis and potential therapeutic candidates since they largely impact the early AMI development. In-depth studies are necessary for understanding the mechanisms of peripheral CD146+ cells in cardiovascular disease.