Valvular heart disease is obtaining growing attention in the cardiovascular field and it is believed that calcific aortic valve disease (CAVD) is the most common valvular heart disease (VHD) worldwide (Iung and Vahanian, 2011; Liu et al., 2017). It is also the predominant causation of aortic valve stenosis (Iung and Vahanian, 2011). CAVD presents with an escalating fibrous calcification remodeling and pathological incrassation of aortic valves, which evolves for a long time, inducing a serious block of cardiac outflow and ultimately reducing the mobility of leaflets (Lindman et al., 2016). Aortic stenosis (AS), the most common clinical manifestation of CAVD, its prevalence increases with age, impacting about 9% of patients older than 80 years. In addition, aortic sclerosis is obviously more widespread, impacting 25–30% of patients older than 65 years, and progresses to AS at a ratio of approximately 2% every year (Coffey et al., 2014; Otto and Prendergast, 2014). It is assumed that CAVD is becoming a major societal and economic burden for the aging world that is likely to be confirmed in the near future (Osnabrugge et al., 2013). In spite of the progress on radical therapies, such as valve replacement and intervention therapy already used for aortic stenosis, still, the specific molecular mechanism of aortic valve calcification remains unclear. Therefore, it is of great necessity to understand the evolution of CAVD and explore the key molecules for better treatment of aortic stenosis.

As is well-known, CAVD develops slowly and the severity of each stage is different. In the beginning, there is only mild valve thickening while at terminal stage, grievous calcification is accompanied by impaired leaflet movement, aortic valve stenosis and clinical symptoms such as heart pits, dyspnea, and angina (Freeman and Otto, 2005; Rajamannan et al., 2007). In the past, CAVD was deemed to be a retrograde procedure owing to the time-conditioned abrasion of valves and calcification deposition passively. At present, lots of convincing evidences have implied that CAVD was an unasked, multifaceted process, which is concerned with the sedimentation of chylomicrons, VLDL, LDL, active valve calcification, ossification of valvular interstitial cells, and so on (Freeman and Otto, 2005). More and more researches suggested that signal transduction channels referred to the progress of aortic calcification consisted of various growth factors, cytokines, and tumor necrosis factors (O’Brien et al., 2002; Jian et al., 2003; Kaden et al., 2005). Since statins and angiotensin-converting enzyme inhibitors failed to retard CAVD progression, there were no effective medical therapies (Lindman et al., 2013; Nishimura et al., 2014). When CAVD progresses to an advanced stage with terrible clinical manifestations, the most effective measures are surgery and transcatheter aortic valve replacement (TAVR), but the actual situation is that these procedures are related to higher costs, unavoidable risk of death, perioperative and long-run complications such as complications of lifelong anti-coagulation therapy and reoperation of artificial valve insufficiency (Lindman et al., 2016).

Hence, it is necessary for us to make a deeper discernment of the latent mechanisms of CAVD and mine certain key genes which perhaps are curative targets. In the recent years, the high-throughput sequencing information acquired via chip techniques helped us confirm the differentially expressed genes (DEGs) and their functions, excavate their pathways related to the advancement of various complex disorders (Zhao et al., 2018). Through comprehensive bioinformatics analysis of public genetic data, we could be able to conduct a secondary data digging and identify biomarkers related to diseases (Lei et al., 2019). Furthermore, we investigated the correlation between key molecules and immune cells to deeply and comprehensively comprehend the immune mechanism among the developing process in CAVD.

Calcific aortic valve disease stands for a disease that covers the process from aortic valve sclerosis to aortic valve stenosis (Freeman and Otto, 2005). Incremental proofs verified that CAVD is an irreversible valvular lesion that remains irrecoverable though tremendous improvement in the surgical treatment strategy (Pawade et al., 2015). CAVD was primitively regarded as a retrograde procedure due to the gradual calcium sedimentation in aortic valve while getting older. It is generally accepted that CAVD is a complicated and active process and various physiological and pathological conditions are involved in the regulation like lipoprotein deposition, inflammatory response, activating RAAS, transformation of ECM, and cellular ossification (Mathieu and Boulanger, 2014; Kostyunin et al., 2019). Previous researches showed that valvular disease was widespread in most industrialized countries, where a decrement in the prevalence of rheumatic heart disease (RHD) has been associated with an enhancement in the popularity of degenerative etiologies (Coffey et al., 2014). Some studies have shown that valvular disease and coronary disease have some common risk factors, including hypertension, diabetes mellitus, dyslipidemia, cigarette, and so on (Nishimura et al., 2014). The pathophysiological process of CAVD principally devastates endothelial valve cells and valvular interstitial cells, exacerbating valvular calcification, making for stenosis of valve orifice, and blocking the left ventricular outflow tract (Dutta and Lincoln, 2018). The loading of valvular disease is anticipated to ascend, in consideration of the prolonged life expectancy and the current insufficiency of effective preventive measures, emphasizing the need to deepen our discernment of the pathophysiology of retrogressive heart valve diseases (Gould et al., 2013; Lindman et al., 2013). In recent decades, gene sequencing and bioinformatics technologies have developed dramatically, so it became possible to further analyze and utilize plentiful sequencing data (Zhao et al., 2020). In the present study, we screened significant DEGs between the aortic stenosis of CAVD and control samples from the GEO database. Furthermore, quite a lot of bioinformatics analysis was performed. The GO function annotation demonstrated that those DEGs were enriched in some BPs such as immune response, signal transduction, inflammatory response, proteolysis, innate immune response, and apoptotic process. KEGG pathway enrichment analysis uncovered that DEGs markedly related to chemokine signaling pathway, cytokine-cytokine receptor interaction, and PI3K-Akt signaling pathway. To explore molecular mechanism of CAVD, ten hub genes (CXCL13, CCL19, CCL8, CXCL8, CXCL16, MMP9, CCL18, CXCL5, VCAM1, and PPBP) connected with CAVD occurrence were explored out based on the STRING and Cytoscape software. We speculate that these important DEGs and their potential functions bring about the progression of CAVD in theory.

Among these hub genes, two kinds of chemokine ligands, CXCLs and CCLs, are very conspicuous. As we all know, the chemokine ligands family contains chemotactic cytokines or ligands and they are significant ingredients of the cross-communication among the assorted cell types in the inflammation or tumor microenvironment (Lacalle et al., 2017). Chemokine ligands mediate their activity by way of interacting with G protein coupled receptors that have unique 7 transmembrane architecture, hence becoming chemokine ligand/receptor pairs (Hussain et al., 2019). Chemokine ligand/receptor interactions include huge signal plasticity and complexity, which are crucial for fine-tuning chemotaxis of specific leukocyte subsets (Borroni et al., 2010). Firstly, the C-X-C motif chemokine ligands CXCL13, CXCL8, CXCL16, and CXCL5 are insiders of the CXC chemokine family. The protein encoded by CXCL13 is a chemokine ligand that plays a role in the homing of B lymphocytes to lymphoid follicles. Activated germinal center helper T cells are expressed at a high level. Cells with an up-regulated expression of CXCL13 receptor CXCL5 can induce chemotaxis. It induces B cells to enter follicles, and induces memory T cells in the paracortical area to return to follicles. Anne et al. reported a crucial function of CXCL13 and its receptor CXCR5 in durability and maintenance of cardiac structure in the case of pressure overload, possibly via modulating small leucine-rich proteoglycans (SLRPs) in ECM (Waehre et al., 2011). CXCL8, also known as interleukin-8, is a cytokine secreted by macrophages and epithelial cells and has a chemotactic effect on neutrophils to achieve its regulation of inflammation. It was reported that this proinflammatory protein may act on coronary artery disease even endothelial dysfunction and CXCL8 classically associates with an effective angiogenic role. The combination of CXCL8 with its receptors (IL-8RB/CXCR2) raises vascular permeability, and the increase of IL-8 level is positively correlated with the severity of prognosis of many diseases. Observations from Kokje et al. (2018) recognized that the high expression of CXCL8 is a clear distinguishing feature among abdominal aortic aneurysm disease (AAA) and atherosclerosis, and proved that there is a transcription mechanism demanding CXCL8 signal transduction in AAA. CXCL16 (C-X-C motif chemokine ligand 16) protein exists in two forms, transmembrane-bound type and soluble secretion type. The two forms have different biological effects and are related to each other. The transmembrane-bound type mainly functions as a scavenger receptor, and the soluble secreted type mainly functions as a chemokine. CXCL16 explicitly combines with Ox LDL, indicating that it probably relates to atherosclerosis (Shimaoka et al., 2000). There was indeed research deeming CXCL16 can have athero-protective effect but might make plaque unstable, yet CXCR6 can prevent myocardial ischemia (Izquierdo et al., 2014). Furthermore, the CXCL16 also induces a strong chemotactic response and calcium mobilization that may have a potential effect on the progression of CAVD. CXCL5 bound to G-protein coupled receptor and transmits cell signals recruiting neutrophils promoting angiogenesis and rebuilding connective tissue. In addition, CXCL5/ENA-78 is a pro-inflammatory chemokine pair that mediates CXCR2-dependent neutrophil transportation in humans (Layhadi et al., 2018). Researchers believed that platelets are the major source of CXCL5 in homeostasis, nevertheless, CXCL5 is mainly generated by lung epithelial cells in fierce infection (Koltsova and Ley, 2010). Performer studies showed that CXCL5 induces EMT to promote CRC cell migration via activating ERK/Elk-1/Snail, and enhances infiltration by activating AKT/GSK3β, thereby inhibiting β-catenin degradation (Zhao et al., 2017). Interestingly, Ravi et al. (2017) found that the plasma CXCL5 level is negatively correlated with CHD severity from clinical evidence.

Besides, the CC cytokines CCL19, CCL8, and CCL18 screened in the network are proteins containing two neighboring cysteine. CCL19 can function in lymphocyte recirculation and homing and is also a significant regulatory factor, which induces T cell activation, immune tolerance, and inflammatory reaction in the course of sequential immune monitoring and homeostasis. It reported that CCL19 and the paired CCR7 were are associated with autoimmune diseases like rheumatoid arthritis and enteropathy (Burman et al., 2005). Damås et al. (2007) found that CCL19 was expressed in the vascular plaques of ApoE -/- mice with atherosclerosis, homo’s carotid plaque and plasma of sufferers with CAD. Moreover, they also testified the plasma level of CCL19 clearly increased in cases with unstable angina pectoris than the sick with stable angina pectoris and healthy control patients (Damås et al., 2007). CCL8 is another member of the CC subfamily, which CCL8 has chemotactic activity on mononuclear cells, leukomonocytes, basophilic granulocytes, and eosinophilic granulocytes. Through enlisting white blood cells to inflammatory places, CCL8 contributes to tumor-interrelated neutrophils infiltration and antivirotic condition in opposition to HIV (Lacalle et al., 2017). The cytokine CCL18 exerts obvious chemoattractant activities on immature T cells, CD4+ and CD8+ T cells, and resting lymphocytes. It recruits immature T lymphocytes to DCs and actuated macrophages in lymph glands and functions in humoral and cellular immune processes (Murphy et al., 2000). Matrix metalloproteinase (MMP) are related to the decomposition of ECM in natural physiological events like fetal development, engendering, and tissue reconstitution, along with in clinical courses like arthrophlogosis and metastatic tumor (Kluger et al., 2013). The function of MMP9 is to debase collagen IV and V. Researches on macaques have shown that MMP9 is concerned with IL-8-elicited mobilization of bone marrow-derived blood ancestral cells and additionally, some studies indicated that MMP9 acted on tumor-related tissue reconstitution (Pruijt et al., 1999). Vascular cell adhesion molecule 1 (VCAM1), belongs to the Ig superfamily, encodes sialoglycoprotein on endothelial cell surface activated by cytokine. VCAM1 is a type I membrane protein as well mediating the adhesion of leukocytes to endothelial cell and signaling transduction and functioning in the development of atherosclerosis and rheumatoid arthritis (Dessein et al., 2005). It is reported that VCAM1/ITGA4/ITGB1 interactivity possibly worked on the pathophysiological process of immunological reactions and leukocyte traffic to inflammatory spots. Pro-platelet basic protein (PPBP), alternately named as CXCL7, is set free abundantly from activated platelets and participates in vascular injury responses (Stankiewicz et al., 2014). PPBP is also an effective chemotactic agent and neutrophil activator. What is more, researched suggested that it stimulated a variety of cellular physiological courses containing DNA synthesis, mitosis, anaerobic glycolysis, cytosolic cAMP amassing, PG-E2 release, and so on (Brown et al., 2017). In a nutshell, it seems that the inflammatory response is very active during the calcification of CAVD.

To search immunocytes infiltration in the microenvironment of CAVD, CIBERSORT was employed to manage a thorough estimation of the data obtained from GEO previously. A growing infiltration of macrophages M0, B cells memory, plasma cells and a dropping infiltration of B cells naïve, NK cells activated, macrophages M2 were identified that may be connected with the incidence and progression of CAVD. Former researchers have demonstrated the existence of inflammatory T cells which accounted for atherosclerotic lesions forming at an early stage of aortic valve disease and the presence of C-reactive protein in valve samples of candidates suffering from aortic stenosis after percutaneous aortic valve replacement illustrating an inflammatory state during calcification (Galante et al., 2001; Rajamannan et al., 2005). Notably, we found that the macrophage0 population were significantly high expression in the calcified valve. This is consistent with previous research results (Cote et al., 2013). In another study, aortic valve interstitial cells (AVICs), the main components of valves, were treated with conditioned media from M1 macrophages. The results displayed that expression of osteogenic calcification markers increased at transcription and translation levels, antibody barricade the inflammatory factors TNF and/or IL-6 decreased the calcification ex vivo indicating a pro-calcific effect of macrophages (Li et al., 2017). Research showed that chronic inflammation with infiltration of lymphocytes, phagocytes, tissue cells, and mastocytes existed in calcific aortic stenosis (Steiner et al., 2012). In addition, it was reported that NK cells gathered in the valve and circulation of patients with CAVD, which is related to the addition of valve pressure gradient (Mazur et al., 2018). From another perspective, correlation analysis in our study showed that the identified key genes CCL8, CCL18, CCL19, CXCL16, and VCAM1 were related to lipid metabolism biomarkers, which were closely related to the occurrence of CAVD from a biochemical point of view. A study on senile calcific aortic valve disease showed that plasma lipoprotein(a) was an independent risk factor for CAVD (Zhanjing et al., 2019). It can be speculated that cytokines and lipid metabolites may also have lipid-cytokine-chemical cascades that is similar to neutrophil-driven joint inflammation in immune complex-mediated arthritis and eosinophilic pneumonia driven by TH2 cells (Sadik and Luster, 2012). Additionally, experiments have shown that the expression of chemokines can directly affect the production of cytokines and cellular immune responses (Murphy et al., 2000). From the results of our study, we reasonably believe that chemokines and immune cells interact with each other during inflammation and even calcification of CAVD. For example, CXCL13 obviously leads to the recruitment of B cells and the production of LT-a β. In turn, LT-a β can stimulate the secretion of CXCL13 (Lalani et al., 1999). In addition, many chemokines secreted by lymphocytes have their own chemotactic effects. The concentration of lymphocytes in the microenvironment can be significantly expanded. Chemokines are also expressed on the surface of immune cells such as T or B cells. The expression of CCR7 causes dendritic cells to accumulate in the lymph. The fluid is infused into the human tube and T cells are distributed in the lymph nodes. Both CCL21 and CCL19 can bind CCR7, they can induce the targeted migration of dendritic cells, and move to a dense area of T cells. On the other hand, dendritic cells chemotaxis through secretion factors CCL18, CCL19, and CCI21 can also recruit natural T cells, Dendritic cells, and activated T cells (Stein et al., 2000). Chemokines provide an effective mechanism for the mutual positioning of immune cells to ensure the smooth progress of the immune response. The migration of immune cells is closely related to the functional role it plays, and chemokine receptors provide better characteristic markers for functional lymphocyte subsets during the progressive inflammatory calcification of AS.

Above all, we found that the chemokines CXCL13, CXCL8, CXCL16, CXCL5, CCL19, CCL8, and CCL18 are the most important markers of CAVD. We detected that regulatory macrophages M0, B cells memory, plasma cells, B cells naïve, NK cells activated, and macrophages M2 possibly referred to the occurrence and progress of CAVD. As the number and status of so many immune cells are significantly changed in CAVD, despite how the immune system affects AV calcification is not clear, we can infer that the immune system produces a major effect on the proceeding of calcification. In addition, these chemokines and immune cells may interact in the development of calcification in CAVD. The limitation of this study is that we did not conduct experimental studies and, thus, a deeper investigation of the immune cells can ascertain the ideal immunotherapy target and heighten autologous immunomodulatory for sufferers with CAVD.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

DW was agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved and drafted the manuscript. BL and TX made substantial contributions to conception and design and revised the manuscript critically for important intellectual content. JW and KX made substantial contributions to acquisition of data. WY made substantial contributions to analysis and interpretation of data. QS gave final approval of the version to be published. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.