Calcific aortic valve disease (CAVD) refers to the fibrosis and calcification of the aortic valve and its surrounding tissues caused by various reasons(10). The disease manifests in the early stage of aortic valve sclerosis, punctate calcification of the valve and thickening of the valve leaflets, without hemodynamic changes (aortic valve flow rate < 2m/s), and no obvious clinical symptoms; as the disease progresses further, the annulus and valve tissue appear thickened, hardened and adhered, leading to fibrosis and calcification of the aortic valve, resulting in aortic stenosis (AS), which in turn leads to a series of hemodynamic changes in the body and Seriously endangers human health (11–13). CAVD is not only the result of aging, but an active pathophysiological process involving multiple factors such as lipid deposition, inflammatory cell infiltration, neovascularization, and cell apoptosis(14).
In the present study, we screened significant DEGs and DEMs between the CAVD and normal samples from the GEO database.Subsequently,through a series of bioinformatics analysis, the miRNA-tF regulatory network was constructed and the genes related to CAVD were determined.The GO analysis demonstrated that those DEGs were enriched in regulation of lymphocyte activation ,leukocyte migration and T cell activation. KEGG analysis revealed that DEGs mostly related to the chemokine signaling pathway, phagosome and natural killer cell mediated cytotoxicity. Finally, three hub genes and two TFs(VCAM1, ITGB2, CD86 ,HLF2 and HIF1A)Identified as key regulators of CAVD.
First of all, In our study, the majority of the genes screened in the network were associated with chemokines, including CCL19, CCR1, CCR2, CCR7, CXCR4.At present, most scholars believe that inflammation is the central link in the pathogenesis of calcified aortic valve disease. Chemokines are a secreted small molecule heparin binding protein, belonging to a superfamily of cytokines, which play an important role in inflammatory response(15). It can chemoattract leukocytes for directional movement, reach the local immune response, and participate in immune regulation and immune pathological response(16). New data shows that CCL19 and CCL21 are involved in inflammatory responses and T cells homing in non-lymphoid tissues(17, 18). Now, more and more evidence shows that CCR is not only related to inflammation, but also related to cardiovascular calcification. Studies have found that SDF-1 can induce platelet aggregation and increase intracellular calcium by binding to the SDF-1 receptor CXCR4 expressed by platelets(19). A clinical study showed that CCR5 polymorphism is related to the degree of heart valve calcification and CCR2 was confirmed with the ability to promote the osteoblastic transformation of valvular interstitial cells(20–22). These strongly suggest that chemokines may regulate pathological cardiovascular calcification. More research is needed to prove the connection between CAVD and CCR and the underlying molecular mechanism.
Secondly, We found that VCAM1 and ITGB2 has important significance in the progress of CAVD.Studies have shown that inflammatory activation of endothelial cells is closely related to calcification in CAVD(23).At the same time, inflammation can induce the early and sustained expression of endothelial cell inflammatory adhesion molecules VCAM-1 and ICAM-1(24).And VCAM-1 has been reported with increased level in endothelium of CAVD, which lead to the main route for tissue leukocyte infiltration and inflammatory process(25–27). Meanwhile, the VCAM-1 interacts with very late antigen-4 on activated lymphocytes and leads to the extravasations of activated CD4 and CD8 lymphocytes into the valve tissue and aggravates inflammatory infiltration.(28). ITGB2 is the beta-2 subunit of the integrin LFA-1, which is also expressed on lymphocytes, especially leukocytes(29). Typically, macrophages gain access to the cardiovascular system via interacting with vascular endothelial cells. Adhesion molecules expressed on the surface of endothelial cells interact with a specific receptor expressed on the surface of macrophages allowing firm and sustained adhesion of macrophages to the vasculature initiating the pro-inflammatory response(30). Meanwhile, Past researchs suggest that MRTF-A may regulate the transport of macrophages by activating the transcription of ITGB2 to promote the pathogenesis of cardiac hypertrophy(31). So, we speculate that the interaction of VCAM1 and ITGB2 triggers an inflammatory response in the aortic valve tissue, which in turn leads to the occurrence of calcification.
Third, CD86 is also one of the very meaningful markers.CD86 is a glycoprotein expressed on antigen-presenting cells which provides costimulatory signals to T cells. Research shows that CD86 can activate regulatory T cells and memory effector T cells express a functional form of CD86 that can costimulate naive T cell responses(32). Studies have shown that there are T cell clonal proliferation and CD4 + and CD8 + T cell infiltration in the calcified aortic valve after surgery, it is confirmed that there is inflammation and damage mediated by adaptive immune response elements in calcified aortic valve(33). Choi et al first confirmed the existence of CD86-expressing dendritic cells in aortic valve(34).All these indicate that CD86 may play a role in CAVD, but its specific mechanism still needs to be explored in depth.
Finally, HIF1A and KLF2 are two important transcription factors discovered in this study. HIF is a hypoxia-inducible factor which is the main regulator of oxygen homeostasis.The increased expression of HIF-targeted genes is associated with many human diseases, including ischemic cardiovascular disease, chronic lung disease, and carcinoma(35). Treatment with the HIF1α inhibitor PX478 significantly reduced calcification of aortic valves in both static and the fibrosa-flow conditions demonstrating their potential as novel anti-CAVD therapeutics(36). In our study, HIF1A is predicted to act as a transcription factor for ITGB2 which is a vital gene in CAVD. So, We speculate that HIF1A-ITGB2 may be one of the important pathways in the pathogenesis of CAVD. KLF2 is a transcription factor induced by laminar flow. Its expression is reduced in calcified human aortic valves and endothelial calcification models, which suggests that KLF2 downregulation may be involved in calcification.Similarly, in our study, we also found low expression of HLF2 in calcified aortic valves.Previous studies have shown that silencing the KLF2 gene can induce the endothelial-mesenchymal transition and lead to calcium phosphate deposition in endothelial cells, which ultimately leads to aortic calcification(37). The above findings indicate that HIF1A and KLF2 are the key molecules in the development of CAVD,which are worthy of our in-depth study.
The miRNA-TF-mRNA regulatory network includes 17 miRNAs, of which 9 miRNAs act on two key TFs respectively. Among them, miRNA-509-5p and miRNA-3121-3p both act on two vital transcription factors(HIF1A,KLF2), however, the interaction between them and the mechanism of action in CAVD have not been reported, and further studies are still needed. MiR-3926 inhibits the proliferation of synovial fibroblasts and the secretion of inflammatory factors by targeting toll-like receptor 5, which suggests that miRNA-3926 may have an impact on the progression of CAVD in the regulation of inflammatory cell function(38). Endothelial cell damage may be the initiating factor in the occurrence of calcified aortic valve stenosis, which is similar to the formation of atherosclerosis(39). miR-186-5p and miR-17-5p are diagnostic biomarkers of atherosclerosis and regulate the proliferation and migration of vascular smooth muscle,which may also mediate the occurrence and progression of CAVD.(40, 41)
Our study has some limitations. First, one limitation of the current research is that the number of samples was relatively small, the input data might still be insufficient to identify and validate key genes in the CAVD development. Second Our study only relies on bioinformatics analysis, and further molecular biology experiments are needed to verify the results of this research. In a follow-up study, the molecular verification experiment will be conducted to verify molecular markers which can be used as a new target for CAVD treatment.