CAVD, a chronic progressive disease, develops gradually from valvular sclerosis and valvular fibrosis to valvular calcification. CAVD eventually leads to stenosis of left ventricular outflow and severely disrupts hemodynamics [27]. As the population ages, CAVD becomes a major health problem due to its high prevalence, morbidity, and mortality rate. With a deeper understanding of CAVD, treatment measures for patients with CAVD will be more precise. Incremental proofs verified that the pathology involved in CAVD is multifactorial, including valve endothelial cells differentiation and damage, inflammation, fibrosis and calcification. In recent years, studies have demonstrated that the inflammatory response plays a pivotal role in occurance and development of CAVD [28, 29]. In the present study, we aimed to screen out important DEIRGs and identify the patterns of infiltrating immune cells in CAVD in detail. 220 DEIRGs were eventually identified in aortic valve tissue samples from patients with CAVD after a detailed integrative analysis that included all relevant gene expression profiles in the GEO database. GO analysis of DEIRGs in CAVD revealed that leukocyte migration, receptor-ligand activity, leukocyte cell-cell adhesion, myeloid leukocyte migration and membrane raft and membrane microdomain were significantly enriched. These biological processes and molecular functions were correlated with immune cells infiltration [29]. Infiltrating immune cells could release inflammatory and fibrotic cytokines and further aggravate inflammatory response. DEIRGs are also involved in the regulation of cytokine receptor activity, cytokine activity and cytokine production in KEGG analysis. The inflammatory factors secreted by invading inflammatory cells, such as IL-1β and NF-κB, promote extracellular matrix remodeling, lipid deposition, fibrosis, ossification and calcification [30]. These findings indicate that DEIRGs in CAVD are involved in the immune and inflammatory processes In the “cytoHubba” plugin, PTPN11, GRB2, SYK, PTPN6 and SHC1 were identified as top 5 key DEIRGs according to the results of mixed character calculation.
Protein tyrosine phosphatase (PTP) is a kind of protein phosphatase, including PTPN1, PTPN2, PTPN6, PTP1 and PTPN22. PTPs function in various important biological processes, including cell circle and cell differentiation, by carrying out phosphorylation and dephosphorylation of tyrosine residues [31]. The role of PTPs in inflammatory response and immune cells infiltration was also gradually revealed [32, 33]. In the present study, PTPN11 was significantly down-regulated, whereas PTPN6 was up-regulated in aortic valves from patients with CAVD. PTPN11 have already been linked to inflammation response, which could inhibit STAT1 pathway and ultimately reduce the level of Th1 cytokine through preventing combination of STAT1 and IFN-γreceptor [34]. Moreover, studies have demonstrated that PTPN11 gene variants are closely associated ulcerative colitis (UC) but not Crohn's disease (CD) [35]. PTPN11 is also an important component in growth factor pathway and closely related to Egfr signaling and formation of valve endothelial cells [36]. In addition, PTPN11 mutation in patients present significantly higher prevalence of pulmonary valve stenosis, named Noonan syndrome (NS) [37]. Interestingly, PTPN11 mutation has also been demonstrated to be harmful to inhibition of myocardial hypertrophy and cardiac fibrotic remodeling via crosstalk with NF-κB pathway and mTOR signaling [38, 39].
PTPN6, another phosphatase of PTPs, specially expressed in the cytoplasm, and prevented excessive autoimmunity in IL-1 dependent inflammatory diseases and pyroptosis dependent inflammatory diseases [40]. Studies have also demonstrated that PTPN6 significantly ameliorates inflammatory disease by decreasing TNF-α, TGF-β and IL-6 [41]. In addition, PTPN6 is important in preventing the harmful effects of pathogens on the host, which is crucial for successful defense mechanisms against invading microorganisms [42]. PTPN6 is known as an important negative regulator of inflammatory response and significantly down regulated in aortic valve tissues from patients with CAVD.
GRB2 is a 25-kDa adaptor protein that functions in modulating and integrating signals from cell membrane surface receptors to intracellular effector proteins [43]. In CAVD, GRB2 was up regulated in aortic valve tissues form patients with CAVD compared with normal individuals [44]. GRB2 is best known in the cardiovascular field for activating Egfr tyrosine kinase and its downstream renin-angiotensin system [45]. Recent studies also demonstrated that GRB2 was involved in the process of development of T cells and Th cells. GRB2-knockout animals have reduced T cells and more prone to inflammatory diseases [46].
Spleen-associated tyrosine kinase (SYK), a member of the none receptor type tyrosine kinase family [47]. SYK was involved in numerous biological functions, including cellular adhesion, vascular development, platelet activation and relaying adaptive immune receptor signaling related to immune cells infiltration [48–50]. SYK, as a proinflammatory molecule, has receiving increasing attention in a variety of diseases. Liang et al. demonstrated that SYK was a crucial biomarker and closely related to the occurrence of coronary heart disease (CHD) as an proinflammatory factor [51]. Researches on the specific role of SYK in CAVD is helpful to better understand the role of inflammatory response and immune infiltration in patients with CAVD.
SHC1, a member of SHC family of adaptor proteins, roles of SHC1 in reactive oxygen species (ROS) production has been widely studied. It is well known that overexpression of SHC1-induce ROS production is related to development of atherosclerosis and CHD [52–53]. Recent evidence suggests that ROS also plays a role in the pathophysiology of CAVD by inducing the differentiation of valvular stromal cells into myofibroblasts, which then become osteoblasts. In addition, ROS directly causes valve endothelial cell damage and lipid deposition in a variety of chronic diseases, including diabetes, and plays an important role in the early stage of CAVD [54]. More studies are needed to determine the involvement of PTPN1, GRB2, PTPN6, SYK, and SHC1 in CAVD.
CIBERSORT algorithm was performed to evaluate immune cells infiltration in aortic valve tissues of patients with CAVD. We found reduced monocytes and NK cells activated, as well as increased neutrophils, T cells CD4 memory activated and macrophages M0, which may be related to the pathogenesis of CAVD. Imbalance of M1 and M2 polarization in macrophages is known to be critical in regulating the intensity of inflammatory responses. Our results are the same as studies have reported before that compared with aortic valves from normal individuals, there were fewer M2 macrophages in calcified aortic valves [55]. In addition, our study has also shown that the macrophages M0 population were significantly elevated in CAVD. Monocytes are the largest white blood cells in volume, which is an important part of the defense system of human bodys. The decrease of monocytes and NK cells in patients with CAVD may be one of reasons for the intensification of inflammatory process and calcification of valve interstitial cells. Neutrophils and C-reactive protein (CRP) are indirect blood markers that roughly reflect the level of inflammation in our bodies, along with some pathological changes in aortic valve tissue [56]. We found that neutrophils was also significantly elevated in valve tissues from patients with CAVD. These results are consistent with previous studies suggesting that calcified aortic stenosis is characterized by chronic inflammation with infiltration of phagocytes, lymphocytes and mammary cells [57].
We also studied the correlation between key DEIRGs and infiltrating immune cells. Based on these results, PTPN1, GRB2, PTPN6, SYK and SHC1 may play a key role in CAVD by modulating immune infiltration. However, roles of various infiltrating immune cells and key DEIRGs in development and progression of CAVD still need to be studied further.