The aim of the present study was to identify protein associations with CHD and CIMT, the latter as a surrogate of atherosclerosis risk. In the age-sex adjusted model, proteomic analysis showed differential abundance of five proteins with CHD and four with CIMT. Following further adjustment of the model for BMI, smoking status, lipid measurements, hypertension, and diabetes status, the quantitative difference of galectin-4 for CHD and NCK1 for CIMT remained significant. Moreover, the association of galectin-4 with CHD was further validated using ELISA-based measurements in an independent study.
Galectin-4 is a member of the beta-galactoside-binding proteins, and has important functions in lipid raft stabilization, protein apical trafficking, cell adhesion, as well as wound healing (28). Galectin-4 may be involved in atherosclerosis by enhancing lipid raft stabilization, which may subsequently affect redox signaling pathways (29). Schroder et al. reported galectin-4 to be correlated with myocardial blood flow reserve, a gold standard diagnostic to clinically assess coronary microvascular dysfunction, in women with angina pectoris and non-obstructive CHD (30). The authors conjectured that galectin-4’s promotion of cell adhesion contributed to the association (30). A Swedish population-based study found galectin-4 to be significantly associated with incident coronary events (hazard ratio (HR) = 1.34, 95% confidence interval (CI) = 1.14–1.57) and incident heart failure (HR = 1.26, 95% CI: 1.03–1.54) (31). Another study compared heart failure patients to controls both recruited in the outpatient clinic at Karolinska University Hospital, finding galectin-4 to be significantly associated with heart failure (HR = 2.6; FDR adjusted p-value 0.005) (32). In addition, galectin-4 has been reported to be associated with hospitalization linked to obesity (33) and ST-segment elevation myocardial infarction (34). All listed reports are in line with our finding of galectin-4’s association with CHD. The pathway analysis of CHD-associated proteins suggests that the interplay of galectin-4 and the predicted activated status of both p38 MAPk signaling and interleukin-1B, representatives of inflammation pathways (35), takes place via the peroxisome proliferator activated receptor alpha (PPARA). PPARG-deficient macrophages have been found to display an elevated production of pro-inflammatory cytokines including interleukin-1B (36).
Among the five CHD-associated proteins using the age-sex adjusted model, two were found to be associated with a higher- and two with a lower-CHD risk. Our reported association of renin with higher CHD-risk might have been lost when using the fully-adjusted model due to the adjustment for hypertension (37). However, renin has been reported to be positively associated with CHD (38, 39). Renin is a member of the renin-angiotensin-aldosterone system, which, via its active peptide angiotensin II, contributes to atherosclerosis development, not only by promoting hypertension but also through multiple direct actions on vessels (40).
Cathepsin H was an additional protein associated with a higher CHD-risk in the age-sex adjusted model. Cathepsin H is a lysosomal cysteine protease important in the overall degradation of lysosomal proteins (41). Its atherogenic role could lead the transformation of LDL to an atherogenic moiety, which in turn induces macrophage foam cell formation (42).
Proteins associated with lower-CHD risk associations included coagulation factor X as well as its active form coagulation factor Xa. A pathogenetic mechanism of CHD includes thrombotic vessel occlusion followed by rupture of an atherosclerotic plaque (3). It is therefore not surprising that constituents and a regulating protein of the coagulation cascade were found significantly associated with CHD in the present study. Factor Xa exerts also non-hemostatic effects by activation of protease-activated receptors-1 (PAR-1) and PAR-2, which have been associated with atherosclerosis, inflammation, and fibrosis (43). Such counterintuitive associations were reported before: where the concentrations of coagulation factor X and prothrombin were lower in blood from patients with CHD having more than 50% stenosis compared with those without CHD (44). Brummel-Ziedins et al. hypothesized that despite the depletion of coagulation factors, the balance between tissue factor and tissue factor pathway inhibitor is the primary driver of a hypercoagulable state in patients with CHD (44).
We also identified proteins to be associated with CIMT. NCK1 was the unique significant protein positively associated with CIMT in the fully-adjusted model. NCK1 is reported to be involved in different pathways leading to the progression of atherosclerosis (45). For instance, it is associated with vascular permeability, which allows the uptake of low-density lipoproteins (LDL) and thereby stimulates inflammation (45, 46). The resulting oxidative stress increase in endothelial cells decreases nitric oxide and thereby supports endothelial cell dysfunction (47). Alfaidi et al. showed in an in-vivo study that NCK1-knockdown reduced NF-κB signaling and thereby inflammation in endothelial cells (45). Additionally, pathway analysis revealed interleukin-9 to indirectly inhibit expression of NCK1, SPTAN1, and CFL1, while the opposite effect was predicted for CMA1. Interleukin-9 has been reported to decrease expression of human NCK1 in MDA-MB-231 human breast cells, and to differentially regulate actin cytoskeleton-related proteins such as NCK1 (48). Interestingly, both SPTAN1 and CFL1 are filamentous cytoskeletal and actin-related proteins, while CMA1 could participate in extracellular matrix degradation (49). We were unable to validate the NCK1 findings using ELISA-measurements due to the lack of CIMT measurements in the validation sample.
We were able to confirm three additional proteins associated with CIMT using our age-sex adjusted model namely GFRA1, IGFBP2 and GHR, which have been all reported to be linked to CVD, mortality, and atherosclerosis (50, 51). IGFBP2 was the unique obtained negative association. The same direction effect has been reported as a strong association with type 2 diabetes in a comparison study of incident type 2 diabetes and coronary heart disease in the KORA cohort (52).
In this study, we identified proteins specifically associated with either CHD or CIMT. Previous reports on KORA F4 have already stated a non-linear relation between CIMT and CHD risk (53). Thus, this complex relationship amongst the two phenotypes could help to explain the specificity of our findings.
A major strength of our study is the proteome-wide approach, which covers proteins at low abundance levels in plasma. By conducting a hypothesis-free analysis, we were able to analyze the association of a wide array of plasma proteins with CHD and CIMT. An additional strength is the availability of an independent sample, which we could use to validate initial results with an alternative measurement technique that provides absolute concentrations. Our study also has limitations. The lack of patient differentiation by CHD severity could, for instance, be attenuating some associations. Manifestations of early-stage CHD differ from the late-stage CHD. In the latter, protein levels change due to myocardial injury and physiological compensation. Additionally, the difference in plaque vulnerability and extent of atherosclerosis between stable and unstable CHD (54), may also impact the plasma proteome. Finally, due to the cross-sectional nature of our study, temporal relations cannot be inferred.
In summary, our proteome-wide study identified a new association of galectin-4 with CHD. Galectin-4 may be involved in atherosclerosis by enhancing lipid raft stabilization, which subsequently affects redox signaling pathways. Moreover, we report the association of NCK1 with CIMT.