The identification of a specific serum biomarker which can provide both an insight to the static state of a pathological process and track its progression (and subsequent regression following appropriate intervention) has proved challenging. This is due to the multimodal nature of the mechanisms which ultimately culminate in stiffening of the aortic valve. However, as a result of various studies of calcification and fibrosis in the heart, there is consensus that four core subprocesses are synergistically responsible in achieving the endpoint of aortic stenosis: valve mineralization (calcification), local inflammation, oxidative stress and ventricular fibrosis [9]. Within the domain of each subprocess, several promising biomarkers have shown acceptable specificity [10]. Here, we hypothesize that an approach using a combination of serum biomarkers and the echocardiographic parameter global longitudinal strain (GLS), a functional marker which will be discussed later, can be used to establish baseline status of fibrocalcific aortic valve disease, predict rate of progression and quantitatively assess any regression of these processes following AVR in patients with T2DM.

Figure 1 provides an overview of the site of expression of relevant biomarkers. BNP (B-type natriuretic peptide), and its prohormone NT-proBNP (n-terminal pro brain natriuretic peptide), are neurohormones and markers of stretching of the heart muscle. BNP is secreted by cardiomyocytes following volume and pressure changes in the left ventricle. NT-proBNP in particular demonstrates an incremental relationship with the severity of aortic stenosis. Although in isolation this biomarker is unable to distinguish between the pathologies of calcification and fibrosis, it is valuable in ascertaining the cumulative consequence of ventricular dysfunction and has predictive value [11].

sST2 (soluble suppression of tumorigenicity 2) is a member of the IL1 (interleukin-1) receptor family and is a marker of ventricular remodeling. sST2 levels increase with myocardial stress and have been demonstrated to correlate quantitatively with myocardial ischemia and heart failure. It is of particular use as it correlates with echocardiographic assessment of diastolic dysfunction and has been demonstrated to correlate with heralding the onset of new symptoms in previously asymptomatic aortic stenosis patients. High levels of sST2 are an independent predictor of mortality following aortic valve replacement surgery [12].

Gal-3 (Galectin-3) is a beta-galactoside-binding lectin and a marker of inflammation and fibrosis of the myocardium, but it is also responsible for other processes such as regulating T-cell function and apoptosis. Gal-3 has a limited role in predicting progression of ventricular stiffening as it is also upregulated in other conditions such as renal dysfunction. However, when considered in tandem with BNP, it has shown to have significant predictive properties in the progression of heart failure [13].

The microRNAs 21 and 19b are specific small RNAs that are 19-25 nucleotides long which are postulated to regulate gene expression related to various cellular mechanisms underpinning cardiovascular function. Although assessment of these micro-RNAs is still in early stages, microRNA-21 has shown good correlation with myocardial fibrosis. MicroRNA-19b has shown promise in determining increased myocardial collagen cross-linking in patients with aortic stenosis [14].

GDF-15 (growth differentiation factor-15) is a stress-responsive cytokine that is sensitive to inflammatory changes and apoptosis. It is abundant in cardiomyocytes, adipocytes and macrophages and has demonstrated good predictive value in heart failure, vascular calcification and endothelial dysfunction. Like Gal-3, it is upregulated in renal dysfunction and additionally in obesity and insulin-resistant states such as diabetes. Although recent work has established GDF-15 as a useful prognostic and diagnostic marker of cardiometabolic disease in patients with diabetes, reference ranges correlating to severity have not yet been established [15].

OPG (osteoprotegerin) is a member of the tumor necrosis factor receptor family and is expressed in early arteriosclerotic lesions and vascular smooth muscle cells. It has an important role in neocalcification, acting as a decoy receptor by binding to the RANKL (receptor activator kappa-B ligand) nuclear factor and inhibiting the receptor activity of RANK (receptor-activated kappa-B). This thus inhibits osteoclastic activity, which ultimately leads to calcification. Modulation of this pathway by the action of anti-Ghrelin antibodies suppressing OPG expression is of particular interest in seceding calcification [16].

Speckle-tracing echocardiography is a relatively novel echocardiographic technique which has been shown to be more sensitive than conventional assessments of ejection fraction (EF) in the assessment of minute, regional wall motion abnormalities (RWMA) and left ventricular function. It correlates well with the presence and magnitude of myocardial fibrosis when compared alongside cardiac magnetic resonance, as assessed through T1-mapping and late gadolinium enhancement as quantification techniques. This noninvasive imaging modality can serve as an additional functional marker alongside the serum biomarkers mentioned above in the assessment of severity of aortic calcification and in the evaluation of reverse-remodeling postoperatively [17].

Figure 2 outlines the process of biomarker testing in the relevant patient cohorts. Gold standard assessments of aortic valve calcification and myocardial fibrosis are direct microscopic inspection of the excised aortic valve, following AVR, and a biopsy specimen of the ventricular myocardium, respectively [18]. Noninvasive imaging techniques done preoperatively, Computed Tomographic (CT) Calcium Scoring for aortic valve calcification and Late Gadolinium Enhancement magnetic resonance imaging (MRI) for myocardial fibrosis, will corroborate the findings of direct inspection. These results will be used to calibrate serum biomarkers and echocardiographic findings for the subset of patients with severe AS undergoing surgery.

Healthy volunteers and patients with milder, asymptomatic forms of the disease will be recruited from outpatient cardiology surveillance clinics to undergo serum biomarker sampling and strain echocardiograms. They will then undergo CT and MRI scans to investigate the relevance of these markers against objective imaging findings. This will enable the establishment of baseline levels in healthy volunteers and grading of the markers in mild, moderate and severe forms of disease.

Finally, postoperative patients will be followed up with in 3 months and 12 months following surgery where they will once again undergo serum biomarker sampling and strain echocardiography to assess for evidence of reverse-remodeling. Again, CT scans for calcium scoring and MRI scans will be repeated to validate the significance of any downregulation of these biomarkers.

The combination of biomarker and echocardiographic data encompassing the entire spectrum of the disease from healthy volunteers to patients with severe disease as well as disease monitoring in postoperative patients will yield a ranking scale for use in the clinic. This scale, once validated for reproducibility in an appropriately powered study, will provide each patient with a fibrocalcific profile which can then be used to recommend an optimal time for intervention.