Unbiased omics identifies mechanistic regulators of calcific aortic valve disease

This editorial refers to ‘DUSP26 induces aortic valve calcification by antagonizing MDM2-mediated ubiquitination of DPP4 in human valvular interstitial cells’, by Y. Wang et al., doi:10.1093/eurheartj/ehab316.

Calcific aortic valve disease (CAVD) contributes to heart valve failure, leading to over 100,000 deaths annually with a global estimate of 1.6 million CAVD patients in 2017.¹ Despite the vast disease burden there are currently no clinically used anti-calcification drugs, and CAVD treatment is limited to surgical or transcatheter valve replacement. Valve replacement is a life-saving procedure, but it is not an option for all CAVD patients, and it can come with medical complications and the need for further interventions. Moreover, it is not widely available in underdeveloped countries, thus leaving many patients with no medical help. The lack of mechanistic understanding of CAVD results in the lack of therapeutic options. Obtaining a better understanding of key regulatory mechanisms in CAVD may identify druggable targets for this critical pathology, thereby increasing patient therapeutic choices and improving cardiovascular outcomes. In this issue of the European Heart Journal, Wang et al.² report an unbiased transcriptomics approach that identified a novel CAVD mechanistic pathway. The authors further found that dual-specificity phosphatase 26 (DUSP26) outcompetes an E3 ubiquitin ligase for binding with dipeptidyl peptidase-4 (DPP4). Through this outcompeting, DUSP26 reduces DDP4 proteasomal degradation, increasing DDP4 protein that in turn promotes valvular interstitial cell (VIC) calcification.

DPP4 is involved in multiple signaling processes, including inflammation and glucose homeostasis in metabolic diseases,³ and DPP4 inhibitors have been developed as therapies for diabetes mellitus. The mechanisms regulating DDP4 levels are not fully understood and may be cell type-dependent. DPP4 was previously shown to be transcriptionally upregulated in VICs from CAVD patients compared to controls via NF-κB binding to the DPP4 promoter.⁴ Inhibition of DPP4, via the anti-diabetic medication, sitagliptin, was also previously shown to suppress calcification in VICs, cardiovascular calcification in endothelial nitric oxide synthase-deficient mice, and valve calcification in rabbits fed a diet with high cholesterol and vitamin D2.⁴ Wang et al.² validate this role of DPP4 in promoting CAVD in vitro and in vivo.

In addition to containing consensus sites for multiple transcription factors driving its expression, DPP4 is also able to interact with various proteins through its cysteine-rich region. In agreement with this binding activity, Wang et al.² found that instead of increased transcriptional regulation of DPP4 in CAVD tissues and experimental models, DUSP26, a DPP4 binding protein, was transcriptionally increased. The authors discovered this change in DUSP26 in human CAVD tissues through an unbiased microarray approach. Furthermore, they demonstrated that increased DUSP26 levels resulted in increased DPP4 protein, via reducing DPP4 protein degradation. The reason for the differences (increased mRNA transcription vs. decreased protein degradation) in DPP4 modulation in different calcification studies is unclear but may suggest multiple means of DPP4 regulation in CAVD. DUSP26 was one of eight genes found to be transcriptionally increased in CAVD tissues compared to controls, and DUSP26 knockdown showed it to be the most promising of these genes in modulating VIC calcification.² Additionally, DUSP26 was observed localized to the aortic surface of valve leaflets, supporting its involvement in calcification,² as that is where calcification initiates. Beyond or perhaps involving its role in reducing DPP4 protein degradation, DUSP26 participates in cell growth, differentiation, inflammation, apoptosis, oxidative stress, and fibrosis,⁵ all of which are processes that associate with ectopic calcification.

The mechanistic findings of Wang et al.² were made through a combination of transcriptomics, histology, and in vitro and in vivo functional assays. We previously proposed and validated a similar cardiovascular calcification discovery pipeline to identify novel mechanisms and disease regulators in an unbiased manner^{6  ,  7} (Graphical Abstract). This unbiased multi-omics approach is a validated platform to identify novel mechanisms of cardiovascular calcification and potential therapeutic targets. As technology progresses, this pipeline could be expanded and modified, but overall elements remain the same: human tissues are analyzed in an unbiased fashion integrating multi-omics techniques and in vivo imaging to identify changes in molecules in disease conditions, which can then be spatially mapped using histology and immunohistochemistry. The functional role of these molecules in calcification can then be tested in vitro and in vivo, and ultimately translated to CAVD patients if drugs targeting these pathways can be developed. This discovery pipeline should be integrated more into CAVD mechanistic discovery.

Graphical Abstract.

CAVD mechanistic pipeline. Integration of imaging of calcification and multi-omics datasets from human CAVD patients and tissues allows for the discovery of novel mechanistic pathways that can be functionally validated in vitro and in vivo to discover anti-calcification drugs to assess clinically.

Targeting DPP4 in CAVD is not without concerns. Single nucleotide polymorphisms in DPP4 that associate with low plasma DPP4 levels may increase the risk for myocardial infarction in coronary artery disease.⁸ However, whether DPP4 inhibitors contribute to increased cardiovascular risk has not been consistently demonstrated in clinical trials. There has been some concern with DPP4 inhibitors increasing cardiovascular risk, including heart failure,⁹ but a DPP4 inhibitor, sitagliptin, did not increase the risk of major cardiovascular events in an outcomes trial.¹⁰ Targeting DPP4 instead via modulating its binding with DUSP26 in CAVD is now an intriguing alternative possibility based on the results of Wang et al.² This approach may allow for normalizing DPP4 activity in CAVD rather than inhibiting it, through some means of blocking DPP4 and DUSP26 interaction.

Overall, the findings of Wang et al.² are very interesting as they offer a novel CAVD regulatory pathway, and further support a greater need to better understand how proteostasis (protein homeostasis) is altered in cardiovascular diseases, including CAVD. While not performed in this study, comparative unbiased multi-omics approaches might help elucidate further CAVD proteostasis mechanisms. Combining unbiased transcriptomics using either whole-cell or single-cell RNA sequencing and proteomics data could reveal additional protein changes that may be independent of direct transcriptional regulation. Ultimately a multi-omics approach to mechanistic discovery could provide clues to additional novel regulatory pathways promoting CAVD.

Funding

This work was supported by National Institutes of Health research grants R01HL136431, R01HL147095, and R01HL141917 (E. A.)

Conflict of Interest. The authors state to conflict of interest.