Abstract
Rationale Aortic valve stenosis (AVS) is a major contributor to cardiovascular death in the elderly population worldwide. MicroRNAs (miRNAs) are highly dysregulated in patients with AVS undergoing surgical aortic valve replacement (SAVR). However, miRNA-dependent mechanisms regulating inflammation and calcification or miRNA-mediated cell-cell crossstalk during the pathogenesis of AVS are still poorly understood. Here, we explored the role of extracellular vesicles (EV)-associated miR-145-5p, which we showed to be highly upregulated upon valvular calcification in AVS in mice and humans.
Methods Human TaqMan miRNA arrays identified dysregulated miRNAs in aortic valve tissue explants from AVS patients compared to non-calcified valvular tissue explants of patients undergoing SAVR. Echocardiographic parameters were measured in association with the quantification of dysregulated miRNAs in a murine AVS model. In vitro calcification experiments were performed to explore the effects of EV-miR-145-5p on calcification and crosstalk in valvular cells. To dissect molecular miRNA signatures and their effect on signaling pathways, integrated OMICS analyses were performed. RNA sequencing (RNA-seq), high-throughput transcription factor (TF) and proteome arrays showed that a number of genes, miRNAs, TFs, and proteins are crucial for calcification and apoptosis, which are involved in the pathogenesis of AVS.
Results Among several miRNAs dysregulated in valve explants of AVS patients, miR-145-5p was the most highly gender-independently dysregulated miRNA (AUC, 0.780, p-value, 0.01). MiRNA arrays utilizing patient-derived- and murine aortic-stenosis samples demonstrated that the expression of miR-145-5p is significantly upregulated and correlates positively with cardiac function based on echocardiography. In vitro experiments confirmed that miR-145-5p is encapsulated into EVs and shuttled into valvular interstitial cells. Based on the integrated OMICs results, miR-145-5p interrelates with markers of inflammation, calcification, and apoptosis. In vitro calcification experiments demonstrated that miR-145-5p regulates the ALPL gene, a hallmark of calcification in vascular and valvular cells. EV-mediated shuttling of miR-145-5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3’ untranslated region to inhibit its translation, thereby diminishing the calcification of target valvular interstitial cells.
Conclusion Elevated levels of pro-calcific and pro-apoptotic EV-associated miR-145-5p contribute to the progression of AVS via the ZEB2-ALPL axis, which could potentially be therapeutically targeted to minimize the burden of AVS.
What is known?
Aortic valve stenosis (AVS) is the most prevalent structural heart valve disease requiring surgical or interventional valve replacement. Currently, no medical treatment option is available to slow, halt, or reverse the progression of the disease.
AVS induces pressure overload on the left ventricle (LV), resulting in concentric hypertrophy and LV dysfunction.
AVS is not an exclusively degenerative disease that leads to fibrosis and calcification of the valve cusps but rather a chronic inflammatory disease, in which mechanical strain and shear stress lead to endothelial dysfunction and immune cell infiltration, which induces chronic inflammation, apoptosis and differentiation of valvular interstitial cells into osteoblast-like cells.
Increasing osteoblastic differentiation and the formation of macrocalcifications are hallmarks of the later stages of AVS.
What is the new information we provide?
During aortic valve stenosis, expression pattern of vesicle-associated regulatory miRNAs is altered.
Patient-derived aortic valve tissue demonstrated an increased expression of miR-145-5p in humans, as well as in aortic valve explants from an experimental murine AVS model.
MiR145-5p contributes to calcification of the aortic valve through ZEB2, a transcriptional repressor of ALPL, in valvular interstitial cells.
Extracellular vesicular shuttling of miR-145-5p contributes to valvular cell-cell crosstalk and plays a role in the pathogenesis of AVS.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
* Co-first authors
Nonstandard Abbreviations and Acronyms
- AVS
- aortic valve stenosis
- CVD
- cardiovascular disease
- ECs
- endothelial cells
- EVs
- extracellular vesicles
- VICs
- vulvular interstial cells
- VECs
- vulvular endothelial cells
- HCAEC
- human coronary artery endothelial cell
- LV
- left ventricle
- LVEF
- left ventricular ejection fraction
- miR
- microRNA
- miRNA
- microRNA
- NGS
- next generation sequencing
- RBP
- RNA-binding protein
- RIP
- RNA immunoprecipitation
- SAVR
- surgical aortic valve replacement
- TAVR
- transcatheter aortic valve replacement
- TEM
- transmission electron microscopy