Abstract
Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.
Highlights
| INTRODUCTIONValvular calcification is a common pathological process which is observed extensively in patients with renal disease, type II diabetes, calcific aortic valve diseases (calcific aortic valve disease, CAVD) including active aortic valve calcification and aortic stenosis.[1–7] the clinical significance of valvular calcification is well-recognized, the mechanisms involved are still not clear
Valvular calcification is a common pathological process which is observed extensively in patients with renal disease, type II diabetes, calcific aortic valve diseases including active aortic valve calcification and aortic stenosis.[1–7] the clinical significance of valvular calcification is well-recognized, the mechanisms involved are still not clear.MicroRNAs have been considered as a nucleus research topic in recent years
Alazarin red S staining (Figure 1A) indicated that calcium deposits were not detected in human aortic valve interstitial cells (hAVICs) cultured in control medium, while positively-stained particles were significantly and time-dependently increased in hAVICs cultured in osteogenic medium. These observations were further confirmed by the quantitative measurement of the intracellular calcium content (Figure 1B) which was dramatically increased in hAVICs cultured in osteogenic medium compared to the control (P < 0.0001)
Summary
Valvular calcification is a common pathological process which is observed extensively in patients with renal disease, type II diabetes, calcific aortic valve diseases (calcific aortic valve disease, CAVD) including active aortic valve calcification and aortic stenosis.[1–7] the clinical significance of valvular calcification is well-recognized, the mechanisms involved are still not clear. MicroRNAs (miRs) have been considered as a nucleus research topic in recent years They are a group of non-coding single-stranded cellular RNAs composed of 18–25 nucleotides, widely involved in the regulation of molecular pathways and trans-differentiation of cell phenotypes.[8–14]. An increasing number of reports indicates that a group of miRs are involved in the differentiation of mesenchymal cells or myofibroblasts into osteoblastic phenotype and heterotopic ossification.[15–26]. Mesenchymal cells, aortic valve interstitial cells, and vascular smooth muscle cells present similarity of differentiation pathways. MiR-29b is involved in the osteoblastic differentiation of vascular smooth muscle cells and mesenchymal stem cells.[30,31,33,37]. We hypothesized that miR-29b could be involved in the osteoblast differentiation of hAVICs and could be an important regulator of heterotopic ossification of the aortic valve. TGF-β3 and activating wnt/β-catenin and RUNX2/ Smad[3] signaling pathways
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