Telocytes-derived extracellular vesicles alleviate aortic valve calcification by carrying miR-30b

Abstract

Aims: Calcific aortic valve disease (CAVD) is frequent in the elderly. Telocytes (TCs) are implicated in intercellular communication by releasing extracellular vesicles (EVs). This study investigated the role of TC-EVs in aortic valve calcification.

Methods and results: TCs were obtained and identified using enzymolysis method and flow cytometry. EVs were isolated from TCs using differential high-speed centrifugation method and identified using transmission electron microscope, western blot, and qNano analysis. The mouse model of CAVD was established. The changes of aortic valve activity-related indicators were analysed by ultrasound, and the expressions of TC markers CD34 and vimentin in mouse valve tissues were detected using RT-qPCR and western blot. The model mice were injected with TC-derived EVs. The expressions of Runx2, osteocalcin, and caspase-3 were detected using RT-qPCR and western blot. The calcification model of valvular interstitial cells (VICs) was established. TC-EVs were co-cultured with calcified VICs, and calcium deposition was detected using alizarin red S staining. miR-30b expression in calcified valvular tissues and cells was detected after EV treatment. miR-30b expression in TCs was knocked down and then EVs were extracted and co-cultured with calcified VICs. The target of miR-30b was predicted through bioinformatics website and verified using dual-luciferase assay. The levels of Wnt/β-catenin pathway-related proteins were detected. ApoE^-/- mice fed with a high-fat diet showed decreased aortic valve orifice area, increased aortic transvalvular pressure difference and velocity, reduced left ventricular ejection fraction, decreased CD34 and vimentin, and increased caspase-3, Runx2, and osteocalcin. The levels of apoptosis- and osteogenesis- related proteins were inhibited after EV treatment. TC-EVs reduced calcium deposition and osteogenic proteins in calcified VICs. EVs could be absorbed by VICs. miR-30b expression was promoted in calcified valvular tissues and cells after EV treatment. Knockdown of miR-30b weakened the inhibitory effects of TC-EVs on calcium deposition and osteogenic proteins. miR-30b targeted Runx2. EV treatment inhibited the Wnt/β-catenin pathway, and knockdown of miR-30b in TCs attenuated the inhibitory effect of TC-EVs on the Wnt/β-catenin pathway.

Conclusion: TC-EVs played a protective role in aortic valve calcification via the miR-30b/Runx2/Wnt/β-catenin axis.

Keywords: Aortic valve calcification; Apoptosis; Extracellular vesicles; Runx2; Telocytes; Wnt/β-catenin; miR-30b.

Figure 1

Incubation of TCs and extraction of EVs. (A) Morphology of TCs was observed under the light microscope on the 4th, 7th, and 14th day; (B) levels of CD34 and CD117 on the surface of primary cells were identified using flow cytometry; (C) level of vimentin in cardiac cells was observed using the laser confocal scanning microscope; (D) morphology of EVs was observed under the TEM; (E) makers of EVs were detected using western blot analysis; (F) diameter of EVs was detected using qNano analysis.

Figure 2

TC‐EVs reduced aortic valve calcification and apoptosis of valve interstitial cells. (A) Aortic valve orifice area, aortic transvalvular pressure difference and velocity, and left ventricular ejection fraction of mice were detected using echocardiography; (B, C) expressions of TC markers CD34 and vimentin in mouse valve were detected using RT‐qPCR and western blot analysis; (D, E) expressions of Runx2, osteocalcin, and caspase‐3 in calcified valves of mice were detected using RT‐qPCR and western blot analysis. The cell experiment was repeated three times independently. N = 6. Data are presented as mean ± standard deviation. Data in panels A/B/C were analysed using t‐test, and data in panels D/E were analysed using one‐way ANOVA, followed by Tukey's multiple comparison test, **P < 0.01.

Figure 3

TC‐EVs prevented aortic valves from transforming into contractile and osteogenic phenotype. (A) Appearance of calcified valve interstitial cells was observed under the light microscope; (B) levels of α‐SMA, vimentin, and vWF in interstitial cells were observed; (C) quantitative analysis of vimentin and α‐SMA‐positive cells in valve interstitial cells was conducted using flow cytometry; (D) cell growth morphology before and after calcification induction was observed; (E) formation of calcium deposition was detected using Alizarin red S staining; (F, G) levels of Runx2 and osteocalcin were detected using RT‐qPCR and western blot analysis. The cell experiment was repeated three times independently. Data are presented as mean ± standard deviation. Data in panel E/F/G were analysed using one‐way ANOVA, followed by Tukey's multiple comparison test, **P < 0.01.

Figure 4

TC‐EVs were the communication media of miR‐30b in aortic valve calcification. (A) Absorption of EVs by valve interstitial cells was observed using immunofluorescence staining; (B) expression of miR‐30b in calcified valve tissues and valve interstitial cells was detected using RT‐qPCR. The cell experiment was repeated three times. Data are expressed as mean ± standard deviation. Data in panel (B) were analysed using t‐test or one‐way ANOVA, followed by Tukey's multiple comparison test, **P < 0.01.

Figure 5

Inhibition of miR‐30b attenuated the protective effect of EVs on aortic valve calcification. (A) Expression of miR‐30b was detected using RT‐qPCR; (B) formation of calcium deposition was detected using Alizarin red S staining; (C) levels of Runx2 and osteocalcin were detected using RT‐qPCR and western blot analysis. The cell experiment was repeated three times. Data are expressed as mean ± standard deviation. Data in panels (A) and (B) were analysed using t‐test, and data in panel (C) were analysed using one‐way ANOVA, followed by Tukey's multiple comparison test, **P < 0.01.

Figure 6

TC‐EVs transferred miR‐30b to inhibit Runx2 and inactivate the Wnt/β‐catenin pathway. (A) Binding relationship between miR‐30b and Runx2 was verified using dual‐luciferase reporter gene assay; (B) levels of the Wnt/β‐catenin pathway‐related proteins in calcified valve tissues and valve interstitial cells were detected using western blot analysis. The cell experiment was repeated three times. Data are expressed as mean ± standard deviation. Data in panel (A) were analysed using t‐test, and data in panel (B) were analysed using one‐way ANOVA, followed by Tukey's multiple comparison test, **P < 0.01.