MicroRNA-204 Deficiency in Human Aortic Valves Elevates Valvular Osteogenic Activity

Figure 1. MiR-204 levels are lower in diseased human aortic valves. (A) Hematoxylin and eosin stain images (10× objective) of normal and calcified human aortic valve leaflets show that calcified aortic valve leaflets loss tri-layered morphology. Calcification nodules (arrows) are present in moderately calcified aortic valve leaflets. Scale bar = 100 µm. (B) Normal and calcified human aortic valve tissue sections were incubated with a full length DIG-labeled LNA probe to miR-204 (a–d) and a DIG-labeled LNA probe specific for the non-coding small nuclear RNA U6 as internal control (e–h). MiR-204 (blue, arrow) is markedly lower in diseased valves (c and d) compared with normal valves (a and b). Original magnification is 4x objective in a, c, e and g, and 20× objective in b, d, f and h. Scale bar = 100 µm. (C) Aortic valve interstitial cells (AVICs) isolated from normal and calcified human aortic valves were analyzed for miR-204 levels by real-time PCR. Quantitative mRNA data confirmed that miR-204 levels are lower in AVICs from calcified valves. Mean ± SE; n = 8 distinct cell isolates; * p < 0.05 vs. AVICs of normal valves.

Figure 2. MiR-204 mimic suppresses the osteogenic activity in AVICs from diseased valves. (A) Human AVICs were infected with lentivirus expressing miR-204 mimic. Representative images show lentiviral infection efficiency (GFP expression) over time in human AVICs. Data of real-time qRT-PCR analysis show markedly elevated miR-204 levels in cultures treated with lentiviral miR-204. Scale bar = 200 μm. Mean ± SE; n = 6 distinct cell isolates; * p < 0.05 vs. Control (culture infected by lentiviral miR-C). (B) AVICs from diseased human aortic valves were treated with miR-204 mimic (5 nM) or irrelevant oligonucleotide (miR-C, 5 nM) for 72 h. Representative immunoblots of 6 separated experiments using distinct cell isolates and densitometric data show that miR-204 mimic suppressed the expression of ALP in AVICs of diseased valves. Mean ± SE; * p < 0.05 vs. normal AVICs; # p < 0.05 vs. untreated calcified valve AVICs; † p < 0.05 vs. calcified valve AVICs+miR-C. (C) AVICs from diseased human aortic valves were untreated or infected with lentivirus that expresses miR-204 mimic or miR-C for seven days, and then cells are incubated in a conditioning medium (growth medium supplemented with 10 mmol/L…

Figure 3. MiR-204 antagomir enhances the expression of osteogenic biomarkers in AVICs from normal aortic valves. AVICs from normal human aortic valves were treated with miR-204 antagomir (50 nM) or irrelevant oligonucleotide (miR-C, 50 nM) for 72 h. Representative immunoblots and densitometric data show that miR-204 antagomir up-regulates the levels of Runx2, Osx and ALP. Mean ± SE; n = 6 distinct cell isolates; * p < 0.05 vs. untreated control; † p < 0.05 vs. cells treated with miR-C.

Figure 4. MiR-204 down-regulates the expression of both Runx2 and Osx expression in human AVICs to suppress pro-osteogenic reprogramming. (A) AVICs from normal aortic valves were treated with miR-204 mimic (5 nM), antagomir (50 nM) or irrelevant oligonucleotide (miR-C, 50 nM) for 24 h and then stimulated with TGF-β1 (0.005 μg/mL) for 24 to 48 h. Representative immunoblots and densitometric data show that miR-204 mimic suppressed, but miR-204 antagomir enhanced the expression of Runx2 and Osx at 24 h, as well as the levels of ALP at 48 h following treatment with TGF-β1. Mean ± SE; n = 6 distinct cell isolates; * p < 0.05 vs. untreated control; # p < 0.05 vs. TGF-β1 alone; † p < 0.05 vs. TGF-β1+miR-C. (B) AVICs from normal aortic valves were treated with specific siRNA (100 nM) for 48 h to knockdown Runx2 or Osx and then stimulated with TGF-β1 (0.005 μg/mL) for 48 h. Controls were pre-treated with scrambled siRNA (100 nM) and then stimulated with TGF-β1. Representative immunoblots and densitometric data show that knockdown of Runx2 markedly reduced the effects of TGF-β1 on ALP levels, whereas knockdown of Osx had a moderate effect. Mean ± SE; n = 6 distinct cell isolates; * p <0.05 vs.…

Figure 5. MiR-204 antagomir promotes aortic valve calcification through up-regulation of Runx2 and Osx expression. (A) Isolated mouse aortic valves were treated with irrelevant miR (control) or miR-204 antagomir for three days. Representative immunofluorescence images of 5 separate experiments show that miR-204 antagomir increases the levels of Runx2 and Osx. Runx2 and Osx are showing in red. The nuclei are showing in blue. Glycoproteins are showing in green to outline tissue and cells. Original magnification 40× objective. (B) Isolated mouse aortic valves were untreated or treated with irrelevant miR or miR-204 antagomir. Additional valves were treated with miR-204 antagomir in the presence or absence of siRNA (100 nM) specific to Runx2 or Osx. Then, all valves were incubated in the conditioning medium for 6 days. Representative images of Alizarin Red S staining and spectrophotometric analysis of Alizarin Red S stains show that miR-204 antagomir augmented the formation of calcium deposits (brick red and black) in mouse aortic valves. Knockdown of Runx2 or Osx attenuated calcium deposit formation caused by antagonizing miR-204. Mean ± SE; n = 5; * p < 0.05 vs. untreated (conditioning…