MiR-425 and miR-155 produced dose-dependent decreases in <i>NPPA</i> expression over a wide range of concentrations.

<p><i>NPPA</i> mRNA levels in human embryonic cell cardiomyocytes (hESC-CMs) transfected with 1 nM, 5 nM, 10 nM, or 20 nM of negative control miRNA (NC), miR-425, or miR-155. Expression is normalized to <i>GAPDH</i> expression and shown relative to expression in cells transfected with negative control miRNA. *<i>P</i><0.01 and **<i>P</i><1x10^-6 versus cells transfected with the same concentration of negative control miRNA. N = 4–8 experiments per miRNA concentration (4–12 replicate wells per condition for each experiment).</p

MiR-425 and miR-155 produced an additive decrease in cGMP levels relative to either miRNA alone.

<p>Cardiomyocytes were transfected with 1 nM (A) or 5 nM (B) of negative control miRNA (NC), miR-425, miR-155, or the combination of miR-425 and miR-155, each at half the concentration of the other miRs. Cyclic GMP levels were measured in NPR1-expressing cells that were incubated for 2 hours with media collected from the cardiomyocytes. Cyclic GMP levels were expressed as picomoles of cGMP per mg of protein and were shown relative to levels in cells exposed to media from cardiomyocytes transfected with negative control miRNA. *<i>P</i><0.01 and **<i>P</i><1x10^-6 versus cells transfected with negative control miRNA. N = 3 experiments (4–12 replicate wells for each condition) for (A) and n = 8 experiments (4 replicate wells per condition) for (B).</p

MiR-425 and miR-155 had an additive repressive effect on cardiomyocyte <i>NPPA</i> expression.

<p><i>NPPA</i> mRNA levels in hESC-CMs that were transfected with either miR-425 or miR-155 alone or with a combination of miR-425 and miR-155. Negative control miRNA (NC) was used as needed to make the total concentration of miRNA constant in each condition. <i>NPPA</i> expression is shown relative to that in cells transfected with negative control miRNA. *<i>P</i><0.01 and **<i>P</i><1x10^-6 versus cells transfected with negative control miRNA. N = 3 experiments (6–12 replicate wells per condition).</p

Development and validation of a biological assay to measure the effects of miRNA-mediated changes in <i>NPPA</i> expression on cGMP levels.

<p>TurboGFP (tGFP) fluorescence in COS7 cells 48 hours after transfection with the NPR1-tGFP expression vector (A). Cell nuclei were visualized using DAPI. The effect of incubating NPR1-expressing COS7 cells with increasing amounts of human ANP on cGMP concentrations (B). Cyclic GMP levels were expressed as picomoles of cGMP per mg of protein, relative to the cGMP levels at baseline (cells not exposed to ANP). The correlation between cGMP levels in NPR1-expressing COS7 cells exposed to media from cardiomyocytes transfected with miRNA(s), and cardiomyocyte <i>NPPA</i> mRNA levels (C) or Nt-proANP levels in the cardiomyocyte media (D).</p

The combination of miR-425 and miR-155 resulted in greater repression of <i>NPPA</i> expression than a higher concentration of either miRNA alone.

<p>Cardiomyocytes were transfected with 1 nM (A) or 5 nM (B) of miR-425, miR-155, or both miR-425 and miR-155, each at half the concentration of either miRNA alone. Two days later, <i>NPPA</i> mRNA levels were measured. <i>NPPA</i> expression was normalized to <i>GAPDH</i> expression, and shown relative to expression in cells transfected with the negative control miRNA. *<i>P</i><0.01 and **<i>P</i><1x10^-6 versus cells transfected with negative control miRNA. N = 4 experiments (4–12 replicate wells per condition) for (A) and n = 8 experiments (4–12 replicate wells per condition) for (B).</p

Restoration of MGP levels decreases calcification of MGP^-/- vascular smooth muscle cells while siRNA-mediated depletion of MGP increases calcification of wild-type vascular smooth muscle cells in a BMP-dependent manner.

<p>Cultured aortic VSMCs isolated from MGP^-/- mice were infected with either (<b>A</b>) a control adenovirus (Ad.GFP) or (<b>B</b>) an adenovirus expressing MGP (Ad.MGP) at a multiplicity of infection of 10 and placed in DMEM supplemented with 10% FBS and 2 mM sodium phosphate. Cultured aortic VSMCs isolated from wild-type mice were transfected with either (<b>C</b>) scrambled siRNA (siSC) or (<b>D & E</b>) siRNA targeting MGP (siMGP) at 20 nM and placed in DMEM supplemented with 10% FBS and 2 mM sodium phosphate. Cells were also treated without (<b>C & D</b>) or with (<b>E</b>) 100 nM LDN-193189 (LDN). Cells were stained after 7 days using the von Kossa method. Serial fields of view were photographed for each condition and von Kossa stain was quantified using image J software after background subtraction (<b>F & G</b>). In (<b>F</b>), *P = 0.03 compared to Ad.GFP. In (<b>G</b>), **P<0.0001 compared to siSC-treated cells. #P = 0.0003 compared to siMGP + control. Restoration of MGP expression reduced phosphate-induced calcification of MGP^-/- VSMCs, while depletion of MGP increased calcification of WT VSMCs and this calcification was partially inhibited by treatment with LDN-193189.</p

Aortic expression of VSMC markers in wild-type and MGP^-/- mice.

<p>RNA was isolated from aortas of WT and MGP^-/- mice and from LDN-193189-treated MGP^-/- mice at 7 and 14 days of age (n = 4–8 in each group). Levels of mRNAs encoding myocardin, α smooth muscle actin (SMA), transgelin, and calponin are depicted. The aortas of 14-day-old MGP^-/- mice have decreased expression of VSMC markers compared to WT mice. Treatment with LDN-193189 did not restore the expression of VSMC markers to WT levels. # P<0.05 compared to 7-day-old MGP^-/- mice.</p

MGP deficiency does not alter basal BMP signaling or responsiveness to BMP-2 in VSMCs.

<p>(<b>A</b>) VSMCs were isolated from the aortas of wild-type and MGP^-/- mice. VSMCs were treated without or with recombinant human BMP-2 (for 2 hours at the indicated doses). Groups were compared using a 2-way ANOVA. Both WT and MGP^-/- VSMCs exhibited similar Id1 mRNA levels, both at baseline and in response to exogenous BMP-2. (<b>B</b>) Cultured aortic VSMCs from wild-type mice were transfected with either scrambled siRNA (siSC) or siRNA targeting MGP (siMGP) at 20 nM. RNA was isolated from cells after 4 days. siMGP decreased MGP mRNA levels in WT VSMCs by >95% compared with siSC-treated cells. However, depletion of MGP in WT VSMCs did not alter Id1 mRNA levels. **P<0.0001 compared to siSC-treated VSMCs. (<b>C</b>) VSMCs isolated from wild-type mice were treated with 20 nM of either scrambled siRNA (siSC) or siRNA specific for MGP (siMGP). Cells were incubated with or without BMP-2 (20 ng/mL) for 1 h prior to protein harvest. Western blots were probed with antibodies specific for phosphorylated Smad 1/5 (P-Smad 1/5) and total Smad 1. Depletion of MGP in WT VSMCs did not alter the ratio of P-Smad 1/5 levels to total Smad 1 levels, both at baseline and in response to exogenous BMP-2.</p

Vascular calcification associated with MGP deficiency occurs in the absence of vascular inflammation.

<p>(<b>A</b>) At 27 days of age, OsteoSense-680 and Prosense-750 were injected via the tail vein of wild-type (WT) and MGP^-/- mice. Aortas were harvested 24 hours later and imaged. Although aortas from MGP^-/- mice exhibited extensive vascular calcification, this calcification was not associated with increased macrophage activity. (<b>B</b>) Aortas were harvested from WT and MGP^-/- mice at 28 days of age, sectioned, and stained for macrophages with an antibody directed towards MAC-2. Aortas from LDLR^-/- mice on a high fat diet were used as a positive control. Nuclei were stained with DAPI. Similar to WT mice, macrophages were not detected by immunohistochemistry in the aortas of MGP^-/- mice.</p

BMP signaling is required for the increased aortic expression of osteogenic markers associated with MGP deficiency.

<p>RNA was isolated from aortas of WT and MGP^-/- mice at 1, 7, 14, and 28 days of age and from LDN-193189-treated MGP^-/- mice at 7, 14, and 28 days of age (n = 4–11 in each group, as indicated). Expression of genes encoding Runx2 and osteopontin (OPN) was measured. MGP^-/- mice had increased levels of aortic Runx2 and OPN mRNA compared to WT mice. Treatment of MGP^-/- mice with LDN-193189 reduced aortic Runx2 and OPN mRNA levels. * P<0.001 compared to WT mice of same age. # P<0.05 compared to age-matched MGP^-/- mice treated with vehicle.</p