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

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

BMP signaling is not increased in aortas of MGP^-/- mice.

<p>(<b>A</b>) Protein lysates were isolated from the aortas of 7-, 14-, and 28-day-old WT and MGP^-/- mice. Each lane represents protein isolated from four pooled aortas. PVDF membranes were incubated with antibodies directed against phosphorylated Smad 1/5 (P-Smad 1/5) and total Smad 1. The ratio of P-Smad 1/5 to total Smad 1 was the same in aortas derived from WT and MGP^-/- mice. (<b>B</b>) RNA was isolated from aortas of WT and MGP^-/- mice at 1, 7, 14, and 28 days of (n = 6–8 in each group, as indicated). No difference in aortic Id1 mRNA levels was observed between MGP^-/- and WT mice.</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