BMP7 signaling is enhanced in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs.

<p>(A) Immunoblots of lysates of WT (Bmpr2^+/+) or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs treated with BMP4 or BMP7 (10 ng/ml) for various times were reacted with antibodies directed against phosphorylated and total Smad1/5/8. Quantification of the ratio of phosphorylated Smad1/5/8 to total Smad1/5/8 (analysis of 3 independent experiments) demonstrated that BMP4 signaling is similar in <i>Bmpr2</i>^<i>+/+</i> or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs, whereas BMP7 signaling is greater in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs. *P<0.05 compared to <i>Bmpr2</i>^<i>+/+</i> PaSMC group treated with BMP7. <i>Id1</i> (B) and <i>Smad6</i> (C) mRNA levels were measured by qPCR in <i>Bmpr2</i>^<i>+/+</i> or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs treated with BMP4 or BMP7 (10 ng/ml) for various times. <i>Id1</i> and <i>Smad6</i> gene expression was normalized to <i>Gapdh</i> and expressed as fold-change relative to control <i>Bmpr2</i>^<i>+/+</i> PaSMC group. *P < 0.01 compared to <i>Bmpr2</i>^<i>+/+</i> PaSMC group treated with BMP7.</p

Bmpr2 expression in PaSMCs obtained from WT or <i>Bmpr2</i>^{<i>Δtd/+</i>} mice.

<p>(A) Levels of <i>Bmpr2</i> mRNA were measured in WT (Bmpr2^+/+) or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs by qPCR using hydrolysis probes for <i>Bmpr2</i> exon junctions 6–7 and 12–13. <i>Bmpr2</i> mRNA levels were normalized to <i>Gapdh</i> and expressed as the fold-change relative to <i>Bmpr2</i>^<i>+/+</i> PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^<i>+/+</i> PaSMCs. (B) Immunoblots prepared from lysates of <i>Bmpr2</i>^<i>+/+</i> and <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were incubated with an antibody directed against the tail domain of Bmpr2 to detect Bmpr2‑WT or with an anti-GFP antibody to detect Bmpr2‑ΔTD. Immunoblots were subsequently incubated with an antibody directed against Gapdh as a control for protein loading. (C) Confocal microscopy image of a PaSMC transiently transfected with a plasmid directing expression of <i>Bmpr2</i>^<i>Δtd</i> and reacted with an anti-GFP antibody showing localization of Bmpr2‑ΔTD at the cell membrane.</p

Bmpr2‑TD attenuates Alk2‑mediated BMP7 signaling in PaSMCs.

<p>Alk3‑deficient <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were transfected with specific siRNA to silence <i>Bmpr2</i>^<i>+</i> (si<i>Bmpr2</i>‑ex12) or <i>Bmpr2</i>^<i>Δtd</i> (si<i>Egfp</i>) transcripts. After 48 h, the ability of BMP7 to induce <i>Id1</i> and <i>Smad6</i> gene expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>}; <i>Alk3</i>^{<i>del/del</i>} PaSMCs treated with siNC. *P < 0.01 compared to control cells (siNC) treated with BMP7. Silencing efficiency was quantified by qPCR.</p

Bmpr2‑ΔTD contributes to BMP7 signaling in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs.

<p>(A) <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were transfected with negative control siRNA (siNC), si<i>Bmpr2</i>‑ex12, or si<i>Egfp</i> (30 nM). After 48 h, the ability of BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> mRNA expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs transfected with siNC. *P < 0.01 compared to siNC group treated with BMP7, ^† P<0.01 compared to si<i>Bmpr2</i>‑ex12 group treated with BMP7. Efficiency of silencing <i>Bmpr2</i>^<i>+</i> (si<i>Bmpr2</i>‑ex12) and <i>Bmpr2</i>^<i>Δtd</i> (si<i>Egfp</i>) transcripts was measured by qPCR. (B) <i>Bmpr2</i>^{<i>Δtd/flox</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs were treated with BMP4 or BMP7 (10 ng/ml) for 30 and 60 minutes, upon which the activation of Smad1/5/8 was evaluated by immunoblotting. Quantification of the Smad1/5/8 activation is plotted as the ratio of pSmad1/5/8 to total Smad1/5/8. (C) The ability of BMP4 or BMP7 to induce <i>Id1</i> and <i>Smad6</i> gene expression in <i>Bmpr2</i>^{<i>Δtd/flox</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to untreated <i>Bmpr2</i>^{<i>Δtd/flox</i>} PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^{<i>Δtd/flox</i>} PaSMC group treated with BMP7.</p

BMP7 preferentially utilizes Alk2 in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs.

<p>(A) The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression, in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs deficient in Alk2 or expressing Alk2 was examined by qPCR. <i>Id1</i> and <i>Smad6</i> gene expression was normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk2</i>^{<i>flox/flox</i>} PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk2</i>^{<i>flox/flox</i>} PaSMCs treated with BMP7. (B) The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression, in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs deficient in Alk3 or expressing Alk3 was measured by qPCR. <i>Id1</i> and <i>Smad6</i> gene expression was normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk3</i>^{<i>flox/flox</i>} PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^{<i>Δtd/+</i>}<i>; Alk3</i>^{<i>flox/flox</i>} PaSMC treated with BMP4.</p

BMP7 signaling in <i>Bmpr2</i>^{<i>Δtd/+</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs does not depend on the presence of Acvr2a.

<p>(A) <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were treated with a siRNA specific for <i>Acvr2a</i> transcripts. The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs treated with siNC. *P < 0.01 compared to siNC within BMP treatment. Silencing efficiency was quantified by measuring <i>Acvr2a</i> mRNA levels. (B) <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs were treated with si<i>Acvr2a</i>. The ability of BMP4 or BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> gene expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs treated with siNC. *P < 0.01 compared to siNC within BMP treatment. <i>Acvr2a</i> silencing efficiency was measured by qPCR.</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

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

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

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