BMP-2–mediated migration of hPASMCs requires activation of RhoA and Rac1. (A) Migration was assessed using the Boyden chamber assay as described in Materials and methods. Cells were starved for 48 h in starvation media (0.1% FBS) before seeding them in Boyden chambers placed in 24-well plates containing either starvation media alone or supplemented with 10 ng/ml BMP-2 or 20 ng/ml PDGF-BB. (B) RhoA (top) and Rac1 (bottom) activation was measured in lysates from cells stimulated with BMP-2 over a period of 6 h using the approach described in Materials and methods. Densitometry values are shown relative to total RhoA and Rac1 in whole cell lysates that were run in different gels. (C) Motility response to BMP-2 was assessed in cells incubated with either Y-27632 at 10, 50, and 100 mM or DMSO for 6 h. Bars represent means ± SEM from n = 3 different cell harvests in triplicate. ***, P < 0.0001 versus control or time 0; and ^##, P < 0.001 versus uninhibited control using one-way ANOVA with the Dunnett (A and B) or Bonferroni (C) tests. CON, control.

Activation of βC is required for BMP-2–mediated hPASMC motility. (A) Levels of active βC in lysates obtained from 10 ng/ml BMP-2–stimulated cells over 6 h were measured by Western immunoblotting (top) and quantified using densitometry (bottom) expressed as the ratio of the OD of βC relative to α-tubulin. (B) RhoA (top) and Rac1 (bottom) activation was measured in lysates from cells transfected with either scrambled or two independent βC siRNAs stimulated with BMP-2 over a period of 6 h using the approach described in Materials and methods. Densitometry values are shown relative to total RhoA and Rac1 in whole cell lysates, which were run in different gels. (C) Boyden chamber assays were performed as previously described to measure the impact of two independent βC versus scrambled (SC) siRNA on hPASMC motility. Bars represent means ± SEM from n = 3. *, P < 0.01; and ***, P < 0.0001 versus control (CON) in A and B were determined using one-way ANOVA with Dunnett’s. ***, P < 0.0001 versus time 0; and ^##, P < 0.001 versus scrambled siRNA in C were determined using one-way ANOVA with Bonferroni’s.

BMP-2 activates βC via pAkt-dependent inhibition of GSK3β and is independent of Smad signaling. (A and B) βC activation in 10 ng/ml BMP-2–stimulated hPASMCs transfected with a nonfunctional pAkt (ΔAkt) in A and a Smad 1 construct (ΔSmad 1) in B was measured by immunoblot analysis and compared with values in cells transfected with vector alone. The pGSK3β activation in A was measured relative to total GSK3β. Bars represent means ± SEM from n = 3. ***, P < 0.0001 versus time 0 in A was determined using one-way ANOVA with Dunnett’s. ***, P < 0.0001 in B were determined using the unpaired t test.

Production and secretion of FN is dependent on BMP-2–mediated activation of βC. (A and B) Quantitative RT-PCR (A) and Western immunoblotting (B) were used to measure changes in levels of FN mRNA and protein, respectively, in hPASMCs stimulated with 10 ng/ml BMP-2 over a period of 6 h. In B, levels of secreted FN were measured in hPASMCs transfected with either scrambled or two independent βC siRNAs. Densitometry values were expressed as the ratio of OD of FN relative to the Ponceau stain of an unrelated band in media samples concentrated by ultrafiltration. Bars represent means ± SEM from n = 3. **, P < 0.001; and ***, P < 0.0001 versus time 0 using one-way ANOVA with Dunnett’s. (C) Representative immunofluorescence images showing FN distribution in scrambled (top) or βC siRNA (bottom)–transfected hPASMCs stimulated with 10 ng/ml BMP-2 over 6 h. DAPI (blue) stain was used to label cell nuclei. Bar, 10 µm.

Activation of integrin-linked kinase 1 (ILK-1) is required for BMP-2–mediated hPASMC motility. (A) ILK-1 kinase assay was performed as described in Materials and methods with GSK3β as the substrate on cells incubated in the presence (right) or absence (left) of 5 µg/ml CS-1. (B and C) Motility in hPASMCs exposed to CS-1 (B) or transfected with either scrambled (SC) or ILK-1–specific siRNA (C) and exposed to 10 ng/ml BMP-2 was measured using the Boyden assay. (D) Levels of active RhoA and Rac1 were measured as described in Materials and methods. Densitometry values are shown relative to total RhoA and Rac1 in whole cell lysates, which were run in different gels. Bars represent means ± SEM from n = 3. In A, *, P < 0.01; and ***, P < 0.0001 were determined using one-way ANOVA with Dunnett’s. In B and C, **, P < 0.001; and ***, P < 0.001 versus baseline; and ^##, P < 0.001; and ^###, P < 0.0001 versus respective scrambled siRNA control using one-way ANOVA with Bonferroni’s. In D, *, P < 0.01; and ***, P < 0.0001 versus time 0 were determined using an unpaired t test. CON, control.

ILK-1–dependent recruitment of Dvl is necessary for BMP-2–mediated hPASMC migration. (A) Confocal microscopy was used to localize ILK-1 (red) and Dvl (green) in hPASMCs stimulated with 10 ng/ml BMP-2. DAPI (blue) was used to label the nuclei. Bars, 10 µm. (B) Co-IP of V5-tagged ILK-1 and WT, ΔDEP, and ΔPRS Dvl-GFP constructs under 10 ng/ml BMP-2 stimulation. Levels of V5-tagged ILK-1 were measured against levels of α-tubulin in whole cell lysates analyzed in a separate gel. (C) RhoA (left) and Rac1 (right) activation in BMP-2–stimulated hPASMCs transfected with either WT, ΔDEP, or ΔPRS Dvl was performed as described in Materials and methods. Levels of active RhoA and Rac1 were measured against levels of total RhoA and Rac1 in whole cell lysates analyzed in a separate gel. (D) Boyden chamber assays were performed as previously described to measure the impact of WT, ΔDEP, and ΔPRS Dvl on BMP-2–induced hPASMC motility. Bars represent means ± SEM from n = 3. **, P < 0.001; and ***, P < 0.0001 versus time 0 in B and C using one-way ANOVA with Dunnett’s. ***, P < 0.0001 versus control (CON) unstimulated; and ^###, P < 0.0001 versus WT stimulation in D was determined using one-way ANOVA with Bonferroni’s. IP, immunoprecipitation. IB, immunoblot.

Presence of ΔDEP Dvl promotes VSMC growth response to PDGF-BB by increasing βC levels. (A) Levels of active βC (left) and pGSK3β (middle) in hPASMCs transfected with either WT or ΔDEP Dvl and stimulated with 10 ng/ml BMP-2 were visualized by Western immunoblotting and quantified by densitometry. Levels of active βC were normalized to α-tubulin, and levels of pGSK3β were normalized to total GSK3β in whole cell lysates. Co-IP of pGSK3β and WT and ΔDEP (right) was carried under BMP-2 stimulation. Levels of pGSK3β were normalized relative to total GSK3β in whole cell lysates analyzed in a separate gel. (B) Cell count studies were performed after addition of PDGF-BB, BMP-2, or both for 72 h. (C) Cell count studies were performed in hPASMCs cotransfected with WT or ΔDEP Dvl and either scrambled or two independent βC siRNAs after addition of PDGF-BB, BMP-2, or both for 72 h. **, P < 0.001; and ***, P < 0.0001 as indicated in A were determined by one-way ANOVA with Dunnett’s. Bars represent mean ± SEM from n = 3. *, P < 0.01; **, P < 0.001; and ***, P < 0.0001 versus baseline; ^#, P < 0.01; and ^##, P < 0.001 versus scrambled counterpart as indicated in B and C using one-way ANOVA with Bonferroni’s. CON, control; IP, immunoprecipitation. IB, immunoblot.

Constitutively active βC induces FN production, RhoA-Rac1, motility, and proliferation in hPASMCs. (A) Levels of secreted FN were measured in hPASMCs transfected with either vector or ΔβC. Densitometry values were expressed as the ratio of OD of FN relative to the Ponceau stain of an unrelated band in media samples concentrated by ultrafiltration. (B) RhoA (left) and Rac1 (right) activation in BMP-2–stimulated hPASMCs transfected with either vector or ΔβC was performed as described in Materials and methods. Levels of active RhoA and Rac1 were measured against levels of total RhoA and Rac1 in whole cell lysates analyzed in a separate gel. (C) Boyden chamber assays were performed as previously described in this paper to measure the impact of vector or ΔβC on hPASMC motility. (D) Cell count studies in hPASMCs transfected with either vector or ΔβC were performed for 72 h. Bars represent means ± SEM from n = 3. ***, P < 0.0001 versus vector using an unpaired t test.

Transfection of ΔDEP leads to increased neointima thickening in stented aortic grafts. Thoracic aortas from C57BL/6J mice were cannulated with a balloon angioplasty catheter and inflated to eight atmospheres for 30 s followed by insertion of a 2.5 × 6–mm metal stent as described in Materials and methods. (A) OCT cross-sectional (top) and longitudinal (bottom) images of the grafts transfected with WT (left) and ΔDEP (right) Dvl were obtained, and intimal thickening was quantified. Values are expressed as percentages of the intimal area measured by comparing the cross-sectional area of the intima relative to the cross sectional area of the stent. The cross-sectional area of the intima is measured as the region above the white stent struts that protrude into the lumen. Yellow lines identify the center of the source for the infrared light in the OCT catheter. Bar, 200 µm. (B) Intimal response to balloon angioplasty and stenting comparing WT Dvl or ΔDEP Dvl. Representative images of histological sections at low power from each group are shown. In both images, black arrowheads denote the vessel wall (endothelium to external elastic lamina), and white arrowheads show the neointima (subendothelial space to internal elastic lamina). Note that in the region illustrated by the arrowheads there is a very small distance between the fragmented internal elastic and the external elastic laminae. An unpaired t test was used to show the difference between WT and ΔDEP Dvl; *, P < 0.05; and **, P < 0.001 (n = 5–6 mice per group). (C) Cross-sectional images of WT (left) and ΔDEP (right) Dvl–transfected grafts stained with DAPI. Insets in the top images represent the areas illustrated on the bottom. Bar, 100 µm.

Proposed models for BMP regulation of hPASMC motility and proliferation by recruitment of Wnt–βC and Wnt–PCP signaling. (A) BMP-2 triggers βC accumulation via pAkt-mediated GSK3β inhibition followed by production and release of FN to the extracellular space (1). By binding to α4β1-integrin, FN activates ILK-1 and induces formation of a complex between ILK-1 and Dvl leading to RhoA and Rac1 activation (2) and simultaneous suppression of βC activation (3). (B) The inability to form or maintain a complex between ILK-1 and Dvl may facilitate hPASMC proliferation in response to growth factors such as PDGF-BB by enhancing βC signaling through ILK-1–mediated GSK3β inhibition. APC, adenomatous polyposis coli.