Tuning BMP-Regulated Cell Differentiation in the Aortic Media by Mutating Matrix Gla Protein

MGP (matrix Gla protein) serves as an inhibitor of vascular calcification by limiting elastin degradation and regulating BMP (bone morphogenetic protein) activity. Mutation of the conserved proline residue 64 to glycine in MGP abolishes BMP binding in vitro. We hypothesized that selective loss of BMP binding would elucidate the contribution of BMP to cell differentiation in Mgp-deficient mice.

Computational analyses using AlphaFold3 and molecular dynamics simulations were performed to determine the structural effects of γ-carboxylation and the proline residue 64 to glycine mutation. The vascular phenotype of Mgp-knockin mice expressing the proline residue 64 to glycine mutation was compared with wild-type (WT) and global Mgp-knockout mice. Proximity ligation assay was performed to assess MGP-BMP4 in aortic cells. Single-cell RNA-sequencing was used to identify cellular alterations in the vascular media.

Molecular dynamics simulation revealed 5 Ca²⁺ ions coordinated by γ-carboxylated Glu residues in the MGP dimer. The proline residue 64 to glycine mutation did not disrupt Ca²⁺ binding but likely suppressed conformational changes required for BMP4 binding. Unlike Mgp-KO mice, Mgp-knockin mice did not develop vascular calcification, elastic lamina proteolysis, and endothelial-mesenchymal transition, but exhibited vascular fibrosis. MGP-BMP4 interaction observed in WT aortic cells was barely detectable in Mgp-knockin aortic cells. Single-cell RNA-sequencing revealed increased fractions of smooth muscle cells and enhanced myofibroblast differentiation in the Mgp-knockin aortas compared with WT. In Mgp-knockin aortas, SMAD2 expression was significantly increased throughout the vessel wall compared with WT aortas. In contrast, SMAD1/5/9 activation in the Mgp-knockout aortas was more confined to the endothelium, whereas it localized to the media-adventitia transition in WT and Mgp-knockin aortas. Both Mgp-knockin and Mgp-knockout mice developed arteriovenous malformations in the lungs, kidneys, and brain.

Our findings suggest that MGP plays a versatile role in preserving vascular integrity, in part serving as an important BMP-trap aimed at directing vascular cell differentiation.