Rationale:: Vascular calcification is a regulated process that involves osteoprogenitor cells and frequently complicates common vascular disease, such as atherosclerosis and diabetic vasculopathy. However, it is not clear whether the vascular endothelium has a role in contributing osteoprogenitor cells to the calcific lesions. Objective:: To determine whether the vascular endothelium contributes osteoprogenitor cells to vascular calcification. Methods and Results:: In this study, we use 2 mouse models of vascular calcification, mice with gene deletion of matrix Gla protein, a bone morphogenetic protein (BMP)-inhibitor, and Ins2 ^Akita/+ mice, a diabetes model. We show that enhanced BMP signaling in both types of mice stimulates the vascular endothelium to contribute osteoprogenitor cells to the vascular calcification. The enhanced BMP signaling results in endothelial–mesenchymal transitions and the emergence of multipotent cells, followed by osteoinduction. Endothelial markers colocalize with multipotent and osteogenic markers in calcified arteries by immunostaining and fluorescence-activated cell sorting. Lineage tracing using Tie2 - Gfp transgenic mice supports an endothelial origin of the osteogenic cells. Enhancement of matrix Gla protein expression in Ins2 ^Akita/+ mice, as mediated by an Mgp transgene, limits the generation of multipotent cells. Moreover, matrix Gla protein–depleted human aortic endothelial cells in vitro acquire multipotency rendering the cells susceptible to osteoinduction by BMP and high glucose. Conclusions:: Our data suggest that the endothelium is a source of osteoprogenitor cells in vascular calcification that occurs in disorders with high BMP activation, such as deficiency of BMP-inhibitors and diabetes mellitus.

Rationale:: Diabetes mellitus is frequently complicated by cardiovascular disease, such as vascular calcification and endothelial dysfunction, which have been associated with bone morphogenetic proteins (BMPs). Objective:: To determine whether hyperglycemia in vitro and diabetes in vivo promote vascular BMP activity and correlate with vascular calcification. Methods and Results:: Increased glucose augmented expression of BMP-2 and BMP-4; the BMP inhibitors matrix Gla protein (MGP) and Noggin; activin-like kinase receptor (ALK)1, -2, -3 and -6; the BMP type 2 receptor; and the vascular endothelial growth factor in human aortic endothelial cells (HAECs). Diabetes induced expression of the same factors in the aortic wall of 3 animal models of diabetes, Ins2 ^Akita/+ mice, db/db mice, and HIP rats (rats transgenic for human islet amyloid polypeptide), representative of types 1 and 2 diabetes. Conditioned media from glucose-treated HAECs increased angiogenesis in bovine aortic endothelial cells, as mediated by BMP-4, and osteogenesis in calcifying vascular cells, as mediated by BMP-2. BMP-4, MGP, ALK1, and ALK2 were predominantly expressed on the endothelial side of the aorta, and small interfering RNA experiments showed that these genes were regulated as a group. Diabetic mice and rats showed a dramatic increase in aortic BMP activity, as demonstrated by SMAD1/5/8 phosphorylation. This was associated with increased osteogenesis and calcium accumulation. These changes were prevented in the Ins2 ^Akita/+ mice by breeding them with MGP transgenic mice, which increased aortic BMP inhibition. Conclusions:: Hyperglycemia and diabetes activate vascular BMP activity, which is instrumental in promoting vascular calcification and may be limited by increasing BMP inhibition.

Arteriovenous malformations (AVMs) in organs, such as the lungs, intestine, and brain, are characteristic of hereditary hemorrhagic telangiectasia (HHT), a disease caused by mutations in activin-like kinase receptor 1 (ALK1), which is an essential receptor in angiogenesis, or endoglin. Matrix Gla protein (MGP) is an antagonist of BMPs that is highly expressed in lungs and kidneys and is regulated by ALK1. The objective of this study was to determine the role of MGP in the vasculature of the lungs and kidneys. We found that Mgp gene deletion in mice caused striking AVMs in lungs and kidneys, where overall small organ size contrasted with greatly increased vascularization. Mechanistically, MGP deficiency increased BMP activity in lungs. In cultured lung epithelial cells, BMP-4 induced VEGF expression through induction of ALK1, ALK2, and ALK5. The VEGF secretion induced by BMP-4 in Mgp^–/– epithelial cells stimulated proliferation of ECs. However, BMP-4 inhibited proliferation of lung epithelial cells, consistent with the increase in pulmonary vasculature at the expense of lung tissue in the Mgp-null mice. Similarly, BMP signaling and VEGF expression were increased in Mgp^–/– mouse kidneys. We therefore conclude that Mgp gene deletion is what we believe to be a previously unidentified cause of AVMs. Because lack of MGP also causes arterial calcification, our findings demonstrate that the same gene defect has drastically different effects on distinct vascular beds.

Rationale:: Endothelial cells have the ability to undergo endothelial–mesenchymal transitions (EndMTs), by which they acquire a mesenchymal phenotype and stem cell–like characteristics. We previously found that EndMTs occurred in the endothelium deficient in matrix γ-carboxyglutamic acid protein enabling endothelial cells to contribute cells to vascular calcification. However, the mechanism responsible for initiating EndMTs is not fully understood. Objective:: To determine the role of specific serine proteases and sex determining region Y-box 2 (Sox2) in the initiation of EndMTs. Methods and Results:: In this study, we used in vivo and in vitro models of vascular calcification to demonstrate that serine proteases and Sox2 are essential for the initiation of EndMTs in matrix γ-carboxyglutamic acid protein–deficient endothelium. We showed that expression of a group of specific serine proteases was highly induced in endothelial cells at sites of vascular calcification in Mgp null aortas. Treatment with serine protease inhibitors decreased both stem cell marker expression and vascular calcification. In human aortic endothelial cells, this group of serine proteases also induced EndMTs, and the activation of proteases was mediated by Sox2. Knockdown of the serine proteases or Sox2 diminished EndMTs and calcification. Endothelial-specific deletion of Sox2 decreased expression of stem cell markers and aortic calcification in matrix γ-carboxyglutamic acid protein–deficient mice. Conclusions:: Our results suggest that Sox2-mediated activation of specific serine proteases is essential for initiating EndMTs, and thus, may provide new therapeutic targets for treating vascular calcification.

Cerebral arteriovenous malformations (AVMs) are common vascular malformations, which may result in hemorrhagic strokes and neurological deficits. Bone morphogenetic protein (BMP) and Notch signaling are both involved in the development of cerebral AVMs, but the cross-talk between the two signaling pathways is poorly understood. Here, we show that deficiency of matrix Gla protein (MGP), a BMP inhibitor, causes induction of Notch ligands, dysregulation of endothelial differentiation, and the development of cerebral AVMs in MGP null (Mgp^−/−) mice. Increased BMP activity due to the lack of MGP induces expression of the activin receptor-like kinase 1, a BMP type I receptor, in cerebrovascular endothelium. Subsequent activation of activin receptor-like kinase 1 enhances expression of Notch ligands Jagged 1 and 2, which increases Notch activity and alters the expression of Ephrin B2 and Ephrin receptor B4, arterial and venous endothelial markers, respectively. Reducing the expression of Jagged 1 and 2 in the Mgp^−/− mice by crossing them with Jagged 1 or 2 deficient mice reduces Notch activity, normalizes endothelial differentiation, and prevents cerebral AVMs, but not pulmonary or renal AVMs. Our results suggest that Notch signaling mediates and can modulate changes in BMP signaling that lead to cerebral AVMs. Significance Cerebral arteriovenous malformations (AVMs) are common vascular abnormalities that may lead to strokes. Signaling by bone morphogenetic proteins (BMPs) and Notch play important roles in the formation of cerebral AVMs, but the cross-talk between the pathways is poorly understood. We report that gene deletion of matrix Gla protein (MGP), a BMP inhibitor, causes cerebral AVMs in mice by activating activin receptor-like kinase 1, a BMP receptor. This activation enhances Notch activity and disrupts endothelial cell differentiation by inducing the Notch ligands Jagged 1 and 2. Reducing Jagged 1 and 2 expression prevents the disruption in differentiation and AVM formation. The findings suggest that MGP maintains the balance between BMP and Notch signaling and promotes a normal brain vasculature.