Differentiation trajectory from ECs to EC-like myofibroblasts and myofibroblasts may pass through a mesenchymal stage. This trajectory exists in a quiescent state in healthy lungs and may be activated when pathological stimuli trigger pulmonary fibrosis. https://bit.ly/3QWfgyS

Vascular calcification (VC) increases morbidity and mortality and constitutes a significant obstacle during percutaneous interventions and surgeries. On a cellular and molecular level, VC is a highly regulated process that involves abnormal cell transitions and osteogenic differentiation, re-purposing of signaling pathways normally used in bone, and even formation of osteoclast-like cells. Endothelial cells have been shown to contribute to VC through a variety of means. This includes direct contributions of osteoprogenitor cells generated through endothelial-mesenchymal transitions in activated endothelium, with subsequent migration into the vessel wall. The endothelium also secretes pro-osteogenic growth factors, such as bone morphogenetic proteins, inflammatory mediators and cytokines in conditions like hyperlipidemia, diabetes, and renal failure. High phosphate levels caused by renal disease have deleterious effects on the endothelium, and induction of tissue non-specific alkaline phosphatase adds to the calcific process. Furthermore, endothelial activation promotes proteolytic destruction of the internal elastic lamina that serves, among other things, as a stabilizer of the endothelium. Appropriate bone mineralization is highly dependent on active angiogenesis, but it is unclear whether the same relationship exists in VC. Through its location facing the vascular lumen, the endothelium is the first to encounter circulating factor and bone marrow-derived cells that might contribute to osteoclast-like versus osteoblast-like cells in the vascular wall. In the same way, the endothelium may be the easiest target to reach with treatments aimed at limiting calcification. This review provides a brief summary of the contributions of the endothelium to VC as we currently know them.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208-215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the new Pi site attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.

Background and aims

Endothelial-mesenchymal transitions (EndMTs) in endothelial cells (ECs) contribute to vascular disease.

Methods

We used ApoE^-/- mice fed a high-fat/high-cholesterol diet.

Results

We reported evidence of EndMT in atherosclerotic lesions contributing to calcification. Stem cell and mesenchymal markers, including sex-determining region Y-box 2 (Sox2), were upregulated in aortic ECs of fat-fed ApoE^-/- mice. Limiting Sox2 decreased marker expression and calcification in ApoE^-/- aortas. Furthermore, a complex of serine proteases was upregulated in ApoE^-/- aortic ECs. Blockade of these proteases reduced expression of Sox2 and atherosclerotic lesion calcification.

Conclusions

Together, our data suggest that EndMTs contribute to atherosclerotic lesion calcification.

Mutations in ABCC6 (ATP-binding cassette, subfamily C, member 6), an orphan transporter expressed in the liver, are the cause of pseudoxanthoma elasticum. Since ABCC6 was reported to affect matrix Gla protein (MGP), an inhibitor of bone morphogenetic proteins (BMPs), we studied BMP signaling and expression in various tissues of mice with and without functional ABCC. Enhanced BMP signaling was found in all examined tissues in the absence of ABCC6. Despite this, the expression of particular BMP proteins varied widely between tissues. Interestingly, the expression of most BMP proteins in the liver moved in the opposite direction to the same BMP proteins in kidneys in response to ABCC6 alterations. Thus, ABCC6 deficiency stimulates BMP signaling by acting on the expression of multiple BMPs.

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.

Endothelial-mesenchymal transition (EndMT) drives the endothelium to contribute to vascular calcification in diabetes mellitus. In our previous study, we showed that glycogen synthase kinase-3β (GSK3β) inhibition induces β-catenin and reduces mothers against DPP homolog 1 (SMAD1) to direct osteoblast-like cells toward endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency. Here, we report that GSK3β inhibition reduces vascular calcification in diabetic Ins2^Akita/wt mice. Cell lineage tracing reveals that GSK3β inhibition redirects endothelial cell (EC)-derived osteoblast-like cells back to endothelial lineage in the diabetic endothelium of Ins2^Akita/wt mice. We also find that the alterations in β-catenin and SMAD1 by GSK3β inhibition in the aortic endothelium of diabetic Ins2^Akita/wt mice are similar to Mgp^-/- mice. Together, our results suggest that GSK3β inhibition reduces vascular calcification in diabetic arteries through a similar mechanism to that in Mgp^-/- mice.

Bone morphogenetic protein (BMP) and Notch signaling are critical for endothelial cell (EC) differentiation in vascular development. Recent studies have shown that excess BMP activity induces Notch signaling in cerebral ECs resulting in arteriovenous malformation (AVMs). However, it is unclear how the crosstalk between BMP and Notch signaling affects cerebral EC differentiation at the gene regulatory level. Here, we report that BMP6 activates the activin receptor-like kinase (ALK) 3, a BMP type 1 receptor, to induce Notch1 receptor and Jagged1 and Jagged2 ligands. We show that increased expression of the Notch components alters the transcriptional regulatory complex in the SRY-Box 2 (Sox2) promoter region so as to induce its expression in cerebral ECs. Together, our results identify Sox2 as a direct target of BMP and Notch signaling and provide information on how altered BMP and Notch signaling affects the endothelial transcriptional landscape.

Notch signaling and Sry-box (Sox) family transcriptional factors both play critical roles in endothelial cell (EC) differentiation in vascularization. Recent studies have shown that excessive Notch signaling induces Sox2 to cause cerebral arteriovenous malformations (AVMs). Here, we examine human pulmonary AVMs and find no induction of Sox2. Results of epigenetic studies also show less alteration of Sox2-DNA binding in pulmonary AVMs than in cerebral AVMs. We identify high expression of ski-interacting protein (Skip) in brain ECs, a Notch-associated chromatin-modifying protein that is lacking in lung ECs. Knockdown of Skip abolished Notch-induction of Sox2 in brain ECs, while restoration of Skip in lung ECs enabled Notch-mediated Sox2 induction. The results suggest that Skip is a key factor for induction of Sox2 in cerebral AVMs.

Matrix Gla protein (MGP) is an antagonist of bone morphogenetic proteins and expressed in vascular endothelial cells. Lack of MGP causes vascular abnormalities in multiple organs in mice. The objective of this study is to define the role of MGP in early endothelial differentiation. We find that expression of endothelial markers is highly induced in Mgp null organs, which, in wild type, contain high MGP expression. Furthermore, Mgp null embryonic stem cells express higher levels of endothelial markers than wild-type controls and an abnormal temporal pattern of expression. We also find that the Mgp-deficient endothelial cells adopt characteristics of mesenchymal stem cells. We conclude that loss of MGP causes dysregulation of early endothelial differentiation.