Fig 1.
EndMTs in aortic tissue of Ins2Akita/+ mice.
(a) Aortic endothelium in Ins2Akita/+ mice was examined by transmission electron microscopy (TEM) (top) and scanning electron microscopy (SEM) (bottom). Magnification for TEM, 3.7×103. Magnification for SEM, 5×102. Lu: lumen; IEL: Internal elastic lamina. (b) Expression of mesenchymal stem cell markers and EC marker VE-cadherin (VE-Cad) in CD31-positive and CD45-negative presorted cells from wild type (WT) and Ins2Akita/+ aortas at 40 weeks of age, as determined by immunoblotting.
Fig 2.
Limiting Sox2 in endothelium decreases EndMTs and calcification in Ins2Akita/+ aortas.
(a-b) Decreased expression of Sox2, stem cell and mesenchymal markers in aortas of Cdh5CreSox2Flox/wtIns2Akita/+, as shown by real-time PCR (a) and immunoblotting (b). VE-cad:VE-cadherin. (c-d) Total aortic calcium and aortic Alizarin red staining of Cdh5CreSox2wt/wtIns2+/+ and Cdh5CreSox2Flox/wtIns2+/+, Cdh5CreSox2wt/wtIns2Akita/+ and Cdh5CreSox2Flox/wtIns2Akita/+ mice. ***p<0.001.
Fig 3.
Sox2 activates CD90 and c-kit in MGP-deficient HAECs.
(a-b) Expression of BMP-4 and MGP in endothelium of Ins2Akita/+ mice (a) and in HAEC (b) treated with different concentrations of glucose. (c-d) MGP expression and pSMAD1/5/8 level in HAECs after depletion of MGP by using CRISPR/Cas9 (MGP CR). (e) Expression of Sox2, c-kit and VE-cadherin (VE-cad) in MGP-depleted HAECs (MGP CR) with or without Noggin treatment. (f) ChIP assay shows abundant Sox2-binding in the promoters of CD90 and c-kit in MGP-depleted HAECs (MGP CR). a-Sox2: anti-Sox2 antibodies. ***, p<0.0001.
Fig 4.
Induction of elastases and kallikreins in Ins2Akita/+ aortic endothelium.
Expression of ELA 1, 2 and KLK 1, 5, 6 in CD31-positive and CD45-negative presorted cells (a) from wild type (WT) and Ins2Akita/+ aortas at 40 weeks of age, and (b) from aortas of WT, Ins2Akita/+, Mgptg/wt and Mgptg/wtIns2Akita/+ mice at 40 weeks of age, as determined by immunoblotting.
Fig 5.
Inhibition of serine proteases decreases EndMTs and vascular calcification.
(a-b) Total aortic calcium of Ins2Akita/+aortas after treatment of (a) diisopropylfluorophosphate (DFP) or (b) serpina1. ***p<0.001. (c) Expression of stem-cell markers in isolated aortic ECs of Ins2Akita/+ mice after treatment with diisopropylfluorophosphate (DFP) or serpina1, as determined by immunoblotting. (d-e) pSMAD1/5/8 levels and Sox2 expression in MGP-depleted HAECs after diisopropylfluorophosphate (DFP) treatment shown by immunoblotting.