Serine Protease Activation Essential for Endothelial-Mesenchymal Transition in Vascular Calcification.
BACKGROUND
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.


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.
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.).
Jiayi
J
Yao
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.).
Pierre J
PJ
Guihard
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.).
Ana M
AM
Blazquez-Medela
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.).
Yina
Y
Guo
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.).
Jeremiah H
JH
Moon
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.).
Medet
M
Jumabay
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.).
Kristina I
KI
Boström
From the Division of Cardiology, David Geffen School of Medicine (J.Y., P.J.G., A.M.B.-M., Y.G., J.H.M., M.J., K.I.B., Y.Y.) and The Molecular Biology Institute (K.I.B.), University of California, Los Angeles; and Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, China (J.Y.). yyao@mednet.ucla.edu.
Yucheng
Y
Yao
D000818:Animals; D002114:Calcinosis; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D004789:Enzyme Activation; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D015151:Immunoblotting; D007610:Kallikreins / Q000235:genetics / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism* / Q000473:pathology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D051879:Twist-Related Protein 1 / Q000235:genetics / Q000378:metabolism; D000097568:Matrix Gla Protein
D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D007610:Kallikreins; D009687:Nuclear Proteins; D010196:Pancreatic Elastase; D055748:SOXB1 Transcription Factors; D012697:Serine Endopeptidases; D051879:Twist-Related Protein 1; D000097568:Matrix Gla Protein; C527783:Sox2 protein, mouse; C067250:Twist1 protein, mouse
endothelium; matrix Gla protein; phenotype; serine proteases; vascular calcification