Serine Protease Activation Essential for Endothelial-Mesenchymal Transition in Vascular Calcification - PubMed (original) (raw)

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

Jiayi Yao et al. Circ Res. 2015.

Abstract

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.

Keywords: endothelium; matrix Gla protein; phenotype; serine proteases; vascular calcification.

© 2015 American Heart Association, Inc.

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Conflict of interest statement

DISCLOSURES

The authors have declared that no conflict of interest exists.

Figures

Figure 1

Figure 1. Lack of MGP causes EndMTs in aortic tissue

(a–b) Mgp+/+ and Mgp−/− aortic endothelium was examined by transmission electron microscopy (TEM) (a) and scanning electron microscopy (SEM) (b). Magnification for TEM, 3.7×103. Magnification for SEM, 5×102. IEL: Internal elastic lamina. Areas of IEL degradation are indicated by arrowheads. (c) Expression of stem-cell and mesenchymal markers in VE-cadherin-positive and CD45-negative presorted cells from Mgp+/+ and Mgp−/− aortas at postnatal day (P) 14 and 28, as determined by real-time PCR. ***p<0.001. (d) Co-expression of the endothelial marker vWF with the stem-cell marker Sox2 and the mesenchymal marker Slug in calcified lesions of Mgp−/− aortas. Scale bars, 100 µm.

Figure 2

Figure 2. Activation of serine proteases in EndMTs and calcification

(a) Global gene expression profile of serine proteases in aortas from wild type (Mgp+/+) and Mgp−/− mice. (b–d) Expression of elastases (ELA) and kallikreins (KLK) in Mgp+/+ and Mgp−/− aortas, determined by real-time PCR and immunoblotting with densitometry. ***p<0.001.

Figure 3

Figure 3. Induction of elastases and kallikreins in Mgp−/− aortic endothelium

(a) Immunostaining to detect co-localization of ELA 1, 2 and KLK 1, 5, 6 with the EC marker vWF. Scale bar: 100 µm. (b) Flow cytometric analysis of co-expression of KLK 1, 5, 6 and VE-Cadherin in Mgp−/− aortic cells.

Figure 4

Figure 4. Inhibition of serine proteases decreases EndMTs and vascular calcification

(a) Survival rate of Mgp−/− mice after treatment with DFP and serpina1 (n=8 per group). (b) Alizarin Red staining of Mgp−/− aortas after 2 weeks of treatment with DFP or serpina1 injections. (c) Aortic calcium in Mgp−/− aortas after injection of DFP (top) or serpina1 (bottom). ***p<0.001. (d) Expression of stem-cell markers in Mgp−/− aortas after treatment with DFP or serpina1 as determined by real-time PCR (top) and immunoblotting with densitometry (bottom). ***p<0.001.

Figure 5

Figure 5. Elastases and kallikreins induce EndMTs in HAECs

(a) Elastase (ELA) 1, 2 and kallikrein (KLK) 1, 5, 6 were induced after depletion of MGP by siRNA (MGP si) in HAEC. The induction was enhanced by BMP-4 or high glucose, and abolished by transfection of siRNA to SMAD1/5/8 as shown by immunoblotting with densitometry. (b) Stem-cell and mesenchymal marker were induced after MGP si depletion in HAECs. The induction was enhanced by BMP-4 or high glucose as shown by immunoblotting with densitometry. (c) Depletion of elastase 1, 2 and kallikrein 1, 5, 6 by siRNA (si) abolished the induction of stem-cell markers in MGP-depleted HAECs as shown by immunoblotting with densitometry. SCR: scrambled siRNA.

Figure 6

Figure 6. Sox2 mediates activation of elastases and kallikreins in the induction of EndMTs

(a) The expression of stem-cell markers was examined by immunoblotting with densitometry in HAECs treated with medium containing elastase 1, 2 (ELAs) and kallikrein 1, 5, 6 (KLKs), and transfected with siRNA (si) to each of the stem-cell markers individually. Ctr: control. SCR: scrambled siRNA. (b) HAECs were depleted of MGP by siRNA (si) and treated with high glucose and BMP-4, then treated with control (lane 1–3), DFP (lane 4–5), serpina1 (lane 6–7), or a combination of DFP, serpina1, and lentivirus (Lv) expressing Sox2 (lane 8–11). Expression of stem-cell markers was examined by immunoblotting with densitometry. (c–f) MGP-depleted HAECs were transfected with siRNAs (si) to elastase (ELA) 1, 2 and kallikrein (KLK) 1, 5, 6, or Sox2, and then treated with control (C), BMP-2 (B), glucose (G), or a combination. Expression of Cbfa1 and Osterix (OSX) was determined by immunoblotting with densitometry (c and d). Calcium accumulation was determined by Alizarin Red and Von Kossa staining (e and f). SCR: Scrambled siRNA.

Figure 7

Figure 7. Limiting Sox2 in endothelium decreases EndMTs and calcification in Mgp−/− aortas

(a) Co-expression of elastase (ELA) 1, 2 and kallikrein (KLK) 1, 5, 6 with EC marker vWF and multipotency marker Sox2 in Mgp−/− aortas as shown by immunostaining. Scale bars: 100 µm. (b) Decreased expression of Sox2 in aortas of Cdh5CreSox2Flox/wtMgp−/−, as shown by immunoblotting with densitometry. (c) Decreased expression of stem-cell and mesenchymal markers in aortas of Cdh5CreSox2Flox/wtMgp−/−, as shown by immunoblotting with densitometry. (d) Aortic calcium in Cdh5CreSox2wt/wtMgp−/− and Cdh5CreSox2Flox/wtMgp−/− mice. ***p<0.001. (e) Alizarin Red staining of Cdh5CreSox2wt/wtMgp−/− and Cdh5CreSox2Flox/wtMgp−/− aortas. (f) Decrease of expression of elastase (ELA) 1, 2 and kallikrein (KLK) 1, 5, 6 in the in aortas of Cdh5CreSox2Flox/wtMgp−/−, as shown by real-time PCR. ***p<0.001.

Figure 8

Figure 8. Mutual regulation of Sox2 and Twist1 in EndMTs in vascular calcification

(a) Increase of Twist1 expression in VE-cadherin+CD45− presorted cells from Mgp−/− aortas, as shown by real-time PCR. ***p<0.001. (b) Co-localization of Twist1 with the EC marker vWF in Mgp−/− aortas, as shown by immunostaining. Scale bars: 100 µm. (c) MGP-depleted HAECs were transfected with siRNAs (si) to Sox2, and then treated with combinations of BMP-4 and glucose. Expression of Twist1 was examined by real-time PCR. ***p<0.001. (d) MGP-depleted HAECs were transfected with siRNAs (si) to Twist1, and then treated with combination of BMP-4 and glucose. Expression of Sox2, Klf4 and c-kit was examined by real-time PCR. ***p<0.001. (e) MGP-depleted HAECs were transfected with siRNAs (si) to Twist1, and then treated with BMP-2 or glucose for 4 days. Expression of Sox2, Klf4 and c-kit was examined by real-time PCR. ***p<0.001. (f) Schematic working model for the induction of serine proteases and Sox2 in EndMTs in vascular calcification.

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