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. 2016 Sep:98:48-57.
doi: 10.1016/j.yjmcc.2016.06.061. Epub 2016 Jun 23.

Mitral valve endothelial cells secrete osteoprotegerin during endothelial mesenchymal transition

Paola Songia  1 , Emanuela Branchetti  2 , Alessandro Parolari  3 , Veronika Myasoedova  4 , Giovanni Ferrari  2 , Francesco Alamanni  5 , Elena Tremoli  4 , Paolo Poggio  6
Affiliations

Mitral valve endothelial cells secrete osteoprotegerin during endothelial mesenchymal transition

Paola Songia et al. J Mol Cell Cardiol. 2016 Sep.

Abstract

Aims: Mitral valve prolapse (MVP) has a prevalence of 3% in the general population, affecting >176 million people worldwide. Despite this, little is known about the molecular and cellular mechanisms involved in the progression of MVP and surgical intervention is the only available option. In this study we investigated the role of osteoprotegerin (OPG) during endothelial to mesenchymal transition (EndMT) in MVP.

Methods and results: VECs and VICs were isolated from posterior mitral valve leaflets of patients undergoing mitral valve repair (n=25). Plasma was collected from 57 subjects (29 controls and 28 MVP patients). Overexpression of OPG during EndMT followed by autocrine effects characterised by reactive oxygen species increment and accelerated migration was documented. In addition, OPG increased VIC proliferation. Finally, OPG plasma levels were significantly higher in MVP patients compared to control subjects and the area under the ROC curve was 0.92.

Conclusion: EndMT has been recognised as a possible pathological mechanism for MVP. For the first time, we report the involvement of OPG in cellular and molecular changes in MVP isolated cells. In addition, we detected elevated circulating OPG levels in MVP patients when compared to controls, which supports the hypothesis that OPG is involved in MVP development and progression.

Keywords: Circulating marker; Endothelial to mesenchymal transition; Mitral valve prolapse; Valve endothelial cells; Valve interstitial cells.

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Figures

Figure 1
Figure 1. Human mitral valve endothelial and interstitial cell characterization
(A–B) Phase contrast imaging and immunofluorescence staining for platelet endothelial cell adhesion molecule (CD31 – Red), smooth muscle actin (SMA – Green), vimentin (VIM – Green) and 4′,6-diamidino-2-phenylindole (DAPI, for nuclei detection – Blue), in human isolated valve endothelial (VEC) and interstitial cells (VIC) from mitral valve prolapse patients. Scale bar, 20 μm.
Figure 2
Figure 2. Endothelial to mesenchymal transition of human mitral valve endothelial cells
(A–B) Quantitative PCR (qPCR) of smooth muscle actin (SMA), bone morphogenetic protein 4 (BMP4), collagen I (Col1A1) and collagen III (Col3A1) of valve endothelial cells (VEC) in absence or in presence of β-glycerophosphate and ascorbic acid (βGAA) for 6 or 12 days (n = 3). (C) Flow cytometry analysis (FACS) of SMA for isotype control, untreated VEC, VEC treated with βGAA for 6 days and activated valve interstitial cells (VIC) as a positive control (n=3). (D) Phase contrast imaging of untreated VECs or treated for 12 days with βGAA. Magnification: 20×. * p <0.05.
Figure 3
Figure 3. Osteoprotegerin expression and secretion during endothelial to mesenchymal transition
(A) Western blot of osteoprotegerin (OPG) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as endogenous control, of mitral valve prolapse (MVP) patients and controls subjects (CTRL). (B) Western blot quantification using ImageJ (n=3). * p < 0.05. (C) Quantitative PCR (qPCR) of osteoprotegerin (OPG) of valve endothelial cells (VEC) in absence or in presence of β-glycerophosphate and ascorbic acid (βGAA) for 6 or 12 days (n = 3). (D) OPG enzyme-linked immunosorbent assay (ELISA) on media of untreated and βGAA treated VEC for 6 days (n = 3). * p < 0.05. (E) Digital PCR analysis of the syndecan family (SDC1, 2, 3 and 4) on nine different VEC populations.
Figure 4
Figure 4. Osteoprotegerin treatment on valve endothelial cells
(A) Quantitative PCR (qPCR) of integrins alpha 5 (αv) and beta 3 (β3), (B) collagen I (Col1A1), collagen III (Col3A1), bone morphogenetic protein 4 (BMP4), smooth muscle actin (SMA), fibroblast specific protein 1 (FSP1) and versican (VCAN) of valve endothelial cells (VEC) untreated or treated for 12 days with 50 ng/ml of osteoprotegerin (OPG) (n=5). * p < 0.05; ** p < 0.001; *** p < 0.0001 (C) Total soluble collagen produced by untreated or osteoprotegerin (OPG - 50 ng/ml) treated VECs for 24 and 48 hours (n = 5); * p < 0.05. (D) Gelatin zymography gel and (E) western blot of matrix metalloproteinase 9 (MMP9) in VECs untreated or treated with OPG (50 ng/ml) (n = 3). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as endogenous control. (F) Western blot of phosphorylated syndecan 4 (pSDC4) and syndecan 4 (SDC4) in VECs after OPG treatment for 5 and 15 minutes. (n = 3). (G) Western blot of pSDC4, SDC4, pERK and ERK, in absence or presence of OPG or Heprianse I (Hep I) pre-treatment (5 U/ml) and OPG (n = 3).
Figure 5
Figure 5. Osteoprotegerin induces ROS and cell migration in endothelial cells
(A) Flow cytometry analysis (FACS) of reactive oxygen species (ROS) for untreated or osteoprotegerin (OPG - 50 ng/ml) treated valve endothelial cells (VEC) for 24 hours (n = 3). (B) Bar graph depicting positive cells to SYTOX® (dead cells) after 24 hours treatment with 50 ng/mL of OPG (n = 3). (C) Representative images of wound healing assay for VECs in presence of OPG (50 ng/mL) or 10 % fetal bovine serum (FBS - positive control). (D) Time course representing the percentage of the area without migrating VECs in presence of OPG (50 ng/mL) or 10 % FBS (n = 5). * p < 0.05; ** p < 0.01. (E) Representative images of wound healing assay for VECs in presence of OPG (50 ng/mL) with or without heparinase I pre-treatment (5 U/mL). (F) Bar graph depicting the percentage of migrating VECs in absence or presence of OPG (50 ng/ml) or heparinase I pre-treatment (5 U/mL) after 24 hours in presence of OPG (50 ng/mL) (n = 3). * p < 0.05 vs. untreated; # p < 0.05 vs. OPG treated VECs.
Figure 6
Figure 6. Osteoprotegerin induces proliferation and proteoglycan expression in interstitial cells
(A) Digital PCR (dPCR) analysis of the syndecan family (SDC1, 2, 3 and 4) on three different valve interstitial cells (VIC) populations. (B) Quantitative PCR (qPCR) of bone morphogenetic protein 4 (BMP4), biglycan (BGN), versican (VCAN), metalloproteinase 2 (MMP2), smooth muscle actin (SMA), osteoprotegerin (OPG), collagen I (Col1A1) and collagen III (Col3A1) of VICs left untreated or OPG treated (50 ng/ml) for 6 days (n = 3). * p < 0.05; ** p < 0.01. (C) Total soluble collagen produced by untreated or osteoprotegerin (OPG - 50 ng/ml) treated VICs for 24 and 48 hours (n = 5); * p < 0.05. (D) Proliferation assay of VICs in absence or presence of osteoprotegerin (OPG - 50 ng/mL) for 6 days (n = 3).
Figure 7
Figure 7. Osteoprotegerin plasma levels identify patients with mitral valve prolapse in a surgical patient population independently of age, sex, and common cardiovascular comorbidities
(A) Osteoprotegerin (OPG) enzyme-linked immunosorbent assay (ELISA) on plasma samples from control subjects (Control) or mitral valve prolapse (MVP) patients. (B) Receiver operating characteristic (ROC) curve and area under the curve (AUC) of OPG. (Control subjects n = 29 and Mitral Valve Patients n = 28) *** p < 0.0001.
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