OxLDL-derived lysophosphatidic acid promotes the progression of aortic valve stenosis through a LPAR1-RhoA-NF-κB pathway

Abstract

Aims: Oxidatively modified lipoproteins may promote the development/progression of calcific aortic valve stenosis (CAVS). Oxidative transformation of low-density lipoprotein (OxLDL) generates lysophosphatidic acid (LPA), a lipid mediator that accumulates in mineralized aortic valves. LPA activates at least six different G protein-coupled receptors, which may play a role in the pathophysiology of CAVS. We hypothesized that LPA derived from OxLDL may promote a NF-κB signature that drives osteogenesis in the aortic valve.

Methods and results: The role of OxLDL-LPA was examined in isolated valve interstitial cells (VICs) and the molecular pathway was validated in human explanted aortic valves and in a mouse model of CAVS. We found that OxLDL-LPA promoted the mineralization and osteogenic transition of VICs through LPAR1 and the activation of a RhoA-NF-κB pathway. Specifically, we identified that RhoA/ROCK activated IκB kinase alpha, which promoted the phosphorylation of p65 on serine 536 (p65 pS536). p65 pS536 was recruited to the BMP2 promoter and directed an osteogenic program not responsive to the control exerted by the inhibitor of kappa B. In LDLR-/-/ApoB100/100/IGFII transgenic mice (IGFII), which develop CAVS under a high-fat and high-sucrose diet the administration of Ki16425, a Lpar1 blocker, reduced by three-fold the progression rate of CAVS and also decreased the osteogenic activity as measured with a near-infrared fluorescent probe that recognizes hydroxyapatite of calcium.

Conclusions: OxLDL-LPA promotes an osteogenic program in the aortic valve through a LPAR1-RhoA/ROCK-p65 pS536 pathway. LPAR1 may represent a suitable target to prevent the progression of CAVS.

Keywords: BMP2; Bone morphogenetic protein 2; Calcific aortic valve disease; Calcific aortic valve stenosis; Calcification; LPAR1; Lysophosphatidic acid; Mineralization; NF-κB; Osteogenic program; OxLDL; RhoA; Valve interstitial cells; p65 serine 536.

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

Figures

Figure 1

LPA-mediated VIC mineralization depends on BMP2. (A) Treatment with Ki16425 prevents OxLDL-induced mineralization of VICs (n = 6) (calcium measured after 7 days). (B) VICs were treated with mineralizing medium in presence of different LPA species (n = 6). (C) VICs were treated with mineralizing medium in presence of increasing LPA 18:1 concentration (n = 6). (D–H) RUNX2, BGLAP, COL1A1, BMP2 genes (n = 6) and ALP activity (n = 6) were increased in VICs following LPA treatment (7 days). (I) Treatment with Noggin inhibits LPA-induced mineralization (n = 6). (J) BMP2 siRNA abrogates LPA-induced mineralization of VICs (n = 6). Values are mean ± SEM. OxLDL: 100 ng/mL, Ki16425: 10 µM, LPA: 10 µM, Noggin: 2.5 µM. * P < 0.05.

Figure 2

LPAR1 mediates LPA response in VICs. (A) LPAR1 expression in VICs (n = 6). (B) Confocal images of LPAR1 in VICs. Scale bar 20 µM, (n = 5). (C) LPAR1 is required for OxLDL and LPA induced mineralization of VICs (n = 6). (D–G) siLPAR1 prevented LPA-mediated rise of BMP2 (D), RUNX2 (E), BGLAP (F) and COL1A1 (G) (n = 6) (measurements at 24 h). (H) LPAR1 mRNA measurement in control non-mineralized (CTL) (n = 31) vs. calcified aortic valves (CAVS) (n = 40). (I) Representative western blot and quantification of LPAR1 in CTL (n = 8) vs. CAVS (n = 8). (J) Epifluorescence image of a calcified aortic valve showing the organization of the tissue in DAPI, scale bar 1000 µM and confocal images showing LPAR1 and vimentin co-expression in the same tissue, scale bar 10 µM (n = 10). (K–M) ROCK activity measurements; kinetics following LPA treatment (n = 6) (K), siLPAR1 negated LPA response (n = 6) (L) and Y27632 abrogated LPA effect (n = 5) (M). (N) Treatment with Y27632 reduces LPA-mediated mineralization in VICs (n = 6). Values are mean ± SEM. LPA: 10 µM, OxLDL: 100 ng/mL, Y27632: 5 µM; * P < 0.05.

Figure 3

LPA-induced VICs mineralization relies on the NF-κB pathway. (A and B) NF-κB reporter assay showing increased NF-κB activity in response to LPA (n = 4) (A) and its inhibition by BAY11-7085 (n = 6) (B). (C) BAY11-7085 prevents LPA-induced mineralization (n = 6). (D–F) NF-κB reporter assay; Y27632 (n = 6) (D) and RhoA N19 (n = 6) (E) inhibits and RhoA L63 (n = 6) (F) mimics LPA effect on NF-κB activity. (G and H) IL-6 mRNA measurements in response to LPA, Y27632 (n = 6) (G) and siLPAR1 (n = 6) (H) decreases LPA-induced IL-6 rise. Values are mean ± SEM. LPA: 10 µM, BAY11-7085: 5 µM, Y27632: 5 µM; * P < 0.05.

Figure 4

LPA-mediated activation of the NF-κB pathway promotes BMP2 expression. (A and B) LPA increases BMP2 promoter activity (n = 4) (A) and BMP2 protein level (n = 6) (B). (C) RhoA N19 inhibits LPA-induced BMP2 promoter activity (n = 6). (D) Y27632 (n = 6) and BAY11-7085 (n = 6) abrogates LPA-mediated rise in BMP2 mRNA. (E and F) p65 is required for LPA regulation of BMP2 level (n = 6) (E) and mineralization (n = 6) (F). Values are mean ± SEM. LPA: 10 µM, Y27632: 5 µM, BAY11-7085; * P < 0.05.

Figure 5

LPA-mediated activation of the NF-κB pathway regulates BMP2 promoter activity. (A) Scheme depicting NRE sites localization in the BMP2 promoter. (B and C) ChIP assays; p65 binds to BMP2 (B) and IL6 (C) promoters in response to LPA (n = 6). (D and E) Western blots on nuclear and cytoplasmic fractions demonstrating the efficiency of leptomycinB (LepB) treatment (D); quantification of WB (n = 4) (E). (F–H) LepB inhibits LPA-mediated rise of IL6 (F) but not of IL8 (G) nor BMP2 (H) (n = 6). (I–K) IκBα super repressor (SS32/36AA) does not block LPA effect on BMP2 (I) and IL8 (J), but blocks its effect on IL6 (K) (n = 6). (L) IκBα SS32/36AA has no effect on LPA-induced mineralization of VICs (n = 6).Values are mean ± SEM. LPA: 10 µM, Leptomycin B: 20 nM; * P < 0.05.

Figure 6

LPA-mediated activation of BMP2 relies on phosphorylation of p65 S536. (A–C) P65 S536A inhibits LPA-mediated rise in BMP2 promoter activity (n = 6) (A) and mineralization (n = 6) (B), while p65 S536E mimics the effect of LPA (n = 6) (C). (D–E) ChIP assays; p65 phosphoS536 binds to BMP2 (D), but not to IL6 (E) promoters in response to LPA (n = 6). (F) LPA induces p65 phosphorylation on S536 (n = 4). (G) Y27632 blocks LPA-induced p65 S536 phosphorylation (n = 4). (H) Representative western blot showing that p65 phosphoS536 is increased in CAVS (n = 12) vs. control (n = 10) tissues. (I) p65 S536 phosphorylation relies on IKKα (n = 4). (J) LPA-mediated IKKα phosphorylation is abrogated by Y27632 (n = 6). Values are mean ± SEM. LPA: 10 µM; * P < 0.05.

Figure 7

Role of the ATX-LPAR1 pathway in vivo. (A) LPA is increased in IGFII mice serum (n = 10). (B–C) LPA level correlates with blood plasma ATX activity (B) and cholesterol level (C) (n = 10). (D–E) Lpar1 is elevated in IGFII mice (D) and it correlates with Bmp2 level (E) (n = 8). (F) Outline of the animal protocol. (G and H) Transaortic velocities (V2) were increased in mice receiving vehicle (G) (n = 12) while they were stable in mice receiving Ki16425 (H) (n = 11). (I) ΔV2 was significantly higher in mice receiving vehicle compared with Ki16425 (n = 23). (J) ΔLVFS was significantly lower in mice receiving vehicle compared with Ki16425 (n = 23). (K) Hydroxyapatite was more abundant in leaflets of mice receiving vehicle compared with Ki16425 (n = 13), scale bar 200 µM. Values are mean ± SEM. Ki16425: 5 mg/kg/day.