Autotaxin inhibition attenuates the aortic valve calcification by suppressing inflammation-driven fibro-calcific remodeling of valvular interstitial cells

Abstract

Background: Patients with fibro-calcific aortic valve disease (FCAVD) have lipid depositions in their aortic valve that engender a proinflammatory impetus toward fibrosis and calcification and ultimately valve leaflet stenosis. Although the lipoprotein(a)-autotaxin (ATX)-lysophosphatidic acid axis has been suggested as a potential therapeutic target to prevent the development of FCAVD, supportive evidence using ATX inhibitors is lacking. We here evaluated the therapeutic potency of an ATX inhibitor to attenuate valvular calcification in the FCAVD animal models.

Methods: ATX level and activity in healthy participants and patients with FCAVD were analyzed using a bioinformatics approach using the Gene Expression Omnibus datasets, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and western blotting. To evaluate the efficacy of ATX inhibitor, interleukin-1 receptor antagonist-deficient (Il1rn^-/-) mice and cholesterol-enriched diet-induced rabbits were used as the FCAVD models, and primary human valvular interstitial cells (VICs) from patients with calcification were employed.

Results: The global gene expression profiles of the aortic valve tissue of patients with severe FCAVD demonstrated that ATX gene expression was significantly upregulated and correlated with lipid retention (r = 0.96) or fibro-calcific remodeling-related genes (r = 0.77) in comparison to age-matched non-FCAVD controls. Orally available ATX inhibitor, BBT-877, markedly ameliorated the osteogenic differentiation and further mineralization of primary human VICs in vitro. Additionally, ATX inhibition significantly attenuated fibrosis-related factors' production, with a detectable reduction of osteogenesis-related factors, in human VICs. Mechanistically, ATX inhibitor prohibited fibrotic changes in human VICs via both canonical and non-canonical TGF-β signaling, and subsequent induction of CTGF, a key factor in tissue fibrosis. In the in vivo FCAVD model system, ATX inhibitor exposure markedly reduced calcific lesion formation in interleukin-1 receptor antagonist-deficient mice (Il1rn^-/-, P = 0.0210). This inhibition ameliorated the rate of change in the aortic valve area (P = 0.0287) and mean pressure gradient (P = 0.0249) in the FCAVD rabbit model. Moreover, transaortic maximal velocity (Vmax) was diminished with ATX inhibitor administration (mean Vmax = 1.082) compared to vehicle control (mean Vmax = 1.508, P = 0.0221). Importantly, ATX inhibitor administration suppressed the effects of a high-cholesterol diet and vitamin D2-driven fibrosis, in association with a reduction in macrophage infiltration and calcific deposition, in the aortic valves of this rabbit model.

Conclusions: ATX inhibition attenuates the development of FCAVD while protecting against fibrosis and calcification in VICs, suggesting the potential of using ATX inhibitors to treat FCAVD.

Keywords: Aortic valve; Autotaxin; Calcification; Fibro-calcific aortic valve disease; Fibrosis.

Fig. 1

The fibro-calcific remodeling in patients with FCAVD is associated with the ATX-LPA axis. A–D Related genes of lipid retention and fibro-calcific changes were compared between healthy (n = 3) and FCAVD patients (n = 3). A Data were obtained from the GSE77287 dataset and visualized using MeV 4.9.0 heatmap software. B The mRNA expression of genes associated with lipid retention and fibro-calcific changes was compared between aortic valvular tissue of patients with FCAVD and age-matched non-FCAVD controls using a two-tailed t-test. C Pearson correlation values were further analyzed to examine the correlation between ATX (ENPP2) and selected fibro-calcific-related genes with up/up gene pairs exhibiting predominantly positive correlation. D Pearson’s correlation coefficient and linear regression array analysis of the correlation between the enhanced ENPP2 and COL1A1, as well as RUNX2 from GSE77287, GSE83453, and GSE51472 datasets. *P < 0.05, **P < 0.01 versus healthy sample. P values were obtained using a two-tailed t-test

Fig. 2

Attenuation of osteogenic transition and osteogenesis-related gene expression in human VICs cultured from FCAVD patients by treatment with ATX inhibitor. A–F Human VICs were cultured in osteogenic differentiation conditions in the presence of BBT-877 or vehicle control. Effects of BBT-877 treatment (1 μM) on ATX activity (A) and LPA species production (B) from VICs during osteogenic differentiation were measured as described in the “Methods” section. C Alkaline phosphatase (ALP) staining and ALP activity of these cells upon exposure to the indicated concentrations of BBT-877 (0, 0.1, 1, 10 μM) after 2 weeks of osteogenic stimulation. D Alizarin red (AR) staining and calcium deposition of the VICs in the presence (0.1, 1, 10 μM) or absence of BBT-877 after 3 weeks of osteogenic stimulation. E The mRNA expression levels of ALPL, RUNX2, SP7, and BGLAP in the VICs in the presence (0.1, 1, 10 μM) or absence of BBT-877. F The mRNA expression levels of FN1, ITGB1, ACTA2, and COL1A1 in VICs in the presence (0.1, 1, 10 μM) or absence of BBT-877. Data are presented as the mean ± SD of triplicates, and a representative set of findings from more than three independent experiments is presented. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control. P values were obtained using a two-tailed t-test

Fig. 3

Effects of ATX inhibition on the production of cytokines related to the infiltration of immune cells, proinflammatory/anti-inflammatory responses, and fibrosis and osteogenesis in human VICs from FCAVD patients. A–F The human VICs were cultured in an osteogenic medium for 3 weeks in the presence or absence of BBT-877 (1 μM), and the indicated cytokines were measured by ELISA. A Chemokines. B Growth factors for immune cells. C Proinflammatory cytokines. D Anti-inflammatory cytokines. E Cytokines related to osteogenesis. F Cytokines related to fibrosis. Data are presented as the mean ± SD of triplicate ELISA tests. A representative set of results from more than three independent experiments is presented. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control. P values were obtained using a two-tailed t-test

Fig. 4

ATX inhibition of human VICs isolated from FCAVD patients causes the downregulation of the TGF-β pathway and related fibrosis processes. A The protein levels of p-Smad2, p-Smad3, Smad2/3, and GAPDH in VICs treated with TGF-β (5 ng/mL) for 0, 5, 10, 30, or 60 min in the presence or absence of BBT-877 (1 μM). B Quantification of phospho-Smad2 levels relative to the total Smad2/3 concentration. Immunoblots were quantified using ImageJ software. C, D Protein levels of p-p38, p38, p-AKT, AKT, p-ERK, ERK, p-JNK, and JNK in VICs treated with TGF-β (5 ng/mL) for 0, 5, 10, 30, or 60 min in the presence or absence of BBT-877 (1 μM). E, F mRNA expression levels of TGF-β receptor 1 (TGFBR1), TGF-β receptor 2 (TGFBR2), and cellular communication network factor 2 (CTGF, CCN2) in VICs after 24 h of TGF-β (5 ng/mL) stimulation in the presence or absence of BBT-877 (1 μM). G CTGF in VICs-conditioned media after 24 h of TGF-β (5 ng/mL) stimulation in the presence or absence of BBT-877 (1 μM). Data are presented as the mean ± SD and the experiments were performed independently in triplicate. **P < 0.01, ***P < 0.001 versus the vehicle control. P values were obtained using a two-tailed t-test

Fig. 5

Alleviation of calcific lesion formation in a mouse FCAVD model, Il1rn^-/- mice by treatment with the ATX inhibitor. A–F Il1rn^-/- mice were orally administrated with BBT-877 (30 mg/kg/day) or vehicle for 8 weeks. A, B LPA production (A) and osteogenesis-related cytokines (B) in mouse serum were measured using ELISA. C, D Calcific lesion formation was determined using molecular imaging after injection of a fluorescence dye for calcification (Osteosense 680EX). Representative images of mice from each group (C) and quantification of fluorescence intensity (D) in all of the mice in each group. E, F Paraffin-embedded serial cross sections of the aortic valves from mice stained with hematoxylin and eosin (H&E) and analyzed by immunohistochemistry (IHC) and Masson’s trichrome staining (red arrow indicates stained positive area in the aortic valve). Antibodies against ATX and α-SMA were used for IHC analyses. Data are presented as the mean ± SD. *P < 0.05, ** P < 0.01, *** P < 0.001 versus the indicated group. P values were obtained using a two-tailed t-test. WT (n = 3); Il1rn^-/- (n = 5); Il1rn^-/-+BBT-877 (n = 5)

Fig. 6

Attenuation of fibrosis and calcification by treatment with the ATX inhibitor in a rabbit model of FCAVD. A Representative 2D echocardiographic images of the aortic valve cusps in the rabbits at 12 weeks after BBT-877 oral administration. B Standard continuity equation of the aortic valve area (AVA). C AVA, transaortic maximal velocity (Vmax) and mean pressure gradient (mean PG) obtained by continuous-wave Doppler analysis through echocardiographic assessment. D–F Paraffin-embedded serial cross sections of the aortic valves from rabbits stained with hematoxylin and eosin (H&E), alizarin red, and analyzed by immunohistochemistry (IHC) and Masson’s trichrome staining. Antibodies against ATX, α-SMA, and F4/80 were used for IHC analyses. Normal diet (n = 3); Chol+VitD, (cholesterol and vitamin D2, disease group, n = 4); Chol+VitD+BBT, (cholesterol and vitamin D2 with BBT-877, BBT-877 group, n = 4). Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus the vehicle control. P values were obtained using a two-tailed t-test

Fig. 7

Graphical abstract. The proposed mechanism by which ATX inhibition regulates the pathological development of FCAVD is illustrated in the schematic diagram. In diseased valves, an inflammatory environment emerges, causing immune cells such as macrophages to infiltrate the valvular tissue and release inflammatory cytokines (e.g., IL-1β). ① This IL-1β upregulates the expression of LPAR1, which strengthens the action of ATX-LPA. In addition, Lp(a) carrying ATX and LPC accumulates in the valves, while activated VICs secrete additional ATX [12, 31]. ② ATX subsequently converts LPC into LPA, which binds to the increased LPA receptor (LPAR), activating the TGF-β signaling pathway [32]. ③ In turn, this activation promotes fibro-calcific remodeling by inducing the expression of CTGF and IL-6 via the TGF-β signaling axis. Importantly, ATX inhibition specifically hinders the activation of ATX-LPA enhancing TGF-β-linked signaling and ultimately alleviating fibrosis and calcium deposition in the activated VICs