Platelet membrane-coated alterbrassicene A nanoparticle inhibits calcification of the aortic valve by suppressing phosphorylation P65 NF-κB

Abstract

Rationale: Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and morbidity with increasing prevalence and incidence. The pathobiology of CAVD involves valvular fibrocalcification, and osteogenic and fibrogenic activities are elevated in aortic valve interstitial cells (VICs) from diseased valves. It has been demonstrated that activated NF-κB pathway was present in the early stage of CAVD process. There is currently no effective clinical drugs targeting NF-κB pathway for CAVD treatment. Therefore, it is of great clinical significance to seek effective treatments for valve calcification. Methods: In this study, we established immortal human valve interstitial cells (im-hVICs) with pGMLV-SV40T-puro lentivirus. Alizarin red staining and western blotting were performed to evaluate the calcification of immortal VICs supplemented with different compounds. The natural fusicoccane diterpenoid alterbrassicene A (ABA) was found to have potential therapeutic functions. Ribonucleic acid sequencing was used to identify the potential target of ABA. Platelet membrane-coated nanoparticle of ABA (PNP-ABA) was fabricated and the IBIDI pump was used to evaluate the adhesion ability of PNP-ABA. Murine wire-induced aortic valve stenosis model was conducted for in vivo study of PNP-ABA. Results: The natural fusicoccane diterpenoid ABA was found to significantly reduce the calcification of human VICs during osteogenic induction via inhibiting the phosphorylation P65. Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein-2 (BMP2) were down regulated with the treatment of ABA in human VICs. Additionally, molecular docking results revealed that ABA bound to RelA (P65) protein. Phosphorylation of P65 (Ser536) was alleviated by ABA treatment, as well as the nuclear translocation of P65 during osteogenic induction in human VICs. Alizarin red staining showed that ABA inhibited osteogenic differentiation of VICs in a dose-dependent manner. PNP-ABA attenuated aortic valve calcification in murine wire-induced aortic valve stenosis model in vivo. Conclusions: The establishment of im-hVICs provides a convenient cell line for the study of CAVD. Moreover, our current research highlights a novel natural compound, ABA, as a promising candidate to prevent the progression of CAVD.

Keywords: NF-κB; calcific aortic valve disease; immortalization; nanoparticle; natural product.

Figure 1

Immortal human VICs function similarly to primary human VICs. (A) The flow chart for establishment of immortal human VICs (im-hVICs); (B, C) Western blotting (WB) analysis and WB quantification of Runx2, BMP2 and OCN protein expression at day 5 of osteogenic induction in both im-hVICs and p-hVICs (n = 6 per group. Data are presented as the mean ± SEM, *P<0.05, **P < 0.01, ***P < 0.001); (D) Immunofluorescent staining was used to examine p-hVICs marker vimentin in both im-hVICs and p-hVICs, and representative crystal violet staining of clone formation is shown (scale bar: 50 µm); (E) Im-hVICs and p-hVICs were stained with alizarin red after osteogenic induction (scale bar: 20 µm).

Figure 2

ABA is identified as a potential anticalcific compound. (A) Ten different compounds (10 µM) were screened by evaluating the effect of anti-calcification treatment with stained alizarin red (scale bar: 20 µm); (B, C) WB analysis and WB quantification of Runx2 and BMP2 protein levels after ten different compounds (10 µM) used in OM-cultured VICs (n = 5 per group. Data are presented as the mean ± SEM, *P<0.05); (D) The chemical structure of compound 6; (E) The effects of the indicated compounds on cell viability were examined in im-hVICs.

Figure 3

ABA induced anticalcific response in im-hVICs involved NF-κB pathway activation. (A) The Venn diagram displays the number of regulated genes shared between ABA compounds and OM groups in upregulation (Red) and downregulation (Blue); (B) KEGG pathway analysis of genes regulated by ABA compounds; (C) Heatmap representing the selected differentially expressed genes (DEGs) across all three groups of samples; (D) ABA docked with P65 and P52; (E) Results of molecular docking score.

Figure 4

ABA reduces the phosphorylation of RelA. (A, B, C) WB analysis and quantification of Runx2, BMP2, P65, P52 and phosphorylation of P65 proteins levels after ABA compounds used in TNF-α cultured im-hVICs (n = 4 per group. Data are presented as the mean ± SEM, ns indicates no significant, *P<0.05, **P < 0.01); (D) The dose-dependent treatment effect of ABA compound was detected by Alizarin Red (scale bar: 20 µm); (E) The alizarin red positive area were quantified in im-hVICs (n = 6 per group. Data are presented as the mean ± SEM, *P<0.05, ***P < 0.001); (F) Immunofluorescence analysis of p65 nuclear translocation (scale bar: 50 µm); (G) The quantification of immunofluorescence analysis. (n = 6 per group. Data are presented as the mean ± SEM, **P < 0.01)

Figure 5

Platelet membrane-coated ABA can better target mVICs. (A) Fabrication process of the PNP-ABA; (B, C) Scanning electron microscope images and size distribution of PNP and PNP-ABA samples (scale bar: 100 nm); (D) WB analysis of PNP and PNP-ABA for characteristic platelet membrane markers; (E) Photograph of the mice heart tissue and valve sample is from left to right. The isolated mVICs were stained with CD31, Vimentin and DAPI (scale bar: 20 µm); (F) The IBIDI instrument was used to compare the extent of binding between ABA and PNP-ABA in mVICs; (G) The remaining compounds were detected and quantified. (n = 6 per group. Data are presented as the mean ± SEM, *P<0.05)

Figure 6

ABA attenuated calcification in murine wire-induced aortic valve stenosis model. (A) Schematic illustrations of wire-induced aortic valve stenosis model; (B) The diagram of the steps of the experiment procedure; (C) Results of echocardiogram in control and PNP-ABA injected groups; (D, E) After ABA and PNP-ABA injected, the concentration of ABA was detected in mouse aortic root in 24 h (n = 6 per group. Data are presented as the mean ± SEM, **P < 0.01); (F) The degrees of calcification were measured by Von Kossa staining (scale bar: 100 µm); (G) Quantitative assessment of peak velocity by echocardiography; (H) The quantification of Von Kossa results; (I) The pP65 and Runx2 was detected by immunofluorescence combined with DAPI staining for nuclei in control and PNP-ABA groups (scale bar: 100 µm). (G, H, J) n = 7 per group. Data are presented as the mean ± SEM, *P<0.05.