KPT-330 Prevents Aortic Valve Calcification via a Novel C/EBPβ Signaling Pathway

Abstract

[Figure: see text].

Keywords: heart valves; mass spectrometry; signal transduction; transcription factors; valve disease.

Figure 1.. In vitro model of VIC calcific nodule formation.

(A) pAVICs were plated on glass to stimulate the formation of calcific nodules. Brightfield (i-iii) and Alizarin Red stained images (iv-vi) of pAVICs at Stage I (~5 days), Stage II (~8–9 days), Stage III (~12–14 days). Arrows depict quiescent (Stage I), pre-calcific (Stage II), and calcific nodules (Stage III) (n=3). (B) Graph showing quantification of the area positive for Alizarin Red staining/microscopic field. The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. Quantification was performed using 3 biological replicates, with 3 different field of views from each replicate. P-value by one-way ANOVA = 6.3E-08. Adjusted p-values using post-hoc pairwise analysis: *Stage I vs Stage II 7.7E-06, *Stage I vs Stage III 1.8E-06, ^#Stage II vs Stage III 0.0004. (C) Graph showing area of positive Alkaline Phosphatase (ALP) reactivity/field at Stages I-III (n=3). P-value by one-way ANOVA = 0.0003. Adjusted p-values using post-hoc pairwise analysis: *Stage I vs Stage II 0.004, *Stage I vs Stage III 0.0003, ^#Stage II vs Stage III 0.02. (D) Immunofluorescence images to detect Runx2 (green), Osteopontin (red), and Cadherin-11 (red) protein expression at Stages I and III. DAPI highlight nuclei in blue (n=3). (E) Percent change in MTT assay (absorbance) of pAVICs harvested at Stages I and III (n=3). *P-value using corrected Mann-Whitney: 0.03. (F) Heat map and (G) Venn diagram of RNA-Seq analysis (n=3). (H) Immunofluorescence images to detect Osteomodulin (red) and Sclerostin (green) protein expression in pAVICs fixed at Stages I, II and III (n=3). (I) qPCR analysis of osteogenic markers: Sclerostin (Sost), Osteomodulin, Osteoglycin, and Osteoclast Stimulating Factor 1 (Ostf1) (n=3). P-values for datasets by one-way ANOVA: Sclerostin n/s, Osteomodulin 0.008, Osteoglycin 0.004, Ostf1 0.027. Adjusted p-values using post-hoc pairwise analysis: Sclerostin: Stage I vs Stage II 0.6, Stage I vs Stage III 0.2, ^#Stage II vs Stage III 0.03. Osteomodulin: *Stage I vs Stage II 0.0006, *Stage I vs Stage III 0.01, Stage II vs Stage III 0.09. Osteoglycin : *Stage I vs Stage II 9.4E-05, *Stage I vs Stage III 0.001, Stage II vs Stage III 0.2,. Ostf1: *Stage I vs Stage II 0.004, Stage I vs Stage III 0.5, Stage II vs Stage III 0.09.

Figure 2.. KPT-330 prevents calcific nodule formation and reduces proliferation.

(A) Experimental timeline. (B) Brightfield (i,ii) and Alizarin Red (iii,iv) images from pAVICs cultured on glass and treated with DMSO or 100nM KPT-330 at Stage I. Arrows, Alizarin Red positive nodules (n=3). (C) Quantification of positive Alizarin Red stained area/field in pAVICs treated with 100nM KPT-330 at Stage I (n=3). *P-value using corrected Mann-Whitney: 0.03. The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. (D) Area of positive ALP reactivity in pAVICs cultured in complete media (CM), osteogenic media (OM), or treated with KPT-330 in the presence of OM (n=3). The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. P-values using corrected Mann-Whitney: *CM vs OM 0.03, ^#OM vs OM+KPT 0.03, CM vs OM+KPT 0.3. (E) Immunofluorescence of pAVICs to detect Sclerostin (green) or Osteomodulin (red) protein expression (arrows showing calcified nodules) after DMSO treatment (n=3). (F) Western Blot analysis and densitometric quantitation (G) to confirm Xpo1 knockdown by Xsi compared to Csi (n=3). P-values using corrected Mann-Whitney: *CM+Csi vs OM+Csi 0.03, *CM+Csi vs CM+Xsi 0.03, ^#OM+Csi vs OM+Xsi 0.03. (H) Alizarin Red staining of pAVICs cultured in CM or OM, and transfected with control (Csi) or siRNA against Xpo1 (Xsi) (n=3). (I) Quantification of Alizarin Red stained area/field from H (n=3). The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. P-values using corrected Mann-Whitney: *CM+Csi vs OM+Csi 0.03, *CM+Xsi vs OM+Xsi 0.03, *CM+Csi vs CM+Xsi 0.03, ^#OM+Csi vs OM+Xsi 0.03. (J) Percent change in Caspase-glo 3/7 luminosity (cell death) in pAVICs treated with DMSO or 100nM KPT-330 plated on glass (n=3). P-value using corrected Mann-Whitney 0.1. (K) Percent change in MTT assay performed in pAVICs treated with DMSO or 100nM KPT-330 plated on glass (n=3). *P-value using corrected Mann-Whitney 0.03. (L) Immunofluorescence of pHH₃ expression in pAVICs cultured in OM and treated with DMSO or 100nM KPT-330 (n=3). (M) Quantification of data presented in U, V. *P-value using corrected Mann-Whitney: 0.03.

Figure 3.. KPT-330 attenuates and mitigates nodule formation in vitro.

(A) Schematic of the experimental timeline to show pAVICs plated on glass and treated with KPT-330 at Stage II. (B) Brightfield (i,ii) and Alizarin Red (iii,iv) images from DMSO or 100nM KPT-330 treated cells at Stage II, plated on glass. Arrows indicate calcific nodules (n=3). (C) Schematic of the experimental timeline utilized in D to show pAVICs plated on glass and treated with KPT-330 at Stage III. (D) Brightfield (i,ii) and Alizarin Red (iii,iv) images from DMSO or KPT-330 treated cells at Stage III, plated on glass (n=6). (E) Quantification of Alizarin red positive area/field in pAVICs before and after KPT-330 treatment (post 72 hours) (n=6). *P-value using corrected Mann-Whitney: 0.0004. (F) qPCR data to show changes in expression of osteogenic markers (Sost, Osteomodulin, Osteoglycin, and Ostf1) between Stage III and Stage III+KPT-330 (n=3). *P-value using corrected Mann-Whitney: Sost 0.03, Osteomodulin 0.03, Osteoglycin 0.03, Osteoclast Stimulating Factor 1 0.03. (G) Schematic of the experimental timeline and accompanying Alizarin Red staining H (n=1). (H) Alizarin Red images of pAVICs from KPT-330 added at Stage III (i) and replated after 72 hours with DMSO treatment (ii) or KPT-330 treatment (iii).

Figure 4.. KPT-330 prevents aortic valve calcification in Klotho^−/− mice.

(A) Alizarin Red staining of pAVICs treated with no phosphate (Pi) (i,v), 1mM Pi (ii,vi) 2.5mM Pi (iii,vii) and 6mM Pi (iv,viii) in the absence (i-iv) and presence of 100nM KPT-330 (v-viii) for 4–5 days. (B) Quantification of positive Alizarin Red stained area/field in untreated and treated pAVICs, *indicates comparison to indicated Pi concentration, # indicates comparison to DMSO-treated at the same Pi concentration (n=3). The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. P-values using corrected Mann-Whitney: *No Pi vs 2.5 mM Pi 0.03, *No Pi vs 6 mM Pi 0.03, *1 mM Pi vs 2.5 mM Pi 0.03, *2.5 mM Pi vs 6 mM Pi 0.03, ^#2.5 mM Pi vs 2.5 mM Pi+KPT 0.03, ^#6 mM Pi vs 6 mM Pi+KPT 0.03. (C) Percent change in MTT assay of untreated and treated pAVICs, *indicates comparison to indicated Pi concentration, ^#indicates comparison to DMSO-treated at the same Pi concentration (n=3). *P-values using corrected Mann-Whitney: No Pi vs No Pi+KPT 0.03, 1 mM Pi vs 1 mM Pi+KPT 0.03, 2.5 mM Pi vs 2.5 mM Pi+KPT 0.03, 6 mM Pi vs 6 mM Pi+KPT 0.03, No Pi+KPT vs 2.5 mM Pi+KPT 0.03, No Pi+KPT vs 6 mM Pi+KPT 0.03. (D) Schematic showing experimental treatment design in Klotho^−/− mice. (E) Low and high magnification of Alizarin Red stained tissue sections from Klotho^−/− mice treated with vehicle. Arrows show presence of annular calcification and arrow heads show vascular calcification. (F) Low and high magnification of Alizarin Red tissue sections from Klotho^−/− mice treated with 30 mg/kg KPT-330. Arrows show absence of annular calcification and arrow heads show vascular calcification (n=6). (G) Quantification of positive Alizarin Red stained area/field within the aortic valve annular region of vehicle and KPT-330 treated Klotho^−/− mice (n=5). *P-value using corrected Mann-Whitney: 0.002. The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J.

Figure 5.. KPT-330 prevents the nuclear export of C/EBPβ in VICs.

C/EBPβ protein intensity from Mass Spectrometry at Stages I vs III (A), Stages III vs Stage I+KPT-330 (prevention) (B) and Stages III vs Stage III+KPT-330 (treatment) (C) n=3. *P-value using corrected Mann-Whitney: Stage I vs Stage III 0.03, Stage III vs Stage I 0.03, Stage III vs Stage III+ KPT 0.03. (D) Western Blot analysis and quantitation (E) of C/EBPβ in cytoplasmic and nuclear lysates from pAVICs cultured on glass and harvested at Stages I (n=3), III (n=5), and III+KPT (n=5). *P-value using corrected Mann-Whitney: 0.08. (F) Western Blot analysis and quantitation (G) of C/EBPβ in cytoplasmic and nuclear lysates from pAVICs cultured in complete media (CM), osteogenic media (OM) or OM + 100nM KPT-330 (n=5). *P-value using corrected Mann-Whitney: 0.006. (H) Western Blot analysis and quantitation (I) of C/EBPβ in nuclear lysates from pAVICs cultured in 2.5mM Phosphate (Pi) in the absence and presence of 100nM KPT-330 (n=3). *P-value using corrected Mann-Whitney: 0.03. (J) Western Blot of nuclear/cytoplasmic fractions of pAVICs in OM, or OM + 100nM KPT-330 with DMSO or MG132 (n=3). (K) Quantification of data presented in (J). P-value using corrected Mann-Whitney: DMSO vs KPT 0.13, DMSO+MG132 vs KPT+MG132 0.2, DMSO vs DMSO+MG132 0.03, KPT vs KPT+MG132 0.3. Molecular weights (MW) are indicated in kDa.

Figure 6.. C/EBPβ overexpression prevents calcification of VICs.

(A) Western Blot analysis and densitometric analysis (B) of Western Blot from pAVICs transfected with pcDNA3-C/EBPβ (pC/EBPβ) or empty pcDNA3 (pEmpty) (n=3). *P-value using corrected Mann-Whitney: 0.03. (C) Immunofluorescence and quantitation (D) of nuclear/cytoplasmic localization of C/EBPβ in pAVICs following transfection with pC/EBPβ or empty pcDNA3 in complete media (CM), (n=3). *P-value using corrected Mann-Whitney: empty pcDNA3 vs pC/EBPβ cytoplasmic 0.03, empty pcDNA3 vs pC/EBPβ nuclear 0.03, empty pcDNA3 vs pC/EBPβ cytoplasmic+nuclear 0.03. (E) Alizarin Red stained images and quantification (F) from pAVICs cultured in OM and transfected with pEmpty or pC/EBPβ (n=3). *P-value using corrected Mann-Whitney: 0.03. The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. (G) Western Blot and densitometric analysis (H) of C/EBPβ levels from pAVICs treated with recombinant C/EBPβ protein (rC/EBPβ) (n=3). *P-value using corrected Mann-Whitney: CM vs 50 0.03, CM vs 100 0.03, CM vs 200 0.03. (I) Alizarin Red images and quantification (J) of positive Alizarin red stained area/field of pAVICs cultured in OM alone, or OM + rC/EBPβ (n=3). *P-value using corrected Mann-Whitney: 0.03. The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. Molecular weights (MW) are indicated in kDa.

Figure 7.. KPT-330 represses canonical Wnt signaling in pAVICs.

(A) RPKM values obtained from RNA-Seq data for Wnt signaling mediator and regulatory genes, *indicates comparison to previous time point, ^#indicates comparison to Stage III (Wnt activators or downstream effectors – Lrp5, Wnt10b, Lef1, Wisp1, Wisp2; Wnt inhibitors- Wif1, Axin1) (n=3). P-values: ≤ 0.01–0.05. (B) Western Blot analysis and quantitation (C) of β-catenin expression in pAVICs harvested at Stage I, or Stage III in the presence and absence of 100nM KPT-330 (n=3). P-value using corrected Mann-Whitney: *Stage I vs Stage III 0.03, ^#Stage III vs Stage III+KPT 0.03, *Stage I vs Stage III + KPT 0.03. (D) Western Blot to show active β-catenin expression in whole pAVIC lysates cultured in complete media (CM), osteogenic media (OM), or OM in the presence of KPT-330. (E) Quantitation of Western Blot shown in (D) (n=3). *P-value using corrected Mann-Whitney: 0.07. (F) Luminescence as %RLU/mg in pAVICs transfected with TOP/FOP Flash plasmids and Renilla plasmid treated with DMSO or 100nM KPT-330 in OM. LiCl treatment was performed for 24 hours to activate Wnt signaling, *indicates comparison to TOP-Flash, ^#indicates comparison to DMSO (n=3). *P-value using corrected Mann-Whitney: OM vs OM+KPT (TOP-Flash) 0.03, OM (FOP-Flash) vs OM (TOP-Flash) 0.03, OM+KPT (FOP-Flash) vs OM+KPT (TOP-Flash) 0.03. (G) Immunofluorescent images of aortic valve annulus (highlighted) stained with β-catenin (red) and DAPI (blue) from Klotho^−/− mice treated with vehicle or KPT-330. (H) Quantitation of β-catenin immunoreactivity (CTCF unit) in the annulus region of Klotho^−/− mice treated with vehicle or KPT-330 (n=3). *P-value using corrected Mann-Whitney: 0.03. The Y axis indicates calibrated arbitrary units of area based on selected montages in Image J. (I) Western blot and densitometric analysis (J) of nuclear β-catenin expression in pAVICS treated with 2.5mM Pi in the absence and presence of 100nM KPT-330 (n=3). *P-value using corrected Mann-Whitney: 0.03. Molecular weights (MW) are indicated in kDa.

Figure 8.. C/EBPβ overexpression represses canonical Wnt signaling in pAVICs.

(A) Western Blot to show active β-catenin expression in whole pAVIC lysates cultured in OM, and in the presence of 400ng/μl rC/EBPβ (n=2). (B) Quantitation of Western Blot shown in (A). (C) Western blot and densitometric analysis (D) of Axin1 from pAVICs transfected with pcDNA3-C/EBPβ (pC/EBPβ) or empty pcDNA3 (n=3). *P-value using corrected Mann-Whitney: 0.03. (E) Luminescence as %RLU/mg in pAVICs transfected with pC/EBPβ or empty pcDNA controls, along with Axin1-Luciferase plasmid, in presence of DMSO or 100nM KPT-330 (n=3). *P-value using corrected Mann-Whitney: empty pcDNA3+DMSO vs empty pcDNA3+KPT 0.03, empty pcDNA3+DMSO vs pC/EBPβ+DMSO 0.03, empty pcDNA3+KPT vs pC/EBPβ+KPT 0.03, pcDNA3+DMSO vs pC/EBPβ+KPT 0.2. (F) Alizarin Red images and quantification (G) of positive Alizarin Red stained area/field in pAVICs cultured in OM or OM with XAV-939 (XAV) (n=3). *P-value using corrected Mann-Whitney: 0.03. (H) Immunofluorescence staining images and quantitation (I) of pHH₃ positive pAVICs cultured in OM or OM with XAV-939 (n=3). *P-value using corrected Mann-Whitney: 0.03.