Inhibitory role of Notch1 in calcific aortic valve disease

Abstract

Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of Sox9 expression has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism.

Figure 1. Expression of Notch1, Hey1 and Hey2 mRNAs in adult murine aortic valves by radioactive in situ hybridization.

(A) Bright field image of transverse section of 12-week old albino mouse heart. Boxed area is shown in high magnification for (B) Notch1 sense; (C) Notch1 antisense; (D) Hey1 antisense; and (E) Hey2 antisense probes. Ao, aorta; LV, left ventricle. Scale bar represents 20 µm.

Figure 2. Calcific aortic valve disease demonstrates ECM disorganization and VIC disarray.

Representative cross sections from control (A) and diseased (B) aortic valve cusps (3 control and diseased valves were examined). (C) and (D) are higher magnification images of boxed areas in (B). (A) Normal cusps have highly organized stratified ECM with fibrosa (F, yellow), spongiosa (S, blue), and ventricularis (V, black). (B, C) Diseased cusps have disorganized and dispersed ECM with increased collagens (yellow) and proteoglycans (blue) and decreased elastic fibers (black). Aortic valve cusp thickness is increased in diseased valves when compared to control valves (A, B). Calcification deposits are found in diseased valves (arrows, B) along with areas of early mineralization (arrowheads, C) and clusters of VICs that populate the margins of overt calcification (arrow, D). In general, the fibrosa appears expanded and calcification occurs in the arterial aspect of the cusp. The fibrosa is oriented upward in all panels, and the scale bars equal 500 microns in (A, B) and 250 microns in (C, D).

Figure 3. Loss of NOTCH1 expression in proximity to calcific nodules in human aortic valves.

(A) Representative sections from control (A,B) and diseased (C-F) aortic valve cusps. (B) is high magnification image of boxed area in (A) and (D,E) are higher magnification of region in (C) while image in (F) shows another calcified aortic valve. Expression of Notch1 intracellular domain (NICD) is found in the thickened fibrosa of diseased aortic valve (C,E) as compared to the acellular fibrosa of control valves (A,B). However, there is significant loss of NICD expression in cells residing adjacent to calcific nodules (D,F). The fibrosa is oriented upward in all panels, and scale bars equal 100 microns (B,D,E,F are at same magnification). Brown signal represents NICD expression while nuclei are counterstained in blue.

Figure 4. Identification of gene expression changes with inhibition of Notch signaling in rat AVICs.

(A) Schematic showing the establishment of AVIC cultures from rat aortic valves. (B) Rat AVICs treated with DAPT demonstrate downregulation of Notch1 intracellular domain (NICD) when compared to untreated cells (DMSO only). Protein amounts were normalized to anti-α-tubulin. (C) RNA from control and DAPT-treated AVICs cultured for 10 days was extracted and analyzed for differential gene expression using Affymetrix Rat 230 2.0 GeneChip microarrays. Table showing fold downregulation of cartilage-specific genes with DAPT treatment of rat AVICs. In blue, direct transcriptional targets of Sox9; (*) genes with multiple hits. Decreased expression of cartilage-specific genes by qRT-PCR in AVICs with DAPT treatment (D) or when cultured in osteogenic media (OM) versus standard media (SM) (E). Expression of osteopontin (Opn) mRNA was upregulated in OM. *, p value<0.05. Experiments were performed in triplicate, and means and standard deviations are shown.

Figure 5. Notch1 regulates Sox9 expression in vitro.

(A) Relative luciferase activity in COS7 cells transfected with Col2a1 luciferase reporter along with indicated amounts of Sox9 and Notch1 intracellular domain (NICD) expression plasmids. *, p value<0.05 comparing transfection of Sox9 or NICD with empty vector; #, p value<0.05 when comparing transfection of Sox9 and NICD with either Sox9 or NICD alone. (B) Transfection of increasing amounts of NICD expression plasmid upregulates Sox9 mRNA in COS7 cells as quantified by qRT-PCR. (C) Following treatment with DAPT, Sox9 mRNA was downregulated by qRT-PCR in rat AVICs after 10 days of culture. Mean and standard deviations are shown. *, p value<0.05. (D) Immunoblot analysis showing expression of Sox9 in rat AVICs over 21 days of culture treated with DAPT or DMSO (untreated). Protein amounts were normalized using α-tubulin antibodies. Experiments in A-C were performed in triplicate and means and standard deviations are shown. Transfected DNA amounts are in nanograms.

Figure 6. Loss of Notch1 and Sox9 expression is concomitant with increased expression of osteogenic markers in porcine AVIC culture.

(A) Notch1 and (B) Sox9 mRNA levels significantly decrease over a 3-week time course as measured by qRT-PCR while increasing levels of (C) Runx2 and (D) alkaline phosphatase (ALP) mRNA were noted. Weeks of culture are noted on x-axis. Time course studies were performed triplicate and means and standard deviations are shown. *, p value < 0.05, when compared to week 3 levels.

Figure 7. Overexpression of Sox9 rescues calcification that occurs with Notch inhibition.

Porcine AVICs were cultured under four conditions: (A,E,I) untreated, (B,F,J) DAPT-treated, (C,G,K) Sox9 overexpression, and (D,H,L) DAPT-treatment following Sox9 overexpression. Bright field image of unstained cells (A-D), and cells stained with von Kossa (E-H) and Alizarin red (I-L) are shown. AVICs demonstrated calcification with calcific nodules in untreated and DAPT treated cells (A, B, E, F, I, J) but no calcification or nodules were found in Sox9 or DAPT/Sox9 treated cells (C, D, G, H, K, L). Three independent experiments were performed and representative images are shown. (M) Quantification of Alizarin red staining is shown. *, p value<0.05. (N) Schematic showing that Notch1-dependent activation of Sox9 is required for proper expression of valve ECM proteins that are essential for normal adult valve function. Conversely, loss of Notch1 or Sox9 results in dysregulated aortic valve ECM and CAVD.