Increased TGFβ1 and SMAD3 Contribute to Age-Related Aortic Valve Calcification

Abstract

Aims: Calcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFβ1 and SMAD3 signaling pathways.

Methods and results: The klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The klotho knockout (Kl ^-/-) mice have shorter life span (8-12 weeks) and develop severe AV calcification. Here, we showed that increased TGFβ1 and TGFβ-dependent SMAD3 signaling were associated with AV calcification in Kl ^-/- mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl ^-/- mice to determine the contribution of TGFβ1 and SMAD3 to the AV calcification in Kl ^-/- mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in Kl ^-/-; Tgfb1 ^± mice compared to Kl ^-/- mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl ^-/- mice compared to the Kl ^-/-; Tgfb1 ^± mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl ^-/-; Tgfb1 ^± and Kl ^-/-; Smad3 ^± mice compared to Kl ^-/- mice. Western blot analysis confirmed that the inhibition of TGFβ canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both Kl ^-/-; Tgfb1 ^± and Kl ^-/-; Smad3 ^± mice.

Conclusion: Overall, inhibition of the TGFβ1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl ^-/- mice. This information is useful in understanding the signaling mechanisms involved in CAVD.

Keywords: CAVD (calcific aortic valve disease); Klotho; Smad3; Tgfb = transforming growth factor beta; aortic valve calcification.

FIGURE 1

The klotho (Kl) genetic deletion leads to calcification in the aortic valve hinge and annulus with upregulation of Tgfb1 and TGFβ-dependent SMAD2 signaling in 10–12-week-old mice. (A–D) Alizarin red staining of wild-type (A) and Kl^–/– (B–D) mice. Scale bars = (A–B) 200 μm; (C) 100 μm; (D) 50 μm. (E–I) Immunohistochemistry showing localized pSMAD2 levels in AV hinge and aortic annulus of wild-type (E,G) and Kl^–/– (F,H). The pSMAD2-stained area was quantified using NIH Image J software (I). (J) qPCR study to quantify Tgfb1 expression in the pooled and micro-dissected tissue samples of AV and annulus from wild-type and Kl^–/– mice. (K) Western blotting analyses showing levels of phosphorylated SMADs (p SMAD2, pSMAD3), total SMADs (SMAD2, SMAD3), and β-actin in micro-dissected and pooled tissue samples containing AV and annulus from wild-type and Kl^–/– mice. (L) Densitometric analysis quantifying band intensities of western blots. The densitometry graphs of pSMAD2 and pSMAD3 were normalized with total or non-phosphorylated form of the proteins or β-actin. Values indicate mean ± SD, and significant “p-values” between wild-type and Kl^–/– groups were given on the top of histograms.

FIGURE 2

Effect of lowering of TGFβ1 and SMAD3 on AV structure and proteoglycan (GAG) distribution in 10- to 12-week-old Kl^–/– mice. (A–D) Hematoxylin and eosin (H&E) staining and (E–H) Alcian blue staining of wild-type, Kl^–/–, Kl^–/–;Tgfb1^±, and Kl^–/–;Smad3^± mice showing aortic valve histology and proteoglycans distribution. A magnified view showing further details of the boxed region is presented for each image. (I–J) Quantitative analysis of AV area in H&E-stained (I) and proteoglycans content in alcian blue-stained (J) images. Quantification of AV area and GAG content was done on multiple serial sections from each animal per group by using NIH- Image J software. Values indicate mean ± SD (n = 5). Scale bars = (A–H) 200 μm.

FIGURE 3

Effect of Tgfb1 and Smad3 heterozygous deletion on collagen and elastin fibers in the aortic valve of 10–12-week-old Kl^–/– mice. (A–H) Verhoeff-Van Gieson (VVG) staining of tissue sections through the aortic valve and annulus of wild-type (A,E), Kl^–/– (B,F), Kl^–/–;Tgfb1^± (C,G), and Kl^–/–;Smad3^± (D,H) mice. Magnified images of the boxed area (A–D) are given in (E–H). Arrows indicate the presence of abnormal elastin fibers in Kl^–/– (F) and Kl^–/–;Tgfb1^± (G) mice. VVG stains elastin in black and collagen fibers in red color. (I) Histogram showing total collagen content in the aortic valve, including leaflets, annulus, and AV hinge. Quantification of collagen fibers content was done by measuring the intensity of collagen staining on multiple serial sections from each animal per group by using NIH-Image J software. Values indicate mean ± SD (n = 5). (J) Histogram showing the average area containing the elastin fibers in the AV hinge. Quantification of elastin fibers content was done by measuring the average area in the AV hinge with elastin staining on multiple serial sections from each animal per group by using NIH-Image J software. Values indicate mean ± SD (n = 5), and significant “p-values” were given on the top of histograms. Scale bars: (A–H) 200 μm.

FIGURE 4

Partial genetic inactivation of Tgfb1 and Smad3 inhibits AV calcification in 10–12-week-old Kl^–/– mice. (A–H) Alizarin red staining of aortic valve in wild-type (A,E), Kl^–/– (B,F), Kl^–/–;Tgfb1^± (C,G), and Kl^–/–;Smad3^± (D,H) mice. Images (E–H) are the magnified views of the corresponding boxed region (A–D). Scale bars = (A–D), 200 μm; (E–F), 50 μm. (I) Quantitative analysis of calcification. Quantification of calcification was done on multiple serial sections from each animal per group by using NIH-Image J software. Values indicate mean ± SD, and significant “p-values” were given on the top of histograms.

FIGURE 5

Effect of partial genetic deletion of Tgfb1 and Smad3 on key genes involved in TGFβ and BMP signaling, and AV calcification. The qPCR analysis of selected genes in micro-dissected pooled tissue samples representing AV leaflets and hinge and aortic annulus of wild-type, Kl^–/–, Kl^–/–;Tgfb1^±, and Kl^–/–;Smad3^± mice. Expression levels of Tgfb1 (TGFβ ligand) (A), Alk2 (BMP Type I receptor) (B), Pai1 (TGFβ target gene) (C), Spp1 (calcification marker) (D), Bmp2 (BMP ligand) (E), and Runx2 (osteoblast differentiation marker) (F) were quantified. Triplicate samples from each of the five different biological replicates (n = 5) were analyzed. Quantitative data are shown as mean ± SD (n = 5). Two-sided Student’s t-test (unpaired) was used. Significant comparisons are indicated on the top of each histogram.

FIGURE 6

Haploinsufficiency of Tgfb1 and Smad3 inhibits canonical and non-canonical TGFβ signaling in the aortic valve of Kl^–/– mice. (A) Western blot analysis on three different micro-dissected pooled tissues, from two to three hearts/sample, representing AV leaflets and hinge area and aortic annulus of 10–12-week-old wild-type, Kl^–/–, Kl^–/–;Tgfb1^±, and Kl^–/–;Smad3^± mice showing protein levels of the phosphorylated forms of canonical and non-canonical pathway molecules (pSMAD2, pSMAD3, pSMAD1/5, pp38, and pERK1/2) and their non-phosphorylated/total forms (SMAD2, SMAD3, SMAD1/5, p38, and ERK1/2). A common and independent β-actin blot (bottom) was also used for normalizing the data. (B) Densitometric quantification of phosphorylated proteins after normalization to total non-phosphorylated proteins or β-actin. Numerical data (mean ± SD) from three pooled samples are presented as scatter plots with bar. The p-values are shown on top of the histograms.

FIGURE 7

The klotho as a mediator of TGFβ1-dependent SMAD3 signaling in AV calcification. SMAD3 can mediate klotho-dependent TGFβ1 signaling. Lowering TGFβ1-dependent SMAD3 signaling also lowers SMAD1/5 signaling. Thus, klotho functions, in part, as a mediator by restraining or attenuating the augmented TGFβ/BMP signaling and TGFβ-regulated transcription of genes involved in CAVD.