Apoptotic Cell Death in Bicuspid-Aortic-Valve-Associated Aortopathy

Abstract

The bicuspid aortic valve (BAV) is the most common cardiovascular congenital abnormality and is frequently associated with proximal aortopathy. We analyzed the tissues of patients with bicuspid and tricuspid aortic valve (TAV) regarding the protein expression of the receptor for advanced glycation products (RAGE) and its ligands, the advanced glycation end products (AGE), as well as the S100 calcium-binding protein A6 (S100A6). Since S100A6 overexpression attenuates cardiomyocyte apoptosis, we investigated the diverse pathways of apoptosis and autophagic cell death in the human ascending aortic specimen of 57 and 49 patients with BAV and TAV morphology, respectively, to identify differences and explanations for the higher risk of patients with BAV for severe cardiovascular diseases. We found significantly increased levels of RAGE, AGE and S100A6 in the aortic tissue of bicuspid patients which may promote apoptosis via the upregulation of caspase-3 activity. Although increased caspase-3 activity was not detected in BAV patients, increased protein expression of the 48 kDa fragment of vimentin was detected. mTOR as a downstream protein of Akt was significantly higher in patients with BAV, whereas Bcl-2 was increased in patients with TAV, assuming a better protection against apoptosis. The autophagy-related proteins p62 and ERK1/2 were increased in patients with BAV, assuming that cells in bicuspid tissue are more likely to undergo apoptotic cell death leading to changes in the wall and finally to aortopathies. We provide first-hand evidence of increased apoptotic cell death in the aortic tissue of BAV patients which may thus provide an explanation for the increased risk of structural aortic wall deficiency possibly underlying aortic aneurysm formation or acute dissection.

Keywords: aneurysm; bicuspid aortic valve; cell signaling; vascular biology; vascular disease.

Figure 1

Statistical analysis of protein expression levels of RAGE and its ligands AGE and S100A6 in whole-tissue lysates of patients with BAV and TAV from the convex (V) and concave (K) regions of the aorta. (A) Relative RAGE protein expression via Western blot analysis in the tissue of patients with BAV (n = 69) and TAV (n = 59) and separated by regions BAV V (n = 33), BAV K (n = 36), TAV V (n = 30) and TAV K (n = 29). GAPDH served as the loading control. (B) Relative AGE protein expression via Western blot analysis in the tissue of patients with BAV (n = 69) and TAV (n = 59) and separated by regions BAV V (n = 33), BAV K (n = 36), TAV V (n = 30) and TAV K (n = 29). GAPDH served as the loading control. (C) Quantification of S100A6 protein expression using ELISA in the tissue of patients with BAV (n = 57) and TAV (n = 89) and separated by regions BAV V (n = 29), BAV K (n = 28), TAV V (n = 42) and TAV K (n = 47). p values of Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.0001.

Figure 2

Statistical analysis of the protein expression levels of the pro-apoptotic proteins caspase-3, vimentin and α-smooth muscle actin in whole-tissue lysates of patients with BAV and TAV from the convex (V) and concave (K) regions of the aorta. (A) Ratio of protein expression of cleaved active caspase-3 and mature inactive caspase-3 via Western blot analysis in the tissue of patients with BAV (n = 97) and TAV (n = 87) and separated by regions BAV V (n = 50), BAV K (n = 47), TAV V (n = 42) and TAV K (n = 45). α-tubulin served as the loading control. (B) Relative vimentin protein expression (57 kDa/48 kDa) via Western blot analysis in the tissue of patients with BAV (n = 99/n = 96) and TAV (n = 94/n = 93) and 48 kDa vimentin protein expression separated by regions BAV V (n = 48), BAV K (n = 48), TAV V (n = 46) and TAV K (n = 47). GAPDH served as the loading control. (C) Relative α-smooth muscle actin protein expression via Western blot analysis in the tissue of patients with BAV (n = 104) and TAV (n = 86) and separated by regions BAV V (n = 52), BAV K (n = 52), TAV V (n = 44) and TAV K (n = 42). GAPDH served as the loading control. p values of Student’s t-test: * p < 0.05, ** p < 0.01, *** p < 0.0001.

Figure 3

Statistical analysis of protein expression levels of the anti-apoptotic proteins Bcl-2, Akt and mTOR in whole-tissue lysates of patients with BAV and TAV from the convex (V) and concave (K) regions of the aorta. (A) Relative Bcl-2 protein expression via Western blot analysis in the tissue of patients with BAV (n = 98) and TAV (n = 85) and separated by regions BAV V (n = 50), BAV K (n = 48), TAV V (n = 43) and TAV K (n = 42). α-tubulin served as the loading control. (B) Relative Akt protein expression via Western blot analysis in the tissue of patients with BAV (n = 103) and TAV (n = 84) and separated by regions BAV V (n = 53), BAV K (n = 50), TAV V (n = 43) and TAV K (n = 41). GAPDH served as the loading control. (C) Relative mTOR protein expression via Western blot analysis in the tissue of patients with BAV (n = 94) and TAV (n = 95) and separated by regions BAV V (n = 49), BAV K (n = 45), TAV V (n = 50) and TAV K (n = 45). GAPDH served as the loading control. p values of Student’s t-test: * p < 0.05, ** p < 0.01.

Figure 4

Statistical analysis of protein expression levels of LC3, p62 and ERK1/2 in whole-tissue lysates of patients with BAV and TAV from the convex (V) and concave (K) regions of the aorta. (A) Ratio of LC3II/LC3-I protein expression via Western blot analysis in the tissue of patients with BAV (n = 56) and TAV (n = 67) and separated by regions BAV V (n = 27), BAV K (n = 29), TAV V (n = 34) and TAV K (n = 32). α-tubulin served as the loading control. (B) Relative p62 protein expression via Western blot analysis in the tissue of patients with BAV (n = 193) and TAV (n = 94) and separated by regions BAV V (n = 49), BAV K (n = 44), TAV V (n = 49) and TAV K (n = 46). GAPDH served as the loading control. (C) Relative ERK1/2 protein expression via Western blot analysis in the tissue of patients with BAV (n = 93) and TAV (n = 99) and separated by regions BAV V (n = 50), BAV K (n = 43), TAV V (n = 52) and TAV K (n = 47). GAPDH served as the loading control. p values of Student’s t-test: ** p < 0.01, *** p < 0.0001.

Figure 5

Excised aortic specimen. (A) Convexity (V) and concavity (K) region in total. (B) Convexity (V) and concavity (K) region after preparation.