Disruption of mechanical stress in extracellular matrix is related to Stanford type A aortic dissection through down-regulation of Yes-associated protein

Abstract

In this study, we assessed whether the down-regulation of Yes-associated protein (YAP) is involved in the pathogenesis of extracellular matrix (ECM) mechanical stress-induced Stanford type A aortic dissection (STAAD). Human aortic samples were obtained from heart transplantation donors as normal controls and from STAAD patients undergoing surgical replacement of the ascending aorta. Decreased maximum aortic wall velocity, ECM disorders, increased VSMC apoptosis, and YAP down-regulation were identified in STAAD samples. In a mouse model of STAAD, YAP was down-regulated over time during the development of ECM damage, and increased VSMC apoptosis was also observed. YAP knockdown induced VSMC apoptosis under static conditions in vitro, and the change in mechanical stress induced YAP down-regulation and VSMC apoptosis. This study provides evidence that YAP down-regulation caused by the disruption of mechanical stress is associated with the development of STAAD via the induction of apoptosis in aortic VSMCs. As STAAD is among the most elusive and life-threatening vascular diseases, better understanding of the molecular pathogenesis of STAAD is critical to improve clinical outcome.

Keywords: Stanford type A aortic dissection; extracellular matrix; mechanical stress; vascular smooth muscle cell; yes-associated protein.

Figure 1

(A) Intraoperative images and CTA results showing the enlarged ascending aorta and typical true and false cavities in STAAD. (B) Echocardiography showing the Vmax of healthy volunteers and patients with STAAD (including aortic wall systolic velocity (red line), late diastolic retraction velocity (blue line) and early diastolic retraction velocity (green line)). (C) The mean aortic wall systolic velocity of the ascending aorta was significantly lower in patients with STAAD than in healthy volunteers (n=5 in healthy volunteer group, n=5 in STAAD group, *p=0.0417). (D) The mean late-diastolic retraction velocity of the ascending aorta was significantly lower in patients with STAAD than in healthy volunteers (n=5 in Healthy volunteer group, n=5 in STAAD group, *p=0.0478). (E) The mean early-diastolic retraction velocity of the ascending aorta was significantly lower in patients with STAAD than in healthy volunteers (n=5 in healthy volunteer group, n=5 in STAAD group, *p=0.0407). (F) TEM showed partly fragmented and reduplicated elastic lamina and abnormal VSMCs, together with an electron-dense amorphous material peripheral cell membrane in the ascending aortic wall of patients with STAAD, H&E and elastin staining showed obvious ascending aorta tissue structure disorganization in patients with STAAD, and immuno-histochemistry showed that cleaved caspase-3 was present at high levels and bcl-2 was present at low levels in the ascending aortic wall of patients with STAAD relative to that of HTDs. (G, H) Confocal fluorescence microscopy showed that the numbers of double stained (TUNEL and α-SMA) cells was higher in the ascending aortic wall of patients with STAAD (n=19 in HTD group, n=23 in STAAD group, *p=0.0009).

Figure 2

(A) Real-time PCR results showing that YAP was significantly down-regulated in the ascending aortic wall of patients with STAAD compared to HTDs (n=19 in HTD group, n=23 in STAAD group, *p=0.0003). (B, C) Western blotting showed that the expression of total YAP proteins was significantly lower in the ascending aortic wall of patients with STAAD compared to HTDs (n=19 in HTD group, n=23 in STAAD group, *p=0.0019). (D) Confocal fluorescence microscopy showing that YAP and α-SMA double stained cells were present at lower numbers and less quantity in patients with STAAD compared to HTDs. (E) Quantified and statistical analyzed of each patient sample's Western blotting result showed that YAP expression was negatively correlated with the ascending aorta diameter (n=10).

Figure 3

(A) Echocardiographic results showing that the area of the ascending aorta without colorful blood flow in the BAPN model suggesting a false cavity of dissection. (B) Gross examination revealing mural hematomas in the BAPN model, suggesting a false cavity of dissection. (C) H&E and elastin staining showing obvious disorganization of the aortic tissue structure in the BAPN model. (D, E) Confocal fluorescence microscopy showing that the cell number of double stained (TUNEL and α-SMA) cells was significantly more in the BAPN-induced ascending aortic dissection model (n=10 in Sham group, n=10 in BAPN group, *p=0.0335).

Figure 4

(A) Real-time PCR showing that YAP was expressed at significantly lower levels in the ascending aortic wall of the BAPN-induced STAAD mouse compared to that of the sham control (n=10 in Sham group, n=10 in BAPN group, *p=0.0337). (B, C) Western blotting showed that the total YAP protein expression was significantly lower in the ascending aortic wall of the BAPN-induced STAAD mouse compared to that of the sham control (n=10 in Sham group, n=10 in BAPN group, *p=0.0088). (D) Confocal fluorescence microscopy showing that YAP and α-SMA double stained cells were present at lower number and less expression of YAP in the ascending aortic wall of BAPN-induced STAAD mice. (E) Elastin staining of the ascending aortas of mice that were treated with BAPN for different times (1, 2 and 3 weeks) showing that the ascending aortas of mice presented significant elastin disorganization after 3 weeks of BAPN administration compared to mice receiving BAPN for 1 or 2 weeks. (F) Western blotting showing that YAP expression in the ascending aorta was lower after feeding with BAPN for 3 weeks than after feeding with BAPN for 2 weeks.

Figure 5

(A) Western blot showing YAP down-regulation in the PLKO-YAP group compared to the scrambled group. (B, C) Flow cytometry showing increased VSMC apoptosis in the PLKO-YAP group compared to the scrambled group (repeated 3 times for statistical analysis, *p=0.0013). (D) Western blotting showing YAP down-regulation in the experimental group compared to the control group after cyclic stretching. (E, F) Flow cytometry showing increased VSMC apoptosis compared to the control group after cyclic stretching in vitro (the lower right quadrant (Q4) represents the apoptotic VSMCs, and repeated 3 times for statistical analysis, *p=0.0010).

Figure 6

Schematic model showing that the disruption of mechanical stress in ECM induces STAAD and is related to the down-regulation of YAP.