Methamphetamine induces thoracic aortic aneurysm/dissection through C/EBPβ

Abstract

Aims: Thoracic aortic aneurysm/dissection (TAAD) is a life-threatening disease with diverse clinical manifestations. Although the association between methamphetamine (METH) and TAAD is frequently observed, the causal relationship between METH abuse and aortic aneurysm/dissection has not been established. This study was designed to determine if METH causes aortic aneurysm/dissection and delineate the underlying mechanism.

Methods and results: A new TAAD model was developed by exposing METH to SD rats pre-treated with lysyl oxidase inhibitor β-aminopropionitrile (BAPN). Combination of METH and BAPN caused thoracic aortic aneurysm/dissection in 60% of rats. BAPN+METH significantly increased the expression and activities of both matrix metalloproteinase MMP2 and MMP9, consistent with the severe elastin breakage and dissection. Mechanistically, METH increased CCAAT-enhancer binding protein β (C/EBPβ) expression by enhancing mothers against decapentaplegic homolog 3 (Smad3) and extracellular regulated protein kinase (ERK1/2) signaling. METH also promoted C/EBPβ binding to MMP2 and MMP9 promoters. Blocking C/EBPβ significantly attenuated METH+BAPN-induced TAAD and MMP2/MMP9 expression. Moreover, BAPN+METH promoted aortic medial smooth muscle cell (SMC) apoptosis through C/EBPβ-mediated IGFBP5/p53/PUMA signaling pathways. More importantly, the expression of C/EBPβ, MMP2/MMP9, and apoptosis-promoting proteins was increased in the aorta of human patients with thoracic aortic dissection, suggesting that the mechanisms identified in animal study could be relevant to human disease.

Conclusions: Our study demonstrated that METH exposure has a casual effect on TAAD. C/EBPβ mediates METH-introduced TAAD formation by causing elastin breakage, medial cell loss and degeneration. Therefore, C/EBPβ may be a potential factor for TAAD clinical diagnosis or treatment.

Keywords: Apoptosis; C/EBPβ; MMP2/9; Methamphetamine; Thoracic aortic aneurysm/dissection.

Figure 1.

METH exposure induced thoracic aortic aneurysm/dissection (TAAD) in BAPN-preconditioned SD rats. (A) Flowchart of animal drug delivery. (B) METH and/or BAPN treatment promoted the formation of TAAD in rat aorta. Red arrows indicate the TAAD in BAPN+METH-treated aorta. (C-D) Prevalence of TAAD in different groups. White area in BAPN+METH group in D denotes the TAAD with aortic rupture. The probability value was adjusted with the Bonferroni method for pairwise comparisons (^▲p<0.05, n=3). (E) H&E and EVG staining of aorta sections from the rectangle areas shown in B. Dissection and elastin fragmentation were observed in METH-treated rats with BAPN-pretreatment.

Figure 2.

METH with BAPN-pretreatment increased MMP9/MMP2 production and activities in rat aorta tissues. (A) Western blot analyses of MMP9 and MMP2 protein expression in aortic tissues from rats treated with vehicle, BAPN, METH, or BAPN+METH. (B-C) Quantification of MMP9 and MMP2 expression by normalized to β-actin (*p<0.05, **p<0.01, ***p<0.005, n=3). (D) Immunostaining of MMP9 in rat aortic tissues exposed to BAPN and/or METH. (E) MMP9 and MMP2 activities in rat aortic tissues were detected by Gelatin-zymography electrophoresis. (F-G) Quantitative analyses of MMP9 and MMP2 activities by normalizing to pro-MMP9 and -MMP2, respectively (**p<0.01, ***p<0.005, n=3). All data were analyzed by factorial design ANOVA followed by LSD post hoc analyses.

Figure 3.

C/EBPβ mediated METH-induced MMP9/MMP2 expression in aorta SMCs. (A) C/EBPβ expression in rat aorta with BAPN and/or METH treatment was examined by Western blot. (B) Quantification of C/EBPβ expression shown in A by normalized to β-actin. (*p<0.05, factorial design ANOVA followed by LSD post hoc analyses, n=3). (C) C/EBPβ expression in rat aorta was detected by IF staining. Arrows indicate SMCs with positive fluorescent signaling. (D) Aortic SMCs were transfected with control (Ctrl siRNA) or C/EBPβ siRNA (siC/EBPβ) and then treated with vehicle or 1.0 mM METH as indicated. C/EBPβ, MMP9, and MMP2 protein expression were detected by Western blot. (E-G) Quantification of protein expression shown in D by normalized to β-actin (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001, one-way ANOVA followed by LSD post hoc analyses, n=3). (H-I) ChIP assay detected C/EBPβ binding to MMP9 and MMP2 promoters in a chromatin setting. (J-K) C/EBPβ binding enrichment on MMP9 and MMP2 promoters, respectively (***p<0.005, Student’s t test, n=3).

Figure 4.

METH regulated C/EBPβ expression through Smad3 and ERK signaling. (A) Smad3 and ERK phosphorylation in aorta of rats treated with BAPN and/or METH were detected by Western blot. (B-C) Quantification of p-Smad3 and p-ERK levels shown in A by normalized to total Smad3 and ERK, respectively (*p<0.05, **p<0.01, ***p<0.005, factorial design ANOVA followed by LSD post hoc analyses, n=3). (D-G) Aortic SMCs were treated with Smad3 inhibitor SIS3 or ERK inhibitor U0126 for 30 min followed by vehicle or METH treatment for 8 hours as indicated. p-SMAD3, p-ERK1/2, and C/EBPβ levels were measured by Western blot. (E-F & H-I) Quantification of p-SMAD3, p-ERK and C/EBPβ levels shown in D or G, respectively (**p<0.01, ***p<0.005, ****p<0.001, one-way ANOVA followed by LSD post hoc analyses, n=3).

Figure 5.

Silencing C/EBPβ inhibited BAPN+METH-induced TAAD and MMP9/2 production in rat aortas. Rats were treated with METH+BAPN along with adenoviral delivery of AD-shGFP or AD-shC/EBPβ as described in methods. (A) Flowchart of animal drug delivery. (B) Representative thoracic aorta with METH and/or BAPN treated. TAAD is indicated by the arrow in BAPN+METH-treated aorta. (C-D) Prevalence of TAAD in different groups, the probability value was adjusted with the Bonferroni method for pairwise comparisons (*p<0.05, n=3). (E) H&E and EVG staining of aorta sections from the rectangle areas shown in B. Aortic dissection and elastin fragmentation displayed in METH/BAPN-treated aorta were blocked in Ad-shC/EBPβ-treated aorta. (F) Western blot analyses of C/EBPβ, MMP9 and MMP2 protein expression in rat aortas with different treatments. (G-I) Quantification of C/EBPβ, MMP2, and MMP9 levels shown in F by normalized to β-actin (*p<0.05, **p<0.01, ***p<0.005, one-way ANOVA followed by LSD post hoc analyses, n=3).

Figure 6.

C/EBPβ mediated METH-induced cell apoptosis by regulating the expression of IGFBP5-P53-PUMA cascade. (A) METH and METH+BAPN induced PARP and caspase3 cleavage in rat aortas as detected by Western blot analyses. (B-C) Quantification of cleaved PARP and caspase3 levels shown in A by normalized to β-actin (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001, factorial design ANOVA followed by LSD post hoc analyses, n=3). (D) METH and METH+BAPN induced cells apoptosis in aorta media as shown by the TUNEL staining. (E) Quantification of apoptotic cells relative to the total cell numbers in aorta sections (***p<0.005, ****p<0.001, factorial design ANOVA followed by LSD post hoc analyses, n=3). (F) Aortic SMC were transfected with control (AD-shGFP) or C/EBPβ siRNA (AD-shC/EBPβ) followed by treatment with vehicle or BAPN+METH (1.0 mM) as indicated. PARP and caspase3 cleavage levels and IGFBP5/PUMA/p53 protein expression were detected by Western blot. (G-K) Quantification of IGFBP5/PUMA/p53 as well as cleaved PARP and caspase3 levels shown in F by normalized to β-actin (*p<0.05, **p<0.01, factorial design ANOVA followed by LSD post hoc analyses, n=3). (L-Q) Knockdown of C/EBPβ attenuated the expression of apoptosis-regulating proteins and decreased PARP and caspase3 cleavage. IGFBP5, p53, PUMA, cleaved-PARP, and cleaved-caspase3 levels were detected by Western blot and quantified by normalized to β-actin (**p<0.01, ***p<0.005, ****p<0.001, factorial design ANOVA followed by LSD post hoc analyses, n=3).

Figure 7.

C/EBPβ expression was increased in human aorta with dissection. (A) Immunostaining of C/EBPβ in human thoracic aorta dissection specimens. Arrow indicates C/EBPβ-positive staining. (B, D, G) C/EBPβ, MMP9, MMP2 protein expression and the cleaved PARP and caspase3 levels in human aorta dissection specimens were detected by Western blot. (C, E, F, H, I) Quantification of C/EBPβ, MMP9, MMP2, cleaved PARP and cleaved caspase3 levels shown in B, D, and G by normalizing to P-actin, respectively (**p<0.01, Student’s t test, n=3).