Fig 2: A causal role for MMP8 in BAPN-induced TAD formation. Three-week-old MMP8-knockout mice (MMP8_KO, ApoE−/−/MMP8−/−) and their wildtype control littermates (WT, ApoE−/−/MMP8+/+) administered with BAPN in drinking water for two (A,B) or four (C–G) weeks, respectively. (A) Thoracic aortic gene expression was analyzed by RT-qPCR. (B) Thoracic aortic MMP8 activity was measured using SensoLyte® 520 MMP-8 Assay Kit. (C) Animal survival rate (Log-rank (Mantel–Cox) test). Images for HE staining (D), EVG staining (E), and the quantitative data of AD incidence (F) and elastin breaks (G) are presented here. Note: Thoracic AD incidence was defined by the mice that died from thoracic aortic rupture, and mice identified with one or more aortic pathologies (aortic intima tear, false lumen, and intramural hematoma). Black or red arrows indicate AD or intima tearing. Data presented here are representative (D,E) or the mean ± SEM of five (A,B) or eleven mice (C–G), respectively (n = 5 or 11 mice). * p < 0.05 (versus WT, unpaired t-test in A,B,G; Chi-square test in F).

Fig 3: MMP8 increased BAPN/Ang II-induced SMC inflammation and apoptosis via ROS generation. (A,B) BAPN and Ang II synergically increased MMP8 gene expression and activity in SMCs. SMCs were treated with 25 µg/mL BAPN with or without 10 nM Ang II for 24 h. Total RNAs and cell culture supernatant were collected and subjected to RT-qPCR analysis (A) and MMP8 activity assay (B), respectively. (C) BAPN and Ang II synergically upregulated inflammatory gene expression. (D) Ang II, and not Ang I, increased inflammatory gene expression in MMP8_KO SMCs. (E) ROS measurement in SMCs were performed using the DCF ROS/RNS Assay Kit. (F) ROS inhibition abolished Ang I-induced inflammatory gene expression in SMCs. Serum-starved WT or MMP8_KO SMCs were incubated with 25 µg/mL BAPN with or without 10 nM Ang I, in the absence or presence of diphenyleneiodonium chloride (DPI, 10 µM), for 24 h. Total RNAs were extracted and subjected to RT-qPCR analysis. (G,H) ROS inhibition abolished BAPN/Ang I-induced SMC apoptosis. Serum-starved WT or MMP8_KO SMCs were subjected to the indicated treatments for 48 h, followed by CCK-8 assays (G) or TUNEL staining analysis (H), respectively. Data presented here are the mean ± SEM of five independent experiments (n = 5). * p < 0.05 (versus vehicle for Ang II in A–C, BAPN/vehicle in D,E, BAPN/vehicle/DMSO in F, or vehicle/DMSO in G,H); # p < 0.05 (versus vehicle for BAPN in A–C, WT in D,E, or BAPN/Ang I/DMSO in F–H); two-way ANOVA with a post-hoc Tukey test.

Fig 4: MMP8-deficiency causes decreased inflammatory cell invasion & transendothelial migration. Bone marrow neutrophils or monocytes were isolated from WT or MMP8_KO mice, and subjected to cell invasion (A) and transendothelial migration (B–E) assays, respectively. (A) The invasion capacity of cell was detected using QCM ECMatrix Cell Invasion Assay. (B) Cell transendothelial migration analysis. Mouse endothelial cells (EC, C166 cells) were pre-cultured onto transwell inserts (pore size: 8 µm) to form an EC monolayer, followed by transendothelial assays in response to 100 ng/mL macrophage inflammatory protein 2 (MIP2) for neutrophils or monocyte chemoattractant protein-1 (MCP-1) for monocytes, respectively. (C) Ang I increased WT but not MMP8_KO monocyte transendothelial migration. (D) Ang II promoted both WT and MMP8_KO monocyte transendothelial migration. (E,F) VCAM1 gene knockdown decreased monocyte transendothelial migration. C166 cells were transfected with control (si-NT) or VCAM1 specific (si-VCAM1) siRNAs, followed by transendothelial migration (E) and RT-qPCR (F) assays, respectively. (G) RT-qPCR analysis of MMP8 gene expressions in C166 cells with indicated treatment for 24 h. (H) MMP8 activity in cell culture supernatant with the indicated treatments for 24 h. (I,J) RT-qPCR analysis of VCAM1 gene expression in C166 cells with indicated treatments (25 μg/mL BAPN with or without 10 nM Ang I/Ang II) for 24 h. Data presented here are Mean ± S.E.M of five independent experiments (n = 5). * p < 0.05 (versus WT, or vehicle); # p < 0.05 (versus vehicle or si-NT); two-way ANOVA with a post-hoc Tukey test.

Fig 5: MMP8 detection in human ascending aorta with (HuAD) or without (HuAA) dissection. (A,B) Immunofluorescent staining showed increased expression levels of MMP8 protein in a dissected human ascending aorta. Representative images (A) and relative mean fluorescence intensity (MFI) (B) of MMP8 or SMA over DAPI staining are presented here. * p < 0.05 (n = 5, versus HuAA) (unpaired t-test). (C) RT-qPCR analysis showed increased MMP8 gene expression in a human ascending aorta with dissection. * p < 0.05 (n = 12 for HuAA or 22 for HuAD, versus HuAA) (Mann–Whitney U test). (D) ELISA analysis showed increased serum MMP8 levels in patients with acute TAD. * p < 0.05 (versus HuAA, n = 26 for HuAA and 18 for HuAD) (Mann–Whitney U test).