MKL1 cooperates with p38MAPK to promote vascular senescence, inflammation, and abdominal aortic aneurysm

Abstract

Abdominal aortic aneurysm (AAA) is a catastrophic disease with little effective therapy. Myocardin related transcription factor A (MRTFA, MKL1) is a multifaceted transcription factor, regulating diverse biological processes. However, a detailed understanding of the mechanistic role of MKL1 in AAA has yet to be elucidated. In this study, we showed induced MKL1 expression in thoracic and abdominal aneurysmal tissues, respectively in both mice and humans. MKL1 global knockout mice displayed reduced AAA formation and aortic rupture compared with wild-type mice. Both gene deletion and pharmacological inhibition of MKL1 markedly protected mice from aortic dissection, an early event in Angiotensin II (Ang II)-induced AAA formation. Loss of MKL1 was accompanied by reduced senescence/proinflammation in the vessel wall and cultured vascular smooth muscle cells (VSMCs). Mechanistically, a deficiency in MKL1 abolished AAA-induced p38 mitogen activated protein kinase (p38MAPK) activity. Similar to MKL1, loss of MAPK14 (p38α), the dominant isoform of p38MAPK family in VSMCs suppressed Ang II-induced AAA formation, vascular inflammation, and senescence marker expression. These results reveal a molecular pathway of AAA formation involving MKL1/p38MAPK stimulation and a VSMC senescent/proinflammatory phenotype. These data support targeting MKL1/p38MAPK pathway as a potential effective treatment for AAA.

Keywords: Aortic aneurysm; Inflammation; MKL1; Senescence; p38MAPK.

Graphical abstract

Fig. 1

MKL1 expression is induced in both AAA and TAA lesions in mice and humans. A, qRT-PCR analysis of Mkl1 in total RNA extracted from aortas of Apoe−/− mice exposed to saline or angiotensin II (Ang II) for 1 week (n = 6). Unpaired two-tailed Student's t-test. B, Representative Western blot images and quantification of MKL1 in aortas of Apoe−/− mice exposed to saline or Ang II for 1 week (n = 8). Unpaired two-tailed Student's t-test with Welch's correction. C and D, Representative confocal microscopy images for immunofluorescence staining of the indicated proteins in cross-sections of the suprarenal aortas from Apoe−/− mice infused with Ang II or saline for 1 week (n = 3). E, Representative confocal microscopy images for immunofluorescence staining of the indicated proteins in cross-sections of the thoracic aortas from C57BL/6 mice infused with high fat diet (HFD) with Ang II or saline for 4 weeks (n = 3). F, Dot plot graph from single-cell RNA-sequencing analysis [27] in aortas of C57BL/6 mice fed with HFD plus Ang II infusion vs C57BL/6 mice fed with chow diet without Ang II infusion (n = 3, pooled). The size of circle represents the percent of cells expressing Mkl1 in each cluster; the color scale indicates the expression level. G, Representative confocal microscopy images for immunofluorescence staining of the indicated proteins in cross-sections of human AAA versus non-aneurysmal aortic tissues (n = 3). H, qRT-PCR analysis of MKL1 in total RNA extracted from control aortic tissues (control, n = 6) from organ donors versus human AAA samples (AAA, n = 24). Mann-Whitney test. I, Western blot image and the quantitation for MKL1 protein in aortic tissues from organ donors (control, n = 10) and patients with ascending thoracic aortic aneurysm (ATAA, n = 16). Mann-Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1

MKL1 expression is induced in both AAA and TAA lesions in mice and humans. A, qRT-PCR analysis of Mkl1 in total RNA extracted from aortas of Apoe−/− mice exposed to saline or angiotensin II (Ang II) for 1 week (n = 6). Unpaired two-tailed Student's t-test. B, Representative Western blot images and quantification of MKL1 in aortas of Apoe−/− mice exposed to saline or Ang II for 1 week (n = 8). Unpaired two-tailed Student's t-test with Welch's correction. C and D, Representative confocal microscopy images for immunofluorescence staining of the indicated proteins in cross-sections of the suprarenal aortas from Apoe−/− mice infused with Ang II or saline for 1 week (n = 3). E, Representative confocal microscopy images for immunofluorescence staining of the indicated proteins in cross-sections of the thoracic aortas from C57BL/6 mice infused with high fat diet (HFD) with Ang II or saline for 4 weeks (n = 3). F, Dot plot graph from single-cell RNA-sequencing analysis [27] in aortas of C57BL/6 mice fed with HFD plus Ang II infusion vs C57BL/6 mice fed with chow diet without Ang II infusion (n = 3, pooled). The size of circle represents the percent of cells expressing Mkl1 in each cluster; the color scale indicates the expression level. G, Representative confocal microscopy images for immunofluorescence staining of the indicated proteins in cross-sections of human AAA versus non-aneurysmal aortic tissues (n = 3). H, qRT-PCR analysis of MKL1 in total RNA extracted from control aortic tissues (control, n = 6) from organ donors versus human AAA samples (AAA, n = 24). Mann-Whitney test. I, Western blot image and the quantitation for MKL1 protein in aortic tissues from organ donors (control, n = 10) and patients with ascending thoracic aortic aneurysm (ATAA, n = 16). Mann-Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Deletion of MKL1 protects mice from Ang II-induced AAA formation and aortic rupture. All mice were infused with saline or Ang II for 4 weeks. A, Representative en face photographs showing aortas from Mkl1^+/+, Apoe^−/− (WT) and Mkl1^−/−, Apoe^−/− (KO) mice. B and C, The incidence of AAA and aortic rupture (B) and Kaplan-Meier survival curve (C) in Ang II-infused WT (n = 20) and KO (n = 17) mice. Chi-square test for (B), Log-rank test for (C). D, Representative Doppler ultrasound images of abdominal aortas before (baseline) and 4 weeks after Ang II infusion (n = 5). E, The maximal abdominal aortic diameter (the width of the maximal expanded portion of suprarenal aorta) quantified from excised aortas as shown in A (n = 6/group for saline controls; n = 12 for WT and n = 13 for KO were included for Ang II infused aortas). Two-way ANOVA followed by Sidak multiple comparison test. F–H, Representative images and quantification (unpaired two-tailed Student's t-test) of hematoxylin and eosin (H&E) (F), Verhoeff-Van Gieson (VVG) (G), and Masson trichrome staining (H) of cross-sections of suprarenal aorta from WT and KO mice (n = 4). Quantifications were performed on at least 3 separate sections from 4 animals from each group, the average value from all the sections of a given animal was used for comparison. Location of extensive elastin degradation was labeled with yellow arrowhead. *P < 0.05, **P < 0.01, ***P < 0.001, NS: not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Depletion of MKL1 inhibits Ang II-induced vascular cell senescence. A and B, Representative images and quantification (unpaired two-tailed Student's t-test) of the senescence-associated beta-galactosidase (SA-β-gal)-stained whole aorta (n = 3) (A) and cross-sections of the suprarenal aorta from Mkl1^+/+, Apoe^−/− (WT) and Mkl1^−/−, Apoe^−/− (KO) mice infused with Ang II for 4 weeks (n = 5) (B). C, Representative Western blot images and quantification of senescence markers in aorta homogenates of WT and KO mice infused with Ang II for 1 week (n = 6 for p53 and p21, unpaired two-tailed Student's t-test; n = 4 for p16, Mann–Whitney test). D and E, qRT-PCR analysis (n = 4) (D) and representative Western blot images and quantification (n = 5) (E) of relative levels of MKL1 and senescence markers from human aortic smooth muscle cells (HASMCs) transduced with control or shMKL1 lentivirus for 72 h followed by Ang II (100 nM) or vehicle treatment. Pair matched two-way ANOVA followed by Sidak multiple comparison test. *P < 0.05, **P < 0.01, NS: not significant.

Fig. 4

Depletion of MKL1 suppresses vascular inflammation. A and B, Representative confocal microscopy images for immunofluorescence staining and quantification (unpaired two-tailed Student's t-test) of the relative fluorescence intensity of the indicated proteins in cross-sections of the suprarenal aorta from Mkl1^+/+, Apoe^−/− (WT) and Mkl1^−/−, Apoe^−/− (KO) mice infused with Ang II for the indicated time (n = 3). C and E, qRT-PCR analysis of relative mRNA level of indicated inflammatory genes from HASMCs transduced with control or shMKL1 lentivirus for 72 h (n = 5, Mann–Whitney test for MKL1 and IL6, Unpaired two-tailed Student's t-test for CCL2 and MMP2) (C) and primary cultured bone marrow derived macrophages (BMDMs) from WT versus KO mice infused with Ang II for 1 week (n = 5–7, Mann–Whitney test for Mkl1 and Cxcl1, Unpaired two-tailed Student's t-test for Il6, Il1b, Mmp14 and Ccl2) (E). D, Heatmap representation of transcriptomic profiling of key inflammatory mediators based on deep RNA-sequencing (RNA-seq) of BMDMs from KO versus WT mice infused with Ang II for 1 week. n = 4 per group. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 5

MKL1 deficiency through gene deletion or inhibitor administration prevents Ang II-induced aortic dissection. A, The incidence of aortic dissection (AD) and abdominal aortic rupture in Mkl1^+/+, Apoe^−/− (WT) (n = 21) and Mkl1^−/−, Apoe^−/− (KO) (n = 12) mice infused with Ang II for 1 week. Chi-square test. B, Representative images and quantification (unpaired two-tailed Student's t-test) of VVG staining of cross-sections of suprarenal aorta from WT and KO mice infused with Ang II for 1 week (n = 4). Location of extensive elastin degradation was labeled with yellow arrowhead. C, Timeline for vehicle or CCG-1423 (1 mg/kg/day) treatment. D, Representative en face photographs showing aortas from vehicle and CCG-1423 treated Apoe^−/− mice (n = 16). E, The incidence of AD and rupture in vehicle and CCG-1423 treated Apoe^−/− mice. Chi-square test. F, Representative images and the quantification (unpaired two-tailed Student's t-test) of VVG staining of cross-sections of suprarenal aorta from vehicle and CCG-1423 treated Apoe^−/− mice (n = 8). G, Representative confocal microscopy immunofluorescence staining of inflammatory mediators and SMC markers in cross-sections of suprarenal aorta from vehicle and CCG-1423 treated Apoe^−/− mice and the quantification (unpaired two-tailed Student's t-test) (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

MKL1 deficiency inhibits p38MAPK activity in VSMCs. A and B, Representative confocal microscopy images for immunofluorescence staining and quantification (unpaired two-tailed Student's t-test) of the relative fluorescence intensity of p-p38 in cross-sections of suprarenal aortas from Mkl1^+/+, Apoe^−/− (WT) and Mkl1^−/−, Apoe^−/− (KO) mice infused with Ang II for the indicated time (A), or from vehicle and CCG-1423 treated Apoe^−/− mice infused with Ang II for 1 week (B) (n = 3/group). Quantifications were performed on at least 3 separate sections from 3 animals from each group, the average value from all the sections of a given animal was used for comparison. C, Representative Western blot images and quantification of indicated proteins in aorta homogenates of WT and KO mice infused with Ang II for 1 week (n = 7). Unpaired two-tailed Student's t-test. D, Representative Western blot images for the indicated proteins of mouse aortic SMCs (MASMCs) isolated from WT or KO mice treated with vehicle (IL1β-) or IL1β (5 ng/ml) for the indicated time, and the quantitation for the groups treated with vehicle and IL1β for 1 h (n = 5). Pair matched two-way ANOVA followed by Sidak multiple comparison test (D). Expression levels of p-STAT3 (C) and p-p38 (D) are normalized with GAPDH and total p38α, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS: not significant.

Fig. 7

MAPK14 deficiency in VSMCs ameliorates AAA formation and vascular senescence and inflammation. A-F, All mice were infused with saline or Ang II for 4 weeks. A, Representative en face photographs showing aortas from Mapk14 WT (Sm22-Cre, Mapk14^+/+, Apoe^−/−) and SMC-Mapk14 KO (Sm22-Cre, Mapk14^f/f, Apoe^−/−) mice. B, The incidence of AAA in Ang II-infused Mapk14 WT (n = 34, including n = 22 of Mapk14^f/f, Apoe^−/− and n = 12 of Sm22-Cre, Mapk14^+/+, Apoe^−/−) and SMC-Mapk14 KO (n = 26) mice. Chi-square test. C, Representative Doppler ultrasound images of abdominal aortas before (baseline) and 4 weeks after Ang II (Ang II 4 wk) (n = 10). D, The maximal abdominal aortic diameter (the width of the maximal expanded portion of suprarenal aorta) quantified from excised aortas as shown in A (n = 7 for Mapk14^f/f, Apoe^−/− and n = 6 for SMC-Mapk14 KO were included for saline control group; n = 24 for Mapk14 WT and n = 17 for SMC-Mapk14 KO were included for Ang II 4wk group). Two-way ANOVA followed by Sidak multiple comparison test. E, Representative images and quantification of elastin degradation (Unpaired two-tailed Student's t-test) from VVG staining of cross-sections of suprarenal aortas (n = 5). Quantifications were performed on at least 3 separate sections from 4 animals from each group, the average value from all the sections of a given animal was used for comparison. F, Representative confocal microscopy images for immunofluorescence staining of indicated proteins in cross-sections of the suprarenal aortas (n = 4). G and H, Western blot images and quantification of the indicated proteins in aorta homogenates from Mapk14 WT and SMC-Mapk14 KO mice infused with Ang II for 1 week (n = 6, G; n = 5, H). Unpaired two-tailed Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, NS: no significant.