Journal: Nature Communications

Article Title: Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis in mice

doi: 10.1038/s41467-024-50036-2

Figure Lengend Snippet: A Representative images of senescence-associated-β-galactosidase (SA-β-gal) staining (upper panel) and quantification (lower panel) in VSMCs transfected with miR-1204 mimics or miR-control (miR-ctl). n = 6 biological replicates. Statistical analysis was performed using two-tailed Student’s t test. Scale bar, 100 μm. B Quantification of mRNA levels of SASP components in VSMCs transfected with miR-1204 mimics or miR-ctl. n = 6 biological replicates. Statistical analysis was performed using two-tailed Student’s t test. C Interleukin 6 (IL-6) concentration in the culture supernatant of VSMCs transfected with miR-1204 mimics or miR-ctl combined with angiotensin II (AngII) treatment. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. D Monocyte chemotactic protein 1 (MCP-1) concentration in the culture supernatant of VSMCs transfected with miR-1204 mimics or miR-ctl combined with AngII treatment. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. E – G Representative plots (upper) and quantification (lower) of immunoblot analysis of contractile markers, α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), and myosin heavy chain 11 (MYH11) in VSMCs transfected with miR-1204 mimics or miR-ctl combined with AngII treatment. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. H Representative images (left) and quantification (right) of immunofluorescence staining of CD68, MYH11, α-SMA, and SM22 in mouse aortas in the indicated groups. n = 17 biological replicates for miR-1204 + AngII group. n = 19 biological replicates for other groups. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test for α-SMA and SM22 quantification, by two-tailed Welch’s ANOVA followed by Dunn’s multiple comparisons for CD68 and MYH11 quantification. Scale bar, 100 μm. Data are presented as mean ± SD. Source data are provided as a source data file.

Article Snippet: Primary human aortic VSMCs (HASMCs) were purchased from ScienCell (Cat. No. 6110, Carlsbad, CA, USA), and cultured in smooth muscle cell medium (SMCM, Cat.No.1101, ScienCell) supplemented with 2% fetal bovine serum (FBS), 1% penicillin/streptomycin, and growth factor.

Techniques: Staining, Transfection, Control, Two Tailed Test, Concentration Assay, Comparison, Western Blot, Immunofluorescence

Journal: Nature Communications

Article Title: Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis in mice

doi: 10.1038/s41467-024-50036-2

Figure Lengend Snippet: A , B Volcano plot showing the mRNA expression of potential miR-1204 targets in VSMCs transfected with miR-1204 mimic or miR-control (miR-ctl) combined with angiotensin II (AngII) treatment ( B ) or not ( A ). n = 3 biological replicates. Statistical analysis was performed using a two-tailed Student’s t test. C Representative plots (upper) and quantification (lower) of MLYK immunoblot analysis in VSMCs transfected with miR-1204 mimic or miR-ctl combined with AngII treatment. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. D Representative images (upper panel) and quantification (lower panel) of MYLK immunofluorescence staining of mouse aortas from in the indicated groups. n = 17 biological replicates for the miR-1204 + AngII group. n = 19 biological replicates for other groups. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. Scale bar represents 100 μm. E Potential hsa-miR-1204 binding site in MYLK (upper panel). Luciferase activity assay confirmed that MYLK is a direct target of miR-1204 (lower panel). n = 6 biological replicates. Statistical analyses were performed using two-tailed Student’s t tests. F Representative images (left) and quantification (right) of MYLK immunofluorescent staining in aortas of healthy participants and patients with AAD. n = 6 (young normal), n = 6 (elder normal), n = 7 (young patient), n = 8 (elder patient). Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. Scale bar represents 20 μm. Data are presented as mean ± SD. Source data are provided as a source data file.

Article Snippet: Primary human aortic VSMCs (HASMCs) were purchased from ScienCell (Cat. No. 6110, Carlsbad, CA, USA), and cultured in smooth muscle cell medium (SMCM, Cat.No.1101, ScienCell) supplemented with 2% fetal bovine serum (FBS), 1% penicillin/streptomycin, and growth factor.

Techniques: Expressing, Transfection, Control, Two Tailed Test, Western Blot, Comparison, Immunofluorescence, Staining, Binding Assay, Luciferase, Activity Assay

Journal: Nature Communications

Article Title: Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis in mice

doi: 10.1038/s41467-024-50036-2

Figure Lengend Snippet: A Representative images of senescence-associated-β-galactosidase (SA-β-gal) staining (left) and quantification (right) in the indicated groups. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. Scale bar represents 100 μm. B Interleukin 6 (IL-6) concentration in the culture supernatant of VSMCs in the indicated groups. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. C Monocyte chemotactic protein 1 (MCP-1) concentration in the culture supernatant of VSMCs in the indicated groups. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. D Quantification of the mRNA levels of SASP components in VSMCs transfected with miR-1204 mimics or miR-ctl in the indicated groups. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. E – G Representative plots (upper panel) and quantification (lower panel) of immunoblot analysis of contractile markers, myosin heavy chain 11 (MYH11), α-smooth muscle actin (α-SMA), and smooth muscle protein 22 (SM22) in the indicated groups. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. H Scheme of AngII-induced aortic aneurysm and dissection (AAD) experiments. MiR-1204 agomir or miR-ctl (10 nmol) was administered by tail vein injection to 4-month-old male and female C57BL/6 mice every 3 d for a total of five times. Then the mice were administered MYLK adenovirus or blank adenovirus via tail vein prior to 4-weeks of AngII-infusion. I AAD incidence. AA aortic aneurysm, AD aortic dissection. Statistical analysis was performed using two-tailed Fisher’s exact test. J Representative photographs of aortas in the indicated groups. Bar indicates 4 mm. K Representative images of SA-β-gal staining in the indicated groups. Scale bar indicates 4 mm. L Representative B-mode ultrasound and doppler ultrasound (DUS) detection of abdominal aortas (upper) and quantification of abdominal aorta diameters (below). n = 17 biological replicates for AngII + miR-1204 group. n = 20 biological replicates for other groups. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. The white arrow indicates an aneurysm. M Representative images of immunofluorescence staining of MYLK, MCP-1, and IL-6 in mouse aortas in the indicated groups. Scale bar represents 100 μm. N Contraction of isolated aortic rings from indicated groups in response to the phenylephrine (Phe) treatment. n = 8 biological replicates. Statistical analysis was performed using two-tailed repeated-measures ANOVA with Bonferroni’s multiple comparison test. Data are presented as mean ± SD. Source data are provided as a source data file.

Article Snippet: Primary human aortic VSMCs (HASMCs) were purchased from ScienCell (Cat. No. 6110, Carlsbad, CA, USA), and cultured in smooth muscle cell medium (SMCM, Cat.No.1101, ScienCell) supplemented with 2% fetal bovine serum (FBS), 1% penicillin/streptomycin, and growth factor.

Techniques: Staining, Comparison, Concentration Assay, Transfection, Western Blot, Dissection, Injection, Two Tailed Test, Immunofluorescence, Isolation

Journal: Nature Communications

Article Title: Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis in mice

doi: 10.1038/s41467-024-50036-2

Figure Lengend Snippet: A Quantification of pri-mir-1204 levels in VSMCs treated with angiotensin II (AngII, 1 × 10 −6 M), hydrogen peroxide (H 2 O 2, 30 μM) or daunorubicin (0.22 μM). n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. B Quantification of pri-mir-1204 levels in VSMCs treated with nutlin-3a with or without silencing TP53. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. C Chromatin immunoprecipitation analysis quantifying DNA fragments co-immunoprecipitated with p53 at the PVT1 response element (PVT1 RE). The P21 3′response element (P21 3′ RE) was used as a positive control. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. D Luciferase activity confirms the interaction between p53 and PVT1 RE. n = 6 biological replicates. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison test. E Representative plots of immunoblot analysis of cellular senescence markers, p53, p21, p16, and p-γH2AX in control VSMCs, VSMCs transfected with miR-1204 mimics alone and VSMCs transfected with miR-1204 mimics plus MYLK adenovirus. F Quantification of cellular senescence markers in the indicated groups by immunoblot analysis. n = 6 biological replicates. Statistical analyses were performed using a one-way ANOVA followed by Tukey’s multiple comparison test expect for MYLK quantification. Statistical analysis was performed using two-tailed Welch’s ANOVA, followed by Dunn’s multiple comparisons for MYLK quantification. G VSMC division was monitored for 72 h in control VSMCs, VSMCs transfected with miR-1204 mimic alone, and VSMCs transfected with miR-1204 mimic and MYLK adenovirus. Scale bar represents 200 μm. H Quantification of VSMC division in the indicated groups. n = 3 biological replicates. Statistical analysis was performed using two-tailed repeated-measures ANOVA with Bonferroni’s multiple comparison test. I KEGG analysis of target genes binding to p53 in VSMCs overexpressing miR-1204. Statistical analyses were performed using hypergeometric tests. J Distribution of reads from ChIP-Seq of SASP genes in miR-1204 overexpressing or control VSMCs. K Phosphorylation of MDM2 in the VSMCs from indicated groups. The experiment was independently repeated three times with similar results. L Expression of MDM2 and p53 in proteins pulled-down by MDM2-Co-IP and input immunoprecipitation in VSMCs from indicated groups. The experiment was independently repeated three times with similar results. Data are presented as mean ± SD. ns not significant. Source data are provided as a source data file.

Article Snippet: Primary human aortic VSMCs (HASMCs) were purchased from ScienCell (Cat. No. 6110, Carlsbad, CA, USA), and cultured in smooth muscle cell medium (SMCM, Cat.No.1101, ScienCell) supplemented with 2% fetal bovine serum (FBS), 1% penicillin/streptomycin, and growth factor.

Techniques: Comparison, Chromatin Immunoprecipitation, Immunoprecipitation, Positive Control, Luciferase, Activity Assay, Western Blot, Control, Transfection, Two Tailed Test, Binding Assay, ChIP-sequencing, Phospho-proteomics, Expressing, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Aging aggravates aortic aneurysm and dissection via miR-1204-MYLK signaling axis in mice

doi: 10.1038/s41467-024-50036-2

Figure Lengend Snippet: Cell senescence induces the expression of miR-1204 through interaction of p53 with the plasmacytoma variant translocation 1response element (PVT1 RE). MiR-1204 directly targets myosin light chain kinase (MYLK), leading to the acquisition of a senescence-associated secretory phenotype (SASP) by vascular smooth muscle cells (VSMCs) and loss of their contractile phenotype. MiR-1204 induces VSMC senescence by forming a positive feedback loop. This figure was created with Bio-Render.com.

Article Snippet: Primary human aortic VSMCs (HASMCs) were purchased from ScienCell (Cat. No. 6110, Carlsbad, CA, USA), and cultured in smooth muscle cell medium (SMCM, Cat.No.1101, ScienCell) supplemented with 2% fetal bovine serum (FBS), 1% penicillin/streptomycin, and growth factor.

Techniques: Expressing, Variant Assay, Translocation Assay