Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol (FORM) suppressed EPO‐induced VSMC senescence in mouse aorta. A) GO enrichment for the intersection genes. B) KEGG enrichment for the intersection genes. C) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of γH2AX (red particles) and αSMA (specific for SMCs, green particles) in ApoE −/− mice receiving vehicle, EPO and EPO+ medium‐dose formoterol treatment, respectively (scale bars = 10 µm). D) Quantitative analysis of γH2AX/αSMC colocalization in (C) ( n = 6 per group). E) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of SIRT1 (green particles) and αSMA (specific for SMCs, red particles) in ApoE −/− mice receiving vehicle, EPO and EPO+medium‐dose formoterol treatment, respectively. (scale bars = 10 µm). F) Quantitative analysis of γH2AX/αSMC colocalization in (E) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol ameliorated aortic senescence via β2AR. A) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of β2AR (green particles) and αSMA (specific for SMCs, red particles) in the aorta of ApoE −/− mice (scale bars = 10 µm). B) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMCs treated with vehicle, EPO, EPO+0.01 nmol mL −1 formoterol, EPO+0.1 nmol mL −1 formoterol, EPO+1 nmol mL −1 formoterol and EPO+10 nmol mL −1 formoterol, respectively. C,D) Quantitative analysis of western blot in (B) ( n = 6 per group). E) Representative sections of SA‐β‐gal staining of VSMCs transfected with β2AR siRNA (siβ2AR) or negative control siRNA (siNC) and treated with vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). F) Quantitative analysis of percentage of VSMCs with positive SA‐β‐gal staining in (E) ( n = 6 per group). G) Representative western blot analysis of protein expression of SIRT1, P21 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO and EPO + 0.1 nmol mL −1 formoterol, respectively. H‐I) Quantitative analysis of western blot in (G) ( n = 6 per group). J) Representative images of immunostaining of SIRT1 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO, and EPO+ 0.1 nmol mL −1 formoterol, respectively. K) Quantitative analysis of colocalization of dapi/SIRT1 in VSMCs in(G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Staining, Transfection, Negative Control, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: cAMP‐regulated the effect of formoterol on VSMC senescence. A) Quantification of intracellular cAMP levels in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. B) Representative images of SA‐β‐gal staining of VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). C) Quantitative analysis of SA‐β‐gal staining in (B) ( n = 6 per group). D) Representative western blot assay of SIRT1, P21 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. E‐F) Quantitative analysis of protein expression of SIRT1, P21 protein levels by western blot in (D), ( n = 6 per group). G) Representative images of immunostaining of SIRT1 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. H) Quantitative analysis of colocalization of dapi/SIRT1 in (G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Control, Staining, Western Blot, Expressing, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Casitas B‐lineage lymphoma (CBL) was essential for EPO‐induced VSMC senescence. A) Representative immunofluorescent analysis of the colocalization (yellow particles) of CBL (red particles) and SIRT1 (green particles) in VSMC receiving vehicle and EPO treatment respectively (scale bars = 10 µm). B) Quantitative analysis of CBL/SIRT1 colocalization in (A) ( n = 6 per group). C) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMC transfected with siCBL or negative control siRNA (siNC) and treated with vehicle and EPO respectively. D‐E) Quantitative analysis of western blot in (C) ( n = 6 per group). F) Representative sections of SA‐β‐gal staining of VSMC transfected with CBL siRNA (siCBL) or negative control siRNA (siNC) and treated with vehicle and EPO respectively (scale bars = 10 µm). G) Quantitative analysis of the percentage of VSMC with positive SA‐β‐gal staining in (F) ( n = 6 per group). ** p < 0.01, *** p < 0.001, unpaired two‐tailed Student's t‐tests with Welch's correction were applied in (B). The other data were analyzed via Two‐way ANOVA followed by the Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Staining, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Schematic diagram showing the mechanism of therapeutic effects of medium‐dose formoterol on EPO‐induced AAA. Formoterol binds to β2AR and activates cAMP, which increases SIRT1 protein expression, leading to suppressed VSMC senescence induced by EPO. In contrast, SIRT1 is downregulated by EPO via activation of CBL, resulting in aggravated VSMC senescence. Thus, medium‐dose formoterol attenuated EPO‐induced AAA via β2AR/cAMP/SIRT1 pathways, which provides a promising medication for the treatment of AAA.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Expressing, Activation Assay