RAAS

Histological analysis staining of three groups. (a) Representative H&E-staining micrographs displaying transverse myocardial section (original magnification ×200). (b) Masson staining of three groups (original magnification ×200). Collagen content (%) of heart tissues were presented as fibrosis (***P < 0.001, n = 7 per group). (c) Cardiac section of three group mice hearts stained with WGA (original magnification ×400) (*P < 0.05, n = 7 per group). (d) The expression of Drp1 significantly increased in DOX group (***P < 0.001, n = 7 per group) and it decreased in DOX + LCZ696 group (**P < 0.01, n = 7 per group).

In vivo effects of candesartan and valsartan in post-MI rats overexpressing adrenal barr1. (A) Trichrome-Masson's staining in myocardial cross-sections from post-MI rats overexpressing barr1 specifically in their adrenals and treated for 7 days with either vehicle or 10 mg/kg body weight/day candesartan or 40 mg/kg body weight/day irbesartan (all via drinking water). Blue denotes collagen fibers, red denotes muscle fibers, and black represents cell nuclei. Representative images are shown from 5–6 rat hearts stained per group, along with staining in sham-operated rat hearts, in which no blue staining was detectable. (B,C) Heart mRNA levels of (B) collagen I (Col1a1) and brain natriuretic peptide (BNP) (C) in these rats at the end of the 7-day drug treatments. Values for post-MI rats receiving AdGFP in their adrenals instead of Adbarr1 (i.e. not overexpressing barr1 in their adrenals) were used as reference. *, p < 0.05, vs. Candesartan (Adbarr1), n=5 rat hearts/group.

Efficacy of ARBs at preventing StAR upregulation in SII-stimulated H295R cells. (A,B) Western blotting for StAR protein levels in H295R cells transfected to overexpress barr1 and treated for 6 hrs with 10 μM SII alone (SII) or in the presence of 10 μM of each of the sartans tested. Representative blots of 3 independent experiments are shown in (A), including blots for barr1 to confirm its overexpression and for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as loading control, and the StAR protein induction (as % of the SII response), as derived by densitometric quantification, is shown in (B). *, p<0.05, n=3 independent experiments/treatment. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). (C,D) Western blotting for StAR protein levels in dominant negative barr1 mutanttransfected H295R cells and treated as in (A-B). Representative blots are shown in (C), including blots for the dominant negative barr1 mutant to confirm its overexpression and for GAPDH as loading control, and the StAR protein induction (as % of vehicle-no stimulation), as derived by densitometric quantification, is shown in (D). No significant differences were observed among treatments, nor did any treatment cause any induction in StAR levels. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). n=3 independent experiments/treatment. LOS: Losartan-; VAL: Valsartan; CAN: Candesartan; OLM: Olmesartan; IRB: Irbesartan.

(B) Dose-response curves of each drug for acute G protein or barr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan.

Efficacy of ARBs at preventing StAR upregulation in SII-stimulated H295R cells. (A,B) Western blotting for StAR protein levels in H295R cells transfected to overexpress barr1 and treated for 6 hrs with 10 μM SII alone (SII) or in the presence of 10 μM of each of the sartans tested. Representative blots of 3 independent experiments are shown in (A), including blots for barr1 to confirm its overexpression and for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as loading control, and the StAR protein induction (as % of the SII response), as derived by densitometric quantification, is shown in (B). *, p <0.05, n=3 independent experiments/treatment. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). (C,D) Western blotting for StAR protein levels in dominant negative barr1 mutanttransfected H295R cells and treated as in (A-B). Representative blots are shown in (C), including blots for the dominant negative barr1 mutant to confirm its overexpression and for GAPDH as loading control, and the StAR protein induction (as % of vehicle-no stimulation), as derived by densitometric quantification, is shown in (D). No significant differences were observed among treatments, nor did any treatment cause any induction in StAR levels. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). n=3 independent experiments/treatment. LOS: Losartan-; VAL: Valsartan; CAN: Candesartan; OLM: Olmesartan; IRB: Irbesartan.

Effect of valsartan and cardamonin on Ang II-induced phosphorylation of p38 MAPK and ERK. Representative results of Western blot.

Ang II-induced MMP-2 protein expression through AT1 receptor in ex vivo cultured IAAA aortic walls (A) Ex vivo cultured aortic pieces from IAAA patients (n = 5) were incubated in the presence of different concentrations of Ang II for 48 h. (B) Ex vivo cultured aneurysmal aorta walls were pre-treated with either candesartan (10 µM, AT1R inhibitor) or PD123319 (10 µM, AT2R antagonist) for 48 h, followed by 1 µM Ang II treatment for 48 h. The group without Ang II incubation was used as control. **P < 0.01 vs. untreated control group; ##P < 0.01 vs. treated with Ang II alone. CA, candesartan; PD, PD123319.

Efficacy of ARBs at preventing StAR upregulation in SII-stimulated H295R cells. (A,B) Western blotting for StAR protein levels in H295R cells transfected to overexpress barr1 and treated for 6 hrs with 10 μM SII alone (SII) or in the presence of 10 mM of each of the sartans tested. Representative blots of 3 independent experiments are shown in (A), including blots for barr1 to confirm its overexpression and for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as loading control, and the StAR protein induction (as % of the SII response), as derived by densitometric quantification, is shown in (B). *, p<0.05, n=3 independent experiments/treatment. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). (C,D) Western blotting for StAR protein levels in dominant negative barr1 mutanttransfected H295R cells and treated as in (A–B). Representative blots are shown in (C), including blots for the dominant negative barr1 mutant to confirm its overexpression and for GAPDH as loading control, and the StAR protein induction (as % of vehicle-no stimulation), as derived by densitometric quantification, is shown in (D). No significant differences were observed among treatments, nor did any treatment cause any induction in StAR levels. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). n=3 independent experiments/treatment. LOS: Losartan-; VAL: Valsartan; CAN: Candesartan; OLM: Olmesartan; IRB: Irbesartan.

(b) ASMCs were incubated with 1 µM candesartan or 10 µM PD123319 for 60 min before stimulation with 0.1 µM Ang II for 24 h in six-well plates. Western blot was used for analyses of the levels of MMP-2 expression in cell lysates. Top panel: Graphs show the relative MMP-2 protein levels normalized to GAPDH relative to control. Bottom panel: Ratio of MMP-2/GAPDH protein expression from a representative Western blot experiment. Shown are mean ± SEM of three individual experiments. **P < 0.01 vs. untreated control cells; ##P < 0.01 vs. cells treated with Ang II

(B) Dose-response curves of each drug for acute G protein or barr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan; EPR: Eprosartan; AZI: Azilsartan; OLM: Olmesartan; CAN: Candesartan; VAL: Valsartan; IRB: Irbesartan.

(B) Dose-response curves of each drug for acute G protein or βarr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan; EPR: Eprosartan; AZI: Azilsartan; OLM: Olmesartan; CAN: Candesartan; VAL: Valsartan; IRB: Irbesartan.

(B) Dose-response curves of each drug for acute G protein or barr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan; EPR: Eprosartan; AZI: Azilsartan; OLM: Olmesartan; CAN: Candesartan; VAL: Valsartan; IRB: Irbesartan.

Histological analysis staining of three groups. (a) Representative H&E-staining micrographs displaying transverse myocardial section (original magnification ×200). (b) Masson staining of three groups (original magnification ×200). Collagen content (%) of heart tissues were presented as fibrosis (***P < 0.001, n = 7 per group). (c) Cardiac section of three group mice hearts stained with WGA (original magnification ×400) (*P < 0.05, n = 7 per group). (d) The expression of Drp1 significantly increased in DOX group (***P < 0.001, n = 7 per group) and it decreased in DOX + LCZ696 group (**P < 0.01, n = 7 per group).

In vivo effects of candesartan and valsartan in post-MI rats overexpressing adrenal barr1. (A) Trichrome-Masson's staining in myocardial cross-sections from post-MI rats overexpressing barr1 specifically in their adrenals and treated for 7 days with either vehicle or 10 mg/kg body weight/day candesartan or 40 mg/kg body weight/day irbesartan (all via drinking water). Blue denotes collagen fibers, red denotes muscle fibers, and black represents cell nuclei. Representative images are shown from 5–6 rat hearts stained per group, along with staining in sham-operated rat hearts, in which no blue staining was detectable. (B,C) Heart mRNA levels of (B) collagen I (Col1a1) and brain natriuretic peptide (BNP) (C) in these rats at the end of the 7-day drug treatments. Values for post-MI rats receiving AdGFP in their adrenals instead of Adbarr1 (i.e. not overexpressing barr1 in their adrenals) were used as reference. *, p < 0.05, vs. Candesartan (Adbarr1), n=5 rat hearts/group.

Efficacy of ARBs at preventing StAR upregulation in SII-stimulated H295R cells. (A,B) Western blotting for StAR protein levels in H295R cells transfected to overexpress barr1 and treated for 6 hrs with 10 μM SII alone (SII) or in the presence of 10 μM of each of the sartans tested. Representative blots of 3 independent experiments are shown in (A), including blots for barr1 to confirm its overexpression and for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as loading control, and the StAR protein induction (as % of the SII response), as derived by densitometric quantification, is shown in (B). *, p<0.05, n=3 independent experiments/treatment. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). (C,D) Western blotting for StAR protein levels in dominant negative barr1 mutanttransfected H295R cells and treated as in (A-B). Representative blots are shown in (C), including blots for the dominant negative barr1 mutant to confirm its overexpression and for GAPDH as loading control, and the StAR protein induction (as % of vehicle-no stimulation), as derived by densitometric quantification, is shown in (D). No significant differences were observed among treatments, nor did any treatment cause any induction in StAR levels. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). n=3 independent experiments/treatment. LOS: Losartan-; VAL: Valsartan; CAN: Candesartan; OLM: Olmesartan; IRB: Irbesartan.

(B) Dose-response curves of each drug for acute G protein or barr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan.

Efficacy of ARBs at preventing StAR upregulation in SII-stimulated H295R cells. (A,B) Western blotting for StAR protein levels in H295R cells transfected to overexpress barr1 and treated for 6 hrs with 10 μM SII alone (SII) or in the presence of 10 μM of each of the sartans tested. Representative blots of 3 independent experiments are shown in (A), including blots for barr1 to confirm its overexpression and for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as loading control, and the StAR protein induction (as % of the SII response), as derived by densitometric quantification, is shown in (B). *, p <0.05, n=3 independent experiments/treatment. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). (C,D) Western blotting for StAR protein levels in dominant negative barr1 mutanttransfected H295R cells and treated as in (A-B). Representative blots are shown in (C), including blots for the dominant negative barr1 mutant to confirm its overexpression and for GAPDH as loading control, and the StAR protein induction (as % of vehicle-no stimulation), as derived by densitometric quantification, is shown in (D). No significant differences were observed among treatments, nor did any treatment cause any induction in StAR levels. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). n=3 independent experiments/treatment. LOS: Losartan-; VAL: Valsartan; CAN: Candesartan; OLM: Olmesartan; IRB: Irbesartan.

Effect of valsartan and cardamonin on Ang II-induced phosphorylation of p38 MAPK and ERK. Representative results of Western blot.

Ang II-induced MMP-2 protein expression through AT1 receptor in ex vivo cultured IAAA aortic walls (A) Ex vivo cultured aortic pieces from IAAA patients (n = 5) were incubated in the presence of different concentrations of Ang II for 48 h. (B) Ex vivo cultured aneurysmal aorta walls were pre-treated with either candesartan (10 µM, AT1R inhibitor) or PD123319 (10 µM, AT2R antagonist) for 48 h, followed by 1 µM Ang II treatment for 48 h. The group without Ang II incubation was used as control. **P < 0.01 vs. untreated control group; ##P < 0.01 vs. treated with Ang II alone. CA, candesartan; PD, PD123319.

Efficacy of ARBs at preventing StAR upregulation in SII-stimulated H295R cells. (A,B) Western blotting for StAR protein levels in H295R cells transfected to overexpress barr1 and treated for 6 hrs with 10 μM SII alone (SII) or in the presence of 10 mM of each of the sartans tested. Representative blots of 3 independent experiments are shown in (A), including blots for barr1 to confirm its overexpression and for GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as loading control, and the StAR protein induction (as % of the SII response), as derived by densitometric quantification, is shown in (B). *, p<0.05, n=3 independent experiments/treatment. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). (C,D) Western blotting for StAR protein levels in dominant negative barr1 mutanttransfected H295R cells and treated as in (A–B). Representative blots are shown in (C), including blots for the dominant negative barr1 mutant to confirm its overexpression and for GAPDH as loading control, and the StAR protein induction (as % of vehicle-no stimulation), as derived by densitometric quantification, is shown in (D). No significant differences were observed among treatments, nor did any treatment cause any induction in StAR levels. Blots shown have been cropped to fit space requirements and run under the same experimental conditions (same gel) (the full length blots are shown in Supplementary Information). n=3 independent experiments/treatment. LOS: Losartan-; VAL: Valsartan; CAN: Candesartan; OLM: Olmesartan; IRB: Irbesartan.

(b) ASMCs were incubated with 1 µM candesartan or 10 µM PD123319 for 60 min before stimulation with 0.1 µM Ang II for 24 h in six-well plates. Western blot was used for analyses of the levels of MMP-2 expression in cell lysates. Top panel: Graphs show the relative MMP-2 protein levels normalized to GAPDH relative to control. Bottom panel: Ratio of MMP-2/GAPDH protein expression from a representative Western blot experiment. Shown are mean ± SEM of three individual experiments. **P < 0.01 vs. untreated control cells; ##P < 0.01 vs. cells treated with Ang II

(B) Dose-response curves of each drug for acute G protein or barr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan; EPR: Eprosartan; AZI: Azilsartan; OLM: Olmesartan; CAN: Candesartan; VAL: Valsartan; IRB: Irbesartan.

(B) Dose-response curves of each drug for acute G protein or βarr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan; EPR: Eprosartan; AZI: Azilsartan; OLM: Olmesartan; CAN: Candesartan; VAL: Valsartan; IRB: Irbesartan.

(B) Dose-response curves of each drug for acute G protein or barr activation derived from the CellKey assay. LOS: Losartan; EXP: EXP3174; TEL: Telmisartan; EPR: Eprosartan; AZI: Azilsartan; OLM: Olmesartan; CAN: Candesartan; VAL: Valsartan; IRB: Irbesartan.