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

Abstract Abdominal aortic aneurysm (AAA) is a life‐threatening vascular disease but effective drugs for treatment of AAA are still lacking. Recently, erythropoietin (EPO) is reported to induce AAA formation in apolipoprotein‐E knock out (ApoE−/−) mice but an effective antagonist is unknown. In this study, formoterol, a β2 adrenergic receptor (β2AR) agonist, is found to be a promising agent for inhibiting AAA. To test this hypothesis, ApoE−/− mice are treated with vehicle, EPO, and EPO plus low‐, medium‐, and high‐dose formoterol, respectively. The incidence of AAA is 0, 55%, 35%,10%, and 55% in these 5 groups, respectively. Mechanistically, senescence of vascular smooth muscle cell (VSMC) is increased by EPO while decreased by medium‐dose formoterol both in vivo and in vitro, manifested by the altered expression of senescence biomarkers including phosphorylation of H2AXserine139, senescence‐associated β‐galactosidase activity, and P21 protein level. In addition, expression of sirtuin 1 (SIRT1) in aorta is decreased in EPO‐induced AAA but remarkably elevated by medium‐dose formoterol. Knockdown of β2AR and blockage of cyclic adenosine monophosphate (cAMP) attenuate the inhibitory role of formoterol in EPO‐induced VSMC senescence. In summary, medium‐dose formoterol attenuates EPO‐induced AAA via β2AR/cAMP/SIRT1 pathways, which provides a promising medication for the treatment of AAA.

kg -1 EPO plus 1mg kg -1 formoterol injection once a day for additional 14 days (n=20), and EPO+high dose formoterol group that received an intraperitoneal injection of 10000 IU kg -1 EPO once a day for 14 days and an intraperitoneal injection of 10000 IU kg -1 EPO plus 3mg kg -1 formoterol injection once a day for additional 14 days (n=20).
The three doses of formoterol were selected based on literature recommendations [24,25] A schematic diagram showing the experiment procedure of ApoE -/-mice was given in Figure S2.
In the second in vivo experiment, 32 eight-week-old male ApoE -/-mice were randomly divided into 4 groups: vehicle group that received an intraperitoneal injection of saline once a day for 28 days, low-dose formoterol group that received an intraperitoneal injection of saline once a day for 14 days and an intraperitoneal injection of saline plus 0.3mg kg -1 formoterol injection once a day for additional 14 days (n=8), medium-dose formoterol group that received an intraperitoneal injection of saline once a day for 14 days and an intraperitoneal injection of saline plus 1mg kg -1 formoterol injection once a day for additional 14 days (n=8), high-dose formoterol group that received an intraperitoneal injection of saline once a day for 14 days and an intraperitoneal injection of saline plus 3mg kg -1 formoterol injection once a day for additional 14 days (n=8).A schematic diagram showing the protocol of this part of experiments was given in Figure S2.

Measurement of blood pressure
A non-invasive tail-cuff system (Softron BP-98A, Tokyo, Japan) was used to measure heart rate and blood pressure, and all mice were trained first to adapt to the device to ensure reproducible measurements.Heart rate and blood pressure were measured between 9:00 a.m. and noon by the same investigator and the values of heart rate and blood pressure in each mouse were recorded as the mean of three consecutive measurements.

Serum lipid assay
At the end of the experiment, all mice were fasted for 12 hours and then blood samples were taken from the cardiac apex of each mouse after euthanasia by an intraperitoneal injection of overdose pentobarbital (80mg kg -1 ).The serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and highdensity lipoprotein cholesterol (HDL-C) were measured by enzymic assays, using an automatic biochemistry analyzer (Chemray 240, Rayto, Shenzhen, China).

Histopathological analyses
After dissection of surrounding connective tissue, aortas were photographed with a ruler aside.An investigator blind to mouse grouping measured the outer diameter of the maximal expanded portion of the abdominal aorta in the images with the utility of ImageJ 1.53k software (Wayne Rasband, USA).
AAA was defined as the increasement in the outer diameter of the abdominal aorta by no less than 50% relative to the vehicle group (4).Then the aortic segment from the ascending aorta to the bifurcation of the common iliac artery were isolated, perfused with 4% PFA for 24h and embedded in paraffin.For characterization of cross sections, the suprarenal region of the abdominal aorta was cut into 5μm-thick sections at an interval of 500 μm and at least 10 sections were analyzed in each mouse.

Histopathological staining
For immunohistochemical (IHC) staining, the aortic tissues of the maximal expanded portion of the abdominal aortas were cut into 5μm-thick sections.After

Zymography
Zymography was performed with an MMP gelatin zymography kit (GenMed Scientific Inc., USA).The protein extracts from the mouse aorta were separated by electrophoresis in SDSPAGE gels containing 0.1% gelatin to measure the activity of MMP2 and MMP9.Gels were washed with renaturing buffer for 1 hour at room temperature and further incubated with developing buffer at 37℃ overnight.After incubation, gels were stained with Coomassie Brilliant Blue and then destained with the buffer until clear white bands appeared on the blue background.The activity of MMP2 and MMP9 was calculated by ImageJ 1.53k software (Wayne Rasband, USA).

TUNEL assay
Cell apoptosis of the aortic tissues was detected by TUNEL using the In Situ Cell Death Detection Kit, (Roche, Germany) following the manufacturer's instructions.5μm-thick sections derived from the maximal expanded portion of the abdominal aorta were used for TUNEL assay.After deparaffinization, rehydration and permeabilization with 0.1% Triton X-100, the sections were incubated with working solution in 37℃ for 1 hour.
The working solution was composed of enzyme solution and label solution at the ratio of 1:9.The slides were visualized by an electric upright microscope (DS-Ri2, Nikon, Japan) and the positive rate of each group was calculated by ImageJ 1.53k software (Wayne Rasband, USA).

Isolation of primary vascular smooth muscle cells
Primary vascular smooth muscle cells (VSMC) were isolated as previously described [7]   .In brief, mice aged 6-8 weeks were euthanatized by injection of an overdose pentobarbital (80mg kg -1 ), and after dissection of surrounding connective tissue, the aortas were immediately transferred to a sterile dish filled with phosphate buffered saline (PBS).The aortic tissues were then cut into pieces and transferred to a sterile dish containing 80% Dulbecco's modified Eagle's medium (DMEM) and 20% fetal bovine serum mixed with 100 μg mL -1 streptomycin and 100U mL -1 penicillin at 37°C in a humidified atmosphere of 5% CO2 and 95% air for at least 5 days.Three to six passages of VSMC at 70-80% confluence was used for the in vitro experiment.
In the second part of the in vitro experiments, to explore the role of endogenous β2AR in the inhibitive effect of formoterol on VSMC senescence, VSMC were transfected with specific siRNA against β2AR or negative control.VSMC were divided into 6 groups, which were treated with siNC transfection + vehicle (PBS), siNC transfection + EPO, siNC transfection + EPO+ 0.1 nmol mL -1 formoterol group, siβ2AR transfection + vehicle (PBS), siβ2AR transfection + EPO, and siβ2AR transfection + EPO+ 0.1 nmol mL -1 formoterol, respectively.Twenty-four hours after transfection, VSMC were treated with vehicle (PBS), EPO and EPO+ 0.1 nmol mL -1 formoterol, respectively, as described above.
In the third part of the in vitro experiments, to unveil the role of cAMP in the mechanism underlying the effects of formoterol, we used the inhibitor of membrane adenylyl cyclase, SQ22535(S8283, Selleck, USA) and dimethyl sulfoxide (DMSO) as solvent control.VSMC were divided into 6 groups ， which were treated with control (DMSO)+vehicle (PBS), control (DMSO)+EPO, control (DMSO)+EPO+0.1 nmol mL - 1 formoterol, SQ22535+PBS, SQ22535+EPO, and SQ22535+EPO+0.1 nmol mL -1 formoterol.The dose of SQ22535 was chosen as 80 μM as suggested by previous experiments (8).The cell treatment described in the first to third part of the in vitro experiments lasted for 24 hours before collection according to our previous study (17).
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.The cell treatment lasted for 4 hours before collection as indicated by a previous study [34] .
In the fifth part of the in vitro experiments, to explore the role of endogenous CBL in the VSMC senescence induced by EPO, VSMC were transfected with siCBL or siNC.
In the sixth part of the in vitro experiments, to explore the effect of different doses of formoterol on β2AR internalization, VSMC were divided into 5 groups treated with vehicle (PBS), 0.01 nmol mL -1 formoterol, 0.1 nmol mL -1 formoterol, 1 nmol mL -1 formoterol and 10 nmol mL -1 formoterol, respectively.The cell treatment lasted for 90 minutes before collection according to a previous study [47] .The entire cellular experiment procedures were illustrated in Figure S6.

Immunofluorescence
VSMC were seeded in 12-well dishes with a cover glass inside.After treatment, cells were washed with PBS and fixed with 4% PFA for 15 minutes at room temperature.
Then the cells were blocked with 5% BSA for 1 hour at room temperature and incubated with the SIRT1 antibody (ab110304, 1:200 dilution; Abcam, UK), αSMA antibody

SA-β-gal activity assay
Cells were stained to measure SA-β-gal activity by a commercial kit (C0602, Beyotime, China) according to the manufacturer's protocols.Briefly, cells seeded on the cover glass in 12-well dishes were washed and fixed with 4% PFA for 15 minutes at room temperature.Next, the cells were incubated at 37°C overnight in darkness with staining solution containing 0.05 mg mL -1 5-bromo-4-chloro-3-indolyl-b-d-galactopyranoside (X-gal).The blue-colored cells were regarded as SA-β-gal positive.The cells were also counterstained with DAPI for nucleus and the SA-β-gal activity-positive cells were detected by light and fluorescence microscopy (DS-Ri2, Nikon, Japan).The results were analyzed by ImageJ 1.53k software (Wayne Rasband, USA).

Measurement of intracellular cAMP level
Intracellular cAMP level was measured using enzyme-linked immunosorbent assay for quantitative detection of mouse-cAMP kits (TAE-239m, Anoric, China) according to the manufacturer's instructions.

Data acquisition and differentially expressed genes (DEGs) analysis
The mRNA expression profiling dataset GSE174556 was downloaded from the GEO database.The samples consisted of the entire aortas isolated from ApoE -/-mice receiving either 5000IU kg -1 EPO (n=3) or vehicle (n=3) treatment.The dataset was sequenced on the Illumina HiSeq 4000 (Mus musculus; Illumina Inc., USA).All differential expression genes (DEGs) were screened with the "edgeR" package of R software 3.6.3for background correction and normalization.The normalization and log2 transformation of gene expression was also conducted with "edgeR" package in R. The genes with false discovery rate (FDR) < 0.05 and |log2(Fold change) | > 1 were regarded as statistically significant DEGs.

Pathway enrichment analysis
The total DEGs as well as the upregulated and downregulated DEGs were enriched with both Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.The enrichment analyses were performed on Metascape [48] and KOBAS [49] .Results obtained from Metascape were visualized with Cytoscape 3.9.0.

Screening of potential targeted drugs against EPO-induced AAA
Connectivity Map (CMap) provides a systematic approach for discovering associations among therapeutic agents, gene expression changes, and biological pathways based on genome wide expression profiling to repurpose the "old drugs" for new use (https://clue.io/data) [22,23].The mechanism of CMap is to find compounds affecting

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ab150080, 1:200 dilution; Abcam, UK) were applied to the sections and incubated for 1hour at room temperature.The negative controls were displayed with sections reacting with non-immune IgG as well as secondary antibodies.The slides were visualized by an electric upright microscope (DS-Ri2, Nikon, Japan) and the Pearson's R value for co-localization was calculated by ImageJ 1.53k software (Wayne Rasband, USA).

Figure S1 .
Figure S1.Weighted correlation network analysis (WGCNA).A) Detection of outlier in sample clustering.The clusters covered all samples and soft threshold power analysis helped to obtain the scale-free-fit index of the network topology (soft threshold power=14).B) Cluster dendrogram of genes based on co-expression relations determined by WGCNA.C) Heatmap of the topological overlap matrix (TOM) of selected genes.The higher degree of overlap, the darker.D) Module-trait relationships of different modules and disease status.E) Co-expression network of genes in the intersection.F) Protein-protein interaction network of genes in the intersection.

Figure S2 .
Figure S2.Schematic diagram showing the in vivo experiment protocol in ApoE -/-mice.

Figure S3 .
Figure S3.Characteristics and biochemical measurements in five groups of ApoE -/- mice.A) Body weight in five groups of ApoE -/-mice (n≥11 per group).B) Heart rate in five groups of ApoE -/-mice (n≥11 per group) C) Systolic, mean and diastolic blood pressure in five groups of ApoE -/-mice (n≥11 per group) D-G) Serum concentrations of TC, TG, LDL-C and HDL-C in five groups of ApoE -/-mice (n=7 per group).**P<0.01,***P<0.001,one-way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Figure S4 .
Figure S4.Photographs of aortic specimens in individual ApoE -/-mice.A) Photographs of all aortic specimens in ApoE -/-mice receiving vehicle, EPO, EPO+low-dose formoterol (FORM), EPO+medium-dose FORM and EPO+high-dose FORM treatment, respectively.The pound sign (#) indicates the specimen used in figures.The pound sign

Figure S5 .
Figure S5.Photographs of all aortic specimens in ApoE -/-mice receiving vehicle, lowdose FORM, medium-dose FORM and high-dose FORM treatment, respectively.

Figure S6 .
Figure S6.Schematic diagram showing the in vitro experimental protocol in VSMC.