Distinct mechanisms of β-arrestin-biased agonist and blocker of AT1R in preventing aortic aneurysm and associated mortality

Abstract

BACKGROUND:

Aortic aneurysm (AA) is a “silent killer” human disease with no effective treatment. While the therapeutic potential of various pharmacological agents have been evaluated, there are no reports of β-arrestin biased AT1R agonist used to prevent the progression of AA.

METHODS:

We tested the hypothesis that β-arrestin-biased AT1R agonist, TRV027 infusion in AngII-induced mouse model of AA prevents AA. High-fat diet fed ApoE-null mice were infused with AngII to induce AA and co-infused with TRV027 and a clinically used AT1R blocker Olmesartan to prevent AA. Aortas explanted from different ligand infusion groups were compared to assess different grades of AA or lack of AA.

RESULTS:

AngII produced AA in ~67% male mice with significant mortality associated with AA rupture. We observed ~13% mortality due to aortic arch dissection without aneurysm in male mice. AngII-induced AA and mortality was prevented by co-infusion of TRV027 or Olmesartan, but through different mechanisms. In TRV027 co-infused mice aortic wall thickness, elastin content, new DNA and protein synthesis were higher than untreated and Olmesartan co-infused mice. Co-infusion with both TRV027 and Olmesartan prevented endoplasmic reticulum stress, fibrosis and vasomotor hyper responsiveness.

CONCLUSIONS:

β-arrestin-biased agonist, TRV027-engaged AT1R prevented AA and associated mortality by distinct molecular mechanisms compared to the AT1R blocker, Olmesartan. Developing novel β-arrestin biased AT1R ligands may yield promising drugs to combat AA.

GRAPHIC ABSTRACT:

A graphic abstract is available for this article.

Graphical Abstract

Introduction

In humans, aortic aneurysm (AA) is a “silent killer” complex disease characterized by progressive expansion of aortic lumen, failing vessel wall associated with inflammation and torn elastic lamina eventually causing blood seepage [1]. AA is caused by a large variety of genetic (mutations in FBN, COL3A1, ACTA2, or βMHC genes) [2–8], and environmental risk factors (age, gender, obesity, smoking) [9]. A major cause of AA mortality is fatal hemorrhage from aortic aneurysm dissection (AAD) accounting for >20,000 deaths per year in the USA [10]. Current standards of care are medical monitoring of the aortic bulge, followed by open or endovascular repair surgery. Unfortunately the 4–8% who survive surgery are at risk of secondary rupture requiring multiple re-intervention [11]. Limited understanding of the mechanisms contributed to no proven pharmacological therapy to prevent AA. Phenotypic manifestations in AA correlate with enhanced signaling by neuro-hormonal GPCRs. Consequently, common medications to ameliorate symptoms include either beta adrenergic receptor blockers, angiotensin II type 1 receptor (AT1R) blockers (ARBs), angiotensin converting enzyme inhibitor (ACEi) or calcium channel blockers [12–17].

Studies of AA mechanisms have relied on animal models in which pharmacological or genetic manipulations produce AA. In three commonly used mouse models, infusion of angiotensin II (AngII) [18–20], intraluminal perfusion of elastase [21], or peri-aortic application of calcium chloride [22] produce AA. AngII-induced experimental AA in apolipoprotein E gene, ApoE-null mice [20] recapitulate many pathological manifestations of human AA including elastin breaks, extracellular matrix degradation, inflammatory cell accumulation, aortic rupture as well as age dependence and gender dimorphism. Genetic ablation of the Agtr1a gene prevents AngII-induced AA [23]. Agtr2-null genotype accelerates AA in mouse model of Marfan syndrome [24]. Likewise, pharmacological intervention using ACEi or ARBs diminishes AngII-induced AA [25] and using AT2R inhibitor PD123319 augments disease severity [26]. These preclinical data suggest ARB repurposing for treating AA in patients. Indeed supporting evidence exists for reduced rate of aneurysm growth in individuals who have hypertension as a comorbid condition and received ARB treatment [12]. Thus, modifying AT1R signaling might help develop a more robust intervention for AA.

Recent pharmacological and structural advances provide a choice of directing AT1R to selectively activate β-arrestin dependent outcomes [27, 28]. Physiological AT1R activation by AngII produces intracellular response through both G protein Gq/11 and β-arrestin. For example, Gq/11–signaling produces acute vasoconstriction followed by β-arrestin signaling which restores normal vascular tone through desensitization, internalization and plasma membrane recycling of AT1R. In addition β-arrestin activation regulates long-term effects of AT1R on protein synthesis, cell-growth and survival without requiring G-protein signaling [29, 30]. Chronic activation of AT1R/G-protein signaling is associated with aberrant molecular mechanisms involved in AA including sustained vasoconstriction, vascular inflammation and fibrosis. That in turn could result in aneurysm formation [31, 32]. ACEi and ARBs prevent the adverse outcomes of G-protein signaling [1, 13, 14, 33], but they also inhibit β-arrestin activation consequently turning off the beneficial pathways. Novel β-arrestin biased agonists including Sar¹-Ile⁴-Ile⁸-AngII (SII) [34] and TRV120027 (TRV027), a derivative with greater efficacy can be used to engage β-arrestin dependent AT1R signaling while inhibiting the G-protein signals [35–37]. Previous studies demonstrated that TRV027 lowered blood pressure similar to ARBs but unlike ARBs, TRV027 preserved cardiac performance and stroke volume [37, 38]. Beneficial effects of TRV027 were also observed on hypertension and vascular function [39]. Interestingly, this biased ligand potentiated water intake and increased salt-aversion behavior in a salt induced hypertension model [40]. The β-arrestin biased AT1R ligands have not been evaluated in a vascular disease model in which the renin-angiotensin system is known to be activated.

We hypothesize that TRV027 co-infusion in an AngII-induced model of AA will block damaging effects of AT1R while sustaining beneficial signals on vessels, such as protein synthesis and long-term survival of vascular cells through preservation of proliferation (DNA synthesis) in the aortic wall. We compared the physiological and molecular effects of TRV027 co-infusion to that of AngII and the ARB, Olmesartan (OLM). We demonstrate that TRV027 inhibited AngII-induced AA similar to OLM in ApoE-null mice. However, in contrast to OLM, vessel preservation is mediated by TRV027 through maintenance of DNA and new protein synthesis without causing endoplasmic reticulum stress and vascular fibrosis. In addition, TRV027 maintained contraction/relaxation response to neuro-hormones. These findings suggest that long-term beneficial effects can result from directing the GPCR signaling towards β-arrestin pathway to promote and preserve vascular renewal.

Materials and Methods

Data Availability:

The authors declare that all supporting data are provided within the article and gel images are available from the corresponding author upon reasonable request.

Animal care and use:

Male and female C57BL/6J and ApoE-null mice (B6.129P2-Apoetm1Unc/J) were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Mice were cared for in accordance with the Guide for the Care and Use of Laboratory Animals [41] in the Biological Resources Unit. All mouse experimental protocols described were reviewed and approved specifically for this project by the Institutional Animal Care and Use Committee at the Cleveland Clinic. Mice were housed (5 animals/cage) with Aspen hardwood chips (#7090A) bedding, Harlan Teklad Global, provided with Reverse Osmosis drinking water system, exposed to 14:10 hrs. of controlled light/dark cycle, with temperature control 18 – 23°C. Mice were fed a normal Harlan Teklad rodent diet with w/w (18.6 % protein + 44.2% carbohydrate + 6.4 fat + 0% cholesterol). In specified experiments, mice were fed high-fat (or Western) rodent diet (TD 88137, Harlan Teklad, Madison, USA) with w/w (17.3% protein + 48.5 % carbohydrate + 21.2 % fat + 0.2 % cholesterol and saturated fat >60%). Mice are cared for and monitored for signs of distress by trained veterinarian and technical staff who are on duty seven days of the week.

Administration of ligands:

To examine the in vivo safety profile of angiotensin peptides for infusion experiments, C57BL/6 mice were used. Thus, we compared:

1. No ligand group: vehicle,

2. Agonist [Sar¹]AngII (1.4 μmol/g, Bachem, Torrance, CA),

3. β-arrestin biased agonist [Sar¹,Phe⁴,dAla⁸]AngII (1.4 μmol/g, LRI peptide synthesis core facility),

4. β-arrestin biased agonist [Sar¹,dAla⁸]AngII (also known as TRV120027 or TRV027) (1.4 μmol/g, LRI peptide synthesis core facility)

5. The ARB, Candesartan (1.4 μmol/g, lot # B01909, Astra-Zeneca, Philadelphia, USA).

Each ligand was infused subcutaneously using osmotic mini pumps (Alzet model 2001, Durect Corporation, Cupertino, CA, USA) for 28 days starting at 8 weeks of age, as described previously [42]. Blood pressure changes and organ weight/body weight ratios were monitored. (Schematic design shown in Fig. S1A).

Ligand administration in AA model:

To examine the effect of the biased ligand in AA model, ApoE−/− mice fed with high fat diet and infused with AngII were used. This experimental model is well established model of AA because it recapitulates some key features of the human disease such as vascular inflammation, macrophage infiltration, medial elastolysis, luminal expansion and thrombus formation [20, 43–45]. Disease progression in the AngII infused group was challenged by co-infusion with either the β-arrestin-biased agonist TRV027 or the ARB, OLM (surrogate for ARB, Candesartan). The morphological, molecular and functional effects on aneurysm formation were monitored.

Male and female ApoE−/− mice at 8-weeks age were shifted to high fat diet and maintained for 42 days on this diet. At 10-weeks of age 6 groups received subcutaneous implantation of Alzet osmotic pumps loaded as follows:

1. No ligand group: Vehicle,

2. The AA group:1.4 μmol/g AngII (Cat # 4006473.0025, Bachem, California, USA);,

3. TRV027 group: 1.4 μmol/g TRV027, (LRI peptide synthesis core facility and Genscript, New Jersey, USA)

4. TRV027 co-infused group:1.4 μmol/g AngII+ 1.4 μmol/g TRV027,

5. OLM group: 1.4 μmol/g OLM (lot # B01909, Daiichi Sankyo Chemical Pharma, New Jersey, USA),

6. OLM co-infused group: 1.4 μmol/g AngII+ 1.4 μmol/g OLM,

Body weights for mice were recorded after recovery from surgery. At the end of 28 days of infusion body weight was recorded, mice were euthanized and aorta and other organs collected for analysis. Osmotic pumps explanted from individual mice were examined for success of ligand infusion in each mice. The aortas underwent into histological, molecular and functional evaluation (Study schematic design Fig. 1A).

Figure 1: Effect of β-arrestin biased ligand co-infusion in Aortic Aneurism prone mouse model:

Study design schematized for placing ApoE^−/− male and female littermates on high-fat diet (HFD) followed by mini pump installation for ligand infusion, blood pressure measurement (BPM) and analysis of vascular pathology, signaling and functions. Eight week old mice were placed on high fat diet and maintained on this diet until 14 weeks old. Osmotic mini pump was installed on day 14 post HFD feeding for infusing different ligands indicated and the animals were monitored for 28 days (A). Percent survival of Apo E−/− mice infused with AngII and co-infused with the β-arrestin biased ligand, TRV027 or the AT1R blocker, Olmesartan (OLM). Mortality in AngII infused males (B1) was 77.5% but none was seen in females (B3) in the 28 day period. The early deaths were mostly due to aortic arch rupture. Majority of deaths recorded were around day 20 of AngII infusion, they were related to AA. Hence, euthanasia of animals was performed on the 15^th day of AngII infusion to obtain more accurate survival graph shown in (B2). Co-infusion with TRV027 or OLM prevented the development of AA, observed as 100% survival in both B1 and B2 groups. Comparison of survival curves were performed using Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test showed ****<0.0001 for male infused with AngII compared to the other groups.

Measuring blood pressure:

Systolic blood pressure (BP) was measured by non-invasive tail-cuff blood volume pressure recording and analysis method using the CODA multi-Channel High Throughput system as reported earlier from our group [46]. Mice in experimental groups were trained for five days to obtain consistent BP reading in the conscious state as described earlier [47, 48]. BP was recorded in a blinded fashion on −7^th day and day prior to osmotic pump implant surgery and on 27–28 days after for each mouse. An average of five readings were recorded for each mouse before and after, in each ligand infusion group.

Morphometry analysis:

Whole aorta from aortic arch to the iliac bifurcation was harvested from mice euthanized by isoflurane. The perivascular adipose tissue was removed, aortas were then pinned down to a wax background respecting their natural resting tension and digitally photographed images were acquired using a Leica MZ16FA stereomicroscope (Leica Microsystems, GmbH, Wetzlar, Germany) equipped with a Retiga SRV camera and QCapture-Pro software (QImaging, Surrey, BC, Canada) using the 5x lens. Maximum diameters of the following 4 regions: aortic arch, thoracic, supra-renal and infra-renal aorta were assessed from the images using Image-Pro Plus software (Media Cybernetics, Inc., Rockville, Maryland, USA). The area of aneurysm was determined in aortas that presented aneurysm using the same software.

Histopathology analysis:

3–4 mm of the supra-renal region of the aorta was dissected and fixed overnight in1X HistoChoice tissue fixative (catalog #H120–1L, VWR Life Science, Illinois, USA).Following fixation, tissues were embedded in paraffin, and transverse sections (5um thick) were prepared for Hematoxylin & Eosin (H&E), Masson’s Trichrome (MT) or Verhoeff van Geison (VVG) staining to assess how different treatments affect cell infiltration profile, collagen and elastin content. H&E staining was automatically performed using a Leica Multistainer ST5020 and Eosin-Phloxine staining solution (catalog #1082, Newcomer Supply, Middleton, USA). The Masson’s trichrome and VVG staining were performed manually using kits (catalog # 9176A and 9116A, Newcomer Supply, Middleton, USA, respectively) as directed by the manufacturer. Images of the stained cross section were acquired using a Leica DMR upright microscope equipped with a Leica DMC4500 camera with LAS X software (Leica Microsystems, GmbH, Wetzlar, Germany) and a Hamamatsu Orca R2 camera (Hamamatsu Photonics, Shizuoka, Japan). Qualitative assessment was performed using H&E staining. The VVG stained images were used to quantitate the adventitia and media thickness as well as the elastin content. The MT stained images were used to measure the fibrosis content. The previous mentioned image analysis was done using Image-Pro Plus (Media Cybernetics, Inc., Rockville, Maryland, USA).

Vascular protein synthesis in vivo by surface sensing of translation (SUnSET):

Puromycin, an aminonucleoside antibiotic produced by Streptomyces alboniger, is used in the SunSET method to label newly synthesized proteins in the vessel wall. The concept behind this strategy is that puromycin has an analog structure of aminoacyl-tRNAs, thus it is incorporated into the nascent polypeptide chain of synthesized proteins, reflecting the rate of mRNA translation in vivo [49–51]. Thus, puromycin was injected in vivo to quantify protein synthesis. 150μL of puromycin (catalog # A11138–03, Gibco, Life Technologies, New York, USA) at a concentration of 0.04 μmol per gram of body mass was injected intraperitoneally. Thirty minutes later, mice were euthanized and their aortas were harvested as described above. Further, perivascular adipose tissue was removed while the aortas were embedded with cold PBS containing a cocktail of protease (catalog # P8340, Sigma, USA) and phosphatase inhibitors (catalog #1862495, Thermo Scientific, USA) at the concentration recommended by the manufacturer. Cleaned abdominal aortas were homogenized in T-PER Tissue Protein Extraction reagent (catalog # 78510, Thermo Scientific, USA) containing protease and phosphatase inhibitors. Supernatants were collected after the homogenates were centrifuged at 10000xg for 10 minutes. Abdominal aneurysm samples were treated to remove hemoglobin captured within the vessel wall. NuGel-HemogloBind (catalog # NP-HO-T-50, Biotechsupport group, New York, USA) was used according to the manufacturer’s protocol. Quantification of the proteins in the supernatants was performed using Bio-Rad Bradford Protein Assay Dye (catalog # 5000006, Bio-Rad Laboratories, USA). To quantify the puromycin incorporation a standard Western blot was performed with 15 μg of protein, incubated overnight at 4°C with 1:10,000 anti-puromycin primary antibody (catalog # MABE343, EMD Millipore Corp, USA) and 1h at room temperature with 1:10,000 anti-mouse secondary antibody (Cell signaling, cat# 7076S). Membranes were developed in ECL substrate and bands visualized on auto radiographic film. Source information regarding primary antibodies used in this study is provided in Table S1.

Protein and DNA synthesis Signaling pathways:

The signaling pathways were assessed using immunoblots on protein extracts. In brief, electrophoresis of 10μg protein of each sample was performed on 4% to 12 % gradient gels, transferred to nitrocellulose membrane and incubated with primary followed by infrared secondary antibody at the recommended dilutions by the manufacturer. Images of the blots were acquired using an Odyssey CLxScanner (LI-COR Biotechnology, Nebraska USA), and densitometry performed using Image Studio software (LI-COR Biotechnology, Nebraska USA).

The following molecules were tested for protein synthesis, autophagy and DNA damage pathways.

Proliferation evaluation by immuno-histochemical detection of Ki67:

Paraffin-embedded formalin-fixed aorta tissue was sectioned at 5¼ thickness. Immuno-histochemical staining was performed using the Discovery ULTRA automated stainer from Roche Diagnostics (Indianapolis, IN). In brief, antigen retrieval was performed using a tris/borate/EDTA buffer (Discovery CC1, 06414575001; Roche), pH 8.0 to 8.5, for 56 minutes at 95°C. Slides were incubated with anti-Ki67 at a 1:300 dilution (NB110–89717PE, Novus Biologicals; Centennial, CO), for 1 hour at room temperature. Bound anti-Ki67 antibody was visualized using the OmniMap anti-Rabbit HRP secondary (05269679001; Roche) and the ChromoMap DAB detection kit (05266645001; Roche). Lastly, the slides were counterstained with hematoxylin and Eosin.

Proteomic evaluation by mass spectrometry:

The plasma and aorta lysate samples were centrifuged at 20,000 x g for 15 minutes. Five μL plasma was taken from each sample and transferred to a new 1.5 ml Eppendorf tube. Forty five μL 8M urea Tris-HCl pH8 lysis buffer with freshly added protease inhibitor cocktail was added into each plasma sample. Protein concentrations of the samples were determined by BCA. Forty μg of protein from each of the samples were taken based on BCA result. In the case of the aorta lysate samples, twenty μg of protein were taken. The samples were reduced by dithiothreitol, alkylated by iodo acetamide and precipitated by cold acetone (−20°C) overnight. Samples were centrifuged at 12000 x g for 8 minutes at 0°C, and the supernatants were removed. Protein pellets were air dried for 30 minutes and dissolved in 40 μL100 mM tri-ethyl ammonium bicarbonate (TEAB) with 0.5 ¼g trypsin per sample. After overnight incubation, digested samples were centrifuged at 20,000 x g for 15 minutes, and 5 ¼g of the digest from each sample was transferred to a new tube and dried down in a Speed-Vac and reconstituted in 25 ¼l of 0.1% formic acid. Ten ¼l sample was mixed with 10 ¼l 0.5x concentration iRT standards and the samples are ready for LCMS analysis.

To build the spectral library plasmas or lysates samples were pooled. The pooled sample was desalted using a Waters Sep-Pak C18 cartridge. The desalted pooled sample was offline fractionated into 16 fractions using high pH reversed phase HPLC method on a Waters XbridgeC18 chromatographic column. Around 5 ¼g peptide from each fraction was transferred to a new tube and dried down in a SpeedVac and reconstituted in 25 ¼l 0.1% formic acid. Ten ¼l sample was mixed with 10 ¼l 0.5x concentration iRT standards and the samples are ready for LCMS analysis.

The LC-MS system was a BrukerTOF pro2 mass spectrometry system interfaced with a Bruker NanoElute HPLC system. The HPLC column was a BurkerFifteen 15 cm x 75 μm id reversed-phase capillary chromatography column. Four μL of the sample were injected and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.3 μL/min were introduced into the source of the mass spectrometer on-line. The micro-electrospray ion source is operated at 1.5 kV. For spectral library generation, the digest was analyzed using a data dependent acquisition (DDA) method, the instrument acquires full scans followed by MS/MS scans of the most abundant ions from the full scans in successive instrument scans. For LC-MS analysis of the samples, a data independent acquisition (DIA) method was used. The instrument acquires full scans followed by MS/MS scans of 32 fixed variable mass windows along the whole LC gradient. The spectral library was generated using Pulsar that is integrated in Spectronaut software package searching the DDA LC-MS data from the 16 fractions of the pooled sample.

The DIA data were analyzed using Spectronaut V16 to search against the spectral library and the mouse UniProtKB protein sequence database for the identification and quantification of proteins and peptides. False discovery rate of protein was set at 1%.

Vasomotor function analysis:

Abdominal aorta cleaned of perivascular tissue were used to cut aortic rings of 2–3mm in length, which were mounted onto 100μm pins in chambers connected to a myograph system (Multi Wire Myograph System, 720 M DMT-USA, Michigan, USA), containing 5 mL physiological salt solution, PSS (130mM NaCl, 4.7mM KCl, 1.18mM KH2PO4, 1.17mM MgSO4–7H2O, 14.9mN NaHCO3, 5.5mM dextrose, 0.026mM CaNa2 versenate, 1.6mM CaCl2) at 37°C, and gassed with 5% CO2 and 95% O2. After stabilization period of one hour at basal tension, vessels were equilibrated at a resting lumen diameter of 0.9×L100 (L100 represents vessel diameter under passive transmural pressure of 100 mmHg). Aortic tension was displayed and recorded with LabChart software (ADINSTRUMENTS, Colorado Springs, USA). Resting tension was determined by applying increased step-wise tension 2 mN to 10 mN. Fifteen minutes later, two potassium-induced constrictions were measured using high concentration potassium solution (74.7mN NaCl, 60mM KCl, 1.18mM KH2PO4, 1.17mM MgSO4–7H2O, 14.9mN NaHCO3, 5.5mM dextrose, 0.026mM CaNa2 versenate, 1.6mM CaCl2 ). Vessels were then pre-constricted with 1μm phenylephrine followed by relaxation with 1μm acetyl-β-methylcholine chloride in pre-constricted vessels to ensure intact endothelial cell layer. Arteries were only used for investigation if they constricted in response to phenylephrine (PE) and dilated in response to acetyl-β-methylcholine chloride.

Concentration–response curves of 5-HT were obtained by exposure of the vessels to 100pM to 300 uM concentrations of 5-HT. Relaxation was induced after pre-contracting the vessel with the EC50 dose of 5-HT. Relaxation-response curves of OLM were obtained by exposure of the vessels to 100 pM to 300 uM concentrations of OLM.

Statistical analysis:

Animal data were obtained from male and female mice in each group. All graphs and data generated in this study were analyzed using GraphPad Prism 9 Software, and are presented as means ± SEM. Means were compared using unpaired t test or analysis of variance with one-way ANOVA followed by Tukey’s multiple comparison test for two or more groups respectively. Survival curves were analyzed by Log-rank (Mantel-Cox) test. A p value < 0.05 was considered statistically significant.

Results:

Novel β-arrestin biased AngII-analogs compared to [Sar1]AngII in C57BL/6 mice

To assess the in vivo effect of β-arrestin biased AngII-analogs we followed the study design schematized Fig.S1. The data indicated that systolic BP was significantly increased in both male and female mice infused with [Sar1]AngII. Decreased body weight, hypertension associated with cardiac hypertrophy was observed in the [Sar1]AngII group. BP, body weight and heart weight were unaffected in the biased ligand, [Sar1, Phe4, dAla8]AngII and TRV027 groups, similar to the no ligand group. No adverse events observed indicate that the novel β-arrestin biased AngII-analogs are as safe as the ARB, Candesartan for using in the chronic infusion studies. Important note, agonist [Sar1]AngII was used because the biased ligands have Sar1 at position 1 of each peptide, which increases AT1R affinity.

Effects of AngII+TRV027 and AngII+OLM co-infusion in ApoE−/− mice

To evaluate effects in a disease model, we chose HFD-fed ApoE-null mice for further experiments infused with physiological agonist AngII, β-arrestin biased agonist, TRV027 and ARB, Olmesartan instead of Candesartan. As indicated in Fig. S1B co-infusion of TRV027 and ARB helped maintained the BP at similar levels as the no ligand group. The body weights were similar between no ligand, AngII+TRV027 and AngII+OLM groups. We could not record changes in BP, body weight and heart weight ratio in AngII-infused ApoE−/− males due to high rate of mortality in the 28 day infusion period. However, it has been broadly reported that AngII infusion increases the BP in ApoE−/− mice [52, 53]. Accordingly, significant increase of cardiac/body weight ratio was observed in AngII infused males. No significant cardiac hypertrophy was observed in female Apo E−/− mice infused with AngII, and upon co-infusion with TRV027 or ARB (Fig. S1B). These observations suggest that co-infusion with TRV027 blocked the hemodynamic effects of AngII similar to well-established effect of the ARB, Olmesartan.

Prevention of AngII-induced AA by TRV027 and OLM

We assessed the effect of TRV027 or OLM on AngII-induced AA in HFD fed ApoE−/− mice, (Fig. 1A). In AngII infused males mortality was ~67% but none in females along the 28 days period. Hundred percent survival was observed in TRV027 or OLM co-infusion groups suggesting that co-infusion strategy prevented the development of AA (Fig. 1B.1). Mortalities observed around day 6 of AngII infusion were mostly due to arch rupture near the aortic root. Deaths around the day 20 of AngII infusion were associated with AA predominantly in the supra renal aortic segment. Therefore, we performed euthanasia at 15^th day of AngII infusion in order to grade aneurysm progression. Consequently, the mortality rate decreased to 39% (Fig B1 and pie-chart in Fig. 2). Female mice did not show any mortality in all treatment groups (Fig B1).

Figure 2: Grades and penetrance of aortic aneurysm in Apo E−/− male mice upon infusion with AT1R ligands:

Aneurysms were graded as I, II, III and IV as described previously, (A). Because most AA related death in male AngII-infused mice occurred around day 20, euthanasia was performed on the 15^th day of AngII infusion and co-infusion groups to obtain distribution of grades of AA shown in the pie-chart in this experiment (B). Co-infusion with TRV027 or OLM significantly prevented the development of AA showing 100% survival in TRV and OLM co-infused groups compared to only 22% with no disease in the AngII group. Pie-chart shows the disease and mortality spectrum.

AngII infusion for 15 days before euthanasia showed a diverse spectrum of aortic disease in male ApoE−/− mice (Fig. 2). The experimental AA severity was classified based on the assessment of morphometry and pathology of the excised aortas as described by Daugherty et al. [54]. Approximately 22% mice presented no features of AA, which was also described in previous studies [49]. Grade I was defined as a dilated lumen in the supra-renal region of the aorta with no thrombus. We did not observe Grade I pathology. Grade II, remodeled tissue in the supra-renal region that frequently contained thrombus was observed in 13%. Grade III, a pronounced bulbous form of grade II that contains a thrombus was observed in 9%. Grade IV a form in which there are multiple aneurysms containing a thrombus, some overlapping, in the suprarenal area of the aorta was observed in 17%. The 39% deaths in males includes rupture of the aortic arch region observed in ~13%, rupture of abdominal AA grade IV in ~22% and grade III in 4% mice (Fig. 2B). In meta-analysis of 33 studies aneurysm incidence reported were 22% grade I, 26% grade II, 29% grade III, and 24% grade IV. However, these studies did not differentiate between males and females. The AA grade distribution observed suggests that there is no correlation between AA grade or length of AngII exposure days and rupture. Co-infusion of AngII with TRV027 prevented rupture of the aorta as well as AA development in male mice.

In contrast, we observed 7% grade III, 7% grade I, 21% grade II in female mice (not shown). Our data was in agreement with the report that female gender is a protective determinant against AA susceptibility [55]. Thus, due to the lower penetrance of aneurysm in females, female groups were not further evaluated in this study.

TRV027 and OLM effects on aortic wall dimension and elastin content

We studied the mechanisms of protection in TRV027 and OLM co-infused groups. When the outer diameter of segments of the aorta were compared, mean diameter of the aortic arch, thoracic aorta, supra-renal aorta were significantly increased (p>0.05) in male AngII infusion group over no ligand, TRV027 or OLM co-infused groups. Diameter of the infra-renal aorta segment was unaffected in all four groups, which served as an internal control for comparison (Fig 3A). Aortic wall area observed in cross sections was significantly increased in TRV027 co-infusion group compared to no ligand and OLM co-infused group (Fig 3A). Similarly, infusion of TRV027 showed increased aortic wall area when compared to OLM group. More specific comparison of thickness of the adventitia, media and intima layers was performed for TRV027 and OLM co-infusion samples. An increase in thickness of media layer was observed in TRV027co-infused group. To evaluate whether elastin content is the basis for increase of aortic area, we evaluated elastin content by two methods. First, elastin area per cross section was quantified by VVG staining. This indeed showed the increase as anticipated (Fig.3B and Fig.4). Second, elastin protein content was evaluated by mass spectrometry. AngII caused increase in elastin content. Elastin content in TRV027 co-infusion group was increased (p=0.05) compared to the no ligand group. The elastin content did not show significant differences in TRV027, OLM, and AngII+OLM infusion groups.

Figure 3: Morphometry and elastin content of the aorta:

After 28 days of infusion with the different ligands, aortas from male Apo E^−/− mice were collected and perivascular fat removed. Diameter of the following aortic segments; the arch, thoracic, supra-renal and infra-renal regions, were measured as indicated. Dilation was observed in the arch, thoracic and supra-renal aorta in the group infused with AngII (A). Deeper analysis was performed to further evaluate the effect of TRV027 versus the Olmesartan (OLM) infusion groups. Thus, cross section areas were evaluated in the co-infused groups. We observed increased cross section area in the TRV027 group compared to no ligand and OLM groups. We also observed increased area in the AngII+TRV027 co-infused group. Elastin content was evaluated by two methods. First, quantification of the area was performed after using VVG staining (see Fig 4). Higher elastin content was observed in the AngII+TRV027 group compared to the no ligand and AngII+OLM groups. Analysis of the elastin protein concentration by proteomics showed increased in elastin concentration in the AngII+TRV027 group compared with the no ligand group (B). Ordinary one-way-ANOVA followed by Tukey’s multiple comparisons test was performed *<0.05, **<0.004, ***<0.0007 ****<0.0001

Figure 4: Histopathology analysis of abdominal aorta:

Evaluation of general histology using hematoxylin and eosin (H&E) staining showed infiltration of immune-cells in the AngII infusion group (→) and not in other groups. Infiltration of red blood cells in the vascular smooth muscle cell layer was observed only in AngII infusion group (→→). The formation of blood vessels was observed in the adventitia layer of only in AngII infusion group (→→→). Thickness of the media layer was decreased in the AngII + OLM co-infused group. In contrast, thickness of the vessel wall was increased in the AngII+TRV027 co-infusion group compared to the no ligand group. Evaluation of Verhoeff–Van Gieson (VVG) staining showed damage to elastin fibers in the AngII group (→→→→) and not in other groups. The elastin fiber thickness was increased in the AngII+TRV027 co-infused group. 5X, 10X, 40X and 100X were the lens magnification used.

Similarly, the collagen stained area per cross section and collagen protein content by mass spectrometry were compared between the groups no ligand, TRV027 and OLM. The collagen area per cross section observed was at similar levels in the co-infusion groups. However the collagen 1 and 3 protein contents by mass spectrometry was higher in the TRV027 co-infused group compared to no ligand and TRV027 (Fig. S2).

Supra-renal region of the aorta susceptible to aneurysm in the ApoE−/− mice were examined by hematoxylin and eosin staining (Fig.4). This showed asymmetrical structural changes only in AngII-infused group. The vessel wall region between the outer elastin layer and the adventitia showed severe thickening due to remodeling with the presence of thrombi and embedded with nuclei indicating infiltration of different inflammatory cells. Hundred times magnification images of the inner media layer showed red blood cells between the elastin fibers in the lamellar unit. Additionally, this region clearly showed elastin fragmentation indicating degradation of medial elastin. Fibrotic collagen deposition was assessed by Masson’s trichrome staining (Fig. S2). The remodeled region of the wall was characterized by fibrosis. The region showed infiltration of immune-cells and red blood cells in the vascular smooth muscle cell layer, as well as formation of blood vessels in the adventitia layer. Thus, two closely connected features, inflammatory remodeling and extracellular matrix degeneration, contributing to weakening of the vessel wall are observed in the AngII-infused group. The apparently “intact wall” region of the aorta seen in the sections, was characterized by lower collagen deposit per area compared to intact wall in no ligand, AngII +TRV027, AngII + OLM groups and also AngII infused mice that did not develop AA. Evaluation of collagen 1 and 3 protein concentration were performed by mass spectrometry. Both collagens were increased in the aortas of the AngII group (Fig S2).

Effect of TRV027 and OLM on nascent protein synthesis

A previous report showed that AT1R/G protein and β-arrestin signaling increase protein synthesis in smooth muscle and HEK293 cells [51]. We evaluated de novo protein synthesis using the SUnSET method as described earlier [49–51]. Minimal amount of puromycin injected into peritoneum and allowed to incorporate into actively synthesized protein pool for 30 min. Puromycin incorporated protein profile in the supra-renal segment of the aorta visualized by immunoblot is shown in Fig.5. The pattern of newly synthesized proteins is impaired in the AngII group, as indicated by increased intensity of some protein bands and simultaneous decrease in other protein bands observed in AngII lane in Fig. 5. In contrast, intensity of diverse molecular weight bands was uniform in the no ligand, TRV027, Olm, AngII+TRV027 and AngII+Olm groups. Puromycin incorporation was slightly increased in the TRV027 group compared to the no ligand and OLM group.

Figure 5: Alteration of protein synthesis and inhibition of LC3 and ER stress by TRV027 and Olmesartan:

AngII infusion caused an impairment in the synthesized levels of some proteins associated with abdominal aortic aneurysm (blot). The overall level of protein synthesis was increased in the group infused with TRV027 compare to Olmesartan (OLM). Furthermore, the co-infusion of TRV027 with AngII or OLM with AngII, prevented the observed impairment caused by AngII. The reduction in protein synthesis found in the AngII group was corroborated by the decreased phosphorylation of AMPK which in turn caused increased phosphorylation of p70s6 and decreased phosphorylation of 4EBP. Increased in eIF2alpha phosphorylation is associated with alteration in the rate of mRNA translation. Concomitantly CHOP and LC3 levels were increased indicating ER stress and an increased in autophagy. ROUT test was run to identify outliers prior to performing One-way-ANOVA followed by paired t-test. * <0.05

Individual ligand effects on regulation of protein synthesis/metabolism was evaluated, by interrogating signaling proteins, AMPK, P70S6, eIF2a, 4EBP, LC3I/II and CHOP (Fig. 5). In AngII-infused males we observed a significant decrease of AMPK phosphorylation which suggests metabolic changes in affected tissue. A significant reduction of p-4EBP downstream of mTORC1 suggests impairment of CAP-dependent mRNA translation. The pAMPK and p-4EBP levels are similar in no ligand and TRV027 co-infusion groups implying normal metabolic state and mRNA translation (Fig. 5).

The phosphorylation of p70s6 and eIF2a protein was increased in AngII-infused males compared to no ligand group. Increased eIF2 phosphorylation could alter efficacy of mRNA translation. In TRV027 co-infused males increase of p70s6 phosphorylation compared to no ligand and OLM groups. We further dissected the mechanism of action of AngII and TRV027 by evaluating the autophagy related protein, microtubule-associated protein I/II-light chain 3 (LC3I/II) as well as the ER-stress marker C/EBP homologous protein (CHOP). The shift of LC3-I/II indicates that autophagic activity is occurring. Increased levels of LC3-II indicate formation of autophagosome and the autophagy cycle that is taking place [56]. Both LC3I/II and CHOP levels are significantly elevated suggesting potential existence of autophagy and ER stress in the AngII-infused aneurysm group. TRV027 co-infusion did not significantly elevate LC3I/II and CHOP levels, which is similar to the OLM and no ligand groups.

Effect of TRV027and OLM on cell proliferation signals

Homeostatic maintenance/renewal of vessels in our body requires cells to duplicate the genome and divide at a low rate. We measured the cell-growth in the aortic wall in no ligand, AngII, AngII+TRV027 and AngII+OLM groups by tracking the proliferation marker Ki67 (Fig. 6B). The total number of cells per cross sections in media and adventitial layers was counted. Interpretation in the AngII group was confounded by presence of blood within the wall, infiltration of inflammatory cells, different grades of AA and atrophy (hence Ki67 data are not presented for AngII group). An increase in Ki67 positive nuclei in TRV027 co-infused group compared to the no ligand group was reproduced in multiple mice. Interestingly, reduced Ki67 positive nuclei in the media in OLM co-infused group was significant (p <0.05) compared to TRV027 co-infused group (Fig 6A). Since the overall phenotype of aorta in these groups did not show inflammation and fibrosis, we did not evaluate infiltration of monocytes and myo-fibroblasts as potential contributing factors for Ki67 signal. Generally, Ki67 positive signal is interpreted as a proliferation indicator in the clinic, in the current state of understanding of this protein’s function and dynamics in proliferative cells. These findings suggest that most cells in the wall remain quiescent but TRV027/AT1R signaling in aorta may promote low level of cell proliferation and OLM may inhibit it.

Figure 6: Comparison of markers for cell proliferation and DNA synthesis:

The number of Ki67 positive cells were counted in the medial and adventitial layers. Which indicated that higher number of positive cells that entered the proliferation stage were found in the AngII + TRV027 group (A). It is important to note that in the adventitia layer, the number of Ki67 positive cells in the AngII+OLM group decreased compared to the AngII+TRV027. To ensure the specificity of Ki67 staining, besides testing the antibody in proliferative tissues such as tonsil, in the process of analysis pictures were split into 3 RGB channels. Brown colors not present in the RGB channel in which nuclei were present, were considered as non-specific and thus removed from the counting. In the AngII group, the presence of blood within the wall created difficulty to accurately count the Ki67 positive aortic wall cells (B). Comparison of markers for DNA synthesis indicated that in the AngII group the expression of Chk2 was downregulated whereas expression of p53 was upregulated. The p53 protein expression was higher in the AngII+TRV027 when compared to AngII+OLM (C). Ordinary one-way ANOVA followed by Tukey’s multiple comparison test was performed. Male and female were grouped per ligand. *<0.05, ** < 0.009.

We evaluated cell cycle regulatory signaling proteins, ATM, check point kinase 2 and p53 in total protein lysates of AngII-infused mice compared to the other co infused ligand groups. The check point kinase 2 signal decreased only AngII group. The p53 signal was inhibited significantly in male OLM co-infused group compared TRV027 and vehicle treated groups (Fig. 6C).

Effect of TRV027 and OLM on liver enzymes and inflammation markers

Mass spectrometry evaluation of the liver enzyme proteins in the plasma were performed. Gamma glutamyl transferase (GGT) and Aspartate amino transferase (AST) were altered. GGT was increased in the AngII group compared to the TRV027, OLM, and the co-infused groups. AST was increased in the AngII group compared to TRV027, OLM, and the co-infused groups.

Evaluation of the matrix metalloproteinases (MMPs) protein content in the plasma was done. Both MMP17 and MMP23 were increased in the AngII group compared to no ligand, TRV027, OLM, and OLM co-infused group. The MMP17 was increased in the AngII+TRV027 group compared to the OLM group. Likewise the MMP23 was higher in the AngII+TRV027 group relative to the no ligand, TRV027 and OLM groups (Fig. S3). The most AA implicated MMP2 and MMP9 proteins were present in our proteomics data, but the levels found are similar in all ligand infusion groups.

Effects of TRV027 and OLM on Vasomotor function

Whether vasomotor dysfunction contributed to AA and AAD pathogenesis is not fully explained. To evaluate the vasomotor response of the aorta to neuro-hormone stimulation after 28 day infusion of different ligands, we measured serotonin (5-hydroxytryptamine, 5-HT)-induced contraction. 5-HT was chosen because it’s coupling to contractile machinery is a Gq-dependent process, but independent of AT1R. Therefore, the 5-HT induced contraction is expected to be unaffected by possible effects of 28 days of AT1R-ligand exposure on receptor desensitization/down regulation (Fig. S4).

In the AngII treated group, significant variability in both agonist sensitivity (EC50) and magnitude of contraction (Emax) was observed compared to the no ligand group (Fig. S4). Normalized 5-HT dose-response curves (Fig. S4A) indicate that the diverse responses were associated with different grades of AA. In grade II AA the 5-HT sensitivity did not significantly change. In grade III AA the EC50 of 5-HT response significantly increased and in grade IV AA, the increase of EC50 value was even higher. These results imply that AA afflicted vessels perform contractile function with reduction of agonist-sensitivity as vessel inflammation and remodeling progress. In contrast, sensitivity of 5-HT response increased in the TRV027 treated animals compared to the no ligand group. Our data is in agreement with a previous report in which 14-day administration of TRV027 was found to increase vascular sensitivity to a variety of agonists including phenylephrine, acetylcholine and sodium nitroprusside [36].

In separate analysis, impact of potential AT1R desensitization/downregulation was evaluated by relaxation response of aorta when exposed to OLM. Aorta explanted from different ligand infusion groups were pre-contracted using the EC50 concentration of 5-HT which was calibrated for each vessel independently. Following the pre-contraction step, increasing doses of OLM were applied to obtain relaxation-response curves (Fig. S4A). Vessels from all treatment groups yielded measurable relaxation response in the range of 25–50%. The IC50 values did not change significantly in different treatment groups, suggesting intact relaxation response in different treatment groups.

Enhanced the acetyl choline-induced, endothelium dependent relaxation in presence of AT1R blockers is known [57], however the exact mechanism is not fully understood. In our study the contraction observed could be the net result of serotonin receptor effect on myofilaments and AT1 receptor sensitization perhaps among others. We speculate that Olmesartan’s inverse agonist character may be responsible for observed relaxation response.

Discussion

In this study we observed that β-arrestin biased ligand, TRV027 co-infusion blocked AngII-induced AA in the ApoE−/− mouse model with efficacy similar to that of the ARB, OLM. The molecular effects of TRV027 in preventing AA are distinct from OLM. Both TRV027 and OLM inhibited aortic lumen dilatation, asymmetric wall thickening, inflammation, vascular fibrosis, elastolysis and seepage of blood. However, TRV027 co-infusion increased the aortic wall area, elastin content, DNA and nascent protein synthesis while OLM co-infusion reduced/inhibited these processes. Consequently, several regulatory molecules involved in AT1R-dependent DNA and protein synthesis are differentially affected by TRV027 and OLM treatments.

We employed a ubiquitously used model, relevant to clinical findings that obesity and hypertension increase the risk of mortality in AA and AAD [58, 59]. The results (Figs. 1, 2 and Fig. S1) confirmed that AngII/AT1R interaction in the context of ApoE−/− genotype and high-fat diet together create the AA and AAD disease state. Therefore, TRV027 and OLM were tested in this model for efficacy to prevent the disease. In the co-infusion experiments, occupancy of AT1R favors TRV027 due to higher affinity conferred by Sar1 in TRV027 compared to AngII. Hence the phenotypes reflect molecular mechanism of the co-infused ligand. Infusion with AngII alone promoted AA quite rapidly (Fig. 2), with significant mortality in the 3–10 days interval as well as late mortality associated with AA rupture as reported by the majority of the groups using this experimental model [19, 54, 60, 61]. The 67% AA mortality in 28 days are similar to that other authors reported in the meta-analysis study of AA in mouse models [62]. We also observed 13% rapid mortality due to aortic arch dissection without aneurysm which has not been reported earlier. In our study, the penetrance of AA in females were 3 times lower than in males, a ratio that is in agreement with previous studies [9]. Although, AngII caused AA in females, mortality was not associated with AA. Spectrum of AA lesion severity observed in male AngII infusion group (Fig. 2) aligns well with reports from other research groups who have used the Daugherty et al. [19] classification of AA grades I-IV. Grades of aneurysm differ in our study for male and female. AA penetrance found in our study differs from the mean values reported in the meta-analysis of 33 experimental AA studies using Daugherty’s criterion. This could be because they did not differentiate between males and females in their report [19]. In the no ligand group we did not observe abdominal or thoracic aneurysms implying that combination of high-fat diet and ApoE−/− genotype do not produce sufficient risk for the disease and AngII/AT1R signaling is necessary. This is also corroborated by a meta-analysis study of 1679 saline-infused ApoE−/− mice controls used in diverse studies [62].

Our data indicated TRV027/AT1R signals produced upon co-infusion of TRV027 prevented development of AA and AAD in males, consequently reducing mortality. Previous ARB co-infusion studies, with losartan (30 mg/kg/day) [14, 26] and valsartan (1 mg/ kg/day) [14] have reported prevention of AA similar to our finding with OLM (0.74mg/kg/day). However, there are no past reports documenting inhibition of AA and AAD by TRV027. Our finding suggests that enhancement of β-arrestin biased AT1R signaling pathways by TRV027 [63] might be a novel therapeutic option. β-arrestin biased AT1R signaling activated by TRV023 was recently reported to promote smooth muscle growth, migration and vascular remodeling in PAH [64]. It has also been reported that genetic deletion of β-arrestin2 in Marfan syndrome [65] and ApoE−/− mouse models [66] diminished AngII-induced AA formation. Further studies will be necessary to clarify the apparent discrepancy between our study and these reports.

Histopathology of AA and AAD observed in the AngII-infused group in our study (Fig.3) is similar to other ApoE−/− mouse model studies [62]. Progressive weakening of the aortic wall occurs as a result of disintegration of the medial and elastic layers. In the media layer, vascular smooth muscle cells (VSMC) are fundamental to aortic structure and function. It was proposed that atrophy of the media layer leads to VSMC stress and contractile dysfunction [67]. In the TRV027 and OLM co-infusion groups remodeling was prevented. TRV027 co-infused group increased the elastin and collagen content which in part contribute to a symmetrically thicker vessel wall compared to the OLM groups in males (Figs. 3 and 4). Healthy contractile VSMC are characterized by their deposition of elastin as well as other extracellular matrix proteins such as collagen to maintain the mechanical integrity of blood vessels (Fig. 4 and S2). Decreased elastin expression in VSMCs indicates a phenotypic switch from contractile state to a synthetic phenotype. It was observed that the elastin levels control the hyper-proliferative state of the VSMC, suggesting inter communication between levels of elastin and the phenotypic state of SMCs. Phenotypic switching is often caused by injury or disease [68, 69].

Mechanism of ligand-specific differences in the phenotype is provided by molecular changes observed that differentially modulate protein and DNA metabolism in mice. Regulatory control molecules of these complex process at multiple levels were examined in the AngII-induced disease group compared to TRV027 and OLM co-infusion groups (Figs. 5). A decrease in pAMPK levels in AngII group indicates metabolic vulnerabilities associated with AA and AAD which is restored to no ligand levels in TRV027 and OLM groups. Physiological AMPK activation plays a key role as a master regulator of cellular energy homeostasis. In other studies AMPK inhibition was reported in AngII-induced hypertension in mice, which could be relieved by Resveratrol [70] or Losartan [71]; both enhance phosphorylation of AMPK. Impairment of mechanistic target of rapamycin (mTOR) pathway downstream of AMPK is observed: (i) by increased phosphorylation of the mTOR substrate S6 kinase (pS6K) that participates in cell growth control through ribosomal translational machinery and overall mRNA translation levels involving the eukaryotic translation initiation factor 2A (eIF2A) [72]. (ii) mTORC1 signaling via 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) is known to be a critical pathway for synthesis of collagen and other matrisomal proteins implicated in the development of fibrosis [73]. 4E-BP is also a critical regulator of fat metabolism, activated under conditions of environmental stress [74].

Potential activation of autophagy indicated by increased level of LC3 I/II and endoplasmic reticulum stress indicated by p-CHOP3 in the AngII group is restored to no ligand level in the TRV027 group. LC3 I/II is a central protein in the autophagy substrate selection and autophagosome biogenesis. Autophagosomes engulf cytosolic proteins and organelles for degradation and salvaging the constituents. Dysregulation of autophagy, reduction or increase in the levels beyond homeostatic fluctuations in a tissue is known to be harmful and lead to abnormal cell growth and/or cell death. For example, different levels of activation of autophagy may impact the extents to which AA progression occurs. Detecting LC3 by immunoblotting has been a reliable method for monitoring autophagy and autophagy-related processes [75]. Accumulation of p-CHOP, which is an AMPK regulated protein suggests ER stress caused by aggregation of misfolded proteins. Efficient functioning of the ER is essential for survival. ER stress may induce cell death and is implicated in the pathophysiology of many cardiovascular diseases [76, 77].

DNA replication/repair and cell division need to occur in the correct order and rate, for cells in our body to renew the tissue they are part of. We observed an inhibitory effect on these processes in the OLM group (Fig. 6). In the TRV027 co-infused group we monitored various regulators which suggest occurrence of tissue maintenance either at the level of no ligand group or slightly higher but not shifted to “over drive” as in the AngII group. For instance, immuno-histochemical staining of markers PCNA and Ki67 is widely used to measure cell proliferation in normal and disease tissues as well as tissue-response to a given therapy [78]. It also serves as a prognostic and predictive indicator for the assessment of biopsies from patients [79]. The tumor suppressor proteins we evaluated, p53 and Chk2 have been previously used to assess DNA replication/damage response by AngII induced oxidative stress [80]. Treatment with candesartan was found to inhibit p53 and Chk2 associated with cell growth in human cancer cells [81].

Increase of liver produced GGT and AST enzymes observed in the AngII group suggests that amino acid metabolism may be impaired in this group but not in other ligand treated groups. GGT catalyzes transfer of γ-glutamyl group from peptides to other amino acids. AST is an enzyme which acts in different organs such as liver, heart skeletal muscle, kidney, brain, red blood cell. It catabolizes amino acids, permitting them to enter the citric acid cycle [82]. Thus, in our study we could infer that treatment with TRV027 or OLM is likely to not cause organ toxicity.

Increase of MMP17 protein observed in the AngII group in our study in contrast to decreases as reported in previous literature [83]. MMP17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall. Absence or downregulation of MMP17 predisposes to AA in mice [84]. Most of the samples evaluated for the MMPs were between the stage 0 and stage 1 of AA in our study. This may be one of the reasons why MMP17 was found increased. With regards to MMP23, this enzymes has been reported in association with other inflammatory diseases [85], but not yet with AA. In our study, the AngII group showed higher levels of MMP23 protein associated with AA of grade I, suggesting the presence of inflammation status. Co-infusion of TRV027 reduced but did not fully block the increase of MMP23.

Past studies have suggested that chronic AngII treatment may cause aneurysms due to decreased aortic smooth muscle cell contractile-protein levels and impaired smooth muscle contractile-unit function. Surprisingly, contractility of aorta did not decrease in AngII-infused mice in our study. Instead aortic hyper-contractility was observed in response to 5-HT stimulation. We speculate that AngII infusion causes degrees of endothelial dysfunction correlated with grades of AA pathologies. This finding is similar to that was observed in ApoE−/− mice due to TGF-β-null effect [86]. However, the reversible OLM relaxation profiles of aorta indicated that AngII-induced endothelial dysfunction may be transient. This observation is consistent with previous finding that Losartan induces endothelial nitric oxide-dependent relaxation of the thoracic aorta rings of Wistar rats [87]. In our study increased 5-HT sensitivity was observed in TRV027 treated aorta; this observation is consistent with previous report that TRV027 increased cardiac contractility [88]. However, β-arrestin biased AT1R ligands did not directly cause vasoconstriction implying that the phenomenon might be an indirect effect of 28 day exposure [89].

In summary, β-arrestin biased AT1R agonist, TRV027 was found to inhibit AngII-induced AA in ApoE-null mice with same efficacy as the ARB, OLM. The structural and dynamic properties of aorta as well as protein and DNA metabolism are better preserved by TRV027 than OLM while preventing AngII-induced remodeling of aorta. Some limitations inherent in this report that need to be addressed in future studies include, (i) testing the efficacy of TRV027 in other aneurysm models, (ii) a deeper understanding of the impact of metabolic changes, abnormal protein synthesis, impairment of autophagy and ER stress in producing different grades of AA and (iii) understanding the basis of difference observed between males and females. These aspects will be explored in depth in future.

Perspectives

Pharmacokinetics and pharmacodynamics properties determined in healthy volunteers indicated that TRV027 successfully reduced adverse events in patients with concomitant hypertension [90]. Preclinical studies have shown that central administration of TRV027 reduced high BP and increased salt-aversion behavior [37]. Also, TRV027 exerted beneficial effects on hypertension and vascular function in hypertensive rats [39]. Guided by the insights from these studies and our data in this report, we propose that the clinical uses of TRV027 must be revisited in many situations associated with elevated RAS activity such as chronic hypertension, obesity induced hypertension and vascular aneurysms. The design of novel therapeutic strategies to target β-arrestin biased AT1R signaling could be the promising future in the treatment of clinical conditions associated with RAS over activation.

NOVELTY AND RELEVANCE.

What is new?

Our results show that AngII-induced AA pathology and AAD mortality are blocked in both male and female AA-prone mice by β-arrestin biased AT1R ligand, TRV027. The aortic wall thickness and elastin content was significantly increased in TRV027 co-infused mice when compared to ARB co-infused mice. Homeostatic levels of new protein and DNA synthesis was maintained in TRV027 co-infused mice while the levels are inhibited in ARB co-infused mice. This finding suggests that mechanism of prevention of AA and AAD by AT1R occupied with β-arrestin biased agonist, TRV027 and ARB, Olmesartan are distinct.

What is Relevant?

It is known that β-arrestin biased AT1R agonists promote cardio-protective effects by diminishing G protein signals and promoting β-arrestin-mediated signals, but nothing is known about their efficacy to prevent vascular aneurism. If β-arrestin biased AT1R agonists and ARBs potently inhibit aneurysm, whether they protect by similar or different mechanisms? Our findings suggest that selective activation of β-arrestin signals of AT1R may be therapeutically applied in future.

Clinical/Pathophysiological Implications?

Pharmacological inhibition of AT1R by ARBs in human is established to protect heart, vasculature and kidney against Ang II–induced injuries. Our findings provide evidence that β-arrestin-biased AT1R signaling is a potential therapeutic avenue to normalize blood pressure control, vascular metabolism, vasomotor function and fibrosis in hypertensive diseases such as AA and AAD. Thus, β-arrestin-mediated AT1 antagonist(s) may have great potential to improve the therapy of hypertensive diseases.

Nonstandard Abbreviations and Acronyms:

AA

aortic aneurysm

AAD

aortic aneurysm dissection

AngII

angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe)

AT1R

angiotensin II type 1 receptor

TRV027

β-arrestin biased AT1R agonist (Sar-Arg-Val-Tyr-Ile-His-Pro-dAla)

GPCR

G protein coupled receptor

ARB

angiotensin II type 1 receptor (AT1R) blocker

OLM

angiotensin receptor blocker, Olmesartan

ACEi

angiotensin converting enzyme inhibitor

ApoE

apolipoprotein E gene

BP

blood pressure

BPM

blood pressure measure

H&E

Hematoxylin & Eosin staining

MT

Masson’s trichrome staining

VVG

Verhoeff-Geison staining

SUnSET

surface sensing of translation

LC3I/II

autophagy marker, microtubule-associated protein I/II-light chain 3

CHOP

ER-stress marker C/EBP homologous protein

AMPK

AMP regulated kinase

mTOR

mechanistic target of rapamycin

5-HT

5-hydroxytryptamine, (Serotonin)

VSMC

vascular smooth muscle cells

4E-BP1

eukaryotic translation initiation factor 4E-binding protein 1

PAH

pulmonary arterial hypertension