Inhibition or deletion of angiotensin II type 1 receptor suppresses elastase-induced experimental abdominal aortic aneurysms

From the Western Vascular Society

Inhibition or deletion of angiotensin II type 1 receptor suppresses elastase-induced experimental abdominal aortic aneurysms Haojun Xuan, MD,a,b Baohui Xu, MD, PhD,a Wei Wang, MD, PhD,a Hiroki Tanaka, MD, PhD,a Naoki Fujimura, MD, PhD,a Masaaki Miyata, MD, PhD,c Sara A. Michie, MD,d and Ronald L. Dalman, MD,a Stanford, Calif; Hangzhou, China; and Kagoshima, Japan

ABSTRACT Objective: Angiotensin (Ang) II type 1 receptor (AT1) activation is essential for the development of exogenous Ang II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic animals. Experimental data derived from this modeling system, however, provide limited insight into the role of endogenous Ang II in aneurysm pathogenesis. Consequently, the potential translational value of AT1 inhibition in clinical AAA disease management remains incompletely understood on the basis of the existing literature. Methods: AAAs were created in wild-type (WT) and AT1a knockout (KO) mice by intra-aortic infusion of porcine pancreatic elastase (PPE). WT mice were treated with the AT1 receptor antagonist telmisartan, 10 mg/kg/d in chow, or the peroxisome proliferator-activated receptor g (PPARg) antagonist GW9662, 3 mg/kg/d through oral gavage, beginning 1 week before or 3 days after PPE infusion. Influences on aneurysm progression as well as mechanistic insights into AT1-mediated pathogenic processes were determined using noninvasive ultrasound imaging, histopathology, aortic gene expression profiling, and flow cytometric analysis. Results: After PPE infusion, aortic enlargement was almost completely abrogated in AT1a KO mice compared with WT mice. As defined by a $50% increase in aortic diameter, no PPE-infused, AT1a KO mouse actually developed an AAA. On histologic evaluation, medial smooth muscle cellularity and elastic lamellae were preserved in AT1a KO mice compared with WT mice, with marked attenuation of mural angiogenesis and leukocyte infiltration. In WT mice, telmisartan administration effectively suppressed aneurysm pathogenesis after PPE infusion as well, regardless of whether treatment was initiated before or after aneurysm creation or continued for a limited or extended time. Telmisartan treatment was associated with reduced messenger RNA levels for CCL5 and matrix metalloproteinases 2 and 9 in aneurysmal aortae, with no apparent effect on PPARg-regulated gene expression. Administration of the PPARg antagonist GW9662 failed to “rescue” the aneurysm phenotype in telmisartan-treated, PPE-infused WT mice. Neither effector T-cell differentiation nor regulatory T-cell cellularity was affected by telmisartan treatment status. Conclusions: Telmisartan effectively suppresses the progression of elastase-induced AAAs without apparent effect on PPARg activation or T-cell differentiation. These findings reinforce the critical importance of endogenous AT1 activation in experimental AAA pathogenesis and reinforce the translational potential of AT1 inhibition in medical aneurysm disease management. (J Vasc Surg 2017;-:1-12.) Clinical Relevance: Clinical trials testing the ability of the angiotensin receptor blocker telmisartan to suppress early abdominal aortic aneurysm disease progression are ongoing. This study investigates the potential mechanisms by which telmisartan may limit abdominal aortic aneurysm disease progression.

From the Department of Surgerya and Department of Pathology,d Stanford

Clinical Trial registration: NCT01683084.

University School of Medicine, Stanford; the Department of Breast Surgery,

Author conflict of interest: none.

Zhejiang Cancer Hospital, Hangzhoub; and the Department of Cardiovascular

Presented at the Thirty-first Annual Meeting of the Western Vascular Society,

Medicine and Hypertension, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima.c

Colorado Springs, Colo, September 24-27, 2016. Additional material for this article may be found online at www.jvascsurg.org.

This work was supported in part by grants from the National Heart, Lung, and

Correspondence: Ronald L. Dalman, MD, Department of Surgery, Stanford

Blood Institute (1R21HL109750-03 and 1R21HL112122-03) and the Japan Soci-

University School of Medicine, Rm P323, MLSL Bldg, Welch Rd, Stanford, CA

ety for the Promotion of Science (26461148). Other support included fellow-

94305 (e-mail: [email protected]).

ships from the China Scholarship Council (W.W.), Keio University (N.F.), and

The editors and reviewers of this article have no relevant financial

the Japan Research Foundation for Clinical Pharmacology (H.T.). In the

relationships to disclose per the JVS policy that requires reviewers to

United States, the Telmisartan in the managEment of abDominal aortic

decline review of any manuscript for which they may have a conflict of

aneurYsm (TEDY) trial is sponsored by Medtronic Inc, Santa Rosa, Calif. Stan-

interest.

ford University is the sole North American TEDY site, and R.L.D. is site principal

0741-5214

investigator for TEDY USA. Medtronic did not provide financial or in-kind sup-

Copyright Ó 2017 by the Society for Vascular Surgery. Published by Elsevier Inc.

port for the current study, however, and had no input on the design, conduct,

http://dx.doi.org/10.1016/j.jvs.2016.12.110

or interpretation of the experiments described.

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Abdominal aortic aneurysm (AAA) is a progressive and lethal degenerative disease of the distal aorta. Although the epidemiology of AAA disease has changed significantly in the last few decades,1 rupture of advanced aneurysms still accounts for thousands of premature deaths annually throughout the developed world.2 Whereas public screening programs frequently detect AAA disease at an early and potentially treatable stage, to date no pharmacologic therapy has proved effective in suppressing aneurysm progression or eventual rupture. Accumulating experimental evidence underscores the significance of the renin-angiotensin system (RAS) in AAA pathogenesis. Nearly all the supporting evidence, however, has been generated in modeling systems incorporating hyperlipidemic mice with whole body deficiency of, or treated with inhibitors to, the angiotensin (Ang) II type 1 receptor (AT1) after exogenous Ang II infusion.3-5 Whereas the exogenous Ang II murine models recapitulate certain clinical and pathologic components of clinical aneurysms, including progressive aortic dilation and transmural inflammation, these are preceded by a process of segmental aortic dissection without known precedent in human AAA disease. In addition, the requirement for exogenous Ang II administration obscures the role that endogenously produced Ang II and AT1 activation play in aneurysm pathogenesis. Modeling systems not dependent on exogenous Ang II administration for AAA creation are available and in wide use.6 We and others have demonstrated that AT1 receptor blockers (ARBs) suppress experimental AAAs formed after intra-aortic infusion of porcine pancreatic elastase (PPE), an exogenous Ang II-independent AAA modeling system, in wild-type (WT) mice.3,6,7 Certain ARBs, such as telmisartan, also manifest peroxisome proliferatoractivated receptor g (PPARg) agonist activity, influence the balance of proinflammatory and anti-inflammatory cytokine-producing T-cell differentiation, and increase immunosuppressive regulatory T-cell (Treg) cellularity.8,9 Whether the AAA-suppressive properties of telmisartan are mechanistically mediated by AT1 antagonism alone or develop in combination with other antiinflammatory influences, such as PPARg agonism, has not been determined to date. In addition, the distinct roles of AT1 activity in promoting aneurysm initiation vs mediating progression of existing aneurysms, if such a difference exists, remain poorly understood. In this study, we used AT1a knockout (KO) mice to determine the significance of AT1 activation in AAA initiation and progression after intra-aortic infusion of PPE. To further examine the temporal consequences of AT1 activation during the progression of experimental AAAs, we employed three distinct, time course-specific telmisartan treatment protocols. Finally, we examined the potential role for non-AT1-related telmisartan activities,

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ARTICLE HIGHLIGHTS d

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d

Type of Research: Prospective experimental study Take Home Message: Angiotensin II type 1 receptor is a likely therapeutic target of abdominal aortic aneurysm. Recommendation: Data presented in this manuscript strongly suggest that inhibition of angiotensin II type 1 receptor attenuates aneurysm progression.

including PPARg agonism or T-cell activation, in suppressing experimental aneurysms.

METHODS Mice. AT1a KO mice (stock #002682), previously backcrossed with C57BL/6J mice for nine generations to acquire the C57BL/6J genetic background, were purchased from the Jackson Laboratory (Bar Harbor, Me). C57BL/6J (stock #000664) mice were used as the WT control strain as recommended by the Jackson Laboratory. All mice were housed and maintained at the Stanford University Research Animal Facility (Stanford, Calif). Only male mice at 10 to 12 weeks of age were used. Experimental and husbandry protocols were conducted in compliance with the Stanford Laboratory Animal Care Guidelines, as approved by the University Administrative Panel on Laboratory Animal Care (protocol #11131). AAA creation. AAAs were created by transient intraaortic infusion of PPE as previously described.3,10,11 Briefly, using inhaled isoflurane anesthesia, the infrarenal aorta was isolated from the level of the left renal vein to the iliac bifurcation through median laparotomy. After exposure, the aorta was temporarily controlled proximally and distally with 6-0 silk suture. Heat-tapered P-10 tubing was inserted into the controlled segment just proximal to the aortic bifurcation. Under constant pressure, 30 mL of type I PPE (1.5 units/mL freshly prepared in phosphatebuffered saline [PBS], catalog #E-1250-100MG; SigmaAldrich, St. Louis, Mo) was infused into the controlled aortic segment for 5 minutes. After infusion and removal of residual infusate, the tubing was withdrawn and the aortotomy closed with 10-0 nylon suture. After laparotomy closure and surgical recovery, mice were housed in separate cages with free access to chow and water. Drug treatment. In all pharmacologic AT1 inhibition experiments, mice were fed telmisartan-supplemented chow (10 mg/kg/d; AK Scientific, Inc, Mountain View, Calif) in protocols B, C, and D or standard chow as control in protocol A (Fig 1).3 Duration of treatment varied, depending on the specific protocol. In the “effect on aneurysm initiation and progression” group, treatment was begun 7 days before and terminated at 14 days after

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technique to determine diameter has consistently demonstrated an intraobserver and interobserver variability rate <2%.3

Fig 1. Treatment regimens for telmisartan and GW9662 administration. Protocol A: Standard chow provided throughout entire experimental period (control for the remaining protocols). Protocol B: To examine the effect of angiotensin (Ang) II type I receptor blocker (ARB) on the initiation and progression of experimental abdominal aortic aneurysms (AAAs), telmisartan-supplemented chow (10 mg/kg/d) was provided throughout the entire experimental period. Protocol C: To examine the effect of ARB on AAA initiation and early progression, telmisartan chow was begun 1 week before and terminated 3 days after porcine pancreatic elastase (PPE) infusion. Protocol D: To examine the effect of ARB on progression of existing AAAs, telmisartan chow was begun day 4 after PPE infusion for 10 days. Protocol E: To isolate potential influence of peroxisome proliferator-activated receptor g (PPARg) agonist properties of telmisartan on AAA progression, chow was supplemented with telmisartan and GW9662 (3 mg/kg/d through oral gavage) begun 1 week before aneurysm initiation for a total of 3 weeks.

PPE infusion (protocol B). In the “effect on aneurysm initiation” group, telmisartan chow was provided beginning 7 days before and continued for 3 days after PPE infusion (protocol C). In the “effect on existing aneurysm” group, telmisartan chow was provided beginning 4 days after recovery from PPE infusion and continued through day 14 (protocol D). The day 4 time point was selected on the basis of past model-specific experience; an AAA, defined as $50% aortic diameter increase compared with baseline, reliably forms within this period after intraaortic elastase infusion.10,11 In experiments examining the potential effects of PPARg agonist activity associated with telmisartan, the PPARg antagonist GW9662 was given to telmisartanfed mice through oral gavage (3 mg/kg/d; Calbiochem, San Diego, Calif) beginning 7 days before PPE infusion and ending 14 days thereafter (protocol E). The dosing of GW9662 was selected on the basis of previously published experience in comparable experimental modeling systems (Supplementary Table I, online only). Aortic diameter determination by serial ultrasound examination. Aortic aneurysm formation and progression were monitored by serial ultrasound-derived diameter measurements at 40 MHz (Model Vevo 770; VisualSonics, Toronto, ON, Canada). An AAA was defined as a $50% increase in aortic diameter over the baseline level. Prior experience with this measurement

Histologic analyses. In all experiments, mice were sacrificed 14 days after PPE or PBS infusion. Aortae were harvested, embedded in OCT medium, sectioned (6-mm thickness), and acetone fixed. Tissue immunostaining was performed using a standard biotin-streptavidin peroxidase procedure as previously described.3,10,11 Primary antibodies included a rabbit anti-mouse smooth muscle cell (SMC) a-actin polyclonal antibody (Lab Vision Corporation, Fremont, Calif), a rat anti-mouse CD31 monoclonal antibody (mAb; clone 390) and a hamster anti-mouse CD3 mAb (clone 145-2C11; BioLegend, San Diego, Calif), and a rat anti-mouse CD68 mAb (clone FA-11; AbD Serotec, Raleigh, NC). Other reagents included biotinylated anti-rabbit, rat, or hamster immunoglobulin G antibodies and streptavidinperoxidase conjugates (Jackson ImmunoResearch Laboratories, Inc, West Glove, Pa) and aminoethylcarbazole peroxidase substrate kit (Vector Laboratories, Inc, Burlingame, Calif). Medial elastin retention was examined using elastin van Gieson staining. Medial elastin degradation and SMC depletion were scored I (mild) to IV (severe) as previously described.11 Aortic mural infiltration of macrophages and T cells was reported as positively stained cells per aortic cross section. Aortic mural neovessels (angiogenesis) were reported as CD31þ blood vessels per aortic cross section. Real-time reverse transcription-polymerase chain reaction (RT-PCR) assays. Total RNA from harvested aortae was extracted using TRIzol reagent. Complementary DNA (cDNA) was synthesized using SuperScript III FirstStand Synthesis System, followed by amplification using SYBR GreenER qPCR SuperMix Universal on the Stratagene MX300P system (Agilent Technologies, Inc, Santa Clara, Calif). b-Actin expression was used as an internal reference. Gene expression levels were expressed as fold changes relative to normal aorta. PCR primers were TAA AAA CCT GGA TCG GAA CCA AA and GCA TTA GCT TCA CAT TTA CGG GT for CCL2; CGA CTG CAA GAT TGG AGC ACT and TTT GCC TAC CTC TCC CTG G for CCL5; GCT TGT TGG CCT CAG TTA AGG and GTA GCT CAG GCG TAC AGA GAT for ABCA1; CTT TCC TAC TCT GTA CCC GAG G and CGG GGC ATT CCA TTG ATA AGG for ABCG1; CTC AAT GCC TGA TGT TTC TCC T and TCC AAC CCT ATC CCT AAA GCA A for LXR; ATG GGC TGT GAT CGG AAC TG and GTC TTC CCA ATA AGC ATG TCT CC for CD36; GAT GTC GCC CCT AAA ACA GAC and CAG CCA TAG AAA GTG TTC AGG T for matrix metalloproteinase 2 (MMP2); GGA CCC GAA GCG GAC ATT G and GAA GGG ATA CCC GTC TCC GT for MMP9; and TAT TGG CAA CGA GCG GTT CC and GGC ATA GAG GTC TTT ACG GAT GT for b-actin. All reagents were purchased from or synthesized at Life Technologies (Grand Island, NY).

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Flow cytometric analysis of cytokine-producing and Treg cells. Single leukocyte suspensions were isolated from mouse spleens at sacrifice, 14 days after PPE infusion. Intracellular cytokine staining was used to detect cytokine-producing CD4þ or CD8þ T cells. Briefly, lymphocytes were stimulated with phorbol myristate acetate (50 ng/mL) and ionomycin (2 mg/mL) for 4 hours in the presence of brefeldin A. These cells were sequentially stained with phycoerythrin (PE)-Cy7-CD4 (clone GK1.5) or CD8 (clone 53-6.7) mAb, fixed with cell fixation buffer, permeabilized, and stained with fluorescein isothiocyanate-interferon-g (IFN-g; clone XMG 1.2), PE-interleukin 10 (IL-10; clone JES5-16E3), and Alexa Fluor 647- interleukin 17 (IL-17; clone TC11-18H1.1) mAbs. Foxp3-expressing regulatory CD4þ T cells were detected by staining with PE-Cy7-CD4, Alexa Fluor 488-CD25 (clone PC61), and PE-Foxp3 (clone MF-14) mAbs using Foxp3 Fix/Perm buffer set. All reagents were purchased from BioLegend and used in accordance with the manufacturer’s instructions. Data on stained samples were acquired and analyzed using FACSCaliber (BD Biosciences, San Jose, Calif) and FlowJo software (version 9.6.4; Tree Star Inc, Eugene, Ore), respectively. Statistical analyses. All continuous data were reported as the mean and standard deviation. The nonparametric Mann-Whitney test and two-way analysis of variance followed by Newman-Keuls post-test were used to determine differences between groups according to data characteristics. Difference in aneurysm incidence was determined by Kaplan-Meier analysis. All statistical analyses were performed using Prism version 6.0g (GraphPad Software Inc, San Diego, Calif). P < .05 was considered statistically significant.

RESULTS Genetic AT1 deficiency prevents elastase-induced AAA formation. In WT mice, aortic enlargement began 3 days after PPE infusion (Fig 2, A and B). All WT mice developed AAAs, as defined as $50% diameter enlargement, within 14 days after PPE infusion. In contrast, PPE-induced aortic enlargement was nearly abolished in AT1a KO mice; in this group, no AAAs formed (Fig 2, A and B). In WT mice, PPE infusion was accompanied by severe aortic medial elastin degradation and substantial SMC depletion (Fig 2, C and D). Mural angiogenesis and aortic macrophage accumulation were prominent in aortic adventitia. Moderate aortic mural T-cell infiltration was also noted. By comparison, in AT1a KO mice, medial elastin degradation and SMC depletion were almost completely abrogated after PPE infusion (Fig 2, C and D). Mural angiogenesis and macrophage and T-cell infiltration were also reduced by 96%, 76%, and 60%, respectively (Fig 2, C and D). Taken together, these results demonstrate that AT1a activation is essential for experimental aneurysm formation, even in the absence of

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exogenous Ang II administration as an initiating mechanism for AAA creation. Telmisartan limits aneurysm initiation and progression. To examine the role of AT1 activation throughout the progression of AAA disease, WT mice were fed either standard chow (protocol A) or telmisartan-supplemented chow (10 mg/kg/d) according to the three protocols (B, C, and D) outlined in the Methods section. In protocol B, 21 days of telmisartan-supplemented chow significantly limited aortic enlargement in all PPE-treated mice (Fig 3, A and B). To isolate the effects of AT1 inhibition on aneurysm initiation, in protocol C, telmisartan treatment was limited to 10 days only, beginning 7 days before PPE infusion. Even after discontinuation of telmisartan chow 3 days after aneurysm initiation in this protocol, further enlargement was still limited compared with control mice (Fig 3, A and B). In protocol D, telmisartan-supplemented chow was initiated 4 days after PPE infusion to study the effect of AT1 inhibition in established aneurysms. In this protocol, further aneurysm progression was completely abolished in these mice despite delayed telmisartan therapy (Fig 3, A and C). Aneurysm incidence ($50% diameter increase) was also significantly reduced in protocol B- and D-treated mice compared with control (Fig 3, A). In protocol C-treated mice, a significant delay in aneurysm onset was apparent, although no difference in incidence was ultimately noted. These results demonstrate that pharmacological inhibition of AT1 activity limits the initiation and progression of, and further expansion of, existing AAAs even in the absence of exogenous Ang II supplementation. Histologic correlates of telmisartan-mediated AAA suppression. In histologic analyses, medial elastin degradation, SMC depletion, and mural angiogenesis were all mitigated in telmisartan-treated mice, regardless of administration protocol, compared with control (Fig 4, A-C). Aortic infiltration of macrophages and T cells was markedly reduced as well (Fig 4, D and E). The degree of aortic preservation and inflammation suppression was related to the duration of telmisartan infusion (protocol B > C or D). These results suggest that telmisartan limits AAA progression by limiting elastin degradation, SMC depletion, mural angiogenesis, and leukocyte accumulation in this experimental model. Expression of aortic CCL5 and MMPs 2 and 9 is downregulated in telmisartan-treated mice. To gain insight into potential mechanisms of AT1 antagonistmediated AAA suppression in this model, real-time RT-PCR assay was performed on aortic tissue harvested from mice in protocols A and B as well as from mice provided with intra-aortic PBS infusion as an additional control (Fig 5). Telmisartan treatment was associated

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Fig 2. Effect of targeted deletion of angiotensin (Ang) II type 1a receptor (AT1a) on aneurysm formation after porcine pancreatic elastase (PPE) infusion. Transient intra-aortic PPE infusion was performed in male wild-type (WT; n ¼ 5) and AT1a knockout (KO; n ¼ 5) mice at 10 to 12 weeks of age. Abdominal aortic aneurysm (AAA) formation and progression were evaluated by serial ultrasound measurements of aortic diameters in vivo followed by histologic analyses at sacrifice. (A) Representative ultrasound images of aortic diameters before and 2 weeks after PPE infusion. (B) Mean and standard deviation of aortic diameters before PPE infusion and indicated days thereafter in individual groups. Two-way analysis of variance followed by Newman-Keuls test, **P < .01 compared with WT mice at corresponding time points. (C) Representative aortic histologic staining of medial elastin (elastin van Gieson), smooth muscle cells (SMCs; a-actin), neoangiogenesis (CD31), macrophages (CD68), and T cells (CD3). (D) Mean and standard deviation of quantitative medial elastin degradation, SMC depletion, neoangiogenesis, and infiltration of macrophages and T cells. Nonparametric Mann-Whitney test, * P < .05 compared with WT mice. ACS, Aortic cross-section.

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Fig 3. Influence of varying telmisartan treatment intervals on abdominal aortic aneurysm (AAA) initiation and progression. Mice were fed either telmisartan-supplemented chow for the indicated interval or standard chow as control. Aneurysm progression was assessed by serial ultrasound-derived infrarenal aortic diameter measurements in vivo. (A) Representative ultrasound images of porcine pancreatic elastase (PPE)-infused mouse aortae after treatment with telmisartan-supplemented chow throughout the observation period (protocol B: day 7 to 13; n ¼ 10) compared with telmisartan treatment during the initiation period only (protocol C: day 7 to 3; n ¼ 10) or after aneurysms were clearly established (protocol D: day 4 to 13; n ¼ 15). Protocol A represents mice fed with standard chow only before and after PPE infusion (n ¼ 15). (B) Telmisartan treatment suppresses both aneurysm initiation and progression (protocols A, B, and C). All data are reported as mean 6 standard deviation of aortic diameters. (C) Telmisartan treatment stabilizes established AAAs (protocols A and D). Data are mean and standard deviation of aortic diameter differences in mice with established AAAs fed telmisartan or standard chow. (D) All telmisartan treatment protocols reduce aneurysm incidence. An AAA was defined as a 50% or greater increase in aortic diameter over baseline levels. Two-way analysis of variance followed by Newman-Keuls test, *P < .05 and **P < .01 between two groups (B, C). Kaplan-Meier analysis, *P < .05 and **P < .01 compared with standard chow group (A).

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Fig 4. Influence of telmisartan treatment status on aortic medial elastin and smooth muscle cell (SMC) retention, mural angiogenesis, and leukocyte accumulation. Aortic frozen sections were prepared from mice differentially fed telmisartan-supplemented or standard chow before or after porcine pancreatic elastase (PPE) infusion, stained for medial elastin (A), SMCs (SMC a-actin; B), neoangiogenesis (CD31; C), macrophages (CD68; D), and T cells (E). Destruction of elastin and SMCs was scored as I (mild) to IV (severe); angiogenesis and aortic leukocytes were quantified as blood vessels or positively stained cells per aortic cross section (ACS). Data are mean and standard deviation for individual groups. Nonparametric Mann-Whitney test, *P < .05 and **P < .01 between two groups (n ¼ 10-15 mice/group).

with suppression of messenger RNA (mRNA) for CCL5, but not CCL2, to a significantly greater extent in PPEthan in PBS-infused aortae. Expression of mRNA for both MMPs 2 and 9 was substantially reduced in telmisartantreated mice as well. Telmisartan-mediated AAA suppression is independent of PPARg agonist activity. Telmisartan is a partial agonist of PPARg, a negative regulator of experimental aneurysm progression in alternative AAA modeling systems.8,12,13 Losartan, an alternative ARB lacking PPARg agonist activity comparable to that of telmisartan, inhibited progression of experimental aneurysms induced by Ang II but not by intra-aortic PPE infusion in previously published work.4,14 To examine the potential contribution of PPARg agonist activity to telmisartan-mediated AAA inhibition, we quantified PPARg-regulated aortic gene expression in differentially treated mice. As shown in Fig 6, A, the

mRNA levels of ABCA1, CD36, and LXR were significantly elevated in aneurysmal aortae. Telmisartan treatment was associated with increased mRNA expression levels of ABCA1 alone. Next, we treated telmisartan-fed mice with the PPARg antagonist GW9662 (3 mg/kg/d) beginning 7 days before PPE infusion for a total of 3 weeks (protocol E). As seen in Fig 6, B, GW9662 failed to “rescue” the aneurysm phenotype in PPE-infused mice receiving telmisartan. These results suggest that telmisartanmediated aneurysm suppression is more likely a function of AT1 inhibition rather than of PPARg activation. Telmisartan does not influence T-cell cytokine production or Treg cells. Prior experimental evidence has demonstrated that pharmacologic RAS inhibition suppresses inflammation through Th1, Th17, and Treg cell-mediated mechanisms.9 Given the involvement of IFN-g and IL-17 in exogenous Ang II-independent experimental aneurysm progression,15-18 we examined the

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Fig 5. Telmisartan treatment reduces expression levels of messenger RNAs (mRNAs) for chemokines and matrix metalloproteinases (MMPs) in aneurysmal aortae. Total RNA was extracted from the aortae of mice infused with phosphate-buffered saline (PBS), porcine pancreatic elastase (PPE), or PPE and fed telmisartan (day 7 to 13) and subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. Message levels reported as fold changes (log scale) relative to expression in control (normal mouse) aorta. Data represent mean and standard deviation from 10 to 15 mice in each group. Nonparametric Mann-Whitney test, *P < .05 and **P < .01 between two groups.

influence of telmisartan treatment on the differentiation of IFN-g-, IL-17-, and IL-10-producing splenic T cells 14 days after PPE infusion (Fig 7). In this construct, telmisartan-supplemented chow had no discernible effect on populations of IFN-g-, IL-10-, or IL-17-producing CD4þ T cells (Fig 7, A and C). Similar trends were noted for IFN-g- or IL-10-producing CD8þ T cells (Fig 7, B and D). In addition, the percentages of splenic Foxp3þ Treg cells in CD4þ cells were comparable between telmisartan and standard chow-fed mice (Fig 7, A and E). These results indicate that the observed experimental aneurysm suppression in protocols B, C, and D was not likely due to telmisartan-mediated influences on T-cell cytokines or Treg cells.

DISCUSSION These experiments underscore the important role played by AT1 in experimental aneurysm pathogenesis, even in modeling systems devoid of supplemental Ang II. This work adds to the growing body of evidence identifying Ang II as a key mediator in experimental and clinical AAA pathogenesis.4,5,19 Smoking and advanced age, two of the most significant risk factors for AAA disease, both modulate RAS activity. Cigarette smoking induces reciprocal effects on levels of proaneurysmal angiotensin-converting enzyme (ACE) and antianeurysmal ACE2 (an Ang II-degrading enzyme) activity and gene expression,20,21 resulting in increased Ang II production. Advanced age is associated with increased arterial expression of the AT1 receptor.22 The current findings confirm and extend prior observations regarding AT1 inhibition in experimental AAAs.3,6,7

Several prior studies have examined the influence of ARBs on experimental aneurysms.3,4,6,7,14 This study, leveraging AT1a KO mice, is the first to examine the significance of AT1 activation by endogenous Ang II in AAA pathogenesis. By varying telmisartan treatment intervals, we also demonstrated the critical role that AT1 activity plays in both AAA initiation and progression. The latter finding, in particular, supports further clinical investigation of telmisartan in the medical management of AAA disease. To date, pharmacologic approaches have proved to be frustratingly ineffective in limiting AAA disease progression. In addition to suppressing existing aneurysms, telmisartan’s long half-time, high receptor affinity, high tissue penetration, and demonstrated clinical safety in treating non-aneurysm-related medical conditions make it an intriguing target for translational investigation in AAA management.8 The Telmisartan in the managEment of abDominal aortic aneurYsm (TEDY) trial, a multicenter, multinational randomized controlled trial, is currently testing the efficacy of 40 mg of telmisartan daily in suppressing early AAA disease progression.23 In addition to limiting AAA progression, further mechanistic insights into the role of the AT1 receptor in experimental aneurysm pathogenesis were enabled by telmisartan in this application. Aortic mural macrophages are known to produce extracellular matrix-destroying proteinases, particularly MMPs 2 and 9.24,25 In the current study, aortic macrophage density was reduced in telmisartan-treated mice, as was mRNA for the chemokine CCL5. MMP production was also suppressed, as evidenced by reduced expression of mRNAs for MMP2 and

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Fig 6. Peroxisome proliferator-activated receptor g (PPARg) agonist activity does not significantly contribute to telmisartan-mediated aneurysm inhibition. (A) Total RNA from the aortae of mice infused with phosphatebuffered saline (PBS), porcine pancreatic elastase (PPE), or PPE and fed telmisartan was analyzed for four PPARg-regulated gene messenger RNA (mRNA) levels by real-time reverse transcription-polymerase chain reaction (RT-PCR) assays. Message levels: fold change (log scale) relative to normal aorta. Nonparametric MannWhitney test, *P < .05 and **P < .01 between two groups (n ¼ 5 mice/group). (B) Serial ultrasound measurements (mean and standard deviation) of aortic diameters in PPE-infused mice fed standard chow or telmisartansupplemented chow and simultaneously treated with a PPARg antagonist GW9662 (3 mg/kg/d). All treatments were begun 1 week before PPE infusion for a total of 3 weeks. Two-way analysis of variance followed by Newman-Keuls test, *P < .05 and **P < .01 between two groups (n ¼ 10 mice/group).

MMP9 and preservation of medial elastin. Previous work has demonstrated attenuation of experimental aneurysms in mice deficient for the CCL2 receptor CCR2 or treated with pharmacologic inhibitors of CCL5.10,26-28 Although the amount of aneurysmal aortic tissue generated in these experiments was not sufficient to determine whether expression of CCL2, CCL5, MMP2, and MMP9 protein was influenced by telmisartan treatment, prior published studies have demonstrated reduced expression of CCL2 and CCL5 protein after exposure to telmisartan and other ARBs in vitro and in vivo (Supplementary Tables II and III, online only). Together, these results suggest that AT1 activation promotes AAA disease in part through upregulation of chemokine

production by vascular constitutive cells or aortic resident leukocytes, increasing monocyte chemotaxis into areas of initial and ongoing aneurysmal aortic degeneration. In addition to producing MMPs, macrophages also mediate pathogenic angiogenesis. Aortic mural angiogenesis, a characteristic pathologic feature of human AAA disease,29 was substantially diminished in AT1a KO mice after PPE infusion and in WT mice treated with telmisartan. Whether this inhibition was the consequence of reduced mural macrophage cellularity, reduced proangiogenic factor production by infiltrating macrophages, or a combination of both could not be determined in these experiments. Aortic macrophage density is also regulated to a certain degree by mural

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Fig 7. Influence of telmisartan treatment status on splenic T-cell cytokine production and regulatory T-cell (Treg) differentiation. Lymphocytes were isolated from the spleens of mice fed telmisartan or control chow beginning 1 week before porcine pancreatic elastase (PPE) infusion and ending 2 weeks thereafter. Interferon-g (IFN-g)-, interleukin 17 (IL-17)-, or interleukin 10 (IL-10)-producing CD4þ T cells and CD8þ T cells or Foxp3expressing cells in CD4þ T cells were stained using the combination of surface and intracellular staining followed by flow cytometric analysis. (A and B) Representative fluorescence-activated cell sorting plots showing cytokine-producing splenic CD4þ T cells (A), Foxp3-expressing splenic CD4þ T cells (A), and cytokine-producing splenic CD8þ T cells (B). FSC, Forward scatter. (C and D) Mean and standard deviation of the percentages of cytokine-producing splenic CD4þ or CD8þ T cells. (E) Mean and standard deviation of the percentages of Foxp3þ Tregs in splenic CD4þ T cells (n ¼ 5-6 mice).

neovascularity. Thus, impaired angiogenesis may be an additional telmisartan-mediated mechanism of aneurysm suppression. PPARg, a nuclear transcription factor, regulates diverse cellular responses relevant to cardiovascular health.30 Although ARBs such as telmisartan and irbesartan may demonstrate partial PPARg agonist activity,8 the extent to which ARB-induced PPARg activity modulates experimental aneurysm progression has been uncertain. PPARg agonists do suppress experimental aneurysms in alternative systems, such as the Ang II infusion in the ApoE-deficient mouse model,12,13,31 and SMC-specific PPARg ablation accelerated aneurysmal degeneration in experimental AAAs induced by adventitial application of calcium chloride. Because losartan, an ARB without PPARg agonist activity, was previously found to be

ineffective in suppressing experimental AAAs created by PPE infusion,14 the efficacy of telmisartan in this application may have otherwise been attributable to PPARgrelated activity. Previous modeling experiments in AT1a KO mice have predominantly linked the antiatherogenic properties of telmisartan to anti-AT1 activity.32,33 In this study, mRNA levels of ABCA1, CD36, and LXR, all regulated by PPARg, were elevated in aneurysmal compared with nonaneurysmal aorta. Expression was not influenced by telmisartan treatment status, however, and in telmisartan-treated mice, concurrent PPARg antagonist administration failed to “rescue” the PPE-induced aneurysm phenotype. Because aneurysms failed to develop after PPE infusion in AT1a KO mice, we were unable to conclusively determine whether “off target” effects of telmisartan, such as

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PPARg modulation, exerted any influence on aneurysm progression. Nonetheless, on the basis of the available evidence, it seems unlikely in this model that telmisartan-mediated aneurysm suppression is dependent on PPARg modulation. RAS suppression by AT1 or ACE inhibitors impairs proinflammatory IFN-g and IL-17 production by T cells and increases Treg cell density in experimental autoimmune inflammation.9 In prior studies, reduced Treg cellularity was associated with augmented experimental AAAs,34 whereas the roles of individual T-cell-derived cytokines have varied.15,17,18,35 In this study, the percentages of IFN-g-, IL-10-, and IL-17-producing cells in either CD4þ or CD8þ T cells were comparable between the telmisartan and standard chow treatment groups. In addition, telmisartan treatment did not alter splenic Treg cellularity. These results indicate that in this construct, telmisartan treatment had no recognizable impact on the differentiation of functional T-cell subsets and that AAA suppression occurred independent of influences on T-cell cytokine production or Treg cellularity. Several limitations exist to this study. Without the availability of a highly specific antimouse Ang II or AT1a antibody, neither aortic AT1a nor aortic or serum Ang II expression or circulating levels could be quantified during or after aneurysm creation in this modeling system. At least one prior study has reported transient increases in aortic mRNA expression levels of ACE, an enzyme responsible for converting Ang I to Ang II, after intraaortic PPE infusion,36 providing at least indirect evidence that endogenous Ang II levels are indeed elevated in this model. Some ARBs also block the “b” subtype of AT1,37,38 raising the possibility that AT1b inhibition may contribute to some of the observed inhibitory effects of telmisartan. Given the nearly complete abrogation of aneurysm formation in AT1a KO mice, however, it is clear that AT1a activity plays a substantive role in AAA formation. If present, additional anti-AT1b activity may play a contributing but not likely essential role in the observed telmisartan effect. Additional pharmacologic effects of telmisartan include suppression of reactive oxygen species production in murine vascular disease models.39 Because deficiency, or inhibition, of reactive oxygen species production also inhibits experimental aneurysms,40 this study design did not exclude the possibility that the observed AAA suppression by telmisartan was not due in part to redox-related effects. In addition, although dose-ranging experiments with the PPARg antagonist GW9662 were not performed, the dose employed was higher than that previously proven to blunt PPARg activity in various rodent disease models (Supplementary Table I, online only).33 The inclusion of a positive PPARg agonist control group, including an agent such as rosiglitazone, was also not performed as further investigations into PPARg-mediated effects on AAA pathogenesis were outside the scope of this study.

CONCLUSIONS These genetic and pharmacologic studies confirm the critical importance of AT1 in the pathogenesis and progression of experimental AAA models, even in the absence of exogenous Ang II supplementation. The aneurysm inhibitory properties of telmisartan are independent of influences on PPARg, T-cell cytokine production and differentiation, and Treg cellularity. These results provide further support for translational investigation of telmisartan in the suppression of early and established clinical AAA disease.

AUTHOR CONTRIBUTIONS Conception and design: BX, MM, SM, RD Analysis and interpretation: HX, BX, WW, HT, NF, MM, SM, RD Data collection: HX, BX, WW, HT Writing the article: BX, RD Critical revision of the article: HX, BX, WW, HT, NF, MM, SM, RD Final approval of the article: HX, BX, WW, HT, NF, MM, SM, RD Statistical analysis: HX, BX, WW, NF, RD Obtained funding: BX, MM, RD Overall responsibility: RD HX and BX contributed equally to this article and share co-first authorship.

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26. Ishibashi M, Egashira K, Zhao Q, Hiasa K, Ohtani K, Ihara Y, et al. Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice. Arterioscler Thromb Vasc Biol 2004;24:e174-8. 27. Daugherty A, Rateri DL, Charo IF, Owens AP, Howatt DA, Cassis LA. Angiotensin II infusion promotes ascending aortic aneurysms: attenuation by CCR2 deficiency in apoE/ mice. Clin Sci (Lond) 2010;118:681-9. 28. MacTaggart JN, Xiong W, Knispel R, Baxter BT. Deletion of CCR2 but not CCR5 or CXCR3 inhibits aortic aneurysm formation. Surgery 2007;142:284-8. 29. Choke E, Cockerill GW, Dawson J, Howe F, Wilson WR, Loftus IM, et al. Vascular endothelial growth factor enhances angiotensin II-induced aneurysm formation in apolipoprotein E-deficient mice. J Vasc Surg 2010;52:159-66.e1. 30. Touyz RM, Schiffrin EL. Peroxisome proliferator-activated receptors in vascular biologydmolecular mechanisms and clinical implications. Vascul Pharmacol 2006;45:19-28. 31. Moran CS, Cullen B, Campbell JH, Golledge J. Interaction between angiotensin II, osteoprotegerin, and peroxisome proliferator-activated receptor-g in abdominal aortic aneurysm. J Vasc Res 2009;46:209-17. 32. Fukuda D, Enomoto S, Hirata Y, Nagai R, Sata M. The angiotensin receptor blocker, telmisartan, reduces and stabilizes atherosclerosis in ApoE and AT1aR double deficient mice. Biomed Pharmacother 2010;64:712-7. 33. Tiyerili V, Becher UM, Aksoy A, Lutjohann D, Wassmann S, Nickenig G, et al. AT1-receptor-deficiency induced atheroprotection in diabetic mice is partially mediated via PPARg. Cardiovasc Diabetol 2013;12:30. 34. Xiao J, Angsana J, Wen J, Smith SV, Park PW, Ford ML, et al. Syndecan-1 displays a protective role in aortic aneurysm formation by modulating T cell-mediated responses. Arterioscler Thromb Vasc Biol 2012;32:386-96. 35. Shimizu K, Shichiri M, Libby P, Lee RT, Mitchell RN. Th2-predominant inflammation and blockade of IFN-g signaling induce aneurysms in allografted aortas. J Clin Invest 2004;114: 300-8. 36. Eagleton MJ, Cho B, Lynch E, Roelofs K, Woodrum D, Stanley JC, et al. Alterations in angiotensin converting enzyme during rodent aortic aneurysm formation. J Surg Res 2006;132:69-73. 37. Zhou Y, Chen Y, Dirksen WP, Morris M, Periasamy M. AT1b receptor predominantly mediates contractions in major mouse blood vessels. Circ Res 2003;93:1089-94. 38. Oliverio MI, Best CF, Kim HS, Arendshorst WJ, Smithies O, Coffman TM. Angiotensin II responses in AT1A receptordeficient mice: a role for AT1B receptors in blood pressure regulation. Am J Physiol 1997;272(Pt 2):F515-20. 39. Takaya T, Kawashima S, Shinohara M, Yamashita T, Toh R, Sasaki N, et al. Angiotensin II type 1 receptor blocker telmisartan suppresses superoxide production and reduces atherosclerotic lesion formation in apolipoprotein E-deficient mice. Atherosclerosis 2006;186:402-10. 40. Emeto TI, Moxon JV, Au M, Golledge J. Oxidative stress and abdominal aortic aneurysm: potential treatment targets. Clin Sci (Lond) 2016;130:301-15.

Submitted Aug 9, 2016; accepted Dec 15, 2016.

Additional material for this article may be found online at www.jvascsurg.org.

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Supplementary Table I (online only). Studies demonstrating the effective dose of peroxisome proliferator-activated receptor g (PPARg) antagonist in various disease models Reference

Model

Species

Cordaro M, et al. Mol Pharmacol 2016;90:549-61

Inflammatory bowel disease

Mouse

1

Left eye

Yoon YS, et al. Mucosal Immunol 2015;8:1031-46

Lung inflammation and fibrosis

Mouse

1

Intraperitoneal

Intracranial aneurysm

Mouse

1

Intraperitoneal

Kriska T, et al. Am J Physiol Heart Circ Physiol 2014;306:H26-32

Hypertension

Mouse

0.05

Intraperitoneal

Kusunoki H, et al. J Am Heart Assoc 2013;2:e000103

Hypertension

Mouse

0.5

Drinking water

Nagahama R, et al. Arterioscler Thromb Vasc Biol 2012;32:2427-34

Hind limb ischemia

Mouse

1

Intraperitoneal

Nagashima A, et al. J Cardiovasc Pharmacol 2012;60:158-64

Myocardial ischemia

Rat

1

Oral gavage

Blood-brain barrier in type 2 diabetes

Mouse

0.35

Drinking water

Artery remodeling

Mouse

0.3, 2

Intraperitoneal

Maejima Y, et al. Lab Invest 2011;91:932-44

Left ventricle remodeling

Mouse

2

Intraperitoneal

Bento AF, et al. Am J Pathol 2011;178:1153-66

Colitis

Mouse

1

Intraperitoneal

Cognitive impairment

Mouse

1

Chow

Myocardial infarct

Rabbit

2

Intravenous

Hasan DM, et al. Hypertension 2015;66:211-20

Min LJ, et al. Hypertension 2012;59:1079-88 Tobiasova Z, et al. Circulation 2011;124:196-205

Washida K, et al. Stroke 2010;41:1798-806 Kobayashi H, et al. Am J Physiol Heart Circ Physiol 2009;296:H1558-65

Dose, mg/kg

Route

Supplementary Table II (online only). In vivo studies demonstrating the suppression of matrix metalloproteinase (MMP) protein level and activity by telmisartan Reference

Model

Species

Dose, mg/kg

Tissue

Assay

MMP

Kono S, et al. J Stroke Cerebrovasc Res 2015;24:537-47

Ischemic stroke

Rat

0.3 or 3

Brain

Immunohistochemistry

MMP9

Araujo AA, et al. J Clin Periodontol 2013;40:1104-11

Periodontitis

Rat

10

Periodontal tissue

Immunohistochemistry

MMP2 MMP9

Hosuge H, et al. J Heart Lung Transplant 2010;29:562-76

Graft arteriosclerosis

Mouse

10

Western blotting

Active MMP2

Right ventricular remodeling

Rat

3

Right ventricle

Western blotting

MMP2 MMP9

Takenaka H, et al. J Mol Cell Cardiol 2006;40:989-97

Left ventricle failure

Rat

5

Left ventricle

Prourokinase assay

MMP2 activity

Grothusen C, et al. Atherosclerosis 2005;182:52-69

Atherosclerosis

ApoE-deficient mouse

1

Aorta

Western blotting Gelatin zymography

MMP9 level and activity

Okada M, et al. J Pharmacol Sci 2009;111:193-200

Cardiac allograft

12.e2

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Supplementary Table III (online only). Studies demonstrating the suppression of CCL2 and CCL5 protein levels by telmisartan or other angiotensin receptor blocker (ARBs) Reference

Model

Species

ARB

Specimen

Assay

Chemokine

Nephropathy

Mouse

Losartan

Kidney

Immunohistochemistry

CCL2

Sato K, et al. J Stroke Cerebrovasc Dis 2014;23:2511-9

Stroke

Rat

Telmisartan

Brain

Immunohistochemistry

CCL2

Sato K, et al. J Stroke Cerebrovasc Dis 2014;23:2580-90

Stroke

Rat

Telmisartan

Brain

Immunohistochemistry

CCL2

Diabetes

Rat

Telmisartan

Aorta

Immunohistochemistry

CCL2

Cardiac injury

Rat

Telmisartan

Heart

ELISA

CCL2

Metabolic syndrome

Rat

Losartan Telmisartan

Urine

ELISA

CCL2

Cell culture

Human

Losartan

ECs

ELISA

CCL2, CCL5

Hyperoxaluria

Rat

Losartan

Kidney

Immunohistochemistry CCL2, CCL5

Li Z et al. Chin J Tramautol 2004;7:56-61

Artery injury

Mouse

Olmesartan

Artery

Western blotting Immunohistochemistry

CCL2

Candido R, et al. Circulation 2004;109:1536-42

Atherosclerosis

Mouse

Irbesartan

Aorta

Immunohistochemistry

CCL2

Ishibashi M, et al. Circ Res 2004;94:1203-10

Hypertension

Mouse

Olmesartan

Aorta

Immunohistochemistry

CCL2

Koh KK, et al. J Am Coll Cardiol 2003;42:905-10

Hypertension

Human Candesartan

Plasma

ELISA

CCL2

Reddy MA, et al. Kidney Int 2014;85:362-73

Guo Z, et al. Cardiovasc Diabetol 2012;11:94 Hadi N, et al. BMC Cardiovasc Disord 2012;12:63 Khan AH, Imig JD. Am J Hypertens 2011;24:816-21 Mateo T, et al. J Immunol 2006;176:5577-86 Tolli JE, et al. Urol Res 33:358-367, 2005

ECs, Endothelial cells; ELISA, enzyme-linked immunosorbent assay.

.