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Published in final edited form as: Exp Gerontol. 2021 May 19;151:111416. doi: 10.1016/j.exger.2021.111416

Senolytic agents lessen the severity of abdominal aortic aneurysm in aged mice

Mojtaba Parvizi a, Federico Franchi b, Bonnie K Arendt a, Sanam Ebtehaj b, Martin Rodriguez-Porcel b, Ian R Lanza a,*
PMCID: PMC11443445  NIHMSID: NIHMS2022612  PMID: 34022272

Abstract

Age is a major risk factor for abdominal aortic aneurysm (AAA), for which treatment options are limited to surgical intervention for large AAA and watchful waiting for small aneurysms. However, the factors that regulate the expansion of aneurysms are unclear. Development of new therapeutic strategies to prevent or treat small aneurysms awaits a more thorough understanding of the etiology of AAA formation and progression with aging. A variety of structural and functional changes have been reported in aging vasculature, but emerging evidence implicates senescent cells in the formation of AAA through their paracrine effects on vascular wall cell populations. Here we show that aging is associated with transcriptional changes in abdominal aortic tissue consistent with loss of smooth muscle cells, leukocyte adhesion, inflammation, and accumulation of senescent cells in the vascular wall and surrounding perivascular adipose tissue. Furthermore, aged mice demonstrated anatomical and histopathological features of AAA development in response to administration of angiotensin II over 28 days. Importantly, in our study we sought to determine if reducing senescent cells could lessen the severity of AAA in aged mice. We find that pretreatment of aged mice with oral senolytic agents (dasatinib + quercetin) reduced senescent cell abundance in the arterial walls and surrounding tissues and lessened the severity of AAA in response to angiotensin II administration. These data provide important preliminary evidence supporting a role of senescent cells in age-related AAA formation and progression and suggest that strategies to reduce senescent cell burden hold promise to lessen AAA severity.

Keywords: Abdominal aortic aneurysm, Senescence, Senolytics, Aging, Angiotensin II

1. Introduction

Abdominal aortic aneurysm (AAA) rupture causes 150,000–200,000 annual deaths worldwide (Golledge, 2019). Current management strategies are limited to surgical repair for large AAAs and “watch-and-wait” for small AAAs. No medical intervention has been proven to prevent or delay the progression of AAA for which age is a major risk factor (Nordon et al., 2011). Although aging is accompanied by structural and functional changes in arteries that make them more susceptible to vascular diseases, the factors that accelerate AAA formation and progression with aging are not entirely understood. The development of novel interventional strategies to treat small aneurysms awaits a more complete understanding of the mechanisms contributing to age-related AAA formation and progression. The influx of inflammatory cells (e.g., macrophages and T-cells) has been implicated in the initiation and progression of AAA. Infiltrating macrophages (MQ) secrete matrix metalloproteases (MMP) that disrupt normal extracellular matrix (ECM) homeostasis (Brown et al., 2014), resulting in increased ECM degradation (Brown et al., 2014; Chen et al., 2016; Brady et al., 2004). In addition, apoptosis of medial smooth muscle cells (SMC) and a shift to synthetic phenotype further contribute to aberrant ECM homeostasis and progression of AAA (Chen et al., 2016; Brady et al., 2004; Daugherty and Cassis, 2004).

Early evidence implicates senescent cells (i.e., stable, irreversible cell cycle arrest) as a factor in the formation and progression of AAA (Golledge, 2019; Katsuumi et al., 2018; Guo et al., 2019; Parvizi et al., 2020). There is growing recognition that senescent cells accumulate with aging, obesity, and diabetes, particularly in adipose tissue pools (Palmer et al., 2019; Tchkonia et al., 2010; Minamino et al., 2009; Tchkonia et al., 2013). A hallmark of senescent cells is their senescent secretory phenotype (SASP), which includes inflammatory cytokines, extracellular matrix modifiers, and reactive oxygen species (Xue et al., 2007; Krtolica and Campisi, 2002; Passos et al., 2010), which lead to remodeling or death of proximal cells and tissues. Senescent cells accumulate in vessel walls with aging, evident from elevated senescence markers p16 and senescence-associated beta-galactosidase (SA-β Gal) in endothelial cells (EC) and vascular smooth muscle cells (VSMC) (Guo et al., 2019; Grootaert et al., 2018). Furthermore, we recently demonstrated that the phenotype and function of vascular wall cells are influenced by the secretome of senescent preadipocytes in a manner similar to what is observed in aneurysmal vessel walls (Parvizi et al., 2020). Furthermore, others have shown that senolytic agents effectively kill senescent endothelial cells in vitro (Zhu et al., 2015).

With accumulating evidence implicating senescent cells in the pathophysiology of age-related AAA formation, it is plausible that strategies to reduce senescent cell burden may hold therapeutic potential to lessen the severity and progression of AAA. The goal of the current study was to determine if oral senolytic agents (dasatinib + quercetin) could reduce senescent cell burden in aortic wall of aged mice and attenuate AAA severity in response to established model of angiotensin II-induced AAA. The main finding is that oral senolytic treatment reduces senescent cell abundance in abdominal aortic wall and lessens the severity of AAA in aged mice. These results suggest that senescent cell burden in vascular wall and surrounding tissues may play a role in age-related AAA formation and that reduction of senescent cells holds promise as a therapeutic strategy for AAA.

2. Materials and methods

2.1. Animal model

All experimental procedures and protocols were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC) and performed in accordance with guidelines and regulations from the Guide for the Care and Use of Laboratory Animals. Male C57Bl/6 background mice were purchased from Jackson laboratory (Bar Harbor, ME) at 19 months of age. Mice were group-housed in a vivarium with ad libitum access to standard chow (PicoLab® 5053 rodent diet) and water and kept on a 12:12 h light:dark cycle. At 26 months of age, mice were implanted with micro-osmotic pumps (model 1004, Alzet, Cupertino, CA) for extended delivery of angiotensin II (Ang-II, 1 μg/kg/min in 0.9% saline for 28 days) for induction of AAA or vehicle (0.9% saline). Pumps were implanted subcutaneously under anesthesia with isoflurane, and mice were pre-medicated with 1 mg/kg buprenorphine for analgesia.

2.2. Senolytic treatments

A subgroup of animals that received Ang-II were co-treated with oral senolytic agents (Dasatinib + quercetin) or vehicle (60% Phosal, 10% ethanol, and 30% PEG-400). Drug treatments were initiated one month prior to osmotic pump implantation and continued throughout the 28 day duration of Ang-II infusion. Dasatinib (5 mg/kg, LC Laboratories, Woburn, MA) plus quercetin (50 mg/kg, Sigma Aldrich, St. Louis, MO) or vehicle were administered by oral gavage for three consecutive days with 14 days between the last dose of one treatment and the first day of the next treatment as described previously (Roos et al., 2016; Xu et al., 2018).

2.3. In vivo ultrasound imaging

In vivo abdominal aorta imaging was performed 28 days following osmotic pump implant using high-resolution ultrasound imaging (MS-400; Vevo 2100, Visual Sonics) using a linear array probe (MS 550D, frequency 22–55 MHz) under anesthesia (1% isoflurane). Cross-sectional images of the abdominal aorta were acquired, and abdominal aortic diameter was measured using the Vevo Strain Software for assessment of the suprarenal aortic diameter.

2.4. Histology and immunohistochemistry

The abdominal aorta was excised under anesthesia at the time of sacrifice. Abdominal aorta specimens were fixed in neutral buffered 10% formalin for histopathology measurements. Formalin-fixed samples were embedded in paraffin, and sections of 4 μm thickness were prepared. Hematoxylin and Eosin (H&E) staining was used for general histology to identify cellular structure and to measure the internal diameter of abdominal aorta. Verhoeff’s–Van Gieson’s (VVG) staining was used to assess the presence of elastin in aortic walls. Paraffin embedded scaffold sections (4 μm) were deparaffinized and antigen retrieval performed with Citrate target retrieval solution pH 6 (Sigma, St. Louis, MO) and steaming for 40 min. Sections were treated by hydrogen peroxide at room temperature for 30 min to remove endogenous peroxidase activity. Sections were washed with PBS three times and incubated with Background Sniper (Biocare Medical, Pacheco, CA) at RT for 10 min. Primary antibody was omitted as background labeling control. Negative and positive tissues served as controls. Ex vivo sample sections were incubated with primary antibodies for detection of senescent cells: rabbit anti-mouse GLB1/Beta-galactosidase (Abcam, Cambridge, UK), rabbit anti-mouse HMGB1 (Abcam, Cambridge, UK) and rabbit anti-mouse alpha SMactin (Cell Signaling, Danvers, MA). Sections were washed with PBS (3× for 5 min) and incubated with secondary antibody (peroxidase labeled polymer anti-rabbit peroxidase labeled polymer (DAKO, Carpinteria, CA)). Staining was performed with chromogen system 3,30-Diaminobenzidine substrate (DAKO). Finally, samples were counterstained in Mayer’s Hematoxylin and mounted in Faramount mounting medium (DAKO). Stained samples were scanned by Motic digital slide scanner and quantified with Aperio ImageScope software (Leica, USA).

2.5. Quantitative reverse transcriptase PCR

Total RNA was isolated using the RNeasy Lipid Tissue Mini Kit according to the manufacturer’s instructions. RNA quantity and purity were assessed by spectrophotometric analysis (Nanodrop). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System for RT-PCR cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. The cDNA equivalent of 5 ng RNA was used for amplification in 384-well microtiter plates in a QuantStudio 7 cycler (Applied Biosystems, CA, USA) using SYBR Green assays. Samples were normalized with the ΔCt method, using the geometrical mean of beta-2-microglobulin (B2M). Fold change in gene expression versus control was calculated using the ΔΔCt method.

2.6. Statistical analysis

All data are expressed as mean ± SD. Statistical analyses were performed using Prism 5.04 software (GraphPad Software, Inc). Comparisons between two groups were analyzed by unpaired Student’s t-test. A value of p < 0.05 was considered statistically significant.

3. Results

3.1. Aging negatively influences the aortic structure and inflammatory components

Abdominal aortic tissues were collected from young (6 months) and older (24 month) mice to assess the effects of aging on histological features and gene expression of abdominal aorta. Gene expression analyses demonstrated decreased mRNA expression of the vascular smooth muscle cell (VSMC) markers calponin 1 (CNN1), actin alpha 1 (ACTA1), and transgelin (TAGLN) in old mice compared to young mice (Fig. 1A, B, C), and a reciprocal increase in expression of vascular cell adhesion molecule 1 (VCAM-1) (Fig. 1D). In support of these age-related reductions in expression of VSMC genes, H&E and elastin staining in a representative pair of young and old mice showed evidence of thinner aortic wall and reduced aortic elastin content and elastin fragmentation with older age (Fig. 1, right side). Abdomen aortic tissue of old mice also exhibited gene expression patterns consistent with a pro-inflammatory state. Aged mice exhibited significantly increased mRNA expression of interleukin 1 beta (IL-1B, Fig. 1E), C-X-C motif chemokine ligand 8 (CXCL8, Fig. 1F), C–C motif chemokine ligand 2 (CCL2, Fig. 1G), and a trend for increased interleukin 6 (IL-6, Fig. 1H) that did not reach statistical significance. The possibility that senescent cells accumulate in abdominal aortic tissue with age was evaluated from gene expression measurements of senescent cell markers cyclin dependent kinase inhibitor 1A and 2A (CDKN1A and CDKN2A). Expression levels of both senescent cell gene markers were significantly increased in aortic tissue of aged mice (Fig. 1I, J). Representative staining of the senescent cell markers galactosidase, beta 1 (GLB-1) and high mobility group box protein 1 (HMGB-1) showed increased senescent cell burden with age in the aortic wall as well as surrounding perivascular adipose tissue (Fig. 1, bottom right). Altogether, these data reveal detrimental effects of aging on abdominal aortic wall structure, inflammation, and senescent cell burden.

Fig. 1.

Fig. 1.

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Age-related structural, histopathological, and gene expression changes in abdominal aortic tissue. Expression of vascular smooth muscle cell genes were decreased in abdominal aorta from older compared to young mice (A–C), while vascular cell adhesion molecule 1 gene expression was increased (E). Expression of inflammatory marker genes were increased in old compared to young aortic tissue (E–H) as were the expression of senescent cell markers (I, J). Supporting histology images (right side) show visual evidence of increased aortic diameter, decreased elastin content (VVG), and immunohistochemical evidence of increased senescent cell populations in aortic wall and surrounding tissues with aging. Elastin positivity measured from VVG staining was lower in older mice (K). Boxplots show the 25th and 75th percentiles (box), median (line), and whiskers defined by largest and smallest values within 1.5 times the interquartile range with individual points representing values beyond this range from N = 4 mice per age group. * denotes significant (P < 0.05) different from young.

3.2. Oral senolytic agents attenuate AAA severity in aged mice

Aged mice that received angiotensin II for 28 days exhibited significantly greater luminal diameter of the suprarenal abdominal aorta compared to control aged mice, based on in vivo ultrasound measurements (Fig. 2A, C) and histological measurements in H&E stained aortic sections (Fig. 2B, D). Pre-treatment of aged mice with oral senolytic agents, dasatinib + quercetin, attenuated the enlargement of the abdominal aorta in response to angiotensin II (Fig. 2).

Fig. 2.

Fig. 2.

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Angiotensin II for AAA induction in aged mice and effects of oral senolytic agents. Angiotensin II (Ang II) increased abdominal aortic luminal diameter in aged mice consistent with AAA induction, measured in vivo by ultrasound (A, C) or by histology (H&E, B, D). Senolytic agents dasatinib and quercetin (D + Q) attenuated the increase in aortic diameter with Ang II. Boxplots show the 25th and 75th percentiles (box), median (line), and whiskers defined by largest and smallest values within 1.5 times the interquartile range with individual points representing values beyond this range from N = 7–13 mice per treatment group. * indicated significantly (P < 0.05) different from Ang II −, D + Q−. ϕ represents indicated significantly (P < 0.05) different from Ang II +, D + Q−.

To further interrogate the effects of senolytic agents on histopathological features of the abdominal aorta and surrounding tissues, we performed immunohistochemical staining and targeted gene expression measurements. Verhoeff’s–Van Gieson’s (VVG) staining demonstrated that content of aortic elastin layers was significantly higher in mice pre-treated with D + Q in advance of angiotensin II (Fig. 3A). An additional qualitative observation was that elastin fragmentation was less evident in D + Q treated mice. This observation, combined with preserved elastin volume, suggests that senolytic treatment preserved elastin integrity in the abdominal aorta. Vascular smooth muscle cells (VSMCs), the main cellular component of the aorta, were stained for alpha-smooth muscle actin (aSMA). Senolytic treatment was associated with significantly higher aSMA positivity compared to Ang-II plus vehicle (Fig. 3B). Furthermore, mRNA expression of VSMC markers (TAGLN, ACTA2, and CNN1) were significantly higher in mice receiving D + Q (Fig. 3F), altogether suggests that senolytics help maintain VSMC component of the aortic wall in the Ang II model of AAA. Matrix metalloproteinases (MMPs) are known to contribute in aneurysm development and progression. Gene expression of MMP-2 and MMP-9 showed significantly lower expression of MMP-2 and MMP-9 in Ang-II + DQ group compared to Ang-II + vehicle treated group (Fig. 3G). Senolytic treatment also reduced inflammatory markers in response to Ang-II administration in aged mice, evident from decreased macrophage infiltration from immunohistochemical staining for CD-68 (Fig. 3C) and decreased mRNA expression of the pro-inflammatory cytokines IL-1B and CCL2 (Fig. 3H). Finally, we evaluated the effects of D + Q senolytics on senescent cell burden in the aortic wall and surrounding tissues. As expected, oral senolytics reduced the abundance of senescent cells in aortic wall and perivascular adipose tissue based on HMGB-1 and GLB-1 immunohistochemical staining (Fig. 3D, E) and gene expression measurements (Fig. 3I).

Fig. 3.

Fig. 3.

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Effects of oral senolytic agents on senescent cells, suprarenal aortic structure, and aortic wall gene expression. VVG staining shows elastin fragmentation with Ang II administration, but less fragmentation and increased elastin component with D + Q treatment (A). Smooth muscle cell marker aSM actin staining showed increased positivity with D + Q treatment (B). Macrophage staining revealed decreased CD68 positivity with D + Q treatment (C). Senescent cell markers HMGB-1 and GLB-1 were abundant in aortic tissue of aged mice treated with Ang II, but reduced with oral senolytics D + Q (D,E). mRNA expression of smooth muscle cell markers TAGLN, ACTA2, and CNN1 were increased in the presence of senolytics (F). mRNA expression of MMPs (MMP9, MMP2) were decreased in aortic tissue of mice given senolytics (G). Gene expression of inflammatory markers (H) and senescent cell markers (I) were decreased with senolytic treatment. Boxplots show the 25th and 75th percentiles (box), median (line), and whiskers defined by largest and smallest values within 1.5 times the interquartile range with individual points representing values beyond this range from N = 10–12 mice per treatment group for immunohistochemistry and 4–6 per group for gene expression data. * indicated significantly (P < 0.05) different from Ang II +, D + Q−.

4. Discussion

Senescent cells accumulate in many tissues with normal organismal aging (Palmer et al., 2019; Tchkonia et al., 2010; Minamino et al., 2009; Tchkonia et al., 2013), and these growth-arrested cells have been suggested to contribute to the formation and progression of AAA (Golledge, 2019; Chen et al., 2016; Katsuumi et al., 2018; Guo et al., 2019; Parvizi et al., 2020; Grootaert et al., 2018), particularly in the context of aging (Daugherty and Cassis, 2004; Fu et al., 2013; Donato et al., 2018). Here we demonstrate that aging is associated with transcriptional changes in abdominal aortic tissue consistent with loss of smooth muscle cells, leukocyte adhesion, inflammation, and accumulation of senescent cells in the vascular wall and surrounding perivascular adipose tissue. The main new finding of this study is that oral senolytic agents effectively reduced senescent cell burden in aortic tissue of aged mice and lessened the severity of abdominal aortic aneurysm in response to angiotensin II administration. These data provide preliminary evidence supporting a role of senescent cells in age-related AAA formation and progression and suggest that senolytic drugs hold promise as an intervention that may lessen AAA severity.

Aging is a major risk factor for abdominal aortic aneurysm (Nordon et al., 2011), likely due to structural and functional changes in arteries that make them more susceptible to vascular diseases (Cuomo et al., 2017; Komutrattananont et al., 2019; Ohyama et al., 2018). Such changes include secretion of MMPs by infiltrating macrophages that promote ECM degradation (Brown et al., 2014; Chen et al., 2016; Brady et al., 2004) and loss of medial smooth muscle cells (Chen et al., 2016; Brady et al., 2004; Daugherty and Cassis, 2004). Indeed, we observed decreased vascular smooth muscle cell markers and heightened expression of pro-inflammatory cytokines in abdominal aorta of aged mice, which, combined with angiotensin II, was sufficient to trigger suprarenal AAA formation within 4 weeks. This follows previous work by Deng and colleagues that showed that osmotic pump delivery of angiotensin II was sufficient to induce AAA in 39% of 7–11 month old C57BL6J mice and at higher percentages (~90%) in hyperlipidemic ApoE deficient mice (Deng et al., 2003). Here we show that angiotensin II administration effectively induces AAA formation in aged mice in the absence of experimental dyslipidemia and is a suitable experimental model for studying age-related AAA in mice.

Senescent cells are apoptosis-resistant cells that reside in a state of irreversible growth arrest. These cells have been found in a wide variety of tissues, including skin, kidney, lung, pancreas, prostate, liver, and adipose tissue (Palmer et al., 2019; Burton, 2009) and linked with a variety of age- and obesity-related pathologies (Palmer et al., 2019; Tchkonia et al., 2010; Tchkonia et al., 2013). Our finding that aged mice exhibit increased senescent cell markers in abdominal aortic tissue is consistent with previous reported evidence of senescent cell accumulation in arterial tissue with aging (Donato et al., 2018; Morgan et al., 2013). We find that mRNA levels of senescent cell markers CDKN1A and CDKN2A were markedly increased in suprarenal abdominal aorta from aged mice. While these gene expression measurements reflect multiple cell types that comprise the abdominal aorta and cannot point to specific cell populations, the supporting immunohistochemical staining (HMGB-1, GLB-1) clearly indicates that senescent cells are present in all three major layers of the artery (intima, media and adventitia) and highly abundant in the surrounding perivascular adipose tissue (PVAT). It is however, important to point out that while HMGB-1 and GLB-1 are commonly-used molecular signatures of senescence (Wagner et al., 2015; Dimri et al., 1995), we cannot ignore the possibility that HMGB-1, being ubiquitous to most cell types, may be released by macrophages when stimulated by inflammatory challenges (Personnaz et al., 2019). Perivascular adipose tissue (PVAT) surrounds the vessel adventitia and provides mechanical support and regulatory functions to vessels. Under pathological conditions, PVAT volume expands and exhibits a phenotype characterized by oxidative stress, reduced production of vasoprotective factors, heightened production of pro-inflammatory cytokines, and infiltration of T cells and macrophages (Brown et al., 2014; Nosalski and Guzik, 2017). It is recognized that PVAT may contribute to vascular dysfunction with aging due to the release of paracrine factors (Pan et al., 2019), and there is published evidence that pro-inflammatory proteins in PVAT are associated with arterial stiffness in aged mice (Fleenor et al., 2014). Here we provide preliminary evidence supporting the notion that PVAT harbors a substantial pool of senescent cells, as observed in other adipose tissue depots with aging (Palmer et al., 2019; Tchkonia et al., 2010; Minamino et al., 2009; Tchkonia et al., 2013). The potential paracrine influence of PVAT senescent cells on neighboring vasculature requires further investigation as a potential contributor to the development and progression of AAA.

The notion that age-related senescent cells may contribute to inflammation and oxidative stress in vascular wall cells through paracrine or systemic effects of their inflammatory secretome is becoming mainstream (Donato et al., 2018). Inasmuch, it is tempting to speculate that clearance or reduction of senescent cells holds promise to lessen the severity of many age-related vascular diseases (Childs et al., 2018; Khosla et al., 2020). To date, pharmacological and genetic approaches to remove senescent cells have been shown to effectively attenuate atherosclerosis (Childs et al., 2016), lessen the severity of cardiac dysfunction after myocardial infarction (Alam et al., 2019), and improve vascular function in aged and atherosclerotic mice (Roos et al., 2016). Although the potential for senolytics as a therapeutic intervention for AAA has been recognized (Dookun et al., 2020), this remains a nascent area of research. Here we show that the senolytic agents, dasatinib and quercetin, attenuated the severity of experimental AAA induction with angiotensin II in aged mice. The evidence supporting this conclusion includes observations that oral senolytics attenuated the increase in suprarenal abdominal aorta luminal diameter. Senolytics also reduced the severity of several key histopathological features of AAA development including elastin loss and fragmentation, recruitment of inflammatory macrophages into the vessel wall, and abundance of senescent cells in the aorta and surrounding tissues. Senolytics also exhibited notable influence on expression patterns of key genes relevant to AAA formation and progression such as VSMC markers, proteolytic enzymes (MMPs), and pro-inflammatory molecules.

Although this study represents an important early step in determining if targeting senescent cells can attenuate AAA progression, it is important to recognize a number of limitations of this work. First, oral administration of dasatinib plus quercetin is a systemic treatment that indiscriminately targets senescent cells in many tissues, not just in the vicinity of the AAA. Inasmuch, we cannot exclude the possibility that the observed effects of senolytics on the anatomy, histopathology, and gene expression patterns of the abdominal aorta may be related to reduced senescent cell burden in other tissues. Although senescent cells are known to exhibit paracrine influence on bystander cells of the vasculature (Parvizi et al., 2020), they are also recognized for their contribution to the milieu of circulating inflammatory molecules that may profoundly influence distal tissues (Tchkonia et al., 2013). Future studies could employ local delivery of senolytics or tissue-specific genetic elimination of senescent cells to further evaluate this possibility. It is also important to highlight that the current study was designed as a prevention study since senolytics were administered to mice in advance of angiotensin II infusion. In future studies it will also be important to include additional control groups such as young animals (+/− AngII, +/− senolytics) and older animals treated with senolytics in the absence of Ang II. Although the combination of dasatinib and quercetin have been shown to have senolytic activity through selective apoptosis of senescent cells (Zhu et al., 2015), it is important to consider that these drugs have off-target effects that include inhibition of tyrosine kinase (TK1) and phosphatidylinositol 3-kinase (PI3K) among other effects (Bruning, 2013; Olave et al., 2010). Along these lines, it is difficult to ignore the possibility that the effects of D + Q on AAA severity may be less direct and mediated by potential effects on cardiometabolic parameters such as insulin sensitivity rather than senolytic action, per se. Although we cannot rule out the potential contributions of these off-target effects on AAA progression, we used an intermittent dosing strategy that takes advantage of the short half-lives of these drugs (Graefe et al., 2001; Christopher et al., 2008) relative to their more durable senolytic effects to increase the likelihood that the observed effects were attributable to clearance of senescent cells rather than acute off-target effects. A logical next step, given the promising early results of this study, is to conduct a treatment study where senolytics are administered after early features of AAA manifest, which would provide opportunity to determine if targeting senescent cells can slow the progression of early AAA. This approach would be more translatable to the human situation where current management strategies are limited to “watch-and-wait” for small AAAs until they become large enough to warrant surgical repair. A treatment option to prevent or delay the progression of small aneurysms would have substantial positive impact.

5. Conclusions

This study demonstrates that abdominal aortic tissue undergoes structural and molecular changes with aging that include loss of smooth muscle cells, inflammation, and accumulation of senescent cells in the vascular wall and surrounding perivascular adipose tissue. The study also provides preliminary evidence that oral senolytic agents effectively reduced senescent cell burden in aortic tissue of aged mice and lessened the severity of experimental abdominal aortic aneurysm induction. These data support a hypothesis that age-related accumulation of senescent cells contribute to AAA formation and suggest that strategies to eliminate senescent cell burden have potential to lessen the severity of AAA. This possibility awaits more extensive studies to better understand the role of senescent cells in age-related AAA formation and the utility of senolytics as a countermeasure.

Acknowledgements

The authors would like to thank Drs. Tamar Tchkonia and James Kirkland for sharing their expertise in the preparation and administration of senolytic agents to mice.

Funding

This work was supported by a Launch Award from the Mayo Clinic Department of Medicine and the Mayo Clinic Center for Biomedical Discovery.

Abbreviations:

AAA

abdominal aortic aneurysm

D + Q

dasatinib + quercetin

MQ

macrophages

MMP

matrix metalloproteases

ECM

extracellular matrix

Ang II

Angiotensin II

SASP

Senescence-associated secretory phenotype

SA-β Gal

senescence-associated beta-galactosidase

EC

endothelial cells

VSMC

vascular smooth muscle cells

H&E

Hematoxylin and Eosin

VVG

Verhoeff’s–Van Gieson’s staining

CNN1

calponin 1

ACTA1

actin alpha 1

TAGLN

transgelin

VCAM1

vascular cell adhesion molecule 1

IL-1B

interleukin 1 beta

CXCL8

C-X-C motif chemokine ligand 8

CCL2

C-C motif chemokine ligand 2

IL-6

interleukin 6

CDKN1A

cyclin dependent kinase inhibitor 1A

CDKN2A

cyclin dependent kinase inhibitor 2A

GLB-1

galactosidase, beta 1

HMGB-1

high mobility group box protein 1

aSMa

alpha-smooth muscle actin

MMP

matrix metalloproteinases

Footnotes

Declaration of competing interest

The authors have no declarations of interests to disclose.

CRediT authorship contribution statement

Mojtaba Parvizi: Conceptualization, Methodology, Investigation, Writing – Original Draft, Writing – Review and Editing, Visualization

Federico Franchi: Methodology, Investigation, Writing – Original Draft, Writing – Review and Editing, Visualization

Bonnie K. Arendt: Methodology, Investigation, Writing – Review and Editing, Visualization

Sanam Ebtehaj: Methodology, Investigation, Writing – Review and Editing

Martin Rodriguez-Porcel: Resources, Writing – Review and Editing, Supervision

Ian R. Lanza: Conceptualization, Resources, Writing – Original Draft, Writing – Review and Editing, Visualization, Supervision, Project administration, Funding acquisition

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