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. Author manuscript; available in PMC: 2018 Apr 10.
Published in final edited form as: Cell Rep. 2017 Nov 7;21(6):1471–1480. doi: 10.1016/j.celrep.2017.10.057

Downregulation of the Apelinergic Axis Accelerates Aging, whereas Its Systemic Restoration Improves the Mammalian Healthspan

Rahul Rai 1,2, Asish K Ghosh 1, Mesut Eren 1,3, Alexander R Mackie 1, Daniel C Levine 2,4, So-Youn Kim 5, Jonathan Cedernaes 4, Veronica Ramirez 1, Daniele Procissi 6, Layton H Smith 7, Teresa K Woodruff 5, Joseph Bass 4, Douglas E Vaughan 1,3,8
PMCID: PMC5892413  NIHMSID: NIHMS917852  PMID: 29117554

SUMMARY

Aging drives the occurrence of numerous diseases, including cardiovascular disease (CVD). Recent studies indicate that blood from young mice reduces age-associated pathologies. However, the “anti-aging” factors in juvenile circulation remain poorly identified. Here, we characterize the role of the apelinergic axis in mammalian aging and identify apelin as an anti-aging factor. The expression of apelin (apln) and its receptor (aplnr) exhibits an age-dependent decline in multiple organs. Reduced apln signaling perturbs organismal homeostasis; mice harboring genetic deficiency of aplnr or apln exhibit enhanced cardiovascular, renal, and reproductive aging. Genetic or pharmacological abrogation of apln signaling also induces cellular senescence mediated, in part, by the activation of senescence-promoting transcription factors. Conversely, restoration of apln in 15-month-old wild-type mice reduces cardiac hypertrophy and exercise-induced hypertensive response. Additionally, apln-restored mice exhibit enhanced vigor and rejuvenated behavioral and circadian phenotypes. Hence, a declining apelinergic axis promotes aging, whereas its restoration extends the murine healthspan.

eTOC Blurb

Rai et al. identify an anti-aging role of the endogenous apelinergic axis. They show that the apelin-apelin receptor axis is downregulated with age, and its absence accelerates the onset and progression of aging. Additionally, restoration of apelin extends murine healthspan.

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INTRODUCTION

Aging perturbs organismal homeostasis and leads to multi-organ dysfunction and frailty (Sahyoun et al., 2001). Identification of pathways regulating aging is, therefore, imperative to prolong human healthspan and lighten the burden of aging on global health. Recent reports demonstrate that plasma from young mice reduces age-associated pathologies in old mice (Villeda et al., 2014, Loffredo et al., 2013, Conboy et al., 2005). Evidently, an age-dependent decline in levels of—sparsely identified—pro-youth factors contribute to the aging process, while their restoration provides anti-aging benefits and rejuvenates aging organs. However, the identities of these pro-youth or anti-aging factor(s)/pathways remain elusive. Further identification of these factor(s) will aid anti-aging drug development and address the lack of aging-associated biomarkers.

Apelin (apln) is primarily an endothelial-derived factor. It is synthesized as 77-amino acid preproapelin and rapidly cleaved into active isoforms- including apln-13 (Kleinz and Davenport, 2004, Barnes et al., 2010). Meanwhile, the apelin receptor (aplnr) is ubiquitously expressed (Pitkin et al., 2010), dictating both local and endocrine effects of systemic apelinergic axis. While few studies have predicted a vasodilatory role of the endogenous apelinergic axis (Japp et al., 2010), its physiological relevance remains unclear as young apln−/− and aplnr−/− mice are normotensive and appear healthy (Kuba et al., 2007, Ishida et al., 2004). However, reduced levels of circulating and tissue apln have been reported in patients suffering from CVDs (Chong et al., 2006, Iwanaga et al., 2006, Przewlocka-Kosmala et al., 2011), CKD (El-Shehaby et al., 2010), chronic pulmonary (Chandra et al., 2011) and metabolic diseases (Zhang et al., 2009, Erdem et al., 2008). Apln−/− mice, when challenged, also exhibits an enhanced neurodegenerative phenotype (Kasai et al., 2011), suggesting a causative or a correlative relationship between reduced apln signaling and age-associated pathologies. We hypothesize that a constitutively active apelinergic signaling is necessary for mammalian homeostasis, and its decline accelerates aging and age-related organ impairments. Additionally, we investigate the anti-aging benefits of systemic apln restoration in 15-month-old WT mice.

RESULTS

Aging and stress-induced senescence downregulates endogenous apelinergic axis

To characterize the effects of aging on the apelinergic axis, we first compared levels of apln and aplnr in multiple organs of 1-month, 12 months and 24 months old wild-type mice. Gene expression and western blot analysis demonstrated an age-dependent decline of aplnr in the aorta, kidneys, lungs, liver and mesenteric fat (m-fat) (Figure 1A-C). Similarly, tissue apln in the heart, kidneys, lungs, and m-fat decreased with age as well (Figures 1D-E and S1A). Aging, however, increased the expression of cardiac aplnr and hepatic apln. Though upregulation of cardiac aplnr and hepatic apln could be compensatory responses to downregulated apln & aplnr, respectively, further investigations are required given the role of aplnr in cardiac hypertrophy (Scimia et al., 2012) and apln in liver fibrosis (Principe et al., 2008). Oxidative stress is widely recognized as an accelerator of tissue aging and degeneration (Munoz-Espin and Serrano, 2014). Using chemical stressors like Nω-Nitro-L-arginine methyl ester (Boe et al., 2013) (L-NAME)—which reduces systemic nitric oxide (NO)—and Angiotensin II (Ang II), we observed that stress-induced premature senescence also reduces cardiovascular apln (Figures 1F and S1B). Induction of cellular senescence by 4-day-treatment of TGF-β downregulated endothelial apelinergic axis as well (Figure 1G). As aging reduced the expression of (prepro)apelin gene and tissue apln protein, we predicted that circulating levels of apln (apln-13, apln-17, apln-36) would decline with age as well. Indeed, a prior report investigating the effects of aging on neuronal dysfunction, measured lower circulating apln-13 in the plasma of old rats (Sauvant et al., 2014). Hence, aging promotes, and is later characterized by a state of multi-organ apelinergic deficiency.

Figure 1. Aging and stress-induced senescence downregulates endogenous apelinergic axis.

Figure 1

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(A) Effect of aging on Aplnr transcript levels, n=4

(B) Immunoblots showing the effect of aging on renal and hepatic aplnr

(C) Quantification of 1B

(D) Effect of aging on cardiac and renal apln protein, n=4. See Figure S1A

(E) Effect of aging on Apln transcript levels, n=4

(F) Apln transcript levels in the heart and aorta of placebo or L-NAME and saline or Ang II treated mice, n= 4 (LNAME heart), n=8 (L-NAME aorta), n=3 (placebo/ saline/ Ang II). See Figure S1B

(G) HCAECs stained with senescence associated-β-gal (SA-β-gal) stain after 4 days of TGF-β treatment. Apln and Aplnr transcript levels were assessed after 4 days, n=3.

All data are shown as the mean ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001.

Aged aplnr−/− and apln−/− mice exhibit accelerated multi-organ aging

As aging reduces multi-organ apln and aplnr, we investigated whether downregulation of apelinergic axis alters the subsequent progression of aging. We hypothesized that apln is an anti-aging factor and abrogation of its signaling promotes the onset and progression of pathological aging. Hence, we compared aging-associated functional and morphological changes in wild type (WT), aplnr−/−, and apln−/− mice. Consistent with prior reports, we did not observe any gross or functional defects in 6 months old aplnr−/− and apln−/− mice (Kuba et al., 2007, Ishida et al., 2004). However, by 8–9 months aplnr−/− and apln−/− females exhibited features of early-onset infertility. Infertility is a robust indicator of reproductive aging and is associated with distinct hormonal changes. We further observed lower levels of inhibin alpha and luteinizing hormone receptor (LHR) immunostaining in aplnr−/− and apln−/− ovarian sections (Figure S1C). As inhibin alpha and LHR are necessary for ovarian homeostasis (Makanji et al., 2014), their patchy expression is consistent with premature ovarian aging (Danforth et al., 1998, Broekmans et al., 2009). With age, aplnr−/− and apln−/− mice displayed other features indicating accelerated pathological aging. In particular, while acetylcholine (ACh) induced a dose-dependent dilation of WT mesenteric arteries, aplnr−/− and apln−/− vessels exhibited an aging-like vasodilatory impairment (Gerhard et al., 1996) (Figure 2A). This response was characterized by reduced ACh responsiveness (Figure S2A), while ligand-receptor affinity (Figure S2B) and vascular compliance remained unchanged (data not shown).

Figure 2. Aged aplnr−/− and apln−/− mice exhibit accelerated multi-organ aging.

Figure 2

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(A) Acetylcholine-mediated dilatory responses of WT, aplnr −/−, and apln −/− mesenteric arteries ex vivo. See Figure S2A-B

(B) Age-dependent changes in systolic blood pressure (SBP) in WT, aplnr−/− and apln−/− mice, n= 3–5. See Figure S2C

(C) (i) Representational Periodic-acid Schiff (PAS)-stained cardiac sections, showing cardiomyocyte morphology and size. (ii) Representational images of PAS-stained renal sections, showing glomerular morphology and (iii) tubular lumens. Protein casts are noticeable in aplnr−/− renal sections (black arrow)

(D) Quantification of cardiomyocytic surface area in WT, aplnr−/− and apln−/− hearts at 15 months

(E) Comparison of heart weight to tibial length (HW/TL) ratio in WT, aplnr−/− and apln−/− mice at 9 and 15 months of age, n=4–6

(F) Quantification of glomerular diameters in 15 months old WT, aplnr−/− and apln−/− kidneys

(G) Comparison of renal weight to tibial length (RW/TL) ratio in WT, aplnr−/− and apln−/− mice at 9 and 15 months of age, n=4–6

(H) Compares urinary protein-to-creatinine ratio at 15 months in WT, aplnr−/− and apln−/− mice, n=3

(I) Shows blood glucose levels at 9 and 15 months in WT, aplnr−/− and apln−/− mice, n= 3–5

All data are shown as the mean ± SEM. *p< 0.05, **p< 0.01

As hypertension (HTN) is a well-known phenotypic manifestation of vascular aging (Gerhard et al., 1996), we next measured murine blood pressures (BPs) by tail-cuff method. While aplnr−/− mice were normotensive at birth, their BPs started rising around 9 months of age, and they became hypertensive by 12 months (SBP= 180 ± 14 mm of Hg, p<0.009) (DBP= 150 ± 14 mm of Hg, p<0.02) (Figures 2B and S2C). In addition to elevated blood pressure, aplnr−/− mice also demonstrated features of cardiac hypertrophy characterized by elevated cardiomyocytic size (Figures 2Ci, 2D-E, and S2D). While cardiac hypertrophy is a hallmark of aging (Loffredo et al., 2013), cardiac dilation has been reported with senescence as well (Leri et al., 2003). Interestingly, in contrast to aplnr−/− mice, aged apln−/− mice demonstrated features of cardiac dilation (Figure S2D, S2E). These divergent cardiac phenotypes of aplnr−/− and apln−/− mice are noteworthy and highlight minor signaling differences between aplnr−/− and apln−/− mice. In addition to changes in wall thickness and diameters, expression analysis also showed upregulation of cardiac p53 in apln−/− and aplnr−/− hearts (Figure S2F-G). After assessing for cardiovascular and reproductive aging, we investigated the effects of repressed apelinergic axis on renal homeostasis. As glomerular atrophy is a well-known hallmark of renal aging (Bolignano et al., 2014), we observed decreased glomerular diameters (Figure 2Cii, 2F-G) in the renal sections of 15-month-old aplnr−/− mice. Aging also disrupts the integrity of the glomerular filtration barrier and enhances urinary protein leak. Hence, urinalysis at 15 months showed significantly higher protein in the urine of aplnr−/− mice (Figure 2Ciii and 2H). These observations suggest that downregulation of apelinergic axis contributes to renal aging and dysfunction. Lastly, as aging is characterized by hyperglycemia and insulin resistance (Kirwan et al., 2001), analysis of blood glucose demonstrated a two-fold increase in aplnr−/− mice (Figure 2I). Therefore, we predict that downregulation of apelinergic axis is not only a marker of senescence but rather accelerates the aging process. Additionally, this multi-morbid phenotype was more severe and systemic in aplnr−/− which could be due to the following: firstly, as other ligands beyond apln bind aplnr (Chng et al., 2013), it is possible that an unknown ligand maintains basal “protective” signaling in apln−/− mice. Secondly, aplnr heterodimerizes with and inactivates Angiotensin II receptor, type 1 (AT1R) (Siddiquee et al., 2013). Hence, aplnr deletion could increase the activation of AT1R, enhancing Ang II signaling and worsening the aplnr−/− senescence phenotype.

Genetic or pharmacological repression of apelinergic signaling induces cellular senescence

Endothelial cells serve as the primary source of local and circulating apln (Kleinz and Davenport, 2004, Barnes et al., 2010), but the effect of declining apln levels on endothelial homeostasis is unclear. Hence, we utilized an endothelial cell culture model to investigate and provide mechanistic insights into the anti-aging effects of the apln signaling. Lentiviral-mediated reduction of apln in human coronary artery endothelial cells (aplnKd) (Figure 3Ai) significantly reduced endothelial proliferation (Figure 3B). Predicting a critical role of apln in endothelial health, aplnKd increased senescence-associated-β-galactosidase (SA-β-gal) positive cells (Figure 3Aii and 3C) and upregulated mRNA (Figure 3D) and protein (Figure 3E) levels of several cell cycle regulators, including p16. Apln reduction also enhanced endothelial susceptibility to oxidative stress, as H2O2 induced a robust upregulation of p16, p21, and p53 (Figure S2H). Confirming that active apln signaling is necessary for endothelial homeostasis, its pharmacological suppression by aplnr antagonist also caused cellular aging, as evidenced by elevation of p16, p21, p53 (Figure 3F and 3G) and SA-β-gal staining (Figure 3H). Additionally, the senescence-promoting effect of apelinergic suppression was not cell-specific as incubation of human cardiac fibroblasts (HCFs) with aplnr antagonist (ML221) also upregulated cellular p21 (Figure 3I) and increased SA-β-gal staining (Figure 3H and S2I). As apln-aplnr interaction on endothelial surface lowers cAMP (Mclean et al., 2012), we measured activities of 20 cAMP-dependent transcription factors (TF) and observed that aplnKd increases activity of senescence-promoting Sp1 (Wu et al., 2007) and decreases activity of proliferation-promoting E4F (Grote et al., 2015) and GATA4 (Figure 3J). Dysregulation of these TFs, either individually or in unison, likely contributes to cellular senescence seen after the genetic or pharmacological suppression of apln signaling. Hence, our data indicate that constitutively active apln signaling is necessary for cellular homeostasis and its suppression is sufficient to induce endothelial—and by extension vascular and organismal—senescence.

Figure 3. Genetic or pharmacological repression of apelinergic signaling induces cellular senescence.

Figure 3

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(A) (i) Shows apln immunostaining in ctrl and apln Kd HCAECs (ii) SA-β-gal staining of ctrl and apln Kd HCAECs

(B) 50,000 ctrl and apln Kd cells were plated on day 0 and counted every 24 hours for 4 days

(C) Quantification of SA-β-gal positive ctrl and apln Kd HCAECs from Figure 3Aii (600 cells were counted)

(D) Apln, p21, p53 and pai1 transcript levels in ctrl and apln Kd HCAECs

(E) Immunoblots showing levels of p53, IGFBP3, p21, p16, β-actin in ctrl and apln Kd HCAECs

(F) Immunoblots showing levels of p16 and β-actin in HCAECs treated with DMSO (0.01 %) or aplnr antagonist (5 µM ML221)

(G) p21, p53 and tgf-β transcript levels in HCAECs treated with DMSO or aplnr antagonist

(H) Quantification of SA-β-gal positive HCAECs and HCFs after four days of incubation with DMSO or aplnr antagonist, (approximately 400 cells counted). See Figure S2I

(I) Apln, aplnr and p21 transcript levels in HCFs treated with DMSO or aplnr antagonist. A to I, n=3 (biological replicates)

(J) DNA- transcription factor (TF) array showing TFs affected by apln Kd in HCAECs. An increase or decrease of ≥ 2-fold was considered significant, n=2 (biological replicates)

All data are shown as the mean ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001.

Systemic restoration of apln ameliorates age-associated pathologies and extends murine healthspan

Based on these findings, we propose that apln reduction deprives the organism of its youth-preserving effects and creates an environment which accelerates the onset and progression of pathological aging. Hence, we hypothesized that exogenous restoration of apln in middle age mice could ameliorate aging-associated organ impairments. To test this hypothesis, we infused WT mice—aged 14–15 months—with apln-13 (1.8 mg/kg/day) for four weeks. As CVDs are the leading cause of death in the age group of 65 years and above—comprising 15% (Wan He, 2015) of the US population—we focused our attention on age-associated cardiovascular pathologies. We observed that apln infusion reduces age-associated cardiac hypertrophy, as evidenced by a significant reduction in the thickness of left ventricular (LV) posterior wall (Figures 4A, S3A, and S3B). Consistent with the echocardiography findings, we also observed a significant reduction in heart weight/ tibial length (HW/TL) ratio after 4 weeks of apln infusion (Figure 4B). The anti-aging effects of apln were further supported by a noticeable, but non-significant, reduction in the cardiomyocytic cross-sectional area (Figure S3C). While our data predict that apln restoration reduces cardiac hypertrophy, apln restoration did not alter left ventricular diameters, cardiac contractility (data not shown), rate and rhythm of the aging heart (Figure S3D). Consistent with prior reports (Japp et al., 2010), we also observed a modest and transient reduction in systolic (SBP) and diastolic blood pressure (DBP) with apln infusion (Figure S3E). As vascular compliance decreases with age (Stern et al., 2003), we investigated the effects of apln restoration on exertional hemodynamics and observed that restoration of apln reduces exercise-induced hypertensive response in mice (Figure 4C). Additionally, apln infusion also tends to lower aortic gene expression of p16 and p21 (Figure S3F). In conclusion, reduction in cardiac hypertrophy and exercise-induced hypertensive response predicts a therapeutic role of apln in pathological cardiovascular aging.

Figure 4. Systemic restoration of apln ameliorates age-associated pathologies and extends murine healthspan.

Figure 4

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(A) Shows changes in left ventricular posterior wall (LVPW) thickness after 4 weeks of apln-infusion in 15-month-old mice

(B) Changes in heart weight-to-tibial length ratio

(C) Shows post-exercise elevation in systolic (SBP) and diastolic blood pressures (DBP). A to C, n=4 (saline), n=6 (apln-13)

(D) Total distance covered by mice during the week of behavioral assessment

(E) Shows total movement during the active and inactive phases

(F) Shows rhythmicity of food consumption. The feeding curve of saline-infused mice is out-of-sync as food consumption during inactive phase is also seen (denoted by arrows). Apln rejuvenates the feeding patterns, curve generated during 3rd to 5th day.

(G) EE-energy expenditure of apln or saline infused mice. No differences are seen during the active phase, while apln lowers EE during the inactive phase, suggesting improved, less-fragmented resting profile. Values normalized by body weight. Mean analysis of 4 day measurements. D to G, n=4.

(H) Co-treatment with apln decreases TGF-β-induced endothelial senescence. HCAECs were treated with either PBS, TGF-β (10ng/ml), apln-13 (1µM) or apln-13+TGF-β for 4 days, and then stained with SA-β-gal

All data are shown as the mean ± SEM. *p< 0.05, ***p< 0.001

As aplnr−/− mice displayed features of multi-organ aging, we next explored the effects of apln restoration on renal and metabolic aging. Post-treatment urinalysis showed that apln modestly reduces age-associated proteinuria in mice (Figure S4A). While the diuretic role of apln is well-known (De Mota et al., 2004), we also observed that apln-infusion reduced water consumption in 15-month-old mice (Figure S4B). As reduced water intake and diuresis can account for reduced proteinuria, future studies need to investigate the effect of apln on glomerular filtration barrier and renal aging. Meanwhile, apln-restoration did not alter glomerular diameters (Figure S4C), renal weight/ tibial length ratio, glomerular and tubular morphology in the aging kidneys (data not shown). Consistent with studies in diabetic mice (Castan-Laurell et al., 2012), restoration of apln also tends to improve the metabolic profile as it reduced aging-associated hyperglycemia (p=0.07) (Figure S4D). Lowering of glucose levels could be due to multiple overlapping factors, including enhanced insulin sensitivity, increased physical activity, and reduced food consumption. As restoration of apln did not alter body weight (Figure S4E), we investigated whether apln-infused mice exhibit any differences in their physical activity—important predictor of systemic well-being—and feeding patterns.

The behavioral analysis was undertaken over a period of seven days and included alternating 14-hour-light (inactive) and 10-hour-dark (active) phases. Confirming the anti-aging benefits of systemic apln, we observed that apln-infused mice displayed enhanced vigor and mobility, covering more distance cumulatively (Figure 4D) and during the active phase of assessment (Figure 4E). Apln-infused mice also spent more time at the center of their cage—rather than the periphery—suggesting a reduction in age-associated anxiety (Byers et al., 2010) (Figure S4F). Interestingly, while no differences were observed in the quantity of food consumption (data not shown), saline-infused mice displayed an irregular feeding pattern, consuming food during both active and inactive phases (Figure 4F-red). This irregular feeding behavior is typical of old age as aging disrupts the rhythmicity of circadian clock and clock-regulated physiological processes, including hunger (Froy, 2011). It was, however, noteworthy that apln-restoration rejuvenated the feeding behavior of mice as food consumption was consolidated during the active phases (Figure 4F-blue). Age-associated disruption of circadian rhythm also alters the onset, quality, and duration of sleep. Predicting that restoration of apln improves the quality of rest (sleep) during the inactive phase, apln-infused mice displayed longer uninterrupted resting periods characterized by lower energy expenditure (Figure 4G) and O2 consumption (Figure S4G). We did not observe any changes in the organismal respiratory exchange ratio (RER) after apln administration (Figure S4H). These behavioral studies demonstrate that systemic phenotype of apln-restored middle aged mice resembles that of young, as they exhibit enhanced vigor, mobility, and restored circadian rhythmicity. A similar senescence-delaying benefit was also observed in vitro as co-treatment with apln reduced TGF-β-induced endothelial senescence, as assessed by SA-β-gal staining (Figure 4H). These changes predict that anti-aging effects of apln extends beyond one organ system and advances the conceptual role of apln deficiency in mammalian aging.

While the reversal of murine aging by young circulation has been well-documented, the anti-aging factors mediating these benefits remain unidentified. We identify apln as an anti-aging factor and show that its decline accelerates the aging pathophysiology. Conversely, apln restoration in mice counteracts the progression of aging and ameliorates age-associated impairment of cardiovascular and behavioral functions. Strong evolutionary conservation of apln and aplnr sequences predict that apln could extend healthspan of other mammals. Lastly, as continuous apln infusion presents practical and clinical challenges, it is necessary to develop orally active and stable aplnr agonists and investigate their effects on healthspan and lifespan extension.

EXPERIMENTAL PROCEDURES

Animals

WT C57BL/6 (male and female) mice were obtained from Jackson Labs, ME. Generation of aplnr−/− and apln−/− mice has been described previously (Charo et al., 2009, Sheikh et al., 2008). Both strains were provided by Dr. Thomas Quertermous, Stanford, CA to LHS. Knockout mice were backcrossed ten generations to ensure 100% C57BL/6 background. Maintenance of mice colonies and all experimental procedures were approved by IACUC, Northwestern Univ., and their guidelines were followed.

Western blot and gene expression analysis

Please refer to supplemental experimental procedures for further details, including antibodies and primer sequences used.

Echocardiography

Left ventricular function was determined using two-dimensional (2D), M, and Doppler modes of echocardiography (Vevo 770, Visualsonics Inc, Canada). After anesthesia, animals were placed supine on warming platform, and parasternal long- and short-axis views were obtained in each mode to assess cardiac function.

Histopathology Analysis

After cardiac perfusion heart and kidneys were harvested. Tissues were then formalin fixed, paraffin embedded, sectioned, and stained with Periodic acid-Schiff. Approximately 200 myocytes were counted per mouse, in 4 random fields for the assessment of myocytic area. For glomerular diameter, around 18–20 glomeruli were analyzed per mouse, in 3 random fields. Please see supplemental experimental procedures for further details.

Pressure myography

The Danish MyoTech (DMT) pressure myography system (Atlanta, GA) was used for assessment of all the vascular parameters. Third-order mesenteric arteries (~240 µm, outer diameter at 80mmHg) were isolated and used for analysis. Please see supplemental experimental procedures for further details.

Blood Pressure Measurements

Blood pressure in conscious mice was measured using the non-invasive tail-cuff device (Volume Pressure Recording, CODA, Kent Scientific, CT). Before initial measurements, the animals underwent three training sessions. On the day of measurement, mice were placed in a specialized restrainer with temperature control for 15 minutes, after which at least three sets of 10 measurements were taken.

Lentiviral shRNA-mediated apln knockdown (aplnkd)

HCAECs were purchased from Cell Applications Inc, San Diego, CA. Cells were maintained in human endothelial cell defined media (EDM, Cell Applications), supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/mL streptomycin (Life Technologies, Carlsbad, CA). Mission Lentiviral transduction particles (Sigma-Aldrich, St. Louis, MO) targeting apln were used for apln knockdown (# TRCN0000358700), while HCAECs transfected with mission non-mammalian shRNA control particles (#SHC002V) served as control. Briefly, on day 1 60–70% confluent HCAECs were incubated with viral particles and 8µg/ml polybrene (cat# sc-134220, Santa Cruz). Next day, media was removed, and cells were cultured in EDM supplemented with 10% FBS. On day 3, fresh media containing 4µg/ml of puromycin (cat# sc-108071, Santa Cruz) was added. Puromycin selection was continued for 72 hours. Apln knockdown was verified by immunostaining and apln expression analysis.

Additional in vitro assays

Please see the supplemental experimental procedure section for cellular proliferation assay, SA-β-gal staining, ML221 treatment and transcription factor array.

Osmotic Mini-Pump Implantation and apln-13 Infusion

Osmotic mini-pump from Braintree Scientific, MA (Cat# AP 1004, Alzet Osmotic Pump) were used. 15-month-old mice were infused with pyr1- apln-13 at 1.8 mg/kg/day (Cat# H-4568, Bachem, CA) or equal volumes of saline for 4 weeks.

Exercise stress test (EST)

EST was undertaken in three 10-minute phases. Firstly, mice were acclimatized to the treadmill (TSE LabMaster) for 10 minutes. Then they were made to run at a speed of 10 meters/ min for 10 min, immediately after which, their blood pressure measured for the next 10–12 minutes using non-invasive tail-cuff device.

TSE LabMaster system

TSE LabMaster system was used to quantify mice movements, mobility, food consumption, energy expenditure and other parameters. Saline and apln treated mice were placed in standard metabolic cages and acclimatized for first 3 days. Data recorded during the next 4–5 days was used for analysis. Please refer to the supplemental experimental procedures for further details.

Statistical Analysis

Data are presented as mean ± SEM. Comparisons between two groups were tested by unpaired, two-tailed Student’s t-test using GraphPad Prism 5 (San Diego, CA), unless noted otherwise. P-value < 0.05 is considered significant.

Supplementary Material

supplement

HIGHLIGHTS.

  • Senescence downregulates endogenous apelinergic axis

  • Aplnr−/− and apln−/− mice, and aplnkd cells, exhibit accelerated senescence

  • Apln reduces cardiac hypertrophy and exercise-induced BP in 15-month-old mice

Acknowledgments

We thank Dr. Thomas Quertermous (Stanford, CA) for providing us with apln−/− and aplnr−/− mice. We acknowledge the support of two NHLBI grants awarded to DEV: 2R01HL051387-18A1 and PPG HLI08795. AKG was supported by AHA grant: 16GRNT31130010. DCL, JC, and JB were supported by NIDDK grant: 5R01DK100814 awarded to JB. SYK and TKW were supported by the Center for Reproductive Health After Disease grant (P50HD076188) from the NIH-NCTRI.

Footnotes

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SUPPLEMENTAL INFORMATION

Supplemental information includes supplemental experimental procedures and four figures.

AUTHOR CONTRIBUTIONS

RR, DEV conceptualized and designed the project. RR performed experiments and contributed data for Figs. 1–4 and Supplementary Figs. 1- 4. RR wrote the original draft. DEV, AKG, JB, TKW, SYK reviewed and edited the original draft. LHS, ME, TKW, JB, DP provided reagents and resources. DEV funded and supervised the study. Experimental contributions: pressure myography- ARM; ovarian immunostaining- SYK; exercise stress test- DCL; mouse behavioral studies- JC, DCL; data acquisition- AKG, ME; echocardiography- VR; EKG and heart rates- DP.

The authors have no conflict of interest to declare.

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