Resveratrol Inhibits Growth of Experimental Abdominal Aortic Aneurysm Associated With Upregulation of Angiotensin-Converting Enzyme 2
Recent evidence suggests an important role for angiotensin-converting enzyme 2 (ACE2) in limiting abdominal aortic aneurysm (AAA). This study examined the effect of ACE2 deficiency on AAA development and the efficacy of resveratrol to upregulate ACE2 in experimental AAA.
Approach and Results
Ace2 deletion in apolipoprotein-deficient mice (ApoE−/−Ace2−/y) resulted in increased aortic diameter and spontaneous aneurysm of the suprarenal aorta associated with increased expression of inflammation and proteolytic enzyme markers. In humans, serum ACE2 activity was negatively associated with AAA diagnosis. ACE2 expression was lower in infrarenal biopsies of patients with AAA than organ donors. AAA was more severe in ApoE−/−Ace2−/y mice compared with controls in 2 experimental models. Resveratrol (0.05/100-g chow) inhibited growth of pre-established AAAs in ApoE−/− mice fed high-fat chow and infused with angiotensin II continuously for 56 days. Reduced suprarenal aorta dilatation in mice receiving resveratrol was associated with elevated serum ACE2 and increased suprarenal aorta tissue levels of ACE2 and sirtuin 1 activity. In addition, the relative phosphorylation of Akt and ERK (extracellular signal-regulated kinase) 1/2 within suprarenal aorta tissue and gene expression for nuclear factor of kappa light polypeptide gene enhancer in B cells 1, angiotensin type-1 receptor, and metallopeptidase 2 and 9 were significantly reduced. Upregulation of ACE2 in human aortic smooth muscle cells by resveratrol in vitro was sirtuin 1-dependent.
This study provides experimental evidence of an important role for ACE2 in limiting AAA development and growth. Resveratrol upregulated ACE2 and inhibited AAA growth in a mouse model.
Abdominal aortic aneurysm (AAA) is an important cause of sudden death because of aortic rupture and responsible for ≈200 000 deaths worldwide each year.1 Most AAAs are identified at an early stage when a medical therapy could be instigated to limit AAA progression. There is great interest in identifying targets for the development of drug therapy that can effectively limit AAA growth and aortic rupture because no proven medical treatment is currently available.2–4
Chronic aortic inflammation is thought to play a significant role in AAA pathogenesis through multiple mechanisms, including the release of proteolytic enzymes from infiltrating leukocytes and the generation of reactive oxygen species that induce an inflammatory phenotype in vascular smooth muscle cells.5 The renin–angiotensin system (RAS) has long been implicated in human AAA pathogenesis.4,5 Angiotensin II (AngII)—the primary effector of the RAS—stimulates proinflammatory activity in the vascular wall.6 Historically, angiotensin-converting enzyme (ACE) and the angiotensin type-1 receptor (Agtr1) have been the focus for clinical interventions targeting the RAS; however, recent studies have demonstrated the importance of ACE2 in maintaining the balance of the RAS.7 The action of ACE2 to metabolize AngII to vascular-protective peptides, such as angiotensin-1 to 7 has identified ACE2 as an important negative regulator of the RAS.8,9
Reduced ACE2 expression has been associated with cardiovascular disease in humans and experimental models,7,10 and therapeutic strategies for augmenting ACE2 expression and activity are thus being developed.11–15 Previous studies have reported that the 3,5,4′-trihydroxy-trans-stilbene resveratrol inhibits experimental aortic aneurysm and dissection development.16–18 Resveratrol is reported to upregulate adipose expression of Ace2 in hyperlipidemic mice.19 Many of the beneficial cardiovascular properties attributed to resveratrol are believed to be mediated either directly or indirectly through activation of the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (Sirt1).20–22 Moreover, it has recently been reported that increased ACE2 expression under conditions of cell stress in vitro is controlled by Sirt1.23 Sirt1 exerts protective effects on several cellular processes, such as gene regulation, the stress response, apoptosis, inflammation, and senescence,24 which are processes implicated in AAA.5,25,26
The present study investigated the regulatory role of ACE2 in AAA by assessing its genetic deletion, firstly, in otherwise unmanipulated apolipoprotein E-deficient (ApoE−/−) mice and then in 2 mouse models of AAA. ACE2 expression was examined in human serum and aortic tissue samples from patients with AAA and controls. Finally, the efficacy of resveratrol in upregulating ACE2 and limiting AAA growth was investigated in a mouse model of established AAA and within in vitro studies using human aortic smooth muscle cells (AoSMC).
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
Ace2-Deficient ApoE−/− Mice Were Predisposed to AAA Development and Aortic Rupture
We examined the suprarenal aorta (SRA) and serum of otherwise unmanipulated ApoE−/− mice deficient in Ace2 (ApoE−/−Ace2−/y) and age- and sex-matched ApoE−/− controls. Median maximum diameter of the SRA in ApoE−/−Ace2−/y mice was significantly larger than that in control mice (Figure 1A). Intriguingly, 2 of the Ace2-deficient animals (but none of the controls) exhibited unexpected marked dilatation of the SRA (Figure 1B). Aortic tissue levels of AngII were greater in ApoE−/−Ace2−/y mice compared with ApoE−/− controls (22±5 pg/mg protein versus 10±2 pg/mg, respectively; n=6, P<0.001). ApoE−/−Ace2−/y mice exhibited markedly upregulated aortic gene expression for the proinflammatory markers interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1, vascular cell adhesion molecule-1, and Mac-1/CD11b (Figure I in the online-only Data Supplement). Aortic gene expression for the extracellular matrix proteins collagen I and III, elastin, fibronectin, connective tissue growth factor, and transforming growth factor β was similar in ApoE−/−Ace2−/y and ApoE−/− mice (Table I in the online-only Data Supplement). Aortic gene expression for osteopontin, osteoprotegerin, and matrix metallopeptidase (Mmp)2 and Mmp9 was significantly greater in ApoE−/−Ace2−/y than ApoE−/− mice (Figure I in the online-only Data Supplement). Lysyl oxidase activity within aortas of ApoE−/−Ace2−/y mice was downregulated compared with ApoE−/− controls (7.1±0.5 relative fluorescence units/ug protein versus 12.8±2.8 relative fluorescence units/ug protein, respectively; n=8, P=0.025). Serum concentration of AngII was significantly higher in ApoE−/−Ace2−/y mice compared with ApoE−/− controls (268±31 versus 135±21 pg/mL, respectively; n=6, P<0.001), whereas serum total cholesterol (10.3±0.6 versus 10.1±0.5 mmol/L) and triglyceride (1.1±0.1 versus 1.1±0.2 mmol/L) concentrations were comparable. Systolic blood pressure was significantly higher in ApoE−/−Ace2−/y mice compared with controls (110±2 versus 98±2 mm Hg, respectively; n=8, P<0.001).
Figure 1. Predisposition of ApoE−/−Ace2−/y mice to aortic dilatation. Larger maximum suprarenal aorta (SRA) diameter (A) and SRA aneurysms (arrow; B) in otherwise unmanipulated ApoE−/−Ace2−/y mice compared with ApoE−/− control mice. Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for maximum SRA diameter (mm; A). C, Kaplan–Meier curves illustrating survival data for AngII-infused ApoE−/−Ace2−/y (open circle) and ApoE−/− mice (closed circle); P value calculated using log-rank (Mantel–Cox) test. D, Incidence of aortic rupture (black) within total number of mice per group (gray); *P=0.001 compared with control by Fisher exact test. E, Ultrasound measurement of infrarenal (IRA) dilatation induced by CaCl2 for 28 d in ApoE−/−Ace2−/y (white bar) compared with ApoE−/− control mice (grey bar). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for maximum diameter (mm); P value calculated for difference between groups by mixed-effects linear regression. F, Increase in IRA diameter induced by CaCl2 in ApoE−/−Ace2−/y mice compared with ApoE−/− controls. Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for percent increase in diameter relative to sham controls.
The effect of Ace2 deficiency was examined in 2 models of experimental AAA. Remarkably, subcutaneous infusion of AngII (1 μg·kg·min) resulted in an 83% rupture rate (10 of 12 mice) in ApoE−/−Ace2−/y mice within 7 days of pump insertion, compared with only 15% (2 of the 13) in ApoE−/− controls during the 28-day AngII infusion period (Figure 1C and 1D; Figure II in the online-only Data Supplement). Ace2 deficiency resulted in more severe aortic dilatation within the calcium chloride (CaCl2) model. After CaCl2 application, a time-dependent increase in infrarenal aortic (IRA) diameter was observed by ultrasound in both ApoE−/− (P<0.001, ANOVA) and ApoE−/−Ace2−/y (P<0.001, ANOVA) mice (Figure 1E). The rate of IRA expansion was markedly greater in ApoE−/−Ace2−/y than control mice (P=0.034, linear mixed-effects; Figure 1E). There were no aortic ruptures in either group of mice within the CaCl2 model. At the end of the study, the aortas were harvested from all mice and used to perform morphometric analyses. A markedly greater percent increase in IRA diameter (relative to corresponding sham controls) was demonstrated in ApoE−/−Ace2−/y mice compared with ApoE−/− controls (Figure 1F; Figure III in the online-only Data Supplement).
Serum ACE2 Activity and Aortic Expression of ACE2 Were Reduced in Patients With AAA
ACE2 activity was measured in serum samples obtained from 548 participants of the HIMS study (Health in Men Study), comprising 205 men who had an AAA and 343 who did not. Participant demographic characteristics are presented in Table II in the online-only Data Supplement. The association of circulating ACE2 activity with AAA diagnosis was assessed using binary logistic regression, including analyses which adjusted for established AAA risk factors (Table III in the online-only Data Supplement). ACE2 activity had a significant inverse association with AAA diagnosis after adjusting for AAA risk factors and potential confounders (P=0.032; Table III in the online-only Data Supplement). A similar trend was observed in the unadjusted analysis although the association was not statistically significant (P=0.087).
Expression of ACE and ACE2, and the level of CpG island methylation in the promoter region of ACE2 (Figure IV in the online-only Data Supplement) were assessed in IRA biopsies collected from patients undergoing open surgery for AAA repair and from heart-beating brain-dead organ donors (controls). Demographic characteristics of the participants are presented in Table IV in the online-only Data Supplement. Expression of ACE was similar in AAA and control biopsies (Figure V in the online-only Data Supplement); however, ACE2 was downregulated 8-fold in AAA biopsies compared with controls (P=0.024; Figure V in the online-only Data Supplement). Hypermethylation of 12 CpG sites was found in all AAA samples, and the mean percentage CpG island methylation was higher in AAA than control aortic biopsies (P=0.032; Figure VI in the online-only Data Supplement).
Growth of Preinduced AAAs Was Inhibited With Dietary Resveratrol Supplementation in a Mouse Model
Two consecutive 28-day subcutaneous pumps were implanted (days 0 and 29) in a cohort of high-fat chow (HFC)-fed ApoE−/− mice to supply continuous AngII (1.0 μg·kg·min) during an 8-week experimental period (Figure VII in the online-only Data Supplement). After an initial 14-day AAA establishment period, surviving mice were allocated to receive either HFC (control; n=15) or HFC+0.05 g resveratrol/100-g HFC (intervention; n=15) diets for the remaining 6 weeks.
Continuous AngII infusion during the intervention period resulted in a mortality rate because of aortic rupture of 27% (4 of 15) in the control (HFC) group versus 7% (1 of 15) in the intervention (HFC+resveratrol) group (Figure VIII in the online-only Data Supplement). The reduced rupture rate in mice receiving resveratrol was not statistically significant (P=0.130, log-rank test; Figure VIII in the online-only Data Supplement). Body weight of surviving mice fed either HFC (P<0.001, ANOVA) or resveratrol-supplemented HFC (P<0.001, ANOVA) diets increased during the 6-week intervention period (Figure 2A). However, the rate of increase in body weight was markedly reduced in mice receiving resveratrol (P<0.001, linear mixed-effects; Figure 2A). The average amount of food consumed during the experimental period was comparable between the groups (Figure IX in the online-only Data Supplement).
Figure 2. Effect of resveratrol (RSV) in an AngII-infused, high-fat fed, ApoE−/− model of established abdominal aortic aneurysm. Bodyweight (A) and ultrasound measurement of the suprarenal aorta (SRA; B) in ApoE−/− mice receiving high-fat chow (HFC; grey) and HFC+0.05 g RSV/100 g HFC (white). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for body weight (g; A) and maximum SRA diameter (mm; B); P values calculated for difference between groups by mixed-effects linear regression. C, Maximum SRA diameter at the end of the study in ApoE−/− mice receiving control diet (HFC) and RSV-supplemented diet (HFC+0.05-g RSV/100-g HFC). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for diameter (mm). D, Serum angiotensin-converting enzyme 2 (ACE2) concentration at baseline, 14 d, and 56 d during an AngII infusion in ApoE−/− mice receiving HFC and at day 56 in ApoE−/− mice receiving HFC supplemented with RSV. Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for concentration (pg/mL); *P=0.005 compared with controls at day 56 by Mann–Whitney U test. Sirtuin 1 (SIRT1) activity (E) and ACE2 (F) measured at the end of the study within SRA tissue of ApoE−/− mice receiving HFC vs HFC supplemented with RSV. Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for deacetylase activity (relative fluorescence units; E) and tissue concentration (pg/μg protein; F).
A time-dependent increase in maximum SRA diameter in response to AngII was observed by ultrasound in control mice during the 6-week period (P<0.001, ANOVA; Figure 2B). In contrast, the maximum SRA diameter in mice administered HFC diet supplemented with resveratrol did not change significantly (P=0.318, ANOVA; Figure 2B). Control mice exhibited a substantially greater rate of SRA expansion during the intervention period compared with mice receiving resveratrol (P=0.034, linear mixed-effects; Figure 2B). Mice receiving resveratrol that reached the end of the experimental period had significantly smaller maximum SRA diameter than controls when measured by morphometry (P<0.001; Figure 2C; Figure X in the online-only Data Supplement).
Serum concentration of ACE2 was determined in all mice at baseline, at the time of allocation to the interventional and control groups (day 14), and at the completion of the intervention period. A decrease in serum ACE2 concentration over time was demonstrated in control mice in response to AngII infusion and HFC diet (P=0.026, ANOVA; Figure 2D). At the end of the study (day 56), the median serum concentration of ACE2 in these mice was significantly lower than at baseline (P=0.015; Figure 2D). Supplementation of the HFC diet with resveratrol inhibited the reduction in serum ACE2 concentration (P=0.075 compared with baseline, ANOVA; Figure 2D), and median concentration of serum ACE2 in mice receiving resveratrol was above baseline and 4-fold higher than in control mice at the end of the study (P=0.005; Figure 2D).
Ace2 gene expression and protein concentration within the SRA was greater in mice receiving resveratrol than controls (Table 1; Figure 2E). Sirt1 expression and Sirt1 deacetylase activity within the SRA was markedly greater in mice receiving resveratrol compared with controls (Table 1; Figure 2F). Protein concentration of total protein kinase B (Akt1) and ERK (extracellular signal-regulated kinase)1/2 kinases within the SRA tissue of mice receiving resveratrol were similar to concentrations in control mice (Figure XI in the online-only Data Supplement). The ratio of phosphorylated-to-total kinase (activity) for both Akt1 and ERK1/2 within SRA tissue from mice receiving resveratrol was significantly lower than that within SRA tissue from mice on the control diet (Figure 3). Gene expression for the inflammatory marker Nfkb1(nuclear factor of kappa light polypeptide gene enhancer in B cells 1) and proteolytic enzymes Mmp9 and Mmp2 within the SRA of mice receiving resveratrol was downregulated 4.5-, 3-, and 2-fold, respectively, compared with controls, whereas gene expression for the Agtr1 was 2-fold lower in mice receiving resveratrol compared with controls (Table 1).
Table 1. Effect of RSV on Gene Expression in Pre-Established, AngII-Induced AAA in Apolipoprotein E-Deficient Mice Fed HFC
Data expressed as median (interquartile range) mRNA expression relative to GAPDH. Ace2 indicates angiotensin-converting enzyme 2; Agtr1, angiotensin II type-1 receptor; AngII, angiotensin II; HFC, high-fat chow; Mmp, matrix metallopeptidase; Nfkb1, nuclear factor of kappa light polypeptide gene enhancer in B cells 1; RSV, resveratrol; and Sirt1, Sirtuin 1.
*2-sided P value for comparison by Mann–Whitney U test.
Figure 3. Resveratrol downregulated aortic Akt and ERK1/2 (extracellular signal-regulated kinase) phosphorylation in AngII-infused, high-fat fed ApoE−/− mice. Ratio of phosphorylated-to-total Akt1 and ERK1/2 in suprarenal aorta (SRA) tissue of mice fed control diet (grey) or resveratrol-supplemented diet (white). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for quantitative relation between phosphorylated and total kinase.
Normalization of ACE2 in Inflammation-Activated Human AoSMC Via Resveratrol-Mediated Sirt1 Activation
The effect of resveratrol (5 μM) on resting human AoSMC and inflammation-activated AoSMC was examined in vitro. The expression of NFKB1, AGTR1, ACE2, and SIRT1 genes in resting AoSMC is shown in Table 2. Although no significant change in basal expression of NFKB1 was seen, incubation of cells with resveratrol for 24 hours resulted in a significant decrease in AGTR1 and increases in ACE2 and SIRT1 expression (Table 2).
Table 2. Effect of RSV During 24 h on Gene Expression for NFκB, AGTR1, ACE2, and SIRT1 in Resting Human Aortic Smooth Muscle Cell In Vitro
(n=6 Repeat Cultures)
(n=6 Repeat Cultures)
Data expressed as median (interquartile range) mRNA expression relative to GAPDH. Ace2 indicates angiotensin-converting enzyme 2; Agtr1, angiotensin II type-1 receptor; NC, no change; Nfkb1, nuclear factor of kappa light polypeptide gene enhancer in B cells 1; RSV, resveratrol; and Sirt1, Sirtuin 1.
*2-sided P value for comparison between control and intervention by Mann–Whitney U test.
Cell stress was modelled in AoSMC via exposure to activated monocyte-derived proinflammatory media for 5 days. Control cells incubated in proinflammatory media alone exhibited marked increases in the expression of NFKB1 and AGTR1, with significant downregulation of ACE2 and SIRT1 (Figure 4A through 4D). The reduction in ACE2 and SIRT1 gene expression correlated with significant reduction in cellular protein and cellular activity of ACE2 and Sirt1, respectively (Figure 4E and 4F). Supplementation of the proinflammatory media with 5-μM resveratrol during the final 24 hours of the 5-day incubation resulted in changes in expression for all 4 genes to within baseline expression ranges (Figure 4A through 4D). Notably, cellular ACE2 protein and Sirt1 activity were upregulated and measured at levels significantly higher than baseline (Figure 4E and 4F).
Figure 4. Effect of resveratrol (RSV) on inflammation-activated human aortic smooth muscle cell (AoSMC). Gene expression for NFκB (A), AGTR1 (B), ACE2 (C), and SIRT1 (D) cellular ACE2 (E) and cellular SIRT1 activity (F) in a model of AoSMC inflammation induced by incubation of cultures in 10% v/v proinflammatory media (PIM) generated from lipopolysaccharide-stimulated monocytic THP-1 cells and in the presence and absence of RSV (5 μM). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for expression relative to GAPDH (A–D) or pg/105 cells (E) and deacetylase activity (relative fluorescence units; F).
The positive effect of resveratrol on gene and protein expression of ACE2 was ablated on targeted knock-down of Sirt1 (Figure 5). Gene silencing of SIRT1 in activated AoSMC performed using small interfering (si) RNA added after 36 hours of incubation in proinflammatory media resulted in 2-fold downregulation in SIRT1 expression (P=0.002; Figure 5A). Supplementation with 5-μM resveratrol 24 hours after SIRT1 silencing failed to correct downregulated ACE2 expression or cellular ACE2 protein levels (Figure 5B and 5C).
Figure 5. Sirt1-dependent upregulation of angiotensin-converting enzyme 2 (ACE2) by resveratrol (RSV) in inflammation-activated human aortic smooth muscle cell (AoSMC). A, siRNA-targeted silencing of SIRT1 gene in a model of AoSMC inflammation induced by incubation of cultures in 10% v/v proinflammatory media (PIM). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for expression relative to GAPDH. Gene (B) and protein (C) expression of ACE2 in inflamed AoSMC (PIM) in the presence and absence of RSV and siRNA for SIRT1 (iSIRT1). Data expressed as median and interquartile range with maximum and minimum data points (whiskers) for expression relative to GAPDH (B) or pg/105 cells (C).
The main finding of this study was that dietary resveratrol supplementation significantly inhibited progression of AAA in a mouse model. The effect of resveratrol was associated with upregulation of ACE2 expression and Sirt1 activity within the aorta. A link between resveratrol-mediated upregulation of ACE2 and increased Sirt1 activity was demonstrated in human AoSMC in vitro.
Preclinical and clinical studies have demonstrated that ACE2 deficiency promotes susceptibility to metabolic and cardiovascular disease;7 however, the importance of ACE2 in AAA has only recently received attention.13,15,27 Thatcher et al13 first reported that Ace2 deficiency in low-density lipoprotein receptor-deficient mice promoted dilatation of the SRA in response AngII infusion. The current study supports and extends these findings by reporting increased propensity to AAA development by ApoE−/−Ace2−/y mice when AAA is induced by either AngII or CaCl2. Notable was the presence of larger SRA diameter and spontaneous aneurysm in otherwise unmanipulated ApoE−/−Ace2−/y mice. We and others have reported that Ace2 deficiency predisposes to vascular inflammation in the ApoE−/− mouse27,28 and results in increased circulating and tissue levels of AngII.28,29 Higher concentration of AngII and increased markers of extracellular matrix inflammation and degeneration within aortic tissue of ApoE−/−Ace2−/y mice was confirmed in the current study. Although there appeared to be no change in collagen synthesis or in the expression of profibrotic mediators like transforming growth factor beta β and connective tissue growth factor, proteolytic enzymes MMP2 and MMP9 were upregulated, and lysyl oxidase (LOX) activity decreased in ApoE−/−Ace2−/y mice. LOX is an extracellular copper enzyme that initiates crosslinking of collagen and elastin to provide vascular tensile strength and elasticity, and its deficiency leads to aneurysm formation and premature death in mice.30,31 Aortic expression of osteoprotegerin—a protein that we and others have shown to promote AAA through stimulating protease release from monocytes and vascular smooth muscle cells32,33—was increased also in ApoE−/−Ace2−/y mice compared with ApoE−/− controls. It is highly likely that these preexisting differences in expression for markers of inflammation and extracellular matrix turnover, AngII concentration, and blood pressure accounted for the increased SRA dilatation in ApoE−/−Ace2−/y mice and predisposed these mice to more severe AngII or CaCl2-induced aortic aneurysms compared with controls.
Previous investigation of ACE2 in human patients with AAA has been limited. Previous studies that measured plasma or serum ACE2 indicate that circulating ACE2 activity is low in healthy subjects, comparably increased in subjects with cardiovascular risk factors, and correlated with cardiovascular disease development.34,35 Here, we measured serum ACE2 activity in men that did and did not have ultrasound screening-detected AAAs and found relative lower levels in men with an AAA after adjusting for other risk factors. This appears in contrast with existing published data on circulating ACE2 and cardiovascular disease. Importantly, ACE2 undergoes cleavage (shedding) to release the catalytically active ectodomain into the extracellular milieu and circulation;36 however, whether the degree of ACE2 activity within the circulation correlates with tissue ACE2 synthesis or ACE2 shedding from tissue remains uncertain and requires further investigation. We also found the hypermethylation of the ACE2 promoter and downregulation of ACE2 expression in human AAA biopsies compared with samples from donor controls. Overall, these findings suggest downregulation of ACE2 in patients with AAA. Interestingly, low-serum ACE2 was recently reported as an independent risk factor and potential prognostic for in-hospital mortality after open repair of ruptured AAA.37
The ACE2/angiotensin/mas receptor axis has emerged within the RAS as a counterbalance to the classical ACE/AngII/AT1R pathway,8,9 and, subsequently, ACE2 as a potential therapeutic target for the treatment of cardiovascular diseases.7,38 Preclinically, therapeutic strategies for ACE2 involve augmenting its activity or expression. To this end, adenovirus-mediated Ace2 gene transfer15 and ACE2 activation (diminazene aceturate)13 have been used in mouse models of AAA with promising outcomes. The current study adopted an alternative approach. The natural dietary 3,5,4′-trihydroxy-trans-stilbene resveratrol has been reported to inhibit AAA development in several rodent models16–18 and in an unrelated study, increase fat tissue Ace2 expression in mice.19 We investigated the efficacy of resveratrol to positively regulate aortic Ace2 expression and limit growth of established AAA in a mouse model.
Resveratrol administration increased SRA and serum levels of ACE2. The upregulation of ACE2 in the SRA of mice receiving resveratrol was associated with concomitant increase in mRNA and activity for Sirt1. Sirt1 is a nicotinamide adenine dinucleotide-dependent protein deacetylase highly expressed in the vasculature.39 An increasing number of vascular-protective actions for Sirt1, including modulation of stress-induced vascular remodeling, are being recognized.40,41 Recent studies suggest that reduced Sirt1 production and activity in vascular smooth muscle links vascular senescence and inflammation to AAA.42 Sirt1 is a putative target for resveratrol with direct and indirect mechanisms described for resveratrol activation of Sirt1.20–22 Upregulation of ACE2 expression by Sirt1 under conditions of energy stress has recently been reported23 and is of particular relevance to the current study. We demonstrated in an in vitro model of inflammation the downregulation of both ACE2 and Sirt1 in human AoSMC. More important was that rescue and upregulation of ACE2 in these cells by resveratrol was abolished on preincubation of AoSMC with Sirt1-targeted siRNA, thus establishing a potential dependant link between resveratrol, Sirt1 activation, and positive regulation of vascular ACE2.
The relative levels of phosphorylated Akt and ERK1/2 within the SRA of ApoE−/− mice have been previously reported to be increased after AngII infusion.43 The Akt and ERK1/2 pathways have been implicated in the activation of extracellular remodeling enzymes and inflammation in AAA.44,45 Notably, ACE2 deficiency has been reported to promote AngII-mediated aortic inflammation and oxidative stress associated with the activation of Akt/ERK/eNOS signaling.46 Resveratrol has been reported to inhibit reactive oxygen species overproduction in vascular smooth muscle through suppression of Akt and p38 MAPK (mitogen-activated protein kinase)/JNK (c-Jun N-terminal kinase)/ERK phosphorylation.47 In the current study, we found marked reduction in the relative levels of phosphorylated Akt and ERK1/2 within SRA samples of mice receiving resveratrol. This finding suggests that resveratrol administration led to downregulation of both the Akt and ERK1/2 signaling pathways. Aortic expression of Nfkb1, Mmp2, Mmp9, and Agtr1 was also downregulated in mice receiving resveratrol suggesting that the abovementioned effect of resveratrol resulted in less aortic inflammation and expression of matrix degrading enzymes.
Several study limitations are acknowledged. First, sample number for the human IRA tissue study were relatively small. Because most AAAs are now managed by endovascular repair, AAA wall biopsies are increasingly difficult to obtain. Similarly, ethical and practical considerations prevent the collection of aortic biopsies from large numbers of control donors; however, sample sizes were similar to other published studies.25,48,49 Second, the organ donor controls were younger than the patients with AAA, and the possibility that the age discrepancy may have contributed to differences in ACE2 expression is recognized. Limited clinical details were available for the organ donor controls, and variation in cardiovascular risk factors between groups could not be adjusted for. However, organ donors are often the only viable source of healthy aortic biopsies. Previous studies have used macroscopically healthy tissues proximal to the AAA sac as matched control tissues50,51; but these were not available for the current study. Third, we did not conclusively demonstrate that the benefits of resveratrol to limit AAA growth resulted from ACE2 upregulation. Ideally a study of the effect of resveratrol in ApoE−/−Ace2−/y mice and ApoE−/− controls would have been performed but was not feasible because of the substantial aortic rupture rate in ApoE−/−Ace2−/y mice in response to AngII. Finally, in vitro studies were conducted only in AoSMC. We acknowledge that resveratrol has been shown to improve endothelial function, and increased expression of ACE2 has been reported to promote positive endothelial responses to inflammation.52,53 It is, therefore, possible that the beneficial effects of resveratrol may have been because of effects on endothelial or even inflammatory cells. However, the current study focused on progression of established AAA. A recognized feature of AAA progression in humans is phenotypic change, accelerated replicative senescence, apoptosis, and subsequent depletion of medial AoSMC.26,54 This and recent evidence that ACE2 deficiency in AoSMC markedly increases reactive oxygen species and apoptosis in response to AngII27 formed the basis for the rationale to focus in vitro studies on AoSMC.
In summary, the current study provides further experimental evidence of an important role for ACE2 in vascular homeostasis and as a modulator of AAA severity. Dietary administration of resveratrol upregulated ACE2 and inhibited AAA progression in a mouse model. The upregulation of aortic ACE2 was paralleled by increased expression and activity of Sirt1 and downregulation of key signaling pathways and markers of inflammation. Resveratrol rescued AoSMC expression and production of ACE2 in a Sirt1-dependent manner in vitro.
Angiotensin-converting enzyme 2 deficiency promoted experimental abdominal aortic aneurysm development and rupture.
Angiotensin-converting enzyme 2 expression was downregulated in human patients with abdominal aortic aneurysm.
Resveratrol inhibited progression of experimental abdominal aortic aneurysm associated with upregulation of aortic angiotensin-converting enzyme 2.
Upregulation of angiotensin-converting enzyme 2 in human aortic smooth muscle cell by resveratrol in vitro was Sirtuin 1 dependent.
Nonstandard Abbreviations and Acronyms
abdominal aortic aneurysm
angiotensin-converting enzyme 2 (ACE2 [human]; Ace2 [mouse])
angiotensin type-1 receptor (AGTR1 [human]; Agtr1 [mouse])
aortic smooth muscle cell
apolipoprotein E-deficient mouse
whole-body Ace2-deficient ApoE−/− mouse
extracellular signal-regulated kinase
nuclear factor of kappa light polypeptide gene enhancer in B cells 1
sirtuin 1 (SIRT1 [human]; Sirt1 [mouse])
Sources of Funding
This work was supported by the National Health and Medical Research Council (grants 1079193, 1079369, and 1098717 and Practitioner Fellowship 1117061), the Office of Health and Medical Research, the Townsville Hospital Private Practice Trust, and Queensland Government. The funding bodies played no role in generation of the data presented in this publication.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.117.310129/-/DC1.
Correspondence to Jonathan Golledge, MChir, Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, College of Medicine and Dentistry, James Cook University Townsville, Queensland, Australia 4811. E-mail email@example.com