Angiotensin Receptor–Neprilysin Inhibition (Sacubitril/Valsartan) Reduces Structural Arterial Stiffness in Middle‐Aged Mice

Abstract

Background

Increasing arterial stiffness is a prominent feature of the aging cardiovascular system. Arterial stiffening leads to fundamental alterations in central hemodynamics with widespread detrimental implications for organ function resulting in significant morbidity and death, and specific therapies to address the underlying age‐related structural arterial remodeling remain elusive. The present study investigates the potential of the recently clinically available dual angiotensin receptor–neprilysin inhibitor (ARNI) sacubitril/valsartan (LCZ696) to counteract age‐related arterial fibrotic remodeling and stiffening in 1‐year‐old mice.

Methods and Results

Treatment of in 1‐year‐old mice with ARNI (sacubitril/valsartan), in contrast to angiotensin receptor blocker monotherapy (valsartan) and vehicle treatment (controls), significantly decreases structural aortic stiffness (as measured by in vivo pulse‐wave velocity and ex vivo aortic pressure myography). This phenomenon appears, at least partly, independent of (indirect) blood pressure effects and may be related to a direct antifibrotic interference with aortic smooth muscle cell collagen production. Furthermore, we find aortic remodeling and destiffening due to ARNI treatment to be associated with improved parameters of cardiac diastolic function in aged mice.

Conclusions

This study provides preclinical mechanistic evidence indicating that ARNI‐based interventions may counteract age‐related arterial stiffening and may therefore be further investigated as a promising strategy to improve cardiovascular outcomes in the elderly.

Nonstandard Abbreviations and Acronyms

ARNI

angiotensin receptor–neprilysin inhibitor

FS

fractional shortening

hAoSMCs

human aortic smooth muscle cells

LSA

left subclavian artery

LVID

left ventricular internal diameter

PARAMETER

Prospective Comparison of Angiotensin Receptor Neprilysin Inhibitor With Angiotensin Receptor Blocker Measuring Arterial Stiffness in the Elderly

PWV

pulse wave velocity

TGF‐β

transforming growth factor‐β

TRF

aortic trifurcation

Research Perspective.

What Is New?

• Angiotensin receptor–neprilysin inhibitor (sacubitril/valsartan) treatment reduces aortic fibrosis and stiffness in middle‐aged mice.

• This effect appears to be partly independent of blood pressure regulation.

What Question Should Be Addressed Next?

• What other mechanisms contribute to aortic destiffening under angiotensin receptor–neprilysin inhibitor therapy, and what is the translational effect of angiotensin receptor–neprilysin inhibitor in elderly patients with increased arterial stiffness?

Despite significant preventative and therapeutic advancements over the past decades, cardiovascular diseases still remain a significant source of morbidity and the leading cause of death worldwide according to the World Health Organization. This is particularly true for the elderly population, where cardiovascular diseases are common and cause >60% of all deaths. ¹

One critical mechanism linking age to increased cardiovascular risk may be age‐related stiffening of conduit arteries, such as the aorta. In fact, a reduction of arterial elastic properties is a prominent feature of the aging cardiovascular system. ² , ³ , ⁴ Pathomechanistically, stiff conduit arteries lose their capability to mechanically buffer the pulsatile cardiac ejections (ie, reduced Windkessel function), which results in widespread augmented hemodynamic stress and end‐organ damage (notably, to the brain, heart, and kidneys). ⁵ As such, increased arterial stiffness has been identified as a strong independent predictor of cardiovascular events and all‐cause death. ⁶ Consequently, reducing age‐related arterial stiffness may be a highly promising intervention to improve cardiovascular outcomes in the elderly and sustain a process of healthy aging. ⁷ However, specific therapies that directly and efficiently target the underlying mechanisms of structural arterial remodeling currently remain elusive.

Vascular fibrosis is a main mechanism contributing to structural stiffening of conduit arteries. ⁵ While (locally) enhanced renin–angiotensin–aldosterone system signaling significantly contributes to arterial fibrosis, ⁸ natriuretic peptides have been identified as antifibrotic regulators in the cardiovascular system. ⁹ , ¹⁰ As such, owing to its dual antifibrotic mechanism, inhibiting renin–angiotensin–aldosterone system signaling as well as augmenting natriuretic peptide abundance, ¹¹ the new class of angiotensin receptor–neprilysin inhibitors (ARNIs) holds significant promise as a modulator of age‐related arterial stiffening.

In this regard, omapatrilat, a combined angiotensin‐converting enzyme–neprilysin inhibitor, has previously been shown to reduce proximal aortic stiffness (assessed by aortic characteristic impedance) in hypertensive patients. ¹² However, the drug was later withdrawn due to safety concerns including increased risk of angioedema. The results of the PARAMETER (Prospective Comparison of Angiotensin Receptor Neprilysin Inhibitor With Angiotensin Receptor Blocker Measuring Arterial Stiffness in the Elderly) study demonstrated that in elderly patients with systolic hypertension and elevated pulse pressure (indicative of increased arterial stiffness) ARNI was more effective than angiotensin receptor blocker (ARB) monotherapy (olmesartan) in reducing central aortic (and brachial) blood pressure. ¹³ However, whether these beneficial blood pressure effects were due to a mitigation or reversal of age‐related structural stiffening of the aortic wall and may therefore qualify as causal therapy remained to be determined.

Our study therefore investigated the potential of the first‐in‐class ARNI sacubitril/valsartan (LCZ696) in comparison with ARB monotherapy (valsartan) to counteract age‐related arterial fibrotic remodeling and stiffening in aged mice.

Methods

The authors declare that all supporting data are available within the article and its online supplementary files.

Animals

Male C57Bl/6 mice were purchased from Taconic, Denmark. One‐year‐old mice were treated with LCZ696‐ABA (57 mg/kg twice daily), valsartan (26 mg/kg twice daily), or vehicle (neat water) via oral gavage for 3 months. Valsartan and sacubitril/valsartan (LCZ696) were provided by Novartis Pharma AG, Switzerland. Drug administration followed specific investigator guidelines provided by the manufacturer ensuring safe and effective dosing and application of LCZ696 as well as administration of equivalent doses of valsartan to those delivered by LCZ696. All animal studies were reviewed and approved by the responsible local animal ethics review boards, and all procedures were in accordance with institutional guidelines.

Pulse Wave Velocity Quantification

Pulse wave velocity (PWV) was assessed as an established in vivo marker of aortic stiffness as previously described. ¹⁴ In brief, the murine aorta was visualized longitudinally using ultrasound imaging. We then recorded pulse waves (via pulse wave Doppler) at 2 distinct aortic locations: at the level of the left subclavian artery (LSA) and more distally at the level of the aortic trifurcation (TRF). Using the R‐wave of the ECG signal as time reference, the transit time of the pulse waves between LSA und TRF (t_TRF‐LSA) was calculated. After quantification of the aortic distance between LSA and TRF (d_LSA‐TRF), PWV was calculated as PWV=d_LSA‐TRF/t_TRF‐LSA.

Pressure Myography

Pressure myography was performed to directly assess the passive aortic mechanics ex vivo as previously described. ¹⁵ In brief, murine thoracic aortae were explanted, placed on specially designed stainless‐steel cannulas, and secured with silk surgical sutures (10–0). The vessel was mounted in the heated chamber of a pressure arteriography system (Model 110P, Danish Myotechnology, Copenhagen, Denmark) and stretched to in vivo length. Physiological saline solution at 37 °C, aerated with 5% CO₂/95% O₂, was used to fill the vessel chamber and for aortic perfusion. After 3 preconditioning cycles, the aortic passive pressure‐diameter relationship was determined by an automated protocol. The artery was pressurized from 0 to 180 mm Hg in 18 mm Hg increments, and the vessel's outer diameter was simultaneously tracked by continuous computer video analysis (MyoVIEW software, Danish Myotechnology, Copenhagen, Denmark). The resting diameter d0 was quantified under 0 mm Hg intraluminal (transmural) pressure and was not significantly different between the experimental groups tested (d0 ~1000 μm).

Echocardiography

Mice were imaged in a supine position on a heated platform. Imaging was performed using a real‐time microvisualization transducer (MS550D) with a frequency of 40 MHz connected to a Vevo 2100 ultrasound system (Visualsonics, Toronto, Canada). For left ventricular (LV) global systolic function assessment fractional shortening (FS) was quantified. Using M‐mode imaging, LV internal diameter (LVID) at end systole) and end diastole were quantified, and FS was calculated as FS=[(LVID at end diastole−LVID at end systole)/LVID at end diastole×100%]. For evaluation of LV diastolic function, pulse wave Doppler and tissue Doppler images were acquired. Pulse wave Doppler images were obtained in the apical 4‐chamber view to record the transmitral flow spectra. The Doppler sample volume was placed in the center of the mitral orifice at the tip level of the mitral leaflets and the peak velocity of early (E) and late (A) transmitral filling was recorded. For tissue Doppler images, the sample volume was placed at the septal side of the mitral annulus to record early (E′) and late (A′) diastolic mitral annulus velocity. Data analysis including calculation of the LV diastolic function indices was performed offline with the use of a commercially available Vevo Analytic Software by a blinded observer. Diastolic dysfunction was defined as E′/A′<1 following a previously published definition. ¹⁶ , ¹⁷

Blood Pressure Measurements

Blood pressure measurements were performed using noninvasive volume‐pressure recording) tail cuff sensor technology (CODA System; Kent Scientific). All mice were acclimated to the restrainer for 10 to 20 minutes per day for at least 3 consecutive days before starting the study. Following this acclimation period, mice typically remained calm and still in the restrainer on the day of testing. To facilitate the acclimation process, the mice were handled gently and not forced to enter the restrainer, and the ambient temperature was maintained at warm room temperature (25–30 °C).

Plasma Brain Natriuretic Peptide Quantification

Plasma BNP (brain natriuretic peptide) levels were measured using the BNP ELISA Kit (Elabscience Biotechnology Co. Ltd, Beijing, China, E‐EL‐M0204) according to the manufacturer's instructions.

Preparation of Mouse Aortic Tissue for RNA Extraction

Immediately following euthanasia, the thoracic aorta was transected and flushed via the left ventricle with ice cold PBS (pH 7.4). The aorta was then dissected from fat and connective tissue under a microscope (Leica, Wetzlar, Germany). Specimens were snap‐frozen in liquid nitrogen and stored at −80 °C before further processing.

RNA Isolation and Quantification

Total RNA was isolated using a TRIzol‐based (Invitrogen, Carlsbad, CA) RNA isolation protocol. RNA was quantified by Nanodrop (Agilent Technologies, Santa Clara, CA), and RNA quality was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies). Samples required 260/280 ratios >1.8, and sample RNA integrity numbers >9 for inclusion. For mRNA, the iScript cDNA synthesis kit (Bio‐Rad Laboratories, Hercules, CA) was used to synthesize first‐strand cDNA according to the manufacturer's protocol. TaqMan real‐time quantitative reverse transcription polymerase chain reaction assay was performed using mouse‐specific primers for Col1a1 (Mm00801666_g1; Thermo Fisher Scientific, Waltham, MA) and Col1a2 (Mm00483888_m1; Thermo Fisher Scientific). All probes were normalized to 18S (4319413E, Thermo Fisher Scientific) as internal control. Amplification took place on a QuantStudio12K Flex (Applied Biosystems, Waltham, MA). All fold changes were calculated by the method of ΔΔCt.

Picrosirius Red and Masson's Trichrome Staining

Aortic cross sections (7 μm) were stained using the Picrosirius Red Stain kit (KTPSRPT; American MasterTech, Lodi, CA) and the Trichrome Stain Kit (ab150686; Abcam, Cambridge, UK) according to manufacturer's instructions. Subsequently, representative images of aortic sections were recorded.

Aortic Collagen Quantification

Total aortic collagen was quantified using a hydroxyproline‐detection–based assay (QuickZyme Total Collagen Assay, QZBTOTCOL; Quickzyme Biosciences, Leiden, The Netherlands) according to the manufacturer's protocol. In brief, dissected thoracic aortae (n=4/group) were dried after removal of all loosely attached perivascular connective tissue, and the dry weight was recorded. Subsequently, aortic tissue was hydrolyzed in 6 M HCl at 95 °C for 20 hours. In the hydrolysate hydroxyproline content was quantified colorimetrically and correlated to a collagen standard curve. Aortic collagen content was expressed as a percentage of dry weight.

Cell Culture

Human aortic smooth muscle cells (hAoSMCs; Lot number 0000369150, age 43, White male; Lonza, Basel, Switzerland) were propagated in growth media (SmGM‐2; Lonza) with 5% fetal bovine serum per standard protocols (passage no. 4–5; Lonza). Before experimental use, hAoSMCs were serum‐starved for 48 hours. Profibrotic stimulation was performed using transforming growth factor‐β (TGF‐β; 5 ng/mL; R&D Systems, Minneapolis, MN). BNP (10 nM; TOCRIS Bioscience, Bristol, UK), valsartan (1 μM; provided by Novartis), and LBQ657 (10 μM; provided by Novartis) were added as indicated 1 hour before stimulation.

Quantification of Soluble Collagen Production In Vitro

For quantification of soluble collagen content, cell supernatant was removed after 96 hours of experimental time, and analysis was performed using the Sirius Red Collagen Detection Kit (9062; Chondrex, Woodinville, WA) according to the manufacturer's instructions.

Immunofluorescence Staining

hAoSMCs were cultured in chamber slides and treated as indicated. The cells were then fixed in 4% paraformaldehyde, washed with PBS, permeabilized with 0.25% Triton‐X, and incubated with anti‐collagen I antibody (ab34710; Abcam) overnight at 4 °C. The cells were then washed and incubated with secondary antibodies (Alexa Fluor, aab150079/83; Abcam) and Hoechst nuclear counterstain (62 249; Thermo Fisher Scientific).

Statistical Analysis

Data are presented as mean±SEM. For comparison of 2 groups, Student's t test (2‐tailed) was performed; multiple‐group (≥3 groups) comparison was accomplished by ANOVA with Bonferroni's posttest. For pressure myography analysis, 2‐way repeated measures ANOVA was used. Normality and homoscedasticity were tested to ensure that parametric testing was appropriate. A value of P<0.05 (2‐sided) was considered statistically significant.

Results

ARNI Treatment Decreases Structural Aortic Stiffness in Aged Mice

To differentially investigate the vascular effects of ARNI in vivo, 1‐year‐old mice were treated with sacubitril/valsartan, valsartan, or vehicle respectively. Expectedly, due to its neprilysin inhibiting effects, sacubitril/valsartan treatment led to significantly increased BNP plasma levels compared with valsartan and vehicle‐treated control groups (Figure S1).

To assess the impact of ARNI treatment on aortic elastic function in vivo, we first measured aortic PWV, the clinical gold standard parameter to quantify arterial stiffness. We found that sacubitril/valsartan‐treated mice exhibit significantly decreased PWV (ie, decreased stiffness) compared with valsartan‐ and vehicle‐treated controls (with no significant difference between valsartan and vehicle) (Figure 1A). In a next step, to better characterize the destiffening effects of ARNI treatment, we performed ex vivo pressure myography in explanted aortic segments. Here, sacubitril/valsartan‐treated aortae exhibited significantly decreased material stiffness indicating that ARNI treatment reduces the structural (passive) stiffness of the aortic wall (Figure 1B).

Figure 1. Sacubitril/valsartan treatment decreases aortic stiffness in aged mice.

A, PWV measured in vivo in Val‐, Sac‐Val–, and Veh‐treated groups (n=5/group). Values are individual data points, presented as mean±SEM. B, Aortic pressure diameter curves derived from mechanical testing of thoracic aorta explanted from Val‐, Sac‐Val–, and Veh‐treated mice (n=5/group). Values are mean±SEM. *P<0.05 vs Veh for pressure levels >54 mm Hg. D/D0 indicates the ratio between aortic diameter at the indicated pressure level (D) and aortic diameter at 0 mmHg pressure (D0) as a metric of aortic strain. n.s. indicates not significant; PWV, pulse wave velocity; Sac‐Val, sacubitril/valsartan; Val, valsartan; and Veh, vehicle.

ARNI Treatment Exerts Antifibrotic Effects in Aged Aortae

To further delineate the mechanisms how ARNI treatment induces aortic structural destiffening, we performed histologic analyses of the aortic wall. Representative images of Picrosirius Red– and Masson's Trichrome–stained aortic sections suggested reduced collagen abundance in the aortic medial layer of sacubitril/valsartan‐treated animals compared with valsartan‐ and vehicle‐treated groups (Figure 2A). Additionally, quantitative assessment revealed reduced total aortic collagen content in sacubitril/valsartan‐treated groups only (Figure 2B).

Figure 2. Sacubitril/valsartan treatment reduces aortic medial fibrosis in aged mice.

A, Aortic cross section from mice treated with Veh, Val, or Sac‐Val as indicated. Representative images of aortic cross section stained with Picrosirius Red (collagen red) or Masson's Trichrome (collagen blue). Original magnifications are ×400, and scale bars are 50 μm. B, Total collagen content per aortic dry weight (n=5/group). Values are mean±SEM. *P<0.05 vs Veh‐ and Val‐treated groups. C, Aortic expression levels of profibrotic genes Col1a1 and Col1a2 in sacubitril/valsartan (Sac‐Val)‐ and valsartan‐treated mice compared with Veh‐treated controls. Values are individual data points, presented as mean±SEM and expressed as fold change relative to the mean expression level in Veh‐treated controls (=1). *P<0.05 vs Veh (n=5/group). Adv indicates adventitia; Int, aortic intimal layer; Med, aortic media; Sac‐Val, sacubitril/valsartan; Val, valsartan; and Veh, vehicle.

On the aortic gene expression level, however, both valsartan and sacubitril/valsartan treatment induced a significant downregulation of the profibrotic matrix genes Col1a1 and Col1a2 (Figure 2C), indicating that the antifibrotic effect of sacubitril/valsartan involves multiple signaling pathways.

Reduced Aortic Stiffness Due to ARNI Treatment Is At Least Partly Independent of Blood Pressure Effects

In the next step, we sought to dissect whether the destiffening mechanism of ARNI treatment is indirectly mediated by blood pressure effects. As such, we compared blood pressure levels between treatment groups using noninvasive tail volume–pressure recording. Indeed, we found that both valsartan and sacubitril/valsartan treatment significantly reduced mean arterial blood pressure levels compared with vehicle‐treated controls (Figure 3). However, mean arterial blood pressure level was not significantly different between valsartan and sacubitril/valsartan groups, suggesting that aortic remodeling and destiffening due to sacubitril/valsartan treatment may be, at least partly, independent of blood pressure effects.

Figure 3. Valsartan and sacubitril/valsartan equally reduce arterial blood pressure in aged mice.

Systolic arterial blood pressure (A), mean arterial blood pressure (B), and heart rate (C) in Val‐, Sac‐Val–, and Veh‐treated groups (n=10/group). Values are individual data points, presented as mean±SEM. BP indicates blood pressure; n.s., not significant; Sac‐Val, sacubitril/valsartan; Val, valsartan; and Veh, vehicle.

ARNI Treatment Reduces Collagen Production in hAoSMCs

To further investigate whether the observed antifibrotic vascular effects of ARNI are due to immediate cellular responses we performed corresponding in vitro experiments in vascular cells. Given that the antifibrotic effects of sacubitril/valsartan were predominantly found in the aortic medial layer, which also critically determines the passive stiffness of conduit vessels, we focused on hAoSMCs, representing the primary cellular component of this compartment.

To principally assess the potential of BNP‐dependent signaling to reduce vascular fibrosis and stiffness we tested the effect of BNP treatment on hAoSMCs under profibrotic (TGF‐β) stimulation. We found that exogeneous BNP reduced collagen production in hAoSMCs (Figure 4A). Further, dissecting the significance of the neprilysin–natriuretic peptides system, we confirmed neprilysin expression in hAoSMCs (Figure S2), thus rendering them susceptible to direct cellular neprilysin‐inhibiting interventions. Finally, to differentially dissect the impact of cellular ARNI intervention, we treated hAoSMCs under basal TGF‐β and BNP costimulation with ARB (valsartan), neprilysin inhibitor (LBQ657; the active metabolite of sacubitril), ARNI (valsartan+LBQ657), or vehicle (control), respectively. In this setup, both valsartan and LBQ657 per se did not significantly reduce collagen production in hAoSMCs. However, combined treatment (ARNI) significantly reduced cellular collagen production in a synergistic fashion (Figure 4B). This phenomenon was further confirmed by immunocytochemical staining of cellular collagen I (Figure 4C and 4D).

Figure 4. ARNI treatment reduces collagen production in human aortic smooth muscle cells (hAoSMCs).

A, Soluble collagen content in supernatant from hAoSMCs treated with TGF‐β±BNP or untreated (ctrl). Data from n=5 independent experiments. Values are individual data points, presented as mean±SEM. B, Soluble collagen content in supernatant from TGF‐β+BNP‐stimulated hAoSMCs cotreated with Val, neprilysin inhibitor LBQ657 (LBQ; active metabolite of sacubitril), valsartan+LBQ657 (Val+LBQ), or untreated (ctrl). Data from n=5 independent experiments. Values are individual data points, presented as mean±SEM. hAoSMCs from corresponding experiments were stained for collagen I (red) (C), and mean fluorescence (normalized to cell number) was quantified in 3 high‐power fields of 5 independent experiments. Values are individual data points, presented as mean±SEM (D). Nuclei are Hoechst stained (blue), and scale bar indicates 100 μm (C). ARNI indicates angiotensin receptor–neprilysin inhibitor; BNP, brain natriuretic peptide; ctrl, control; hAoSMCs, human aortic smooth muscle cells; n.s., not significant; TGF‐β, transforming growth factor‐β; and Val, valsartan.

ARNI‐Treated Animals Exhibit Improved Cardiac Diastolic Function

Increased arterial stiffness has been associated with both heart failure (HF) with reduced ejection fraction as well as HF with preserved ejection fraction. As such, in a final set of experiments we investigated both entities in our treatment groups using cardiac ultrasound. FS was quantified as a parameter of global LV systolic function. All groups (vehicle, valsartan, and sacubitril–valsartan) exhibited physiological FS without significant differences between treatments (Figure 5A). Diastolic dysfunction is a hallmark of HF with preserved ejection fraction. Thus, we assessed mitral anulus tissue velocity profiles (E′/A′) as a parameter of diastolic function. ¹⁸ Here, aged mice (control group) exhibited signs of impaired LV relaxation as indicated by an E′/A′ ratio <1 (Figure 5B). However, in sacubitril/valsartan‐treated animals, in contrast to valsartan‐treated mice, we found a significantly increased E′/A′ ratio indicating improved LV diastolic function (Figure 5B). Improved diastolic function in in sacubitril/valsartan‐treated animals was further indicated by additional markers that are commonly used in a clinical context (elevated E′, reduced E/E′ ratio; Figure S3).

Figure 5. Sacubitril/valsartan‐treated animals exhibit signs of improved diastolic function.

A, Fractional shortening was assessed as an indicator of global systolic LV function in Val‐, Sac‐Val–, and Veh‐treated groups. B, E′/A′ ratio was quantified as a metric of diastolic LV function. Values <1 (dotted line) indicate restrictive LV physiology. *P<0.05 vs Veh (n=5/group). Values are individual data points, presented as mean±SEM. LV indicates left ventricular; n.s., not significant; Sac‐Val, sacubitril/valsartan; Val, valsartan; and Veh, vehicle.

Discussion

As a major finding of the present study, we report that ARNI treatment with sacubitril/valsartan reduces aortic stiffness in aged mice significantly compared with ARB monotherapy (valsartan) and vehicle controls (Figure 1). Importantly, in addition to our in vivo experiments that used PWV as the clinically most relevant marker of arterial stiffness, mechanical testing of explanted aortic segments ex vivo enabled us to demonstrate that the destiffening effect of ARNI treatment results from active structural remodeling of the aortic wall (and is not simply due to passive, blood pressure–dependent mechanical unloading of the vessel). These results may appear somewhat in contrast to the findings of the EVALUATE‐HF (Effects of Sacubitril/Valsartan vs. Enalapril on Aortic Stiffness in Patients With Mild to Moderate HF With Reduced Ejection Fraction) trial that reported no statistically significant effect of sacubitril/valsartan treatment on proximal aortic stiffness (aortic characteristic impedance) in heart failure patients. ¹⁹ However, apart from obvious discrepancies that may arise out of interspecies comparisons, it is worth noting that the patients included in the trial did not exhibit elevated levels of proximal aortic stiffness at baseline and treatment duration was rather short (12 weeks). Yet another study of 15 patients with dilated cardiomyopathy did not find significant effects of sacubitril/valsartan on arterial stiffness also after 6 months of treatment. ²⁰

Focusing further on the structural basis of the observed aortic destiffening following ARNI administration, our histologic and quantitative analyses revealed significantly reduced fibrotic remodeling of the load‐bearing medial layer in sacubitril/valsartan‐treated aortae compared with valsartan‐ and vehicle‐treated controls (Figure 2). These findings are consistent with clinical data demonstrating a reduction of circulating profibrotic biomarkers in patients with HF treated with sacubitril/valsartan and other animal studies reporting antifibrotic cardiac effects following ARNI administration. ²¹ , ²² Mechanistically, this may be partly due to suppression of aortic gene expression of fibril‐forming type I collagen (Figure 2C). However, both valsartan and sacubitril/valsartan exhibited similar inhibitory effects on both genes. This also underlines the requirement of a diverse (renin–angiotensin–aldosterone system– and natriuretic peptide–dependent) antifibrotic signaling ⁸ , ⁹ , ¹⁰ to explain the added benefits of ARNI on vascular collagen deposition.

As arterial stiffness and arterial blood pressure are closely intertwined, we monitored blood pressure levels using noninvasive tail volume–pressure recording. Expectedly, we found that valsartan and sacubitril/valsartan both reduced mean arterial blood pressure levels compared with vehicle‐treated controls (Figure 3). Interestingly, however, we did not find significant blood pressure differences between valsartan‐ and sacubitril/valsartan‐treated groups, which is in agreement with reports from other preclinical studies. ²³ These data indicate that ARNI treatment may modulate aortic structure and stiffness, at least in part, independently of blood pressure regulation. This prompted us investigate the immediate effects of ARNI treatment on vascular cells. Here, in our in vitro setting, we found that combined angiotensin receptor antagonism and neprilysin inhibition directly and synergistically reduces collagen production in hAoSMCs (Figure 4). This finding recapitulates previous studies using cardiac cell types. ²²

One may speculate that reduced vascular fibrosis under ARNI medication may lead to a decrease in peripheral vascular resistance, which in turn may decrease blood pressure. However, as noted above, we did not observe significant blood pressure differences between valsartan‐ and sacubitril/valsartan‐treated groups. This phenomenon may be related to the fundamental differences between elastic conduit arteries (such as the aorta) and peripheral muscular arteries. Unlike elastic conduit arteries, whose mechanical properties are largely defined by the composition of the extracellular matrix, muscular arterial stiffness is rather related to smooth muscle cell tone. ²⁴ As such, the effects of antifibrotic interventions (eg, ARNI) may be functionally less effective/relevant in muscular arteries and may therefore not necessarily translate in decreased peripheral vascular resistance and blood pressure.

Age‐related arterial stiffening decreases aortic Windkessel properties and thereby augments cardiac afterload. Accordingly, aortic stiffness has been identified as a strong risk factor and contributor to incident HF, including both entities HF with reduced ejection fraction and HF with preserved ejection fraction. ²⁵ , ²⁶ Thus, we were interested to determine whether ARNI induced aortic destiffening was associated with improved cardiac performance indices. Here, we found that sacubitril/valsartan treatment did not affect global systolic LV function, which was physiologic in all treatment groups (Figure 5A). However, aged mice exhibited signs of impaired LV relaxation (E′/A′ ratio <1) that was improved by sacubitril/valsartan treatment (Figure 5B). Of note, ARNI's cardiac effects have been extensively characterized and this study may not discern whether improved diastolic function is causally linked to restored aortic compliance in aged mice or rather related to ARNI's direct effects on cardiomyocytes. Still, given the heterogeneous benefit of ARNI interventions observed in HF with preserved ejection fraction patient subgroups, that may well reflect the phenotypic and pathophysiological heterogeneity of the syndrome, ²⁷ our findings may stimulate further investigations of potential ARNI benefits in patient subpopulations with HF with preserved ejection fraction who exhibit increased aortic stiffness.

Limitations

Focusing on arterial aging, this study investigates the effect of ARNI on structural aortic stiffness in 1‐year‐old mice. It should be noted that this age more precisely corresponds to middle‐aged mice. This study also does not investigate the effects of ARNI in young mice since we do not envision a comparable treatment of young patients in a clinical context when arterial stiffness is not increased. Only male mice were studied, limiting the generalizability of the results.

Although the results of the study suggest that the effects of ARNI on age‐related arterial stiffness are mediated mainly by antifibrotic properties, this does not exclude other contributing mechanisms that are related to vascular stiffness (eg, improvement of endothelial function ²⁸ ). For example, as a limitation of the study, effects of ARNI treatment on wall thickness was not specifically investigated. While aortic wall thickness appeared grossly unaffected by ARNI treatment in representative histological images (Figure 2A), aortic wall thinning (eg, due to less collagen deposition in the aortic wall) may contribute to the destiffening effects of ARNI therapy.

Conclusions

In summary, the results of our study indicate that ARNI interventions (sacubitril/valsartan), in contrast to ARB monotherapy (valsartan), may attenuate and even reverse age‐related aortic fibrotic remodeling and structural aortic stiffening. These effects appear to be (at least in part) independent of indirect antihypertensive mechanisms but rather due to direct interference with cellular collagen production within the aortic wall. Furthermore, we find aortic remodeling and destiffening due to ARNI treatment to be associated with improved parameters of cardiac diastolic function in aged mice.

In conclusion, this study provides preclinical mechanistic evidence indicating that ARNI‐based interventions may be sufficient to counteract age‐related arterial stiffening as a comprehensive independent risk factor for cardiovascular morbidity and death.

Sources of Funding

This work was supported by a research grant from Novartis Pharma GmbH, Germany. Valsartan, sacubitril/valsartan (LCZ696), and LBQ657 were provided by Novartis Pharma AG, Switzerland.

Disclosures

Drs Schellinger and Raaz are cofounders of Angiolutions GmbH, which is an academic spin‐off company developing vascular devices for aneurysm diseases. The remaining authors have no disclosures to report.

Supporting information

Figures S1–S3.