Sacubitril-valsartan improves conduit vessel function and functional capacity and reduces inflammation in heart failure with reduced ejection fraction

Abstract

The Prospective comparison of ARNI with angiotensin-converting enzyme inhibitor to Determine Impact on Global Mortality and morbidity in Heart Failure trial identified a marked reduction in the risk of death and hospitalization for heart failure in patients with heart failure with reduced ejection fraction (HFrEF) treated with sacubitril-valsartan (trade name Entresto), but the physiological processes underpinning these improvements are unclear. We tested the hypothesis that treatment with sacubitril-valsartan improves peripheral vascular function, functional capacity, and inflammation in patients with HFrEF. We prospectively studied patients with HFrEF (n = 11, 10 M/1 F, left ventricular ejection fraction = 27 ± 8%) on optimal, guideline-directed medical treatment who were subsequently prescribed sacubitril-valsartan (open-label, uncontrolled, and unblinded). Peripheral vascular function [brachial artery flow-mediated dilation (FMD, conduit vessel function) and reactive hyperemia (RH, microvascular function)], functional capacity [six-minute walk test (6MWT) distance], and the proinflammatory biomarkers tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18) were obtained at baseline and at 1, 2, and 3 mo of treatment. %FMD improved after 1 mo of treatment, and this favorable response persisted for months 2 and 3 (baseline: 3.25 ± 1.75%; 1 mo: 5.23 ± 2.36%; 2 mo: 5.81 ± 1.79%; 3 mo: 6.35 ± 2.77%), whereas RH remained unchanged. 6MWT distance increased at months 2 and 3 (baseline: 420 ± 92 m; 1 mo: 436 ± 98 m; 2 mo: 465 ± 115 m; 3 mo: 460 ± 110 m), and there was a sustained reduction in TNF-α (baseline: 2.38 ± 1.35 pg/mL; 1 mo: 2.06 ± 1.52 pg/mL; 2 mo: 1.95 ± 1.34 pg/mL; 3 mo: 1.92 ± 1.37 pg/mL) and a reduction in IL-18 at month 3 (baseline: 654 ± 150 pg/mL; 1 mo: 595 ± 140 pg/mL; 2 mo: 601 ± 176 pg/mL; 3 mo: 571 ± 127 pg/mL). This study provides new evidence for the potential of this new drug class to improve conduit vessel function, functional capacity, and inflammation in patients with HFrEF.

NEW & NOTEWORTHY We observed an approximately twofold improvement in conduit vessel function (brachial artery FMD), increased functional capacity (6MWT distance), and a reduction in inflammation (TNF-α and IL-18) following 3 mo of sacubitril-valsartan therapy. These findings provide important new information concerning the physiological mechanisms by which this new drug class provokes favorable changes in HFrEF pathophysiology.

INTRODUCTION

Sacubitril-valsartan (trade name Entresto) is the first agent in a new class of drugs called angiotensin receptor-neprilysin inhibitors (ARNI), approved for the treatment of patients with heart failure with reduced ejection fraction (HFrEF) (1). The PARADIGM-HF [Prospective comparison of ARNI with angiotensin-converting enzyme inhibitor (ACEI) to Determine Impact on Global Mortality and morbidity in Heart Failure] trial demonstrated that sacubitril-valsartan was superior to enalapril (an ACEI) in reducing the risk of death from cardiovascular causes and hospitalization for heart failure in patients with HFrEF (2). Consequently, current heart failure guidelines have recommended the use of sacubitril-valsartan in place of an ACEI or an angiotensin receptor blocker (ARB) (3). However, despite the clear benefits of sacubitril-valsartan, the physiological processes underpinning these improvements are currently not fully understood (4).

Although several putative physiological mechanisms may be responsible for the favorable effects of sacubitril-valsartan, changes in peripheral vascular function appear to be a likely candidate. Indeed, endothelium-dependent vasodilation, as assessed by flow-mediated dilation (FMD), has been well documented to be attenuated in patients with HFrEF (5–9), a maladaptation that likely contributes to fatigue, dyspnea upon exertion, and exercise intolerance in this patient group (10–12). Specifically, in the presence of endothelial dysfunction, the ability to vasodilate the exercising skeletal muscle vasculature is impaired, thereby resulting in sustained peripheral vasoconstriction, attenuated blood flow and oxygen delivery to the myocardium and exercising skeletal muscles, and reduced physical capacity (12, 13). In an animal model of HFrEF, treatment with an ARB has been demonstrated to improve endothelium-dependent vasodilation of the aorta (14–16). Interestingly, although animals with HFrEF treated with an ARB exhibited a similar improvement in endothelium-dependent vasodilation of the aorta compared with those treated with sacubitril-valsartan, the improvement appeared to subside over time only in animals treated with an ARB, suggesting that sacubitril-valsartan was superior to an ARB in sustaining the improvement in vascular health in this preclinical work (16). Given that both conduit vessel dysfunction and microvascular dysfunction predict mortality risk in patients with HFrEF (10, 17, 18), the possibility that improved clinical outcomes in patients with HFrEF treated with sacubitril-valsartan could be mediated via improvements in peripheral vascular health is an attractive hypothesis. To our knowledge, no studies, to date, have examined conduit vessel and microvascular function following treatment with sacubitril-valsartan in patients in HFrEF.

In addition to peripheral vascular dysfunction, functional capacity is reduced in patients with HFrEF and is associated with poor quality of life and a worse prognosis (19, 20). The six-minute walk test (6MWT) is a well-tolerated and highly reproducible assessment that has been widely used to estimate functional capacity in patients with HFrEF (21), and an increase in the 6MWT distance has been related to an improvement in self-perceived symptoms of HFrEF (22). There is evidence that treatment with sacubitril-valsartan may improve the 6MWT distance (23) and exercise tolerance (24) in patients with HFrEF, although the time course and mechanisms responsible for these beneficial changes remain uncertain.

Over the past 20 years, evidence has accumulated supporting the importance of disease-related increases in cardiac and systemic inflammation in the pathophysiology of HFrEF (25), with preclinical data suggesting a beneficial effect of treatment with sacubitril-valsartan on proinflammatory biomarkers. Indeed, in an animal model of diabetic cardiomyopathy (DCM), treatment with sacubitril-valsartan reduced the expression of proinflammatory cytokines and biomarkers of heart failure, suggesting that this treatment promotes anti-inflammatory and anti-heart failure effects (26). Understanding the potential of treatment with sacubitril-valsartan as a targeted anti-inflammatory therapy in patients with HFrEF is of clinical significance (27), given that inflammation and the associated production of reactive oxygen species have been implicated in the pathogenesis of peripheral vascular dysfunction in this patient group (28). Thus, although these preclinical data support the possibility that sacubitril-valsartan may exert an anti-inflammatory effect, whether treatment with this novel drug class can mitigate inflammation in patients with HFrEF remains to be elucidated.

Thus, the current single-arm, open-label, uncontrolled, and unblinded prospective study sought to determine the impact of a 3-mo treatment with sacubitril-valsartan on peripheral vascular function, functional capacity, and inflammation in patients with HFrEF who were on guideline-directed medical treatment. We hypothesized that treatment with sacubitril-valsartan would improve peripheral vascular function, increase functional capacity and reduce inflammation in patients with HFrEF.

METHODS

Ethical Approval

All experimental procedures and protocols were approved by the University of Utah and the Salt Lake City Veterans Affairs Institutional Review Board (IRB_00084575). All aspects of the study conformed to the standards set by the Declaration of Helsinki, except for registration in a database. All experimental procedures were explained to participants in writing and verbally, and written informed consent was obtained from all participants before study participation.

Subjects

Eleven patients with class II-III HFrEF were recruited in the Heart Failure Clinic at the University of Utah. The inclusion criteria were diagnosis of chronic HFrEF with left ventricular ejection fraction ≤35%, ongoing guideline-directed medical treatment for heart failure for at least 6 mo before study enrollment, and a clinical decision to initiate treatment with sacubitril-valsartan. Seven patients were transitioned to sacubitril-valsartan from an ACEI, and four patients from an ARB. Exclusion criteria for patients with HFrEF included atrial flutter, uncorrected primary valvular disease, smokers, orthopedic limitations, hormonal replacement therapy, dementia, severe cardiopulmonary disease, end-stage renal disease, type I diabetes, insulin therapy, morbid obesity (body mass index >40 kg/m²), uncontrolled hypertension, severe renal insufficiency, or end-stage malignancy.

Experimental Design

All data collection took place with patients in the supine position in a thermoneutral environment. All patients reported to the laboratory at least 4 h postprandial, having consumed a low-fat, standardized breakfast and abstained from caffeine, alcohol, and exercise for 24 h prior. Data collection included anthropometrics, venous blood samples, blood pressure, peripheral vascular function [brachial artery FMD and reactive hyperemia (RH, microvascular function)], and functional capacity via the 6MWT. Venous blood samples were processed using standard clinical procedures at the Salt Lake City Veterans Affairs Medical Center. Following the completion of baseline data collection, the patients taking ACEI were instructed to withhold their ACEI medication for 36 h before initiating treatment with sacubitril-valsartan, as concomitant use of an ACEI and sacubitril-valsartan is contraindicated due to an increased risk of angioedema (29). The starting dose was 24 mg/26 mg of sacubitril-valsartan twice daily, up-titrated at monthly intervals to a maximally tolerated dose of 97 mg/103 mg, twice daily, and patient safety and treatment tolerability were closely monitored by the clinical team in accordance with established guidelines (29). After treatment with sacubitril-valsartan had been initiated, patients were re-evaluated on a monthly basis, with a total of three follow-up visits (i.e., 1 mo, 2 mo, and 3 mo of treatment).

Brachial Artery Flow-Mediated Dilation and Reactive Hyperemia Measurements and Analyses

FMD testing for the assessment of conduit vessel function was performed in accordance with established guidelines (30, 31). Briefly, a blood pressure cuff was positioned on the right forearm (below the antecubital crease) distal to the ultrasound Doppler probe. The brachial artery was insonated approximately midway between the antecubital and axillary regions, and measurements of brachial artery diameter and intensity-weighted mean blood velocity were acquired (Logiq 7, GE Medical Systems, Milwaukee, WI). After resting measurements of brachial artery diameter and mean blood velocity were performed, a blood pressure cuff was inflated to 250 mmHg for 5 min. After cuff release, data were recorded continuously for 2 min. Mean blood velocity was automatically calculated using commercially available software (Logiq 7). End-diastolic, ECG R-wave gated images were collected via video output from the Logiq 7 for off-line analysis of brachial artery diameter using automated edge-detection software (Medical Imaging Applications, Coralville, IA).

FMD was quantified as the maximal change in brachial artery diameter following cuff release, expressed as a percentage increase from pre-occlusion values (%FMD). Shear rate was calculated as follows: shear rate (s⁻¹) = blood velocity × 8/vessel diameter. Forearm blood flow was calculated according to the equation: forearm blood flow (mL/min) = [blood velocity × π × (vessel diameter/2)² × 60]. Cumulative area-under-the-curve (AUC) values for shear rate and blood flow were integrated via the trapezoid rule and calculated as: (Σ{y_i[x_{(i + 1)} − x_i] + (1/2)[y_{(i + 1)} − y_i][x_{(i + 1)} − x_i]}) (x is time, y is shear rate, x_i is initial time point, y_i is initial blood velocity) (30, 32). Shear stimulus was quantified as cumulative shear rate AUC until peak vasodilation. Normalized FMD was calculated by dividing %FMD by the cumulative shear rate AUC from cuff release to peak brachial artery vasodilation. In addition, RH was quantified as brachial artery blood flow AUC for the 2-min period following cuff release (5). Importantly, RH is a phenomenon that occurs as a result of a complex interaction between myogenic and locally released vasoactive substances within skeletal muscle resistance vessels, and thus, RH provides an index of microvascular function (33–35). The same investigators performed both the FMD measurements and analyses, and they were blinded to the measurement time points during the analysis. To maintain continuity between exams, investigators confirmed a similar distance between the antecubital fossa and the ultrasound probe on each study visit and ensured sample volume size and placement, as well as sampling frequency, were similar between exams.

Six-Minute Walk Test

The 6MWT was conducted following a standardized protocol (22, 23). Briefly, the patients were instructed to cover the maximum distance possible in 6 min, at a self-selected walking speed, and were unaccompanied and without verbal encouragement, to avoid any social influence on the walking speed. The patients were able to rest, as needed, in a flat, obstacle-free corridor, and time remaining was called out every second minute (22, 23). The 6MWT distance is used to estimate functional capacity and is highly reproducible in patients with HFrEF, and changes in the 6MWT distance are sensitive to changes in self-perceived symptoms of heart failure (22).

Blood Biomarker Assays

Blood samples were obtained from the antecubital vein upon arrival to the laboratory, centrifuged to collect plasma and serum, and stored at −80°C until analysis. Plasma concentrations of tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), and interleukin-6 (IL-6) were determined using a high-sensitivity enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). A commercially available enzyme-linked immunosorbent assay was used to analyze plasma concentrations of interleukin-18 (IL-18) (MBL Life Science, Japan), interleukin-1β (IL-1β) (Invitrogen, Waltham, MA), human N-terminal probrain natriuretic peptide (NT-proBNP) (Abcam, Cambridge, MA), as well as human B-type natriuretic peptide (BNP) and troponin T (Thermo Fisher Scientific, Frederick, MD). A lipid panel and complete blood cell count were assessed by standard clinical techniques. Lipid peroxidation was evaluated as a biomarker of oxidative stress, assessed by the malondialdehyde (MDA) concentration using colorimetry (OxisResearch, Burlingame, CA). Protein carbonyl, a biomarker of oxidant damage, was determined with a high-sensitivity enzyme-linked immunosorbent assay (Northwest Life Science Specialties, Vancouver, WA). Total antioxidant capacity was assessed by the ferric-reducing ability of plasma (FRAP) assay (36), and endogenous antioxidant activity was assessed by determining catalase and superoxide dismutase (SOD) (Cayman Chemical Company, Ann Arbor, MI). Plasma concentrations of endothelin-1 and human N-terminal pro-atrial natriuretic peptide (NT-proANP) were analyzed in duplicate and then averaged, utilizing commercially available enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MS). Plasma concentrations of bradykinin were assessed using a colorimetric assay (Abcam, Cambridge, MA).

Statistical Analyses

Statistics were performed using commercially available software (SigmaPlot 13, Systat Software Inc., Point Richmond, CA). Data were checked for normality using the Shapiro–Wilk test. For normally distributed data, a one-way repeated-measures ANOVA was used to identify the time-course responses, whereas non-normally distributed data were analyzed using Friedman’s repeated-measures ANOVA on ranks. The Tukey method was used for α adjustment and post hoc analysis. Pearson’s correlational analyses were used to assess the relationship between variables. All data were expressed as means ± SD. Significance was established at P < 0.05.

RESULTS

Patient Characteristics and Sacubitril-Valsartan Tolerance

Patient characteristics and clinical variables at baseline as well as during treatment with sacubitril-valsartan are documented in Table 1. Prior to initiating treatment with sacubitril-valsartan, 64% of the patients received previous treatment with an ACEI, whereas 36% of the patients were previously treated with an ARB. Disease-related characteristics and pharmacological information for the patients with HFrEF are documented in Table 2. There were no significant changes in any of these patient characteristics or clinical variables, except for reductions in potassium and creatinine concentrations (P < 0.05, Table 1). Furthermore, treatment with sacubitril-valsartan for 3 mo was well tolerated in all patients.

Table 1.

Patient characteristics and clinical variables at baseline and after 1, 2, and 3 mo of treatment with sacubitril-valsartan

	Baseline	1 mo	2 mo	3 mo	P value
Age, yr	69 ± 8	–	–	–
Height, cm	176.9 ± 9.9	–	–	–
Weight, kg	90.9 ± 15.2	91.2 ± 14.6	91.9 ± 14.4	92.5 ± 14.3	0.099
Body mass index, kg/m2	29.0 ± 3.5	29.1 ± 3.4	29.3 ± 3.5	29.5 ± 3.6	0.061
Systolic blood pressure, mmHg	113 ± 19	103 ± 15	99 ± 14	101 ± 15	0.089
Diastolic blood pressure, mmHg	69 ± 10	66 ± 10	66 ± 7	66 ± 10	0.508
Mean arterial pressure, mmHg	84 ± 11	78 ± 11	77 ± 9	78 ± 10	0.154
Heart rate, beats/min	68 ± 10	69 ± 11	69 ± 8	68 ± 9	0.513
Total cholesterol, mg/dL	164 ± 74	180 ± 108	176 ± 101	165 ± 61	0.497
HDL cholesterol, mg/dL	42 ± 13	41 ± 11	38 ± 8	41 ± 10	0.144
LDL cholesterol, mg/dL	83 ± 36	85 ± 31	85 ± 31	88 ± 26	0.766
Triglycerides, mg/dL	207 ± 222	289 ± 476	276 ± 392	235 ± 304	0.863
Glucose, mg/dL	113 ± 42	124 ± 47	109 ± 24	119 ± 44	0.214
Hemoglobin, g/dL	14.5 ± 1.5	15.0 ± 1.4	14.6 ± 1.6	14.6 ± 1.9	0.150
Hematocrit, %	44.4 ± 4.1	45.6 ± 4.5	44.5 ± 5.2	44.2 ± 6.0	0.390
Erythrocytes, M/µL	4.74 ± 0.40	4.87 ± 0.36	4.75 ± 0.40	4.74 ± 0.61	0.380
Leukocytes, K/µL	6.85 ± 2.15	6.26 ± 1.67	6.37 ± 1.72	6.29 ± 1.60	0.299
Albumin, g/dL	4.36 ± 0.22	4.35 ± 0.37	4.26 ± 0.22	4.20 ± 0.20	0.073
Potassium, mmol/L	4.6 ± 0.4	4.3 ± 0.3	4.2 ± 0.4*	4.3 ± 0.5	0.041
Creatinine, mg/dL	1.46 ± 0.40	1.38 ± 0.43	1.29 ± 0.42*	1.35 ± 0.44	0.028
eGFR, mL/min/1.73 m2	52 ± 20	55 ± 19	60 ± 20	57 ± 21	0.053

Data are means ± SD (n = 11 patients). eGFR, estimated glomerular filtration rate; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

*P < 0.05 vs. baseline.

Table 2.

Patient disease-specific characteristics and medications

	HFrEF
Disease-specific characteristics
Left ventricular ejection fraction (%)	27 ± 8
Diagnosis
Ischemic cardiomyopathy	5/11
Nonischemic cardiomyopathy	6/11
NYHA class II	8/11
NYHA class III	3/11
Type 2 diabetes	3/11
Coronary artery disease	7/11
Medications
β-Blocker	10/11
Angiotensin-converting enzyme inhibitor	7/11
Angiotensin receptor inhibitor	4/11
Statin	9/11
Diuretic	10/11
Aldosterone antagonist	10/11
Antiarrhythmic	1/11
Anticoagulant	4/11
Metformin	3/11

Data are means ± SD or number of cases (n = 11 patients). HFrEF, heart failure with reduced ejection fraction; NYHA, New York Heart Association.

Vascular Measurements

Treatment with sacubitril-valsartan did not alter resting brachial artery diameter (baseline: 4.67 ± 0.71 mm; 1 mo: 4.71 ± 0.74 mm; 2 mo: 4.71 ± 0.72 mm; 3 mo: 4.71 ± 0.74; main effect: P = 0.183) or resting forearm blood flow (baseline: 82 ± 37 mL; 1 mo: 92 ± 33 mL; 2 mo: 99 ± 38 mL; 3 mo: 96 ± 32 mL; main effect: P = 0.121). Compared with baseline, peak brachial artery diameter following cuff release was higher at all the monthly follow-up visits (baseline: 4.81 ± 0.67 mm; 1 mo: 4.94 ± 0.71 mm; 2 mo: 4.98 ± 0.69 mm; 3 mo: 4.99 ± 0.70 mm; main effect: P = 0.005). Importantly, treatment with sacubitril-valsartan improved brachial artery FMD, both in terms of percentage increase (baseline: 3.25 ± 1.75%; 1 mo: 5.23 ± 2.36%; 2 mo: 5.81 ± 1.79%; 3 mo: 6.35 ± 2.77%) and absolute change in brachial artery diameter (baseline: 0.14 ± 0.06 mm; 1 mo: 0.24 ± 0.08 mm; 2 mo: 0.27 ± 0.07 mm; 3 mo: 0.29 ± 0.10 mm) (P < 0.05, Fig. 1, A and B). There was no treatment effect on shear rate AUC in any of the follow-up visits (P > 0.05, Fig. 1C). Although %FMD was positively correlated with shear rate AUC (r = 0.38, P = 0.012), even after normalizing for this increasing shear stimulus, the increases in %FMD still remained significant after 2 and 3 mo of treatment with sacubitril-valsartan (P < 0.05, Fig. 1D). Interestingly, treatment with sacubitril-valsartan did not alter microvascular function, as determined by RH (main effect: P > 0.05, Fig. 2).

Figure 1.

Brachial artery flow-mediated dilation (FMD) (A, B, and D) and shear rate area under the curve (AUC) from cuff release until peak dilation (C) at baseline and after 1, 2, and 3 mo of treatment with sacubitril-valsartan in patients with heart failure with reduced ejection fraction (n = 11). The FMD data are expressed as percentage change (A), absolute change (B), and %FMD/shear rate AUC (D). Data are presented as mean and individual data (white dots). *Significantly different from baseline, P < 0.05. #Significantly different from 1 mo, P < 0.05.

Figure 2.

Reactive hyperemia following 5-min cuff occlusion in patients with heart failure with reduced ejection fraction at baseline and after 1, 2, and 3 mo of treatment with sacubitril-valsartan (n = 11). Data are presented as means ± SD.

Functional Capacity

The 6MWT distance increased from baseline at months 2 and 3 of treatment with sacubitril-valsartan (baseline: 420 ± 92 m; 1 mo: 436 ± 98 m; 2 mo: 465 ± 115 m; 3 mo: 460 ± 110 m) (P < 0.05, Fig. 3).

Figure 3.

Six-minute walk test distance in patients with heart failure with reduced ejection fraction at baseline and after 1, 2, and 3 mo of treatment with sacubitril-valsartan (n = 11). Data are presented as mean and individual data (white dots). *Significantly different from baseline, P < 0.05.

Blood Biomarkers

The quantitative assessment of blood biomarkers in the patients with HFrEF is documented in Table 3 and TNF-α and IL-18 is highlighted in Fig. 4. Although there were no treatment effects on the majority of these blood biomarkers (P > 0.05, Table 3), sacubitril-valsartan evoked a sustained reduction in TNF-α (baseline: 2.38 ± 1.35 pg/mL; 1 mo: 2.06 ± 1.52 pg/mL; 2 mo: 1.95 ± 1.34 pg/mL; 3 mo: 1.92 ± 1.37 pg/mL) (P < 0.05, Fig. 4A) as well as a reduction in IL-18 at month 3 of the treatment in the patients with HFrEF (baseline: 654 ± 150 pg/mL; 1 mo: 595 ± 140 pg/mL; 2 mo: 601 ± 176 pg/mL; 3 mo: 571 ± 127 pg/mL) (P < 0.05, Fig. 4B).

Table 3.

Blood biomarkers in patients with heart failure with reduced ejection fraction at baseline and after 1, 2, and 3 mo of treatment with sacubitril-valsartan

	n	Baseline	1 mo	2 mo	3 mo	P value
Inflammation
CRP, ng/mL	10	2,168 ± 1,611	1,764 ± 892	1,689 ± 1,078	1,565 ± 1,058	0.430
IL-6, pg/mL	10	4.54 ± 2.59	4.28 ± 1.88	4.88 ± 2.17	4.62 ± 2.51	0.948
IL-1β, pg/mL	10	1.37 ± 0.44	1.41 ± 0.38	1.35 ± 0.37	1.41 ± 0.42	0.953
Oxidative stress
MDA, µM	10	3.92 ± 1.55	3.88 ± 1.66	3.91 ± 1.78	3.86 ± 1.73	0.996
Protein carbonyl, nmoL/mg	10	0.20 ± 0.09	0.19 ± 0.06	0.21 ± 0.07	0.20 ± 0.06	0.840
Antioxidant capacity
FRAP, mM	10	6.61 ± 2.37	6.66 ± 2.33	6.31 ± 2.22	6.26 ± 2.24	0.232
Free radical scavenging
Catalase, mmoL/min/mL	10	36.7 ± 18.7	49.6 ± 23.5	44.5 ± 19.2	42.0 ± 18.7	0.189
SOD, U/mL	10	3.12 ± 1.38	3.76 ± 1.61	3.44 ± 0.86	4.37 ± 1.91	0.163
Clinical biomarkers
NT-proBNP, pg/mL	10	1,404 ± 1,073	1,052 ± 1,031	1,168 ± 1,097	1,222 ± 1,004	0.118
BNP, pg/mL	10	114 ± 118	87 ± 94	115 ± 144	92 ± 106	0.095
NT-proANP, ng/mL	11	27.4 ± 11.1	25.8 ± 14.4	28.2 ± 13.2	29.9 ± 12.9	0.487
Troponin T, pg/mL	10	305 ± 287	295 ± 204	270 ± 186	270 ± 204	0.524
Bradykinin, pg/mL	11	2,653 ± 1,919	2,681 ± 2,164	1,644 ± 1,045	1,976 ± 1,685	0.086
Endothelin-1, pg/mL	11	2.03 ± 0.81	1.78 ± 0.49	1.81 ± 0.46	2.34 ± 1.03	0.263

Data are means ± SD. BNP, B-type natriuretic peptide; CRP, C-reactive protein; FRAP, ferric-reducing ability of plasma; IL-6, interleukin-6; IL-1β, interleukin-1 β; MDA, malondialdehyde; NT-proANP, N-terminal proatrial natriuretic peptide; NT-proBNP, N-terminal probrain natriuretic peptide; SOD, superoxide dismutase.

Figure 4.

Plasma tumor necrosis factor-α (TNF-α) (A) and interleukin-18 (IL-18) (B) in patients with heart failure with reduced ejection fraction at baseline and after 1, 2, and 3 mo of treatment with sacubitril-valsartan (n = 9 and n = 10, respectively). Data are presented as mean and individual data (white dots). *Significantly different from baseline, P < 0.05.

Correlational Analyses

Improvements in the 6MWT distance were not correlated with the improvement in FMD (%FMD: r = 0.07, P = 0.694; %FMD/shear rate AUC: r = −0.14, P = 0.433). Furthermore, reductions in TNF-α were not correlated with the improvement in FMD (%FMD: r = 0.12, P = 0.518; %FMD/shear rate AUC: r = 0.20, P = 0.297) or the 6MWT distance (r = 0.08, P = 0.693). Reductions in IL-18 were also not correlated with the improvement in FMD (%FMD: r = −0.06, P = 0.792; %FMD/shear rate AUC: r = −0.25, P = 0.285) or the 6MWT distance (r = 0.08, P = 0.671).

DISCUSSION

There are several notable findings from the present open-label, uncontrolled, and unblinded prospective study that provide novel insight into the physiological underpinnings of the beneficial effects of treatment with sacubitril-valsartan in patients with HFrEF. First, in patients with HFrEF who were on optimal, guideline-directed medical treatment, treatment with sacubitril-valsartan improved conduit vessel function, as determined by FMD testing, after 1 mo of treatment, and this response persisted in months 2 and 3 of treatment. Second, functional capacity, assessed by the 6MWT distance, improved at months 2 and 3 of treatment with sacubitril-valsartan. Finally, these favorable changes in conduit vessel function and functional capacity were accompanied by a marked (≈20%) and persistent reduction in inflammation, as determined by TNF-α (across the course of treatment) and by IL-18 (at month 3 of treatment), although other biomarkers of clinical status remained unchanged. In addition, RH was unchanged across all study time points, suggesting that microvascular function was unaffected by drug treatment. Taken together, these findings suggest that the treatment of patients with HFrEF with sacubitril-valsartan improves conduit vessel function, increases functional capacity, and reduces inflammation, providing new information regarding the beneficial effects of ARNI therapy in this patient group.

Impact of Treatment with Sacubitril-Valsartan on Peripheral Vascular Function

The presence of vascular dysfunction in the peripheral circulation is a well-known sequela of HFrEF. Indeed, previous works from our group (5, 9) and others (17, 37) have reported marked reductions in %FMD in patients with HFrEF, which may be particularly relevant given the known relationship between FMD and mortality risk in this patient group (10, 17). To our knowledge, this is the first study to evaluate conduit vessel function following the initiation of treatment with sacubitril-valsartan in humans with HFrEF. %FMD improved as early as 1 mo (+69%) after initiating treatment with sacubitril-valsartan compared with baseline, and this favorable change was sustained at months 2 (+92%) and 3 (+90%) (Fig. 1, A and B). Importantly, the improvement in conduit vessel function persisted even after accounting for the increase in shear rate, the laminar shear force across the vascular endothelial cells that is the primary stimulus for the FMD response (38), that accompanied the increase in %FMD (r = 0.38, P = 0.012) (Fig. 1D). In an experimental animal model of HFrEF, although 12-wk treatment with sacubitril-valsartan improved ex vivo vascular function, as measured by aortic vasorelaxation responses to acetylcholine and sodium nitroprusside, similarly to those of animals treated with valsartan, the improvement in vascular function tended to decline between weeks 8 and 12 in animals treated with valsartan but was sustained in animals treated with sacubitril-valsartan (16). Thus, the present study extends this preclinical work, by demonstrating, for the first time, to our knowledge, a beneficial effect of this new drug class on %FMD in patients with HFrEF. Importantly, all patients in this study exhibited very low %FMD values before treatment with sacubitril-valsartan despite being well medicated on standard heart failure pharmacotherapy that included β-blockade, as well as antihypertensive and statin drug regimens (Table 2). Considering the proven efficacy of ACEI (39), ARB (40), and statins (41) to improve %FMD in patients with HFrEF, the marked improvement in conduit vessel function following treatment with sacubitril-valsartan in the present study is particularly noteworthy and demonstrates the potential added value of ARNI therapy on vascular health in the presence of optimized pharmacotherapy.

The assessment of FMD also afforded the opportunity to examine microvascular function via the determination of RH (i.e., forearm blood flow AUC following cuff release) before and during the course of treatment with sacubitril-valsartan. RH is prognostic for adverse cardiovascular outcomes in populations with heightened cardiovascular disease risks (42, 43) and, like FMD, is predictive of mortality risk and cardiovascular hospitalization in patients with HFrEF (18). In contrast to the favorable effect on %FMD, there were no changes in RH, suggesting no effect of sacubitril-valsartan on microvascular function (Fig. 2). This lack of improvement could be explained, in part, by the lack of a clear deficit in microvascular function in patients with HFrEF before treatment. Indeed, RH values of 500–600 mL were observed in patients with HFrEF (Fig. 2), values that are similar to that reported in older, healthy adults (561 ± 65 mL) (9). Furthermore, the differential vasodilatory mechanistic underpinnings of RH versus FMD may also explain why RH was not further improved following treatment with sacubitril-valsartan. Conduit vessel function, assessed via brachial artery FMD, has been demonstrated to be mediated, at least in part, by nitric oxide (NO) (44). In contrast, Crecelius et al. (33) have recently reported that microvascular function, assessed via RH, is primarily mediated by activation of inwardly rectifying potassium channels and sodium-potassium-ATPase, though this still remains a topic of ongoing debate (45). Importantly, previous studies have found that inhibition of the renin-angiotensin-aldosterone system pathway (46, 47) and exogenous atrial natriuretic peptide (48) both resulted in an increase in NO bioavailability, in part, via reductions in inflammation and oxidative stress; thus, this may explain why we observed an improvement in conduit vessel function, and not microvascular function, in the present study. Taken together, these %FMD and RH responses provide important new evidence for the potential of short-term treatment with sacubitril-valsartan to improve conduit vessel function, but not microvascular function, in patients with HFrEF.

Impact of Treatment with Sacubitril-Valsartan on Functional Capacity

The 6MWT distance is a well-tolerated and highly reproducible test that has been widely utilized to assess overall functional capacity and exercise tolerance in patients with HFrEF (21, 49). In the present study, treatment with sacubitril-valsartan increased the 6MWT distance from baseline by +16 m after 1 mo, +45 m after 2 mo, and +40 m after 3 mo in patients with HFrEF (Fig. 3). Although we cannot exclude the possibility of a “learning effect” as a consequence of performing multiple tests across time (50), the magnitude of improvement in the current study is well beyond what might be expected as a result of repeated testing. Indeed, Guyatt et al. (51) reported only a ≈4% (≈+15 m) improvement in walk distance in patients with HFrEF who performed the 6MWT six times over a 12-wk period, suggesting only a modest effect of repeat testing on walk distance. Even if this potential ≈4% improvement as a consequence of serial measurements is taken into account (equivalent to +17 m in the present study), patients with HFrEF still improved their walking distance by a magnitude of +23–28 m following treatment with sacubitril-valsartan. Considering that the minimal clinically important change in the 6MWT distance ranges between +14 m and 31 m (52), the improvement in 6MWT observed in the present study after sacubitril-valsartan is likely to represent a significant improvement in physical capacity.

In support of our findings, Beltrán et al. (23) recently reported an increase in the 6MWT distance (+42 m to 56 m) following 1 mo of treatment with sacubitril-valsartan in patients with HFrEF (New York Heart Association class II-IV). Our data extend this previous finding by identifying a significant increase in functional capacity that is maintained across 3 mo of treatment with sacubitril-valsartan. This is particularly noteworthy given that a reduction in functional capacity has been associated with poor quality of life and a worse prognosis in patients with HFrEF (19, 20). Conversely, an improvement in functional capacity, assessed via the 6MWT distance, has been related to an improvement in self-perceived symptoms of HFrEF (22). Therefore, it is possible that the potential benefits of the treatment with sacubitril-valsartan in terms of the reduced risk of death from cardiovascular causes and hospitalization for heart failure in patients with HFrEF, as documented in the PARADIGM-HF trial (2), may also stem, at least in part, from improved functional capacity.

The mechanisms by which treatment with sacubitril-valsartan improved functional capacity in patients with HFrEF remain unclear and may be multifactorial. Indeed, the decline in functional or physical capacity in patients with HFrEF is, in part, a consequence of endothelial dysfunction affecting both the coronary and peripheral arteries, leading to an impaired ability to vasodilate, thereby reducing coronary and skeletal muscle blood flow, as well as nutrient and oxygen delivery (12, 13). Furthermore, evidence from our group (53) and others (54, 55) have demonstrated a clear relationship between brachial artery FMD and coronary endothelial function (assessed via acetylcholine-induced, endothelium-dependent vasodilatory ability), suggesting that brachial artery FMD represents a surrogate for coronary endothelial function and that endothelial dysfunction may occur simultaneously in both coronary and peripheral arteries. There is also evidence supporting an association between brachial artery FMD and functional capacity in patients with HFrEF (11, 56), suggesting that an improved brachial FMD may indicate a systemic improvement in endothelial function that could be relevant for more downstream portions of the arterial tree. In support of this rationale, other pharmacological interventions that have favorably improved conduit vessel function in patients with HFrEF also observed concomitant improvements in functional capacity in this patient group (57, 58). However, in the present study, a significant correlation between improvements in FMD and 6MWT distance was not present, which may be attributed to a relatively small sample size. Beyond improvements in endothelial function, favorable changes in cardiac and skeletal muscle structure/function, which are known to contribute to functional capacity and exercise tolerance in patients with HFrEF (59), may also be improved, although assessments of cardiac and skeletal muscle function are beyond the scope of the present study. Nevertheless, findings from long-term studies (12 mo) have reported the beneficial effects of sacubitril-valsartan on cardiac function and remodeling in patients with HFrEF (60–62), which may partly explain why we observed an improvement in functional capacity in the present study. In contrast, in hypertensive obese patients, no changes in lipolysis, substrate utilization, and energy expenditure during exercise were observed following 2 mo of treatment with sacubitril-valsartan (63), suggesting that the beneficial effects of sacubitril-valsartan may not be related to changes in metabolic function.

Impact of Treatment with Sacubitril-Valsartan on Inflammation

An elevation in inflammatory biomarkers is a hallmark feature of the HFrEF clinical syndrome, having been documented both during heart failure progression (64) and in established, chronic HFrEF (65). In the present study, we observed a marked reduction in the proinflammatory cytokines TNF-α across the treatment period (Fig. 4A) and IL-18 after 3 mo of treatment (Fig. 4B), suggesting that this new drug class may possess anti-inflammatory properties. Given that high circulating levels of TNF-α (66) and IL-18 (67) are associated with increased risks of cardiovascular mortality and morbidity, our findings of the reductions in these proinflammatory cytokines lend support to the potential mechanism by which sacubitril-valsartan reduces the risk of death cardiovascular causes and hospitalization for heart failure in patients with HFrEF in the PARADIGM-HF trial (2). In contrast to TNF-α and IL-18, we did not observe any significant reductions in other proinflammatory cytokines CRP, IL-6, and IL-1β, which may be attributable, in part, to small sample size, assay sensitivity issues, and/or the complexity and pleiotropy of proinflammatory cytokines in patients with HFrEF. Although the impact of sacubitril-valsartan on inflammation has not been previously reported in human HFrEF, preclinical studies provide initial evidence supporting the potential pleiotropic effect of treatment with sacubitril-valsartan. Indeed, in an animal model of diabetic cardiomyopathy (DCM), 16 wk of sacubitril-valsartan reduced the expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α as well as a biomarker of heart failure (NT-proBNP) in the serum of diabetic mice (26). Thus, this treatment exerts anti-inflammatory and anti-heart failure effects in an experimental model of DCM. Furthermore, in a rat model of chronic kidney disease, sacubitril-valsartan ameliorated biomarkers of inflammation and fibrosis and improved renal function (68), lending further support to the capacity of this treatment to mitigate disease-related inflammation. Thus, the current findings extend these previous observations in animal disease models, documenting, for the first time, to our knowledge, a reduction in the proinflammatory biomarker TNF-α in as little as 4 wk following initiation of sacubitril-valsartan therapy in patients with HFrEF.

Chronic systemic inflammation has been implicated in the pathogenesis of conduit vessel dysfunction in patients with HFrEF due, in part, to elevated oxidative stress, i.e., an excess production of free radicals relative to antioxidant defenses (28). Free radicals such as superoxide contribute to conduit vessel dysfunction by scavenging nitric oxide (NO), thereby diminishing NO bioavailability and attenuating endothelium-dependent vasodilation (28). Given the interdependent relationship between inflammation and oxidative stress (28), any improvement in inflammatory status should theoretically be accompanied by a decrement in oxidative stress. To our surprise, the reductions in proinflammatory cytokines TNF-α and IL-18 were not accompanied by decrements in biomarkers of oxidative stress, MDA and protein carbonyl, which may be attributable, in part, to unchanged antioxidant capacity (FRAP) and free radical scavenging (catalase and SOD). Furthermore, we did not identify a significant correlation between the reductions in proinflammatory cytokines TNF-α and IL-18 and improvements in FMD following short-term treatment with sacubitril-valsartan. This lack of apparent improvement in redox balance supports a non-antioxidant mechanism of action and may suggest that a longer duration of treatment is needed to further reduce circulating levels of proinflammatory cytokines and subsequently improve redox balance in patients with HFrEF.

Strategies that mitigate vascular inflammation may also lead to improved function capacity through enhanced vasodilatory ability of the exercising skeletal muscle (12, 13). Indeed, an association between TNF receptors I and II and peak oxygen uptake has been documented in patients with HFrEF (69), highlighting the role of inflammation as a contributing factor to exercise intolerance in this patient group. In the present study, the reductions in proinflammatory cytokine TNF-α was not correlated with improvements in 6MWT distance following treatment with sacubitril-valsartan, possibly due, in part, to small sample size. It should also be noted that TNF receptors I and II, which are soluble TNF receptors reflective of the activity of TNF-α, are thought to be more sensitive than TNF-α in predicting prognosis in patients with HFrEF (70, 71). Thus, this assay sensitivity issue may partly explain the lack of correlation between TNF-α and 6MWT distance in the present study. In addition, given previous findings of an association between IL-18 and physical activity levels (72), the lack of significant correlation between the reductions in proinflammatory cytokine IL-18 and improvements in 6MWT distance following short-term treatment with sacubitril-valsartan in the present study may also stem from similar sample size/assay sensitivity issues and/or may require a longer treatment duration.

Impact of Treatment with Sacubitril-Valsartan on Clinical Biomarkers

The marked improvement in patients with HFrEF treated with sacubitril-valsartan in the PARADIGM-HF trial (2) was attributed, in part, to the combined effect of neprilysin and angiotensin receptor inhibition on established, prognostic clinical biomarkers (73). Indeed, treatment with sacubitril-valsartan increased plasma BNP concentrations, whereas plasma NT-proBNP concentrations (the biologically inactive, N-terminal fragment) were lower, indicating reduced myocardial wall stress (74). In addition to reduced myocardial wall stress, treatment with sacubitril-valsartan has also been reported to lower plasma troponin T concentrations, reflecting reduced myocardial injury in patients with HFrEF (74), and to reduce plasma concentrations of vasoactive neurohumoral peptides such as endothelin-1 and angiotensin II (75). In the present study, we did not observe any alterations in plasma concentrations of NT-proBNP, BNP, NT-proANP, Troponin T, bradykinin, or endothelin-1 across the 3 mo of treatment with sacubitril-valsartan (Table 3), which may be due to differences in sample size, drug dosing, and duration of treatment between the prior and current studies. However, the marked improvement in conduit vessel function and inflammation in the presence of unchanged biomarkers of myocardial function highlights the favorable noncardiac effects of ARNI therapy, thereby lending support to the accumulating evidence that the peripheral circulation does indeed represent a “crucial target in fighting heart failure” (28, 76).

Perspectives and Significance

The overwhelming benefits of treatment with sacubitril-valsartan with respect to reducing the risk of mortality from cardiovascular causes and hospitalization for heart failure in patients with HFrEF in the PARADIGM-HF trial (2) have influenced the current guidelines for the treatment of heart failure. Specifically, it is now recommended to use sacubitril-valsartan in place of an ACEI or an ARB (3). Although the mechanisms mediating improved clinical status are not fully understood, findings from the present study, while preliminary in nature, suggest that such benefits may be partially attributable to improvements in conduit vessel function, increased functional capacity, and reduced inflammation in patients with HFrEF, endpoints that are established predictors of mortality and morbidity in this patient population (10, 17, 19, 20, 77). In addition, treatment with sacubitril-valsartan reduced potassium and creatinine concentrations, coupled with a tendency for increased estimated glomerular filtration rate in patients with HFrEF, suggesting preserved renal function (78, 79). Taken together, these findings provide new, mechanistic insight regarding the physiological underpinnings of the beneficial effects in patients with HFrEF who transition to sacubitril-valsartan. This information may serve to inform future clinical trials investigating the efficacy of this new drug class on patient outcomes.

Experimental Considerations

We acknowledge experimental considerations that may be perceived as limitations to this prospective study. First, we only studied patients with HFrEF who were instructed to initiate treatment with sacubitril-valsartan based on their cardiologist’s clinical decision; thus, this approach enabled the opportunity to study these patients in a real-world setting. Second, we did not include a placebo group or condition to demonstrate the temporal stability of serial FMD measures; rather, a longitudinal design was utilized whereby patients with HFrEF served as their own control (day 0). The use of a single-arm, open-label, prospective study design has been recognized as an appropriate method of testing that allows insight into the efficacy of an intervention using a small number of patients and repeated measurements, whereby patients serve as their own controls (80). Importantly, this type of study design, which provides evidence-based findings that set the stage for subsequent trials that will involve more rigorous study designs (80), has been used in several clinical studies to evaluate the efficacy of an intervention in various patient populations (81–83). It is noted that this study includes a relatively small sample size, which may limit the generalizability of our findings. We also acknowledge the significant weaknesses of small, open-labeled studies with no control group and with no blinding of participant, assessor, or analyst, since this allows for potential introduction of considerable conscious and unconscious bias that can inflate positive findings significantly. Thus, our findings may not be definitive and would benefit by confirmation in a larger patient cohort. The present study was not adequately powered to detect whether the presence of type 2 diabetes (84), concomitant use of statins (85), or disease severity (86) differentially affected the magnitude of responses to treatment, and we therefore cannot exclude the potential influence of these covariates on study outcomes. It is also noteworthy that the coefficient of variation for FMD and RH assessments across serial study visits has not been systematically evaluated in our laboratory, though adherence to established methodological guidelines minimizes concern regarding excessive test-retest variability (87). As cardiac and skeletal muscle functions were not evaluated in the present study, we cannot exclude the role of these parameters on the observed improvement in functional capacity. Finally, the present study included only one female patient, which limits our ability to infer about potential sex differences from our findings.

Summary

Treatment with sacubitril-valsartan improves conduit vessel function, increases functional capacity, and reduces inflammation in patients with HFrEF, providing new information regarding the beneficial effects of ARNI therapy in this patient group. Together, these preliminary findings provide rationale for a larger, randomized, placebo-controlled study aimed at evaluating the favorable peripheral vascular effects of treating patients with HFrEF with sacubitril-valsartan, with the potential to inform contemporary, guideline-directed heart failure therapy.

GRANTS

This work was funded in part by the National Institutes of Health (R01 HL118313, to D.W.W.; R56 AG057584, to R.S.R.; T32 HL139451, to K.B.), the US Department of Veterans Affairs (RX001311, to D.W.W.; E6910-R, E1697-R, to R.S.R.; E1433-P, to R.S.R.; E9275-L, to R.S.R.; and E1572-P, to R.S.R.), and the American Heart Association (18POST33960192, to K.B.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.B., S.M.R., and D.W.W. conceived and designed research; K.B., S.M.R., J.K.A., S.H.P., and C.L.J. performed experiments; K.B. and S.M.R. analyzed data; K.B., J.S., A.S.S., and R.S.R. interpreted results of experiments; K.B. prepared figures; K.B. and D.W.W. drafted manuscript; K.B., S.M.R., J.K.A., S.H.P., C.L.J., J.S., A.S.S., R.S.R., and D.W.W. edited and revised manuscript; K.B., S.M.R., J.K.A., S.H.P., C.L.J., J.S., A.S.S., R.S.R., and D.W.W. approved final version of manuscript.