Heart failure (HF) is a widespread health problem and represents a major cause of cardiac morbidity and mortality representing the second cause of hospitalization in subjects >65 years old, despite optimal treatment (Tavazzi et al., 2013; Armentaro et al., 2021). Among affected patients, HF with reduced ejection fraction (HFrEF) represents about 50% of cases (Iorio et al., 2019).

Notably, endothelial dysfunction is a frequent condition in patients affected by HF and in this setting it predicts major cardiovascular (CV) events and mortality. Endothelial dysfunction is characterized by reduced bioavailability of nitric oxide (NO), in response to increased reactive oxygen species (ROS) production, with an imbalance between vasoconstrictor and vasodilator factors, predisposing to a pro-atherogenic and pro-trombotic phenotype, characterized by leukocyte adhesion, mitogenesis, platelet activation and vascular inflammation (Dhananjayan et al., 2016; Alem, 2019). Endothelial dysfunction may also result from abnormal electrical property and the changed expression profile of ion channels (Fan et al., 2022a).

It is known that inflammation and oxidative stress, characterized by increased 8,12-isoprostane F (2α) concentration, low endothelium-bound extracellular superoxide dismutase activity and increased endothelium-bound xanthine oxidase (XO) activity and cytokines production, play a central role in pathogenesis of both endothelial dysfunction and HF (Bulua et al., 2011; Odegaard et al., 2016; Alem, 2019; Cassano et al., 2022). Moreover, endothelial dysfunction attracts circulating inflammatory cells through Intercellular adhesion molecules (ICAMs) and inflamed endothelium interacts with cardiomyocytes and platelets (El-Battrawy et al., 2016b). In addition, increased ROS levels are one of the causes of the redox imbalance through many molecular mechanisms, as for example augmented secretion of proinflammatory cytokines (El-Battrawy et al., 2017) (Fan et al., 2022b).

Arterial stiffness, resulting from reduced elasticity of the arterial wall, increases with age and different cardiometabolic abnormalities promoting endothelial dysfunction and representing an independent predictor of CV events and HF (Husmann et al., 2015; Cassano et al., 2020). According with this, recently, many studies investigated mechanistic pathways of endothelial dysfunction and arterial stiffness in HF with the aim to provide the rationale for new drugs development and to improve the application of existing therapy (Alem, 2019).

It is known that, arterial stiffness and endothelial dysfunction are strongly interconnected recognizing similar pathophysiological mechanisms. In particular, arterial stiff-ness is correlated with increased activity of angiotensin II, which activates matrix metalloproteinases (MMP) and pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Inflammatory cytokines promote the production of C reactive protein (CRP) by vascular smooth muscle cells, which increases vascular inflammation and reduces endothelium-dependent vasodilation (Park and Lakatta, 2012). The increased vascular inflammation promotes smooth muscle cells proliferation, vascular fibrosis and impair endothelial vasodilatation, which leads to worsening of arterial stiffness, the last in turn, by inducing a reduction in vascular shear stress, may further reduce NO bioavailability.

Sacubitril/Valsartan is the first angiotensin receptor-neprilysin inhibitor (ARNI) acting by reducing the degradation of natriuretic peptides and blocking selective AT1-receptor with positive effects on CV system (Vardeny et al., 2014). In HFrEF outpatients, the PARA-DIGM-HF study demonstrated that Sacubitril/Valsartan was associated with a lower risk of composite endpoint of CV mortality or first hospitalization for HF compared to enalapril, during a twenty-seven months median of follow-up. In addition, in patients with acute HF, Sacubitril/Valsartan resulted more efficacious than enalapril in reducing both N-terminal pro-brain natriuretic peptide (NT pro-BNP) levels and rehospitalization and CV death, in an exploratory analysis (Velazquez et al., 2019). Data from real life showed that Sacubitril/Valsartan is associated with improvement in cardiac remodelling, New York Heart Association (NYHA) functional class and exercise tolerance with consequently hospital readmissions reduction (Armentaro et al., 2021). In addition, a study conducted by Abumayyaleh demonstrated improvement in cardiac valvular insufficiency, after 24 months of treatment with sacubitril/valsartan in patients with HFrEF (Abumayyaleh et al., 2022).

Moreover, a post hoc analysis from PARADIGM-HF demonstrated that subjects with type 2 diabetes mellitus (T2DM) in treatment with Sacubitril/Valsartan presented an improved glycaemic profile, compared to patients in treatment with enalapril, after a follow-up of 12 months (Seferovic et al., 2017). In addition, results from a post-hoc analysis of PARAGON-HF study demonstrated that subjects with HF and preserved ejection fraction (HFpEF), in treatment with Sacubitril/Valsartan, presented significant reduction in triglycerides and increase in HDL cholesterol. Therefore, the inhibition of renin angiotensin aldosterone system (RAAS) and neprilysin by Sacubitril/Valsartan results also in metabolic improvement as recently demonstrated in real life setting (Armentaro et al., 2022). But the benefits of Sacubitril/Valsartan are not limited to the treatment of HFrEF through modulation of the RAAS and enhancement of natriuretic peptides, an anti-arrhythmic effect is also observed due to the reduction of pro-arrhythmic remodelling and/or a direct anti-arrhythmic effect on cardiomyocytes (Sutanto et al., 2021) (Abumayyaleh et al., 2021). However, there are controversial data regarding the efficacy of sacubitril/valsartan in the treatment of ventricular tachycardia and/or ventricular fibrillation. A study conducted by El-Battrawy et al. 2019c demonstrated in HFrEF patinets, that treatment with sacubitril/valsartan does not reduce the risk of ventricular tachyarrhythmias, after 12 months of follow- up. By contrast, recently, a study showed a significant reduction in ventricular arrhythmias in HFrEF patients, in treatment with sacubitril/valsartan (El-Battrawy et al., 2019a).

Taken together, all these findings could suggest a possible direct protective effect of Sacubitril/Valsartan on CV system by modulating inflammation and oxidative stress. According with this, a recent study conducted by Qing Ge et al. (2019) in an animal model, demonstrated that Sacubitril/Valsartan could protect against diabetic cardiomyopathy (DCM) through the inhibition of inflammatory mediators, oxidative stress and apoptosis, without adverse effects.

By the way, in literature no study investigated the possible effect of treatment with Sacubitril/Valsartan on endothelial dysfunction and arterial stiffness in patients with HF.

With the aim to better understand pathophysiological mechanisms underlying cardiovascular protective effects of Sacubitril/Valsartan, and in agreement with evidence al-ready in the literature, the aim of the present study was to evaluate the effect of Sacubitril/Valsartan on endothelial dysfunction and arterial stiffness in patients with HFrEF evaluated at baseline and after 6 months of treatment. Moreover, we evaluated the effects of Sacubitril/Valsartan on oxidative stress levels and platelets activation and the correlation with arterial stiffness and endothelial dysfunction.

To the best of our knowledge, this is the first study to demonstrate the possible effect of Sacubitril/Valsartan, a combination of Ang II receptor blocker and neprilysin inhibitor, on endothelial function and arterial stiffness in HF patients.

The endothelium is a monolayer of cells covering the inner surface of blood vessels and acts as structural and functional barrier between vessels wall and blood. It is known that the endothelium is involved in the control of thrombosis and thrombolysis, leukocytes adhesion and platelets aggregation, and the regulation of vascular tone by balancing vasoactive substances production (Marti et al., 2012) (El-Battrawy et al., 2016a) (El-Battrawy et al., 2019b).

In particular, endothelium-derived NO is released in response to physical stimuli, hormones, and platelet-derived substances and it induces vascular relaxation and platelet function inhibition.

Endothelial dysfunction, characterized by a shift in the properties of the endothelium toward impaired vasodilation promoting a pro-inflammatory and pro-thrombic state, is associated with reduced NO bioavailability (Elkayam et al., 2002). It has a crucial role in the pathogenesis of CVD and it is correlated to HF predicting major clinical events in this setting of patients (Zuchi et al., 2020) (El-Battrawy and Akin, 2022).

Notably, endothelium-dependent vasodilation is impaired in patients with both reduced and preserved HF (Kubo et al., 1991; Ramsey et al., 1995; Morgan et al., 2004; Katz et al., 2005). In particular, the dysfunction of vascular endothelium can be demonstrated in several vascular beds. According with this, several studies high-lighted a significant reduction in endothelial-dependent vasodilation in peripheral arteries in patients with chronic HF (Kubo et al., 1991; Ramsey et al., 1995; Morgan et al., 2004; Katz et al., 2005).

Of interest, endothelial dysfunction can be found not only in patients with ischemic but also non-ischemic HF, affecting non-coronary vascular beds (Husmann et al., 2015; Cassano et al., 2020). Moreover, a study conducted by Maio et al. (2021), demonstrated that impaired endothelium-dependent vasodilatation is able to predict development of HF in hypertensive patients, allowing to hypothesize its role in the CV continuum, from hypertension to clinical events.

The endothelial dysfunction observed in HF patients is amply justified by increased formation of superoxide radicals and other oxidant substances in the vascular beds. The increase in oxidative stress promotes a direct inactivation of NO thus favouring a reduced endothelium dependent vasodilation (Paolocci et al., 2001; Zuchi et al., 2010; Breitenstein et al., 2017). Furthermore, previous studies demonstrated that endothelial function and NO bioavailability can affect arterial stiffness, an independent predictor value for CVD (Westergren et al., 2016). Multiple mechanisms potentially underlie in-creased arterial stiffness including both functional and structural vascular wall changes so as collagen deposition, smooth muscle hypertrophy and changes in endothelial per-meability. Neurohormonal activation, in particular RAAS, that characterizes HF patients has been linked to endothelial dysfunction and increased arterial stiffness, by diminishing vascular elasticity, by modulating extracellular matrix growth and by inducing smooth muscle cells proliferation (Incalza et al., 2018).

Present data obtained from this longitudinal, one-center study, demonstrated that six months treatment with Sacubitril/Valsartan had positive effects on the improvement of endothelial dysfunction and arterial stiffness in a consistent cohort HFrEF symptomatic patients, as well as a significant improvement of clinical, biochemical and haemodynamic parameters, confirming previous evidence (Armentaro et al., 2021; Pelaia et al., 2022). In accordance with a study conducted by Abumayyaleh et al. (2021), conducted in patients HFrEF and stratified by age, our enrolled population is defined older (>65 years old) (mean age 69.7 ± 7.7), moreover, a further stratification of the population according to gender (82 male and 18 female) showed that there was no difference in age (male mean age 69.6 ± 7.7 and female mean age 70.4 ± 7.8, p = 0.663). Therefore, the entire population of our study can be considered older.

The novelty of our study is the significant betterment of inflammatory profile, as evidenced by the decreased values of IL-6, TNF-α, hs-CRP, and a reduction of oxidative stress and platelets activation biomarkers, after 6 months of treatment with Sacubitril/Valsartan; all these mechanisms may account for the improvement in vascular function.

In details, data obtained from our study showed a significant decrease in biomarkers of oxidative stress at follow-up, and a significant correlation with vascular function parameter (ΔRHI and ΔPWV). The decrease in oxidative stress levels could be due to the ability of Sacubitril/Valsartan to block angiotensin II receptor, as well as the reduction of inflammatory indices. In fact, it is known that, in patients suffering from HF, Angiotensin II, pro-inflammatory cytokines and other pathological stimuli such as hypercholesterolemia and hyperglycaemia, significantly stimulate the expression and activity of NAD(P)H oxidase (Incalza et al., 2018). In particular, Nox-2 isoform of NAD(P)H contributes to reduced bioavailability of NO and plays a crucial role in vascular disease, promoting inflammation, endothelial dysfunction and consequently arterial stiffness, via excessive production of ROS (Drummond et al., 2011; Ismaeel et al., 2018).

Furthermore, previous studies demonstrated that oxidative stress might lead to platelets hyperactivity; in fact, ROS over production is relevant for increase in important markers of platelets activation including sP-selectin, glycoprotein VI (GPVI) and, thromboxane B2 (TXB2) (Fuentes et al., 2019). Platelets activation has a crucial role for CVD development; in fact, platelets constitute atherothrombotic plaques, whose rupture is capable of triggering arterial thrombosis and infarction. The key role that platelets activity has in thrombotic events and in the onset and progression of atherosclerosis, might at least in partly justify the association between arterial stiffness and CVD. Our results demonstrated a significant reduction in sP-selectin and GPVI levels, after six months of Sacubitril/Valsartan treatment, indicating a reduction in platelets activity; the correlation analysis between ΔRHI, ΔPWV and platelets activity biomarkers may also explain the improvement in endothelial function and arterial stiffness observed in our enrolled patients. Reduction in platelet activation in our cohort of patients is also related to improvement in inflammatory profile; an in vitro study conducted by De Biase et al. (2003) proved that TNF-α may promote platelets oxidative stress, by activating NADPH enzyme oxidase. Furthermore, Pignatelli et al. (2005) demonstrated that, in HF patients, circulating levels of TNF-α may account for platelet hyperfunction because TNF-α is able to activate platelets through stimulation of the arachidonic acid pathway.

In addition, our results demonstrated a significant improvement in metabolically in-dices such as FPG, FPI, HbA1c and HOMA, and these data are in agreement with our previous evidence (Armentaro et al., 2022). Many studies demonstrated that hyperglycemia triggers vascular damage by creating imbalance between NO availability and ROS accumulation (Kaur et al., 2018; Cassano et al., 2022). Moreover, chronic hyperglycaemia injures vascular wall by many cellular mechanisms, including an increased production of intracellular advanced glycation end products (AGEs), raised expression of AGE receptors (RAGE) and ligands, activation of protein kinase C (PKC), augmented polyol and hexosamine flux and over activation of the hexosamine pathway. In our study, a linear correlation analysis showed a significant and inversely correlation between ΔRHI, ΔPWV, and ΔHOMA, indicating that with the reduction of the HOMA index there is an improvement in the endothelial function parameter and in arterial stiffness. This is clinically relevant because HOMA index is more strongly associated with CVD than glucose or insulin concentration, in non-diabetic patients (Gast et al., 2012). Furthermore, a study conducted by Westergren et al. demonstrated that HOMA index was associated with endothelial dysfunction in non-diabetic patients with myocardial ischemia and that high values of HOMA index correlated with higher IL-6 levels (Westergren et al., 2016).

The increased levels of IGF-1 observed in our cohort of patients, at six months follow-up, confirm the amelioration of metabolic profile; according with this lower IGF-1 levels have previously been shown to correlate with endothelial dysfunction (Sesti et al., 2014), so the increased levels of IGF-1 observed in our study may also explain the improvement of vascular function. In addition, also the reduction of uric acid levels observed in our enrolled patients, may contribute to improve endothelial dysfunction and arterial stiffness. In fact, it is known that hyperuricemia has negative effects on CVD, by reducing NO bioavailability and promoting oxidative stress and vascular inflammation, thus favouring endothelial dysfunction (Cassano et al., 2020). Moreover, uric acid may negatively affect insulin-sensitivity by modulating the intracellular insulin signalling pathway. All these mechanisms are responsible for subclinical organ damage, indicating that higher value of uric acid are associated with arterial stiffness (Perticone et al., 2013). Finally, it is important to remark that Sacubitril/Valsartan significantly improved vascular parameters, in particular endothelial function and arterial stiff-ness, known predictors of CV outcome. This improvement, as evidenced by the regression analysis, is linked to the improvement of oxidative stress and inflammation indices. Notably, RHI and PWV follow-up values were comparable to those of newly diagnosed hypertensive patients without diabetes or CV complications (Sciacqua et al., 2012; Cassano et al., 2021). Similarly, the indicators of inflammation, in particular hs-CRP, and of oxidative stress significantly improved with Sacubitril/Valsartan to such an extent that they were comparable to those of normotolerant hypertensive patients (Cassano et al., 2022) (Figure 4).

In conclusion, the present study strengthens scientific evidence supporting the use of Sacubitril/Valsartan in HFrEF patients and confirms its beneficial effects on hemodynamic, clinical, biochemical and metabolic profile. Moreover, the novelty of our study is that six months treatment with Sacubitril/Valsartan is able to improve endothelial dysfunction and arterial stiffness in HFrEF patients, likely through the reduction of oxidative stress, platelet activation and inflammation biomarkers circulating levels with the improvement of cardio-metabolic burden.