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Angiopoietins, vascular endothelial growth factors and secretory phospholipase A2 in heart failure patients with preserved ejection fraction

  • Author Footnotes
    ⁎ Co-first authors.
    Gilda Varricchi
    Correspondence
    Corresponding author at: Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy.
    Footnotes
    ⁎ Co-first authors.
    Affiliations
    Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy

    Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, 80131, Naples, Italy

    World Allergy Organization (WAO), Center of Excellence, 80131, Naples, Italy

    Institute of Experimental Endocrinology and Oncology "G. Salvatore" (IEOS), National Research Council (CNR), 80131, Naples, Italy
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  • Author Footnotes
    ⁎ Co-first authors.
    Remo Poto
    Footnotes
    ⁎ Co-first authors.
    Affiliations
    Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy

    World Allergy Organization (WAO), Center of Excellence, 80131, Naples, Italy

    Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161, Rome, Italy
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  • Anne Lise Ferrara
    Affiliations
    Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy

    World Allergy Organization (WAO), Center of Excellence, 80131, Naples, Italy

    Institute of Experimental Endocrinology and Oncology "G. Salvatore" (IEOS), National Research Council (CNR), 80131, Naples, Italy
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  • Giuseppina Gambino
    Affiliations
    Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy
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  • Gianni Marone
    Affiliations
    Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy

    Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, 80131, Naples, Italy

    World Allergy Organization (WAO), Center of Excellence, 80131, Naples, Italy

    Institute of Experimental Endocrinology and Oncology "G. Salvatore" (IEOS), National Research Council (CNR), 80131, Naples, Italy
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  • Giuseppe Rengo
    Affiliations
    Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy

    Istituti Clinici Scientifici Maugeri SpA Società Benefit, 82037, Telese, (BN), Italy
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  • Author Footnotes
    § Co-senior authors.
    Stefania Loffredo
    Footnotes
    § Co-senior authors.
    Affiliations
    Department of Translational Medical Sciences, University of Naples Federico II, 80131, Naples, Italy

    Center for Basic and Clinical Immunology Research (CISI), University of Naples Federico II, 80131, Naples, Italy

    World Allergy Organization (WAO), Center of Excellence, 80131, Naples, Italy

    Institute of Experimental Endocrinology and Oncology "G. Salvatore" (IEOS), National Research Council (CNR), 80131, Naples, Italy
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  • Author Footnotes
    § Co-senior authors.
    Leonardo Bencivenga
    Footnotes
    § Co-senior authors.
    Affiliations
    Department of Advanced Biomedical Sciences, University of Naples Federico II, 80131, Naples, Italy

    Gèrontopole de Toulouse, Institut du Vieillissement, CHU de Toulouse, 31000, Toulouse, France
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  • Author Footnotes
    ⁎ Co-first authors.
    § Co-senior authors.
Published:October 22, 2022DOI:https://doi.org/10.1016/j.ejim.2022.10.014

      Highlights

      • Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for about 50% of all HF patients.
      • Angiopoietin 1 (ANGPT1) was decreased in HF with reduced ejection fraction (HFrEF).
      • ANGPT2 levels were increased in both HFpEF and HFrEF compared to controls.
      • VEGF-D was increased in both HFpEF and HFrEF compared to controls.
      • Secretory phospholipase A2 was increased in HFrEF but not in HFpEF compared to controls.

      Abstract

      Background

      Heart failure (HF) is a growing public health burden, with high prevalence and mortality rates. A proportion of patients with HF have a normal ventricular ejection fraction (EF), referred to as HF with preserved EF (HFpEF), as opposed to patients with HF with reduced ejection fraction (HFrEF). HFpEF currently accounts for about 50% of all HF patients, and its prevalence is rising. Angiopoietins (ANGPTs), vascular endothelial growth factors (VEGFs) and secretory phospholipases A2 (sPLA2s) are proinflammatory mediators and key regulators of endothelial cells. Methods: The aim of this study was to analyze the plasma concentrations of angiogenic (ANGPT1, ANGPT2, VEGF-A) and lymphangiogenic (VEGF-C, VEGF-D) factors and the plasma activity of sPLA2 in patients with HFpEF and HFrEF compared to healthy controls.

      Results

      The concentration of ANGPT1 was reduced in HFrEF compared to HFpEF patients and healthy controls. ANGPT2 levels were increased in both HFrEF and HFpEF subjects compared to controls. The ANGPT2/ANGPT1 ratio was increased in HFrEF patients compared to controls. The concentrations of both VEGF-A and VEGF-C did not differ among the three groups examined. VEGF-D was increased in both HFrEF and HFpEF patients compared to controls. Plasma activity of sPLA2 was increased in HFrEF but not in HFpEF patients compared to controls.

      Conclusions

      Our results indicate that three different classes of proinflammatory regulators of vascular permeability and smoldering inflammation are selectively altered in HFrEF or HFpEF patients. Studies involving larger cohorts of these patients will be necessary to demonstrate the clinical implications of our findings.

      Keywords

      Abbreviations:

      ANGPT (angiopoietin), BMI (body mass index), BNP (B-type natriuretic peptide), COPD (chronic obstructive pulmonary disease), DM (diabetes mellitus), DOPC (dioleoylphosphatidylcholine), EF (ejection fraction), GFR (glomerular filtration rate), HF (heart failure), HFpEF (heart failure with preserved ejection fraction), HFrEF (heart failure with reduced ejection fraction), IHF (ischemic heart failure), LVEF (left ventricular ejection fraction), RFU (relative fluorescent unit), sPLA2 (secretory phospholipases A2), VEGF (vascular endothelial growth factor)

      1. Introduction

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      ]. A deeper knowledge of the molecular and immunological mechanisms involved in this complex pathophysiology is needed for the identification of novel biomarkers and therapeutic targets to stratify prognosis and drive decision-making processes.
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      Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions.
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      The effect of angiopoietin-1 upregulation on the outcome of acute ischaemic stroke in rodent models: A meta-analysis.
      ,
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      • Fu T.I.
      • et al.
      Angiopoietin 1 influences ischemic reperfusion renal injury via modulating endothelium survival and regeneration.
      ]. ANGPT2, stored in Weibel-Palade bodies in endothelial cells [
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      • Ergun S.
      • Schumacher U.
      • et al.
      In vitro differentiation of endothelial cells from AC133-positive progenitor cells.
      ], is rapidly released in response to inflammatory stimuli [
      • Fiedler U.
      • Scharpfenecker M.
      • Koidl S.
      • et al.
      The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies.
      ] and competitively inhibits ANGPT1/Tie2 interaction [
      • Maisonpierre P.C.
      • Suri C.
      • Jones P.F.
      • et al.
      Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis.
      ,
      • Saharinen P.
      • Eklund L.
      • Alitalo K.
      Therapeutic targeting of the angiopoietin-TIE pathway.
      ], resulting in vascular instability and leakage [
      • Roviezzo F.
      • Tsigkos S.
      • Kotanidou A.
      • et al.
      Angiopoietin-2 causes inflammation in vivo by promoting vascular leakage.
      ]. ANGPT2 is a proinflammatory molecule [
      • Fiedler U.
      • Reiss Y.
      • Scharpfenecker M.
      • et al.
      Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.
      ], and its concentrations are increased in patients with acute coronary syndrome [
      • Lee K.W.
      • Lip G.Y.
      • Blann A.D.
      Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes.
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      • Poss J.
      • Fuernau G.
      • Denks D.
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      Angiopoietin-2 in acute myocardial infarction complicated by cardiogenic shock–a biomarker substudy of the IABP-SHOCK II-Trial.
      ], hypertension [
      • Patel J.V.
      • Lim H.S.
      • Varughese G.I.
      • et al.
      Angiopoietin-2 levels as a biomarker of cardiovascular risk in patients with hypertension.
      ,
      • David S.
      • Kumpers P.
      • Lukasz A.
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      Circulating angiopoietin-2 in essential hypertension: relation to atherosclerosis, vascular inflammation, and treatment with olmesartan/pravastatin.
      ], congestive HF [
      • Chong A.Y.
      • Caine G.J.
      • Freestone B.
      • et al.
      Plasma angiopoietin-1, angiopoietin-2, and angiopoietin receptor tie-2 levels in congestive heart failure.
      ] and congenital HF [
      • Lukasz A.
      • Beutel G.
      • Kumpers P.
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      Angiopoietin-2 in adults with congenital heart disease and heart failure.
      ]. It is a prognostic biomarker of adverse cardiovascular events after percutaneous coronary intervention (PCI) [
      • Jian W.
      • Li L.
      • Wei X.M.
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      Prognostic value of angiopoietin-2 for patients with coronary heart disease after elective PCI.
      ,
      • Zeng Z.Y.
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      Effects of percutaneous coronary intervention on serum angiopoietin-2 in patients with coronary heart disease.
      ] in myocardial infarction [
      • Iribarren C.
      • Phelps B.H.
      • Darbinian J.A.
      • et al.
      Circulating angiopoietins-1 and -2, angiopoietin receptor Tie-2 and vascular endothelial growth factor-A as biomarkers of acute myocardial infarction: a prospective nested case-control study.
      ] and HFpEF [
      • Chirinos J.A.
      • Orlenko A.
      • Zhao L.
      • et al.
      Multiple plasma biomarkers for risk stratification in patients with heart failure and preserved ejection fraction.
      ].
      The vascular endothelial growth factor (VEGF) family includes VEGF-A, VEGF-B, VEGF-C, and VEGF-D [
      • Varricchi G.
      • Loffredo S.
      • Galdiero M.R.
      • et al.
      Innate effector cells in angiogenesis and lymphangiogenesis.
      ]. VEGFs and their receptors on blood and lymphatic endothelial cells play critical roles in inflammatory and tumor angiogenesis [
      • Varricchi G.
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      Future needs in mast cell biology.
      ]. VEGF-A, the most potent proangiogenic factor [
      • Sammarco G.
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      Mast cells, angiogenesis and lymphangiogenesis in human gastric cancer.
      ], is also known for its powerful permeabilizing activity [
      • Varricchi G.
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      Innate effector cells in angiogenesis and lymphangiogenesis.
      ,
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      ,
      • Varricchi G.
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      ,
      • Varricchi G.
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      ]. VEGF-C and VEGF-D are major modulators of inflammatory and tumor lymphangiogenesis [
      • Fankhauser M.
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      • Potin L.
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      ,
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      ]. Elevated levels of circulating VEGF-A have been found in patients with myocardial infarction [
      • Lee K.W.
      • Lip G.Y.
      • Blann A.D.
      Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes.
      ,
      • Heeschen C.
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      ,
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      ,
      • Kawamoto A.
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      ,
      • Kranz A.
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      ]. The roles of VEGF-A, VEGF-C and VEGF-D in HFpEF remain unclear or totally unexplored.
      Phospholipase A2s (PLA2s) hydrolyze the fatty acids from membrane phospholipids releasing arachidonic acid and lysophospholipids [
      • Granata F.
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      ,
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      ,
      • Murakami M.
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      ]. Secreted or extracellular PLA2 (sPLA2) modulates vascular permeability [
      • Rizzo M.T.
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      Secreted phospholipase A(2) induces vascular endothelial cell migration.
      ] and activates inflammatory cells [
      • Loffredo S.
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      Secreted phospholipases A2 in hereditary angioedema with C1-inhibitor deficiency.
      ,
      • Loffredo S.
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      ]. Circulating levels of sPLA2 predict coronary events in patients with coronary artery disease [
      • Kugiyama K.
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      Circulating levels of secretory type II phospholipase A(2) predict coronary events in patients with coronary artery disease.
      ], in apparently healthy men and women [
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      ] and increase the risk of early atherosclerosis [
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      Elevated Type II secretory phospholipase A2 increases the risk of early atherosclerosis in patients with newly diagnosed metabolic syndrome.
      ]. Serum sPLA2 levels also predict long-term mortality for HF after myocardial infarction [
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      Serum secretory phospholipase A2-IIa (sPLA2-IIA) levels in patients surviving acute myocardial infarction.
      ].
      Despite some evidences are available on ANGPTs, VEGF isoforms, and sPLA2 involvement in ischemic heart disease [
      • Varricchi G.
      • Loffredo S.
      • Bencivenga L.
      • et al.
      Angiopoietins, vascular endothelial growth factors and secretory phospholipase A2 in ischemic and non-ischemic heart failure.
      ], to the best of our knowledge, no study has focused on their role in the different HF phenotypes. Thus, the aim of the present study was to evaluate the circulating levels of ANGPTs, VEGFs and sPLA2 activity in patients with HFpEF or HFrEF compared to healthy controls.

      2. Materials and methods

      2.1 Study population

      The study population consisted of Caucasian patients suffering from HF admitted to the Department of Translational Medical Sciences of the University of Naples Federico II. The primary objective of this study was to analyze the plasma concentrations of angiogenic (ANGPT1, ANGPT2, VEGF-A) and lymphangiogenic (VEGF-C, VEGF-D) factors and the plasma activity of sPLA2 in patients with HFpEF and HFrEF compared to healthy controls. Inclusion criteria listed: age ≥ 18 years, diagnosis of HF from at least 6 months [
      • Ponikowski P.
      • Voors A.A.
      • Anker S.D.
      • et al.
      2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC.
      ], stable clinical condition during the month prior to inclusion, optimal guideline-based pharmacotherapy from at least 3 months. Exclusion criteria were represented by chronic obstructive pulmonary disease (COPD), diabetes mellitus (DM), immune disorders (rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjögren syndrome, vasculitis, psoriatic arthritis, dermatomyositis, ankylosing spondylitis), malignancies (also past), obesity as assessed through Body Mass Index (BMI) more than 30 Kg/m2, dialysis-dependent kidney failure, acute coronary syndromes and/or coronary revascularization in the previous 6 months, and inability to provide informed consent. The control group was represented by Caucasian subjects consecutively referred to the Department of Translational Medical Sciences of the University of Naples Federico II without HF and in accordance with the exclusion criteria, who accepted to participate in the research protocol. The sample size was set assuming as primary outcome the assessment of ANGPT2 plasma levels in HF patients compared to healthy controls, an alpha error equal to 5% was set, together with a statistical power equal to 80%. Expecting the mean concentrations of ANGPT2 to be 500 pg/ml in healthy individuals, according to previous evidence [
      • Chong A.Y.
      • Caine G.J.
      • Freestone B.
      • et al.
      Plasma angiopoietin-1, angiopoietin-2, and angiopoietin receptor tie-2 levels in congestive heart failure.
      ,
      • Varricchi G.
      • Loffredo S.
      • Bencivenga L.
      • et al.
      Angiopoietins, vascular endothelial growth factors and secretory phospholipase A2 in ischemic and non-ischemic heart failure.
      ] a minimum of 30 individuals per group were considered necessary to reveal as significant a ±25% difference in plasma concentration between controls and HF patients. All patients underwent medical history evaluation and demographic/clinical data collection, including age, gender, BMI, cardiovascular risk factors and comorbidities. Clinical examination, transthoracic echocardiography and serum BNP determination were performed at the time of the enrolment. In the present study, the population was divided into two groups based on EF: HFrEF (EF < 50%) or HFpEF (EF ≥ 50%). IHD was established based on either previous documented myocardial infarction and/or significant coronary artery disease with indication to cardiac revascularization. This study was approved by the Ethics Committee of the University of Naples Federico II (protocol number 124/17). All participants were carefully informed and signed a written consent to participate in the study.

      2.2 Blood sample collection

      Blood samples were collected during routine diagnostic procedures, and the remaining plasma sample was labeled with a code that was documented in a data sheet. Blood was collected by a clean venipuncture and minimal stasis using sodium citrate 3.2% as anticoagulant and centrifuged (2000 g for 20 min at 22°). Plasma was aliquoted into 1.5 ml cryovials, stored at −80 °C and subsequently thawed for this study. Technicians who performed the assays were blinded to the patients’ history.

      2.3 Plasma measurement of ANGPTs and VEGFs

      Concentrations of the following proteins, ANGPT1, ANGPT2, VEGF-A, VEGF-C, and VEGF-D were measured using commercially available ELISA kits (R&D System, Minneapolis, USA) according to kit specifications. The ANGPT1/ANGPT2 ratio, a parameter of vascular permeability, was calculated as previously described [
      • Loffredo S.
      • Bova M.
      • Suffritti C.
      • et al.
      Elevated plasma levels of vascular permeability factors in C1 inhibitor-deficient hereditary angioedema.
      ]. The ELISA sensitivity was 156.25 – 10,000 pg/ml for ANGPT1, 31.1 – 4,000 pg/ml for ANGPT2, 31.1 – 2,000 pg/ml for VEGF-A, 62.5 – 4,000 pg/ml for VEGF-C, and 31.3 – 2,000 pg/m for VEGF-D.

      2.4 Plasma measurement of phospholipase A2 activity

      Plasma PLA2 activity was measured using a Life Technologies EnzChek®phospholipase A2 assay. A PLA2 substrate cocktail consisting of 7-hydroxycoumarinyl-arachidonate (0.3 mM), 7-hydroxycoumarinyl-linolenate (0.3 mM), hydroxycoumarinyl 6-heptenoate (0.3 mM), dioleoylphosphatidylcholine (DOPC) (10 mM), and dioleoylphosphatidylglycerol (DOPG) (10 mM) was prepared in ethanol. Liposomes were formed by gradually adding 77 µl substrate/lipid cocktail to 10 ml of PLA2 buffer (50 mM Tris–HCl, 100 mM NaCl, 1 mM CaCl2) while stirring rapidly over 1 min using a magnetic stirrer. Fluorescence (excitation at 360 nm and emission at 460 nm) was measured and specific activity [relative fluorescent units (RFU)/ml] for each sample was calculated. Plasma (50 µl) was added to 96-well plates, and PLA2 activity was evaluated by adding 50 µl of substrate cocktail.

      2.5 Statistics

      Categorical data were reported as absolute observations with percentages and compared using Pearson's χ2 test. Continuous variables were expressed as mean ± standard deviation (SD) or median and interquartile range (IQR) and compared through Student's t-test or one-way ANOVA and Bonferroni's multiple comparison tests, or the Mann–Whitney U-test according to data distribution. Spearman's rank r correlation was computed to assess the relationship between variables. Pearson's r correlation coefficient was employed to evaluate the relationship between each mediator and EF. Plasma concentrations of VEGFs and ANGPTs and the activity of sPLA2 were shown as the median (horizontal black line), the 25th and 75th percentiles (boxes) and the 5th and 95th percentiles (whiskers) of HFrEF or HFpEF patients and controls. As statistical significance threshold, a p < 0.05 was employed for all analyzes performed through the SPSS 26 software (IBM, USA).

      3. Results

      3.1 Clinical and demographic characteristics of the overall population

      The study population comprises 47 patients with HFrEF, 31 patients with HFpEF and 47 healthy controls, carefully selected according to inclusion/exclusion criteria. Demographic and clinical characteristics of the study population are summarized in Table 1. As expected, HFpEF patients were older, more prevalently female, and showed higher left ventricular EF (LVEF) and glomerular filtration rate (GFR), lower BNP levels and IHD etiology compared to HFrEF patients.
      Table 1Demographic and clinical characteristics of HFpEF, HFrEF and control groups.
      VariableControls (n = 47)HFrEF (n = 47)HFpEF (n = 31)p-value
      Age (yrs)71.17 ± 12.8569.23 ± 11.5675.80 ± 9.080.049
      Gender, male (%)18 (38.30)29 (61.70)12 (38.71)0.042
      BMI (kg/m2)25.89 ± 3.9525.59 ± 3.7026.21 ± 4.650.801
      BNP (pg/mL)50.58 ± 32.06958.11 ± 763.94550.5 ± 681.71<0.001
      Leukocytes (x 103/mm3)7.43 ± 2.728.48 ± 3.528.26 ± 3.490.407
      GFR (mL/min)71.43 ± 22.5551.93 ± 29.0357.75 ± 30.060.032
      EF (%)61.55 ± 5.5034.20 ± 7.161.82 ± 6.04<0.001
      Smoking (%)7 (14.89)19 (40.42)5 (16.12)0.007
      Hypertension (%)31 (65.95)30 (63.8)26 (83.87)0.146
      Coronary artery disease (%)none20 (42.55)9 (29.03)<0.001
      Hyperlipidemia (%)14 (29.78)17 (36.17)19 (61.29)0.076
      Atrial fibrillation (%)6 (12.77)17 (36.17)12 (38.71)0.013
      Diuretics (%)5 (10.63)35 (74.47)10 (32.26)<0.001
      ACEIs (%)8 (17.02)21 (44.68)8 (28.81)0.045
      ARBs (%)12 (25.53)10 (21.28)7 (22.58)0.908
      Beta-blockers (%)15 (31.91)37 (78.72)18 (58.06)<0.001
      Data are expressed as mean values for continuous variables and percentage (%) for categorical variables.
      BMI: Body Mass Index; BNP: B-type natriuretic peptide; GFR: glomerular filtration rate (assessed through CKD-EPI equation); LVEF: left ventricular ejection fraction. Assessment of comorbidities, such as hypertension and dyslipidemia, was based on clinical history and examination of chronic therapies for each participant.

      3.2 Plasma concentrations of ANGPT1, ANGPT2 and their ratio in patients with HFrEF or HFpEF and healthy controls

      Fig. 1A shows that lower concentrations of ANGPT1 were detected in HFrEF patients compared to healthy controls and HFpEF subjects; the plasma levels of ANGPT1 in HFpEF patients were similar to controls. Plasma concentrations of ANGPT2 were significantly higher in both HF groups than controls (panel B). ANGPT2/ANGPT1 ratio was higher in HFrEF but not in HFpEF patients, compared to controls (panel C). There were no differences in ANGPT1 or ANGPT2 between male and female values in both controls and groups of patients. Moreover, no correlations were observed between the age of patients and the concentrations of the different mediators examined.
      Fig 1
      Fig. 1(A) Plasma concentrations of ANGPT1 in patients with HFrEF, HFpEF and healthy controls; (B) Plasma concentrations of ANGPT2 in patients with HFrEF, HFpEF and healthy controls; (C) ANGPT2/ANGPT1 ratio in patients with HFrEF, HFpEF and healthy controls.
      *p < 0.05; **p < 0.01; ***p < 0.001.
      The strongest significant correlation was observed between cardiac function assessed through EF and PLA2 (r = −0.435; p < 0.01) (Table 2). Weaker significant coefficient correlations were found between ANGPT1 (r = 0.307; p < 0.01) orANGPT2/ANGPT1 (r = −0.244; p = 0.01) and EF; contrarywise, no association was observed with the other examined biomarkers.
      Table 2Correlations between plasma concentrations of angiogenic, lymphangiogenic and proinflammatory mediators and ejection fraction (EF).
      MediatorEFp value
      ANGPT10.307<0.01*
      ANGPT2−0.1530.12
      ANGPT2/ANGPT1−0.2440.01*
      VEGF-A−0.0060.94
      VEGF-C0.0170.86
      VEGF-D−0.0950.349
      PLA2−0.435<0.01*
      p value corresponds to Pearson's r correlation coefficient.

      3.3 Plasma concentrations of VEGF-A, VEGF-C, and VEGF-D in patients with HFrEF or HFpEF and healthy controls

      The concentrations of the vasopermability [
      • Senger D.R.
      • Galli S.J.
      • Dvorak A.M.
      • et al.
      Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.
      ] and angiogenic factor [
      • Sammarco G.
      • Varricchi G.
      • Ferraro V.
      • et al.
      Mast cells, angiogenesis and lymphangiogenesis in human gastric cancer.
      ] VEGF-A were also analyzed. The mean plasma concentrations of VEGF-A were essentially similar in patients with HFrEF, HFpEF and controls (Fig. 2A). VEGF-C and VEGF-D are the main lymphangiogenic factors [
      • Randolph G.J.
      • Ivanov S.
      • Zinselmeyer B.H.
      • et al.
      The lymphatic system: integral roles in immunity.
      ,
      • Zheng W.
      • Aspelund A.
      • Alitalo K.
      Lymphangiogenic factors, mechanisms, and applications.
      ]. The mean plasma concentrations of VEGF-C did not differ in all patients with different HF phenotypes and healthy donors (Fig. 2B). By contrast, the plasma concentrations of VEGF-D were increased in both HFrEF and HFpEF patients compared to controls (Fig. 2C). There was a trend in HFpEF to display a further increase in VEGF-D compared to HFrEF patients. There were no differences in VEGF-A, VEGF-C, and VEGF-D concentrations between male and female values in both controls and HF patients. Moreover, the age of patients and the examined VEGF concentrations did not correlate.
      Fig 2
      Fig. 2(A) Plasma concentrations of VEGF-A in HFrEF and HFpEF patients and healthy controls; (B) Plasma concentrations of VEGF-C in HFrEF and HFpEF patients and healthy controls; (C) Plasma concentrations of VEGF-D in HFrEF and HFpEF patients and healthy controls.
      **p < 0.01.

      3.4 Plasma concentrations of sPLA2 activity in patients with HFrEF or HFpEF and healthy controls

      sPLA2 represents an important class of vasodilatory factors [
      • Rizzo M.T.
      • Nguyen E.
      • Aldo-Benson M.
      • et al.
      Secreted phospholipase A(2) induces vascular endothelial cell migration.
      ] involved in several inflammatory processes [
      • Granata F.
      • Staiano R.I.
      • Loffredo S.
      • et al.
      The role of mast cell-derived secreted phospholipases A2 in respiratory allergy.
      ,
      • Loffredo S.
      • Ferrara A.L.
      • Bova M.
      • et al.
      Secreted phospholipases A2 in hereditary angioedema with C1-inhibitor deficiency.
      ,
      • Loffredo S.
      • Marone G.
      Hereditary angioedema: the plasma contact system out of control: comment.
      ]. Fig. 3 shows that the plasma activity of sPLA2 was significantly increased in HFrEF patients compared to HFpEF and healthy donors. There were no differences in sPLA2 activity between male and female values in both controls and patients. Moreover, no correlations were observed between the age of patients and the concentration of sPLA2 activity.
      Fig 3
      Fig. 3Plasma sPLA2 activity in HFrEF, HFpEF patients and healthy controls.
      ****p < 0.0001.

      3.5 Correlations between altered mediators in patients with HFrEF or HFpEF

      The correlations among the altered mediators in patients with HFrEF or HFpEF were also analyzed. Fig. 4A shows an inverse correlation between plasma concentrations of ANGPT2 and ANGPT1 in HFrEF patients. In addition, there was a negative correlation between sPLA2 activity and ANGPT1 (Fig. 4B) and between ANGPT2 and VEGF-D (Fig. 4C) in HFrEF patients. Fig. 4D shows a positive correlation between PLA2 activity and ANGPT2 in HFrEF patients. No correlation was observed between ANGPT1 and VEGF-D (Fig. 4E) and sPLA2 activity and VEGF-D in HFrEF patients (Fig. 4F). Moreover, no correlation was observed between ANGPT2 and VEGF-D plasma concentrations of HFpEF (Fig. 4G).
      Fig 4
      Fig. 4(A) Correlations between the plasma concentrations of ANGPT2 and ANGPT1 in HFrEF patients; (B) Correlation between circulating sPLA2 activity and the concentration of ANGPT1 in HFrEF patients; (C) Correlation between the plasma concentrations of VEGF-D and ANGPT2 in HFrEF patients; (D) Correlation between the sPLA2 activity and ANGPT2 in HFrEF patients. (E) Correlation between the plasma concentration of VEGF-D and ANGPT1 in HFrEF patients; (F) Correlation between circulating sPLA2 activity and VEGF-D in HFrEF patients; (D) Correlation between the plasma concentration of VEGF-D and ANGPT2 in HFpEF patients. Spearman's correlation coefficients (r) were calculated and are shown in the panels.

      4. Discussion

      ANGPT1, which maintains endothelial integrity [
      • Jeansson M.
      • Gawlik A.
      • Anderson G.
      • et al.
      Angiopoietin-1 is essential in mouse vasculature during development and in response to injury.
      ,
      • Thurston G.
      • Rudge J.S.
      • Ioffe E.
      • et al.
      Angiopoietin-1 protects the adult vasculature against plasma leakage.
      ] and exerts anti-inflammatory effects [
      • Gamble J.R.
      • Drew J.
      • Trezise L.
      • et al.
      Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions.
      ], was selectively downregulated in HFrEF patients compared to HFpEF and controls. By contrast, ANGPT2, involved in endothelial dysfunction [
      • Roviezzo F.
      • Tsigkos S.
      • Kotanidou A.
      • et al.
      Angiopoietin-2 causes inflammation in vivo by promoting vascular leakage.
      ,
      • Fiedler U.
      • Reiss Y.
      • Scharpfenecker M.
      • et al.
      Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.
      ,
      • Schmaier A.A.
      • Pajares Hurtado G.M.
      • Manickas-Hill Z.J.
      • et al.
      Tie2 activation protects against prothrombotic endothelial dysfunction in COVID-19.
      ], was upregulated in both HFrEF and HFpEF patients. Elevated circulating levels of ANGPT2 have been reported in acute coronary syndromes [
      • Lee K.W.
      • Lip G.Y.
      • Blann A.D.
      Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes.
      ,
      • Poss J.
      • Fuernau G.
      • Denks D.
      • et al.
      Angiopoietin-2 in acute myocardial infarction complicated by cardiogenic shock–a biomarker substudy of the IABP-SHOCK II-Trial.
      ], and it has been proposed as a negative prognostic marker after myocardial infarction [
      • Iribarren C.
      • Phelps B.H.
      • Darbinian J.A.
      • et al.
      Circulating angiopoietins-1 and -2, angiopoietin receptor Tie-2 and vascular endothelial growth factor-A as biomarkers of acute myocardial infarction: a prospective nested case-control study.
      ] and after percutaneous coronary intervention [
      • Jian W.
      • Li L.
      • Wei X.M.
      • et al.
      Prognostic value of angiopoietin-2 for patients with coronary heart disease after elective PCI.
      ,
      • Zeng Z.Y.
      • Gui C.
      • Li L.
      • et al.
      Effects of percutaneous coronary intervention on serum angiopoietin-2 in patients with coronary heart disease.
      ]. ANGPT2 is associated with a greater risk of cardiovascular mortality in the general population [
      • Lorbeer R.
      • Baumeister S.E.
      • Dorr M.
      • et al.
      Circulating angiopoietin-2, its soluble receptor Tie-2, and mortality in the general population.
      ], and with higher mortality in patients suffering from myocardial infarction and cardiogenic shock [
      • Poss J.
      • Fuernau G.
      • Denks D.
      • et al.
      Angiopoietin-2 in acute myocardial infarction complicated by cardiogenic shock–a biomarker substudy of the IABP-SHOCK II-Trial.
      ,
      • Link A.
      • Poss J.
      • Rbah R.
      • et al.
      Circulating angiopoietins and cardiovascular mortality in cardiogenic shock.
      ]. ANGPT2 is overexpressed in endothelial cells at the border of infarct area after ischemic injury in mice [
      • Lee S.J.
      • Lee C.K.
      • Kang S.
      • et al.
      Angiopoietin-2 exacerbates cardiac hypoxia and inflammation after myocardial infarction.
      ]. In the remodeling phase after myocardial infarction, endothelial- and macrophage-derived ANGPT2 promoted abnormal vascular remodeling and exacerbated inflammation. A recent study reported a significant correlation between serum concentrations of ANGPT2 and NT-proBNP in more than 200 patients who had undergone diagnostic cardiac catheterization, including patients with IHD, one of the main etiologies leading to HFrEF [
      • Jian W.
      • Mo C.H.
      • Yang G.L.
      • et al.
      Angiopoietin-2 provides no incremental predictive value for the presence of obstructive coronary artery disease over N-terminal pro-brain natriuretic peptide.
      ]. In addition, Chirinos and coworkers found that ANGPTs were associated with incident risk of all-cause death or HF-related hospital admission in HFpEF patients [
      • Chirinos J.A.
      • Orlenko A.
      • Zhao L.
      • et al.
      Multiple plasma biomarkers for risk stratification in patients with heart failure and preserved ejection fraction.
      ]. Our results extend the previous findings by showing that plasma levels of ANGPT2 are increased in both HFpEF and HFrEF patients. Interestingly, in our study the ANGPT2/ANGPT1 ratio, a prognostic biomarker of endothelial activation, [
      • Loffredo S.
      • Bova M.
      • Suffritti C.
      • et al.
      Elevated plasma levels of vascular permeability factors in C1 inhibitor-deficient hereditary angioedema.
      ], was increased in HFrEF but not in HFpEF patients. Future studies on larger patient cohorts will give the possibility to verify if the ANGPT2/ANGPT1 ratio is a selective biomarker of HFpEF.
      Our results may have clinical implications in patients suffering from HF. First, the evaluation of plasma concentrations of ANGPT1, ANGPT2 and their ratio may recognize different pathophysiological patterns underlying HFrEF and HFpEF. Second, the unique role of the ANGPTs/Tie2 signaling pathway in vascular stability suggests that it could serve as a target for therapeutic intervention in diseases whose pathophysiology comprises the alteration of vascular integrity [
      • Saharinen P.
      • Eklund L.
      • Alitalo K.
      Therapeutic targeting of the angiopoietin-TIE pathway.
      ,
      • Parmar D.
      • Apte M.
      Angiopoietin inhibitors: a review on targeting tumor angiogenesis.
      ], such as HF. Recently, it has been demonstrated that specific Angpt2 deletion or the use of an anti-ANGPT2 antibody markedly reduced cardiac hypoxia, proinflammatory macrophage polarization, adverse vascular remodeling and the consequent progression of HF after myocardial infarction in mice [
      • Lee S.J.
      • Lee C.K.
      • Kang S.
      • et al.
      Angiopoietin-2 exacerbates cardiac hypoxia and inflammation after myocardial infarction.
      ]. The latter results contribute to elucidate the roles of ANGPT2 in the pathogenesis of post-ischemic cardiovascular remodeling. These fascinating experimental results designate ANGPT2 as a promising therapeutic target to prevent/ameliorate HF. The latter findings might have translational relevance. Several anti-ANGPT2 strategies are now available and under development [
      • Parmar D.
      • Apte M.
      Angiopoietin inhibitors: a review on targeting tumor angiogenesis.
      ]. Moreover, ANGPT2 appears to be a potential therapeutic option in experimental HF [
      • Lee S.J.
      • Lee C.K.
      • Kang S.
      • et al.
      Angiopoietin-2 exacerbates cardiac hypoxia and inflammation after myocardial infarction.
      ]. Future studies should investigate the possibility that ANGPT2 antagonists could be effective in the treatment of both HFrEF and HFpEF patients.
      Plasma concentrations of both VEGF-A and VEGF-C in both HFrEF and HFpEF patients were similar to those in healthy controls. VEGF-A is a potent permeability factor [
      • Senger D.R.
      • Galli S.J.
      • Dvorak A.M.
      • et al.
      Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.
      ] and a major proangiogenic and proinflammatory mediator [
      • Sammarco G.
      • Varricchi G.
      • Ferraro V.
      • et al.
      Mast cells, angiogenesis and lymphangiogenesis in human gastric cancer.
      ,
      • Detoraki A.
      • Staiano R.I.
      • Granata F.
      • et al.
      Vascular endothelial growth factors synthesized by human lung mast cells exert angiogenic effects.
      ]. Several studies have found elevated circulating levels of VEGF-A in myocardial infarction [
      • Lee K.W.
      • Lip G.Y.
      • Blann A.D.
      Plasma angiopoietin-1, angiopoietin-2, angiopoietin receptor tie-2, and vascular endothelial growth factor levels in acute coronary syndromes.
      ,
      • Heeschen C.
      • Dimmeler S.
      • Hamm C.W.
      • et al.
      Prognostic significance of angiogenic growth factor serum levels in patients with acute coronary syndromes.
      ,
      • Hojo Y.
      • Ikeda U.
      • Zhu Y.
      • et al.
      Expression of vascular endothelial growth factor in patients with acute myocardial infarction.
      ,
      • Kawamoto A.
      • Kawata H.
      • Akai Y.
      • et al.
      Serum levels of VEGF and basic FGF in the subacute phase of myocardial infarction.
      ,
      • Kranz A.
      • Rau C.
      • Kochs M.
      • et al.
      Elevation of vascular endothelial growth factor-A serum levels following acute myocardial infarction. Evidence for its origin and functional significance.
      ]. Differently from acute vascular injuries, plasma levels of VEGF-A are not altered in the overall HF population and in both HFrEF and HFpEF patients. Thus, it is possible to speculate on the different roles exerted by this mediator in acute versus chronic settings of myocardial injury. Although VEGF-A is released by several circulating immune cells, such as neutrophils [
      • Loffredo S.
      • Borriello F.
      • Iannone R.
      • et al.
      Group V secreted phospholipase A2 induces the release of proangiogenic and antiangiogenic factors by human neutrophils.
      ,
      • Braile M.
      • Cristinziano L.
      • Marcella S.
      • et al.
      LPS-mediated neutrophil VEGF-A release is modulated by cannabinoid receptor activation.
      ], basophils [
      • de Paulis A.
      • Prevete N.
      • Fiorentino I.
      • et al.
      Expression and functions of the vascular endothelial growth factors and their receptors in human basophils.
      ,
      • Marone G.
      • Borriello F.
      • Varricchi G.
      • et al.
      Basophils: historical reflections and perspectives.
      ], and eosinophils [
      • Loffredo S.
      • Borriello F.
      • Iannone R.
      • et al.
      Group V secreted phospholipase A2 induces the release of proangiogenic and antiangiogenic factors by human neutrophils.
      ], plasma concentrations of VEGF-A were not increased in both HFrEF and HFpEF patients compared to healthy individuals. These findings do not exclude the possibility that these immune cells participate in the pathogenesis of HFrEF and HFpEF by releasing other proinflammatory mediators.
      Although circulating levels of VEGF-C were similar in patients with HFrEF and HFpEF and controls, the concentrations of VEGF-D were increased in both groups of patients compared to controls. The differential alterations of VEGF-C and VEGF-D in these patients are intriguing but not surprising. In fact, recent evidence demonstrates that VEGF-C and VEGF-D differently modulate the immune system [
      • Fankhauser M.
      • Broggi M.A.S.
      • Potin L.
      • et al.
      Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma.
      ].
      VEGF-C and -D are mainly produced by tissue-resident macrophages [
      • Granata F.
      • Staiano R.I.
      • Loffredo S.
      • et al.
      The role of mast cell-derived secreted phospholipases A2 in respiratory allergy.
      ,
      • Staiano R.I.
      • Loffredo S.
      • Borriello F.
      • et al.
      Human lung-resident macrophages express CB1 and CB2 receptors whose activation inhibits the release of angiogenic and lymphangiogenic factors.
      ,
      • Braile M.
      • Fiorelli A.
      • Sorriento D.
      • et al.
      Human lung-resident macrophages express and are targets of thymic stromal lymphopoietin in the tumor microenvironment.
      ] and mast cells [
      • Varricchi G.
      • Loffredo S.
      • Borriello F.
      • et al.
      Superantigenic activation of human cardiac mast cells.
      ,
      • Detoraki A.
      • Staiano R.I.
      • Granata F.
      • et al.
      Vascular endothelial growth factors synthesized by human lung mast cells exert angiogenic effects.
      ,
      • Varricchi G.
      • Rossi F.W.
      • Galdiero M.R.
      • et al.
      Physiological roles of mast cells: collegium internationale allergologicum update 2019.
      ,
      • Cristinziano L.
      • Poto R.
      • Criscuolo G.
      • et al.
      IL-33 and superantigenic activation of human lung mast cells induce the release of angiogenic and lymphangiogenic factors.
      ,
      • Marcella S.
      • Petraroli A.
      • Braile M.
      • et al.
      Vascular endothelial growth factors and angiopoietins as new players in mastocytosis.
      ]. In a mouse model of HF, VEGF-C and VEGF-D were upregulated in the early stages of the disease, with levels returning afterward to baseline [
      • Huusko J.
      • Lottonen L.
      • Merentie M.
      • et al.
      AAV9-mediated VEGF-B gene transfer improves systolic function in progressive left ventricular hypertrophy.
      ]. VEGF-C levels are increased in patients with ischemic or non-ischemic cardiomyopathy [
      • Abraham D.
      • Hofbauer R.
      • Schafer R.
      • et al.
      Selective downregulation of VEGF-A(165), VEGF-R(1), and decreased capillary density in patients with dilative but not ischemic cardiomyopathy.
      ] and in an animal model of ischemic cardiomyopathy [
      • Park J.H.
      • Yoon J.Y.
      • Ko S.M.
      • et al.
      Endothelial progenitor cell transplantation decreases lymphangiogenesis and adverse myocardial remodeling in a mouse model of acute myocardial infarction.
      ] and in human atherosclerotic lesions [
      • Rutanen J.
      • Leppanen P.
      • Tuomisto T.T.
      • et al.
      Vascular endothelial growth factor-D expression in human atherosclerotic lesions.
      ]. Recent evidence indicates that lymphangiogenesis [
      • Henri O.
      • Pouehe C.
      • Houssari M.
      • et al.
      Selective stimulation of cardiac lymphangiogenesis reduces myocardial edema and fibrosis leading to improved cardiac function following myocardial infarction.
      ] and VEGF-C improve cardiac functions after experimental myocardial infarction [
      • Klotz L.
      • Norman S.
      • Vieira J.M.
      • et al.
      Cardiac lymphatics are heterogeneous in origin and respond to injury.
      ]. Another study has suggested a cardioprotective role for macrophage-derived VEGF-C during acute myocardial infarction [
      • Glinton K.E.
      • Ma W.
      • Lantz C.
      • et al.
      Macrophage-produced VEGFC is induced by efferocytosis to ameliorate cardiac injury and inflammation.
      ]. VEGF-C is released from activated human cardiac mast cells [
      • Varricchi G.
      • Loffredo S.
      • Borriello F.
      • et al.
      Superantigenic activation of human cardiac mast cells.
      ], and proinflammatory and profibrotic mediators released from mast cells [
      • Guimbal S.
      • Cornuault L.
      • Rouault P.
      • et al.
      Mast cells are the trigger of small vessel disease and diastolic dysfunction in diabetic obese mice.
      ] and macrophages [
      • Shen J.L.
      • Xie X.J.
      Insight into the pro-inflammatory and profibrotic role of macrophage in heart failure with preserved ejection fraction.
      ,
      • Hulsmans M.
      • Sager H.B.
      • Roh J.D.
      • et al.
      Cardiac macrophages promote diastolic dysfunction.
      ] have recently been implicated in cardiac microvessel disease and diastolic dysfunction in models of HFpEF. By contrast, the circulating levels of VEGF-D, which does not possess cardioprotective effects, were increased in both HFrEF and HFpEF patients compared to controls. Further studies are necessary to understand the role of cardiac mast cells and macrophages as possible critical components in HFpEF pathogenesis.
      PLA2 modulates endothelial cell migration and vascular permeability in vitro and in humans [
      • Loffredo S.
      • Ferrara A.L.
      • Bova M.
      • et al.
      Secreted phospholipases A2 in hereditary angioedema with C1-inhibitor deficiency.
      ,
      • Rizzo M.T.
      • Nguyen E.
      • Aldo-Benson M.
      • et al.
      Secreted phospholipase A(2) induces vascular endothelial cell migration.
      ,
      • Loffredo S.
      • Marone G.
      Hereditary angioedema: the plasma contact system out of control: comment.
      ,
      • David S.
      • Kumpers P.
      • Hellpap J.
      • et al.
      Angiopoietin 2 and cardiovascular disease in dialysis and kidney transplantation.
      ,
      • Lambeau G.
      • Gelb M.H.
      Biochemistry and physiology of mammalian secreted phospholipases A2.
      ]. Circulating levels of sPLA2 predict coronary events in patients with coronary artery disease [
      • Kugiyama K.
      • Ota Y.
      • Takazoe K.
      • et al.
      Circulating levels of secretory type II phospholipase A(2) predict coronary events in patients with coronary artery disease.
      ] and even in apparently healthy men and women [
      • Boekholdt S.M.
      • Keller T.T.
      • Wareham N.J.
      • et al.
      Serum levels of type II secretory phospholipase A2 and the risk of future coronary artery disease in apparently healthy men and women: the EPIC-Norfolk Prospective Population Study.
      ]. Moreover, serum sPLA2 levels also predict readmission for HF after myocardial infarction [
      • Xin H.
      • Chen Z.Y.
      • Lv X.B.
      • et al.
      Serum secretory phospholipase A2-IIa (sPLA2-IIA) levels in patients surviving acute myocardial infarction.
      ]. Finally, elevated levels of sPLA2 were associated with the risk of early atherosclerosis [
      • Sun C.Q.
      • Zhong C.Y.
      • Sun W.W.
      • et al.
      Elevated Type II secretory phospholipase A2 increases the risk of early atherosclerosis in patients with newly diagnosed metabolic syndrome.
      ]. Our study is, to our knowledge, the first to demonstrate that high plasma concentrations of PLA2 activity can be found in HFrEF patients only. These intriguing results designate sPLA2 as a promising circulating biomarker to differentiate HFrEF from HFpEF. Several immune cells produce sPLA2 [
      • Hallstrand T.S.
      • Lai Y.
      • Hooper K.A.
      • et al.
      Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.
      ,
      • Murakami M.
      • Yamamoto K.
      • Miki Y.
      • et al.
      The Roles of the secreted phospholipase A2 gene family in immunology.
      ,
      • Triggiani M.
      • Giannattasio G.
      • Calabrese C.
      • et al.
      Lung mast cells are a source of secreted phospholipases A2.
      ], which modulates vascular permeability [
      • Rizzo M.T.
      • Nguyen E.
      • Aldo-Benson M.
      • et al.
      Secreted phospholipase A(2) induces vascular endothelial cell migration.
      ] and activates several human inflammatory cells [
      • Loffredo S.
      • Ferrara A.L.
      • Bova M.
      • et al.
      Secreted phospholipases A2 in hereditary angioedema with C1-inhibitor deficiency.
      ,
      • Loffredo S.
      • Marone G.
      Hereditary angioedema: the plasma contact system out of control: comment.
      ,
      • Lambeau G.
      • Gelb M.H.
      Biochemistry and physiology of mammalian secreted phospholipases A2.
      ]. Overexpression of this vasoactive and proinflammatory factor seems to be a rather specific biomarker of HFrEF. Future studies from larger cohorts will evaluate the role of sPLA2 assay in the differential diagnosis of HFrEF vs. HFpEF.
      It is widely recognized that coronary heart disease represents the predominant cause of HFrEF [
      • Sacks D.
      • Baxter B.
      • Campbell B.C.V.
      • et al.
      Multisociety consensus quality improvement revised consensus statement for endovascular therapy of acute ischemic stroke.
      ]. HFpEF pathophysiology is more heterogeneous [
      • Hahn V.S.
      • Knutsdottir H.
      • Luo X.
      • et al.
      Myocardial gene expression signatures in human heart failure with preserved ejection fraction.
      ,
      • Hedman A.K.
      • Hage C.
      • Sharma A.
      • et al.
      Identification of novel pheno-groups in heart failure with preserved ejection fraction using machine learning.
      ,
      • Cogliati C.
      • Ceriani E.
      • Gambassi G.
      • et al.
      Phenotyping congestion in patients with acutely decompensated heart failure with preserved and reduced ejection fraction: the Decongestion duRing therapY for acute decOmpensated heart failure in HFpEF vs HFrEF- DRY-OFF study.
      ], due to several etiologic factors, frequently concurrent [
      • Sharma K.
      • Kass D.A.
      Heart failure with preserved ejection fraction: mechanisms, clinical features, and therapies.
      ,
      • Mishra S.
      • Kass D.A.
      Cellular and molecular pathobiology of heart failure with preserved ejection fraction.
      ,
      • Pfeffer M.A.
      • Shah A.M.
      • Borlaug B.A.
      Heart failure with preserved ejection fraction in perspective.
      ]. Advanced age, female sex and comorbidities, such as obesity [
      • Lam C.S.
      • Donal E.
      • Kraigher-Krainer E.
      • et al.
      Epidemiology and clinical course of heart failure with preserved ejection fraction.
      ,
      • Borlaug B.A.
      • Anstrom K.J.
      • Lewis G.D.
      • et al.
      Effect of inorganic nitrite vs placebo on exercise capacity among patients with heart failure with preserved ejection fraction: The INDIE-HFpEF randomized clinical trial.
      ], diabetes mellitus [
      • Lam C.S.
      • Donal E.
      • Kraigher-Krainer E.
      • et al.
      Epidemiology and clinical course of heart failure with preserved ejection fraction.
      ], hypertension [
      • Ho J.E.
      • Enserro D.
      • Brouwers F.P.
      • et al.
      Predicting heart failure with preserved and reduced ejection fraction: the international collaboration on heart failure subtypes.
      ], and atrial fibrillation [
      • Mishra S.
      • Kass D.A.
      Cellular and molecular pathobiology of heart failure with preserved ejection fraction.
      ] are common among patients with HFpEF. Although none of these factors discriminate patients with HFpEF from HFrEF, the presence of these comorbid conditions is believed to ultimately lead to low-grade inflammation [
      • Sharma K.
      • Kass D.A.
      Heart failure with preserved ejection fraction: mechanisms, clinical features, and therapies.
      ,
      • Mishra S.
      • Kass D.A.
      Cellular and molecular pathobiology of heart failure with preserved ejection fraction.
      ,
      • Paulus W.J.
      • Tschope C.
      A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation.
      ]. Systemic inflammation could lead to endothelial dysfunction supported by higher expression of vascular cell adhesion molecules such as VCAM-1, E-selectin, and ROS [
      • Mishra S.
      • Kass D.A.
      Cellular and molecular pathobiology of heart failure with preserved ejection fraction.
      ,
      • Westermann D.
      • Lindner D.
      • Kasner M.
      • et al.
      Cardiac inflammation contributes to changes in the extracellular matrix in patients with heart failure and normal ejection fraction.
      ,
      • Sanders-van Wijk S.
      • Tromp J.
      • Beussink-Nelson L.
      • et al.
      Proteomic evaluation of the comorbidity-inflammation paradigm in heart failure with preserved ejection fraction: results from the PROMIS-HFpEF study.
      ]. An increasingly popular theory about HFpEF is that the syndrome reflects systemic and/or myocardial inflammation [
      • Mishra S.
      • Kass D.A.
      Cellular and molecular pathobiology of heart failure with preserved ejection fraction.
      ,
      • Gallet R.
      • de Couto G.
      • Simsolo E.
      • et al.
      Cardiosphere-derived cells reverse heart failure with preserved ejection fraction (HFpEF) in rats by decreasing fibrosis and inflammation.
      ,
      • Suetomi T.
      • Willeford A.
      • Brand C.S.
      • et al.
      Inflammation and NLRP3 inflammasome activation initiated in response to pressure overload by Ca(2+)/Calmodulin-dependent protein kinase II delta signaling in cardiomyocytes are essential for adverse cardiac remodeling.
      ,
      • Voors A.A.
      • Kremer D.
      • Geven C.
      • et al.
      Adrenomedullin in heart failure: pathophysiology and therapeutic application.
      ]. The greatest evidence for inflammatory conditions is HFpEF comes from analyses of circulating biomarkers (e.g., IL-1β, IL-6, IL-10, IL-11, TNF-α, etc.) in patients [
      • Chirinos J.A.
      • Orlenko A.
      • Zhao L.
      • et al.
      Multiple plasma biomarkers for risk stratification in patients with heart failure and preserved ejection fraction.
      ,
      • Guimbal S.
      • Cornuault L.
      • Rouault P.
      • et al.
      Mast cells are the trigger of small vessel disease and diastolic dysfunction in diabetic obese mice.
      ,
      • Corden B.
      • Adami E.
      • Sweeney M.
      • et al.
      IL-11 in cardiac and renal fibrosis: Late to the party but a central player.
      ] as well as in mouse models of HFpEF [
      • Schiattarella G.G.
      • Altamirano F.
      • Tong D.
      • et al.
      Nitrosative stress drives heart failure with preserved ejection fraction.
      ]. Our results identify different patterns of expression of several vascular permeability and inflammatory mediators in the clinical setting of HFpEF and HFrEF. It is tempting to speculate that these different patterns of inflammatory and vasoactive mediators reflect pathophysiological differences between HFpEF and HFrEF.

      5. Limitations

      This study has several limitations that should be pointed out. The limited number of subjects enrolled represents the main limitation of the present investigation. However, it is important to point out that in order to identify specific differences between HFrEF and HFpEF, the study protocol included stringent exclusion criteria to reduce potential interference with the inflammatory and angiogenic patterns explored in the study. Indeed, prevalent comorbidities such as COPD, DM, immune disorders, malignancies, and severe obesity were excluded from the study, greatly limiting the number of patients that were included in the analysis. In fact, despite the two examined cohorts were rather small, the patients examined in our study presented clinical characteristics consistent with the main typical features of individuals suffering from the different phenotypes of HF, being mean age and prevalence of female higher in HFpEF participants, while HFrEF individuals suffered from more IHD, impaired renal function and showed higher BNP levels [
      • Marra A.M.
      • Bencivenga L.
      • D'Assante R.
      • et al.
      Heart failure with preserved ejection fraction: Squaring the circle between comorbidities and cardiovascular abnormalities.
      ]. Similarly, the selection of healthy controls was burdened by the exclusion criteria, limiting the possibility to match them with HF participants according to demographic and antropometric features.
      The results of this preliminary study will have to be extended in future multicenter trials examining larger cohorts of HFrEF and HFpEF patients. In addition, it will be important to evaluate circulating biomarkers of angiogenesis/lymphangiogenesis during stable and unstable clinical conditions of HFpEF and HFrEF patients. Moreover, HFpEF and HFrEF patients were treated with several drugs (i.e., statins, diuretics, ACEIs, ARB, and beta-blockers), and we cannot exclude the possibility that some of them may have directly or indirectly affected some of our results. The results of our cross-sectional study do not allow to establish a cause-effect relationship between alterations of angiogenic/lymphangiogenic factors and HFpEF or HFrEF. Finally, this study was performed on a Caucasian population and future studies should be extended to other ethnic groups.

      6. Concluding remarks

      To the best of our knowledge, this is the first study reporting significant and distinct alterations of plasma concentrations of three different classes of proinflammatory mediators essential for vascular development, integrity and remodeling (i.e., angiopoietins, VEGFs, and secretory phospholipase A2) in patients with HFrEF or HFpEF compared to controls. These findings could pave the way for the identification of new inflammatory biomarkers underlying different forms of HF pathophysiology and novel therapeutic targets.

      Ethical Statement

      This study was approved by the Ethics Committee of the University of Naples Federico II (protocol number 124/17). All participants were carefully informed and signed a written consent to participate in the study. All the procedures involved in this study were in accordance with the ethical standards of the Helsinki Declaration and its later amendments.

      Funding

      This work was supported in part by grants from the CISI-Lab Project (University of Naples Federico II), TIMING Project (Regione Campania), and Campania Bioscience.

      CRediT authorship contribution statement

      Gilda Varricchi: Conceptualization, Resources, Supervision, Formal analysis, Writing – review & editing. Remo Poto: Conceptualization, Supervision, Formal analysis, Visualization, Methodology, Writing – review & editing. Anne Lise Ferrara: Supervision, Formal analysis, Visualization, Methodology, Writing – review & editing. Giuseppina Gambino: Supervision, Formal analysis, Visualization, Methodology, Writing – review & editing. Gianni Marone: Project administration, Resources, Supervision, Writing – review & editing. Giuseppe Rengo: Conceptualization, Supervision, Formal analysis, Visualization, Methodology, Writing – review & editing. Stefania Loffredo: Project administration, Supervision, Formal analysis, Methodology, Writing – review & editing. Leonardo Bencivenga: Project administration, Supervision, Formal analysis, Visualization, Methodology, Writing – review & editing.

      Declaration of Competing Interest

      All authors declare they have no conflict of interest.

      Acknowledgments

      Dr. Leonardo Bencivenga has been supported by a research grant provided by the CardioPaTh PhD program, the research grant provided by the FDIME and the STAR PLUS Research Grant provided by the University of Naples Federico II and Compagnia San Paolo. The authors thank Dr. Gjada Criscuolo for her excellent managerial assistance in preparing this manuscript and the administrative staff (Dr. Roberto Bifulco, Dr. Anna Ferraro and Dr. Maria Cristina Fucci), without whom it would not be possible to work as a team.

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