TYPE Original Research PUBLISHED 03 November 2022 DOI 10.3389/fcvm.2022.939625 New factors in heart failure OPEN ACCESS pathophysiology: Immunity cells EDITED BY Kenneth Scott Campbell, University of Kentucky, United States release of extracellular vesicles REVIEWED BY Raquel Guillamat-Prats, Alba Vilella-Figuerola1,2 , Teresa Padró1,3 , Eulàlia Roig4 , LMU Munich University Hospital, Germany Sònia Mirabet3,4† and Lina Badimon1,3,5*† Diego Vinicius Santinelli Pestana, 1 Cardiovascular-Program ICCC, IR-Hospital Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain, Brigham and Women’s Hospital 2 Department of Biochemical and Molecular Biology, Universitat Autònoma de Barcelona, and Harvard Medical School, Barcelona, Spain, 3 Centro de Investigación Biomédica en Red Cardiovascular, Instituto de Salud United States Carlos III, Madrid, Spain, 4 Heart Failure Group, Department of Cardiology, Hospital Santa Creu i Sant Takehiro Funamizu, Pau, Barcelona, Spain, 5 UAB-Chair Cardiovascular Research, Barcelona, Spain Juntendo University, Japan *CORRESPONDENCE Lina Badimon lbadimon@santpau.cat † These Leukocyte-shed extracellular vesicles (EVs) can play effector roles in the authors have contributed equally to this work pathophysiological mechanisms of different diseases. These EVs released by SPECIALTY SECTION membrane budding of leukocytes have been found in high amounts locally This article was submitted to in inflamed tissues and in the circulation, indicating immunity cell activation. Heart Failure and Transplantation, a section of the journal These EVs secreted by immune cell subsets have been minimally explored Frontiers in Cardiovascular Medicine and deserve further investigation in many areas of disease. In this study we RECEIVED 09 May 2022 have investigated whether in heart failure there is innate and adaptive immune ACCEPTED 12 September 2022 cell release of EVs. Patients with chronic heart failure (cHF) (n = 119) and PUBLISHED 03 November 2022 in sex- and age-matched controls without this chronic condition (n = 60). CITATION Vilella-Figuerola A, Padró T, Roig E, Specifically, EVs were quantified and phenotypically characterized by flow Mirabet S and Badimon L (2022) New cytometry and cell-specific monoclonal antibodies. We observed that even factors in heart failure pathophysiology: Immunity cells in well medically controlled cHF patients (with guideline-directed medical release of extracellular vesicles. therapy) there are higher number of blood annexin-V+ (phosphatidylserine+ )- Front. Cardiovasc. Med. 9:939625. doi: 10.3389/fcvm.2022.939625 EVs carrying activated immunity cell-epitopes in the circulation than in controls (p < 0.04 for all cell types). Particularly, EVs shed by monocytes and COPYRIGHT © 2022 Vilella-Figuerola, Padró, Roig, neutrophils (innate immunity) and by T-lymphocytes and natural-killer cells Mirabet and Badimon. This is an (adaptive immunity) are significantly higher in cHF patients. Additionally, EVs- open-access article distributed under the terms of the Creative Commons shed by activated leukocytes/neutrophils (CD11b+ , p = 0.006; CD29+ /CD15+ , Attribution License (CC BY). The use, p = 0.048), and T-lymphocytes (CD3+ /CD45+ , p < 0.02) were positively distribution or reproduction in other forums is permitted, provided the correlated with cHF disease severity (NYHA classification). Interestingly, cHF original author(s) and the copyright patients with ischemic etiology had the highest levels of EVs shed by owner(s) are credited and that the original publication in this journal is lymphocytes and neutrophils (p < 0.045, all). In summary, in cHF patients cited, in accordance with accepted there is a significant immune cell activation shown by high-release of EVs that academic practice. No use, distribution is accentuated by clinical severity of cHF. These activated innate and adaptive or reproduction is permitted which does not comply with these terms. immunity cell messengers may contribute by intercellular communication to the progression of the disease and to the common affectation of distant organs in heart failure (paracrine regulation) that contribute to the clinical deterioration of cHF patients. KEYWORDS extracellular microvesicles (EVs), chronic heart failure, immune cells, inflammation, liquid biopsies, extracellular vesicle (EV) Frontiers in Cardiovascular Medicine 01 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 Introduction phosphatidylserine-rich phospholipid bilayer that exposes transmembrane proteins and receptors from their parental Leukocyte-shed extracellular vesicles (EVs) can play effector cells, EVs are released after physiological stimuli such as cell roles in the pathophysiological mechanisms of different diseases. activation, damage, stress, apoptotic or necrotic damage (14, These EVs released by membrane budding of leukocytes have 15). EVs play a role in physiology and pathophysiology since been found in high amounts locally in inflamed tissues and in they are involved in various processes such as cell adhesion, the circulation, indicating immunity cell activation. These EVs communication (they carry miRNA, proteins and lipids), secreted by immune cell subsets have been minimally explored apoptosis, immune response, vascular function, remodeling, and deserve further investigation in many areas of disease (1). hemostasis or thrombosis (16). Their cargo reflects their Heart failure (HF) is a complex syndrome that accounts for parental cell condition and metabolic state and its numbers a high proportion of cardiovascular death worldwide (2, 3). have been correlated with disease progression and severity It is characterized by an impairment of the cardiac function (14, 16, 17). In cardiovascular disease, they have been related due to genetic and environmental insults and comorbidities to several pathologies (18) including atherosclerosis (19–21), such as ischemic disease, that induce significant alterations coronary artery disease (14), ischemia and myocardial infarction of heart structure (left ventricle hypertrophy or dilation, (22–24) and stroke (25). fibrosis, maladaptive remodeling) and function (decreased To gain more insight in the pathophysiology of chronic cardiac output, increased end-diastolic pressure and diastolic HF (cHF), we aimed to investigate whether immune cells or systolic dysfunction) (4). HF can be classified in HF were activated in patients, even though treated according with preserved ejection fraction (HFpEF), HF with reduced to guideline-directed medical therapy. Several studies ejection fraction (HFrEF) and in HF with mildly reduced have investigated endothelial-derived EVs in relation ejection fraction (HFmrEF), according to their echographic with cardiovascular risk factors and cardiovascular disease values of left ventricular ejection fraction (LVEF) (2, 5). pathophysiology (26–29). However, to our knowledge, there Although these groups present common symptomatology, their are no studies investigating the type of immune cells that are pathophysiological mechanisms and therapeutic approaches are activated and shed EVs in cHF. slightly different. In that sense, although this is not universal, the In this study we have investigated whether in chronic heart vast majority of HFrEF have underlying ischemic etiology, while failure there is innate and adaptive immune cell release of EVs HFpEF patients tend to develop HF due to hypertensive or heart and whether specific circulating EV patterns associate with the valve disease (6, 7). underlying cHF etiology (ischemic and non-ischemic) or the The systemic inflammation of these patients represents a disease severity in cHF patients. chronic and non-resolving health impairment with the presence of elevated levels of circulating cytokines or pro-inflammatory mediators, such as TNFα, IL-β1, IL-6, or CRP (4, 8). Further Materials and methods to the raised levels of inflammatory biomarkers, another source of organ damage is the defective equilibrium between Clinical study population pro-inflammatory and anti-inflammatory immune-cell subsets, such as monocytes/macrophages, neutrophils, lymphocytes (B, A total of 119 ambulatory patients with clinical diagnosis Treg, Th), that often results in impaired repair processes (9– of cHF and under guideline-directed medical therapy were 11). Several clinical trials have tried to target the on-going prospectively recruited in the outpatient HF unit of the Hospital inflammation with neutral results (12, 13). The failure of these de la Santa Creu i Sant Pau (Barcelona, Spain) between approaches suggests that the potentially involved pathways September 2016 and July 2018 and were followed-up until are not well understood, and that further research in the October 2019. During follow-up patients were treated according pathophysiology of the disease is needed. Indeed, HF is a to international guidelines and at clinician discretion in the spectrum of overlapping phenotypes that need to be more outpatient unit of our hospital. A control group was also accurately classified. included, comprising 60 sex- and age-matched controls without Circulating extracellular microvesicles (EVs) are a cHF. Baseline demographic data, classical cardiovascular risk heterogeneous population of extracellular vesicles with factors and background medication of the study population a size range between 20 and 1,000 nm. Composed of a (cHF and control groups) are shown in Table 1. Exclusion criteria were: HF with mildly reduced ejection fraction (40–50% of left ventricular ejection fraction [LVEF]), past Abbreviations: AV, annexin V; cHF, chronic heart failure; EVs, extracellular microvesicles; HF-Isch, heart failure with an ischemic underlying history of cancer, inflammatory disorders, sepsis or infection. etiology; HF-Nisch, heart failure with a non-ischemic underlying Pregnant women were also excluded. Characteristics of the etiology; HFpEF, heart failure with preserved ejection fraction; HFrEF, heart failure with reduced ejection fraction; LVEF, left ventricle ejection cHF patients including biochemical and hematological data, fraction; NYHA, New York Heart Association. cHF-etiology and cHF-severity (according to the New York Frontiers in Cardiovascular Medicine 02 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 TABLE 1 Patients and controls baseline characteristics. Controls cHF P-value n = 60 n = 119 controls-cHF Demographic characteristics; mean ± SD Male/Female, n 34/26 81/38 0.133 Age, years 67.1 ± 7.1 67 ± 11.8 0.553 Systolic blood pressure, mmHg 144.8 ± 20.2 120.4 ± 19.1 0.000 Diastolic blood pressure, mmHg 83.7 ± 11.3 73.9 ± 11.1 0.000 Left ventricular ejection fraction,% 52–74† 45.59 ± 18.98 – Risk factors; n (%) Smokers 10 (16) 13 (10.9) 0.278 Hypertension 33 (55) 82 (68.9) 0.067 Pulmonary hypertension – 49 (41.1) – Diabetes mellitus 10 (16) 53 (44.5) 0.000 Dyslipidaemia 45 (75) 64 (53.7) 0.004 Chronic kidney disease 2 (3.3) 46 (38.6) 0.000 Atrial fibrillation – 50 (42) – Background medication; n (%) Angiotensin-converting-enzyme inhibitors 19 (31.6) 48 (40.3) 0.333 Angiotensin II receptor blockers 9 (15) 35 (29.4) 0.054 Beta-blockers 2 (3.3) 100 (84) 0.000 Aldosterone antagonists – 66 (55.4) – Diuretics‡ 6 (10) 104 (87.3) 0.000 Angiotensin receptor neprilysin inhibitors – 17 (14.2) – Ivabradine – 14 (11.7) – Statins 37 (61.6) 77 (64.7) 0.690 Insulin 3 (5) 16 (13.4) 0.083 Anti-diabetic drugs 8 (13.3) 40 (33.6) 0.004 Antiplatelet agents 12 (20) 46 (38.6) 0.012 Anticoagulants 2 (3.3) 61 (51.2) 0.000 Anti-arrhythmic drugs – 26 (21.8) – † LVEF normal range (excerpted from https://www.ncbi.nlm.nih.gov/books/NBK459131/ on 18/01/2021). ‡ Includes: furosemide, hydrochlorothiazide, torasemide and indapamide. SD, standard deviation; cHF, chronic heart failure. The statistically significant p-values are in bold. Heart Association Classification [NYHA]) are given in Table 2 common trunk or left anterior descending artery, as well as and Supplementary Tables 1, 2. NYHA classification system when stenosis was equal or greater than 75% in two or more of considers cardiac functionality and disease severity, dividing the principal coronary arteries. cHF in four categories (5). Briefly, NYHA I patients do not The ethics committee at the Hospital de la Santa Creu i present limitations in their physical activity and daily activity Sant Pau in Barcelona (Spain) approved the study (Ref 16/44) does not cause breathlessness, fatigue or palpitations. NYHA and it was conducted under the principles of the Declaration II patients have a slight limitation of physical activity, and of Helsinki. A written informed consent was obtained from all whereas they are comfortable at rest, ordinary activity produces participants prior recruitment. breathlessness, fatigue or palpitations. Patients in NYHA III present a marked limitation of physical activity. Although they are comfortable at rest, less than ordinary physical activity Blood sampling, circulating results in breathlessness, fatigue or palpitations. Finally, NYHA extracellular vesicles isolation and IV patients are unable to carry on any physical activity without characterization discomfort and can have symptoms at rest. In patients with ischemic etiology, ischemia was defined as history of a previous Venous blood was withdrawn from the cubital vein without myocardial infarction, or in the absence of an acute ischemic tourniquet using a 20-gauge needle after 10–14 h of fasting into event, if there was stenosis equal or greater than 75% in the 3.8% sodium citrate tubes (BD Vacutainer, Becton Dickinson). Frontiers in Cardiovascular Medicine 03 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 TABLE 2 Clinical characteristics of cHF patients at baseline (n = 119). Eppendorf) to avoid in vitro platelet activation. Platelet-poor Patients characteristics plasma (PPP) was carefully aspirated, leaving about a 1 mm undisturbed layer on top of cells. A second centrifugation Clinical history; n(%) cHF etiology step was then performed at 1,500 g for 10 min at 20◦ C Ischemic 43 (36.1) (Eppendorf 5415R centrifuge, FA45-24-11 rotor, Eppendorf) to Non-ischemic 76 (63.9) ensure the complete removal of cells and obtain the platelet- Hypertensive cardiomyopathy 19 (15.9) free plasma (PFP). PFP aliquots were stored at −80◦ C until flow Dilated cardiomyopathy 24 (20.1) cytometry studies. Hypertrophic cardiomyopathy 12 (10) The EVs fraction was isolated from PFP by a two- Heart valve disease 17 (14.2) step high-speed centrifugation, according to the procedure Other 4 (3.3) previously described (20, 24, 30). Specifically, PFP was thawed New York Heart Association cHF stage and centrifuged at 1,500 g for 10 min at 20◦ C (Eppendorf NYHA I 0 (0) 5417R centrifuge, FA45-24-11 rotor, Eppendorf). Next, 250 µl NYHA II 52 (43.7) of PFP were collected from the upper part of the vial NYHA III 66 (55.5) and transferred to a new tube to pellet the EVs, and then NYHA IV 1 (0.8) centrifuged at 20,000 g for 30 min at 20◦ C (Eppendorf 5417R Hospitalizations in the 6 months prior study initiation 49 (41.1) centrifuge, FA45-24-11 rotor, Eppendorf). The supernatant Re-events in the year prior study initiation 9 (7.6) (225 µl) was discarded, and the EVs-enriched pellet was Percutaneous coronary intervention 4 (44.5) washed with 225 µl of citrate-phosphate buffered saline (PBS) Coronary artery bypass grafting 2 (22.2) solution (citrate-PBS; 1.4 mmol/L phosphate, 154 mmol/L NaCl, Medical treatment 3 (33.3) 10.9 mmol/L trisodium citrate, pH 7.4) before a second equal Biochemistry; mean ± SD centrifugation (20,000 g, 30 min, 20◦ C) was pursued. Finally, Hemoglobin, mg/dl 129.8 ± 18.5 the remaining pellets were resuspended in a final volume of Creatinine, mg/dl 1.3 ± 0.54 100 µl citrate-PBS. C-Reactive Protein, mg/ml 7.63 ± 11 NT-proBNP, pg/ml 2954.8 ± 4211.3 High-sensitive troponin T, ng/l 27.7 ± 20.9 Flow cytometric analysis of Erythrocytes, 106 /mm3 3.98 ± 0.74 extracellular vesicles Platelets, 103 /mm3 173.2 ± 56.89 Leukocytes, mm3 7426.1 ± 1950 Extracellular vesicles were phenotypically characterized by Neutrophils, 109 /L 4.74 ± 1.54 three-label flow cytometric analysis, performed as described Monocytes, 109 /L 0.75 ± 0.54 previously (20, 24, 30). Washed EVs suspensions were diluted Major outcomes during follow-up; n (%) in PBS containing 2.5 mmol of CaCl2 (Annexin binding buffer Cardiovascular event† 26 (21.8) [ABB], BD Biosciences, San Jose). Afterward, combinations Stroke 8 (6.7) of CFBlue-conjugated annexin V (AV) (Immunostep, Aortic dissection 1 (0.8) Salamanca, Spain) to detect phosphatidylserine, and two AMI + Cardiogenic shock 3 (2.5) specific monoclonal antibodies (mAb) (Supplementary HTx/HTx waiting list 10 (8.4)/1 (0.8) Table 3) labeled with fluorescein isothiocyanate (FITC) and CV death 7 (5.8) phycoerythrin (PE), or the isotype-matched control mAb, were Emergency hospital admission for cHF 16 (13.4) added. Samples were incubated for 20 min at 20◦ C in the dark Rehospitalisation for cHF 57 (47.8) and diluted with ABB before being immediately analyzed on Aortic aneurism 1 (0.8) a FACSCantoIITM (Becton Dickinson, Franklin Lakes, NJ, Other death causes‡ 10 (8.4) USA) flow cytometer. † Includes patients that suffered a stroke, an aortic dissection, an AMI, a cardiogenic Sample acquisition was performed during 1 min per sample shock, a CV death (mainly due to cHF) or were admitted to the emergency department. at “low flow” rate. Forward scatter (FSC), side scatter (SSC), It does not include patients that underwent a HTx. ‡ Includes patients that died due to a septic shock, a hemorrhage or a non-successful HTx. and fluorescence data were obtained with the settings in the AMI, acute myocardial infarction; CV, cardiovascular; cHF, chronic heart failure; HTx, logarithmic scale. Gate limits were established as follows with heart transplantation; NT-proBNP, N-terminal pro-hormone of brain natriuretic peptide; the same criteria previously described (20, 24, 30). The upper NYHA, New York Heart Association; SD, standard deviation. threshold for FSC was set with the Megamix-Plus FSC beads (BioCytex, Marseille, France). Megamix-Plus FSC beads for All samples were processed identically and within the first cytometer setting are a mix of beads of the following bead- 2 h. Blood was centrifuged at 1,560 g for 20 min at 20◦ C equivalent diameters: 0.1, 0.3, 0.5, and 0.9 µm. According to (Eppendorf 5810R GLOOB04932 centrifuge, A-4-81 rotor, the beads signal, the lower detection limit was placed as a Frontiers in Cardiovascular Medicine 04 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 threshold above the electronic background noise of the flow were performed to identify threshold concentrations of EVs cytometer for FSC and the second logarithm for SSC. EVs within able to discriminate between cHF NYHA stages, or between the established gate limits (>0.1 to 1 µm) were identified and severity in ischemic patients, and the corresponding area under quantified based on their binding to AV and reactivity to cell- the curve (AUC) with its 95% confidence interval (CI) was specific mAb. To identify positive marked events, thresholds calculated. Binary logistic regression models were pursued of fluorescence were also set based on samples incubated with to estimate predicted probabilities for NYHA severity using the same final concentration of isotype-matched control mAb combinations of EVs and/or the currently used biomarker after titration experiments. AV binding level was corrected NT-proBNP. A p < 0.05 was considered statistically significant. for auto-fluorescence using fluorescence signals obtained with Sample size was determined using the GRANMO sample size EVs in a calcium-free buffer (PBS). Additional controls to calculator (version 7.12, April 2012). To detect mean differences correct for FITC-, PE- and CFBlue-fluorescence were also in the number of EVs, a total of 126 subjects (controls and cHF) pursued (unstained and single-stained controls), as well as would be needed to complete the study (α risk = 0.05, beta serial dilutions to ensure proper event detection and to prevent risk = 0.2, two-sided test). swarming. Finally, to corroborate the presence of EVs in the EVs suspension, 5% saponin-treated controls were performed (see Supplementary Figure 1). To reduce background noise, Results buffers were prepared on the same day and filtered through 0.22 µm pore-size filters under vacuum. EVs markers observed Clinical characteristics of the study in cHF patients by flow cytometry were validated by western population blot as described in Supplementary material and shown in Supplementary Figure 2. Mean age of cHF was 67 ± 12 years (68% of men). Data was analyzed with BD FACSDivaTM Software (version Sixty cHF patients presented reduced ejection fraction (HFrEF; 6.1.3, Becton Dickinson, Franklin Lakes, NJ, USA). The LVEF < 40%; n = 60) while 59 (49.6%) had cHF with preserved concentration (number of EVs per µl of plasma) was ejection fraction (HFpEF; LVEF > 50%). Additionally, 52 determined according to Nieuwland’s procedure (31), based on (43.7%) cHF presented a less severe cHF symptomatology sample’s volume, flow cytometer’s flow rate and the number according to the NYHA classification (class II), while 66 (55.5%) of fluorescence-positive events (N), as follows: EVs/µl = N and 1 (0.8%) were in the higher severity spectrum, being x (Vf/Va) x (Vt/FR) x (1/Vi), where Vf(µl) = final volume in the NYHA stages III and IV, respectively. Demographics, of washed EVs suspension, Va(µl) = volume of washed EVs classical risk factors and background medication are shown suspension used for each labeling analysis, Vt(µl) = volume in Table 1, while etiology, biochemistry data and follow-up of EVs suspension before fluorescence-activated cell sorting events and outcomes are listed in Table 2. Demographical, analysis, FR(µl/min) = flow rate of the cytometer at low mode pharmacological and classical risk factor data of cHF according (the average volume of EVs suspension analyzed in 1 min), 1 is to their ejection fraction or severity (NYHA classification) is the µl unit of volume, and Vi(µl) = original volume of plasma disclosed in Supplementary Tables 1, 7. Underlying etiology used for EVs isolation. classification and clinical data of cHF according to these groups are listed in Supplementary Tables 2, 8. Statistical analysis Immunity cell-derived circulating Statistical analysis was performed using SPSS Statistical extracellular vesicles: Cell-origin and Analysis System (version 26.0, IBM Corp. Armonk, NY). activation Normality of variables was assessed with Saphiro–Wilks test. Descriptive analysis for qualitative variables was performed Total levels of EVs-AV+ shed by leukocytes were using number of cases and percentages, while for quantitative significantly higher in cHF patients than in controls. As shown variables, mean ± SD were used except when specified. in Figure 1A, EVs carrying the pan-leukocyte marker CD45+ , Frequencies of qualitative variables (clinical outcomes, risk the T-lymphocyte co-receptor (CD3+ ) or both (CD3+ /CD45+ ), factors, and medications) were compared between groups were significantly increased in cHF (p < 0.0001). In addition, using Chi-squared analysis. Median values of quantitative patients had significantly higher amounts of monocyte- and variables were contrasted with non-parametric tests. Statistical neutrophil-derived AV+ -EVs carrying CD16+ (receptor Fcγ significances between groups were determined with U III) and CD15+ (sialyl Lewis X), respectively. EVs shed by Mann–Whitney tests. Correlation analyses were pursued natural-killer cells carrying the neural cell adhesion molecule-1 using the Spearman correlation test. Receiver operating (CD56+ ) were significantly elevated in cHF compared to characteristic (ROC) curve analyses for predicted probabilities controls. Frontiers in Cardiovascular Medicine 05 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 FIGURE 1 Cellular origin and distribution of AV+ EVs in cHF and controls. (A) Distribution of EVs from total leukocytes (CD45+ ), T-lymphocytes (CD3+ and CD3+ /CD45+ ), neutrophils (CD15+ ), monocyte/macrophage (CD16+ ) and natural-killer cells (CD56+ ). (B) Distribution of EVs from activated neutrophils (CD29+ /CD15+ ) and from activated leukocytes (CD11b+ ). A p < 0.05 was considered significant (U Mann–Whitney test). AV+ , annexin V+ ; cHF, chronic heart failure; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; mEVs, monocyte-derived EVs; nEVs, neutrophil-derived EVs; NKC-EVs, natural-killer cells-derived EVs; PFP, platelet-free plasma. In addition, cHF patients had significantly higher levels Immune cell-derived circulating of EVs derived from activated immune cells (identified for extracellular vesicles signature and the shedding of activation markers in EVs). Specifically, AV+ - chronic heart failure severity EVs from activated leukocytes (integrin α-M; CD11b+ ) and activated neutrophils (integrin β-1/sialyl Lewis X; Chronic heart failure patients with more severe form of CD29+ /CD15+ ) were significantly increased in patients disease (a higher score in the NYHA classification [NYHA compared to controls (Figure 1B). III-IV]) had higher values of EVs derived from leukocytes The activation of the immunity cells measured by EVs did (CD45+ /AV+ ) and specifically those shed by T-lymphocytes not correlate with the number of blood leukocytes or CRP levels, (CD3+ /AV+ , CD3+ /CD45+ /AV+ ). In addition, a significant indicating different pathophysiological pathways. Interestingly, increase in EVs from activated leukocytes (CD11b+ /AV+ ) and circulating EVs from T-lymphocytes (CD3+ /CD45+ /AV+ ) activated neutrophils (CD29+ /CD15+ /AV+ ) was observed in significantly correlated with the cardiac damage markers hsTnT these patients, compared to those with NYHA II (Figure 2A). and NT-proBNP, while levels of EVs from activated neutrophils Interestingly, these differences were maintained when both (CD29+ /CD15+ /AV+ ) and hsTnT and NT-proBNP showed a groups were compared with controls (Supplementary Table 5). trend toward significance (Supplementary Table 4). cHF and Distribution of the cHF population according to disease control groups did not differ in the percentages of EVs shed by severity (according to the NYHA classification) is given platelets (CD41a+ ; αIIbβ 3-integrin). in Table 2. Patients’ characteristics according to degree of Frontiers in Cardiovascular Medicine 06 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 FIGURE 2 Distribution of AV+ -EVs of immune-cell origin considering disease severity according to NYHA. (A) Distribution of EVs from total leukocytes (CD45+ ), T-lymphocytes (CD3+ and CD3+ /CD45+ ), activated leukocytes (CD11b+ ) and from activated neutrophils (CD29+ /CD15+ ). A p < 0.05 was considered significant (U Mann–Whitney test). (B) ROC curve analyses to evaluate EVs and NT-proBNP association to NYHA severity, with AUC indicated along its 95% CI. A p < 0.05 was considered significant. AUC, area under the curve; AV+ , annexin V+ ; cHF, chronic heart failure; CI, confidence interval; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; nEVs, neutrophil-derived EVs; NYHA, New York Heart Association; PFP, platelet-free plasma; ROC, receiver operating characteristic curve. severity (NYHA II and NYHA III-IV stages) are shown in Instead, as expected, a negative correlation of NT-proBNP Supplementary Tables 1, 2. Disease severity score according to and LVEF was observed (rho = −0.222, p = 0.015). These NYHA correlated positively with hsTnT (rho = 0.200; p = 0.031) results indicate that immune cell activation is a common and NT-proBNP (rho = 0.313; p = 0.001). feature in patients with both HFpEF and HFrEF. Demographic, Leukocyte-derived EVs subtypes showed a significant biochemical and clinical classification of the patients according association to NYHA severity, evidencing their involvement to their LVEF is shown in Supplementary Tables 7–9. in disease pathophysiology. By C-statistics analysis, the clustering of CD11b+ /AV+ and CD45+ /AV+ showed an AUC (AUC = 0.658 ± 0.050 [95% CI: 0.559–0.757]; Circulating extracellular vesicles and p = 0.003) (Supplementary Table 6) similar to NT-proBNP chronic heart failure etiology: Ischemic (AUC = 0.676 ± 0.050 [95% CI: 0.578–0.774]; p = 0.001), and non-ischemic disease hormone secreted by cardiomyocytes in the heart ventricles in response to stretching caused by increased ventricular blood Chronic heart failure patients with ischemic etiology (HF- volume that signals for a different pathophysiological pathway Isch) presented increased levels of EVs. Specifically, EVs derived to that mapped by EVs (Figure 2B and Supplementary Table 6). from T-lymphocytes (CD3+ /AV+ , p = 0.045) or carrying Interestingly, no differences in immune cell-derived EVs the leukocyte activation marker integrin β-1 (CD29+ /AV+ , levels/phenotypes were detected in HFpEF and HFrEF patients. p = 0.025) were significantly increased in HF-Isch compared Frontiers in Cardiovascular Medicine 07 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 to non-ischemic patients (HF-NIsch). Moreover, EVs from when the immune reparative processes become impaired. The activated neutrophils (CD29+ /CD15+ /AV+ ) displayed higher present study has aimed at obtaining information on the levels in HF-Isch compared to HF-NIsch, although this trend activation state of different immunity cells in cHF patients. We was non-significant (p = 0.079) (Figure 3A). have demonstrated that cHF patients present differential EVs Within HF-Isch, different EVs profiles associated to disease profiles in comparison to control subjects, observing increased severity (NYHA class). There were significantly higher levels levels of EVs derived from leukocytes in cHF, reflecting the of EVs derived from leukocytes (CD45+ /AV+ ; p = 0.013), chronic activation state of these cells. T-lymphocytes (CD3+ /AV+ and CD45+ /CD3+ /AV+ ; We have specifically detected EVs derived from p < 0.024), and activated leukocytes, monocytes and lymphocytes, neutrophils, monocytes/macrophages and neutrophils (CD11b+ /AV+ , CD11b+ /CD14+ /AV+ and natural-killer cells in cHF compared to controls. In addition, CD29+ /CD15+ /AV+ , respectively; p < 0.05) in severe disease we have shown that numbers of these EVs, except those shed patients. No differences associated to severity were detected by natural-killer cells and monocytes, correlated with the in HF-NIsch (Figure 4). C-statistics analyses to assess the severity of the symptomatology. The combination of EVs involvement of EVs in the pathophysiology and severity CD11b+ /AV+ and CD45+ /AV+ was able to discriminate of disease in HF-Isch, showed that activated leukocytes between NYHA stages II and III-IV, with an AUC of (CD11b+ /AV+ ) had the best discrimination power, with an 0.658, indicating the implication of the activation of these AUC of 0.848 ± 0.058 (95% CI: 0.734–0.961; p < 0.001), immunity cells in disease severity pathophysiology. We followed by activated neutrophils (CD29+ /CD15+ /AV+ ), also found that EVs could discriminate the etiology of with and AUC of 0.811 ± 0.065 (95% CI: 0.683–0.939; cHF, with those patients with underlying ischemic etiology p = 0.001). Again, in an order of magnitude comparable to presenting significantly increased immune cell shed EVs. the contribution of disease showed by NT-proBNP, with an Further, we observed that in these patients EVs from AUC of 0.816 + 0.071 (95% CI: 0.676–0.955; p < 0.001). When activated leukocytes in combination with those from analyses were performed with the combined probabilities of activated neutrophils, had a high discriminative potential EVs from activated leukocytes and neutrophils (CD11b+ /AV+ in assessing cHF severity similar to NT-proBNP that and CD29+ /CD15+ /AV+ ) the AUC increased to 0.858 ± 0.056 measures another component of disease pathophysiology. (95% CI: 0.747–0.968; p < 0.001), with a sensitivity and We did not observe, however, differences in immune-cell- specificity of 80.0 and 83.3%, respectively. The addition of derived EVs profiles depending on the LVEF of cHF patients, NT-proBNP to the analysis did not improve discrimination indicating that both HFpEF and HFrEF patients have activation (AUC = 0.858 ± 0.056 [0.747–0.968]; p < 0.001) (Figure 3B of immunity cells. and Supplementary Table 10) indicating two types of Several studies have shown how EVs can signal contribution to disease progression of EVs and NT-proBNP, pathophysiological pathways of cardiovascular disease respectively. progression. For instance, our group has recently demonstrated Extracellular vesicles shedding levels, considering elapsed that EVs can predict the presence of atherosclerotic time between any ischemic event and sample collection, was plaques and discriminate plaque composition in familial similar in those patients with longer elapsed time between hypercholesterolemia (21, 34). Further, it is widely accepted ischemic event and blood withdrawal (more than 1 year: that EVs can identify severity of various pathologies (30, 7 ± 7 years) and those with lower time-periods (less than a year: 35) and predict cardiovascular events (24, 36, 37), including 5 ± 2 months) (Supplementary Table 11). cardiovascular death (22, 30). In fact, some studies have already observed increased levels of EVs in cHF, such as EVs from endothelial cells carrying the marker CD144+ (29). Discussion Here, we have shown in patients under guideline-directed medical therapy that immunity cells are activated. The Heart failure is a complex syndrome where the heart is amount of EVs shed by neutrophils (CD29+ /CD15+ /AV+ ), unable to maintain a correct blood supply to ensure that T-lymphocytes (CD3+ /AV+ and CD3+ /CD45+ /AV+ ) or the metabolic needs of the body are met (2). This can carrying the activation marker CD11b+ /AV+ , positively occur due to structural and functional defects, which at the correlates with disease severity. Further, we have shown for the same time are the result of major pathogenic mechanisms first time that immune-cell-derived EVs associate to NYHA like hemodynamic overload, ischemia-related dysfunction, or disease stage, and that immune cells are similarly activated in ventricular remodeling among others (32). cHF patients have HFpEF and HFrEF patients. A non-resolving inflammation shown in diverse studies to have a chronic and non-resolving is a reported characteristic of HF (11, 38); in addition, our inflammatory state (11, 33). results indicate that there is an activated state of the innate and The immune system has an important role in the setting adaptive immune cells that may need be the target of future of structural and functional changes in many organs, especially therapeutic interventions. Frontiers in Cardiovascular Medicine 08 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 FIGURE 3 Distribution of AV+ -EVs from immune-cell origin considering disease etiology. (A) Distribution of EVs from T-lymphocytes (CD3+ ), activated leukocytes (CD29+ ) and from activated neutrophils (CD29+ /CD15+ ). A p < 0.05 was considered significant (U Mann–Whitney test). (B) ROC curve analyses were used to evaluate the impact of EVs and combinations of EVs with NT-proBNP on HF severity (NYHA) in ischemic patients (AUC indicated along its 95% CI). A p < 0.05 was considered significant. AUC, area under the curve; AV+ , annexin V+ ; cHF, chronic heart failure; CI, confidence interval; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; nEVs, neutrophil-derived EVs; NYHA, New York Heart Association; PFP, platelet-free plasma; ROC, receiver operating characteristic curve. Patients with cHF of ischemic origin have significantly As study limitations we should first mention that the higher numbers of EVs from T-lymphocytes and neutrophils. relatively small cohort size included in the study has impeded This is a pathophysiological indication of the increase in the adjustment for comorbidities and medications; however, neutrophils in the myocardium after an initial insult (10). this is a hypothesis generating study. Second, by experimental Neutrophils are of the first cells mobilized to the damaged design, we did not include patients with HF with mildly myocardium, where they clear diseased cells, start the reparative reduced ejection fraction, which could have provided additional macrophages (M1) induction and promote inflammation, tissue insights in the pathophysiology of the disease. Third, it is reparation and fibrosis (10, 11, 39). Following the neutrophil also worth mentioning that the CD11b marker (integrin α- and macrophage wave, T-lymphocytes infiltrate the heart and M) is expressed by activated cells and indicates the type of promote wound healing. However, if this pro-inflammatory activated cell when is associated with another CD marker. state is prolonged in time and is not switched to a more anti- Additionally, due to methodological issues we could not inflammatory phenotype, maladaptive remodeling and fibrosis analyze EVs released by B lymphocytes, nor determine cell occur, promoting HF (11, 39). This crosstalk among cells in counts of all white blood cells analyzed. However, in view the damaged myocardium is mapped by the phenotypically of our results, further studies addressing EVs from B cells characterized EVs. are warranted. Frontiers in Cardiovascular Medicine 09 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 FIGURE 4 Distribution of AV+ -EVs of immune-cell origin considering disease severity in ischemic patients. Distribution of EVs of total leukocytes (CD45+ ), T-lymphocytes (CD3+ and CD3+ /CD45+ ), activated leukocytes (CD11b+ ) and from activated monocytes (CD11b+ /CD14+ ) and activated neutrophils (CD29+ /CD15+ ). A p < 0.05 was considered significant (U Mann–Whitney test). AV+ , annexin V+ ; EVs, extracellular microvesicles; LEVs, leukocyte-derived EVs; lEVs, lymphocyte-derived EVs; mEVs, monocyte-derived EVs; nEVs, neutrophil-derived EVs; NYHA, New York Heart Association; PFP, platelet-free plasma. Conclusion acting as distant cell function regulators of targeted-receptor cells, even in distant organs. In conclusion, HF is a complex syndrome where multiple pathophysiological pathways converge. In well treated chronic heart failure patients, immunity cells, both innate and adaptive Data availability statement immunity cells, are activated and release high number of circulating EVs. The released EVs may amplify the underlying The data that support the findings of this study are inflammatory processes in these patients, affecting different available from the corresponding author upon reasonable tissues. Indeed, EVs are involved in crosstalk with other cells, request. Frontiers in Cardiovascular Medicine 10 frontiersin.org Vilella-Figuerola et al. 10.3389/fcvm.2022.939625 Ethics statement to recruitment. This work has been performed under the framework of the Doctoral Program in Biomedicine, The studies involving human participants were reviewed Biochemistry and Molecular Biology of the Universitat and approved by Ref 16/44; Ethics Committee of the Autònoma de Barcelona. Hospital de la Santa Creu i Sant Pau in Barcelona. The patients/participants provided their written informed consent to participate in this study. Conflict of interest LB declares to have acted as SAB member of Sanofi, to have a Author contributions Research Grant of AstraZeneca, to have received speaker fees of Sanofi and Bayer and to have founded the Spin-offs Glycardial LB, TP, ER, and SM designed the research. AV-F performed Diagnostics SL and Ivastatin Therapeutics S (all unrelated to this the experiments. AV-F, TP, and LB analyzed the results. AV-F, work). TP declares to be co-founder of the Spin-offs Glycardial TP, SM, and LB wrote and revised the manuscript. All authors Diagnostics SL and Ivastatin Therapeutics S (all unrelated to this contributed to the article and approved the submitted version. work). The remaining authors declare that the research was conducted in the absence of any commercial or financial Funding relationships that could be construed as a potential conflict of interest. This work was supported by a Research Grant of the Spanish Society of Cardiology (SEC-2016) to LB; Spanish Ministry of Economy and Competitiveness of Science “Agencia Estatal Publisher’s note de Investigación (AEI)” Proj Ref AEI/10.13039/501100011033- (PID2019-107160RB-I00) to LB; Institute of Health Carlos III All claims expressed in this article are solely those of the (ISCIII) (Red RICORS TERAV- RD21/0017/0013 to LB; FIS authors and do not necessarily represent those of their affiliated PI19/01687 to TP); and cofounded by FEDER “Una Manera organizations, or those of the publisher, the editors and the de Hacer Europa”. AV-F was recipient of a research contract reviewers. Any product that may be evaluated in this article, or from the Cardiovascular Program-ICCC (IR-HSCSP). We claim that may be made by its manufacturer, is not guaranteed thank Fundación Jesus Serra and Fundación de Investigación or endorsed by the publisher. 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