New factors in heart failure pathophysiology: Immunity cells release of extracellular vesicles

Abstract

Leukocyte-shed extracellular vesicles (EVs) can play effector roles in the pathophysiological mechanisms of different diseases. These EVs released by membrane budding of leukocytes have been found in high amounts locally in inflamed tissues and in the circulation, indicating immunity cell activation. These EVs secreted by immune cell subsets have been minimally explored and deserve further investigation in many areas of disease. In this study we have investigated whether in heart failure there is innate and adaptive immune cell release of EVs. Patients with chronic heart failure (cHF) (n = 119) and in sex- and age-matched controls without this chronic condition (n = 60). Specifically, EVs were quantified and phenotypically characterized by flow cytometry and cell-specific monoclonal antibodies. We observed that even in well medically controlled cHF patients (with guideline-directed medical therapy) there are higher number of blood annexin-V+ (phosphatidylserine+)-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 neutrophils (innate immunity) and by T-lymphocytes and natural-killer cells (adaptive immunity) are significantly higher in cHF patients. Additionally, EVs-shed by activated leukocytes/neutrophils (CD11b+, p = 0.006; CD29+/CD15+, p = 0.048), and T-lymphocytes (CD3+/CD45+, p < 0.02) were positively correlated with cHF disease severity (NYHA classification). Interestingly, cHF patients with ischemic etiology had the highest levels of EVs shed by lymphocytes and neutrophils (p < 0.045, all). In summary, in cHF patients there is a significant immune cell activation shown by high-release of EVs that is accentuated by clinical severity of cHF. These activated innate and adaptive 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.

Platelet-Released Extracellular Vesicle Characteristics Differ in Chronic and in Acute Heart Disease

“…A second centrifugation at 1,500 g for 10 minutes at 20°C (Eppendorf 5415R centrifuge, FA45–24–11 rotor, Eppendorf) was then performed to completely remove the remaining cells and obtain platelet-free plasma (PFP), according to a standardized procedure for studies of EV by flow cytometry. 32 33 PFP aliquots were stored at −80°C until flow cytometry studies.…”

“…A two-step high-speed centrifugation was performed to isolate the EV fraction from PFP, as previously described. 32 33 Specifically, PFP was thawed and centrifuged at 1,500 g for 10 minutes at 20°C (Eppendorf 5417R centrifuge, FA45–24–11 rotor, Eppendorf) before 250 µL was collected from the upper part of the vial and centrifuged to pellet the EVs at 20,000 g for 30 minutes at 20°C (Eppendorf 5417R centrifuge, FA45–24–11 rotor, Eppendorf). The supernatant was then discarded, and the EV-containing pellet was washed with citrate-phosphate buffered saline (PBS) solution (citrate-PBS; 1.4 mmol/L phosphate, 154 mmol/L NaCl, 10.9 mmol/L trisodium citrate, pH 7.4).…”

“…Three-label flow cytometric analysis was performed as we have previously reported. 32 33 In brief, EV suspensions were diluted in PBS containing 2.5 mmol of CaCl 2 (Annexin Binding Buffer [ABB], BD Biosciences, San Jose) and then labeled with combinations of CFBlue-conjugated annexin V (AV) (Immunostep, Salamanca, Spain), to detect PS, and two specific monoclonal antibodies (mAbs) labeled with phycoerythrin (PE) and fluorescein isothiocyanate (FITC), or the isotype-matched control mAb ( Supplementary Table S2 , available in the online version). Samples were incubated in the dark for 20 minutes at 20°C and then diluted with ABB before being immediately analyzed on a FACSCantoII (Becton Dickinson, Franklin Lakes, New Jersey, United States) flow cytometer.…”

“…Gate limits were set following the criteria previously described. 32,33 The FSC upper threshold was established with the Megamix-Plus FSC beads (BioCytex, Marseille, France). Megamix-Plus FSC beads for cytometer setting in EV analyses are a mixture of beads with diameters of 0.1, 0.3, 0.5, and 0.9 µm.…”

Systems Biology in Chronic Heart Failure—Identification of Potential miRNA Regulators

“…Similarly, the TGF-β and PI3K-AKT pathways have been related to cardiac fibrosis [ 49 , 50 , 51 ]. Further, HF patients are characterised for presenting an unresolved inflammatory state, where chemokine levels are increased [ 52 ] and innate immunity cell increase their release of extracellular vesicles [ 53 ].…”