Extracellular Vesicles in Sickle Cell Disease: Plasma Concentration, Blood Cell Types Origin Distribution and Biological Properties

Abstract

Prototype of monogenic disorder, sickle cell disease (SCD) is caused by a unique single mutation in the β-globin gene, leading to the production of the abnormal hemoglobin S (HbS). HbS polymerization in deoxygenated condition induces the sickling of red blood cells (RBCs), which become less deformable and more fragile, and thus prone to lysis. In addition to anemia, SCD patients may exhibit a plethora of clinical manifestations ranging from acute complications such as the frequent and debilitating painful vaso-occlusive crisis to chronic end organ damages. Several interrelated pathophysiological processes have been described, including impaired blood rheology, increased blood cell adhesion, coagulation, inflammation and enhanced oxidative stress among others. During the last two decades, it has been shown that extracellular vesicles (EVs), defined as cell-derived anucleated particles delimited by a lipid bilayer, and comprising small EVs (sEVs) and medium/large EVs (m/lEVs); are not only biomarkers but also subcellular actors in SCD pathophysiology. Plasma concentration of m/lEVs, originated mainly from RBCs and platelets (PLTs) but also from the other blood cell types, is higher in SCD patients than in healthy controls. The concentration and the density of externalized phosphatidylserine of those released from RBCs may vary according to clinical status (crisis vs. steady state) and treatment (hydroxyurea). Besides their procoagulant properties initially described, RBC-m/lEVs may promote inflammation through their effects on monocytes/macrophages and endothelial cells. Although less intensely studied, sEVs plasma concentration is increased in SCD and these EVs may cause endothelial damages. In addition, sEVs released from activated PLTs trigger PLT-neutrophil aggregation involved in lung vaso-occlusion in sickle mice. Altogether, these data clearly indicate that EVs are both biomarkers and bio-effectors in SCD, which deserve further studies.

Keywords: coagulation; endothelial dysfunction; extracellular vesicles; inflammation; oxidative stress; sickle cell disease.

Figure 1

Mechanisms of production and size of the different extracellular vesicles types. (A) Exosomes are formed within multivesicular bodies (MVBs) and released upon fusion of MVBs with plasma membrane. Exosomes (sEVs) exhibit a narrow diameter ranging between 30 and 150 nm. (B) Microparticles (m/lEVs) diameter ranges from 100 to 1,000 nm. MPs derive from the cytoplasmic membrane of activated or apoptotic cells. Cell activation induces an increase of intracellular Ca²⁺ concentration leading to the translocation of phosphatidylserine (PS) to the outer leaflet of the cytoplasmic membrane and the activation of proteases that cleave the cytoskeleton, weaken its interaction with the cytoplasmic membrane, ultimately leading to the release of m/lEVs. (C) Apoptotic bodies are the largest EV subtypes exhibiting the wider size distribution (100–5,000 nm). They result from cell fragmentation and decomposition of the cell membrane of apoptotic cells.

Figure 2

Biological properties and pathophysiological consequences of extracellular vesicles in sickle cell disease. Extracellular vesicles (EVs) partly cause the hypercoagulant and prothrombotic state known in sickle cell disease (SCD). m/lEVs generated in vitro from stimulated monocytes, RBCs or platelets are able to trigger thrombin generation through TF-dependent and TF-independent mechanisms. Intrinsic coagulation pathway activation by RBC- and platelet-derived m/lEVs relies on the exposure of phosphatidylserine at their outer membrane leaflet. EVs also contribute to the inflammatory state of SCD patients. m/lEVs produced in vitro from sickle RBCs can be internalized by monocytes leading to the secretion of several proinflammatory cytokines and can increase the adhesion of monocytes to the endothelium. m/lEVs generated in vitro by sickle RBCs have been shown to promote renal vaso-occlusion in sickle cell mice and to induce endothelial cell apoptosis and ROS production. The high level of PS exposed at the surface of these vesicles, as well as their content in heme, could play a role in their deleterious effects on the vascular function. RBC-m/lEVs directly isolated from SCD patients' blood samples, promote the expression of adhesion molecules (ICAM-1, E-Selectin) and the production of pro-inflammatory cytokines by cultured endothelial cells. The endothelial activation mediated by these EVs involves the TLR4 signaling pathway. These proinflammatory properties are considerably reduced for m/lEVs obtained from patients treated with HU, which exhibit low PS externalization. In contrast, m/lEVs collected from patients during vaso-occlusive crisis exhibit high PS exposure and have deleterious effects on endothelial cells. RBC-m/lEVs could decrease NO bioavailability through their scavenging effects. In addition, both externalized PS and heme exposed by RBC-derived m/lEVs obtained using a calcium ionophore, have been shown to activate complement system on endothelial cell membranes. In humanized SCD mice, the stimulation of platelets leads to the release of sEVs highly loaded with IL-1β and caspase-1, which bind to neutrophils and promote platelet-neutrophil aggregation. VOC, vaso-occlusive crisis; NO, nitric oxide; ROS, reactive oxygen species; HU, hydroxyurea; PS, phosphatidylserine; TF, tissue factor; TLR4, toll like receptor 4; Casp.1, caspase 1; PLT, platelets; RBC, red blood cells.