Comparative histological analysis of spleens in pediatric patients with hemolytic anemias: Insights into the pathophysiological mechanisms of spleen destruction in sickle cell anemia.

While sickle cell anemia (SCA) and hereditary spherocytosis (HS) share common features of increased spleen erythrophagocytosis due to increased red blood cell (RBC) turnover, SCA is specifically characterized by susceptibility to infections. In this study, histological lesions in the spleens of pediatric patients with SCA were analyzed, in close correlation with past clinical history and comparatively to HS, healthy and transfused ß-thalassemia patients (TDT). An evaluation of red pulp elementary lesions (red pulp fibrosis, iron deposition, number of Gandy-Gamna, and RBC trapping) combined into a severity score was established, as well as B-cell follicles analysis. Quantification on digitalized slides of iron deposition, RBC trapping, and red pulp fibrosis was additionally performed. Spleens from 22 children with SCA, eight with HS, eight with TDT, and three healthy controls (HC) were analyzed. Median age at splenectomy was not different between SCA and HS patients, 6.05 years (range: 4.5-16.0) versus 4.75 (range: 2.2-9.5). Marked heterogeneity was found in SCA spleens in contrast to other conditions. Contrary to previous reports, B-cell follicles were generally preserved in SCA. While RBC trapping was significantly increased in both SCA and HS (compared to TDT and HC), quantitative fibrosis and overall red pulp severity score were significantly increased in SCA spleens compared to other conditions. Moreover, there was an inverse correlation between quantitative fibrosis and number of B-cell follicles, linking these two compartments as well as spleen fibrosis to infectious susceptibility in SCA, potentially through impaired red pulp macrophage scavenging and B-cell subpopulations defects.

Red Blood Cells: A Newly Described Partner in Central Retinal Vein Occlusion Pathophysiology?

Central retinal vein occlusion (CRVO) is a frequent retinal disorder inducing blindness due to the occlusion of the central vein of the retina. The primary cause of the occlusion remains to be identified leading to the lack of treatment. To date, current treatments mainly target the complications of the disease and do not target the primary dysfunctions. CRVO pathophysiology seems to be a multifactorial disorder; several studies did attempt to decipher the cellular and molecular mechanisms underlying the vessel obstruction, but no consensual mechanism has been found. The aim of the current review is to give an overview of CRVO pathophysiology and more precisely the role of the erythroid lineage. The review presents emerging data on red blood cell (RBC) functions besides their role as an oxygen transporter and how disturbance of RBC function could impact the whole vascular system. We also aim to gather new evidence of RBC involvement in CRVO occurrence.

Fetal hemoglobin rescues ineffective erythropoiesis in sickle cell disease.

While ineffective erythropoiesis has long been recognized as a key contributor to anemia in thalassemia, its role in anemia of sickle cell disease (SCD) has not been critically explored. Using in vitro and in vivo derived human erythroblasts we assessed the extent of ineffective erythropoiesis in SCD. Modeling the bone marrow hypoxic environment, we found that hypoxia induces death of sickle erythroblasts starting at the polychromatic stage, positively selecting cells with high levels of fetal hemoglobin (HbF). Cell death was associated with cytoplasmic sequestration of heat shock protein 70 and was rescued by induction of HbF synthesis. Importantly, we document that in the bone marrow of SCD patients similar cell loss occurs during the final stages of terminal differentiation. Our study provides evidence for ineffective erythropoiesis in SCD and highlights an anti-apoptotic role for HbF during the terminal stages of erythroid differentiation. These findings imply that the beneficial effect on anemia of increased HbF levels is not only due to the increased life span of red cells but also a consequence of decreased ineffective erythropoiesis.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

OBJECTIVE: Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. APPROACH AND RESULTS: Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6-/- mice contain less inflammatory (CCR2hiCX3CR1lo) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2hiCX3CR1lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). CONCLUSIONS: This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2hiCX3CR1lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis.

Hydroxycarbamide decreases sickle reticulocyte adhesion to resting endothelium by inhibiting endothelial lutheran/basal cell adhesion molecule (Lu/BCAM) through phosphodiesterase 4A activation.

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4ß1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

Vascular Gas6 contributes to thrombogenesis and promotes tissue factor up-regulation after vessel injury in mice.

Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (-/-) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5 µM ADP and 10 µM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6(-/-) mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6(-/-) endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism.

Extracellular RNA Induces Neutrophil Recruitment Via Toll-Like Receptor 3 During Venous Thrombosis After Vascular Injury.

BACKGROUND: Venous thromboembolism is associated with endothelial cell activation that contributes to the inflammation-dependent activation of the coagulation system. Cellular damage is associated with the release of different species of extracellular RNA (eRNA) involved in inflammation and coagulation. TLR3 (toll-like receptor 3), which recognizes (viral) single-stranded or double-stranded RNAs and self-RNA fragments, might be the receptor of these species of eRNA during venous thromboembolism. Here, we investigate how the TLR3/eRNA axis contributes to venous thromboembolism. METHODS AND RESULTS: Thrombus formation and size in wild-type and TLR3 deficient (-/-) mice were monitored by ultrasonography after venous thrombosis induction using the ferric chloride and stasis models. Mice were treated with RNase I, with polyinosinic-polycytidylic acid, a TLR3 agonist, or with RNA extracted from murine endothelial cells. Gene expression and signaling pathway activation were analyzed in HEK293T cells overexpressing TLR3 in response to eRNA or in human umbilical vein endothelial cells transfected with a small interference RNA against TLR3. Plasma clot formation on treated human umbilical vein endothelial cells was analyzed. Thrombosis exacerbated eRNA release in vivo and increased eRNA content within the thrombus. RNase I treatment reduced thrombus size compared with vehicle-treated mice (P<0.05). Polyinosinic-polycytidylic acid and eRNA treatments increased thrombus size in wild-type mice (P<0.01 and P<0.05), but not in TLR3-/- mice, by reinforcing neutrophil recruitment (P<0.05). Mechanistically, TLR3 activation in endothelial cells promotes CXCL5 (C-X-C motif chemokine 5) secretion (P<0.001) and NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation (P<0.05). Finally, eRNA triggered plasma clot formation in vitro (P<0.01). CONCLUSIONS: We show that eRNA and TLR3 activation enhance venous thromboembolism through neutrophil recruitment possibly through secretion of CXCL5, a potent neutrophil chemoattractant.

Oxidative Stress in Healthy and Pathological Red Blood Cells.

Red cell diseases encompass a group of inherited or acquired erythrocyte disorders that affect the structure, function, or production of red blood cells (RBCs). These disorders can lead to various clinical manifestations, including anemia, hemolysis, inflammation, and impaired oxygen-carrying capacity. Oxidative stress, characterized by an imbalance between the production of reactive oxygen species (ROS) and the antioxidant defense mechanisms, plays a significant role in the pathophysiology of red cell diseases. In this review, we discuss the most relevant oxidant species involved in RBC damage, the enzymatic and low molecular weight antioxidant systems that protect RBCs against oxidative injury, and finally, the role of oxidative stress in different red cell diseases, including sickle cell disease, glucose 6-phosphate dehydrogenase deficiency, and pyruvate kinase deficiency, highlighting the underlying mechanisms leading to pathological RBC phenotypes.

Differential modulation of adhesion molecule expression by hydroxycarbamide in human endothelial cells from the micro- and macrocirculation: potential implications in sickle cell disease vasoocclusive events.

BACKGROUND: All the cellular partners of the vascular system and especially endothelial cells are involved in the pathophysiology of the vasoocclusive crises associated with sickle cell disease. In sickle cell disease, circulating cells adhere abnormally to endothelial cells in a chronic pro-inflammatory context. Hydroxycarbamide is the only drug with demonstrated efficacy to reduce the frequency of vasoocclusive crises. Here, we investigated the effects of hydroxycarbamide and/or cytokines on the expression of genes related to adhesion events in endothelial cells from three different vascular sites. DESIGN AND METHODS: Endothelial cells representative of the macro- (HUVEC) or microcirculation (TrHBMEC and HPMEC) were grown in the presence or absence of hydroxycarbamide and/or cytokines (TNFα and IFNγ). Expression of genes encoding adhesion proteins was analyzed by RQ-PCR, ELISA, flow cytometry, in situ ELISA for extracellular matrix proteins, and Western blot. RESULTS: In cells from the microcirculation, expression of TSP-1, vWF, and PECAM-1 genes was decreased by hydroxycarbamide and/or cytokine treatment at the mRNA level. In the macro-circulation their expression was unaffected or increased. Hydroxycarbamide significantly decreased vWF incorporated in the TrHBMEC extracellular matrix. CD36 mRNA was strongly down-regulated by cytokines in HPMEC, the only cell type in which it is expressed. Hydroxycarbamide decreased soluble PECAM-1 in HUVEC supernatants. CONCLUSIONS: Our results highlight the heterogeneity of vascular endothelial cell responses to hydroxycarbamide and/or cytokines depending upon their origin. They also suggest that hydroxycarbamide has an anti-adhesogenic effect on endothelial cells, but by mechanisms which could vary according to their macro- or microcirculation and organ origin.

Hydroxycarbamide stimulates the production of proinflammatory cytokines by endothelial cells: relevance to sickle cell disease.

BACKGROUND AND OBJECTIVE: The clinical hallmarks of sickle cell disease (SCD) are vaso-occlusive crises (VOC) triggered by red blood cells (RBC) stiffening and abnormal adhesion to vascular endothelial cells (VEC) in the context of chronic inflammation, cell activation, and vascular tone abnormalities. Hydroxycarbamide (HC) is the only drug with a proven efficacy in decreasing VOC frequency. HC decreases RBC stiffening, modulates adhesion protein expression by RBC and VEC, and reduces endothelin-1 production by VEC. Our objective was to test whether HC could also affect inflammation through its action on VEC. METHODS: We used microarrays to study the effect of HC on the transcriptome of transformed human bone marrow endothelial cell, a cell line derived from bone marrow microcirculation (the predilection site of VOC), in basal and proinflammatory conditions. Microarray results were confirmed by real-time quantitative PCR and protein analysis on transformed human bone marrow endothelial cell (TrHBMEC) and on two other VEC types in the primary culture: human pulmonary microcirculation endothelial cell (HPMEC) and human umbilical vein endothelial cell (HUVEC a classical model for the macrocirculation). RESULTS: HC had a significant effect on the expression of genes of the 'inflammation pathway'. Strikingly, it stimulates the expression of proinflammatory genes such as IL1A, IL1B, IL6, IL8, CCL2, CCL5, CCL20, and CCL8 in all the tested VEC types. CONCLUSION: Our study confirms that VECs are significant targets of HC in the context of SCD and identifies its earlier unsuspected action on another major component of SCD pathophysiology, that is, the 'inflammation pathway'.

Growth arrest-specific gene 6 (gas6) and vascular hemostasis.

Gas6 (growth arrest-specific 6) belongs structurally to the family of plasma vitamin K-dependent proteins. Gas6 has a high structural homology with the natural anticoagulant protein S, sharing the same modular composition. Interestingly, despite the presence of a γ-carboxyglutamic acid domain in its structure, no role in the coagulation cascade has been identified for gas6. Gas6 has been shown to be involved in vascular homeostasis and more precisely is involved in proliferation, apoptosis, efferocytosis, leukocyte migration, and sequestration and platelet aggregation. It is also involved in the activation of different cell types, from platelets to endothelial and vascular smooth muscle cells. Thus, it has been shown to play a role in several pathophysiological processes such as atherosclerosis, cancer, and thrombosis. Interestingly, studies using gas6 null mice highlighted that gas6 may represent a novel potential target for anticoagulant therapy, because these animals are protected from lethal venous thromboembolism without excessive bleeding. However, the mechanism in thrombus occurrence remains to be further explored. In the present review, we will focus on the role of gas6 in innate immunity, atherosclerosis, thrombosis, and cancer-related events.

Abnormal flow adhesion of sickle red blood cells to human placental trophoblast extracellular matrix.

Pregnancy in sickle cell disease (SCD) has been associated with increased complications such as vaso-occlusive crises, severe anemia and foetal loss. It has been proposed that the sickling of red blood cells (RBCs) inside the placenta circulation could participate to these complications. The present study investigated the adhesion of sickle RBCs on human trophoblast-derived cell and its extracellular matrix. Results demonstrated 1) similar adhesion of sickle RBCs and healthy RBCs to trophoblast but 2) a greater adhesion of sickle RBCs to the extracellular matrix of trophoblasts as compared with healthy RBCs. This greater adhesion could partly involve the Lu/BCAM glycoproteins and could participate to the complications reported in SCD pregnant women.