Molecular and cellular effects of in vivo chronic intravascular hemolysis and anti-inflammatory therapeutic approaches.

Intravascular hemolysis (IVH) occurs in numerous inherited and acquired disorders, including sickle cell disease (SCD), malaria and sepsis. These diseases display unique symptoms, but often share complications, such as vasomotor dysfunction and pulmonary hypertension. Consequently, in vivo models are needed to study the effects of continuous intravascular hemolytic processes, independently of the molecular alteration or extrinsic factor that leads to erythrocyte destruction. We gave twice-weekly low-dose phenylhydrazine (LDPHZ) to C57BL/6 J mice for 4 weeks, and measured parameters indicative of anemia, hemoglobin-clearance pathways, inflammation and iron turnover, comparing these to those of a murine model of SCD, which displays associated IVH. LDPHZ administration provoked discreet anemia in mice and significant reticulocytosis, in association with hemoglobin/heme-clearance pathway protein depletion. Mice subjected to chronic hemolysis displayed elevated leukocyte counts and plasma levels of interleukin (IL)-1ß, TNF-α, IL-6, soluble ICAM-1, endothelin-1 and anti-inflammatory IL-10, closely emulating alterations indicative of systemic inflammatory and endothelial activation in SCD, and confirming chronic IVH in itself as a serious complication. Discreet accelerations in hepatic and splenic iron turnover also occurred in LDPHZ mice, without alterations in liver damage markers. Examining the effects of two therapies on hemolysis-induced inflammation, the administration of hydroxyurea (and to a lesser extent, l-glutamine) significantly abrogated hemolytic inflammation in mice, without apparent inhibition of hemolysis. In conclusion, the isolation of chronic IVH, a common disease mechanism, using this model, may allow the study of hemolysis-specific sequelae at the cellular and systemic level, and the investigation of candidate agents that could potentially counter hemolytic inflammation.

Role of Macrophages in Sickle Cell Disease Erythrophagocytosis and Erythropoiesis.

Sickle cell disease (SCD) is an inherited blood disorder caused by a ß-globin gene point mutation that results in the production of sickle hemoglobin that polymerizes upon deoxygenation, causing the sickling of red blood cells (RBCs). RBC deformation initiates a sequence of events leading to multiple complications, such as hemolytic anemia, vaso-occlusion, chronic inflammation, and tissue damage. Macrophages participate in extravascular hemolysis by removing damaged RBCs, hence preventing the release of free hemoglobin and heme, and triggering inflammation. Upon erythrophagocytosis, macrophages metabolize RBC-derived hemoglobin, activating mechanisms responsible for recycling iron, which is then used for the generation of new RBCs to try to compensate for anemia. In the bone marrow, macrophages can create specialized niches, known as erythroblastic islands (EBIs), which regulate erythropoiesis. Anemia and inflammation present in SCD may trigger mechanisms of stress erythropoiesis, intensifying RBC generation by expanding the number of EBIs in the bone marrow and creating new ones in extramedullary sites. In the current review, we discuss the distinct mechanisms that could induce stress erythropoiesis in SCD, potentially shifting the macrophage phenotype to an inflammatory profile, and changing their supporting role necessary for the proliferation and differentiation of erythroid cells in the disease. The knowledge of the soluble factors, cell surface and intracellular molecules expressed by EBI macrophages that contribute to begin and end the RBC's lifespan, as well as the understanding of their signaling pathways in SCD, may reveal potential targets to control the pathophysiology of the disease.

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 µM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

TGF-ß1 Reduces Neutrophil Adhesion and Prevents Acute Vaso-Occlusive Processes in Sickle Cell Disease Mice.

Sickle cell disease (SCD) patients experience chronic inflammation and recurrent vaso-occlusive episodes during their entire lifetime. Inflammation in SCD occurs with the overexpression of several inflammatory mediators, including transforming growth factor beta-1 (TGF-ß1), a major immune regulator. In this study, we aimed to investigate the role played by TGF-ß1 in vascular inflammation and vaso-occlusion in an animal model of SCD. Using intravital microscopy, we found that a daily dose of recombinant TGF-ß1 administration for three consecutive days significantly reduced TNFα-induced leukocyte rolling, adhesion, and extravasation in the microcirculation of SCD mice. In contrast, immunological neutralization of TGF-ß, in the absence of inflammatory stimulus, considerably increased these parameters. Our results indicate, for the first time, that TGF-ß1 may play a significant ameliorative role in vascular SCD pathophysiology, modulating inflammation and vaso-occlusion. The mechanisms by which TGF-ß1 exerts its anti-inflammatory effects in SCD, however, remains unclear. Our in vitro adhesion assays with TNFα-stimulated human neutrophils suggest that TGF-ß1 can reduce the adhesive properties of these cells; however, direct effects of TGF-ß1 on the endothelium cannot be ruled out. Further investigation of the wide range of the complex biology of this cytokine in SCD pathophysiology and its potential therapeutical use is needed.

Reduced blood pressure in sickle cell disease is associated with decreased angiotensin converting enzyme (ACE) activity and is not modulated by ACE inhibition.

BACKGROUND: Sickle cell disease (SCD) incurs vaso-occlusive episodes and organ damage, including nephropathy. Despite displaying characteristics of vascular dysfunction, SCD patients tend to present relatively lower systemic blood pressure (BP), via an unknown mechanism. We investigated associations between BP and renin-angiotensin-system (RAS) components in SCD and determined whether an inhibitor of angiotensin converting enzyme (ACE; often used to slow SCD glomerulopathy) further modulates BP and RAS components in a murine model of SCD. METHODS: BP was compared in human subjects and mice with/without SCD. Plasma angiotensin II, ACE and renin were measured by immunoassay. BP was reevaluated after treating mice with enalapril (25 mg/kg, 5x/week) for 5 weeks; plasma and organs were stored for angiotensin II and ACE activity measurement, and quantitative real-time PCR. RESULTS: Diastolic BP and systolic BP were significantly lower in patients and mice with SCD, respectively, compared to controls. Reduced BP was associated with increased plasma renin and markers of kidney damage (mice) in SCD, as well as significantly decreased plasma ACE concentrations and ACE enzyme activity. As expected, enalapril administration lowered BP, plasma angiotensin II and organ ACE activity in control mice. In contrast, enalapril did not further reduce BP or organ ACE activity in SCD mice; however, plasma angiotensin II and renin levels were found to be significantly higher in enalapril-treated SCD mice than those of treated control mice. CONCLUSION: Relative hypotension was confirmed in a murine model of SCD, in association with decreased ACE concentrations in both human and murine disease. Given that ACE inhibition has an accepted role in decreasing BP, further studies should investigate mechanisms by which ACE depletion, via both Ang II-dependent and alternative pathways, could contribute to reduce BP in SCD and understand how ACE inhibition confers Ang II-independent benefits on kidney function in SCD.

Introduction to the thematic issue: Recognition of women leaders in Science.

This thematic issue of Experimental Biology and Medicine is dedicated to the incredibly important contributions made by women leaders in the biomedical sciences throughout recent history. Scientists from many disciplines have contributed papers, both original research and state of the art reviews, to demonstrate the type of work being performed every day by women leaders committed to advancing scientific knowledge in their respective fields of specialization. In this introduction, we provide readers with a brief highlight of the information to be found in the invited papers.

Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway.

Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells, in turn triggering inflammatory processes. This study investigated the in vitro effects of heme, a major inflammatory DAMP, on the adhesive properties of isolated human neutrophils. Heme (20 and 50 µM) significantly increased the adhesion of neutrophils to fibronectin and to recombinant ICAM-1, under static conditions, even more efficiently than the potent pro-inflammatory cytokine, tumor necrosis factor-α (TNF); a microfluidic assay confirmed that heme stimulated neutrophil adhesion under conditions of shear stress. Heme-induced neutrophil adhesion was associated with the increased activities, but not expressions, of the Mac-1 and LFA-1 integrin subunits, CD11b and CD11a, on the cell surface. Notably, heme (50 µM) significantly induced NFκB translocation in neutrophils, and inhibition of NFκB activity with the BAY11-7082 molecule abolished heme-induced cell adhesion to fibronectin and significantly decreased CD11a activity. Flow cytometric analysis demonstrated major reactive oxygen species (ROS) generation in neutrophils following heme stimulation that could be inhibited by the antioxidant, α-tocopherol, and by BAY11-7082. Furthermore, co-incubation with α-tocopherol abrogated both heme-stimulated neutrophil adhesion and CD11a/CD11b activation. Thus, our data indicate that heme, at clinically relevant concentrations, is a potent activator of neutrophil adhesion, increasing the ligand affinity of the ß2 integrins via a mechanism that may be partially mediated by an NFkB-dependent pathway and the generation of ROS. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in SCD vaso-occlusion and other vascular inflammatory processes, our findings provide further evidence that cell-free heme is a major therapeutic target in the hemolytic diseases.

Vasculopathy in Sickle Cell Disease: From Red Blood Cell Sickling to Vascular Dysfunction.

Sickle cell disease (SCD) is a hereditary disorder that leads to the production of an abnormal hemoglobin, hemoglobin S (HbS). HbS polymerizes in deoxygenated conditions, which can prompt red blood cell (RBC) sickling and leaves the RBCs more rigid, fragile, and prone to hemolysis. SCD patients suffer from a plethora of complications, ranging from acute complications, such as characteristic, frequent, and debilitating vaso-occlusive episodes to chronic organ damage. While RBC sickling is the primary event at the origin of vaso-occlusive processes, other factors that can further increase RBC transit times in the microcirculation may also be required to precipitate vaso-occlusive processes. The adhesion of RBC and leukocytes to activated endothelium and the formation of heterocellular aggregates, as well as increased blood viscosity, are among the mechanisms involved in slowing the progress of RBCs in deoxygenated vascular areas, favoring RBC sickling and promoting vascular occlusion. Chronic inflammatory processes and oxidative stress, which are perpetuated by hemolytic events and ischemia-reperfusion injury, result in this pan cellular activation and some acute events, such as stroke and acute chest syndrome, as well as chronic end-organ damage. Furthermore, impaired vasodilation and vasomotor hyperresponsiveness in SCD also contribute to vaso-occlusive processes. Treating SCD as a vascular disease in addition to its hematological perspective, the present article looks at the interplay between abnormal RBC physiology/integrity, vascular dysfunction and clinical severity in SCD, and discusses existing therapies and novel drugs in development that may ameliorate vascular complications in the disease. © 2021 American Physiological Society. Compr Physiol 11:1785-1803, 2021.

Sickle cell vaso-occlusion: The dialectic between red cells and white cells.

The pathophysiology of sickle cell anemia, a hereditary hemoglobinopathy, has fascinated clinicians and scientists alike since its description over 100 years ago. A single gene mutation in the HBB gene results in the production of abnormal hemoglobin (Hb) S, whose polymerization when deoxygenated alters the physiochemical properties of red blood cells, in turn triggering pan-cellular activation and pathological mechanisms that include hemolysis, vaso-occlusion, and ischemia-reperfusion to result in the varied and severe complications of the disease. Now widely regarded as an inflammatory disease, in recent years attention has included the role of leukocytes in vaso-occlusive processes in view of the part that these cells play in innate immune processes, their inherent ability to adhere to the endothelium when activated, and their sheer physical and potentially obstructive size. Here, we consider the role of sickle red blood cell populations in elucidating the importance of adhesion vis-a-vis polymerization in vaso-occlusion, review the direct adhesion of sickle red cells to the endothelium in vaso-occlusive processes, and discuss how red cell- and leukocyte-centered mechanisms are not mutually exclusive. Given the initial clinical success of crizanlizumab, a specific anti-P selectin therapy, we suggest that it is appropriate to take a holistic approach to understanding and exploring the complexity of vaso-occlusive mechanisms and the adhesive roles of the varied cell types, including endothelial cells, platelets, leukocytes, and red blood cells.

Thromboinflammatory mechanisms in sickle cell disease - challenging the hemostatic balance.

Sickle cell disease (SCD) is an inherited hemoglobinopathy that is caused by the presence of abnormal hemoglobin S (HbS) in red blood cells, leading to alterations in red cell properties and shape, as the result of HbS dexoygenation and subsequent polymerization. SCD pathophysiology is characterized by chronic inflammatory processes, triggered by hemolytic and vaso-occlusive events, which lead to the varied complications, organ damage and elevated mortality seen in individuals with the disease. In association with activation of the endothelium and leukocytes, hemostatic alterations and thrombotic events are well-documented in SCD. Here we discuss the role for inflammatory pathways in modulating coagulation and inducing platelet activation in SCD, due to tissue factor activation, adhesion molecule expression, inflammatory mediator production and the induction of innate immune responses, amongst other mechanisms. Thromboinflammatory pathways may play a significant role in some of the major complications of SCD, such as stroke, venous thromboembolism and possibly acute chest syndrome, besides exacerbating the chronic inflammation and cellular interactions that trigger vaso-occlusion, ischemia-reperfusion processes, and eventually organ damage.

CXCR4hi effector neutrophils in sickle cell anemia: potential role for elevated circulating serotonin (5-HT) in CXCR4hi neutrophil polarization.

Leukocyte recruitment and heterocellular aggregate formation drive the inflammatory vaso-occlusive processes associated with sickle cell anemia (SCA). We characterized neutrophils in a population of patients with SCA and investigated whether platelet-derived molecules can induce phenotypic alterations in this cell type. Imaging flow cytometry analysis demonstrated that the frequency of circulating CXCR4hi neutrophils was significantly higher in steady-state SCA individuals than in healthy control individuals and that these cells presented increased CD11b activation and toll-like receptor-4 expression. SCA neutrophils display increased neutrophil-platelet aggregation, and CXCR4hi neutrophils demonstrated augmented neutrophil-platelet aggregate frequency with a higher mean number of platelets adhered per neutrophil. Importantly, incubation of neutrophils with platelets significantly elevated their CXCR4 expression, while SCA plasma was found to induce CXCR4hi neutrophil polarization significantly more than control plasma. SCA individuals had significantly increased plasma levels of serotonin (5-HT), and serotonin molecule and SCA plasma induced neutrophil CXCR4 expression in a serotonin-receptor-dependent manner. Thus, the augmented CXCR4hi neutrophil population may contribute to mechanisms that promote vaso-occlusion in SCA; furthermore, circulating serotonin, derived from platelet activation, may play a role in the polarization of neutrophils, suggesting that serotonin-receptor antagonists or serotonin reuptake inhibitors could represent therapeutic approaches to reduce neutrophil activation in SCA.