RESUMO
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.
Assuntos
Anemia Falciforme , Cálcio , Humanos , Cálcio/metabolismo , Laminina/metabolismo , Eritrócitos/metabolismo , Eritrócitos Anormais/metabolismoRESUMO
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.
Assuntos
Anemia Falciforme , Hemólise , Camundongos , Animais , Camundongos Endogâmicos C57BL , Anemia Falciforme/tratamento farmacológico , Hemoglobinas/metabolismo , Hemoglobinas/uso terapêutico , Inflamação/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Ferro/uso terapêuticoRESUMO
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.
Assuntos
Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/fisiopatologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Peptidil Dipeptidase A/biossíntese , Adolescente , Adulto , Angiotensina II/metabolismo , Animais , Diástole , Modelos Animais de Doenças , Feminino , Humanos , Hidroxiureia/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Renina/sangue , Sistema Renina-Angiotensina , Sístole , Adulto JovemRESUMO
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.