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.

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.

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.

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.

Expanding the Knowledge on Lignocellulolytic and Redox Enzymes of Worker and Soldier Castes from the Lower Termite Coptotermes gestroi.

Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

Chronic treatment with resveratrol improves overactive bladder in obese mice via antioxidant activity.

The objective of the present work was to evaluate whether oral intake with resveratrol ameliorates overactive bladder in high-fat fed mice. Male C57BL6 mice fed with standard chow or high-fat diet to induce obesity received a two-week therapy with resveratrol (100mg/kg, given as a daily gavage). Weight and metabolic profile, together with cystometry and in vitro bladder contractions were evaluated. Measurements of gp91phox and SOD1 mRNA expressions and reactive-oxygen species (ROS) in bladder tissues, and serum TBARS were performed. Obese mice exhibited increases in body weight and epididymal fat mass, which were significantly reduced by oral treatment with resveratrol. Cystometric study in obese mice showed increases in non-voiding contractions, post-voiding pressure and voiding frequency that were reversed by resveratrol treatment. Likewise, the in vitro bladder overactivity in response to electrical-field stimulation (80V, 1-32Hz) or carbachol (1nM to 10mM) were normalized by resveratrol. The gp91phox and SOD1 mRNA expressions in bladder tissues were markedly higher in obese mice compared with lean group. In addition, ROS levels in bladder tissues and serum lipid peroxidation (TBARS assay) were markedly higher in obese compared with lean mice, all of which were reduced by resveratrol treatment. In lean group, resveratrol had no effect in any parameter evaluated. Our results show that two-week therapy of obese mice with resveratrol reduces the systemic and bladder oxidative stress, and greatly ameliorated the cystometry alterations and in vitro bladder overactivity. Resveratrol treatment could be an option to prevent obesity-associated overactive bladder.

Reduced rate of sickle-related complications in Brazilian patients carrying HbF-promoting alleles at the BCL11A and HMIP-2 loci.

The presence of high levels of fetal haemoglobin (HbF) provides well-validated clinical benefits to patients with sickle cell anaemia (SCA). Nevertheless it has been difficult to show clear direct effects of the known genetic HbF modifiers, such as the enhancer polymorphisms for haematopoietic transcription factors BCL11A and MYB, on SCA severity. Investigating SCA patients from Brazil, with a high degree of European genetic admixture, we have detected strong effects of these variants on HbF levels. Critically, we have shown, for the first time, that the presence of such HbF-promoting variants leads to a reduced rate of SCA complications, especially stroke.

Key endothelial cell angiogenic mechanisms are stimulated by the circulating milieu in sickle cell disease and attenuated by hydroxyurea.

As hypoxia-induced inflammatory angiogenesis may contribute to the manifestations of sickle cell disease, we compared the angiogenic molecular profiles of plasma from sickle cell disease individuals and correlated these with in vitro endothelial cell-mediated angiogenesis-stimulating activity and in vivo neovascularization. Bioplex demonstrated that plasma from patients with steady-state sickle cell anemia contained elevated concentrations of pro-angiogenic factors (angiopoietin-1, basic fibroblast growth factor, vascular endothelial growth factor, vascular endothelial growth factor-D and placental growth factor) and displayed potent pro-angiogenic activity, significantly increasing endothelial cell proliferation, migration and capillary-like structure formation. In vivo neovascularization of Matrigel plugs was significantly greater in sickle cell disease mice than in non-sickle cell disease mice, consistent with an up-regulation of angiogenesis in the disease. In plasma from patients with hemoglobin SC disease without proliferative retinopathy, anti-angiogenic endostatin and thrombospondin-2 were significantly elevated. In contrast, plasma from hemoglobin SC individuals with proliferative retinopathy had a pro-angiogenic profile and more significant effects on endothelial cell proliferation and capillary formation than plasma from patients without retinopathy. Hydroxyurea therapy was associated with significant reductions in plasma angiogenic factors and inhibition of endothelial cell-mediated angiogenic mechanisms and neovascularization. Thus, individuals with sickle cell anemia or hemoglobin SC disease with retinopathy present a highly angiogenic circulating milieu, capable of stimulating key endothelial cell-mediated angiogenic mechanisms. Combination anti-angiogenic therapy to prevent the progression of unregulated neovascularization and associated manifestations in sickle cell disease, such as pulmonary hypertension, may be indicated; furthermore, the benefits and drawbacks of the potent anti-angiogenic effects of hydroxyurea should be clarified.

Reduced plasma angiotensin II levels are reversed by hydroxyurea treatment in mice with sickle cell disease.

AIMS: Sickle cell disease (SCD) pathogenesis leads to recurrent vaso-occlusive and hemolytic processes, causing numerous clinical complications including renal damage. As vasoconstrictive mechanisms may be enhanced in SCD, due to endothelial dysfunction and vasoactive protein production, we aimed to determine whether the expression of proteins of the renin-angiotensin system (RAS) may be altered in an animal model of SCD. MAIN METHODS: Plasma angiotensin II (Ang II) was measured in C57BL/6 (WT) mice and mice with SCD by ELISA, while quantitative PCR was used to compare the expressions of the genes encoding the angiotensin-II-receptors 1 and 2 (AT1R and AT2R) and the angiotensin-converting enzymes (ACE1 and ACE2) in the kidneys, hearts, livers and brains of mice. The effects of hydroxyurea (HU; 50-75mg/kg/day, 4weeks) treatment on these parameters were also determined. KEY FINDINGS: Plasma Ang II was significantly diminished in SCD mice, compared with WT mice, in association with decreased AT1R and ACE1 expressions in SCD mice kidneys. Treatment of SCD mice with HU reduced leukocyte and platelet counts and increased plasma Ang II to levels similar to those of WT mice. HU also increased AT1R and ACE2 gene expression in the kidney and heart. SIGNIFICANCE: Results indicate an imbalanced RAS in an SCD mouse model; HU therapy may be able to restore some RAS parameters in these mice. Further investigations regarding Ang II production and the RAS in human SCD may be warranted, as such changes may reflect or contribute to renal damage and alterations in blood pressure.

Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi.

BACKGROUND: Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production. RESULTS: The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite's digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch-degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis. CONCLUSIONS: Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29).