Altered Plasma Membrane Lipid Composition in Hypertensive Neutrophils Impacts Epithelial Sodium Channel (ENaC) Endocytosis.

Biological membranes are composed of a lipid bilayer with embedded proteins, including ion channels like the epithelial sodium channel (ENaC), which are critical for sodium homeostasis and implicated in arterial hypertension (HTN). Changes in the lipid composition of the plasma membrane can significantly impact cellular processes related to physiological functions. We hypothesized that the observed overexpression of ENaC in neutrophils from HTN patients might result from alterations in the structuring domains within the plasma membrane, disrupting the endocytic processes responsible for ENaC retrieval. This study assessed the structural lipid composition of neutrophil plasma membranes from HTN patients along with the expression patterns of key elements regulating ENaC at the plasma membrane. Our findings suggest alterations in microdomain structure and SGK1 kinase activity, which could prolong ENaC presence on the plasma membrane. Additionally, we propose that the proteasomal and lysosomal degradation pathways are insufficient to diminish ENaC presence at the plasma membrane in HTN. These results highlight the importance of understanding ENaC retrieval mechanisms and suggest that targeting these mechanisms could provide insights for developing drugs to prevent and treat HTN.

Differential Gene Expression Pattern of Importin ß3 and NS5 in C6/36 Cells Acutely and Persistently Infected with Dengue Virus 2.

The establishment of persistent dengue virus infection within the cells of the mosquito vector is an essential requirement for viral transmission to a new human host. The mechanisms involved in the establishment and maintenance of persistent infection are not well understood, but it has been suggested that both viral and cellular factors might play an important role. In the present work, we evaluated differential gene expression in Aedes albopictus cells acutely (C6/36-HT) and persistently infected (C6-L) with Dengue virus 2 by cDNA-AFLP. We observed that importin ß3 was upregulated in noninfected cells compared with C6-L cells. Using RT-qPCR and plaque assays, we observed that Dengue virus levels in C6-L cells essentially do not vary over time, and peak viral titers in acutely infected cells are observed at 72 and 120 h postinfection. The expression level of importin ß3 was higher in acutely infected cells than in persistently infected cells; this correlates with higher levels of NS5 in the nucleus of the cell. The differential pattern of importin ß3 expression between acute and persistent infection with Dengue virus 2 could be a mechanism to maintain viral infection over time, reducing the antiviral response of the cell and the viral replicative rate.

Association between genetic variants of membrane transporters and the risk of high-grade hematologic adverse events in a cohort of Mexican children with B-cell acute lymphoblastic leukemia.

Background: Advances in the understanding of the pathobiology of childhood B-cell acute lymphoblastic leukemia (B-ALL) have led towards risk-oriented treatment regimens and markedly improved survival rates. However, treatment-related toxicities remain a major cause of mortality in developing countries. One of the most common adverse effects of chemotherapy in B-ALL is the hematologic toxicity, which may be related to genetic variants in membrane transporters that are critical for drug absorption, distribution, and elimination. In this study we detected genetic variants present in a selected group genes of the ABC and SLC families that are associated with the risk of high-grade hematologic adverse events due to chemotherapy treatment in a group of Mexican children with B-ALL. Methods: Next generation sequencing (NGS) was used to screen six genes of the ABC and seven genes of the SLC transporter families, in a cohort of 96 children with B-ALL. The grade of hematologic toxicity was classified according to the National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) version 5.0, Subsequently, two groups of patients were formed: the null/low-grade (grades 1 and 2) and the high-grade (grades 3 to 5) adverse events groups. To determine whether there is an association between the genetic variants and high-grade hematologic adverse events, logistic regression analyses were performed using co-dominant, dominant, recessive, overdominant and log-additive inheritance models. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated. Results: We found two types of associations among the genetic variants identified as possible predictor factors of hematologic toxicity. One group of variants associated with high-grade toxicity risk: ABCC1 rs129081; ABCC4 rs227409; ABCC5 rs939338, rs1132776, rs3749442, rs4148575, rs4148579 and rs4148580; and another group of protective variants that includes ABCC1 rs212087 and rs212090; SLC22A6 rs4149170, rs4149171 and rs955434. Conclusion: There are genetic variants in the SLC and ABC transporter families present in Mexican children with B-ALL that can be considered as potential risk markers for hematologic toxicity secondary to chemotherapeutic treatment, as well as other protective variants that may be useful in addition to conventional risk stratification for therapeutic decision making in these highly vulnerable patients.

Clinical Application of Epithelial Sodium Channel (ENaC) as a Biomarker for Arterial Hypertension.

Arterial hypertension (HTN) is a global public health concern and an important risk factor for cardiovascular diseases and renal failure. We previously reported overexpression of ENaC on the plasma membrane of human platelets is a hallmark of HTN. In this double-blinded study of an open population (n = 167), we evaluated the sensitivity and specificity of a diagnostic assay based on gold nanoparticles (AuNPs) conjugated to an antibody against epithelial sodium channel (ENaC) expressed on platelets, which is detected using a fluorescent anti-ENaC secondary antibody and spectrofluorometry. Using the cutoff value for the AuNP-anti-ENaC assay, we confirmed the diagnosis for 62.1% of patients with clinical HTN and detected 59.7% of patients had previously undiagnosed HTN. Although some shortcomings in terms of accurately discriminating healthy individuals and patients with HTN still need to be resolved, we propose this AuNP-anti-ENaC assay could be used for initial screening and early diagnosis to critically improve opportune clinical management of HTN.

Differentially expressed proteins in platelets derived from patients with hypertension.

Hypertension (HTN) causes end-organ damage and is a major cause of morbidity and mortality globally. Recent studies suggested blood cells participate in the maintenance of HTN. Platelets-anucleated cell fragments derived from megakaryocytes-exert diverse functions, including their well-characterized role in the formation of hemostatic clots. However, platelets from patients with HTN exhibit altered membrane lipid and protein compositions that impact platelet function and lead to formation of aggregates and vascular obstructions. Here, for the first time, we have identified, by proteomic analyses, the most relevant 11 proteins that show the greatest difference in their expression in platelets derived from patients with HTN, in comparison with those from normotensive individuals. These proteins are involved in cytoskeletal organization and the coagulation cascade that contributes to platelet activation, release of granule contents, and aggregation, which culminate in thrombus formation. These results have important implications in our understanding of the molecular mechanisms associated with the development of HTN, and in consequence, the development of new strategies to counteract the cardiovascular disorders associated with constitutive activation of platelets in HTN.

Data on the characterization of a platelet lysate and of the system in which it is included (nanoparticles/hydrogel), intended for the treatment of wounds.

Platelet lysate has attracted attention for different biomedical applications, including biological processes where cellular proliferation and migration have been altered. Spectroscopic properties of a platelet lysate obtained from human platelets were performed in order to be incorporated in polymeric nanoparticles and then into a PluronicⓇ F127 hydrogel, intended for wound healing (more details can be found at https://doi.org/10.1016/j.ejps.2020.105231 [1]). The platelet lysate (PL) was assessed by ultraviolet, infrared and circular dichroism spectroscopy. The developed hydrogel was also analyzed by infrared spectroscopy to evaluate if the PluronicⓇ F127 structure was maintained when the nanoparticles or platelet lysate-loaded nanoparticles were included. The sol-gel transition temperature of the hydrogel was determined through its thermal behavior and by dynamic light scattering.

Alterations to the contents of plasma membrane structural lipids are associated with structural changes and compartmentalization in platelets in hypertension.

Arterial hypertension (HTN) can lead to serious organ damage. Several mechanisms have been implicated in the pathogenesis of HTN including constitutive activation of platelets, which increases the risk of aggregation and clot formation. We recently demonstrated the plasma membranes of platelets from patients with HTN exhibit modified structural and physicochemical properties; Raman and Fourier transform infrared by attenuated total reflectance (FTIR-ATR) spectroscopy also indicated lipid content and protein structure alterations. This study aimed to precisely quantify the constituents of the main structural phospholipids and cholesterol in the plasma membranes of platelets from patients with HTN and normotensive individuals. We also assessed the consequence of these alterations on platelet structure and function. Liquid chromatography coupled to triple quadrupole mass spectrometry revealed the plasma membranes of HTN platelets contained less cholesterol and phosphatidylcholine, more phosphatidylserine and phosphatidylethanolamine and had similar sphingosine contents. Atomic force microscopy revealed HTN platelets exhibited increased surface roughness and more pleats. Transmission electron microscopy revealed diminution of the internal membranous structures in HTN platelets. Our findings strongly suggest plasma membrane lipid content alterations-including cholesterol depletion-occur in HTN, and these alterations may induce morphological and physiological abnormalities that participate in the functional changes associated with hypertension.

Alterations to plasma membrane lipid contents affect the biophysical properties of erythrocytes from individuals with hypertension.

Genetic and environmental factors may contribute to high blood pressure, which is termed essential hypertension. Hypertension is a major independent risk factor for cardiovascular disease, stroke and renal failure; thus, elucidation of the etiopathology of hypertension merits further research. We recently reported that the platelets and neutrophils of patients with hypertension exhibit altered biophysical characteristics. In the present study, we assessed whether the major structural elements of erythrocyte plasma membranes are altered in individuals with hypertension. We compared the phospholipid (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingosine) and cholesterol contents of erythrocytes from individuals with hypertension (HTN) and healthy individuals (HI) using LC/MS-MS. HTN erythrocytes contained higher phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine contents and a lower cholesterol content than HI erythrocytes. Furthermore, atomic force microscopy revealed important morphological changes in HTN erythrocytes, which reflected the increased membrane fragility and fluidity and higher levels of oxidative stress observed in HTN erythrocytes using spectrophotofluorometry, flow cytometry and spectrometry. This study reveals that alterations to the lipid contents of erythrocyte plasma membranes occur in hypertension, and these alterations in lipid composition result in morphological and physiological abnormalities that modify the dynamic properties of erythrocytes and contribute to the pathophysiology of hypertension.

Enhanced expression of the Epithelial Sodium Channel in neutrophils from hypertensive patients.

Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules. Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process.

Role of α-Dystrobrevin in the differentiation process of HL-60 cells.

The α-Dystrobrevin gene encodes at least five different protein isoforms, expressed in diverse tissues. The α-Dystrobrevin-1 isoform (α-Db-1) is a member of the cytoplasmic dystrophin-associated protein complex, which has a C-terminal extension comprising at least three tyrosine residues susceptible to phosphorylation in vivo. We previously described α-Db in stem-progenitor cells and blood neutrophils as playing a scaffolding role and, in association with kinesin and microtubules, α-Db promotes platelet-granule trafficking. Additionally, the microtubules must establish a balanced interaction with the lamina A/C network for appropriate nuclear morphology. Considering that the most outstanding feature during neutrophil differentiation is nuclei lobulation, we hypothesized that α-Db might possess a pivotal function during the neutrophil differentiation process. Western Blot (WB) and confocal microscope assays evidenced a differential pattern expression and a subcellular redistribution of α-Db in neutrophils derived from HL-60 cells. At the end of the differentiation process, we detected an important diminution in the expression of tubulin, kinesin, and α-Db-1. Knockdown of α-Db prevented nuclei lobulation, increased Lamin A/C and syne1 expression and augmented the roughness of derived neutrophil membrane and disturbed filopodia assembly. Our results suggest that HL-60 cells undergo extensive cytoskeletal reorganization including α-Db in order to possess lobulated nuclei when they further differentiate into neutrophils.

A role for dystroglycan in the pathophysiology of acute leukemic cells.

AIMS: Previous reports have demonstrated that alterations or reduced expression of Dystroglycan (Dg) complex (αDg and ßDg subunits) are related to progression and severity of neoplastic solid tissues. Therefore we determined the expression pattern and subcellular distribution of Dg complex in Acute Myeloid Leukemia (AML) primary blasts (M1, M2, and M3 phenotypes), as well as HL-60 and Kasumi-1 leukemia cell lines. Additionally, we evaluated the relative expression of the main enzymes controlling α-Dg glycosylation to ascertain the post-translational modifications in the leukemia cell phenotype. MAIN METHODS: Primary leukemia blasts and leukemia cell lines were processed by confocal analysis to determine the subcellular distribution of α-Dg, ß-Dg, and phosphorylated ß-Dg (Y892), to evaluate the expression pattern of the different Dg species we performed Western Blot (WB) assays, while the messenger RNA (mRNA) expression of enzymes involved in α-Dg glycosylation, such as POMGnT1, POMT1, POMT2, LARGE, FKTN, and FKRP, were evaluated by qualitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Finally, in an attempt to ameliorate the leukemia cell phenotype, we transfected leukemia cells with a plasmid expressing the Dg complex. KEY FINDINGS: The Dg complex was altered in leukemia cells, including decreased mRNA, protein, and α-Dg glycosylated levels, mislocalization of ß-Dg, and a diminution of mRNA expression of LARGE in patients leukemia blasts and in cell lines. Interestingly, the exogenous expression of Dg complex promoted filopodial formation, differentiation, and diminished proliferation, attenuating some HL-60 and Kasumi cells characteristics. SIGNIFICANCE: Dg complex integrity and balance are required for a proper hematopoietic cell function, in that its disruption might contribute to leukemia pathophysiology.

Dystroglycan Depletion Impairs Actin-Dependent Functions of Differentiated Kasumi-1 Cells.

BACKGROUND: Dystroglycan has recently been characterised in blood tissue cells, as part of the dystrophin glycoprotein complex involved in the differentiation process of neutrophils. PURPOSE: In the present study we have investigated the role of dystroglycan in the human promyelocytic leukemic cell line Kasumi-1 differentiated to macrophage-like cells. METHODS: We characterised the pattern expression and subcellular distribution of dystroglycans in non-differentiated and differentiated Kasumi-1 cells. RESULTS: Our results demonstrated by WB and flow cytometer assays that during the differentiation process to macrophages, dystroglycans were down-regulated; these results were confirmed with qRT-PCR assays. Additionally, depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated Kasumi-1 cells, including morphology, migration and phagocytic activities although secretion of IL-1ß and expression of markers of differentiation are not altered. CONCLUSION: Our findings strongly implicate dystroglycan as a key membrane adhesion protein involved in actin-based structures during the differentiation process in Kasumi-1 cells.

Association of membrane/lipid rafts with the platelet cytoskeleton and the caveolin PY14: participation in the adhesion process.

Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and ß-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete ß-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for ß-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

Haemostatic role of intermediate filaments in adhered platelets: importance of the membranous system stability.

The role of platelets in coagulation and the haemostatic process was initially suggested two centuries ago, and under appropriate physiological stimuli, these undergo abrupt morphological changes, attaching and spreading on damaged endothelium, preventing bleeding. During the adhesion process, platelet cytoskeleton reorganizes generating compartments in which actin filaments, microtubules, and associated proteins are arranged in characteristic patterns mediating crucial events, such as centralization of their organelles, secretion of granule contents, aggregation with one another to form a haemostatic plug, and retraction of these aggregates. However, the role of Intermediate filaments during the platelet adhesion process has not been explored. J. Cell. Biochem. 114: 2050-2060, 2013. © 2013 Wiley Periodicals, Inc.

In-vitro model for the ultrastructural study of the formation of thrombi in human platelets.

Platelets are cell fragments with dynamic properties involved in clot formation after tissue damage. Platelet activation causes a change in shape, secretion of intracellular granules and aggregation with each other through the cytoskeleton components and biochemical changes. Platelet adhesion, considered as the major event in haemostasis, has been studied in several in-vitro and in-vivo models to evaluate the feasible thrombogenicity of some materials, the dynamics of specific receptors, as well as the effect of different buffers and inhibitors in this process. In spite of the numerous reports about platelet activation, to date there is no information available about the fine structure of the platelet-platelet and platelet-substrate interactions. In the present report we describe an in-vitro system that allows the visualization of these interactions: platelets are adhered to an inert substrate, and interactions with suspended platelets as a process to initiate the formation of thrombi was followed by ultramicrotomy and transmission electron microscopy.