Erythrocyte Volume Homeostasis Research Articles

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Piezo-Type Mechanosensitive Ion Channel Component 1 (Piezo1): A Promising Therapeutic Target and Its Modulators.

Piezo1 is a member of the mechanosensitive piezo ion channel family, which transduces various mechanical stimulations into electrochemical signals. Piezo1 is closely implicated in different physiological processes ranging from erythrocyte volume homeostasis to lymphatic vessel formation and bone homeostasis. Aberrant Piezo1 functions caused by gain-of-function or loss-of-function mutations are associated with various pathological conditions. Due to the significant contribution on the recognition of Piezo ion channels for sensing mechanical stress, Ardem Patapoutian received the 2021 Nobel Prize in Physiology or Medicine (jointly). Strategies of targeting and modulating Piezo1 have shown potential to produce significant therapeutic effects, thus validating Piezo1 as a promising drug target for diseases. In this Perspective, we review the cryo-EM structure, mechanogating mechanism, and physiological profiles of Piezo1, together with the latest advances in the development of its modulators. Limitations and challenges as well as future development of Piezo1 modulators are discussed as well.

TRANSMEMBRANE ION AND WATER TRANSPORTS IN ERYTHROCYTE VOLUME HOMEOSTASIS: AN OVERVIEW OF THE PHYSIOLOGIC PROCESSES

Background: The intraerythrocytic concentrations of ions (Na+, K+, Cl- and HCO3-) play key roles in maintaining erythrocyte volume homeostasis. Anisosmotic and isosomotic changes of these ion concentrations challenge erythrocyte volume to either shrink or swell, thereby sending signals to activate regulatory volume mechanisms that are mediated by ion transporters. Ion fluxes directed inwardly or outwardly with obliged water movements do restore the erythrocyte volume to its steady state. These physiological phenomena prevent the erythrocyte from becoming overhydrated or dehydrated with the consequence of intravascular haemolysis or senescent changes associated with eryptosis. Objectives: To review the literature on the physiological processes associated with transmembrane ion and water transports during erythrocyte volume homeostasis. Method: Offline and online libraries were searched with indexing tools using keywords derived from the subject area of review. Conclusions: The review has highlighted the physiological concepts involved in erythrocyte volume homeostasis in relation to the engaged transmembrane ion and water transport systems, which can influence experimental designs to study ion and water channel blockers and channelopathies of erythrocytes.

Inside Out Integrin Activation Mediated by PIEZO1 Signaling in Erythroblasts

The non-selective mechanosensitive ion channel PIEZO1 controls erythrocyte volume homeostasis. Different missense gain-of-function mutations in PIEZO1 gene have been identified that cause Hereditary Xerocytosis (HX), a rare autosomal dominant haemolytic anemia. PIEZO1 expression is not limited to erythrocytes and expression levels are significantly higher in erythroid precursors, hinting to a role in erythropoiesis. During erythropoiesis, interactions between erythroblasts, central macrophages, and extracellular matrix within erythroblastic islands are important. Integrin α4β1 and α5β1 present on erythroblasts facilitate such interactions in erythroblastic islands. Here we found that chemical activation of PIEZO1 using Yoda1 leads to increased adhesion to VCAM1 and fibronectin in flowing conditions. Integrin α4, α5, and β1 blocking antibodies prevented this PIEZO1-induced adhesion suggesting inside-out activation of integrin on erythroblasts. Blocking the Ca2+ dependent Calpain and PKC pathways by using specific inhibitors also blocked increased erythroid adhesion to VCAM1 and fibronectins. Cleavage of Talin was observed as a result of Calpain and PKC activity. In conclusion, PIEZO1 activation results in inside-out integrin activation, facilitated by calcium-dependent activation of PKC and Calpain. The data introduces novel concepts in Ca2+ signaling during erythropoiesis with ramification on erythroblastic island homeostasis in health and disease like Hereditary Xerocytosis.

Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters.

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-β-cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP's response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure-response curve, increased channel latency in response to mechanical stimuli, and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM superresolution imaging revealed that, at the nanoscale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of polyunsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane composition and lateral organization of membrane cholesterol domains, which coordinate the activity of clustered PIEZO1 channels.

Cholesterol-Dependent Piezo1 Clusters are Essential for Efficient Cellular Mechanotransduction

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis [1,2,3]. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment, depletion (methyl-β-Cyclodextrin; MBCD) and the disruption of membrane cholesterol organization (Dynasore) affects PIEZO1's response to mechanical force. STORM super-resolution imaging revealed that, at the nano-scale, PIEZO1 channels in the membrane associate as clusters which increase in number upon cholesterol addition. Both cluster size and diffusion rates were profoundly affected by treatment with MBCD (5 mM). In addition, electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1 pressure-response curve and increased channel latency in response to mechanical stimuli. Our results indicate that PIEZO1 function is directly dependent on the amount and lateral organization of membrane cholesterol which is crucial for the concerted inactivation of PIEZO1 clusters. 1 Cahalan SM, et al. Piezo1 links mechanical forces to red blood cell volume. eLife. 2015;4. 2 Rode B, et al. Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance. Nat Commun. 2017;8. 3 Li J, et al. Piezo1 integration of vascular architecture with physiological force. Nature, 2014. Supported by the Australian Research Council and the National Health and Medical Research Council of Australia.

Human Piezo1 Membrane Localization and Gating Kinetics are Modulated by Cholesetrol Levels

The human mechanosensitive ion channel Piezo1 gates in response to membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently little is known regarding theplasma membrane localization and organization of Piezo1, but previous work suggests that membrane cholesterol content is a key determinant of Piezo1 function. Using a previously characterised Piezo1-GFP fusion protein (hP1-1591-GFP[1]), we investigated the effect of the cholesterol depleting agent methyl-β-Cyclodextrin (mβCD) on the membrane organization and the response of Piezo1 to mechanical force in HEK-293 cells.

New insights on hereditary erythrocyte membrane defects.

After the first proposed model of the red blood cell membrane skeleton 36 years ago, several additional proteins have been discovered during the intervening years, and their relationship with the pathogenesis of the related disorders have been somewhat defined. The knowledge of erythrocyte membrane structure is important because it represents the model for spectrin-based membrane skeletons in all cells and because defects in its structure underlie multiple hemolytic anemias. This review summarizes the main features of erythrocyte membrane disorders, dividing them into structural and altered permeability defects, focusing particularly on the most recent advances. New proteins involved in alterations of the red blood cell membrane permeability were recently described. The mechanoreceptor PIEZO1 is the largest ion channel identified to date, the fundamental regulator of erythrocyte volume homeostasis. Missense, gain-of-function mutations in the PIEZO1 gene have been identified in several families as causative of dehydrated hereditary stomatocytosis or xerocytosis. Similarly, the KCNN4 gene, codifying the so called Gardos channel, has been recently identified as a second causative gene of hereditary xerocytosis. Finally, ABCB6 missense mutations were identified in different pedigrees of familial pseudohyperkalemia. New genomic technologies have improved the quality and reduced the time of diagnosis of these diseases. Moreover, they are essential for the identification of the new causative genes. However, many questions remain to solve, and are currently objects of intensive studies.

Mutations in the Gardos channel (KCNN4) are associated with hereditary xerocytosis

AbstractHereditary xerocytosis (HX; MIM 194380) is an autosomal-dominant hemolytic anemia characterized by primary erythrocyte dehydration. In many patients, heterozygous mutations associated with delayed channel inactivation have been identified in PIEZO1. This report describes patients from 2 well-phenotyped HX kindreds, including from one of the first HX kindreds described, who lack predicted heterozygous PIEZO1-linked variants. Whole-exome sequencing identified novel, heterozygous mutations affecting the Gardos channel, encoded by the KCNN4 gene, in both kindreds. Segregation analyses confirmed transmission of the Gardos channel mutations with disease phenotype in affected individuals. The KCNN4 variants were different mutations in the same residue, which is highly conserved across species and within members of the small-intermediate family of calcium-activated potassium channel proteins. Both mutations were predicted to be deleterious by mutation effect algorithms. In sickle erythrocytes, the Gardos channel is activated under deoxy conditions, leading to cellular dehydration due to salt and water loss. The identification of KCNN4 mutations in HX patients supports recent studies that indicate it plays a critical role in normal erythrocyte deformation in the microcirculation and participates in maintenance of erythrocyte volume homeostasis.

Disorders of erythrocyte volume homeostasis.

Inherited disorders of erythrocyte volume homeostasis are a heterogeneous group of rare disorders with phenotypes ranging from dehydrated to overhydrated erythrocytes. Clinical, laboratory, physiologic, and genetic heterogeneities characterize this group of disorders. A series of recent reports have provided novel insights into our understanding of the genetic bases underlying some of these disorders of red cell volume regulation. This report reviews this progress in understanding determinants that influence erythrocyte hydration and how they have yielded a better understanding of the pathways that influence cellular water and solute homeostasis.

Endothelial-Specific Knockdown Of Caveolin-1 Lowers Circulating Protein Disulfide Isomerase Levels and Is Associated With Changes In Erythrocyte Volume Homeostasis

We have recently reported that Endothelin-1 (ET1), a potent vasoconstrictor peptide, is implicated in the pathophysiology of Sickle Cell Disease (SCD) via increased circulating Protein Disulfide Isomerase (PDI) activity (Prado, 2013 FASEB J). PDI is a multifunctional enzyme of the thioredoxin superfamily that mediates redox modifications, catalyzes disulfide interchange reactions in the plasma membrane, regulates KCNN4 channel and erythrocyte volume and is up-regulated under hypoxic conditions as commonly observed in SCD. In erythrocytes, ET1 stimulates PDI activity via activation of ET1 receptor B (ETRB). However, the precise mechanisms by which ET1 leads to increases in PDI are not entirely clear. There is evidence that activation of endothelial cells leads to increased PDI secretion and that ETRBs form a complex with caveolin-1 (CAV1) within caveolae to mediate ET1's cellular effects. We tested the hypothesis that reduction of CAV1 would alter PDI secretion. We studied the in vivo effects of endothelial-specific CAV1 knockdown on circulating PDI activity in mice. We optimized conditions to measure circulating PDI using fluorescently labeled GSSG conversion to GSH. We now report that circulating plasma PDI levels were significantly decreased in CAV1 knockdown mice when compared to wild-type littermates (WT) (7.44±0.70 vs 10.93±2.66, n=7, P<0.05). In addition and consistent with our report showing a role for PDI in erythrocyte volume regulation, we also observed lower cell hemoglobin concentration mean (CHCM) and hemoglobin distribution width (HDW) that was associated with increased erythrocyte and reticulocyte mean cell volume (MCV) in blood from CAV1 knockdown mice when compared to WT (n=13 and n=19, respectively, P<0.005). We then isolated early cultures of mouse aortic endothelial cells (MAEC) from these mice and measured PDI activity following 24 hrs of incubation in 0.4% fetal bovine serum. Our results show that MAEC from CAV1 knockdown mice had lower PDI secretion when compared to cells from WT mice (99.4±16 vs 129.9±35, n=5, P<0.03). We then studied the effects of ET1 on PDI secretion from human endothelial cells. We detected PDI and ETRB by western blot analyses in membranes from the human endothelial cell line, EA.hy926 (EA). We observed that incubation of EA cells for 60 mins with 10-7 M ET1 was associated with increased extracellular PDI activity (15.97±7.22 to 34.07±8.89 [RFU/mg protein], n=3, P<0.011) that was sensitive to preincubation with BQ788, a specific ETRB receptor antagonist (15.97±7.22 to 7.97±3.25 (RFU/mg protein), n=3, P<0.02). Similar increases in PDI were observed when cells were treated with the specific ETRB agonist, IRL1620 (1143±137 to 1593 207 RFU/mg protein). In addition, PDI siRNA knockdown was associated with reduced ET1-stimulated PDI activity when compared to scrambled siRNA transfected cells (1731±147 to 757±141 RFU, n=2). We then tested the effects of methyl-β-cyclodextrin to disrupt caveolae in these cells and observed a blunted IRL1620–stimulated PDI response (288±40 to 171±14 RFU/mg protein, n=3, P<0.025). We also characterized the effects of ET1 on PDI expression in EA cells, using quantitative RT-PCR with ABI TaqMan probes and β-actin as an endogenous control and observed that stimulation of EA cells with 10-8 M ET1 for 4 hr was associated with increased PDI mRNA expression levels that were 1.89 fold greater than vehicle treated cells (n=6, P<0.04). Thus our results provide evidence for a heretofore unrecognized role of endothelial specific CAV1 in erythrocyte volume and circulating PDI levels. Supported by NIH R01HL090632 (AR) and R01HL104032 (LHP). Disclosures:No relevant conflicts of interest to declare.

Piezo1 plays a role in erythrocyte volume homeostasis

Mechanosensitivity is an inherent property of virtually all cell types, allowing them to sense and respond to physical environmental stimuli. Stretch-activated ion channels represent a class of mechanosensitive proteins which allow cells to respond rapidly to changes in membrane tension; however their identity has remained elusive. The piezo genes have recently been identified as a family of stretch-activated mechanosensitive ion channels. We set out to determine the role of piezo1 during zebrafish development. Here we report that morpholino-mediated knockdown of piezo1 impairs erythrocyte survival without affecting hematopoiesis or differentiation. Our results demonstrate that piezo1 is involved in erythrocyte volume homeostasis, disruption of which results in swelling/lysis of red blood cells and consequent anemia.

Disorders of red cell volume regulation

Regulation of erythrocyte volume homeostasis is critical for survival of the erythrocyte. Inherited or acquired disorders that perturb this homeostasis jeopardize the erythrocyte, leading to its premature destruction. This report reviews recent insights into pathways that influence cellular water and solute homeostasis and cell volume. The molecular and genetic bases of primary disorders of erythrocyte hydration are beginning to be revealed. Recent studies have implicated roles for a new protein PIEZO1, a long sought after mammalian mechanosensory protein; GLUT1, the glucose transporter; SLC4A1, the anion transporter; RhAG, the Rh-associated glycoprotein; and ABCB6, an ATP-binding cassette family member. Secondary disorders associated with perturbed cellular volume and volume regulation include sickle cell disease, thalassemia, and hereditary spherocytosis, in which dehydration contributes to disease pathology and clinical complications. Advances in understanding the mechanisms regulating erythrocyte solute and water content, particularly associated with mechanotransduction pathways, have revealed novel mechanisms controlling erythrocyte hydration. Understanding these processes may provide innovative strategies to maintain normal erythrocyte volume in disorders associated with primary or secondary cellular dehydration. Understanding the mechanisms controlling erythrocyte volume regulation will serve as a paradigm for other cells and may reveal new therapeutic targets for disease prevention and treatment.

Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis

AbstractHereditary xerocytosis (HX, MIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. Copy number analyses, linkage studies, and exome sequencing were used to identify novel mutations affecting PIEZO1, encoded by the FAM38A gene, in 2 multigenerational HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all affected persons in both kindreds. All patients were heterozygous for FAM38A mutations, except for 3 patients predicted to be homozygous by clinical and physiologic studies who were also homozygous at the DNA level. The FAM38A mutations were both in residues highly conserved across species and within members of the Piezo family of proteins. PIEZO proteins are the recently identified pore-forming subunits of channels that mediate mechanotransduction in mammalian cells. FAM38A transcripts were identified in human erythroid cell mRNA, and discovery proteomics identified PIEZO1 peptides in human erythrocyte membranes. These findings, the first report of mutation in a mammalian mechanosensory transduction channel-associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte volume homeostasis.