The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells.

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Adverse effects of delta-9-tetrahydrocannabinol on sickle red blood cells.

THC triggers a pronounced entry of Ca2+ , which may be deleterious, into sickle cell red blood cells via activation of the TRPV2 channel.

A high-throughput electrophysiology assay to study the response of PIEZO1 to mechanical stimulation.

PIEZO1 channels are mechanically activated cation channels that play a pivotal role in sensing mechanical forces in various cell types. Their dysfunction has been associated with numerous pathophysiological states, including generalized lymphatic dysplasia, varicose vein disease, and hereditary xerocytosis. Given their physiological relevance, investigating PIEZO1 is crucial for the pharmaceutical industry, which requires scalable techniques to allow for drug discovery. In this regard, several studies have used high-throughput automated patch clamp (APC) combined with Yoda1, a specific gating modifier of PIEZO1 channels, to explore the function and properties of PIEZO1 in heterologous expression systems, as well as in primary cells. However, a combination of solely mechanical stimulation (M-Stim) and high-throughput APC has not yet been available for the study of PIEZO1 channels. Here, we show that optimization of pipetting parameters of the SyncroPatch 384 coupled with multihole NPC-384 chips enables M-Stim of PIEZO1 channels in high-throughput electrophysiology. We used this approach to explore differences between the response of mouse and human PIEZO1 channels to mechanical and/or chemical stimuli. Our results suggest that applying solutions on top of the cells at elevated pipetting flows is crucial for activating PIEZO1 channels by M-Stim on the SyncroPatch 384. The possibility of comparing and combining mechanical and chemical stimulation in a high-throughput patch clamp assay facilitates investigations on PIEZO1 channels and thereby provides an important experimental tool for drug development.

Cardiac competence of the paraxial head mesoderm fades concomitant with a shift towards the head skeletal muscle programme.

The vertebrate head mesoderm provides the heart, the great vessels, some smooth and most head skeletal muscle, in addition to parts of the skull. It has been speculated that the ability to generate cardiac and smooth muscle is the evolutionary ground-state of the tissue. However, whether indeed the entire head mesoderm has generic cardiac competence, how long this may last, and what happens as cardiac competence fades, is not clear. Bone morphogenetic proteins (Bmps) are known to promote cardiogenesis. Using 41 different marker genes in the chicken embryo, we show that the paraxial head mesoderm that normally does not engage in cardiogenesis has the ability to respond to Bmp for a long time. However, Bmp signals are interpreted differently at different time points. Up to early head fold stages, the paraxial head mesoderm is able to read Bmps as signal to engage in the cardiac programme; the ability to upregulate smooth muscle markers is retained slightly longer. Notably, as cardiac competence fades, Bmp promotes the head skeletal muscle programme instead. The switch from cardiac to skeletal muscle competence is Wnt-independent as Wnt caudalises the head mesoderm and also suppresses Msc-inducing Bmp provided by the prechordal plate, thus suppressing both the cardiac and the head skeletal muscle programmes. Our study for the first time suggests a specific transition state in the embryo when cardiac competence is replaced by skeletal muscle competence. It sets the stage to unravel the cardiac-skeletal muscle antagonism that is known to partially collapse in heart failure.

Improved PIEZO1 agonism through 4-benzoic acid modification of Yoda1.

BACKGROUND AND PURPOSE: The protein PIEZO1 forms mechanically activated, calcium-permeable, non-selective cation channels in numerous cell types from several species. Options for pharmacological modulation are limited and so we modified a small-molecule agonist at PIEZO1 channels (Yoda1) to increase the ability to modulate these channels. EXPERIMENTAL APPROACH: Medicinal chemistry generated Yoda1 analogues that were tested in intracellular calcium and patch-clamp assays on cultured cells exogenously expressing human or mouse PIEZO1 or mouse PIEZO2. Physicochemical assays and wire myography assays on veins from mice with genetic disruption of PIEZO1. KEY RESULTS: A Yoda1 analogue (KC159) containing 4-benzoic acid instead of the pyrazine of Yoda1 and its potassium salt (KC289) have equivalent or improved reliability, efficacy and potency, compared with Yoda1 in functional assays. Tested against overexpressed mouse PIEZO1 in calcium assays, the order of potency (as EC50 values, nM) was KC289, 150 > KC159 280 > Yoda1, 600). These compounds were selective for PIEZO1 over other membrane proteins, and the physicochemical properties were more suited to physiological conditions than those of Yoda1. The vasorelaxant effects were consistent with PIEZO1 agonism. In contrast, substitution with 2-benzoic acid failed to generate a modulator. CONCLUSION AND IMPLICATIONS: 4-Benzoic acid modification of Yoda1 improves PIEZO1 agonist activity at PIEZO1 channels. We suggest naming this new modulator Yoda2. It should be a useful tool compound in physiological assays and facilitate efforts to identify a binding site. Such compounds may have therapeutic potential, for example, in diseases linked genetically to PIEZO1 such as lymphatic dysplasia.

Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens.

Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond.

Blue-LED-Light Photobiomodulation of Inflammatory Responses and New Tissue Formation in Mouse-Skin Wounds.

Background: Recent studies evidence that blue-LED-light irradiation can modulate cell responses in the wound healing process within 24 h from treatment. This study aims to investigate blue-light (410-430 nm) photobiomodulation used in a murine wound model within six days post-treatment. Methods: A superficial wound was made in 30 CD1 male mice. The injuries were treated with a blue LED light (20.6 J/cm2), and biopsies were collected at 24, 72, and 144 h. Histology, fluorescence analysis, and advanced microscopy techniques were used. Results: We can observe an increase in the cellular infiltrate response, and in mast-cell density and their degranulation index correlated to the expression of the major histocompatibility complex after 24 h. Furthermore, after six days, the vessel density increases with the expression of the platelet-derived growth factor in the mast cells. Finally, collagen deposition and morphology in the treated wounds appear more similar to unwounded skin. Conclusions: Blue-light photobiomodulation stimulates several cellular processes that are finely coordinated by mast cells, leading to more rapid wound healing and a better-recovered skin morphology.

The role of mast cells in cellular modifications evoked by Exendin-4 in treated wounds: a preclinical study.

OBJECTIVE: To assess the response of cellular infiltration in wounds treated with Exendin-4. METHOD: In this study, 16 mice were used. On each mouse, two wounds were produced, one above the other, in order to study the effects of the various treatments carried out. The wounds then received an intradermal injection of either saline (20µl; Group 1) or Exendin-4 (Exe4, 62ng; Group 2) in the upper and lower wounds, respectively. The mice were euthanised in order to collect the wounds at time of abrasion (T0), at 48 hours (T1), 96 hours (T2) and 144 hours (T3). The expression of the glucagon-like peptide-1 receptor (GLP-1R) was evaluated by Western blot in wound lysates. Histological and histochemistry methods were applied in cryosections. RESULTS: In T2 and T3 treated wounds, the mast cells degranulation index increased while GLP-1R expression, tumour necrosis factor (TNF)-α, or heat shock protein (HSP)47 antigens were detected in their cytoplasm. These cells interacted with dendritic cells, vessels or granulocytes. The density of dendritic cells increased progressively, and intercellular connections were found between these cells and vessels. Among the dendritic cells at T2, only M2 macrophages increased. However, M1 cells expressed transforming growth factor (TGF)-ß and both interacted with either fibroblasts or with vessels. The number of plasmacytoid dendritic cells increased and established close contacts with regulatory T cells. CONCLUSION: We propose that after treatment with Exe4, early activation of mast cells is critical in wound healing acceleration. This is crucial in understanding the potential effect of this drug for viable clinical therapies. DECLARATION OF INTEREST: No potential conflict of interest was reported by the authors.

Data describing the effects of potassium channels modulators on outward currents measured in human lymphoma cell lines.

In the present work, applying the whole-cell patch-clamp technique in voltage clamp mode, we have investigated the effects of different drugs, such as riluzole, Psora-4 and Tram-34, on the potassium currents in four human lymphoma cell lines. We focused on outward currents mediated by two potassium channels (Kv1.3 and KCa3.1), which are known to play a key physiological role in lymphoid cells. The currents were evoked by voltage ramps ranging from -120 mV to +40 mV and the conductance of the two potassium channels was measured between +20 mV and +40 mV, both in the absence and in the presence of the specific blockers Psora-4 (Kv1.3; 1 µM) and Tram-34 (KCa3.1; 1 µM). The effect of the latter was tested after KCa3.1 channels were activated by riluzole 10 µM. Taken together, these data could be useful as an indication of the functional characteristics of the potassium channels in human lymphomas and represent a starting point for the study of potassium conductance in cellular models of these tumors.

ALA-PDT exerts beneficial effects on chronic venous ulcers by inducing changes in inflammatory microenvironment, especially through increased TGF-beta release: A pilot clinical and translational study.

A cohort of 19 patients affected by chronic venous ulcers was recruited from our centre. A 4-mm punch biopsy from wound bed was taken before application of ALA 20% gel and repeated one hour after the first PDT irradiation. We observed a significant and progressive reduction of wounds mean volumes right after three ALA-PDT sessions (once per week; 4479.9 +/- 345.5 mm3 vs 34599 +/- 190.3 mm3, p < .01). On immunofluorescence staining from biopsy specimens, we observed a change in all tested stains of post treatment specimens compared to pre-treatment ones. An increase of plasmacytoid dendritic cells (from 699 +/- 22 cells/0.018 mm2 to 1369 +/- 27 cells/0.018 mm2, p < .0001); MHC-II expression (260.39 +/- 99.7 Red, Green, Blue [RGB 0-255] to 370.2 +/- 162.6 RGB (0-255), p < .01), TNF-alpha positive mast cells expression (49 +/- 0.3 cells/0.018 mm2 to 69 +/- 0.4 cells/0.018 mm2, p < .001), TGF-beta expression (59.89 +/- 23.2 RGB (0-255)/cell vs 137.39 +/- 56.6 RGB (0-255)/cell, p < .01) and CD4+/CD25+ Treg cells (39 +/- 1 cells/0.018 mm2 vs 209 +/- 10 cells/0.018 mm2, p < .001) was observed. An increase of TGF-beta was correlated in a statistical significant manner with a reduction of wounds' mean volumes.