Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Pharmacology of PIEZO1 channels.

PIEZO1 is a eukaryotic membrane protein that assembles as trimers to form calcium-permeable, non-selective cation channels with exquisite capabilities for mechanical force sensing and transduction of force into effect in diverse cell types that include blood cells, endothelial cells, epithelial cells, fibroblasts and stem cells and diverse systems that include bone, lymphatics and muscle. The channel has wide-ranging roles and is considered as a target for novel therapeutics in ailments spanning cancers and cardiovascular, dental, gastrointestinal, hepatobiliary, infectious, musculoskeletal, nervous system, ocular, pregnancy, renal, respiratory and urological disorders. The identification of PIEZO1 modulators is in its infancy but useful experimental tools emerged for activating, and to a lesser extent inhibiting, the channels. Elementary structure-activity relationships are known for the Yoda series of small molecule agonists, which show the potential for diverse physicochemical and pharmacological properties. Intriguing effects of Yoda1 include the stimulated removal of excess cerebrospinal fluid. Despite PIEZO1's broad expression, opportunities are suggested for selective positive or negative modulation without intolerable adverse effects. Here we provide a focused, non-systematic, narrative review of progress with this pharmacology and discuss potential future directions for research in the area.

Endothelial force sensing signals to parenchymal cells to regulate bile and plasma lipids.

How cardiovascular activity interacts with lipid homeostasis is incompletely understood. We postulated a role for blood flow acting at endothelium in lipid regulatory organs. Transcriptome analysis was performed on livers from mice engineered for deletion of the flow-sensing PIEZO1 channel in endothelium. This revealed unique up-regulation of Cyp7a1, which encodes the rate-limiting enzyme for bile synthesis from cholesterol in hepatocytes. Consistent with this effect were increased gallbladder and plasma bile acids and lowered hepatic and plasma cholesterol. Elevated portal fluid flow acting via endothelial PIEZO1 and genetically enhanced PIEZO1 conversely suppressed Cyp7a1. Activation of hepatic endothelial PIEZO1 channels promoted phosphorylation of nitric oxide synthase 3, and portal flow-mediated suppression of Cyp7a1 depended on nitric oxide synthesis, suggesting endothelium-to-hepatocyte coupling via nitric oxide. PIEZO1 variants in people were associated with hepatobiliary disease and dyslipidemia. The data suggest an endothelial force sensing mechanism that controls lipid regulation in parenchymal cells to modulate whole-body lipid homeostasis.

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.

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.

Biological Effects of Modifications of the Englerin A Glycolate.

Modifications at the glycolate moiety of englerin A were made to explore variations at the most sensitive site on the molecule for activity in the NCI 60 screen, wherein englerin A is highly potent and selective for renal cancer cells. Replacement of the glycolate by other functionalities as well as esterification of the glycolate hydroxyl yielded compounds which displayed excellent selectivity and potency compared with the natural product. TRPC4/5 ion channel experiments with five compounds showed delayed or reduced agonism with TRPC5, at much higher concentrations than englerin A. With TRPC4, these compounds all had no effect at 10 µM. The same compounds were not detectable in mouse serum after a single oral dose of 12.5 mg/kg. At 100 mg/kg p.o., no toxicity was observed, and blood levels were barely detectable. Intravenous administration led to toxicity but at substantially lower doses than for englerin A.

Global PIEZO1 Gain-of-Function Mutation Causes Cardiac Hypertrophy and Fibrosis in Mice.

PIEZO1 is a subunit of mechanically-activated, nonselective cation channels. Gain-of-function PIEZO1 mutations are associated with dehydrated hereditary stomatocytosis (DHS), a type of anaemia, due to abnormal red blood cell function. Here, we hypothesised additional effects on the heart. Consistent with this hypothesis, mice engineered to contain the M2241R mutation in PIEZO1 to mimic a DHS mutation had increased cardiac mass and interventricular septum thickness at 8-12 weeks of age, without altered cardiac contractility. Myocyte size was greater and there was increased expression of genes associated with cardiac hypertrophy (Anp, Acta1 and ß-MHC). There was also cardiac fibrosis, increased expression of Col3a1 (a gene associated with fibrosis) and increased responses of isolated cardiac fibroblasts to PIEZO1 agonism. The data suggest detrimental effects of excess PIEZO1 activity on the heart, mediated in part by amplified PIEZO1 function in cardiac fibroblasts.

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity.

Piezo1 forms mechanically activated nonselective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial cell-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial cell apoptosis, with no effect on TSP1, a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without an effect on Tsp1. In endothelial cells, Piezo1 was required for normal expression of endothelial NO synthase. The data suggest an endothelial cell-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability.

Immunotherapy for Epstein-Barr virus-associated malignancies.

Epstein-Barr virus (EBV) is present in the malignant cells of several human cancers including post-transplant lymphoproliferative disease (PTLD), Hodgkin's lymphoma, nasopharyngeal carcinoma, natural killer/T lymphomas and Burkitt's lymphoma. Yet in > 90% of the world's adult population, who carry EBV as a lifelong asymptomatic infection, the oncogenic potential of this virus is controlled by a strong virus-specific T-cell response. Accordingly, EBV-associated malignancies represent good candidates for a T-cell-based therapy and provide an important model for developing such therapies for other human cancers. This review summarizes the impressive results seen with T-cell therapy for PTLD and discusses, in the light of recent technological advances, the prospects for developing more effective approaches for other EBV-associated cancers.

Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells.

Mechanical force is a determinant of Notch signalling but the mechanism of force detection and its coupling to Notch are unclear. We propose a role for Piezo1 channels, which are mechanically-activated non-selective cation channels. In cultured microvascular endothelial cells, Piezo1 channel activation by either shear stress or a chemical agonist Yoda1 activated a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), a Ca2+-regulated transmembrane sheddase that mediates S2 Notch1 cleavage. Consistent with this observation, we found Piezo1-dependent increase in the abundance of Notch1 intracellular domain (NICD) that depended on ADAM10 and the downstream S3 cleavage enzyme, γ-secretase. Conditional endothelial-specific disruption of Piezo1 in adult mice suppressed the expression of multiple Notch1 target genes in hepatic vasculature, suggesting constitutive functional importance in vivo. The data suggest that Piezo1 is a mechanism conferring force sensitivity on ADAM10 and Notch1 with downstream consequences for sustained activation of Notch1 target genes and potentially other processes.

Aryl Hydrocarbon Receptor Interacting Protein Maintains Germinal Center B Cells through Suppression of BCL6 Degradation.

B cell lymphoma-6 (BCL6) is highly expressed in germinal center B cells, but how its expression is maintained is still not completely clear. Aryl hydrocarbon receptor interacting protein (AIP) is a co-chaperone of heat shock protein 90. Deletion of Aip in B cells decreased BCL6 expression, reducing germinal center B cells and diminishing adaptive immune responses. AIP was required for optimal AKT signaling in response to B cell receptor stimulation, and AIP protected BCL6 from ubiquitin-mediated proteasomal degradation by the E3-ubiquitin ligase FBXO11 by binding to the deubiquitinase UCHL1, thus helping to maintain the expression of BCL6. AIP was highly expressed in primary diffuse large B cell lymphomas compared to healthy tissue and other tumors. Our findings describe AIP as a positive regulator of BCL6 expression with implications for the pathobiology of diffuse large B cell lymphoma.

Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state.

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naïve' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro Amongst these are the pluripotency-linked genes Klf4 and Lin28b The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human.